# Patent

## Title
Proteomic analysis of active multiple sclerosis lesions

## Abstract

The invention provides methods for treating demyelinating inflammatory diseases by administering to the subject an effective amount of an agent that provides activated protein C activity, where the dose is effective to reduce the adverse clinical indicia of the disease. In some embodiments, the patient being treating is of the chronic active plaque type.

## Background
BACKGROUND OF THE INVENTION
Multiple sclerosis (MS) is an inflammatory and degenerative disease of the central nervous system (CNS) with diverse clinical presentations and heterogeneous histopathological features. Understanding the neuropathology of MS is essential to develop improved therapies. MS lesions or “plaques” in the CNS white matter have distinct histological and immunocytological characteristics depending on disease activity. This heterogeneity implies that there are discrete molecular events at different pathogenetic stages of MS. Therefore, identification of targets specific to pathological types of MS lesions may have therapeutic benefits during different stages of disease.
For example, Lucchinetti et al. (2000) Annals of Neurology 47(6):707-717 determined four patterns of disease into which MS lesions fit. All had inflammatory infiltrates by T lymphocytes and macrophages in common but segregated on the basis of plaque geography, distribution of myelin protein loss, evidence of immunoglobulin and complement deposition, and oligodendrocyte death.
Although patterns of demyelination were heterogeneous between patients, multiple lesions within a patient all manifested the same phenotype, suggesting that lesion patterns are distinct mechanisms present in subgroups of MS patients. Understanding the pathogenetic mechanisms in demyelinating lesions has significant implications for developing and implementing appropriate therapies. In particular, understanding the relative loss or sparing of oligodendrocytes will determine what therapeutic strategies have potential efficacy in an individual MS patient.
In MS, myelin reactive T cells enter into the brain and spinal cord and mediate destruction of the myelin sheath surrounding neurons resulting in progressive motor dysfunction and eventual paralysis. Current treatment strategies include switching the pro-inflammatory Th1 T cell phenotype to an anti-inflammatory Th2 response, preventing encephalitogenic T cells from extravasating into the brain, inducing T cell tolerance, anergy or apoptosis, and repairing or replacing damