# Patent

## Title
Purified and isolated platelet calcium channel nucleic acids and polypeptides and therapeutic and screening methods using same

## Abstract

Isolated and purified platelet voltage dependent calcium channel (VDCC) α1 subunit polypeptides, and nucleic acid molecules encoding the same. Recombinant host cells, recombinant nucleic acids, and recombinant proteins are also disclosed, along with methods of producing each. Isolated and purified antibodies to platelet VDCC α1 subunit polypeptides, and methods of producing the same, are also disclosed. Platelet VDCC α1 subunit polypeptides have biological activity in calcium transport. Thus, therapeutic and diagnostic methods involving this activity are also disclosed.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a continuation-in-part of U.S. patent application Ser. No. 10/029,413, filed Dec. 20, 2001, now U.S. Pat. No. 7,041,475 which itself is based on and claims priority to U.S. Provisional Patent Application Ser. No. 60/258,169 filed Dec. 22, 2000, the entire contents of both of which are herein incorporated by reference.


GRANT STATEMENT
This work was supported by NIH grants I-P20-DE123474 and I-P60-DE 13079. Thus, the U.S. Government has certain rights in the presently claimed subject matter.


TECHNICAL FIELD
The presently claimed subject matter relates generally to isolated and purified proteins and nucleic acids. In one embodiment, the presently claimed subject matter relates to modulating phosphatidylserine exposure on the surface of a cell.










Table of Abbreviations


















2D
two-dimensional



32Pi
radioactive inorganic phosphate



ACD
acid-citrate-dextrose



ADP
adenosine diphosphate



ASO
allele-specific oligonucleotide



A-T
ataxia-telangiectasia



ATP
adenosine triphosphate



b.i.d.
bis in die; twice a day



bp
basepair (s)



BSA
bovine serum albumin



[Ca2+]i
Ca2+ concentration (intracellular)



cAMP
cyclic adenosine monophosphate



CDR
complementarity-determining region



cmRNA
complementary mRNA



DHP
dihydropyridine



ECL
electrochemiluminescence



ELISA
enzyme-linked immunosorbent assay



EM
electron microscope/microscopy



EST
expressed sequence tag



FACS
fluorescence activated cell sorting



FISH
fluorescence in situ hybridization



FITC
fluorescein isothiocyanate



fl
full length



hα1D
human platelet VDCC α1D subunit



hα1S
human platelet VDCC α1S subunit



HAT
cell culture media comprising hypoxanthine,




aminopterin, and thymidine



IMAGE
integrated molecular analysis of genomes and




their expression



kDa
kilodalton (s)



KLH
keyhole limpet hemocyanin



kV
kilovolt (s)



L
liter (s)



LAT
ligation-activated translation



LCR
ligase chain reaction



MAb
monoclonal antibody



MAb 1A
Monoclonal Antibody 1A



MCF
mean cell fluorescence



μg
microgram (s)



mL
milliliter (s)



MTD
maximally tolerated dose



NASBA TM
nucleic acid sequence-based amplification



nm
nanometer (s)



nt
nucleotide (s)



OCS
open canalicular system



OLAs
oligonucleotide ligation assays



ORF
open reading frame



P2x
an adenosine diphosphate receptor



pα1D
porcine platelet VDCC α1D subunit



pα1S
porcine platelet VDCC α1S subunit



PADGEM
platelet activation-dependent granule-external




membrane protein; P-selectin



PAGE
polyacrylamide gel electrophoresis



PBS
phosphate buffered saline



PCR
polymerase chain reaction



PGI2
prostaglandin I2



PLSCR1
phospholipid scramblase 1



PRP
platelet-rich plasma



PS
phosphatidylserine



PVD
polyvinyl derivative



RACE
rapid amplification of conserved ends



RCR
repair chain reaction



RFLP
restriction fragment length polymorphism



r.t.
room temperature



RT-PCR
reverse transcription polymerase chain reaction



SDA
strand displacement activation



SDS
sodium dodecyl sulfate



SDS-PAGE
sodium dodecyl sulfate polyacrylamide gel




electrophoresis



SNPs
single nucleotide polymorphisms



SR
sarcoplasmic reticulum



SSC
standard saline citrate; 150 mM NaCl/15 mM




sodium citrate, pH 7.0



SSCP
single strand conformation polymorphism



SV40
Simian Virus 40



TLC
thin layer chromatography



VDCC(s)
voltage dependent calcium channel (s)



vWf
von Willebrand factor



wt
wild type










BACKGROUND ART
Calcium influx into the platelet controls important processes during platelet activation. Sage, 1997; Mills, 1996. The pathway for this calcium entry is not well understood. Recently, gating of an ADP receptor (P2x) has been suggested to be responsible for rapid calcium (Ca2+) influx into the platelet. Mackenzie et al., 1996; Sun et al., 1998. Indeed, several ions, including Ca2+, Mg2+, and Na+, enter when this receptor is activated. However, calcium entry via this receptor is believed not to be sufficient for the processes that take place during platelet activation. Jin & Kanapuli 1998; Kanapuli, 1998. P2x is an ionotropic channel, and its gating in other cell types, such as skeletal muscle cells, has an excitatory depolarizing effect that activates voltage dependent calcium channels (VDCCs). Bean, 1993; Surprenant et al., 1995; Abbrachio et al., 1994. It is the activation of these calcium selective VDCCs that triggers calcium dependent events inside these cells, such as secretion and contraction. Ashcroft, 2000; Boyd, 1992; Hille 1992; Armstrong & Hille, 1998; Berridge, 1997.
The presence of VDCCs in platelets is controversial (Sage, 1997), and heretofore their expression has not been investigated at the molecular level. Thus, in view of the role of calcium influx in controlling processes during platelet activation, the identification of a VDCC polypeptide in platelets represents a long-felt and continuing need in the art.
SUMMARY
This Summary lists several embodiments of the presently claimed subject matter, and in many cases lists variations and permutations of these embodiments. This Summary is merely exemplary of the numerous and varied embodiments. Mention of one or more representative features of a given embodiment is likewise exemplary. Such an embodiment can typically exist with or without the feature(s) mentioned; likewise, those features can be applied to other embodiments of the presently disclosed subject matter, whether listed in this Summary or not. To avoid excessive repetition, this Summary does not list or suggest all possible combinations of such features.
The presently claimed subject matter provides a method of screening candidate substances for an ability to modulate phosphatidylserine exposure on the surface of a cell. In one embodiment, the method comprises (a) establishing a test sample comprising a platelet voltage dependent calcium channel (VDCC) α1 subunit polypeptide; (b) administering a candidate substance to the test sample; and (c) measuring the interaction, effect, or combination thereof, of the candidate substance on the test sample to thereby determine the ability of the candidate substance to modulate phosphatidylserine exposure on the surface of the cell. In one embodiment, the candidate substance is a candidate polypeptide, in another embodiment an antibody, in another embodiment a nucleic acid, and in yet another embodiment a chemical compound.
In another embodiment of the present method, the method further comprises isolating a gene encoding the candidate polypeptide. In one embodiment, the test sample comprises a nucleic acid molecule encoding a platelet voltage dependent calcium channel (VDCC) α1 subunit polypeptide. In one embodiment, the platelet voltage dependent calcium channel (VDCC) α1 subunit polypeptide is contained within a cell in cell culture. In one embodiment, the measuring comprises (a) determining a first level of phosphatidylserine exposure on a surface of the test sample before the administering step (b); (b) determining a second level of phosphatidylserine exposure on the surface of the test sample after the administering step (b); and (c) comparing the first level and the second level, wherein a difference between the first level and the second level is indicative of the ability of the candidate substance to modulate phosphatidylserine exposure on the surface of the cell. In one embodiment, the determining comprises (a) contacting the test sample with an antibody comprising a detectable moiety, wherein the antibody binds to phosphatidylserine; and (b) quantitating an amount of the antibody bound to the test sample, wherein the amount of the antibody bound to the test sample is indicative of an amount of phosphatidylserine on the surface of the test sample. In one embodiment, the detectable moiety is selected from the group consisting of a radiolabel, a fluorescent label, a chemiluminescent label, and an enzyme. In one embodiment, the quantitating is by a method selected from the group consisting of radioactive detection, fluorescence detection, chemiluminescent detection, calorimetric detection, Western blotting, immunoprecipitation, and fluorescence-activating cell sorting (FACS).
In one embodiment of the present method, the interaction, effect, or combination thereof, of the candidate substance on the test sample results in an increase in phosphatidylserine exposure on the surface of the cell. In another embodiment, the interaction, effect, or combination thereof, of the candidate substance on the test sample results in a decrease in phosphatidylserine exposure on the surface of the cell.
The presently claimed subject matter also provides a method of screening candidate substances for an ability to modulate phosphatidylserine exposure on a surface of a cell comprising (a) establishing a test sample comprising a modulatable transcriptional regulatory sequence of a platelet voltage dependent calcium channel (VDCC) α1 subunit polypeptide-encoding gene and a reporter gene which is capable of producing a detectable signal; (b) administering a candidate substance to the test sample; and (c) measuring the detectable signal produced as a result of an interaction between the candidate substance and the test sample, wherein a candidate substance as a modulator of phosphatidylserine exposure on a surface of a cell is based on the amount of signal produced in relation to a control sample. In one embodiment, the reporter gene encodes a platelet voltage dependent calcium channel (VDCC) α1 subunit polypeptide.
The presently claimed subject matter also provides a method of screening candidate substances for an ability to modulate platelet voltage dependent calcium channel (VDCC) α1 subunit biological activity. In one embodiment, the method comprises (a) establishing a test sample comprising a platelet voltage dependent calcium channel (VDCC) α1 subunit polypeptide; (b) administering a candidate substance to the test sample; and (c) measuring the interaction, effect, or combination thereof, of the candidate substance on the test sample to thereby determine the ability of the candidate substance to modulate platelet voltage dependent calcium channel (VDCC) α1 subunit biological activity. The candidate substance is in one embodiment a candidate polypeptide, in another embodiment an antibody, in another embodiment a nucleic acid, and in still another embodiment a chemical compound. In one embodiment, the method further comprises the step of purifying and isolating a gene encoding the candidate polypeptide.
In one embodiment of the present method, the test sample comprises a nucleic acid molecule encoding a platelet voltage dependent calcium channel (VDCC) α1 subunit polypeptide. In one embodiment, the platelet voltage dependent calcium channel (VDCC) α1 subunit polypeptide is contained within cells in cell culture. In another embodiment of the present method, the measuring comprises (a) determining a first level of phosphatidylserine exposure in the test sample before the administering step (b); (b) determining a second level of phosphatidylserine exposure in the test sample after the administering step (b); and (c) comparing the first level and the second level, wherein a difference between the first level and the second level is indicative of the ability of the candidate substance to modulate platelet voltage dependent calcium channel (VDCC) α1 subunit biological activity.
In still another embodiment of the present method, the determining comprises (a) contacting the test sample with an antibody comprising a detectable moiety, wherein the antibody binds to phosphatidylserine; and (b) quantitating an amount of the antibody bound to the test sample, wherein the amount of the antibody bound to the test sample is indicative of the ability of the candidate substance to modulate platelet voltage dependent calcium channel (VDCC) α1 subunit biological activity. In one embodiment, the detectable moiety is selected from the group consisting of a radiolabel, a fluorescent label, a chemiluminescent label, and an enzyme. In still another embodiment of the present method, the quantitating is by a method chosen from the group consisting of radioactive detection, fluorescence detection, chemiluminescent detection, calorimetric detection, Western blotting, immunoprecipitation, and fluorescence-activating cell sorting (FACS).
The presently claimed subject matter also provides a method of screening candidate substances for an ability to modulate platelet voltage dependent calcium channel (VDCC) α1 subunit biological activity. In one embodiment, the method comprises (a) establishing a test sample comprising a modulatable transcriptional regulatory sequence of a platelet voltage dependent calcium channel (VDCC) α1 subunit polypeptide-encoding gene and a reporter gene which is capable of producing a detectable signal; (b) administering a candidate substance to the test sample; and (c) measuring the detectable signal produced as a result of an interaction between the candidate substance and the test sample, wherein a candidate substance as a modulator of platelet voltage dependent calcium channel (VDCC) α1 subunit biological activity is based on the amount of signal produced in relation to a control sample. In one embodiment, the reporter gene encodes a platelet voltage dependent calcium channel (VDCC) α1 subunit polypeptide. The presently claimed subject matter also provides a method of modulating platelet voltage dependent calcium channel (VDCC) α1 subunit polypeptide biological activity in a cell, the method comprising administering to the cell an effective amount of a substance capable of modulating activity of a platelet voltage dependent calcium channel (VDCC) α1 subunit polypeptide in the cell to thereby modulate platelet voltage dependent calcium channel (VDCC) α1 subunit polypeptide biological activity in the cell. In one embodiment, the cell is a platelet or a megakaryocyte. In another embodiment, the cell comprises a cell in a vertebrate subject. In one embodiment, the vertebrate subject is a mammal.
In one embodiment of the presently claimed subject matter, the administering further comprises administering an effective amount of a substance that modulates expression of a platelet voltage dependent calcium channel (VDCC) α1 subunit polypeptide-encoding nucleic acid molecule in the cell. The substance that modulates the platelet voltage dependent calcium channel (VDCC) α1 subunit biological activity comprises in one embodiment an anti-platelet voltage dependent calcium channel (VDCC) α1 subunit polypeptide antibody, in another embodiment a polypeptide, in another embodiment a chemical compound, and in still another embodiment a nucleic acid. In one embodiment, the nucleic acid comprises an antisense oligonucleotide. In one embodiment, the polypeptide or chemical compound comprises a ligand for a modulatable transcriptional regulatory sequence of a platelet voltage dependent calcium channel (VDCC) α1 subunit polypeptide-encoding nucleic acid molecule.
The presently claimed subject matter also provides a method of modulating phosphatidylserine exposure on a surface of a cell, the method comprising administering to the cell an effective amount of a substance capable of modulating the activity of a platelet voltage dependent calcium channel (VDCC) α1 subunit polypeptide in the cell to thereby modulate phosphatidylserine exposure on the surface of the cell. In one embodiment, the cell is a platelet, and in another embodiment the cell is a megakaryocyte. In one embodiment, the cell comprises a cell in a vertebrate subject. In one embodiment, the vertebrate subject is a mammal. In one embodiment of the present method, the administering further comprises administering an effective amount of a substance that modulates expression of a platelet voltage dependent calcium channel (VDCC) α1 subunit polypeptide-encoding nucleic acid molecule in the cell.
In one embodiment of the present method, the substance capable of modulating the activity of a platelet voltage dependent calcium channel (VDCC) α1 subunit polypeptide in the cell comprises an anti-platelet voltage dependent calcium channel (VDCC) α1 subunit polypeptide antibody, in another embodiment a polypeptide, in another embodiment a chemical compound, and in still another embodiment a nucleic acid. In one embodiment, the nucleic acid comprises an antisense oligonucleotide. In one embodiment, the polypeptide or chemical compound comprises a ligand for a modulatable transcriptional regulatory sequence of a platelet voltage dependent calcium channel (VDCC) α1 subunit polypeptide-encoding nucleic acid molecule.
The presently claimed subject matter also provides a method for modulating phosphatidylserine exposure on a surface of a cell comprising introducing to the cell a construct comprising a nucleic acid sequence encoding a platelet voltage dependent calcium channel (VDCC) α1 subunit polypeptide operatively linked to a promoter, wherein production of the platelet voltage dependent calcium channel (VDCC) α1 subunit polypeptide in the cell results in modulation of phosphatidylserine exposure on a surface of the cell. In one embodiment, the construct further comprises a vector selected from the group consisting of a plasmid vector and a viral vector. In another embodiment, the construct further comprises a liposome complex. In one embodiment, the cell is a platelet or a megakaryocyte. In another embodiment, the cell comprises a cell in a vertebrate subject. In one embodiment, the vertebrate subject is a mammal.
The presently claimed subject matter also provides a method for modulating thrombosis in a subject, the method comprising administering to the subject an effective amount of a substance capable of modulating the activity of a platelet voltage dependent calcium channel (VDCC) α1 subunit polypeptide in the cell, wherein thrombosis in the subject is modulated. In one embodiment, the subject is a mammal. In one embodiment, the mammal is a human.
In one embodiment of the present method, the administering further comprises administering an effective amount of a substance that modulates expression of a platelet voltage dependent calcium channel (VDCC) α1 subunit polypeptide-encoding nucleic acid molecule in the cell. The substance that modulates expression of a platelet voltage dependent calcium channel (VDCC) α1 subunit polypeptide-encoding nucleic acid molecule in the cell comprises in one embodiment an anti-platelet voltage dependent calcium channel (VDCC) α1 subunit polypeptide antibody, in another embodiment a polypeptide, in another embodiment a chemical compound, and in still another embodiment a nucleic acid. In one embodiment, the nucleic acid comprises an antisense oligonucleotide. In one embodiment, the polypeptide or chemical compound comprises a ligand for a modulatable transcriptional regulatory sequence of a platelet voltage dependent calcium channel (VDCC) α1 subunit polypeptide-encoding nucleic acid molecule.
The presently claimed subject matter also provides a method for modulating thrombosis in a subject, the method comprising introducing into a cell present within the subject a construct comprising a nucleic acid sequence encoding a platelet voltage dependent calcium channel (VDCC) α1 subunit polypeptide operatively linked to a promoter, wherein production of the platelet voltage dependent calcium channel (VDCC) α1 subunit polypeptide in the cell results in modulation of thrombosis in the subject. In one embodiment, the construct further comprises a vector selected from the group consisting of a plasmid vector and a viral vector. In another embodiment, the construct further comprises a liposome complex. In one embodiment, the cell is a platelet or a megakaryocyte. In one embodiment, the subject is a mammal. In one embodiment, the mammal is a human.
The presently claimed subject matter also provides a method for modulating calcium transport in a cell, the method comprising introducing to the cell a construct comprising a nucleic acid sequence encoding a platelet voltage dependent calcium channel (VDCC) α1 subunit polypeptide operatively linked to a promoter, wherein production of the platelet voltage dependent calcium channel (VDCC) α1 subunit polypeptide in the cell results in modulation of calcium transport. In one embodiment, the construct further comprises a vector selected from the group consisting of a plasmid vector or a viral vector. In another embodiment, the construct further comprises a liposome complex. In one embodiment, the cell is a platelet and in another embodiment the cells is a megakaryocyte. In still another embodiment, the cell comprises a cell in a vertebrate subject. In one embodiment, the vertebrate subject is a mammal.
The presently claimed subject matter also provides a method of modulating apoptosis in a cell selected from the group consisting of a platelet and a megakaryocyte and comprising a voltage dependent calcium channel (VDCC) α1 subunit polypeptide. In one embodiment, the method comprises (a) identifying a candidate substance that modulates the exposure of phosphatidylserine on a surface of the platelet or megakaryocyte; and (b) contacting the cell with the candidate substance identified in step (a), whereby apoptosis in the cell is modulated. In one embodiment, the exposure of phosphatidylserine on a surface of the cell is increased, thereby enhancing apoptosis in the cell. In another embodiment, the exposure of phosphatidylserine on a surface of the cell is decreased, thereby inhibiting apoptosis in the cell. In one embodiment, the cell is a platelet.
The presently claimed subject matter also provides pharmaceutical compositions of the modulators identified by the disclosed screening methods. In one embodiment, a pharmaceutical composition comprises a therapeutically effective amount of a modulator of phosphatidylserine exposure on a surface of a cell and a pharmaceutically acceptable diluent or vehicle. In one embodiment, the modulator of phosphatidylserine exposure on a surface of a cell binds a platelet voltage dependent calcium channel (VDCC) α1 subunit polypeptide, or a fragment or derivative thereof. In another embodiment, a pharmaceutical composition comprises a therapeutically effective amount of a modulator of thrombosis and a pharmaceutically acceptable diluent or vehicle, wherein the modulator of thrombosis binds a platelet voltage dependent calcium channel (VDCC) α1 subunit polypeptide, or a fragment or derivative thereof. In still another embodiment, a pharmaceutical composition comprises a therapeutically effective amount of a modulator of a biological activity of a platelet voltage dependent calcium channel (VDCC) α1 subunit polypeptide and a pharmaceutically acceptable diluent or vehicle. In one embodiment, the modulator binds a platelet voltage dependent calcium channel (VDCC) α1 subunit polypeptide, or a fragment or derivative thereof.
The presently claimed subject matter also provides a method of increasing a storage life of a cell selected from the group consisting of a platelet and a megakaryocyte. In one embodiment, the method comprises (a) identifying a candidate substance that decreases the exposure of phosphatidylserine on the surface of the cell; and (b) contacting the cell with the candidate substance identified in step (a), whereby the storage life of the cell is increased. In one embodiment, the cell is a platelet.
The presently claimed subject matter also provides recombinant cell lines suitable for use in the claimed methods.
An object of the presently claimed subject matter having been stated herein above, other objects will become evident as the description proceeds when taken in connection with the accompanying Figures and Examples as best described herein below.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A is a schematic presentation of platelet VDCC α1 subunit polypeptides cloned from human megakaryocytes and porcine platelets. A human platelet VDCC α1S subunit clone (hα1S, 3130 bp) from human megakaryocytes encodes a polypeptide between III S4 and the CO2− end of the human α1S. A 400 bp platelet VDCC α1 subunit clone encodes the intra-cytoplasmic loop between motifs II-III. A human platelet VDCC α1D subunit (hα1D, 2682 bp) clone from human megakaryocytes encodes a polypeptide between IIIP and CO2− end of the human α1D subunit. A porcine platelet VDCC α1S subunit polypeptide (pα1S, 1031 bp) clone from porcine platelets encodes a polypeptide region between IV S3 and amino acid 1531 of α1S. A porcine platelet VDCC α1D subunit polypeptide (pα1D, 210 bp) clone from porcine platelets encodes a polypeptide between II S3 and II S5. ●=glutamate residue in cloned regions; +=positively charged residue in cloned regions; P=pore in cloned regions; Δ=residues that contribute to dihydropyridine (DHP) binding.
FIG. 1B is a schematic depicting sequences absent from human α1S and α1D and porcine α1S in cloned regions (shaded areas).
FIG. 1C depicts a photograph of gel electrophoresis of a 1033 basepair (bp) polymerase chain reaction (PCR) product from human megakaryocytes that was producing using oligonucleotides 1 and 2 (Table 3) as primers to amplify cDNA regions. The cDNA clones obtained in this reaction had two different sequences. Clones 1 and 2 (lanes 1 and 2) were α1S, clone 3 (lane 3) was α1D. Left lane, DNA low molecular weight markers VI (2.1-0.5 kb, Boehringer Mannheim, Indianapolis, Ind., United States of America)
FIG. 1D depicts an autoradiograph of a Northern blot of mRNA from human megakaryocytes. 10 μg of mRNA was loaded per lane and probed with riboprobes synthesized from the PCR products described for FIG. 1C. An antisense mRNA (hereinafter “complementary mRNA” or “cmRNA”) synthesized from clone 1 (α1S) was used to probe the mRNA on the left lane and cmRNA from clone 3 (α1D) was synthesized and used to probe the mRNA on the right lane. Arrows point to a 6.5 kilobase (kb) transcript in α1S and a 10.8 kb transcript in α1D. In addition, a 2.5 kb band hybridized with both probes, which are homologous but not identical. Bars on right are 9.4 kb, 7.46 kb, 4.4 kb, and 2.37 kb molecular weight standards.
FIG. 2A depicts an autoradiograph of a Western blot on cultured megakaryocytes, which depicts that α1S and α1D subunits are expressed at the protein level in platelets and megakaryocytes. Lane a: the antiserum generated in rabbit against the peptide NEELRAIIKKIWKRTSMKLL (SEQ ID NO: 27) located in the putative carboxyl end of α1 subunits (arrow in FIG. 1) recognized its determinant in a 170 kilodalton (kDa) polypeptide. Lane b: a monoclonal antibody, Mab 1A, previously described to recognize its determinant in α1S from skeletal muscle, was used to confirm that the 170 kDa polypeptide is α1S. Lane c: normal rabbit IgG. Lane d: β cells from pancreas confirmed that the anti-peptide antibody recognized its determinant in α1D (208 kDa) in β cells. Lane e: the monoclonal antibody Mab 1A did not recognize a determinant in α1D. Lane f: normal non-immune rabbit serum. Bars on the right depict the positions of 218 kDa and 125 kDa molecular weight standards.
FIG. 2B depicts an autoradiograph of an immunoblot of immunoprecipitations on porcine platelets. The anti-peptide antibody precipitated determinants in polypeptides with electrophoretic mobilities of 208 kDa and 170 kDa (lane a, arrows). The primary antibody was replaced with non-immune rabbit IgG in the immunoprecipitation control condition (lane b). Bars on right depict the positions of 218 kDa and 125 kDa molecular weight standards.
FIGS. 3A-3F depicts a nucleotide sequence alignment of a human platelet VDCC α1S subunit gene fragment of SEQ ID NO: 5 and three closely related sequences: GENBANK® Accession No. XM—001910 (SEQ ID NO: 9; FIGS. 3A-3B), GENBANK® Accession No. NM—000069 (SEQ ID NO: 11; FIGS. 3C-3D), and GENBANK® Accession No. L33798 (SEQ ID NO: 13; FIGS. 3E-3F).
FIG. 4 depicts a nucleotide sequence alignment of a porcine platelet VDCC α1S subunit gene fragment of SEQ ID NO: 7 and a human VDCC α1S subunit fragment sequence: GENBANK® Accession No. XM—001910 (SEQ ID NO: 9).
FIGS. 5A-5F depict a nucleotide sequence alignment of a human platelet VDCC α1D subunit fragment of SEQ ID NO: 6 and three closely related sequences: GENBANK® Accession No. XM—003238 (SEQ ID NO: 15; FIGS. 5A and 5B), GENBANK® Accession No. NM—000720 (SEQ ID NO: 17; FIGS. 5C and 5D), and GENBANK® Accession No. M83566 (SEQ ID NO: 19; FIGS. 5E and 5F).
FIG. 6 depicts the effects of monoclonal antibody MA3-920 on intracellular platelet calcium concentration ([Ca2+]i) at various thrombin concentrations (p<0.003; student's t test).
FIG. 7 depicts the effects of the antagonist nitrendipine on platelet [Ca2+]i at various thrombin concentrations.
FIG. 8 depicts the effects of the agonist Bay K(−) on platelet [Ca2+ ]i at various thrombin concentrations.



BRIEF DESCRIPTION OF THE SEQUENCE LISTING
SEQ ID NOs: 1 and 2 are nucleic acid and deduced amino acid sequences, respectively, of a human platelet VDCC family member (ha1S).
SEQ ID NOs: 3 and 4 are nucleic acid and deduced amino acid sequences, respectively, of a human platelet VDCC family member (ha1D).
SEQ ID NO: 5 is a nucleic acid sequence of a partial cDNA for a human platelet VDCC family member (clone 990312Ca50ha1S1181-1392).
SEQ ID NO: 6 is a nucleic acid sequence of a partial cDNA for a human platelet VDCC family member (clone 990303Ca61ha1D1294-1490).
SEQ ID NO: 7 is a nucleic acid sequence of a partial cDNA for a porcine platelet VDCC family member (clone 990208Cb18pa1 S1172-1365).
SEQ ID NO: 8 is a nucleic acid sequence of a partial cDNA for a porcine platelet VDCC family member (clone 990210Db29pa1S1511-1325).
SEQ ID NOs: 9 and 10 are the nucleic acid and deduced amino acid sequences, respectively for CACNA1S, a human calcium channel, voltage-dependent, L type, alpha 1S subunit (GENBANKTM Accession No. XM—001910).
SEQ ID NOs: 11 and 12 are the nucleic acid and deduced amino acid sequences, respectively for CACNA1S, a human calcium channel, voltage-dependent, L type, alpha 1S subunit (GENBANKTM Accession No. NM—000069).
SEQ ID NOs: 13 and 14 are the nucleic acid and deduced amino acid sequences, respectively for CACNL1A3, a human dihydropyridine-sensitive L-type calcium channel alpha-1 subunit (GENBANKTM Accession No. L33798).
SEQ ID NOs: 15 and 16 are the nucleic acid and deduced amino acid sequences, respectively for CACNA1D, a human calcium channel, voltage-dependent, L type, alpha 1D subunit (GENBANKTM Accession No. XM—003238).
SEQ ID NOs: 17 and 18 are the nucleic acid and deduced amino acid sequences, respectively, for CACNA1D, a human calcium channel, voltage-dependent, L type, alpha 1 D subunit (GENBANKTM Accession No. NM—000720).
SEQ ID NOs: 19 and 20 are the nucleic acid and deduced amino acid sequences, respectively, for HUMCACH1A, a human neuroendocrine/beta-cell-type calcium channel alpha-1 subunit (GENBANKTM Accession No. M83566).
SEQ ID NOs: 21 and 22 are the nucleic acid and deduced amino acid sequences, respectively, for OCDHPR, a rabbit skeletal muscle dihydropyridine (DHP) receptor (GENBANKTM Accession No. X05921).
SEQ ID NO: 23 is the 57 nucleotide sequence that is missing in the human platelet VDCC family member (ha1S; SEQ ID NO: 1) when compared to the human calcium channel, voltage-dependent, L type, alpha 1S subunit (SEQ ID NO: 11).
SEQ ID NO: 24 is the amino acid sequence deduced from SEQ ID NO: 23.
SEQ ID NO: 25 is the 45 nucleotide sequence that is missing in the human platelet VDCC family member (ha1D; SEQ ID NO: 3) when compared to the human calcium channel, voltage-dependent, L type, alpha 1D subunit (SEQ ID NO: 15).
SEQ ID NO: 26 is the amino acid sequence deduced from SEQ ID NO: 25.
SEQ ID NO: 27 is the amino acid sequence of a peptide antigen (NEELRAIIKKIWKRTSMKLL), which corresponds to the sequence of amino acids 1487-1506 (arrow in FIG. 1A) in the putative intracytoplasmic carboxyl-terminal region of adult rabbit and human calcium channel, voltage-dependent, L type, alpha 1S subunit polypeptide as disclosed in Tanabe et al., 1987 and Hogan et al., 1994.
SEQ ID NO: 28 is the nucleic acid sequence of the platelet ha1D junction.
SEQ ID NO: 29 is the nucleic acid sequence of the platelet ha1S junction.
DETAILED DESCRIPTION
Disclosed herein is the first evidence for the expression of VDCC α1 subunits in platelets. The presently claimed subject matter teaches novel members of the VDCC gene family that are expressed in platelets. Representative embodiments are set forth in SEQ ID NOs: 1-8, 28, and 29.
Thus, the presently claimed subject matter pertains to isolated and purified nucleic acids encoding platelet VDCC α1 subunit polypeptides, to isolated and purified platelet VDCC α1 subunit polypeptides, to the characterization of the role played by the platelet VDCC α1 subunit polypeptides in modulating calcium levels within and outside cells, and to the characterization of upstream or downstream processes affected by such modulation (e.g., inside-out and/or outside-in signaling and phosphatidylserine exposure on the surface of a cell).
Summarily, the identification of sequences that encode platelet VDCC α1 subunit polypeptides, the cloning of the corresponding cDNAs, and the expression of the corresponding proteins affords the molecular tools required for modulating calcium homeostasis in platelets, and has application in the development of diagnostic, pharmacological, and/or therapeutic applications, including treatments for various bleeding, thrombotic, and related disorders in human and animal subjects.
Following long-standing patent law convention, the terms “a” and “an” mean “one or more” when used in this application, including the claims.
A. General Considerations
Diversity among VDCCs, expressed in their biophysical and pharmacological properties, has led to their classification into several categories (Birnbaumer et al., 1994). The long-activating (L-type) dihydropyridine (DHP)-sensitive VDCC is such a channel. cDNA cloning of its major, pore forming α1 subunit from skeletal muscle, α1S, has led to a proposed structural model for all α1 subunits. Tanabe et al., 1987; see also FIG. 1. This model predicts that the encoded polypeptide contains four homologous but not identical tandem motifs (I-IV) which are made up of six transmembrane regions (S1-S6) each. This α1 subunit contains sequences that convey to the channel calcium selectivity, voltage and pharmacological sensitivities, gating properties, and susceptibility to bind with other subunits and neighboring proteins. Hille, 1992; Peres-Reyes & Schneider, 1995. While α1S is the major subunit from L-type VDCC in skeletal muscle, α1D is that from neuroendocrine cells. Birnbaumer et al., 1994; Seino et al., 1992.
The expression and ultrastructural localization of the VDCCs in platelets and their progenitor cell, the megakaryocyte, has been investigated. The novel polypeptides and polynucleotides disclosed herein represent the first identification of a VDCC in platelets and megakaryocytes.
The gene structure of platelet VDCC α1 subunits derived from human megakaryocytes and porcine platelets is depicted in FIG. 1. The characteristic feature of platelet VDCC α1 subunits as compared with known VDCC α1 subunits is indicated as a missing sequence in the IV S3-S4 linker. FIG. 2 demonstrates that platelet VDCC α1S and α1D subunit polypeptides are expressed in platelets and megakaryocytes.
In accordance with the presently disclosed subject matter, the functionality of recently described L-type voltage dependent calcium channels (VDCC) in platelets has also been characterized. There are many agonists and antagonists of the DHP-sensitive VDCC that have been developed for skeletal and cardiac muscle. As discussed in more detail herein below, several of these modulators were selected to investigate the role of VDCCs in thrombin activated human platelets. These modulators included a monoclonal antibody to the alpha 1 subunit of the DHP receptor (MA3-920), nitrendipine, and two stereoisomers of Bay K8644 (Bay K(−) and Bay K(+)).
B. Polypeptides and Polynucleotides
As used in the following detailed description and in the claims, the term “platelet VDCC” includes nucleic acids and polypeptides encoding calcium channels in platelets and megakaryocytes. Platelet VDCC α1S subunit nucleic acid sequences are expressed in platelets and megakaryocytes, and optionally in other tissues. The term “platelet VDCC” specifically refers to a VDCC α1 subunit characterized by the IV S3-S4 linker. Particularly, a platelet VDCC α1S subunit polypeptide and a platelet VDCC α1D subunit polypeptide are disclosed herein. Representative platelet VDCC α1S subunits from human and porcine sources are disclosed in SEQ ID NOs: 1, 2, 5, and 28 and SEQ ID NOs: 7-8, respectively. Representative human platelet VDCC α1D subunits are disclosed in SEQ ID NOs: 3, 4, 6, and 29.
The term “platelet VDCC” further comprises vertebrate homologues of platelet calcium channel family members including, but not limited to mammalian and avian homologues. Representative mammalian homologues of platelet calcium channel family members include, but are not limited to porcine and human homologues. VDCC α1 subunit homologues are characterized by missing sequences that encode the IV S3-S4 linker region of VDCC α1S polypeptides, as disclosed herein for human VDCC α1S subunit, porcine VDCC α1S subunit, and human VDCC α1D subunit.
As used herein, the term “polypeptide” means any polymer comprising any of the 20 protein amino acids, regardless of its size. Although “protein” is often used in reference to relatively large polypeptides, and “peptide” is often used in reference to small polypeptides, usage of these terms in the art overlaps and varies. The term “polypeptide” as used herein refers to peptides, polypeptides, and proteins, unless otherwise noted. As used herein, the terms “protein”, “polypeptide”, and “peptide” are used interchangeably herein when referring to a gene product.
The terms “platelet VDCC α1 subunit gene product”, “platelet VDCC α1 subunit protein”, and “platelet VDCC α1 subunit polypeptide” refer to peptides having amino acid sequences which are substantially identical to native amino acid sequences from the organism of interest and which are biologically active in that they comprise the amino acid sequence of a platelet VDCC α1 subunit polypeptide, or cross-react with antibodies that specifically bind a platelet VDCC α1 subunit polypeptide, or retain all or some of the biological activity of the native amino acid sequence or protein. Such biological activity can include immunogenicity. As disclosed herein below, an important feature of platelet VDCC α1 subunits is the absence of sequences encoding a portion of the IV S3-S4 linker of VDCC α1 subunit polypeptides.
The terms “platelet VDCC α1 subunit gene product”, “platelet VDCC α1 subunit protein”, and “platelet VDCC α1 subunit polypeptide” are meant to encompass a platelet VDCC α1S subunit polypeptide or a platelet VDCC α1D subunit polypeptide including, but not limited to those disclosed herein as SEQ ID NOs: 2 and 4. Indeed, the definitions and descriptions of “platelet VDCC α1 subunit gene product”, “platelet VDCC α1 subunit protein”, and “platelet VDCC α1 subunit polypeptide” presented herein are also meant to be applied to a platelet VDCC α1S subunit polypeptide or a platelet VDCC α1D subunit polypeptide.
The terms “platelet VDCC α1 subunit gene product”, “platelet VDCC α1 subunit protein”, and “platelet VDCC α1 subunit polypeptide” also include analogs of a platelet VDCC α1 subunit polypeptide. By “analog” it is intended that a DNA or peptide sequence can contain alterations relative to the sequences disclosed herein, yet retain all or some of the biological activity of those sequences. Analogs can be derived from nucleotide sequences as are disclosed herein or from other organisms, or can be created synthetically. Those skilled in the art will appreciate that other analogs, as yet undisclosed or undiscovered, can be used to design and/or construct calcium channel analogs. There is no need for a “platelet VDCC α1 subunit gene product”, “platelet VDCC α1 subunit protein”, and “platelet VDCC α1 subunit polypeptide” to comprise all or substantially all of the amino acid sequence of a native platelet VDCC α1 subunit polypeptide gene product. Shorter or longer sequences are anticipated to be of use in the presently claimed subject matter; shorter sequences are herein referred to as “segments”. Thus, the terms “platelet VDCC α1 subunit gene product”, “platelet VDCC α1 subunit protein”, and “platelet VDCC α1 subunit polypeptide” also include fusion or recombinant platelet VDCC α1 subunit polypeptides and proteins comprising sequences of the presently claimed subject matter. Methods of preparing such proteins are known in the art.
The terms “platelet VDCC α1 subunit gene”, “platelet VDCC α1 subunit gene segment”, “platelet VDCC α1 subunit gene sequence”, “platelet VDCC α1 subunit polynucleotide”, “platelet VDCC α1 subunit nucleic acid molecule”, and “platelet VDCC α1 subunit nucleic acid sequence” refer to any nucleic acid sequence (e.g., a DNA sequence) that is substantially identical to a polynucleotide sequence encoding a platelet VDCC α1 subunit gene product, platelet VDCC α1 subunit protein, or platelet VDCC α1 subunit polypeptide as defined above, and can also comprise any combination of associated control sequences. The terms also refer to RNA, or antisense sequences, complementary to such DNA sequences. As used herein, the term “DNA segment” refers to a DNA molecule that has been isolated free of total genomic DNA of a particular species. Furthermore, a DNA segment encoding a platelet VDCC α1 subunit polypeptide refers to a DNA segment that contains platelet VDCC α1 subunit coding sequences, yet is isolated away from, or purified free from, total genomic DNA of a source species, such as Homo sapiens. Included within the term “DNA segment” are DNA segments and smaller fragments of such segments, and also recombinant vectors, including, for example, plasmids, cosmids, phages, viruses, and the like.
The terms “platelet VDCC α1 subunit gene”, “platelet VDCC α1 subunit gene segment”, “platelet VDCC α1 subunit gene sequence”, “platelet VDCC α1 subunit polynucleotide”, “platelet VDCC α1 subunit nucleic acid molecule”, and “platelet VDCC α1 subunit nucleic acid sequence” are meant to encompass a polynucleotide encoding a platelet VDCC α1S subunit polypeptide or a platelet VDCC α1D subunit polypeptide, including but not limited to those disclosed herein as SEQ ID NOs: 1, 3, 5-8, 28, and 29. Indeed, the definitions and explanations of the terms “platelet VDCC α1 subunit gene”, “platelet VDCC α1 subunit gene segment”, “platelet VDCC α1 subunit gene sequence”, “platelet VDCC α1 subunit polynucleotide”, “platelet VDCC α1 subunit nucleic acid molecule”, and “platelet VDCC α1 subunit nucleic acid sequence” presented herein are also meant to be applied to a platelet VDCC α1S subunit polypeptide or a platelet VDCC α1D subunit polypeptide.
A characteristic feature of the platelet VDCC α1S subunits of the presently claimed subject matter is the absence of sequences that encode a portion of the IV S3-S4 linker of known VDCC α1S subunits. The sequences that are lacking in the disclosed platelet VDCC α1S subunits are set forth as SEQ ID NOs: 23-24 (α1S) and SEQ ID NOs: 25-26 (α1D). The absence of these sequences from the platelet VDCC α1S subunits of the presently claimed subject matter is depicted in FIGS. 1A, 1B, and 3-5.
FIGS. 1A and 1B show a schematic representation of the sequences that are absent from human and porcine platelet VDCC α1 subunits in the region of the IV S3-S4 linker.
FIG. 3 depicts a nucleotide sequence alignment of a human platelet VDCC α1S subunit fragment of SEQ ID NO: 5 and three closely related sequences, GENBANK®) Accession No. XM—001910 (SEQ ID NO: 9), GENBANK® Accession No. NM—00069 (SEQ ID NO: 11), and GENBANK® Accession No. L33798 (SEQ ID NO: 13). The nucleotide sequence of SEQ ID NO: 5 represents an individual sequence read of a cloned human platelet VDCC α1S subunit fragment. When using this fragment as a query sequence, BLAST analysis indicated the three above-mentioned GENBANK® sequences as having the highest level of sequence identity with platelet VDCC α1S subunit sequences of the presently claimed subject matter. The alignment further reveals the absence of an about 57 base pair sequence (SEQ ID NO: 23) in the human platelet VDCC α1S subunit fragment of SEQ ID NO: 5 compared to known VDCC α1S subunit sequences. The deleted sequence is revealed by disruption of each alignment between contiguous base pairs 48 and 49 of the query sequence, and substantial sequence conservation is observed over the remainder of the query sequence.
FIG. 4 depicts a nucleotide sequence alignment of a porcine platelet VDCC α1S subunit gene fragment of SEQ ID NO: 7 and a human VDCC α1S subunit fragment sequence, GENBANK®) Accession No. XM—001910 (SEQ ID NO: 9). The nucleotide sequence of SEQ ID NO: 7 represents an individual sequence read of a cloned porcine platelet VDCC α1S subunit fragment. When using this fragment as a query sequence, BLAST analysis indicated human VDCC α1S subunit gene (GENBANK® Accession No. XM—001910, SEQ ID NO: 9) and rabbit dihydropyridine receptor (GENBANK® Accession No. X05921, SEQ ID NO: 21) as being the most closely related sequences (Table 1). The alignment further reveals the absence of an about 57 base pair sequence (SEQ ID NO: 23) in the porcine platelet VDCC α1S subunit fragment of SEQ ID NO: 7, as the alignment is disrupted between base pairs 60 and 67 base pairs of the query sequence, but substantial sequence conservation is observed over the remainder of the query sequence.












TABLE 1





SEQ
Applicants'
Best





ID NO.
Reference
BLAST hit
Score
E value
Identities







7
990210Db29
XM
761
0.0
516/599



pa1S1511
001910


92%


8
990208Cb18
X05921
563
0.0
395/432



pa1S1172



91%









A comparison of the predicted amino acid sequences encoded by the human and porcine platelet VDCC α1S subunit fragments demonstrates that the deleted sequence, relative to known VDCC α1S subunit sequences, is similarly positioned in human and porcine platelet VDCC α1S subunit sequences (FIGS. 1A and 1B).
FIG. 5 depicts a nucleotide sequence alignment of a human platelet VDCC α1D subunit gene fragment of SEQ ID NO: 6 and three closely related sequences: GENBANK® Accession No. XM—003238 (SEQ ID NO: 15), GENBANK® Accession No. NM—000720 (SEQ ID NO: 17), and GENBANK®) Accession No. M83566 (SEQ ID NO: 19). The nucleotide sequence of SEQ ID NO: 6 represents an individual sequence read of a cloned human platelet VDCC α1D subunit fragment. When using this fragment as a query sequence, BLAST analysis indicated the three above-mentioned GENBANK® sequences as having the highest level of sequence identity with platelet VDCC α1D subunit sequences of the presently claimed subject matter. The alignment further reveals the absence of an about 48 basepair sequence (SEQ ID NO: 25) in the human platelet VDCC α1D subunit fragment of SEQ ID NO: 6 as compared to known sequences. The deleted sequence is revealed by disruption of each alignment between contiguous base pairs 44 and 49 of the query sequence. Substantial sequence conservation is observed over the remainder of the query sequence.
B.1. Sequence Similarity and Identity
As used herein, the term “substantially similar” means that a particular sequence varies from a platelet VDCC α1 subunit nucleic acid sequence, or a platelet VDCC α1 subunit amino acid sequence, by one or more deletions, substitutions, or additions, the net effect of which is to retain at least some of biological activity of the natural gene, gene product, or sequence. For example, a “substantially similar” polypeptide can exhibit decreased or increased biological activity, as in a pathological or disease- or dysfunction-causing condition. Such sequences include “mutant” or “polymorphic” sequences, or sequences in which the biological activity is altered to some degree but retains at least some of the original biological activity. A critical measure of substantial identity of a platelet VDCC α1 subunit is the absence of nucleotide sequences encoding and amino acid sequences comprising (SEQ ID NOs: 23-26) a region of the IV S3-S4 linker of VDCC α1 subunit polypeptides.
Nucleic acids that are substantially identical to SEQ ID NOs: 1, 3, 5-8, 28, and 29 are representative platelet VDCC α1 subunit sequences; e.g., allelic variants, genetically altered versions of the gene, etc., bind to the provided platelet VDCC α1 subunit sequences under stringent hybridization conditions. By using probes, particularly labeled probes of DNA sequences, one can isolate homologous or related genes. The source of homologous genes can be any species, e.g., primate species, rodents (such as rats and mice), canines, felines, bovines, equines, etc.
Between mammalian species, for example human and pig, homologues have substantial sequence similarity; in one embodiment at least 75% sequence identity between nucleotide sequences and in another embodiment at least 90% sequence identity. Sequence similarity is calculated based on a reference sequence, which can be a subset of a larger sequence, such as a conserved motif, coding region, flanking region, etc. A reference sequence will usually be at least about 18 nucleotides (nt) long, more usually at least about 30 nt long, and can extend to the complete sequence that is being compared. Algorithms for sequence analysis are known in the art, such as BLAST, described in Altschul et al., 1990. In determining nucleic acid sequences, all subject nucleic acid sequences capable of encoding substantially similar amino acid sequences are considered to be substantially similar to a reference nucleic acid sequence, regardless of differences in codon sequences or substitution of equivalent amino acids to create biologically functional equivalents.
Percent identity or percent similarity of a DNA or peptide sequence can be determined, for example, by comparing sequence information using the GAP computer program, available from the Accelrys, Inc. (San Diego, Calif., United States of America). The GAP program utilizes the alignment method of Needleman et al., 1970, as revised by Smith et al., 1981. Briefly, the GAP program defines similarity as the number of aligned symbols (i.e., nucleotides or amino acids) which are similar, divided by the total number of symbols in the shorter of the two sequences. Representative parameters for the GAP program are the default parameters, which do not impose a penalty for end gaps. See Schwartz et al., 1979; Gribskov et al., 1986.
The term “similarity” is contrasted with the term “identity”. Similarity is defined as above; “identity”, however, means a nucleic acid or amino acid sequence having the same amino acid at the same relative position in a given family member of a gene family. Homology and similarity are generally viewed as broader terms than the term identity. Biochemically similar amino acids, for example leucine and isoleucine or glutamate and aspartate, can be present at the same position—these are not identical per se, but are biochemically “similar.” As disclosed herein, these are referred to as conservative differences or conservative substitutions. This differs from a conservative mutation at the DNA level, which changes the nucleotide sequence without making a change in the encoded amino acid, e.g., TCC to TCA, both of which encode serine.
As used herein, DNA analog sequences are “substantially identical” to specific DNA sequences disclosed herein if: (a) the DNA analog sequence is derived from coding regions of the nucleic acid sequence shown in any of SEQ ID NOs: 1, 3, 5-8, 28, and 29 and lacks sequences of SEQ ID NOs: 23 and 25; or (b) the DNA analog sequence is capable of hybridization to any of SEQ ID NOs: 1, 3, 5-8, 28, and 29 under stringent conditions, lacks sequences of SEQ ID NOs: 23 and 25, and encodes a biologically active gene product of the nucleic acid sequence shown in any of SEQ ID NOs: 1, 3, 5-8, 28, and 29; or (c) the DNA sequences are degenerate as a result of alternative genetic code to the DNA analog sequences defined in (a) and/or (b). Substantially identical analog proteins will be greater than about 60% identical to the corresponding sequence of the native protein. Sequences having lesser degrees of identity but comparable biological activity are considered to be equivalents.
As used herein, “stringent conditions” means conditions of high stringency, for example 6× standard saline citrate (SSC), 0.2% polyvinylpyrrolidone, 0.2% Ficoll, 0.2% bovine serum albumin, 0.1% sodium dodecyl sulfate, 100 g/mL salmon sperm DNA and 15% formamide at 68° C. For the purposes of specifying additional conditions of high stringency, exemplary conditions are salt concentration of about 200 mM and temperature of about 45° C. One example of such stringent conditions is hybridization at 4×SSC at 65° C., followed by a washing in 0.1×SSC at 65° C. for one hour. Another exemplary stringent hybridization scheme uses 50% formamide, 4×SSC at 42° C. Stringent features are understood to be able to detect a remoter address.
In contrast, nucleic acids having sequence similarity are detected by hybridization under lower stringency conditions. Thus, sequence identity can be determined by hybridization under lower stringency conditions, for example, at 50° C. or higher and 0.1×SSC (15 mM NaCl/1.5 mM sodium citrate) and the sequences will remain bound when subjected to washing at 55° C. in 1×SSC.
Thus, in certain embodiments, the presently claimed subject matter concerns the use of platelet VDCC α1 subunit genes and gene products that include within their respective sequences a sequence which is essentially that of a platelet VDCC α1 subunit gene, or the corresponding protein. The term “a sequence essentially as that of a platelet VDCC α1 subunit gene”, means that the sequence is substantially identical to a portion of a platelet VDCC α1 subunit gene and contain a minority of bases or amino acids (whether DNA or protein) which are not identical to those of a platelet VDCC α1 subunit protein or a platelet VDCC α1 subunit gene, or which are not a biologically functional equivalent. The term “biologically functional equivalent” is well understood in the art and is further defined in detail herein.
Nucleotide sequences are “substantially identical” where they have in one embodiment between about 70% and about 80%, in another embodiment between about 81% and about 90%, and in still another embodiment between about 91% and about 99%, sequence identity for nucleic acid residues which are identical to the nucleotide sequence of a platelet VDCC α1 subunit gene. Gene structure is also a useful measure of substantially identical sequences. In this case, the omission of sequences encoding a region of the IV S3-S4 linker of VDCC α1 subunit polypeptides is a critical feature of the disclosed sequences.
Peptide sequences which have in one embodiment from 35% to 45%, in another embodiment from 45 to 55%, in another embodiment from 55 to 65%, or in yet another embodiment 65% or greater, amino acids which are identical or functionally equivalent or biologically functionally equivalent to the amino acids of a platelet VDCC α1 subunit polypeptide will be sequences which are “substantially similar”. Peptide structure is also a useful indicator of substantially identical sequences. The presently claimed subject matter discloses sequences that have a shorter IV S3-S4 linker compared to known VDCC α1 subunit polypeptides. Thus, this is an important feature when considering substantial similarity among VDCC α1 subunit sequences.
Platelet VDCC α1 subunit gene products and platelet VDCC α1 subunit-encoding nucleic acid sequences which have functionally equivalent codons are also covered by the presently claimed subject matter. The term “functionally equivalent codon” is used herein to refer to codons that encode the same amino acid, such as the ACG and AGU codons for serine (Table 2). Thus, when referring to the sequence examples presented in SEQ ID NOs: 1, 3, 5-8, 28, and 29 applicants contemplate substitution of functionally equivalent codons of Table 2 into the sequence examples of SEQ ID NOs: 1, 3, 5-8, 28, and 29. Thus, applicants are in possession of amino acid and nucleic acids sequences which include such substitutions but which are not set forth herein in their entirety for convenience.







TABLE 2







Functionally Equivalent Codons










Amino Acid
3-Letter
1-Letter
mRNA Codons





Alanine
Ala
A
GCA GCC GCG GCU


Arginine
Arg
R
AGA AGG CGA CGC CGG CGU


Asparagine
Asn
N
AAC AAU


Aspartic Acid
Asp
D
GAC GAU


Cysteine
Cys
C
UGC UGU


Glutamic Acid
Glu
E
GAA GAG


Glutamine
Gln
Q
CAA CAG


Glycine
Gly
G
GGA GGC GGG GGU


Histidine
His
H
CAC CAU


Isoleucine
lie
I
AUA AUC AUU


Leucine
Leu
L
UUA UUG CUA CUC CUG CUU


Lysine
Lys
K
AAA AAG


Methionine
Met
M
AUG


Proline
Pro
P
CCA CCC CCG CCU


Phenylalanine
Phe
F
UUC UUU


Serine
Ser
S
ACG AGU UCA UCC UCG UCU


Threonine
Thr
T
ACA ACC ACG ACU


Tryptophan
Trp
W
UGG


Tyrosine
Tyr
Y
UAC UAU


Valine
Val
V
GUA GUC GUG GUU









It will also be understood by those of skill in the art that amino acid and nucleic acid sequences can include additional residues, such as additional N- or C-terminal amino acids or 5′ or 3′ nucleic acid sequences, and yet still be essentially as set forth in one of the sequences disclosed herein, so long as the sequence retains biological protein activity where protein expression is concerned. The addition of terminal sequences particularly applies to nucleic acid sequences which can, for example, include various non-coding sequences flanking either of the 5′ or 3′ portions of the coding region or can include various internal sequences such as introns, which are known to occur within genes.
The presently claimed subject matter also encompasses the use of nucleotide segments that are complementary to the sequences of the presently claimed subject matter. Nucleic acid sequences which are “complementary” are those which are base-paired according to the standard Watson-Crick complementarity rules. As used herein, the term “complementary sequences” means nucleic acid sequences which are substantially complementary, as can be assessed by the same nucleotide comparison set forth above, or is defined as being capable of hybridizing to the nucleic acid segment in question under moderately stringent conditions such as those described herein. A particular example of a provided complementary nucleic acid segment is an antisense oligonucleotide. In this case, complementary is defined by both cross-hybridization and the lack of coding sequences of SEQ ID NOs: 23 and 25.
Hybridization can be used to assess complementary sequences and/or to isolate complementary nucleotide sequences. Nucleic acid hybridization will be affected by such conditions as salt concentration, temperature, or organic solvents, in addition to the base composition, length of the complementary strands, and the number of nucleotide base mismatches between the hybridizing nucleic acids, as will be readily appreciated by those skilled in the art. Stringent temperature conditions will generally include temperatures in one embodiment in excess of about 30° C., in another embodiment in excess of about 37° C., and in still another embodiment in excess of about 45° C. Stringent salt conditions will in one embodiment be less than about 1,000 mM, in another embodiment less than about 500 mM, and in still another embodiment less than about 200 mM. However, the combination of parameters is much more important than the measure of any single parameter. (See e.g., Wetmur & Davidson, 1968). Determining appropriate hybridization conditions to identify and/or isolate sequences containing high levels of identity is well known in the art. (See e.g., Sambrook et al., 1989).
Representative moderate stringency conditions comprise, for example, hybridization at 50° C. and in 10×SSC (1.5 M NaCl/0.15 M sodium citrate), wherein the hybridized nucleic acid molecules remain bound when subjected to washing at 55° C. in 1×SSC. Sequence identity can be further determined by hybridization under more stringent conditions, for example, at 50° C. or higher and 0.1×SSC (15 mM NaCl/1.5 mM sodium citrate), as described below. Typically, under “stringent conditions” a probe will hybridize specifically to its target sequence, but to no other sequences.
For the purposes of specifying conditions of high stringency, exemplary conditions are salt concentration of about 200 mM and temperature of about 45° C. One example of such stringent conditions is hybridization at 4×SSC, at 65° C., followed by a washing in 0.1×SSC at 65° C. for one hour. Another representative stringent hybridization scheme uses 50% formamide, 4×SSC at 42° C. As used herein, “stringent conditions” can also mean conditions of high stringency, for example 6×SSC, 0.2% polyvinylpyrrolidone, 0.2% Ficoll, 0.2% bovine serum albumin, 0.1% sodium dodecyl sulfate, 100 μg/mL salmon sperm DNA and 15% formamide at 60° C.
Nucleic acids that are substantially identical to the provided platelet VDCC α1 subunit sequences, e.g., allelic variants, genetically altered versions of the gene, polymorphic sequences, etc., bind to the provided platelet VDCC α1 subunit sequences under stringent hybridization conditions. By using probes, particularly labeled probes of DNA sequences, one can isolate homologous or related genes. The source of homologous genes can be any species, for example, primate species, rodents (such as rats and mice), canines, felines, bovines, ovines, equines, etc.
Between mammalian species, (for example, human and pig), homologues display similar gene structure, have substantial sequence similarity: in one embodiment at least 75% sequence identity between nucleotide sequences and in another embodiment greater than 90% sequence similarity, and specifically lack nucleotide sequences set forth as SEQ ID NOs: 23 and 25. Sequence similarity is calculated based on a reference sequence, which can be a subset of a larger sequence, such as a conserved motif, coding region, flanking region, etc. A reference sequence is in one embodiment at least about 18 nt long and in another embodiment at least about 30 nt long, and can extend to the complete sequence that is being compared. Algorithms for sequence analysis are known in the art, such as BLAST, described in Altschul et al., 1990. Another commonly used alignment program is entitled CLUSTAL W and is described in Thompson et al., 1994, among other places. The sequences provided herein are useful for recognizing platelet VDCC α1 subunit related and homologous proteins in database searches.
At a biological level, identity is just that, i.e. the same amino acid at the same relative position in a given family member of a gene family. Homology and similarity are generally viewed as broader terms. For example, biochemically similar amino acids, for example leucine and isoleucine or glutamate/aspartate, can be present at the same position—these are not identical per se, but are biochemically “similar”. As disclosed herein, these are referred to as conservative differences or conservative substitutions. This differs from a conservative mutation at the DNA level, which changes the nucleotide sequence without making a change in the encoded amino acid, e.g., TCC to TCA, both of which encode serine.
The platelet VDCC α1 subunit genes disclosed herein are thus homologous proteins, but when percentages are referred to herein, it is meant to refer to percent identity.
Probe sequences can also hybridize specifically to duplex DNA under certain conditions to form triplex or other higher order DNA complexes. The preparation of such probes and suitable hybridization conditions are disclosed herein and are known in the art.
As used herein and in the claims, the term “gene” refers to both genomic sequences and cDNA sequences. “Isolated substantially away from other coding sequences” means that the gene of interest, in this case, a platelet VDCC α1 subunit gene, forms the significant part of the coding region of the DNA segment, and that the DNA segment does not contain large portions of naturally-occurring coding DNA, such as large chromosomal fragments or other functional genes or cDNA coding regions. Of course, this refers to the DNA segment as originally isolated, and does not exclude genes or coding regions later added to the segment by the hand of man.
Thus, in particular embodiments, the presently claimed subject matter concerns isolated DNA segments and recombinant vectors incorporating DNA sequences which encode a platelet VDCC α1 subunit polypeptide that includes within its amino acid sequence an amino acid sequence of the presently claimed subject matter. In other particular embodiments, the presently claimed subject matter concerns recombinant vectors incorporating DNA segments which encode a protein comprising the amino acid sequence of a human platelet VDCC α1 subunit polypeptide protein.
B.2. Biologically Functional Equivalents
As mentioned above, modifications and changes can be made in the structure of the platelet VDCC α1 subunit polypeptide proteins and peptides described herein and still constitute a molecule having like or otherwise desirable characteristics. For example, certain amino acids can be substituted for other amino acids in a protein structure without appreciable loss of interactive capacity with, for example, structures in the nucleus of a cell. Since it is the interactive capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid sequence substitutions can be made in a protein sequence (or the nucleic acid sequence encoding it) to obtain a protein with the same, enhanced, or antagonistic properties. Such properties can be achieved by interaction with the normal targets of the native protein, but this need not be the case, and the biological activity of the presently claimed subject matter is not limited to a particular mechanism of action. It is thus provided in accordance with the presently claimed subject matter that various changes can be made in the sequence of the platelet VDCC α1 subunit polypeptide proteins and peptides or underlying nucleic acid sequence without appreciable loss of their biological utility or activity.
Biologically functional equivalent peptides, as used herein, are peptides in which certain, but not most or all, of the amino acids can be substituted. Thus, when referring to the sequence examples presented in SEQ ID NOs: 1, 3, 5-8, 28, and 29, applicants envision substitution of codons that encode biologically equivalent amino acids as described herein into the sequence examples of SEQ ID NOs: 1, 3, 5-8, 28, and 29. Thus, applicants are in possession of amino acid and nucleic acids sequences which include such substitutions but which are not set forth herein in their entirety for convenience.
Alternatively, functionally equivalent proteins or peptides can be created via the application of recombinant DNA technology, in which changes in the protein structure can be engineered, based on considerations of the properties of the amino acids being exchanged, e.g., substitution of Ile for Leu. Changes designed by man can be introduced through the application of site-directed mutagenesis techniques, e.g., to introduce improvements to the antigenicity of the protein or to test platelet VDCC α1 subunit mutants in order to examine platelet VDCC α1 subunit calcium transport activity, or other activity at the molecular level.
Amino acid substitutions, such as those which might be employed in modifying the platelet VDCC α1 subunit polypeptide proteins and peptides described herein, are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. An analysis of the size, shape, and type of the amino acid side-chain substituents reveals that arginine, lysine, and histidine are all positively charged residues; that alanine, glycine, and serine are all of similar size; and that phenylalanine, tryptophan, and tyrosine all have a generally similar shape. Therefore, based upon these considerations, arginine, lysine, and histidine; alanine, glycine, and serine; and phenylalanine, tryptophan, and tyrosine; are defined herein as biologically functional equivalents. Other biologically functionally equivalent changes will be appreciated by those of skill in the art.
In making biologically functional equivalent amino acid substitutions, the hydropathic index of amino acids can be considered. Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics, these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine (+2.5); methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine (−4.5).
The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is generally understood in the art (Kyte & Doolittle, 1982, incorporated herein by reference). It is known that certain amino acids can be substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity. In one embodiment, amino acids for which the hydropathic indices are within ±2 of the original value are chosen, in another embodiment those for which the hydropathic indices are within ±1 of the original value are chosen, and in still another embodiment those for which the hydropathic indices are within ±0.5 of the original value are chosen when making amino acid substitutions based on hydropathic indices.
It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. U.S. Pat. No. 4,554,101, incorporated herein by reference, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigenicity, i.e. with a biological property of the protein. It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent protein.
As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4); proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4).
In making changes based upon similar hydrophilicity values, the substitution of amino acids whose hydrophilicity values are within ±2 of the original value are chosen in one embodiment, those that are within ±1 of the original value are chosen in another embodiment, and those within ±0.5 of the original value are chosen in still another embodiment.
While discussion has focused on functionally equivalent polypeptides arising from amino acid changes, it will be appreciated that these changes can be effected by alteration of the encoding DNA, taking into consideration also that the genetic code is degenerate and that two or more codons can code for the same amino acid.
Thus, it will also be understood that this presently claimed subject matter is not limited to the particular nucleic acid and amino acid sequences of SEQ ID NOs: 1-8, 28, and 29. Recombinant vectors and isolated DNA segments can therefore variously include the platelet VDCC α1 subunit polypeptide-encoding region itself, coding regions bearing selected alterations or modifications in the basic coding region, or larger polypeptides which nevertheless comprise platelet VDCC α1 subunit polypeptide-encoding regions or can encode biologically functional equivalent proteins or peptides. Biological activity of a platelet VDCC α1 subunit polypeptide can be determined, for example, by any of the assays disclosed herein below in Section G.2.
In particular embodiments, the presently claimed subject matter concerns gene therapy methods that use isolated DNA segments and recombinant vectors incorporating DNA sequences which encode a protein comprising an amino acid sequence of SEQ ID NO: 2 or 4. In other particular embodiments, the presently claimed subject matter concerns isolated DNA sequences and recombinant DNA vectors incorporating DNA sequences which encode a protein comprising an amino acid sequence of a platelet VDCC α1 subunit polypeptide protein from human or pig. In certain other embodiments, the presently claimed subject matter concerns isolated DNA segments and recombinant vectors that comprise a nucleic acid sequence essentially as set forth in any of SEQ ID NOs: 1, 3, 5-8, 28, and 29.
The nucleic acid segments of the presently claimed subject matter, regardless of the length of the coding sequence itself, can be combined with other DNA sequences, such as promoters, enhancers, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length can vary considerably. Thus, a nucleic acid fragment of almost any length can be employed, with the total length being limited in one embodiment by the ease of preparation and use in the intended recombinant DNA protocol. For example, nucleic acid fragments can be prepared which include a short stretch complementary to a nucleic acid sequence set forth in any of SEQ ID NOs: 1, 3, 5-8, 28, and 29, in one embodiment about 10 nucleotides, and which are up to 10,000 or 5,000 base pairs in length, with segments of 3,000 being an exemplary embodiment. DNA segments with total lengths of about 4,000, 3,000, 2,000, 1,000, 500, 200, 100, and about 50 base pairs in length are also useful.
The DNA segments of the presently claimed subject matter encompass biologically functional equivalent platelet VDCC α1 subunit polypeptides. Such sequences can rise as a consequence of codon redundancy and functional equivalency that are known to occur naturally within nucleic acid sequences and the proteins thus encoded. Alternatively, functionally equivalent proteins or peptides can be created via the application of recombinant DNA technology, in which changes in the protein structure can be engineered, based on considerations of the properties of the amino acids being exchanged. Changes can be introduced through the application of site-directed mutagenesis techniques, e.g., to introduce improvements to the antigenicity of the protein or to test platelet VDCC α1 subunit mutants in order to examine activity in the modulation of calcium transport, or other activity at the molecular level. Site-directed mutagenesis techniques are known to those of skill in the art and are disclosed herein.
The presently claimed subject matter further encompasses fusion proteins and peptides wherein the platelet VDCC α1 subunit polypeptide coding region is aligned within the same expression unit with other proteins or peptides having desired functions, such as for purification or immunodetection purposes.
Recombinant vectors form important further aspects of the presently claimed subject matter. Particularly useful vectors are those in which the coding portion of the DNA segment is positioned under the control of a promoter. The promoter can be that naturally associated with the platelet VDCC α1 subunit polypeptide gene, as can be obtained by isolating the 5′ non-coding sequences located upstream of the coding segment or exon, for example, using recombinant cloning and/or PCR technology and/or other methods known in the art, in conjunction with the compositions disclosed herein.
In other embodiments, certain advantages will be gained by positioning the coding DNA segment under the control of a recombinant, or heterologous, promoter. As used herein, a recombinant or heterologous promoter is a promoter that is not normally associated with a platelet VDCC α1 subunit polypeptide gene in its natural environment. Such promoters can include promoters isolated from bacterial, viral, eukaryotic, or mammalian cells. Representative heterologous promoters can thus comprise PF4, αIIb/β3 (GPIIb/IIIa), P-selectin, or GPIb promoter, which have been disclosed in the art. Naturally, it will be important to employ a promoter that effectively directs the expression of the DNA segment in the cell type chosen for expression. The use of promoter and cell type combinations for protein expression is generally known to those of skill in the art of molecular biology (for example, see Sambrook et al., 1989, specifically incorporated herein by reference). The promoters employed can be constitutive or inducible and can be used under the appropriate conditions to direct high level expression of the introduced DNA segment, such as is advantageous in the large-scale production of recombinant proteins or peptides. Appropriate promoter systems provided for use in high-level expression include, but are not limited to, the vaccinia virus promoter and the baculovirus promoter.
In an alternative embodiment, the presently claimed subject matter provides an expression vector comprising a polynucleotide that encodes a biologically active platelet VDCC α1 subunit polypeptide in accordance with the presently claimed subject matter. In one embodiment, an expression vector of the presently claimed subject matter comprises a polynucleotide that encodes human or pig platelet VDCC α1 subunit polypeptide gene product. In another embodiment, an expression vector of the presently claimed subject matter comprises a polynucleotide that encodes a polypeptide comprising an amino acid residue sequence of SEQ ID NO: 2 or 4. In another embodiment, an expression vector of the presently claimed subject matter comprises a polynucleotide comprising the nucleotide sequence of any of SEQ ID NOs: 1, 3, 5-8, 28, and 29. In another embodiment, an expression vector of the presently claimed subject matter comprises a polynucleotide operatively linked to an enhancer-promoter. In still another embodiment, an expression vector of the presently claimed subject matter comprises a polynucleotide operatively linked to a prokaryotic promoter. Alternatively, an expression vector of the presently claimed subject matter can comprise a polynucleotide operatively linked to an enhancer-promoter that is a eukaryotic promoter and the expression vector further comprises a polyadenylation signal that is positioned 3′ of the carboxy-terminal amino acid and within a transcriptional unit of the encoded polypeptide. The expression vector can further comprise an intronic sequence from a platelet VDCC α1 subunit gene, another platelet or megakaryocytic intronic sequence, or any other intronic sequence, as it has been shown that the inclusion of an intronic sequence into an expression vector can increase levels of expression.
In yet another embodiment, the presently claimed subject matter provides a recombinant host cell transfected, infected, or adsorbed with a polynucleotide that encodes a biologically active platelet VDCC α1 subunit polypeptide in accordance with the presently claimed subject matter. SEQ ID NOs: 1-8, 28, and 29 set forth nucleotide and amino acid sequences from representative vertebrates, human and pig. Also provided by the presently claimed subject matter are homologous or biologically functionally equivalent polynucleotides and platelet VDCC α1 subunit polypeptides found in other vertebrates, including particularly dog and bovine homologues. In one embodiment, a recombinant host cell of the presently claimed subject matter is transfected with the polynucleotide that encodes human or pig platelet VDCC α1 subunit polypeptide. In another embodiment, a recombinant host cell of the presently claimed subject matter is transfected with the polynucleotide sequence encoding or set forth in any of SEQ ID NOs: 1-8, 28, and 29. In another embodiment, a recombinant host cell is a mammalian cell. In still another embodiment, a recombinant host cell is a platelet or a megakaryocyte.
In another aspect, a recombinant host cell of the presently claimed subject matter is a prokaryotic host cell, including parasitic and bacterial cells. In one embodiment, a recombinant host cell of the presently claimed subject matter is a bacterial cell, in one embodiment a strain of Escherichia coli. In another embodiment, a recombinant host cell comprises a polynucleotide under the transcriptional control of regulatory signals functional in the recombinant host cell, wherein the regulatory signals appropriately control expression of the platelet VDCC α1 subunit polypeptide in a manner to enable all necessary transcriptional and post-transcriptional modification.
In yet another embodiment, the presently claimed subject matter provides a method of preparing a platelet VDCC α1 subunit polypeptide comprising transfecting a cell with polynucleotide that encodes a biologically active platelet VDCC α1 subunit polypeptide in accordance with the presently claimed subject matter, to produce a transformed host cell, and maintaining the transformed host cell under biological conditions sufficient for expression of the polypeptide. The polypeptide can be isolated if desired, using any suitable technique. The host cell can be a prokaryotic or eukaryotic cell. In one embodiment, the prokaryotic cell is a bacterial cell of Escherichia coli. In one embodiment, a polynucleotide transfected into the transformed cell comprises the nucleotide base sequence of any of SEQ ID NOs: 1, 3, 5-8, 28, and 29. SEQ ID NOs: 1-8, 28, and 29 set forth nucleotide and amino acid sequences for representative vertebrates, human and pig. Also provided by the presently claimed subject matter are homologues or biologically equivalent platelet VDCC α1 subunit polypeptide polynucleotides and polypeptides found in other vertebrates, particularly warm-blooded vertebrates, more particularly mammals, and even more particularly bovine and rat homologues.
As mentioned above, in connection with expression embodiments to prepare recombinant platelet VDCC α1 subunit polypeptide proteins and peptides, it is contemplated that longer DNA segments will most often be used, with DNA segments encoding the entire platelet VDCC α1 subunit polypeptide protein, functional domains, or cleavage products thereof being representative examples. However, it will be appreciated that the use of shorter DNA segments to direct the expression of platelet VDCC α1 subunit polypeptides or core regions, such as can be used to generate anti-platelet VDCC α1 subunit polypeptide antibodies, also falls within the scope of the presently claimed subject matter.
DNA segments which encode peptide antigens in one embodiment from about 15 to about 50 amino acids in length and in another embodiment from about 15 to about 30 amino acids in length are contemplated to be particularly useful. DNA segments encoding peptides will generally have a minimum coding length in the order of about 45 to about 150, or to about 90 nucleotides. DNA segments encoding full length proteins can have a minimum coding length on the order of about 4,000 or 5,000 nucleotides for a protein in accordance with any of SEQ ID NOs: 1, 3, 5-8, 28, and 29. DNA segments of the presently claimed subject matter can contain 300, 400, 500, 1,000, 1,500, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500, or up to 5,000 nucleotides. Peptides of the presently claimed subject matter can contain 10, 20, 50, 100, 200, 300, 400, 500, 750, 1,000, or up to 1,500 amino acids.
B.3. Sequence Modification Techniques
Modifications to the platelet VDCC α1 subunit proteins and peptides described herein can be carried out using techniques known in the art, including site directed mutagenesis. Site-specific mutagenesis is a technique useful in the preparation of individual peptides, or biologically functional equivalent proteins or peptides, through specific mutagenesis of the underlying DNA. The technique further provides a ready ability to prepare and test sequence variants; for example, incorporating one or more of the foregoing considerations, by introducing one or more nucleotide sequence changes into the DNA. Site-specific mutagenesis allows the production of mutants through the use of specific oligonucleotide sequences which encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed. In one embodiment, a primer of about 17 to 30 nucleotides in length is employed, with about 5 to 10 residues on both sides of the junction of the sequence being altered.
In general, the technique of site-specific mutagenesis is well known in the art as exemplified by publications (see e.g., Adelman et al., 1983; Sambrook et al., 1989) and can be achieved in a variety of ways generally known to those of skill in the art.
B.4. Other Structural Equivalents
The knowledge of the structure of the platelet VDCC α1 subunit polypeptide of the presently claimed subject matter provides a tool for investigating the mechanism of action of these proteins in a subject. For example, binding of these proteins to various substrate molecules can be predicted by various computer models. Upon discovering that such binding in fact takes place, knowledge of the protein structure then allows design and synthesis of small molecules which mimic the functional binding of the platelet VDCC α1 subunit polypeptide to the substrate. This is the method of “rational” drug design, also described below.
Use of the isolated and purified platelet VDCC α1 subunit polypeptide of the presently claimed subject matter in rational drug design is thus provided in accordance with the presently claimed subject matter. Additional rational drug design techniques are described in U.S. Pat. Nos. 5,834,228 and 5,872,011, herein incorporated in their entirety.
Thus, in addition to the peptidyl compounds described herein, other sterically similar compounds can be formulated to mimic the key portions of the peptide structure. Such compounds can be used in the same manner as the peptides of the presently claimed subject matter and hence are also functional equivalents. The generation of a structural functional equivalent can be achieved by the techniques of modeling and chemical design known to those of skill in the art. It will be understood that all such sterically similar constructs fall within the scope of the presently claimed subject matter.
C. Introduction of Gene Products
In accordance with the presently claimed subject matter, where a platelet VDCC α1 subunit gene itself is employed to introduce a platelet VDCC α1 subunit gene product, a convenient method of introduction will be through the use of a recombinant vector that incorporates the desired gene, together with its associated control sequences. The preparation of recombinant vectors is well known to those of skill in the art and described in many references, such as, for example, Sambrook et al., 1989, incorporated herein by reference in its entirety.
C.1. Vector Construction
It is understood that the DNA coding sequences to be expressed, in this case those encoding the platelet VDCC α1 subunit gene products, are positioned in a vector adjacent to and under the control of a promoter. It is understood in the art that to bring a coding sequence under the control of such a promoter, one generally positions the 5′ end of the transcription initiation site of the transcriptional reading frame of the gene product to be expressed between about 1 and about 50 nucleotides “downstream” of (i.e., 3′ of) the chosen promoter. One can also desire to incorporate into the transcriptional unit of the vector an appropriate polyadenylation site (e.g., 5′-AATAAA-3′), if one was not contained within the original inserted DNA. Typically, these poly-A addition sites are placed about 30 to 2000 nucleotides “downstream” of the coding sequence at a position prior to transcription termination.
While in one embodiment the control sequences of the specific gene is employed, other control sequences can be employed, so long as they are compatible with the genotype of the cell being treated. Thus, one can mention other useful promoters by way of example, including, e.g., a Simian Virus 40 (SV40) early promoter, a long terminal repeat promoter from retrovirus, an actin promoter, a heat shock promoter, a metallothionein promoter, and the like. Representative platelet specific promoters include but are not limited to PF4, αIIb (GPIIb), P-selectin or GPIb.
As is known in the art, a promoter is a region of a DNA molecule typically within about 100 nucleotide pairs upstream of (i.e., 5′ to) the point at which transcription begins (i.e., a transcription start site). That region typically contains several types of DNA sequence elements that are located in similar relative positions in different genes.
Another type of discrete transcription regulatory sequence element is an enhancer. An enhancer imposes specificity of time, location and expression level on a particular coding region or gene. A major function of an enhancer is to increase the level of transcription of a coding sequence in a cell that contains one or more transcription factors that bind to that enhancer. An enhancer can function when located at variable distances from transcription start sites so long as a promoter is present.
As used herein, the phrase “enhancer-promoter” means a composite unit that contains both enhancer and promoter elements. An enhancer-promoter is operatively linked to a coding sequence that encodes at least one gene product. As used herein, the phrase “operatively linked” means that an enhancer-promoter is connected to a coding sequence in such a way that the transcription of that coding sequence is controlled and regulated by that enhancer-promoter. Means for operatively linking an enhancer-promoter to a coding sequence are well known in the art; the precise orientation and location relative to a coding sequence of interest is dependent, inter alia, upon the specific nature of the enhancer-promoter.
An enhancer-promoter used in a vector construct of the presently claimed subject matter can be any enhancer-promoter that drives expression in a cell to be transfected. By employing an enhancer-promoter with well-known properties, the level and pattern of gene product expression can be optimized. The vector can further comprise an intronic sequence from a platelet VDCC α1 subunit gene, another platelet or megakaryocytic intronic sequence, or any other intronic sequence, as it has been shown that the inclusion of an intronic sequence into an vector can increase levels of expression, particularly in the case of a gene therapy vector.
For introduction of, for example, a human platelet VDCC α1 subunit gene, a vector construct that will deliver the gene to the affected cells is desired. Viral vectors can be used. In exemplary embodiments, these vectors can be an adenoviral, a retroviral, a vaccinia viral vector, adeno-associated virus or Lentivirus; these vectors have been successfully used to deliver desired sequences to cells and tend to have a high infection efficiency. Suitable vector-platelet VDCC α1 subunit gene constructs are adapted for administration as pharmaceutical compositions, as described herein below. Viral promoters can also be of use in vectors of the presently claimed subject matter, and are known in the art.
Commonly used viral promoters for expression vectors are derived from polyoma, cytomegalovirus, Adenovirus 2, and Simian Virus 40 (SV40). The early and late promoters of SV40 virus are particularly useful because both are obtained easily from the virus as a fragment that also contains the SV40 viral origin of replication. Smaller or larger SV40 fragments can also be used, provided there is included the approximately 250 bp sequence extending from the Hind III site toward the Bgl I site located in the viral origin of replication. Further, it is also possible, and often desirable, to utilize promoter or control sequences normally associated with the desired gene sequence, provided such control sequences are compatible with the host cell systems.
The origin of replication can be provided either by construction of the vector to include an exogenous origin, such as can be derived from SV40 or other viral source, or can be provided by the host cell chromosomal replication mechanism. If the vector is integrated into the host cell chromosome, the latter is often sufficient.
Where a platelet VDCC α1 subunit gene itself is employed it will be most convenient to simply use a wild type platelet VDCC α1 subunit gene directly. However, certain regions of a platelet VDCC α1 subunit gene can be employed exclusively without employing an entire wild type platelet VDCC α1 subunit gene. It is proposed that it will ultimately be advantageous to employ the smallest region needed to modulate biological activity so that one is not introducing unnecessary DNA into cells which receive a platelet VDCC α1 subunit gene construct. The biological activity of these regions can easily be determined by the assays reported herein.
C.2. Genetically Modified Cell Lines and Animals
It is also within the scope of the presently claimed subject matter to prepare a genetically modified cell line (e.g., platelet or megakaryocyte) and/or a genetically modified animal (e.g., a transgenic non-human animal) that expresses a platelet VDCC α1 subunit gene of the presently claimed subject matter, that does not express a platelet VDCC α1 subunit gene, or that has a modified expression of a platelet VDCC α1 subunit gene of the presently claimed subject matter. Representative transgenic animals include, but are not limited to mice, pigs, and dogs.
Techniques for the preparation of genetically modified cell lines are disclosed herein above, and are generally known in the art. Modified megakaryocyte and platelet cell lines have utility in the study of the biological activity and in the preparation and development of laboratory and medical applications for platelets, including enhanced ability to store or otherwise manipulate platelets.
Techniques for the preparation of transgenic animals are known in the art. Exemplary techniques are described in U.S. Pat. No. 5,489,742 (transgenic rats); U.S. Pat. Nos. 4,736,866, 5,550,316, 5,614,396, 5,625,125 and 5,648,061 (transgenic mice); U.S. Pat. No. 5,573,933 (transgenic pigs); U.S. Pat. No. 5,162,215 (transgenic avian species) and U.S. Pat. No. 5,741,957 (transgenic bovine species), the entire contents of each of which are herein incorporated by reference.
With respect to a representative method for the preparation of a transgenic pig, cloned recombinant or synthetic DNA sequences or DNA segments encoding a platelet VDCC α1 subunit polypeptide gene product are injected into fertilized eggs. The injected eggs are implanted in pseudo pregnant females and are grown to term to provide transgenic pigs whose cells express a platelet VDCC α1 subunit polypeptide gene product.
Additionally, a genetically modified animal of the presently claimed subject matter can comprise a pig with targeted modification of the platelet VDCC α1 subunit polypeptide gene. Pig strains with complete or partial functional inactivation of a platelet VDCC α1 subunit polypeptide gene in megakaryocytes and/or in platelets are generated using standard techniques of site-specific recombination in embryonic stem cells. Capecchi, 1989; Thomas & Capecchi, 1990; Delpire, 1999. Procedures analogous to those employed in the generation of a “knock-out” animal can be applied in the generation of a “knock-out” cell line.
Alternatives include the use of anti-sense or ribozyme VDCC constructs, driven by a universal or tissue-specific promoter, to reduce levels of a platelet VDCC α1 subunit polypeptide in platelets or megakaryocytes, thus achieving a “knock-down” of individual isoforms (Luyckx et al., 1999). The presently claimed subject matter also provides the generation of animal strains with conditional or inducible inactivation of individual or multiple VDCC genes (Sauer, 1998). For example, pigs are created which lack expression of any platelet VDCC α1 subunit polypeptide in platelets or megakaryocytes through the sequential mating of pig strains with lox-P-flanked VDCC genes with a transgenic line expressing Cre-recombinase in platelets or megakaryocytes, using a platelet specific promoter, such as PF4, αIIb (GPIIb), P-selectin, or GPIb (Ding et al., 1997).
The presently claimed subject matter also provides animal strains with specific “knocked-in” modifications in a platelet VDCC α1 subunit polypeptide gene. This includes animals with genetically (Forlino et al., 1999) and functionally (Kissel et al., 2000) relevant point mutations in the VDCC genes, in addition to manipulations such as the insertion of disease-specific repeat expansions (White et al., 1997).
D. Generation of Antibodies
In still another embodiment, the presently claimed subject matter provides an antibody that specifically binds a polypeptide of the presently claimed subject matter. In one embodiment, an antibody of the presently claimed subject matter is a monoclonal antibody. Techniques for preparing and characterizing antibodies are well known in the art (See e.g., Harlow & Lane, 1988).
The phrase “specifically (or selectively) binds to an antibody”, or “specifically (or selectively) immunoreactive with”, when referring to a protein or peptide, refers to a binding reaction which is determinative of the presence of the protein in a heterogeneous population of proteins and other biological materials. Thus, under designated immunoassay conditions, the specified antibodies bind to a particular protein and do not show significant binding to other proteins present in the sample. Specific binding to an antibody under such conditions can require an antibody that is selected for its specificity for a particular protein. For example, antibodies raised to a protein with an amino acid sequence encoded by any of the nucleic acid sequences of the presently claimed subject matter can be selected to obtain antibodies specifically immunoreactive with that protein and not with other proteins.
Briefly, a polyclonal antibody is prepared by immunizing an animal with an immunogen comprising a polypeptide or polynucleotide of the presently claimed subject matter, and collecting antisera from that immunized animal. A wide range of animal species can be used for the production of antisera. Because of the relatively large blood volume of rabbits, a rabbit is a representative choice for production of polyclonal antibodies.
As is well known in the art, a given polypeptide or polynucleotide can vary in its immunogenicity. It is often necessary therefore to couple the immunogen (e.g., a polypeptide or polynucleotide) of the presently claimed subject matter) with a carrier. Exemplary carriers are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albumins such as ovalbumin, mouse serum albumin or rabbit serum albumin can also be used as carriers.
Reagents for conjugating a polypeptide or a polynucleotide to a carrier protein are well known in the art and include glutaraldehyde, N-maleimidobenzoyl-N-hydroxysuccinimide ester, carbodiimide, and bis-diazotized benzidine.
As is also well known in the art, immunogenicity to a particular immunogen can be enhanced by the use of non-specific stimulators of the immune response known as adjuvants. Exemplary adjuvants include complete Freund's adjuvant, incomplete Freund's adjuvants, and aluminum hydroxide adjuvant.
The amount of immunogen used of the production of polyclonal antibodies varies, inter alia, upon the nature of the immunogen as well as the animal used for immunization. A variety of routes can be used to administer the immunogen, e.g., subcutaneous, intramuscular, intradermal, intravenous, and intraperitoneal. The production of polyclonal antibodies is monitored by sampling blood of the immunized animal at various points following immunization. When a desired level of immunogenicity is obtained, the immunized animal can be bled and the serum isolated and stored.
In another aspect, the presently claimed subject matter provides a method of producing an antibody that specifically binds a platelet VDCC α1 subunit polypeptide, the method comprising: (a) transfecting recombinant host cells with a polynucleotide that encodes that polypeptide; (b) culturing the host cells under conditions sufficient for expression of the polypeptide; (c) recovering the polypeptide; and (d) preparing antibodies to the polypeptide. In one embodiment, the platelet VDCC α1 subunit polypeptide is capable of modulating calcium levels within or outside of cells in accordance with the presently claimed subject matter.
A monoclonal antibody of the presently claimed subject matter can be readily prepared through use of well-known techniques such as the hybridoma techniques exemplified in U.S. Pat. No. 4,196,265 and the phage-displayed techniques disclosed in U.S. Pat. No. 5,260,203, the contents of which are herein incorporated by reference.
A typical technique involves first immunizing a suitable animal with a selected antigen (e.g., a polypeptide or polynucleotide of the presently claimed subject matter) in a manner sufficient to provide an immune response. Rodents such as mice and rats are representative animals. Spleen cells from the immunized animal are then fused with cells of an immortal myeloma cell. Where the immunized animal is a mouse, an exemplary myeloma cell is a murine NS-1 myeloma cell.
The fused spleen/myeloma cells are cultured in a selective medium to select fused spleen/myeloma cells from the parental cells. Fused cells are separated from the mixture of non-fused parental cells, for example, by the addition of agents that block the de novo synthesis of nucleotides in the tissue culture media. This culturing provides a population of hybridomas from which specific hybridomas are selected. Typically, selection of hybridomas is performed by culturing the cells by single-clone dilution in microtiter plates, followed by testing the individual clonal supernatants for reactivity with an antigen-polypeptides. The selected clones can then be propagated indefinitely to provide the monoclonal antibody.
By way of specific example, to produce an antibody of the presently claimed subject matter, mice are injected intraperitoneally with between about 1-200 μg of an antigen comprising a polypeptide of the presently claimed subject matter. B lymphocyte cells are stimulated to grow by injecting the antigen in association with an adjuvant such as complete Freund's adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis). At some time (e.g., at least two weeks) after the first injection, mice are boosted by injection with a second dose of the antigen mixed with incomplete Freund's adjuvant.
A few weeks after the second injection, mice are tail bled and the sera titered by immunoprecipitation against radiolabeled antigen. In one embodiment, the method of boosting and titering is repeated until a suitable titer is achieved. The spleen of the mouse with the highest titer is removed and the spleen lymphocytes are obtained by homogenizing the spleen with a syringe.
Mutant lymphocyte cells known as myeloma cells are obtained from laboratory animals in which such cells have been induced to grow by a variety of well-known methods. Myeloma cells lack the salvage pathway of nucleotide biosynthesis. Because myeloma cells are tumor cells, they can be propagated indefinitely in tissue culture, and are thus “immortal”. Numerous cultured cell lines of myeloma cells from mice and rats, such as murine NS-1 myeloma cells, have been established.
Myeloma cells are combined under conditions appropriate to foster fusion with the normal antibody-producing cells from the spleen of the mouse or rat injected with the antigen/polypeptide of the presently claimed subject matter. Fusion conditions include, for example, the presence of polyethylene glycol. The resulting fused cells are hybridoma cells. Like myeloma cells, hybridoma cells grow indefinitely in culture.
Hybridoma cells are separated from unfused myeloma cells by culturing in a selection medium such as HAT media (hypoxanthine, aminopterin, and thymidine). Unfused myeloma cells lack the enzymes necessary to synthesize nucleotides from the salvage pathway because they are killed in the presence of aminopterin, methotrexate, or azaserine. Unfused lymphocytes also do not continue to grow in tissue culture. Thus, only cells that have successfully fused (hybridoma cells) can grow in the selection media.
Each of the surviving hybridoma cells produces a single antibody. These cells are then screened for the production of the specific antibody that specifically binds an antigen/polypeptide of the presently claimed subject matter. Single cell hybridomas are isolated by limiting dilutions of the hybridomas. The hybridomas are serially diluted many times and, after the dilutions are allowed to grow, the supernatant is tested for the presence of the monoclonal antibody. The clones producing that antibody are then cultured in large amounts to produce an antibody of the presently claimed subject matter in convenient quantity.
By use of a monoclonal antibody of the presently claimed subject matter, specific polypeptides and polynucleotide of the presently claimed subject matter can be recognized as antigens, and thus identified. Once identified, those polypeptides and polynucleotide can be isolated and purified by techniques such as antibody-affinity chromatography. In antibody-affinity chromatography, a monoclonal antibody is bound to a solid substrate and exposed to a solution containing the desired antigen. The antigen is removed from the solution through an immunospecific reaction with the bound antibody. The polypeptide or polynucleotide is then easily removed from the substrate and purified.
E. Detecting a Polynucleotide or a Polypeptide
Alternatively, the presently claimed subject matter provides a method of detecting a polypeptide of the presently claimed subject matter, wherein the method comprises immunoreacting the polypeptides with antibodies prepared according to the method described above to form antibody-polypeptide conjugates, and detecting the conjugates.
In yet another embodiment, the presently claimed subject matter provides a method of detecting messenger RNA transcripts that encode a polypeptide of the presently claimed subject matter, wherein the method comprises hybridizing the messenger RNA transcripts with polynucleotide sequences that encode the polypeptide to form duplexes; and detecting the duplex. Alternatively, the presently claimed subject matter provides a method of detecting DNA molecules that encode a polypeptide of the presently claimed subject matter, wherein the method comprises hybridizing DNA molecules with a polynucleotide that encodes that polypeptide to form duplexes; and detecting the duplexes.
The detection and screening assays disclosed herein can optionally be used as a prognosis tool and/or diagnostic aid. Platelet VDCC α1 subunit polypeptides and nucleic acids can be readily used in clinical setting as a prognostic and/or diagnostic indicator for screening for levels of expression of platelet VDCC α1 subunit polypeptides, or alterations in native sequences. The nucleotide sequences of the subject presently claimed subject matter can be used to detect differences in gene or gene product sequences between normal, carrier, or affected individuals. As discussed herein above, such differences can consist of single-nucleotide changes or multiple changes, deletions, or additions in the native sequence which result in altered transcription, translation, or activity or biological activity or properties of the gene or gene product. These differences can be readily detected using the compositions of the presently claimed subject matter and techniques known in the art, including but not limited to single strand conformation polymorphism (SSCP) analysis, restriction fragment length polymorphism (RFLP) analysis, or other PCR- or nucleotide-based analyses.
DNA segments of the presently claimed subject matter or RNA having the sequence of, or a sequence complementary to, SEQ ID NOs: 1, 3, 5-8, 28, and 29 can be used. Such polynucleic acids can comprise 10, 20, 40, 50, 70, 100, 250, 300, 400, 500, or 1,000 nucleotides or up to the full length of SEQ ID NOs: 1, 3, 5-8, 28, and 29. Such polynucleic acids can, but need not, encode polypeptides which retain some or all of the biological activity of the native gene or gene product.
The presently claimed subject matter provides a method of screening a biological sample for the presence of a platelet VDCC α1 subunit polypeptide. A biological sample to be screened can be a biological fluid such as extracellular or intracellular fluid, or a cell or tissue extract or homogenate. A biological sample can also be an isolated cell (e.g., in culture) or a collection of cells such as in a tissue sample or histology sample. A tissue sample can be suspended in a liquid medium or fixed onto a solid support such as a microscope slide.
In accordance with a screening assay method, a biological sample is exposed to an antibody that specifically binds the polypeptide whose presence is being assayed. Typically, exposure is accomplished by forming an admixture in a liquid medium that contains both the antibody and the candidate polypeptide. Either the antibody or the sample with the polypeptide can be affixed to a solid support (e.g., a column or a microtiter plate). Additional details of methods for such assays are known in the art. The presence of polypeptide in the sample is detected by evaluating the formation and presence of antibody-polypeptide conjugates. Techniques for detecting such antibody-antigen conjugates or complexes are well known in the art and include but are not limited to centrifugation, affinity chromatography and the like, and binding of a secondary antibody to the antibody-candidate receptor complex.
In one embodiment, detection is accomplished by detecting an indicator affixed to the antibody. Exemplary and well-known indicators include radioactive labels (e.g., 32P, 125I, 14C), a second antibody, or an enzyme such as horseradish peroxidase. Techniques for affixing indicators to antibodies are known in the art.
In another aspect, the presently claimed subject matter provides a method of screening a biological sample for the presence of antibodies that specifically bind a platelet VDCC α1 subunit polypeptide. In one embodiment, the antibody so identified has activity in the modulation of platelet VDCC α1 subunit polypeptide biological activity in accordance with the presently claimed subject matter. In accordance with such a method, a biological sample is exposed to a platelet VDCC α1 subunit polypeptide under biological conditions and for a period of time sufficient for antibody-polypeptide conjugate formation and the formed conjugates are detected.
A DNA or RNA molecule and particularly a DNA segment or polynucleotide can be used for hybridization to a DNA or RNA source or sample suspected of encoding a platelet VDCC α1 subunit polypeptide of the presently claimed subject matter; such molecules are referred to as “probes”, and such hybridization is “probing”. Such probes can be made synthetically. The probing is usually accomplished by hybridizing the oligonucleotide to a DNA source suspected of possessing a platelet VDCC α1 subunit polypeptide gene. In some cases, the probes constitute only a single probe, and in others, the probes constitute a collection of probes based on a certain amino acid sequence or sequences of the polypeptide and account in their diversity for the redundancy inherent in the genetic code.
Other molecules which are neither DNA nor RNA but are capable of hybridizing in a similar manner and which are designed structurally to mimic the DNA or RNA sequence of a platelet VDCC α1 subunit polypeptide gene are also provided. Here, a suitable source to examine is capable of expressing a polypeptide of the presently claimed subject matter and can be a genomic library of a cell line of interest. Alternatively, a source of DNA or RNA can include total DNA or RNA from the cell line of interest. Once the hybridization method of the presently claimed subject matter has identified a candidate DNA segment, a positive clone can be confirmed by further hybridization, restriction enzyme mapping, sequencing and/or expression and testing.
Alternatively, such DNA molecules can be used in a number of techniques including their use as: (1) diagnostic tools to detect normal and abnormal DNA sequences in DNA derived from patient's cells; (2) reagents for detecting and isolating other members of the polypeptide family and related polypeptides from a DNA library potentially containing such sequences; (3) primers for hybridizing to related sequences for the purpose of amplifying those sequences; (4) primers for altering native platelet VDCC α1 subunit DNA sequences; as well as (5) other techniques which rely on the similarity of the sequences of interest to those of the sequences herein disclosed.
As set forth above, in certain aspects, DNA sequence information provided by the presently claimed subject matter allows for the preparation of probes that specifically hybridize to encoding sequences of a selected platelet VDCC α1 subunit gene. In these aspects, probes of an appropriate length are prepared based on a consideration of the encoding sequence for a polypeptide of this presently claimed subject matter. The ability of such probes to specifically hybridize to other encoding sequences lends them particular utility in a variety of embodiments. Most importantly, the probes can be used in a variety of assays for detecting the presence of complementary sequences in a given sample. However, other uses are envisioned, including the use of the sequence information for the preparation of mutant species primers, or primers for use in preparing other genetic constructions.
To provide certain of the advantages in accordance with the presently claimed subject matter, an exemplary nucleic acid sequence employed for hybridization studies or assays includes probe sequences that are complementary to or mimic at least a 14 to 40 or so long nucleotide stretch of a nucleic acid sequence of the presently claimed subject matter, such as a sequence shown in any of SEQ ID NOs: 1, 3, 5-8, 28, and 29. A size of at least 14 nucleotides in length helps to ensure that the fragment is of sufficient length to form a duplex molecule that is both stable and selective. Molecules having complementary sequences over stretches greater than 14 bases in length are generally employed, though, to increase stability and selectivity of the hybrid, and thereby improve the quality and degree of specific hybrid molecules obtained. One will generally design nucleic acid molecules having gene-complementary stretches of 14 to 20 nucleotides, or even longer where desired, such as 30, 40, 50, 60, 100, 200, 300, or 500 nucleotides or up to the full length of any of SEQ ID NOs: 1, 3, 5-8, 28, and 29. Such fragments can be readily prepared by, for example, directly synthesizing the fragment by chemical synthesis, by application of nucleic acid amplification technology, such as the PCR technology of U.S. Pat. No. 4,683,202, herein incorporated by reference, or by introducing selected sequences into recombinant vectors for recombinant production.
Accordingly, a nucleotide sequence of the presently claimed subject matter can be used for its ability to selectively form duplex molecules with complementary stretches of the gene. Depending on the application envisioned, one employs varying conditions of hybridization to achieve varying degrees of selectivity of the probe toward the target sequence. For applications requiring a high degree of selectivity, one typically employs relatively stringent conditions to form the hybrids. For example, one selects relatively low salt and/or high temperature conditions, such as provided by 15 to 300 mM salt (e.g., NaCl), in one embodiment 200 mM salt, at temperatures of 50° C. to 70° C., in one embodiment a temperature of about 55° C., in another embodiment about 60° C. and in still another embodiment about 65° C. Such conditions are particularly selective, and tolerate little, if any, mismatch between the probe and the template or target strand.
Of course, for some applications, less stringent hybridization conditions are typically needed to allow formation of the heteroduplex; one of skill in the art will know how to adjust the hybridization conditions for optimizing particular procedures. For example, it is generally appreciated that conditions can be rendered more stringent by the addition of increasing amounts of formamide, which serves to destabilize the hybrid duplex in the same manner as increased temperature. Thus, hybridization conditions can be readily manipulated by one of skill in the art using known methods to carry out the desired function or experiment, without undue experimentation.
In another aspect, the presently claimed subject matter provides assay kits for detecting the presence of a polypeptide of the presently claimed subject matter in biological samples, where the kits comprise a first antibody capable of immunoreacting with the polypeptide. In one embodiment, the assay kits of the presently claimed subject matter further comprise a second container containing a second antibody that immunoreacts with the first antibody. In another embodiment, the antibodies used in the assay kits of the presently claimed subject matter are monoclonal antibodies. In another embodiment, the first antibody is affixed to a solid support. In still another embodiment, the first and second antibodies comprise an indicator, and, in one embodiment, the indicator is a radioactive label or an enzyme.
The presently claimed subject matter also provides an assay kit for screening agents. Such a kit can contain a polypeptide of the presently claimed subject matter. The kit can additionally contain reagents for detecting an interaction between an agent and a polypeptide of the presently claimed subject matter.
In an alternative aspect, the presently claimed subject matter provides assay kits for detecting the presence, in biological samples, of a polynucleotide that encodes a polypeptide of the presently claimed subject matter, the kits comprising a first container that contains a second polynucleotide identical or complementary to a segment of at least 10 contiguous nucleotide bases of, as representative examples, any of SEQ ID NOs: 1, 3, 5-8, 28, and 29. In another embodiment, the presently claimed subject matter provides assay kits for detecting the presence, in a biological sample, of antibodies that specifically binds a polypeptide of the presently claimed subject matter, the kits comprising a platelet VDCC α1 subunit polypeptide that immunoreacts with the antibodies.
F. Mapping and Polynucleotide and Polypeptide Screening
In another embodiment of the presently claimed subject matter, the nucleic acid sequences which encode a platelet VDCC α1 subunit polypeptide can also be used to generate hybridization probes which are useful for mapping naturally occurring genomic sequences and/or disease loci. The sequences can be mapped to a particular chromosome or to a specific region of the chromosome using well-known techniques. Such techniques include, but are not limited to fluorescence in situ hybridization (FISH), fluorescence activated cell sorting (FACS), or artificial chromosome constructions, such as yeast artificial chromosomes, bacterial artificial chromosomes, bacterial P1 constructions or single chromosome cDNA libraries as reviewed in Price, 1993 and Trask, 1991.
F.1. Mapping
FISH (as described in Verma et al., 1988) can be correlated with other physical chromosome mapping techniques and genetic map data. Examples of genetic map data can be found in the 1994 Genome Issue of Science (volume 265, page 1981f). Correlation between the location of the gene encoding a platelet VDCC α1 subunit polypeptide on a physical chromosomal map and a specific disease, or predisposition to a specific disease, can help delimit the region of DNA associated with that genetic disease. The nucleotide sequences of the subject presently claimed subject matter can be used to detect differences in gene sequences between normal, carrier, or affected individuals.
In situ hybridization of chromosomal preparations and physical mapping techniques such as linkage analysis using established chromosomal markers can be used for extending genetic maps. Often the placement of a gene on the chromosome of another mammalian species, such as mouse, reveals associated markers also found in other mammals such as humans even if the number or arm of a particular human chromosome is not known. New sequences can be assigned to chromosomal arms, or parts thereof, by physical mapping. This provides valuable information to investigators searching for disease genes using positional cloning or other gene discovery techniques. Once the disease or syndrome has been crudely localized by genetic linkage to a particular genomic region, for example, ataxia-telangiectasia (A-T) to 11q22-23 (Gatti et al., 1988), any sequences mapping to that area can represent associated or regulatory genes for further investigation. The nucleotide sequences of the presently claimed subject matter can thus also be used to detect differences in the chromosomal location due to translocation, inversion, etc. among normal, carrier, or affected individuals.
The mapping methods of the presently claimed subject matter also employ genomic clones of the exons of a platelet VDCC α1 subunit gene. Sequences for a human platelet VDCC α1 subunit polypeptide gene are set forth in SEQ ID NOs: 1-6, 28, and 29. Thus, the presently claimed subject matter also provides genetic assays based on the genomic sequence of the human platelet VDCC α1 subunit polypeptide genes. An intronic sequence flanking an individual exon encoding a platelet VDCC α1 subunit polypeptide is employed in the design of oligonucleotide primers suitable for the mutation analysis of human genomic DNA. Thus, intronic primers can be used to screen for genetic variants by a number of PCR-based techniques, including single-strand conformation polymorphism (SSCP) analysis (Orita et al., 1989), SSCP/heteroduplex analysis, enzyme mismatch cleavage, and direct sequence analysis of amplified exons (Kestila et al., 1998; Yuan et al., 1999). Similar techniques can be applied to putative 5′-regulatory regions, e.g., the putative promoters 5′ of a platelet VDCC α1 subunit gene.
F.2. Polynucleotide Screening
Automated methods can also be applied the large-scale characterization of single nucleotide polymorphisms (SNPs; Brookes, 1999; Wang et al., 1998) within and near a platelet VDCC α1 subunit gene. Once genetic variants have been detected in specific patient populations, the presently claimed subject matter provides assays to detect the mutation by methods such as allele-specific hybridization (Stoneking et al., 1991), or restriction analysis of amplified genomic DNA containing the specific mutation. Again, these detection methods can be automated using existing technology (Wang et al., 1998). In the case of genetic disease or human phenotypes caused by repeat expansion (Lafreniere et al., 1997; Timchenko & Caskey, 1996), the presently claimed subject matter provides an assay based on PCR of genomic DNA with oligonucleotide primers flanking the involved repeat.
As used herein and in the claims, the term “polymorphism” refers to the occurrence of two or more genetically determined alternative sequences or alleles in a population. A polymorphic marker is the locus at which divergence occurs. Representative markers have at least two alleles, each occurring at frequency of greater than 1%. A polymorphic locus can be as small as one base pair.
The provided nucleic acid molecules can be labeled according to any technique known in the art, such as with radiolabels, fluorescent labels, enzymatic labels, sequence tags, etc. Such molecules can be used as allele-specific oligonucleotide probes. Body samples can be tested to determine whether a platelet VDCC α1 subunit gene contains a polymorphism. Suitable body samples for testing include those comprising DNA, RNA, or protein obtained from biopsies, including bone marrow biopsies; or from blood.
In one embodiment of the presently claimed subject matter two pairs of isolated oligonucleotide primers are provided. These sets of primers are optionally derived from a platelet VDCC α1 subunit exon. The oligonucleotide primers are useful, for example, in detecting a polymorphism of a platelet VDCC α1 subunit gene. The primers direct amplification of a target polynucleotide prior to sequencing. In another embodiment of the presently claimed subject matter isolated allele specific oligonucleotides (ASO) are provided. The allele specific oligonucleotides are also useful in detecting a polymorphism of a platelet VDCC α1 subunit gene.
The terms “substantially complementary to” or “substantially the sequence of” refer to sequences which hybridize to the sequences provided (e.g., SEQ ID NOs: 1, 3, 5-8, 28, and 29) under stringent conditions as disclosed herein and/or sequences having sufficient identity with any of SEQ ID NOs: 1, 3, 5-8, 28, and 29, such that the allele specific oligonucleotides of the presently claimed subject matter hybridize to the sequence. The term “isolated” as used herein includes oligonucleotides substantially free of other nucleic acids, proteins, lipids, carbohydrates or other materials with which they can be associated, such association being either in cellular material or in a synthesis medium. A “target polynucleotide” or “target nucleic acid” refers to the nucleic acid sequence of interest e.g., a platelet VDCC α1 subunit-encoding polynucleotide. Other primers which can be used for primer hybridization are readily ascertainable to those of skill in the art based upon the disclosure herein.
The primers of the presently claimed subject matter embrace oligonucleotides of sufficient length and appropriate sequence so as to provide initiation of polymerization on a significant number of nucleic acids in the polymorphic locus. Specifically, the term “primer” as used herein refers to a sequence comprising in one embodiment two or more deoxyribonucleotides or ribonucleotides, in another embodiment more than three, in another embodiment more than eight, and in still another embodiment at least about 20 nucleotides of a platelet VDCC α1 subunit exonic or intronic region as are disclosed herein. Such oligonucleotides can be between ten and thirty bases in length. Such oligonucleotides can optionally further comprises a detectable label.
Environmental conditions conducive to synthesis include the presence of nucleoside triphosphates and an agent for polymerization, such as DNA polymerase, and a suitable temperature and pH. The primer can be single stranded for maximum efficiency in amplification, but can be double stranded. If double stranded, the primer is first treated to separate its strands before being used to prepare extension products. The primer must be sufficiently long to prime the synthesis of extension products in the presence of the inducing agent for polymerization. The exact length of primer will depend on many factors, including temperature, buffer, and nucleotide composition. The oligonucleotide primer typically contains 12-20 or more nucleotides, although it can contain fewer nucleotides.
Primers of the presently claimed subject matter are designed to be “substantially” complementary to each strand of the genomic locus to be amplified. This means that the primers must be sufficiently complementary to hybridize with their respective strands under conditions which allow the agent for polymerization to perform. In other words, the primers should have sufficient complementarity with the 5′ and 3′ sequences flanking the transition to hybridize therewith and permit amplification of the genomic locus.
Oligonucleotide primers of the presently claimed subject matter are employed in the amplification method which is an enzymatic chain reaction that produces exponential quantities of polymorphic locus relative to the number of reaction steps involved. Typically, one primer is complementary to the negative (−) strand of the polymorphic locus and the other is complementary to the positive (+) strand. Annealing the primers to denatured nucleic acid followed by extension with an enzyme, such as the large fragment of DNA polymerase I (Klenow) and nucleotides, results in newly synthesized + and − strands containing the target polymorphic locus sequence. Because these newly synthesized sequences are also templates, repeated cycles of denaturing, primer annealing, and extension results in exponential production of the region (i.e., the target polymorphic locus sequence) defined by the primers. The product of the chain reaction is a discreet nucleic acid duplex with termini corresponding to the ends of the specific primers employed.
The oligonucleotide primers of the presently claimed subject matter can be prepared using any suitable method, such as conventional phosphotriester and phosphodiester methods or automated embodiments thereof. In one such automated embodiment, diethylphosphoramidites are used as starting materials and can be synthesized as described in Beaucage et al., 1981. One method for synthesizing oligonucleotides on a modified solid support is described in U.S. Pat. No. 4,458,066.
Any nucleic acid specimen, in purified or non-purified form, can be utilized as the starting nucleic acid or acids, providing it contains, or is suspected of containing, a nucleic acid sequence containing the polymorphic locus. Thus, the method can amplify, for example, DNA or RNA, including messenger RNA, wherein DNA or RNA can be single stranded or double stranded. In the event that RNA is to be used as a template, enzymes, and/or conditions optimal for reverse transcribing the template to DNA would be utilized. In addition, a DNA-RNA hybrid which contains one strand of each can be utilized. A mixture of nucleic acids can also be employed, or the nucleic acids produced in a previous amplification reaction herein, using the same or different primers can be so utilized. The specific nucleic acid sequence to be amplified, i.e., the polymorphic locus, can be a fraction of a larger molecule or can be present initially as a discrete molecule, so that the specific sequence constitutes the entire nucleic acid. It is not necessary that the sequence to be amplified be present initially in a pure form; it can be a minor fraction of a complex mixture, such as contained in whole human DNA.
DNA utilized herein can be extracted from a body sample, such as blood, tissue material (e.g., bone marrow tissue), and the like by a variety of techniques such as that described by Maniatis et al., 1982. If the extracted sample is impure, it can be treated before amplification with an amount of a reagent effective to open the cells, or animal cell membranes of the sample, and to expose and/or separate the strand(s) of the nucleic acid(s). This lysing and nucleic acid denaturing step to expose and separate the strands will allow amplification to occur much more readily.
The deoxyribonucleotide triphosphates dATP, dCTP, dGTP, and dTTP are added to the synthesis mixture, either separately or together with the primers, in adequate amounts and the resulting solution is heated to about 90-100° C. from in one embodiment about 1 to 10 minutes and in another embodiment from 1 to 4 minutes. After this heating period, the solution is allowed to cool, which allows for the primer to hybridize. To the cooled mixture is added an appropriate agent for effecting the primer extension reaction (called herein “agent for polymerization”), and the reaction is allowed to occur under conditions known in the art. The agent for polymerization can also be added together with the other reagents if it is heat stable. This synthesis (or amplification) reaction can occur at room temperature up to a temperature above which the agent for polymerization no longer functions. Thus, for example, if DNA polymerase is used as the agent, the temperature is generally no greater than about 40° C. Most conveniently the reaction occurs at room temperature.
The agent for polymerization can be any compound or system which will function to accomplish the synthesis of primer extension products, including enzymes. Suitable enzymes for this purpose include, for example, E. coli DNA polymerase I, Klenow fragment of E. coli DNA polymerase, polymerase muteins, reverse transcriptase, other enzymes, including heat-stable enzymes (i.e., those enzymes which perform primer extension after being subjected to temperatures sufficiently elevated to cause denaturation), such as Taq polymerase. Suitable enzyme will facilitate combination of the nucleotides in the proper manner to form the primer extension products which are complementary to each polymorphic locus nucleic acid strand. Generally, the synthesis will be initiated at the 3′ end of each primer and proceed in the 5′ direction along the template strand, until synthesis terminates, producing molecules of different lengths.
The newly synthesized strand and its complementary nucleic acid strand will form a double-stranded molecule under hybridizing conditions described herein and this hybrid is used in subsequent steps of the method. In the next step, the newly synthesized double-stranded molecule is subjected to denaturing conditions using any of the procedures described above to provide single-stranded molecules.
The steps of denaturing, annealing, and extension product synthesis can be repeated as often as needed to amplify the target polymorphic locus nucleic acid sequence to the extent necessary for detection. The amount of the specific nucleic acid sequence produced will accumulate in an exponential fashion. McPherson et al., 1991.
The amplification products can be detected by Southern blot analysis with or without using radioactive probes. In one such method, for example, a small sample of DNA containing a very low level of the nucleic acid sequence of the polymorphic locus is amplified, and analyzed via a Southern blotting technique or similarly, using dot blot analysis. The use of non-radioactive probes or labels is facilitated by the high level of the amplified signal. Alternatively, probes used to detect the amplified products can be directly or indirectly detectably labeled, for example, with a radioisotope, a fluorescent compound, a bioluminescent compound, a chemiluminescent compound, a metal chelator or an enzyme. Those of ordinary skill in the art will know of other suitable labels for binding to the probe, or will be able to ascertain such, using routine experimentation.
Sequences amplified by the methods of the presently claimed subject matter can be further evaluated, detected, cloned, sequenced, and the like, either in solution or after binding to a solid support, by any method usually applied to the detection of a specific DNA sequence such as dideoxy sequencing, PCR, oligomer restriction (Saiki et al., 1985), allele-specific oligonucleotide (ASO) probe analysis (Conner et al., 1983), oligonucleotide ligation assays (OLAs; Landgren et al., 1988a), and the like. Molecular techniques for DNA analysis have been reviewed (Landgren et al., 1988b).
In one embodiment, the method of amplifying is by PCR, as described herein and in U.S. Pat. Nos. 4,683,195; 4,683,202; and 4,965,188 each of which is hereby incorporated by reference; and as is commonly used by those of ordinary skill in the art. Alternative methods of amplification have been described and can also be employed as long as a VDCC locus amplified by PCR using primers of the presently claimed subject matter is similarly amplified by the alternative means. Such alternative amplification systems include but are not limited to self-sustained sequence replication, which begins with a short sequence of RNA of interest and a T7 promoter. Reverse transcriptase copies the RNA into cDNA and degrades the RNA, followed by reverse transcriptase polymerizing a second strand of DNA.
Another nucleic acid amplification technique is nucleic acid sequence-based amplification (NASBATM) which uses reverse transcription and T7 RNA polymerase and incorporates two primers to target its cycling scheme. NASBATM amplification can begin with either DNA or RNA and finish with either, and amplifies to about 108 copies within 60 to 90 minutes.
Alternatively, nucleic acid can be amplified by ligation-activated transcription (LAT). LAT works from a single-stranded template with a single primer that is partially single-stranded and partially double-stranded. Amplification is initiated by ligating a cDNA to the promoter oligonucleotide and within a few hours, amplification is about 108- to about 109-fold. The QB replicase system can be utilized by attaching an RNA sequence called MDV-1 to RNA complementary to a DNA sequence of interest. Upon mixing with a sample, the hybrid RNA finds its complement among the specimen's mRNAs and binds, activating the replicase to copy the tag-along sequence of interest.
Another nucleic acid amplification technique, ligase chain reaction (LCR), works by using two differently labeled halves of a sequence of interest which are covalently bonded by ligase in the presence of the contiguous sequence in a sample, forming a new target. The repair chain reaction (RCR) nucleic acid amplification technique uses two complementary and target-specific oligonucleotide probe pairs, thermostable polymerase and ligase, and DNA nucleotides to geometrically amplify targeted sequences. A 2-base gap separates the oligo probe pairs, and the RCR fills and joins the gap, mimicking normal DNA repair.
Nucleic acid amplification by strand displacement activation (SDA) utilizes a short primer containing a recognition site for Hinc II with short overhang on the 5′ end which binds to target DNA. A DNA polymerase fills in the part of the primer opposite the overhang with sulfur-containing adenine analogs. Hinc II is added but only cuts the unmodified DNA strand. A DNA polymerase that lacks 5′ exonuclease activity enters at the site of the nick and begins to polymerize, displacing the initial primer strand downstream and building a new one which serves as more primer.
SDA produces greater than about a 107-fold amplification in 2 hours at 37° C. Unlike PCR and LCR, SDA does not require instrumented temperature cycling. Another amplification system useful in the method of the presently claimed subject matter is the QB Replicase System. Although PCR is a representative method of amplification of the presently claimed subject matter, these other methods can also be used to amplify a platelet VDCC α1 subunit locus as described herein. Thus, the term “amplification technique” as used herein and in the claims is meant to encompass all the foregoing methods.
In another embodiment of the presently claimed subject matter a method is provided for identifying a subject having a polymorphism of a platelet VDCC α1 subunit gene, comprising sequencing a target nucleic acid of a sample from a subject by dideoxy sequencing, in one embodiment following amplification of the target nucleic acid.
In another embodiment of the presently claimed subject matter a method is provided for identifying a subject having a polymorphism of a platelet VDCC α1 subunit gene, comprising contacting a target nucleic acid of a sample from a subject with a reagent that detects the presence of a platelet VDCC α1 subunit polymorphism and detecting the reagent. A number of hybridization methods and conditions are well known to those skilled in the art and are disclosed herein. Many of them are useful in carrying out the presently claimed subject matter.
Accordingly, a nucleotide sequence of the presently claimed subject matter can be used for its ability to selectively form duplex molecules with complementary stretches of a platelet VDCC α1 subunit gene. Depending on the application envisioned, one employs varying conditions of hybridization to achieve varying degrees of selectivity of the probe toward the target sequence. For applications requiring a high degree of selectivity, one typically employs relatively stringent conditions to form the hybrids. For example, one selects relatively low salt and/or high temperature conditions, such as provided by 15-300 mM salt at temperatures of about 50° C. to about 70° C. including in one embodiment a temperature of about 55° C., in another embodiment about 60° C., and in still another embodiment of about 65° C. Such conditions are particularly selective, and tolerate little, if any, mismatch between the probe and the template or target strand.
Of course, for some applications, for example, where one desires to prepare mutants employing a mutant primer strand hybridized to an underlying template or where one seeks to isolate polypeptide coding sequences from related species, functional equivalents, or the like, less stringent hybridization conditions are typically needed to allow formation of the heteroduplex. Under such circumstances, one employs conditions such as 0.15 M-0.9 M salt, at temperatures ranging from about 20° C. to about 55° C., including in one embodiment a temperature of about 25° C., in another embodiment of about 37° C., in another embodiment of about 45° C., and in yet another embodiment of about 50° C. Cross-hybridizing species can thereby be readily identified as positively hybridizing signals with respect to control hybridizations. In any case, it is generally appreciated that conditions can be rendered more stringent by the addition of increasing amounts of formamide, which serves to destabilize the hybrid duplex in the same manner as increased temperature. Thus, hybridization conditions can be readily manipulated, and thus will generally be a method of choice depending on the desired results.
In certain embodiments, it is advantageous to employ a nucleic acid sequence of the presently claimed subject matter in combination with an appropriate means, such as a label, for determining hybridization. A wide variety of appropriate indicator reagents are known in the art, including radioactive, enzymatic or other ligands, such as avidin/biotin, which are capable of giving a detectable signal. In exemplary embodiments, one likely employs an enzyme tag such a urease, alkaline phosphatase, or peroxidase instead of radioactive or other environmentally undesirable reagents. In the case of enzyme tags, calorimetric indicator substrates are known which can be employed to provide a reagent visible to the human eye or spectrophotometrically, to identify specific hybridization with complementary nucleic acid-containing samples.
In general, it is envisioned that the hybridization probes described herein are useful both as reagents in solution hybridization as well as in embodiments employing a solid phase. In embodiments involving a solid phase, the sample containing test DNA (or RNA) is adsorbed or otherwise affixed to a selected matrix or surface. This fixed, single-stranded nucleic acid is then subjected to specific hybridization with selected probes under desired conditions. The selected conditions depend inter alia on the particular circumstances based on the particular criteria required (depending, for example, on the G+C contents, type of target nucleic acid, source of nucleic acid, size of hybridization probe, etc.). Following washing of the hybridized surface so as to remove nonspecifically bound probe molecules, specific hybridization is detected, or even quantified, by means of the label.
The materials for use in the method of the presently claimed subject matter are ideally suited for the preparation of a screening kit. Such a kit can comprise a carrier having compartments to receive in close confinement one or more containers such as vials, tubes, and the like, each of the containers comprising one of the separate elements to be used in the method. For example, one of the containers can comprise an amplifying reagent for amplifying a platelet VDCC α1 subunit DNA, such as the necessary enzyme(s) and oligonucleotide primers for amplifying target DNA from the subject.
The oligonucleotide primers include primers having a sequence derived from the group including, but not limited to: SEQ ID NOs: 1, 3, 5-8, 28, and 29, or primer sequences substantially complementary or substantially homologous thereto. Oligonucleotide primers comprising target flanking 5′ and 3′ polynucleotide sequence have substantially the sequence set forth in the flanking 5′ and 3′ portions of any of SEQ ID NOs: 1, 3, 5-8, 28, and 29, and sequences substantially complementary or homologous thereto. Other oligonucleotide primers for amplifying a platelet VDCC α1 subunit will be known or readily ascertainable to those of skill in the art given the disclosure of the presently claimed subject matter presented herein.
A kit in accordance with the presently claimed subject matter can further comprise solutions, buffers, or other reagents for extracting a nucleic acid sample from a biological sample obtained from a subject. Any such reagents as would be readily apparent to one of ordinary skill in the art fall within the scope of the presently claimed subject matter. By way of particular example, a suitable lysis buffer for the tissue or cells along with a suspension of glass beads for capturing the nucleic acid sample and an elution buffer for eluting the nucleic acid sample off of the glass beads comprise a reagent for extracting a nucleic acid sample from a biological sample obtained from a subject.
Other examples include commercially available extraction kits, such as the GENOMIC ISOLATION KIT A.S.A.P.TM (Boehringer Mannheim, Indianapolis, Ind., United States of America), Genomic DNA Isolation System (GIBCO BRL, Gaithersburg, Md., United States of America), ELU-QUIKTM DNA Purification Kit (Schleicher & Schuell, Keene, N.H., United States of America), DNA Extraction Kit (Stratagene, La Jolla, Calif., United States of America), TURBOGENTM Isolation Kit (Invitrogen, San Diego, Calif., United States of America), and the like. Use of these kits according to the manufacturer's instructions is generally acceptable for purification of DNA prior to practicing the methods of the presently claimed subject matter.
F.3. Polypeptide Screening
A method of screening for a disorder affecting calcium homeostasis in platelets is also provided. The method comprises: (a) obtaining a biological sample from a subject; (b) determining an amount of a platelet VDCC α1 subunit polypeptide present in the biological sample; (c) determining the activity of a platelet VDCC α1 subunit polypeptide present in the biological sample; and (d) detecting variations in calcium transport activity between a wild type platelet VDCC α1 subunit polypeptide and an isolated platelet VDCC α1 subunit polypeptide, any calcium transport activity variations between the wild type platelet VDCC α1 subunit polypeptide and the isolated platelet VDCC α1 subunit indicating the possibility of a disorder affecting calcium homeostasis in platelets. Detecting an amount of a platelet VDCC α1 subunit polypeptide present in the biological sample can also be carried out in assessing a response to medication in a subject.
The nucleic acid sequences shown in SEQ ID NOs: 1, 3, 5-8, 28, and 29 comprise platelet VDCC α1 subunit polypeptide encoding sequences that are isolated from wild type cells. The sequence represents the platelet VDCC α1 subunit nucleic acid sequence occurring in nature and existing without mutation. Therefore, wild type cells, as referred to herein, are those cells occurring in nature that contain non-mutated platelet VDCC α1 subunit nucleic acid sequences. The wild type sequence is the native nucleic acid sequence and is the sequence against which assessments of polymorphism and mutation are made.
In another embodiment, the presently claimed subject matter provides an antibody that specifically binds a platelet VDCC α1 subunit polypeptide. In one embodiment, an antibody of the presently claimed subject matter is a monoclonal antibody. In one embodiment, an antibody distinguishes between a wild type form and a mutant or polymorphic form of a platelet VDCC α1 subunit polypeptide. Techniques for preparing such antibodies are disclosed herein. The antibodies can be used to screen for the presence of a mutant or polymorphic form of a platelet VDCC α1 subunit polypeptide in a manner analogous to that set forth above with respect to polynucleotide screening
G. Screening for Modulators of VDCC Biological Activity
In yet another aspect, the presently claimed subject matter provides a method of screening substances for their ability to affect or modulate the biological activity of platelet VDCC α1 subunit gene products, and for their ability to affect or modulate in vivo platelet VDCC α1 subunit levels. This modulation can affect platelet activation and other biological functions of platelets. Compounds identified via the screening methods of the presently claimed subject matter have application as anti-thrombotic agents or as agents for modulation of other biological events mediated by platelets.
Utilizing the methods and compositions of the presently claimed subject matter, screening assays for the testing of candidate substances are performed. A candidate substance is a substance which potentially can promote or inhibit the biological activity of gene product by binding or other intermolecular interaction with a platelet VDCC α1 subunit gene or gene product or control sequence.
G.1. Method of Screening for Modulators of Platelet VDCC α1 Subunit Biological Activity
A representative method of screening candidate substances for their ability to modulate platelet VDCC α1 subunit biological activity comprises: (a) establishing replicate test and control samples that comprise a biologically active platelet VDCC α1 subunit polypeptide; (b) administering a candidate substance to test samples; (c) measuring the biological activity of the platelet VDCC α1 subunit polypeptide in the test and the control samples; and (d) determining whether the candidate substance modulates platelet VDCC α1 subunit biological activity relative to an appropriate control. By “modulate” it is intended an increase, decrease, preservation, maintenance, or other effect of any or all biological activities or properties of a platelet VDCC. By way of additional example, a candidate substance identified according to the screening assay described herein has an ability to facilitate preservation of stored platelets. Thus, a candidate substance identified according to the screening assay described herein has an ability to modulate platelet VDCC α1 subunit biological activity.
Such a candidate compound has utility in the treatment of disorders and conditions associated with the biological activity of a platelet VDCC α1 subunit. Candidate compounds are typically about 500-1,000 daltons, and can be hydrophobic, polycyclic, or both, molecules. Such compounds should be considered as candidates for therapeutic intervention in accordance with the methods described herein below. Thus, compounds identified via the screening methods of the presently claimed subject matter have application as anti-thrombotic agents or as agents for modulation of other biological events mediated by platelets. Dosages of test agents can be determined by deriving dose-response curves, such as those disclosed in U.S. Pat. No. 5,849,578, herein incorporated by reference.
In a cell-free system, the method comprises establishing a control system comprising a platelet VDCC α1 subunit polypeptide and a ligand to which the platelet VDCC α1 subunit polypeptide is capable of binding, establishing a test system comprising the platelet VDCC α1 subunit polypeptide, the ligand, and a candidate compound, and determining whether the candidate compound modulates platelet VDCC α1 subunit activity in a cell-free system. A representative ligand comprises a monoclonal antibody, and in this embodiment, the biological activity or property screened includes binding affinity. Additionally, the platelet VDCC α1 subunit polypeptide can be provided in a lipid bi-layer in accordance with techniques disclosed by Malouf et al., 1987 to facilitate the simulation of in vivo conditions in a cell-free setting.
In another embodiment of the presently claimed subject matter, a platelet VDCC α1 subunit polypeptide (e.g., platelet VDCC α1S subunit or platelet VDCC α1D subunit) or catalytic or immunogenic fragment or oligopeptide thereof, can be used for screening libraries of compounds in any of a variety of drug screening techniques. The fragment employed in such screening can be free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly. The formation of binding complexes, between the platelet VDCC α1 subunit polypeptide and the agent being tested, can be measured.
Another technique for drug screening which can be used provides for high throughput screening of compounds having suitable binding affinity to the protein of interest as described in published PCT application WO 84/03564, herein incorporated by reference. In this method, as applied to a platelet VDCC α1 subunit polypeptide, large numbers of different small test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. The test compounds are reacted with a platelet VDCC α1 subunit polypeptide, or fragments thereof, and washed. Bound platelet VDCC α1 subunit polypeptide is then detected by methods well known in the art. Purified platelet VDCC α1 subunit polypeptide can also be coated directly onto plates for use in the aforementioned drug screening techniques. Alternatively, non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support.
As is well known in the art, a screening assay can provide a cell under conditions suitable for testing the modulation of VDCC biological activity and/or levels of calcium channels. These conditions include but are not limited to pH, temperature, tonicity, the presence of relevant metabolic factors (e.g., metal ions such as for example Ca2+, growth factor, interleukins, or colony stimulating factors), and relevant modifications to the polypeptide such as glycosylation or prenylation. A polypeptide of the presently claimed subject matter can be expressed and utilized in a prokaryotic or eukaryotic cell. The host cell can also be fractionated into sub-cellular fractions where the receptor can be found. For example, cells expressing the polypeptide can be fractionated into the nuclei, the endoplasmic reticulum, vesicles, or the membrane surfaces of the cell. U.S. Pat. Nos. 5,837,479; 5,645,999; 5,786,152; 5,739,278; and 5,352,660 also describe exemplary screening assays, and the entire contents of each are herein incorporated by reference.
In one embodiment, a screening assay is designed to be capable of discriminating candidate substances having selective ability to interact with or modulate one or more of the genes or gene products of the presently claimed subject matter but which substances are without a substantially overlapping activity with another gene or gene product. For example, a substance can modulate the biological activity of a platelet VDCC α1S subunit but have no effect, or a diminished effect, on a platelet VDCC α1D subunit. Such selective effect can comprise in one embodiment a 30% greater effect on one test sample versus another, or in another embodiment 100% or greater effect.
A method of identifying modulators of a platelet calcium channel polypeptide by rational drug design is provided in accordance with the presently claimed subject matter. The method comprises the steps of designing a potential modulator for a platelet calcium channel polypeptide that will form non-covalent bonds with amino acids in the substrate binding site based upon the structure of a platelet VDCC α1 subunit polypeptide; synthesizing the modulator; and determining whether the potential modulator modulates the activity of a calcium channel. Modulators can be synthesized using techniques known in the art. The determination of whether the modulator modulates the biological activity of a calcium channel is made in accordance with the screening methods disclosed herein, or by other screening methods known in the art.
A screening assay of the presently claimed subject matter can also involve determining the ability of a candidate substance to modulate, i.e. preserve, inhibit, or promote platelet VDCC α1 subunit biological activity and in one embodiment to thereby modulate the biological activity of calcium channels in target cells. Target cells can be either naturally occurring cells known to contain a polypeptide of the presently claimed subject matter or transformed cells produced in accordance with a method of transformation set forth herein above. The test samples can further comprise a cell or cell line that expresses a platelet VDCC α1 subunit polypeptide; the presently claimed subject matter also provides a recombinant cell line suitable for use in the exemplary method. Such cell lines can be mammalian, or human, or they can from another organism, including but not limited to yeast. Exemplary assays include, but are not limited to genetic screening assays and molecular biology screens such as a yeast two-hybrid screen that will effectively identify platelet VDCC α1 subunit-interacting genes important for calcium transport or other platelet VDCC α1 subunit-mediated cellular method. One version of the yeast two-hybrid system has been described (Chien et al., 1991) and is commercially available from Clontech (Palo Alto, Calif., United States of America).
G.2. Assays for Biological Activity
Biological activity of a platelet VDCC α1 subunit polypeptide can be determined, for example, by an assay disclosed immediately below. Indeed, the biological activity of a platelet VDCC α1 subunit polypeptide of the presently claimed subject matter can also be monitored as an indicator of drug therapy, such anti-thrombotic therapy or other therapy directed at platelet activity.
G.2.a. Measurement of Cytoplasmic Calcium Signal
The conduction of calcium by the VDCC is measured in vitro by measuring changes in cytoplasmic free (ionized) calcium concentrations. Cytoplasmic calcium concentrations are measured by performing flow cytometric analysis with the calcium indicators fluo-3 and fura red as detailed by Novak & Rabinovitch, 1994. Briefly, platelets in whole blood, platelet rich plasma or biological buffers are incubated with esters of fluo-3 and fura red, and then aspirated into the flow cytometric instrument for measurement of calcium-dependent fluorescent changes. Cytoplasmic calcium concentrations are calculated from the ratio or fluo-3 and fura red relative fluorescence intensities. Experiments are performed with increasing doses of VDCC inhibitors with platelets that are in the resting state and subjected to different concentrations of activation agonists and VDCC antagonists and agonists.
G.2.b. Platelet Activation Reactions
The following methods are used to analyze the effect of VDCC channel inhibition, potentiation, and/or structural manipulation on platelet function.
1. Activation time courses. Platelets are incubated at 37° C. with [3H]serotonin and 32PO4 for one hour to respectively load dense granules with a secretion marker and label the cytoplasmic ATP pool for protein phosphorylation studies. During this time period, some samples are incubated with increasing concentrations of prostaglandin I2 (PGI2) to elevate intracellular cyclic AMP (cAMP). The cells are exposed with agitation to different concentrations of thrombin, ADP, epinephrine, collagen, phorbol ester, and A23187. Samples are withdrawn, quenched, and analyzed to characterize the time course of aggregation, secretion, morphological changes, and clot retraction.
2. Aggregation. Aggregation is followed with flow cytometry by analyzing forward and side scatter patterns. Samples are be examined with light microscopy and scored morphometrically and subjected to optical aggrenometry.
3. Secretion. Platelets are diluted and centrifuged during the activation time course to obtain a supernatant with granule contents. Dense, alpha and lysozomal granule secretion are analyzed respectively by measuring [3H]serotonin, thrombospondin, and beta-N-acetylglucose aminadase levels in the supernatants. P-selectin expression on the surface membrane is quantified with flow cytometry after incubation of the cells with FITC-labeled monoclonal antibody to P-selectin (e.g., anti-P-selectin, Sigma Chemical Corp, St. Louis, Mo., United States of America).
4. Ultrastructural analysis. Platelet samples are subjected to scanning and transmission electron microscopy to study shape change, pseudopodia extension, granule centralization, microfilament organization, and microtubule structure.
5. Clot retraction. Microscale clot retraction analysis is performed in platelet-free plasma in siliconized glass capillary tubes with 10 μL samples. Time courses are initiated by adding thrombin for 1 unit/mL and calcium to 10 mM. The time course of retraction is followed by photographing the capillary sample every 10 seconds, and then the size of the clots are measured morphometrically.
6. Intracellular stimulus response coupling. Protein kinase activities are followed by performing two-dimensional (2D) isoelectric focusing, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and autoradiography as has been detailed elsewhere (Fischer et al., 2000; White et al., 1990). The identity of tyrosine, threonine and serine protein kinase substrates is confirmed with Western analysis.
7. Analysis of cytoplasmic nucleotide pool. Platelets are incubated with radioactive inorganic phosphate (32Pi) to incorporate a radiolabel into cytoplasmic nucleotides. Cytoplasmic levels of ATP and ADP are measured with polyethyleneimine thin layer chromatography (TLC) and autoradiography as has been detailed elsewhere (Fischer et al., 2000). Ethanol solubility is used to determine the ratio of free versus protein bound nucleotides (Holmsen, 1972). The intracellular nucleotide pool are examined before cross-linking, after cross-linking, and after lyophilization and rehydration. TLC analysis and sample preparation follow the procedure of Crabtree & Henderson, 1971. Briefly, TLC plates are developed with 2 M sodium formate, pH=3.4, and then subjected to autoradiography. ATP, ADP, and monophosphate spots are scraped and subjected to liquid scintillation counting to measure cytoplasmic levels of the nucleotides.
8. GPIIb-IIIa function: fibrinogen binding and trafficking. The density of unligated GPIIb-IIIa on the platelet surface is determined by measuring RGD-inhabitable 125I-fibrinogen binding as detailed by Sanders et al., 1996. Also, the amount of fibrinogen that is present on the surface of platelets from alpha-granule secretion is measured by quantifying fluorescein isothiocyanate (FITC)-conjugated anti-fibrinogen monoclonal antibody binding with fluorescence. The 125I-fibrinogen and anti-fibrinogen monoclonal antibody-FITC binding studies are conducted with standards so as to respectively yield the number of unoccupied and fibrinogen-ligated GPIIb-IIIa receptor per cell. The functionality of clathrin-dependent trafficking of surface bound fibrinogen to alpha-granules and clathrin-independent internalization to lyososomes (see e.g., Benke, 1992) is followed with confocal microscopy as described by Merricks et al., 1998.
9. GPIb-IX function: von Willebrand binding The function of the von Willebrand receptor complex is analyzed by performing binding studies with 125I-labeled von Willebrand factor (vWf) as detailed elsewhere (Khandelwal et al., 1997). Briefly, platelets are incubated with the radio labeled ligand, washed and then subjected to liquid scintillation counting. Binding studies are performed in the presence and absence of an inhibitory anti-GPIb monoclonal antibody (GPIb-mAb, Immunotech, Inc., Westbrook, Me., United States of America) and/or ristocetin.
G.3. Method of Screening for Modulators of In Vivo Platelet VDCC α1 Subunit Levels
In accordance with the presently claimed subject matter there are also provided methods for screening candidate compounds for the ability to modulate in vivo platelet VDCC α1 subunit levels and/or activity. Representative modulators of platelet VDCC α1 subunit levels can comprise modulators of platelet VDCC α1 subunit transcription or expression. Pharmaceuticals that increase or decrease the transcription or expression of platelet VDCC α1 subunit encoding genes have important clinical application for the modulation of the biological activity of calcium channels. This modulation can affect calcium homeostasis in platelets.
This presently claimed subject matter thus includes a method for discovery of compounds that modulate the expression levels of platelet VDCC α1 subunit encoding genes, including not only the platelet VDCC α1 subunit genes of the presently claimed subject matter but also other calcium channel polypeptide-encoding genes, and describes the use of such compounds. The general approach is to screen compound libraries for substances which increase or decrease expression of platelet VDCC α1 subunit-encoding genes. Exemplary techniques are described in U.S. Pat. Nos. 5,846,720 and 5,580,722, the entire contents of each of which are herein incorporated by reference.
While the following terms are believed to be well understood by one of skill in the art, the following definitions are set forth to facilitate explanation of the presently claimed subject matter.
“Transcription” means a cellular method involving the interaction of an RNA polymerase with a gene that directs the expression as RNA of the structural information present in the coding sequences of the gene. The method includes, but is not limited to the following steps: (a) the transcription initiation, (b) transcript elongation, (c) transcript splicing, (d) transcript capping, (e) transcript termination, (f) transcript polyadenylation, (g) nuclear export of the transcript, (h) transcript editing, and (i) stabilizing the transcript. “Expression” generally refers to the cellular methods by which a biologically active polypeptide is produced from RNA.
“Transcription factor” means a cytoplasmic or nuclear protein which binds to such gene, or binds to an RNA transcript of such gene, or binds to another protein which binds to such gene or such RNA transcript or another protein which in turn binds to such gene or such RNA transcript, so as to thereby modulate expression of the gene. Such modulation can additionally be achieved by other mechanisms; the essence of “transcription factor for a gene” is that the level of transcription of the gene is altered in some way.
In accordance with the presently claimed subject matter there is provided a method of identifying a candidate compound or molecule that is capable of modulating the transcription level of a gene encoding a platelet VDCC α1 subunit polypeptide and thus is capable of acting as a therapeutic agent in the modulation of platelet VDCC α1 subunit polypeptide effects. This modulation can affect calcium homeostasis in platelets, platelet activation, and other biological functions of platelets, and can also affect platelet storage or production of platelet products. Such modulation can be direct, i.e., through binding of a candidate molecule directly to the nucleotide sequence, whether DNA or RNA transcript, or such modulation can be achieved via one or more intermediaries, such as proteins other than a platelet VDCC α1 subunit polypeptide which are affected by the candidate compound and ultimately modulate platelet VDCC α1 subunit polypeptide transcription by any mechanism, including direct binding, phosphorylation or dephosphorylation, etc.
This method comprises contacting a cell or nucleic acid sample with a candidate compound or molecule to be tested. These samples contain nucleic acids which can contain elements that modulate transcription and/or translation of a platelet VDCC α1 subunit gene, such as a platelet VDCC α1 subunit promoter or putative upstream regulatory region, or other VDCC α1 subunit promoter or putative upstream regulatory region, and a DNA sequence encoding a polypeptide which can be detected in some way. Thus, the polypeptide can be described as a “reporter” or “marker”. In one embodiment, the candidate compound directly and specifically transcriptionally modulates expression of the platelet VDCC α1 subunit polypeptide-encoding gene. Such have therapeutic or pharmaceutical uses in treating platelet VDCC α1 subunit polypeptide-related diseases and/or disorders, in platelet-based medicine and laboratory efforts, and in preserving and transporting platelets.
The DNA sequence is coupled to and under the control of the promoter, under conditions such that the candidate compound or molecule, if capable of acting as a transcriptional modulator of a gene encoding platelet VDCC α1 subunit polypeptide, causes the polypeptide to be expressed and so produces a detectable signal, which can be assayed quantitatively and compared to an appropriate control. Candidate compounds or molecules of interest can include those that increase or decrease, i.e., modulate, transcription from a platelet VDCC α1 subunit promoters. The reporter gene can encode a reporter known in the art, such as luciferase, or it can encode a platelet VDCC α1 subunit.
In certain embodiments of the presently claimed subject matter the polypeptide so produced is capable of complexing with an antibody or is capable of complexing with biotin. In this case the resulting complexes can be detected by methods known in the art. The detectable signal of this assay can also be provided by messenger RNA produced by transcription of said reporter gene. Exactly how the signal is produced and detected can vary and is not the subject of the presently claimed subject matter; rather, the presently claimed subject matter provides the nucleotide sequences and/or putative regulatory regions of a platelet VDCC α1 subunit for use in such an assay. The molecule to be tested in these methods can be a purified molecule, a homogenous sample, or a mixture of molecules or compounds. Further, in the method of the presently claimed subject matter, the DNA in the cell can comprise more than one modulatable transcriptional regulatory sequence.
In accordance with the presently claimed subject matter there is also provided a rapid and high throughput screening method that relies on the methods described above. This screening method comprises separately contacting each of a plurality of substantially identical samples. In such a screening method, the plurality of samples comprises in one embodiment more than about 104 samples and in another embodiment comprises more than about 5×104 samples.
G.4. Method of Screening for Modulators of Phosphatidylserine Exposure on the Surface of a Cell
In accordance with the presently claimed subject matter there are also provided methods for screening candidate compounds for the ability to modulate phosphatidylserine exposure on the surface of a cell. In one embodiment, the method comprises (a) establishing a test sample comprising a platelet voltage dependent calcium channel (VDCC) α1 subunit polypeptide; (b) administering a candidate substance to the test sample; and (c) measuring the interaction, effect, or combination thereof, of the candidate substance on the test sample to thereby determine the ability of the candidate substance to modulate phosphatidylserine exposure on the surface of the cell.
In one embodiment, a candidate compound can be a polypeptide, in another embodiment an antibody, in another embodiment a nucleic acid, and in still another embodiment a chemical compound (for example, a small molecule). In one embodiment, a candidate modulator can be a polypeptide encoded by a gene present in an organism: for example, a human gene. In this embodiment, the gene encoding the polypeptide might be known (i.e. the sequence of the gene has been published or is present in a publicly available database) or might be novel. In one embodiment, the present method further comprises isolating the gene encoding the candidate polypeptide. The cloning of nucleic acid sequences encoding polypeptides is known in the art, and can include the use of degenerate primers in the polymerase chain reaction (PCR). Briefly, degenerate primers are synthesized, the sequences of which are based upon the amino acid sequence of the candidate polypeptide. These degenerate primers can then be used in a PCR reaction to amplify a region of the genome of an appropriate organism (for example, the human genome) or a cDNA library from the organism (for example, a cDNA library made from mRNA isolated from a tissue of the organism believed to express the candidate polypeptide). After an amplified nucleic acid fragment is isolated, the full length gene or a full length cDNA can be isolated using the amplified nucleic acid fragment (if necessary). Techniques for the generation of amplified nucleic acid fragments by PCR and the isolation of full length genes and sequences using amplified nucleic acid fragments are known in the art. For an example of the use of degenerate primers to clone the human erythropoietin gene, see U.S. Pat. No. 4,703,008.
In one embodiment of the present method, the test sample can comprise a nucleic acid molecule encoding a platelet voltage dependent calcium channel (VDCC) α1 subunit polypeptide. In one embodiment, the platelet voltage dependent calcium channel (VDCC) α1 subunit polypeptide is contained within a cell in cell culture.
Representative modulators of phosphatidylserine exposure can also comprise modulators of platelet VDCC α1 subunit gene transcription and/or expression, or can comprise modulators of platelet VDCC α1 subunit polypeptide activity. Pharmaceuticals that increase or decrease the transcription, expression, or activity of platelet VDCC α1 subunit encoding genes and/or polypeptides can have clinical application for the modulation of the biological activity of calcium channels. This modulation can affect calcium homeostasis in platelets.
As disclosed herein, the concentration of intracellular calcium ([Ca2+]i) has been found to correlate with the level of exposure of phosphatidylserine on the surface of the platelets. As [Ca2+]i increases, the level of phosphatidylserine exposed on the surface of the platelet increases. Similarly, as [Ca2+ ]i decreases, phosphatidylserine exposure also decreases. As phosphatidylserine exposure has been associated with such processes as apoptosis (Fadok et al., 1992; Kunzelmann-Marche et al., 2000; Zhou et al., 2002) and platelet activation (Thiagarajan & Tait, 1990; Kunzelmann-Marche et al., 2000; Zhou et al., 2002), the modulation of phosphatidylserine exposure on the surface of platelets can be used to modulate the biological activities of platelets (for example, apoptosis and thrombosis).
This presently claimed subject matter thus includes a method for discovery of compounds that modulate the exposure of phosphatidylserine on the surface of a cell (for example, a platelet), and describes the use of such compounds. The general approach is to screen compound libraries for substances that increase or decrease the exposure of phosphatidylserine on the surface of a cell.
Differences phosphatidylserine exposure on the surface of a cell can be measured using any standard technique. In one embodiment, the measuring comprises (a) determining a first level of phosphatidylserine exposure on a surface of the test sample before the administering of the test substance; (b) determining a second level of phosphatidylserine exposure on the surface of the test sample after the administering the test substance; and (c) comparing the first level and the second level, wherein a difference between the first level and the second level is indicative of the ability of the candidate substance to modulate phosphatidylserine exposure on the surface of the cell.
The levels of exposure of phosphatidylserine on the surface of a cell can be measured, for example, using antibodies that are specific for phosphatidylserine. The antibodies can be employed in techniques that are known in the art including, but not limited to Western blotting, immunoprecipitation, and fluorescence-activated cell sorting (FACS). The level of exposure of phosphatidylserine on the surface of the cell can be determined, for example, by employing an antibody comprising a detectable label (for example, a radiolabel, a fluorescent label, a chemiluminescent label, or a calorimetric label such as an enzyme). The level of exposure of phosphatidylserine on the surface of the cell can be determined before and after exposure to a candidate substance, and a comparison of the before and after levels can indicate whether or not the candidate substance has modulated the level of phosphatidylserine exposure. In one embodiment, the determining comprises (a) contacting the test sample with an antibody comprising a detectable moiety, wherein the antibody binds to phosphatidylserine; and (b) quantitating an amount of the antibody bound to the test sample, wherein the amount of the antibody bound to the test sample is indicative of an amount of phosphatidylserine on the surface of the test sample.
The presently disclosed subject matter also provides a method of screening candidate substances for an ability to modulate phosphatidylserine exposure on a surface of a cell comprising (a) establishing a test sample comprising a modulatable transcriptional regulatory sequence of a platelet voltage dependent calcium channel (VDCC) α1 subunit polypeptide-encoding gene and a reporter gene which is capable of producing a detectable signal; (b) administering a candidate substance to the test sample; and (c) measuring the detectable signal produced as a result of an interaction between the candidate substance and the test sample, wherein a candidate substance as a modulator of phosphatidylserine exposure on a surface of a cell is based on the amount of signal produced in relation to a control sample. In one embodiment, the reporter gene encodes a platelet voltage dependent calcium channel (VDCC) α1 subunit polypeptide, although any reporter gene that produces a detectable signal can be employed. Other exemplary reporter genes include, but are not limited to genes encoding fluorescent proteins (for example, genes encoding green fluorescent protein (GFP), enhanced green fluorescent protein, or red fluorescent protein), chloramphenicol acetyl transferase, β-galactosidase, luciferase, and aquorlin secretory alkaline phosphatase (SEAP).
G.5. Animal Models
In addition, animal-based systems can be used to identify compounds capable of modulating platelet VDCC α1 subunit biological activity. Such animal models can be used for the identification of drugs, pharmaceuticals, therapies, and interventions that can be effective in modulating platelet VDCC α1 subunit polypeptide biological activity. For example, animal models can be exposed to a compound that is suspected of exhibiting an ability to modulate platelet VDCC α1 subunit polypeptide biological activity at a sufficient concentration and for a time sufficient to elicit such modulation of platelet VDCC α1 subunit polypeptide biological activity in the exposed animals. The response of the animals to the exposure can be monitored by assessing in vivo platelet VDCC α1 subunit polypeptide expression levels and activity, or by testing biological samples from the animal. As in the methods described above, the mechanism by which a compound modulates a platelet VDCC α1 subunit polypeptide activity or achieves therapeutic effects can vary; the utility of the presently claimed subject matter does not depend on the precise mechanism by which an effect is achieved.
For example, an animal model of the presently claimed subject matter can comprise a pig with targeted modification of a pig platelet VDCC α1 subunit polypeptide gene, as described herein above.
H. Modulation of Platelet VDCC α1 Subunit Biological Activity in a Laboratory or Clinical Setting
An aspect of the presently claimed subject matter encompasses any treatments that alter any aspect of platelet VDCC α1 subunit polypeptide biological activity. Such methods of modulating the biological activity of a platelet calcium channel polypeptide are applicable in the laboratory and/or clinical setting to enhance the capability to store, freeze dry, dehydrate, or otherwise manipulate platelets or platelet products, as well as being applicable in therapeutic intervention in a subject. Therapeutic intervention in a subject can encompass, for example, anti-thrombotic therapy or therapy via modulation of other biological events mediated by platelets. Representative anti-thrombotic therapy comprises treatment or prevention of arterial thromboses, e.g., the blood clots that cause heart attacks and strokes. Another representative therapeutic application comprises increasing platelet function in bleeding disorders (e.g., hemophilia), whether acquired or inherited.
As used herein, the terms “activity” and “biological activity” are meant to be synonymous and are meant to refer to any biological activity of a platelet VDCC α1 subunit polypeptide (e.g., platelet VDCC α1S subunit or platelet VDCC α1D subunit). Representative biological activities of a platelet VDCC α1 subunit include calcium transport or other biological activity in accordance with the presently claimed subject matter.
With respect to the therapeutic methods of the presently claimed subject matter, an exemplary subject is a vertebrate subject. A representative vertebrate is warm-blooded; a representative warm-blooded vertebrate is a mammal. A representative mammal is a pig or, in one embodiment a human. As used herein and in the claims, the term “patient” is contemplated to include both human and animal patients. Thus, veterinary diagnostic and therapeutic uses are provided in accordance with the presently claimed subject matter and comprise an exemplary embodiment of the presently claimed subject matter.
Contemplated is the treatment of mammals such as humans, as well as those mammals of importance due to being endangered, such as Siberian tigers; of economic importance, such as animals raised on farms for consumption by humans; and/or animals of social importance to humans, such as animals kept as pets or in zoos. Examples of such animals include but are not limited to: carnivores such as cats and dogs; swine, including pigs, hogs, and wild boars; ruminants and/or ungulates such as cattle, oxen, sheep, giraffes, deer, goats, bison, and camels; and horses. Also contemplated is the treatment of birds, including the treatment of those kinds of birds that are endangered and/or kept in zoos, as well as fowl, and more particularly domesticated fowl, i.e., poultry, such as turkeys, chickens, ducks, geese, guinea fowl, and the like, as they are also of economic importance to humans. Thus, contemplated is the treatment of livestock, including, but not limited to, domesticated swine, ruminants, ungulates, horses, poultry, and the like.
H.1. Modulation of Platelet VDCC α1 Subunit Polypeptide Biological Activity
In one embodiment, the present inventive method comprises administering to a cell a substance that modulates, i.e., inhibits or promotes a biological activity of a platelet VDCC α1 subunit polypeptide or modulates the level of phosphatidylserine exposure on the surface of a cell. Such a substance can be identified according to any of the screening assays set forth above, either in vitro or in vivo. Representative cells include platelets and megakaryocytes. The cell can be in an in vitro setting or can be in a subject to be treated, such as a warm-blooded vertebrate as described herein above.
The method comprises treating a vertebrate subject suffering from a disorder associated with or mediated by platelet VDCC α1 subunit polypeptide biological activity (for example, thrombosis) by administering to the subject an effective amount of a substance identified according to a screening assay described above. By the term “modulating”, it is meant that the substance can either promote or inhibit a biological activity of a platelet VDCC α1 subunit, depending on the disorder to be treated, and can affect one or several of the platelet VDCC α1 subunit polypeptides, including the platelet VDCC α1D subunit polypeptide or the platelet VDCC α1S subunit polypeptide, as well as other ion transporters, or other unrelated genes or gene products. The modulation of the biological activity of the platelet VDCC α1 subunit can result in a modulation of the exposure of phosphatidylserine on the surface of a cell (for example, a platelet), thereby modulating the biological activity of the cell (for example, thrombosis).
Therapeutic treatment can comprise the administration of antibodies against a chosen region of a platelet VDCC α1 subunit polypeptide, the administration of a protein that enhances activity, or the administration of a protein that inhibits the transcription of the platelet VDCC α1 subunit polypeptide. Such administration can provide treatment of disorders which can be caused or exacerbated by platelet VDCC α1 subunit polypeptide-mediated mechanisms (for example, thrombosis).
Insofar as a modulator of platelet VDCC α1 subunit polypeptide activity can take the form of a polypeptide or of an anti-platelet VDCC α1 subunit polypeptide monoclonal antibody or fragment thereof, it is to be appreciated that the potency can vary, and therefore a “therapeutically effective” amount can vary. However, as shown by the present assay methods, one skilled in the art can readily assess the potency and efficacy of a candidate platelet VDCC α1 subunit polypeptide biological activity modulator of the presently claimed subject matter and adjust the therapeutic regimen accordingly. A modulator of platelet VDCC α1 subunit biological activity can be evaluated by a variety of techniques, including through the use of a responsive reporter, which drives expression of a reporter gene; interaction of platelet VDCC α1 subunit polypeptides with a monoclonal antibody as described herein; and other assays known in the art and described herein.
The monoclonal antibodies or polypeptides of the presently claimed subject matter can be administered parenterally by injection or by gradual infusion over time. Although the tissue to be treated can typically be accessed in the body by systemic administration and therefore most often treated by intravenous administration of therapeutic compositions, other tissues and delivery means are contemplated where there is a likelihood that the tissue targeted contains the target molecule and are known to those of skill in the art. The compositions are formulated in an appropriate manner and administered in a manner compatible with the dosage formulation.
H.2. Monoclonal Antibodies
The presently claimed subject matter describes, in one embodiment, platelet VDCC α1 subunit polypeptide modulators in the form of monoclonal antibodies which were elicited in response to platelet VDCC α1 subunit but which can immunoreact with any platelet VDCC α1 subunit polypeptide, or with a specific isoform of a platelet VDCC α1 subunit polypeptide, and bind the platelet VDCC α1 subunit polypeptide to modulate biological activity. The presently claimed subject matter also describes cell lines that produce the antibodies, methods for producing the cell lines, and methods for producing the monoclonal antibodies.
The term “antibody” or “antibody molecule” refers collectively to a population of immunoglobulin molecules and/or immunologically active portions of immunoglobulin molecules, i.e., molecules that contain a paratope. A paratope is the portion or portions of an antibody(ies) that is or are responsible for that antibody binding to an antigenic determinant, or epitope.
Representative antibodies for use in the presently claimed subject matter are intact immunoglobulin molecules, substantially intact immunoglobulin molecules, single chain immunoglobulins or antibodies, those portions of an immunoglobulin molecule that contain the paratope, including antibody fragments. Indeed, it is within the scope of the presently claimed subject matter that a monovalent modulator can optionally be used. Thus, the terms “modulate”, “modulating”, and “modulator” are intended to encompass such a mechanism.
The term “monoclonal antibody” refers to a population of antibody molecules that contain only one species of paratope and thus typically display a single binding affinity for any particular epitope with which it immunoreacts; a monoclonal antibody can have a plurality of antibody combining sites, each immunospecific for a different epitope, e.g., a bispecific monoclonal antibody. Methods of producing a monoclonal antibody, a hybridoma cell, or a hybridoma cell culture are described above.
It is also possible to determine, without undue experimentation, if a monoclonal antibody has the same or equivalent specificity or immunoreaction characteristics as a monoclonal antibody of this presently claimed subject matter by ascertaining whether the former prevents the latter from binding to a preselected target molecule. If the monoclonal antibody being tested competes with the monoclonal antibody of the presently claimed subject matter, as shown by a decrease in binding by the monoclonal antibody of the presently claimed subject matter in standard competition assays for binding to the target molecule when present in the solid phase, then it is likely that the two monoclonal antibodies bind to the same, or a closely related, epitope.
Still another way to determine whether a monoclonal antibody has the specificity of a monoclonal antibody of the presently claimed subject matter is to pre-incubate the monoclonal antibody of the presently claimed subject matter with the target molecule with which it is normally reactive, and then add the monoclonal antibody being tested to determine if the monoclonal antibody being tested is inhibited in its ability to bind the target molecule. If the monoclonal antibody being tested is inhibited then, in all likelihood, it has the same, or functionally equivalent, epitopic specificity as the monoclonal antibody of the presently claimed subject matter.
An additional way to determine whether a monoclonal antibody has the specificity of a monoclonal antibody of the presently claimed subject matter is to determine the amino acid residue sequence of the complementarity-determining regions (CDRs) regions of the antibodies in question. “CDRs” mean the three subregions of the light or heavy chain variable regions which have hypervariable sequences and form loop structures that are primarily responsible for making direct contact with antigen. Antibody molecules having identical, or functionally equivalent, amino acid residue sequences in their CDR regions have the same binding specificity. Methods for sequencing polypeptides are well known in the art. Further, other ways of determining whether antibodies have similar immunospecificities are known in the art and can be useful in practicing the presently claimed subject matter.
The immunospecificity of an antibody, its target molecule binding capacity, and the attendant affinity the antibody exhibits for the epitope are defined by the epitope with which the antibody immunoreacts. The epitope specificity is defined at least in part by the amino acid residue sequence of the variable region of the heavy chain of the immunoglobulin that comprises the antibody, and in part by the light-chain-variable-region amino acid residue sequence. Use of the terms “having the binding specificity of” or “having the binding preference of” indicates that equivalent monoclonal antibodies exhibit the same or similar immunoreaction (binding) characteristics and compete for binding to a preselected target molecule.
Humanized monoclonal antibodies offer particular advantages over monoclonal antibodies derived from other mammals, particularly insofar as they can be used therapeutically in humans. Specifically, human antibodies are not cleared from the circulation as rapidly as “foreign” antigens, and do not activate the immune system in the same manner as foreign antigens and foreign antibodies. Methods of preparing “humanized” antibodies are generally well known in the art, and can readily be applied to the antibodies of the presently claimed subject matter.
The use of a molecular cloning approach to generate antibodies, particularly monoclonal antibodies, and more particularly single chain monoclonal antibodies, is also provided. The production of single chain antibodies has been described in the art, see e.g., U.S. Pat. No. 5,260,203, the contents of which are herein incorporated by reference. For this approach, combinatorial immunoglobulin phagemid libraries are prepared from RNA isolated from the spleen of the immunized animal, and phagemids expressing appropriate antibodies are selected by panning on endothelial tissue. The advantages of this approach over conventional hybridoma techniques are that approximately 104 times as many antibodies can be produced and screened in a single round, and that new specificities are generated by H and L chain combination in a single chain, which further increases the chance of finding appropriate antibodies. Thus, an antibody of the presently claimed subject matter, or a “derivative” of an antibody of the presently claimed subject matter, pertains to a single polypeptide chain binding molecule which has binding specificity and affinity substantially similar to the binding specificity and affinity of the light and heavy chain aggregate variable region of an antibody described herein.
H.3. Other Modulators
Given the disclosure of the platelet VDCC α1 subunit polypeptide activity in tissues herein, chemical compounds (e.g., small molecule mimetics) can be used to modulate platelet VDCC α1 subunit polypeptide activity in tissues in accordance with the methods of the presently claimed subject matter. The identification of such compounds is facilitated by the description of screening assays directed to platelet VDCC α1 subunit polypeptide activity in tissues presented above. Such compounds are typically about 500-1,000 daltons, and can be hydrophobic, polycyclic, or both, molecules.
H.4. Gene Therapy
Platelet VDCC α1 subunit polypeptide genes can be used for gene therapy in accordance with the presently claimed subject matter. Exemplary gene therapy methods, including liposomal transfection of nucleic acids into host cells, are described in U.S. Pat. Nos. 5,279,833; 5,286,634; 5,399,346; 5,646,008; 5,651,964; 5,641,484; and 5,643,567, the contents of each of which are herein incorporated by reference.
Briefly, gene therapy directed toward modulation of platelet VDCC α1 subunit polypeptide levels, to thereby affect or modulate the biological activity of platelet VDCC α1 subunit polypeptide in a target cell is described. This modulation can affect calcium transport, to thereby affect platelet activation (i.e. thrombosis) or other biological effect (for example, apoptosis). In one embodiment, a therapeutic method of the presently claimed subject matter provides a method for modulation of platelet VDCC α1 subunit polypeptide levels comprising the steps of: (a) delivering to the cell an effective amount of a DNA molecule comprising a polynucleotide that encodes a polypeptide that modulates the biological activity of one or more than one platelet VDCC α1 subunit polypeptide; and (b) maintaining the cell under conditions sufficient for expression of said polypeptide.
In one embodiment, the delivered polypeptide comprises the sequence of SEQ ID NO: 2 or 4. Delivery can be accomplished by injecting the DNA molecule into the cell. Where the cell is in a subject, administering comprises: (a) providing a vehicle that contains the DNA molecule; and (b) administering the vehicle to the subject.
A vehicle is in one embodiment a cell transformed or transfected with the DNA molecule or a transfected cell derived from such a transformed or transfected cell. An exemplary transformed or transfected cell is a lymphocyte or a tumor cell from the tumor being treated. Methods for transforming or transfecting a cell with a DNA molecule of the presently claimed subject matter are set forth above.
Alternatively, the vehicle is a virus or an antibody that specifically infects or immunoreacts with an antigen of the target tissue or tumor. An advantage of a viral infection system is that it allows for a very high level of infection into the appropriate recipient cell. Also, antibodies have been used to target and deliver DNA molecules.
It is also envisioned that this embodiment of the presently claimed subject matter can be practiced using alternative viral or phage vectors, including retroviral vectors, adenoviral vectors and vaccinia viruses whose genome has been manipulated in alternative ways so as to render the virus non-pathogenic. Methods for creating such a viral mutation are set forth in detail in U.S. Pat. No. 4,769,331, incorporated herein by reference.
In one embodiment, the vector is a recombinant vector comprising: (a) a sequence of genomic viral DNA showing affinity for host cells; (b) a DNA sequence encoding a platelet VDCC α1 subunit polypeptide and operatively linked to said sequence of genomic viral DNA; and (c) a selectable marker.
H.5. Method of Modulating Phosphatidylserine Exposure on a Surface of a Cell
In one embodiment, the presently claimed subject matter provides a method of modulating phosphatidylserine exposure on a surface of a cell, the method comprising administering to the cell an effective amount of a substance capable of modulating the activity of a platelet voltage dependent calcium channel (VDCC) α1 subunit polypeptide in the cell to thereby modulate phosphatidylserine exposure on the surface of the cell. In one embodiment, the cell comprises a cell in a vertebrate subject. In one embodiment, the vertebrate subject is a mammal, for example, a human. In one embodiment, the cell is a platelet or a megakaryocyte.
In one embodiment of the present method, the administering further comprises administering an effective amount of a substance that modulates expression of a platelet voltage dependent calcium channel (VDCC) α1 subunit polypeptide-encoding nucleic acid molecule in the cell. In one embodiment, the substance capable of modulating the activity of a platelet voltage dependent calcium channel (VDCC) α1 subunit polypeptide in the cell comprises an anti-platelet voltage dependent calcium channel (VDCC) α1 subunit polypeptide antibody, a polypeptide, a chemical compound, or a nucleic acid. In one embodiment, the nucleic acid comprises an antisense oligonucleotide. In one embodiment, the polypeptide or chemical compound comprises a ligand for a modulatable transcriptional regulatory sequence of a platelet voltage dependent calcium channel (VDCC) α1 subunit polypeptide-encoding nucleic acid molecule. In one embodiment, the substance capable of modulating the activity of a platelet is supplied in the form of a pharmaceutical composition of the presently claimed subject matter, as discussed in more detail herein below.
In another embodiment, the presently claimed subject matter provides a method for modulating phosphatidylserine exposure on a surface of a cell, the method comprising introducing to the cell a construct comprising a nucleic acid sequence encoding a platelet voltage dependent calcium channel (VDCC) α1 subunit polypeptide operatively linked to a promoter, wherein production of the platelet voltage dependent calcium channel (VDCC) α1 subunit polypeptide in the cell results in modulation of phosphatidylserine exposure on a surface of the cell. In one embodiment, the construct further comprises a vector selected from the group consisting of a plasmid vector and a viral vector. In another embodiment, the construct further comprises a liposome complex. In one embodiment, the cell comprises a cell in a vertebrate subject. In one embodiment, the vertebrate subject is a mammal. In another embodiment, the cell is a platelet or a megakaryocyte.
H.6. Method of Modulating In Vivo Platelet VDCC α1 Subunit Polypeptide Levels in the Treatment of Related Diseases and Disorders
A method for transcriptionally modulating in a cell or in a multicellular organism the expression of a gene encoding a platelet VDCC α1 subunit polypeptide to modulate platelet VDCC α1 subunit polypeptide biological activity in the cell or organism is also provided in accordance with the presently claimed subject matter. This method comprises administering to cell or to the organism a compound at a concentration effective to transcriptionally modulate expression of platelet VDCC α1 subunit polypeptide or co-transporters. Representative cells include platelets and megakaryocytes. The cell can be in an in vitro setting or can be in an organism to be treated, such as a warm-blooded vertebrate as described herein above.
In accordance with the presently claimed subject matter, the provided compound can optionally comprise an antibody or polypeptide prepared as described above and which transcriptionally modulates expression of platelet VDCC α1 subunit polypeptides. Optionally, the antibody or polypeptide directly binds to DNA or RNA, or directly binds to a protein involved in transcription.
Particular chemical entities (e.g., small molecule mimetics) for use in accordance with the presently claimed subject matter do not naturally occur in any cell, whether of a multicellular or a unicellular organism. Even more particularly, the chemical entity is not a naturally occurring molecule, e.g., it is a chemically synthesized entity. Optionally, the compound can bind a modulatable transcription sequence of the gene. For example, the compound can bind a promoter region upstream of a nucleic acid sequence encoding a platelet VDCC α1 subunit polypeptide.
In the methods above, modulation of transcription results in either upregulation or downregulation of expression of the gene encoding the protein of interest, depending on the identity of the molecule that contacts the cell.
H.7. Modulation of Thrombosis
The presently claimed subject matter also provides a method for modulating thrombosis in a subject, the method comprising administering to the subject an effective amount of a substance capable of modulating the activity of a platelet voltage dependent calcium channel (VDCC) α1 subunit polypeptide in the cell, wherein thrombosis in the subject is modulated. In one embodiment, the subject is a mammal. In one embodiment, the mammal is a human.
In one embodiment of the present method, the administering further comprises administering an effective amount of a substance that modulates expression of a platelet voltage dependent calcium channel (VDCC) α1 subunit polypeptide-encoding nucleic acid molecule in the cell. In one embodiment, the substance that modulates expression of a platelet voltage dependent calcium channel (VDCC) α1 subunit polypeptide-encoding nucleic acid molecule in the cell comprises an anti-platelet voltage dependent calcium channel (VDCC) α1 subunit polypeptide antibody, a polypeptide, a chemical compound, or a nucleic acid. In another embodiment, the substance that modulates expression of a platelet voltage dependent calcium channel (VDCC) α1 subunit polypeptide-encoding nucleic acid molecule in the cell comprises a nucleic acid comprising an antisense oligonucleotide. In still another embodiment, the substance that modulates expression of a platelet voltage dependent calcium channel (VDCC) α1 subunit polypeptide-encoding nucleic acid molecule in the cell comprises a polypeptide or chemical compound comprising a ligand for a modulatable transcriptional regulatory sequence of a platelet voltage dependent calcium channel (VDCC) α1 subunit polypeptide-encoding nucleic acid molecule.
The presently claimed subject matter also provides a method for modulating thrombosis in a subject, the method comprising introducing into a cell present within the subject a construct comprising a nucleic acid sequence encoding a platelet voltage dependent calcium channel (VDCC) α1 subunit polypeptide operatively linked to a promoter, wherein production of the platelet voltage dependent calcium channel (VDCC) α1 subunit polypeptide in the cell results in modulation of thrombosis in the subject. In one embodiment, the construct further comprises a vector selected from the group consisting of a plasmid vector and a viral vector. In another embodiment, the construct further comprises a liposome complex. In one embodiment, the cell is a platelet or a megakaryocyte. In one embodiment, the subject is a mammal, and in one embodiment the mammal is a human.
H.8. Modulation of Apoptosis
The presently claimed subject matter also provides a method of modulating apoptosis in a cell selected from the group consisting of a platelet and a megakaryocyte and comprising a voltage dependent calcium channel (VDCC) α1 subunit polypeptide comprising (a) identifying a candidate substance that modulates the exposure of phosphatidylserine on a surface of the platelet or megakaryocyte; and (b) contacting the cell with the candidate substance identified in step (a), whereby apoptosis in the cell is modulated. In one embodiment of the present method, the exposure of phosphatidylserine on a surface of the cell is increased, thereby enhancing apoptosis in the cell. In another embodiment, the exposure of phosphatidylserine on a surface of the cell is decreased, thereby inhibiting apoptosis in the cell. In one embodiment, the cell is a platelet.
H.9. Increasing the Storage Life of a Cell
The presently claimed subject matter also provides a method of increasing a storage life of a cell (for example, a platelet or a megakaryocyte) comprising (a) identifying a candidate substance that decreases the exposure of phosphatidylserine on the surface of the cell; and (b) contacting the cell with the candidate substance identified in step (a), whereby the storage life of the cell is increased. For example, a candidate substance identified by any one of the disclosed methods can be added to a platelet preparation (for example, as a component of a preservative) in an amount sufficient to decrease the rate of platelet cell death that occurs during storage of the cells.
H.10. Antisense Oligonucleotide Therapy
It is also provided according to the presently claimed subject matter that expression of a platelet VDCC α1 subunit polypeptide can be modulated in a vertebrate subject through the administration of an antisense oligonucleotide derived from a nucleic acid molecule encoding a platelet VDCC α1 subunit polypeptide, such as those described in SEQ ID NO: 2 and 4. Therapeutic methods utilizing antisense oligonucleotides have been described in the art, for example, in U.S. Pat. Nos. 5,627,158 and 5,734,033, the contents of each of which are herein incorporated by reference.
H.11. Pharmaceutical Compositions
The presently disclosed subject matter also provides a pharmaceutical composition comprising a therapeutically effective amount of a modulator of thrombosis and a pharmaceutically acceptable diluent or vehicle, wherein the modulator of thrombosis binds a platelet voltage dependent calcium channel (VDCC) α1 subunit polypeptide, or a fragment or derivative thereof.
H. 12. Dosages
As used herein, an “effective” dose refers to one that is administered in doses tailored to a particular application in which calcium transport modulation or other modulation of platelet VDCC α1 subunit biological activity is sought. For example, after review of the disclosure herein of the presently claimed subject matter, one of ordinary skill in the art can tailor the dosages to an individual patient, taking into account the particular formulation and method of administration to be used with the composition as well as patient height, weight, severity of symptoms, and stage of the disorder to be treated.
An effective dose and a therapeutically effective dose are generally synonymous. However, compounds can be administered to patients having reduced symptoms or even administered to patients as a preventative measure. Hence, the composition can be effective in therapeutic treatment even in the absence of symptoms of the disorder.
A unit dose can be administered, for example, 1 to 4 times per day. In one embodiment, the unit dose is administered twice a day (b.i.d.). The dose depends on the route of administration and the formulation of a composition containing the compound or compounds. Further, it will be appreciated by one of ordinary skill in the art after receiving the disclosure of the presently claimed subject matter that it can be necessary to make routine adjustments or variations to the dosage depending on the combination of agents employed, on the age and weight of the patient, and on the severity of the condition to be treated.
Such adjustments or variations, as well as evaluation of when and how to make such adjustments or variations, are well known to those of ordinary skill in the art of medicine. Evaluation parameters and techniques can vary with the patient and the severity of the disease. Particularly useful evaluative techniques are disclosed in the Examples.
H12.a Gene Therapy Vector Construct Dosing
Maximally tolerated dose (MTD) of vector construct when administered directly into the affected tissue is determined. Primary endpoints are: 1) the rate of transduction in abnormal and/or normal cells, 2) the presence and stability of this vector in the systemic circulation and in affected cells, and 3) the nature of the systemic (fever, myalgia) and local (infections, pain) toxicities induced by the vector. A secondary endpoint is the clinical efficacy of the vector construct.
For example, a 4 mL serum-free volume of viral (e.g., adenoviral, retroviral, etc.) vector construct (containing up to 5×107 viral particles in AIM V media) is administered daily per session. During each session, 1 mL of medium containing the appropriate titer of vector construct is injected into 4 regions of the affected tissue for a total of 4 mL per session in a clinical examination room. This is repeated daily for 4 days (4 sessions). This 16 mL total inoculum volume over 4 days is proportionally well below the one safely tolerated by nude mice (0.5 mL/20 g body weight).
Patient evaluation includes history and physical examination prior to initiation of therapy and daily during the 4-day period of vector construct injection. Toxicity grading is done using the ECOG Common Toxicity Criteria. CBC, SMA-20, urinalysis, and conventional studies are performed daily during this period.
H.12.b. Dose Escalation and MTD
Patients are treated with 3×106 viral particles×4. Once they have all recovered from all grade 2 or less toxicities (except alopecia), and as long as grade 3-4 toxicity is not encountered, a subsequent dose level is initiated in patients. As one grade 3 or 4 toxicity occurs at a given dose level, a minimum of 6 patients are enrolled at that level. As only 1 of 6 patients has grade 3 or 4 toxicity, dose escalation continues. The MTD of vector construct is defined as the dose where 2 of 6 patients experience grade 3 or 4 toxicity. If 2 of 3, or if 3 of 6 patients experience grade 3 or 4 toxicity, the MTD is defined as the immediately lower dose level.
The following escalation schema is followed: 1) level 1, 3×106 viral particles; 2) level 2, 1×107; 3) level 3, 3×107; 4) level 4, 5×107. Patients with measurable disease are evaluated for a clinical response to vector construct. Histology and local symptoms are followed.
H.13. Formulation of Therapeutic Compositions
The platelet VDCC α1 subunit polypeptide biological activity modulating substances, gene therapy vectors, substances that inhibit or promote expression of a platelet VDCC α1 subunit polypeptide encoding nucleic acid segment, and substances that modulate the expression of phosphatidylserine on the surface of a cell described above are adapted for administration as a pharmaceutical compositions as described above. Additional formulation and dose preparation techniques have been described in the art, see for example, those described in U.S. Pat. No. 5,326,902 issued to Seipp et al. on Jul. 5, 1994, U.S. Pat. No. 5,234,933 issued to Marnett et al. on Aug. 10, 1993, and PCT Publication WO 93/25521 of Johnson et al. published Dec. 23, 1993, the entire contents of each of which are herein incorporated by reference.
For the purposes described above, the identified substances can normally be administered systemically or partially, usually by oral or parenteral administration. The doses to be administered are determined depending upon age, body weight, symptom, the desired therapeutic effect, the route of administration, and the duration of the treatment, etc.; one of skill in the art of therapeutic treatment will recognize appropriate procedures and techniques for determining the appropriate dosage regimen for effective therapy. Various compositions and forms of administration are provided and are generally known in the art. Other compositions for administration include liquids for external use, and endermic liniments (ointment, etc.), suppositories, and pessaries which comprise one or more of the active substances and can be prepared by known methods.
Thus, the presently claimed subject matter provides pharmaceutical compositions comprising a polypeptide, polynucleotide, or molecule or compound of the presently claimed subject matter and a physiologically acceptable carrier. In one embodiment, a pharmaceutical composition comprises a compound discovered via the screening methods described herein below.
A composition of the presently claimed subject matter is typically administered parenterally in dosage unit formulations containing standard, well-known nontoxic physiologically acceptable carriers, adjuvants, and vehicles as desired. The term “parenteral” as used herein includes intravenous, intramuscular, intra-arterial injection, or infusion techniques.
Injectable preparations, for example sterile injectable aqueous or oleaginous suspensions, are formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation can also be a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol.
Among the acceptable vehicles and solvents that can be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or di-glycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.
Representative carriers include neutral saline solutions buffered with phosphate, lactate, Tris, and the like. Of course, one purifies the vector sufficiently to render it essentially free of undesirable contaminants, such as defective interfering adenovirus particles or endotoxins and other pyrogens such that it does not cause any untoward reactions in the individual receiving the vector construct. An exemplary means of purifying the vector involves the use of buoyant density gradients, such as cesium chloride gradient centrifugation or column chromatography.
A transfected cell can also serve as a carrier. By way of example, a liver cell can be removed from an organism, transfected with a polynucleotide of the presently claimed subject matter using methods set forth above and then the transfected cell returned to the organism (e.g., injected intra-vascularly).
EXAMPLES
The following Examples have been included to illustrate modes of the presently claimed subject matter. Certain aspects of the following Examples are described in terms of techniques and procedures found or contemplated by the present inventors to work well in the practice of the presently claimed subject matter. These Examples are exemplified through the use of standard laboratory practices of the inventors. In light of the present disclosure and the general level of skill in the art, those of skill will appreciate that the following Examples are intended to be exemplary only and that numerous changes, modifications and alterations can be employed without departing from the spirit and scope of the presently claimed subject matter.
Example 1
Isolation of Fresh Platelets and Cultured Megakaryocytes
Due to the paucity of undegraded mRNA in stored human platelets, mRNA was isolated from platelet precursor cells, megakaryocytes, in culture. The findings disclosed herein were confirmed in freshly isolated porcine platelets. Freshly isolated human platelets were used for the ultrastructural studies.
Blood was collected in sodium citrate (9:1 v/v) from informed and consenting healthy volunteers and from pigs according to the National Institute of Health (NIH) human and animal utilization guidelines and approved by University of North Carolina-Chapel Hill (UNC-CH) committees. Platelet-rich plasma (PRP) was obtained by centrifugation of the blood at 800 g for 5 minutes at room temperature (r. t.), and platelets were isolated at 2000 g centrifugation for 15 minutes at r. t. The platelets were immediately used for mRNA or protein isolations.
Meg 01 cells (a human megakaryocytic cell line available from the American Type Culture Collection (ATCC) in Manassas, Va., United States of America) were grown in RPMI (GIBCO BRL, Gaithersburg, Md., United States of America) supplemented with 10% fetal calf serum in 10% CO2 at 37° C. Cells were harvested by pelleting. Human megakaryocytes were obtained from discarded bone marrow smears with permission from the (UNC—CH) Internal Review Board (IRB) committee.
Example 2
Preparation of mRNA for Northern Blots and RT-PCR
Platelets or megakaryocytes were lysed in a reagent sold under the registered trademark TRIPURE® by Boehringer Manheim (Indianapolis, Ind., United States of America) via repetitive pipeting. Total RNA was isolated according to the specifications supplied by the vendor. The total RNA was treated with 20 units of RNase-free DNAse per 100 μg of total RNA. mRNA was enriched using a mRNA kit sold under the registered trademark OLIGOTEX® (Qiagen, Inc. Valencia, Calif., United States of America). Approximately 10 μg of mRNA was size fractionated on gels (Ambion Inc., Austin, Tex., United States of America) in formaldehyde denaturing conditions for Northern blots. See FIG. 4. mRNA was transferred onto a membrane by capillary action for Northern blots.
Antisense riboprobes of 1033 nucleotides (nt) were synthesized to correspond to the region between IV S3 and amino acid 1531 (aa1531) for α1S and between IV S3 and amino acid 1663 (aa1663) for α1D (Seino et al., 1992). These probes were obtained from the PCR reaction using oligonucleotides 1 and 2 (Table 3). The probes were transcribed as the antisense strand from the linearized PCR-TOPO® cloning vector (Invitrogen Corporation Carlsbad, Calif., United States of America) that contained the PCR product as an insert.
First strand cDNA was synthesized from 0.2 μg mRNA with Oligo (dT) or random hexamers as primers. A reverse transcriptase enzyme sold under the trademark SUPERSCRIPT IITM by Life Technologies, Inc. (Rockville, Md., United States of America) was used, and the reaction was carried out according to specifications provided by the vendor. Following the synthesis of the first strand, the mRNA template was removed with RNase treatment. The cDNA obtained was ethanol precipitated and then used for PCR amplification. PCR primers were designed based upon regions of VDCC α1S (Hogan et al., 1994) and α1D (Seino et al., 1992) subunits, such that isoform specific intervening sequences containing regions previously reported to have functional importance would be amplified. Primers (Table 3) were used at a concentration of 100 picomoles per reaction.
PCR was carried out according to standard techniques. The PCR products were examined by agarose gel electrophoresis and cloned into a vector sold under the registered trademark PCR II-TOPO® by Invitrogen Corporation (Carlsbad, Calif., United States of America). The clones were screened by PCR, and the desired cDNA was prepared using a DNA Miniprep kit (Qiagen, Inc. Valencia, Calif., United States of America) for sequencing by the UNC-CH Automated DNA Sequencing Facility.












TABLE 3








Oligonucleotide







Combination for

Predicted


Oligonucleotide
5′
3′
PCR

Polypeptide Region


No.
position
position
Amplification
ref.
Amplified




















1
3763
3785
1 × 2
α1S
IV S3-aa1531





(see FIG. 1)


2
4796
4760
1 × 2
α1D
IV S3-aa1633





(see FIG. 1)


3
4684
4705
3 × 4
α1S
aa1483-end of the







polypeptide


4
5845
5824


5
6661
6633
3 × 5
α1D
aa1586-end of the







polypeptide


6
3240
3265
6 × 7
α1D
III P–IV P


7
4220
4194


8
2221
2243
8 × 9
α1S
Intracytoplasmic loop







II–III


9
2622
2601


10
2949
2972
10 × 11
α1S
III S4–IV S3









Oligonucleotide primers were used in PCR reactions to amplify regions of α1S and α1D from platelets and megakaryocytes. The 5′ and 3′ positions are numbered according to the cDNAs accessed in GENBANK® under Accession Nos. L33798 (Hogan et al., 1994) and M83566 (Seino et al., 1992) for the human α1S and α1D cDNAs, respectively. The predicted polypeptide position of the encoded protein is designated. P=pore.
Example 3
Antibodies and Immunoprecipitation
A rabbit antibody was generated against the synthetic peptide NEELRAIIKKIWKRTSMKLL (SEQ ID NO: 27) which corresponds to the sequence amino acids 1487-1506 (arrow in FIG. 1A) in the putative intracytoplasmic carboxyl-terminal region of adult rabbit and human α1S (Tanabe et al., 1987; Hogan et al., 1994). This sequence is common to many L-type calcium channel α1 subunits from various species. A tetravalent multiple antigenic peptide was synthesized by the protein chemistry laboratories of UNC—CH and was used for immunization at a concentration of 500 μg for the initial dose. A boosting dose of 100 μg of peptide was administered twice at 3 week intervals. Antibody response was detected with ELISA using the synthetic peptide as antigen and on Western blots using megakaryocytes solubilized with Laemmli sample buffer and fractionated by SDS-PAGE electrophoresis.
Pig platelets were used at a concentration of 1.5×1011/mL. They were isolated from 1 L of fresh whole blood, pelleted, and washed twice in RPMI supplemented with a cocktail of protease inhibitors. Washed platelets were then lysed in a lysis buffer (Boehringer Manheim, Indianapolis, Ind., United States of America) which contained 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P40, 0.5% sodium deoxycholate, and a cocktail of protease inhibitors (supplied by Boehringer Manheim, Indianapolis, Ind., United States of America). The platelet lysate was transferred to a pre-chilled Dounce homogenizer and homogenized by approximately 10-15 repeated strokes using a type B pestle. After clearing with centrifugation at 12,000 g, the supernatant was pre-absorbed onto protein A-agarose suspension overnight at 4° C. The beads were discarded, and the supernatant was incubated overnight at 4° C. with the purified rabbit anti-peptide IgG at a concentration of 200 μg/mL. Controls were incubated with a non-immune rabbit IgG at the same concentration (Dako Corp., Carpinteria, Calif., United States of America).
The immune-complexes were captured with 100 μg of protein A-agarose beads overnight, at 4° C. After extensive washings, the immunoprecipitated proteins were separated on SDS-PAGE after eluting the immune complex with Laemmli sample buffer at 100° C. for 3-4 minutes. Western blots were performed after transfer of proteins onto nitrocellulose or polyvinyl derivative (PVD) membranes. The anti-peptide antiserum was used as a primary antibody on western blots at a dilution of 1:500. A goat anti-rabbit antibody labeled with alkaline phosphatase was used as a secondary antibody. The substrate for the immunoprecipitation reactions was provided in the electrochemiluminescence (ECL) reaction (Amersham Biosciences Corp., Piscataway, N.J., United States of America).
To confirm that the anti-peptide antibody recognized α1S and α1D in human bone marrow megakaryocytes, it was incubated for 60 minutes at r. t. on human bone marrow smears. The antiserum was used in 1:1000 dilution. A fluorescent-labeled goat anti-rabbit antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, Pa., United States of America) was used as a secondary antibody.
To preserve the antigenicity of the determinant recognized by our antipeptide antibody, human platelets were processed using the post-embedding method described by Madden, 1998. Fresh human platelets were allowed to settle and adhere on glass coverslips for ten minutes at r. t. Following gentle rinsing with PBS, the platelets were fixed in 2% paraformaldehyde, 0.5% glutaraldehyde in 0.1 M sodium cacodylate, pH 7.4, and irradiated using a laboratory microwave oven (Ted Pella, Inc., Redding, Calif., United States of America). The cells were postfixed in 1% buffered osmium tetroxide for 10 minutes at r. t. They were dehydrated in acetone and infiltrated with L.R. White resin (London Resin Co., Ltd., Reading, England) using microwave irradiation. After polymerization with ultraviolet light (365 nm wavelength), the monolayers were sectioned en face at a thickness of 80 nm and mounted on 300 mesh nickel grids.
For immunocytochemical labeling, all steps were carried out at r. t. The anti-peptide antibody was diluted in 0.1 M phosphate-buffered saline with globulin-free 0.1% bovine serum albumin, pH 7.4, and the secondary immunogold labeled antibody (Amersham Life Science Inc., Arlington Heights, Ill., United States of America) was diluted in 0.1 M Tris-buffered saline with 0.1% bovine serum albumin, pH 8.2. Grid-mounted sections were etched in 5% aqueous sodium metaperiodate for 30 minutes, then incubated in 0.2 M glycine in PBS/BSA for 5 minutes. The sections were blocked in 5% normal goat serum in PBS/BSA for 10 minutes before incubation in the anti-peptide antibody (1:5 dilution) for 2 hours. After rinsing with PBS/BSA, the sections were incubated for 1 hour in goat anti-rabbit IgG 10 nm colloidal gold (1:25 dilution). Negative controls were performed concurrently by deleting primary antiserum and/or incubating in normal rabbit IgG at 1:5 dilution (Dako Corp. of Carpinteria, Calif., United States of America).
The immunogold-labeled sections were post-fixed in 1% glutaraldehyde in phosphate-buffered saline (PBS) followed by uranyl acetate and lead citrate treatment. The grids were observed and photographed using a LEO EM-910 transmission electron microscope (LEO Electron Microscopy, Thornwood, N.Y., United States of America) at 80 kilovolts (kV). Platelets were also prepared by conventional electron microscopy using 0.1% tannic acid in the fixative so as to obtain the optimal ultrastructural morphology of the surface-connected membranous open canalicular system.
Example 4
Two L-type VDCC α1 Subunit cDNAs are Expressed in Platelets and Megakaryocytes
Reverse transcription-polymerase chain reaction (RT-PCR) was used to amplify, clone, and sequence flanking regions of VDCC α1 subunit cDNA from cultured human megakaryocytes (Meg 01, ATCC) and from fresh porcine platelets. Two different isoforms were found to be expressed in human megakaryocytes. One exhibits sequence identity to the L-type VDCC α1 subunit sequence from human skeletal muscle α1S (Hogan et al., 1994), and is presented herein as SEQ ID NOs: 1, 2, 5, and 28. The other exhibits sequence identity to the human neuroendocrine cells α1D sequence (Seino et al., 1992; see also FIGS. 1A and 1B), and is presented herein as SEQ ID NOs: 3, 4, 6, and 29. These partial cDNAs encode regions between III S4 and the carboxyl-end of α1S, and between III S5 and the carboxyl-end of α1D, respectively (FIGS. 1A and 1B). Both cDNAs are missing nucleotides that encode a peptide in the putative extracellular linker between IV S3 and IV S4 (FIG. 1B). The intracytoplasmic loop between motifs II-III of the human α1S was also sequenced (FIGS. 1A and 1B).
Similarly, a cDNA was obtained from circulating porcine platelets that corresponds to an encoded peptide in the region between IV S3 and the amino acid 1531 of α1S (FIGS. 1A and 1B). This platelet cDNA has 91% sequence identity with the rabbit α1S skeletal muscle subunit (Tanabe et al., 1987), is presented herein as SEQ ID NOs: 7 and 8 and demonstrates the same missing sequence as the above human sequence in the putative IV S3 and IV S4 linker (FIGS. 1A and 1B). Another porcine platelet cDNA which encodes the region between II S3 and II S6 of the α1 subunit was sequenced. This partial porcine cDNA has 88% sequence identity to the human neuroendocrine α1D cDNA.
The cloned cDNAs indicate that two different α1 subunits of L-type voltage dependent calcium channels are expressed in the platelet and their megakaryocytic precursors and that they encode polypeptides having sequence identity with the α1 subunits of VDCCs from human skeletal muscle and neuroendocrine cells. Importantly, the encoded polypeptides have regions of known active roles in the VDCC functions.
The cloned cDNAs predict the following structural and functional regions:
(a) pore lining segments in III S5-S6 and IV S5-S6 linkers (FIGS. 1A and 1B): VDCCs pores are lined by the amphipathic loops in the S5-S6 linkers in each of the four motifs, where a glutamate residue in equivalent positions conveys calcium selectivity to the ion-conducting channel;
(b) voltage sensing segments in transmembrane regions III S4 and IV S4 where the positively charged residues in every third or fourth position sense depolarization and induce conformational changes responsible for channel gating;
(c) dihydropyridine binding sites in III S6 and IV S6 characteristic of L-type calcium channels (Striessnig et al., 1998); and
(d) an intracytoplasmic loop between II S6 and III S1 that is unique to α1S and believed to be specific for the type of excitation-contraction coupling in skeletal muscle.
The missing sequence in the IV S3-S4 linker in every cDNA cloned is envisioned to be a characteristic for the VDCC function in the platelets. A skipped exon has been detected in the same region in other L-type VDCCs. Peres-Reyes & Schneider, 1995; Snutch et al., 1991; Diebold et al., 1992. Isoform specific riboprobes that correspond to the nucleotides between 3763 and 4796 of the human α1S and the corresponding sequence from α1D (FIG. 1C) were synthesized as cmRNA and used to probe separate mRNA blots from human megakaryocytes. A 6.5 kb transcript hybridized with the α1S specific cmRNA, and a 10.8 kb transcript hybridized with the α1D specific cmRNA on Northern blots (FIG. 1D). The sizes of these transcripts are consistent with those published for α1S and α1D from skeletal muscle and neuroendocrine mRNA respectively. Tanabe et al., 1987; Seino et al., 1992.
Example 5
α1S and α1D VDCC Subunits are Expressed at the Protein Level in Platelets and Megakaryocytes
An anti-peptide antibody was generated in a rabbit against the peptide sequence amino acids 1486-1506 (Tanabe et al., 1987; see also FIG. 1 arrow). This sequence is conserved in all published L-type VDCC α1 subunits. This antibody recognizes its antigenic determinant in a 170 kDa polypeptide on Western blots from human megakaryocytes resolved by SDS-PAGE (FIG. 2A). This electrophoretic mobility is consistent with that of α1S on SDS-PAGE previously described from skeletal muscle by Tanabe et al., 1987. That the 170 kDa polypeptide is indeed α1S was confirmed with a monoclonal antibody (Mab 1A) previously published to recognize its determinant in α1S from skeletal muscle (FIG. 2A), kindly provided by Dr. S. Froehner, Department of Cell Physiology, UNC-CH and described by Morton & Froehner, 1987.
Suspecting that α1D is expressed at a level below the detection threshold on direct Western blots, the subunits were enriched by immuno-precipitation using the anti-peptide antibody described in Example 3 above. Polypeptides with 208 kDa and 170 kDa electrophoretic mobilities were immunoprecipitated from porcine platelets. When the primary antibody in the immunoprecipitation reaction was replaced with non-immune IgG, these polypeptides were not present. See FIG. 2B.
That the anti-peptide antibody recognizes the 208 kDa as α1D was confirmed by Western blots on pancreatic β cells in culture resolved by SDS-PAGE, as shown in FIG. 2A. Pancreatic β cells in culture were kindly provided by Dr. Michael Freemark, Duke University Department of Pediatrics. The antibody recognized a determinant in a polypeptide with electrophoretic mobility of 208 kDa in the pancreatic β cells. Mab 1A did not recognize a determinant in these cells. The determinant recognized by Mab 1A has not been mapped and thus, might be specific to α1S.
The expression of the α1 subunits in in vivo megakaryocytes was demonstrated on human bone marrow smears with the anti-peptide antibody as a primary antibody and an FITC labeled anti-rabbit secondary antibody. A discrete speckled pattern was detected on the megakaryocytes and was interpreted to be α1 subunits. Thus, the anti-peptide antibody recognized its determinant in a speckled pattern. The anti-peptide antibody was also replaced with non-immune rabbit IgG as a negative control. In the analysis, original magnification was 60×.
Thus, the polypeptide that has an electrophoretic mobility of 170 kDa corresponds to the 6.5 kb transcript identified on Northern blots and represents the α1S subunit from human megakaryocytes and porcine platelets (SEQ ID NOs: 1, 2, and 7-8, respectively). In contrast, the 208 kDa polypeptide corresponds to the 10.8 kb transcript on Northern blots and represents the α1D subunit from human megakaryocytes and porcine platelets.
Example 6
The Platelet VDCC α1 Subunit Polypeptide α1 Subunit is Localized in a Tight Membranous Network of the Open Canalicular System
The site of probable function of the VDCC in the platelet was investigated at the ultrastructural level using the anti-peptide antibody to localize the antigenic determinant in unstimulated human platelets. An anti-rabbit secondary antibody labeled with 10 nm gold particles showed that the epitope recognized by the anti-peptide antibody was present in a tightly branching membranous network inside the platelets. This network forms a tortuous cribriform vesicular membranous system previously described by conventional electron microscopy to be continuous with the surface-connected open canalicular system (White, 1970; White, 1972).
In the examination of localization of VDCC, human platelets were post-embedded and incubated with the anti-peptide antibody as primary antibody. Ten nm gold particle labeled goat anti-rabbit antibody was the secondary antibody. Electron dense gold particles represented antigenic sites recognized by the anti-peptide antibody. This determinant was present in a membranous network continuous with the surface-connected open canalicular system. This network forms tightly branching vacuoles that extend deep inside the platelet. A sample was prepared by conventional electron microscopy (EM) to maintain the morphological integrity of the surface-connected open canalicular system. No gold particles were present when the anti-peptide antibody was replaced with normal rabbit IgG in a control experiment. In the analysis, original magnification was 16,000× and 500×.
Discussion of Examples 1-6
L-type VDCCs are generally co-localized with specialized intracellular organelles of which the function is calcium concentration dependent (Berridge, 1997; Bokvist et al., 1995; López-López et al., 1995; Shirokova et al., 1998). Activation of the VDCC in the cell membrane generates highly localized signals in the inner microenvironment that trigger responses strictly from the underlying organelles. Indeed, the L-type VDCC in striated muscle is preferentially localized in the t-tubule membrane, a sequestered surface membrane that invaginates from the cell membrane deep into the muscle cell. There, the VDCC is in close proximity with the calcium storing sarcoplasmic reticulum (SR) of muscle. Activation of the L-type VDCC in the t-tubule triggers the primary signal that causes release of Ca2+ from the SR lumen (Hille, 1992; López-López et al., 1995; Shirokova et al., 1998). This then generates the global Ca2+ signal that results in muscle contraction (Berridge, 1997).
Similarly, the L-type VDCC is clustered in the cell membrane of insulin secreting β cells in a domain that is adjacent to the highest density of secretory granules inside the cell (Bokvist et al., 1995). Activation of the L-type VDCCs in the cell membrane results in a localized intracellular Ca burst that initiates exocytosis and release of insulin from these granules.
These morphological relationships in muscle and endocrine cells between the VDCC in surface membrane domains and specialized intracellular organelles appear structurally homologous to those observed in the platelet, as discussed herein above. The surface-connected open canalicular system allows the platelet cell membrane to extend deeply into the platelet. There, some of its components are juxtaposed with the dense tubular system, the calcium storing compartment of the platelet (White, 1970). Others act as a conduit in the platelet secretory pathway (White, 1972). The finding of a preferential localization of the VDCC α1 subunit in this membranous network implicates the VDCC at these sites in specific functions of the platelet, such as contraction and secretion.
That the L-type voltage dependent calcium channel is physiologically active in platelets is supported by a study on the effects of the dihydropyridine antagonists on platelet aggregation (Palés et al., 1991). When used in a nanomolar range, these L-type calcium channel blockers inhibit platelet aggregation in a dose-dependent manner. Inhibition of Ca2+ channels at these submicromolar concentrations is believed to be selective for the L-type voltage dependent calcium channel (Lee et al., 1983). Also, and of interest, is the unexplained finding that platelet aggregation time is prolonged in hypertensive patients receiving an L-type dihydropyridine calcium channel blocker for control of their hypertension (Sinzinger et al., 1992; Gebara et al., 1996; Tison et al., 1994).
Other studies, namely electrophysiological patch experiments, have not supported the role of VDCC in Ca2+ entry in the platelets (Mahaut-Smith et al., 1992) while other electrophysiologic studies have confirmed in phospholipid bilayers the presence of a calcium selective channel in platelet vesicles (Zschauer et al., 1998). The preferential localization of the VDCC, as disclosed herein for the first time, in membranes deep inside the platelets could have masked these channels from patch experiments. Furthermore, the binding and antagonistic effects of dihydropyridines on the L-type VDCC are known to depend on the gated state of the channel. In the resting state this channel has a low affinity for these pharmacological agents (Bean, 1984). This gated dependent property could complicate the interpretation of results from experiments that use channel blockers on platelet aggregation and could explain some of the discrepancies reported on their effects on platelet function in different assays.
Tissue-specific expression of VDCC α1 subunits confers a unique function to that tissue or organ. The expression of α1D in neuroendocrine cells reflects the characteristics of the VDCC in excitation-secretion coupling in these cells. Similarly, the expression of α1S in skeletal muscle (Birnbaumer et al., 1994; Tanabe et al., 1987) or α1C in cardiac muscle (Birnbaumer et al., 1994; Mikami et al., 1989) reflects the characteristics of the corresponding VDCC in excitation-contraction coupling in skeletal (Shirokova et al., 1998) and cardiac (López-López et al., 1995) muscles respectively. Extrapolating the expression of α1D and α1S subunits to the function of the platelet, the corresponding channels are implicated in excitation-secretion and excitation-contraction couplings in platelets.
Summary of Examples 7-9
An inhibitory monoclonal antibody to the alpha 1 subunit of the DHP receptor (MA3-920), two antagonists (nitrendipine and Bay K(+)), and one agonist (Bay K(−)), were used to characterize the functionality of VDCCs in thrombin-activated platelets. Using these reagents, it was possible to show that intracellular calcium concentrations can be modulated in thrombin-activated platelets. MA3-920 was the most potent inhibitor of [Ca2+]i, reducing PS exposure by 21%. Nitrendipine reduced [Ca2+]i under certain conditions and also decreased annexin V binding by 22%.
A striking observation was consistently observed: alterations in [Ca2+]i occurred concomitantly with platelet surface PS exposure. Bay K(−) caused a trend towards increased [Ca2+ ]i and a 56% increase in annexin V binding. Considered together, these observations suggest one function of the platelet VDCC is control of platelet surface PS exposure.
Upon activation, there is exposure of phosphatidylserine on the surface of platelets. Also, other negatively charged phospholipids migrate to the outer membrane leaflet as well (See Sims & Wiedmer, 2001; Zhou et al., 2002). This not only forms a pro-coagulant surface, but also might depolarize the membrane enough to activate VDCCs. It is possible that the VDCC is involved with platelet surface phospholipid scrambling during activation. Interestingly, a recently reported knockout mouse for phospholipid scramblase 1 (PLSCR1) showed normal platelet function as measured by normal bleeding time and factor V binding reflecting PS exposure (Zhou et al., 2002). The authors suggest that this apparent discrepancy might be due to redundancy of function among the four known PLSCR genes. Likewise, redundancy of function between platelet α1D and α1S VDCC might account for normal platelet aggregation with ablated [Ca2+ ]i seen in these Examples.
The absence or varied results in platelet aggregation suggest that VDCCs might have little or no role in platelet aggregation under these experimental conditions. Little or no change was seen in the expression of activated GPIIb/IIIa on the platelet surface (Table 4). The binding of fibrinogen to the activated GPIIb/IIIa receptor is required for platelet aggregation. Alternatively, the activity of VDCCs in platelet aggregation might be slightly delayed and have more of an influence on platelet secretion of granular contents. Furthermore, since VDCCs are encrypted or deep in the OCS, initial activation might cause depolarization of the membrane which leads to opening of VDCCs, causing release of calcium from the dense tubular system and further activation of the platelet.
Example 7
MA3-920 Blocks Calcium Influx
As shown in FIG. 6, a 1:200 dilution of MA3-920 completely blocked the rise of [Ca2+ ]i over the entire range of thrombin. MA3-920-treated platelets exhibited a 21% reduction in annexin V binding consistent with decreased phosphatidylserine exposure (see Table 4). As shown in Table 4, this antibody increased PAC1 and CD62 surface expression by 12% and 16%, respectively. Its effects on the rate of platelet aggregation varied, giving anywhere from a 27% increase to a 70% decrease. The rise in platelet aggregation receptors and α-granule release could account for the variability seen in aggregation experiments.







TABLE 4







Effect of VDCC Blockers on Platelet Activation


Markers Following Thrombin Stimulationa













Annexin V
PAC1
CD62







Bay K (−)
56% ↑
 9% ↓
 4% ↓



MA3-920
21% ↓
12% ↑
16% ↑



Nitrendipine
22% ↓
—
 2% ↓



Bay K (+)
 4% ↓
 7% ↓
 5% ↓







aPlatelets were analyzed by flow cytometry to detect changes in the following cell surface markers of activation: Annexin-V (phosphatidylserine); PAC1 (activated GPIIb/IIIa); CD62 (PADGEM; P-selectin).






Example 8
Effects of Nitrendipine Treatment on Calcium Influx and Annexin V Binding
As shown in FIG. 7, 50 nM nitrendipine (a DHP antagonist) showed a decrease in [Ca2+]i ratio at the two highest concentrations of thrombin: 0.167 and 0.5 units/ml. Additionally, nitrendipine caused a 22% decrease in annexin V binding. It had minimal effect on the surface expression of PAC1 and CD62 (Table 4). It had no effect on the rate of platelet aggregation.
Example 9
Effects of Bay K8644 Stereoisomers
5 μM Bay K(−) caused an increase in [Ca2+ ]i at one minute with thrombin concentrations ranging from 0.0167 to 0.5 units/ml (FIG. 8). Bay K(−) produced a 56% increase in annexin V binding consistent with a significant increase in platelet surface phosphatidylserine (PS) exposure (Table 4). In contrast, Bay K(−) produced a 9% decrease in PAC1 and a 4% decrease in CD62, suggesting that the reduction in GPIIb/IIIa receptors and platelet alpha granule release was much less pronounced than platelet surface PS exposure. Bay K(+), on the other hand, showed no significant effect on [Ca2+]i, platelet surface activation markers, or platelet aggregation.
Discussion of Example 9
Bay K(+) has been shown to be an agonist of VDCC activity in the following tissues: frog skeletal muscle, guinea pig smooth muscle cells and papillary muscle, rat aorta and heart, pituitary cells, canine ventricular muscle, mice brain, and rabbit aorta and heart (Franckowiak et al., 1985; Kass, 1987; O'Neill & Bolger, 1988; Ferrante et al., 1989; Saha et al., 1989; van Amsterdam et al., 1989; Enyeart et al., 1990; O'Neill et al., 1990; Ravens & Schopper, 1990; Gil-Longo et al., 1992; Usowicz et al., 1995; Oz et al., 2000). Although one might infer that the stereoisomer Bay K(−) would produce an opposite effect, this has not been the case in the literature cited or in the instant findings in platelets. In platelets, Bay K(−) produced variable results as seen in the other tissues described in the references disclosed hereinabove. The reason for these apparent discrepancies is unknown, but might relate to differences in the extent of membrane depolarization, ion concentration, pH, or other unknown variables.
Materials and Methods for Examples 7-9
A. Calcium Channel Modulators and Platelet Activation Markers
Monoclonal antibody MA3-920 was obtained from Affinity Bioreagents, Inc. (Golden, Colo., United States of America). Nitrendipine and stereoisomers of Bay K8644, termed BK(+) and BK(−) were obtained from Sigma-RBI (St. Louis, Mo., United States of America). Fura 2-AM and Fluo 3-AM were from Molecular Probes, inc. (Eugene, Oreg., United States of America). Thrombin was purchased from Chrono-log Corp. (Havertown, Pa., United States of America) and CaCl2 from Diagnostica Stago, Inc. (Parsippany, N.J., United States of America). Annexin-V-Fluos staining kit was from Roche Diagnostics Corp., Roche Applied Science (Indianapolis, Ind., United States of America) and PAC-1 FITC, CD61 PerCP, and CD62 PE were from BD Biosciences (San Jose, Calif., United States of America).
B. Fresh Platelets
Human blood was collected in acid-citrate-dextrose (ACD, 85 mM trisodium citrate, 70 mM citric acid, 111 mM dextrose). Platelet rich plasma (PRP) was prepared by centrifugation of anticoagulated blood at 25° C. for 8 minutes at 200×g. Platelets were separated from plasma via gel filtration by applying the PRP to a 2.7×10 cm SEPHAROSETM CL4B column (Amersham Biosciences Corp., Piscataway, N.J., United States of America) equilibrated with modified Tyrode's buffer (145 mM NaCl, 4 mM KCl, 1 mM MgSO4, 6 mM glucose, 10 mM Na-HEPES). Gel filtered platelets were collected and adjusted to a concentration of about 200×106 platelets/ml.
C. Flow Cytometric Analysis of [Ca2+]i 
PRP (prepared as above) was incubated with 5 μM Fura 2-AM and 5 μM Fluo 3-AM for 20 minutes at 37° C. Gel filtration followed, as above, and platelets were adjusted to a concentration of about 200×106 platelets/ml. Gel filtered platelets were then incubated for 2 hours with 1:200, 500, and 1000 MA3-920, 50, 20, and 10 nM nitrendipine, or 5, 1, and 0.5 μM Bay K(+) or Bay K(−). Platelets were then diluted 1:20 in modified Tyrode's and CaCl2 was added to a final concentration of 1 μM. Thrombin was then added to a final concentration of 0.0167, 0.05, 0.167 or 0.5 units/ml and mean cell fluorescence (MCF) of Fura 2 and Fluo 3 was measured at 1 minute on a FACSCANTM (BD Biosciences, San Jose, Calif., United States of America) flow cytometer as per Novak & Rabinovitch, 1994. The ratio of fluo 3 to fura 2 MCF was calculated and compared to controls for each experimental condition.
D. Cell Surface Markers of Platelet Activation
Platelets were stained for cell surface activation markers according to “Flow Cytometric Analysis of Platelets”. Monoclonal Antibodies Source Book. Vol. Section 2.19 (Becton, Dickinson And Co., Franklin Lakes, N.J., United States of America). Briefly, after treatment with agonists or antagonists as above, cells were activated with 0.167 units/ml thrombin (concentration chosen based on [Ca2+ ]i studies) for 1 minute in the presence of 1 μM CaCl2, and then stained with the combination of CD61 PerCP and Annexin V FITC, or PAC-1 FITC, CD61 PerCP, and CD62 PE for 15 minutes at room temperature in the dark. The reaction was then quenched with cold 1% paraformaldehyde in PBS. Cells were analyzed on a FACSCANTM flow cytometer. Mean cell fluorescence (MCF) was compared to control platelets (thrombin only) for each experimental condition. CD61 PerCP was used as the platelet marker for gating purposes. CD61 recognizes the GPIIIa (the β subunit of the GPIIb/IIIa complex) and the vitronectin receptor. It is found on all normal resting and activated platelets. Annexin V has a high binding affinity for phosphatidylserine. PAC-1 recognizes an epitope of GPIIb/IIIa on activated platelets at or near the fibrinogen receptor. CD62 is a marker of platelet a granule release (PADGEM: platelet activation-dependent granule-external membrane protein; also called P-selectin).
E. Platelet Aggregation
Gel-filtered platelets at about 200×106/ml were incubated for 2 hours with 1:200 MA3-920, 50 nM nitrendipine, 5 μM Bay K(−), or 5 μM Bay K(+). These concentrations exhibited the greatest effect on [Ca2+ ]i by flow cytometry. CaCl2 was added to a final concentration of 1 μM and aggregation was observed at 37° C. with various concentrations of thrombin on a Chrono-log aggregometer. Results were analyzed with the AGGROLINK® software (Chrono-log Corp.).
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It will be understood that various details of the presently claimed subject matter can be changed without departing from the scope of the presently claimed subject matter. Furthermore, the foregoing description is for the purpose of illustration only, and not for the purpose of limitation—the presently claimed subject matter being defined by the claims.

## Claims

1. A method of screening candidate substances for an ability to modulate phosphatidylserine exposure on the surface of a platelet, the method comprising:
(a) providing a test sample comprising a platelet voltage dependent calcium channel (VDCC) comprising a VDCCα1 subunit polypeptide on the surface of a platelet, wherein said VDCCα1 subunit polypeptide is selected from the group consisting of SEQ ID NO: 2 or SEQ ID NO: 4,
(b) administering a candidate substance to the test sample, and
(c) measuring the effect of said candidate substance on phosphatidylserine exposure on the surface of the platelet by determining a first level of phosphatidylserine exposure on the surface of the platelet before the administering step (b); determining a second level of phosphatidylserine exposure on the surface of the platelet after the administering step (b); and comparing the first level and the second level, wherein a difference between the first level and the second level indicates the ability of the candidate substance to modulate phosphatidylserine exposure on the surface of a platelet.

2. The method of claim 1, wherein the candidate substance is a polypeptide, an antibody, a nucleic acid, or a chemical compound.

3. The method of claim 1, wherein the platelet expresses a nucleic acid molecule encoding the platelet VDCCα1 subunit.

4. The method of claim 3 wherein the nucleic acid molecule encoding the platelet VDCCα1 subunit polypeptide is selected from the group consisting of SEQ ID NO: 1 or SEQ ID NO: 3.

5. The method of claim 1, wherein said determining of phosphatidylserine exposure comprises contacting the platelet with an antibody comprising a detectable moiety, wherein the antibody binds to phosphatidylserine; and quantitating an amount of the antibody bound to the platelet, wherein the amount of the antibody bound to the platelet is indicates the amount of phosphatidylserine on the surface of the platelet.

6. The method of claim 5, wherein the detectable moiety is selected from the group consisting of a radiolabel, a fluorescent label, a chemiluminescent label, and an enzyme.

7. The method of claim 5, wherein the quantitating is by a method selected from the group consisting of radioactive detection, fluorescence detection, chemiluminescent detection, calorimetric detection, Western blotting, immunoprecipitation, and fluorescence-activating cell sorting (FACS).

8. The method of claim 1 wherein the platelets are human platelets.

9. The method of claim 1 wherein the platelets are porcine platelets.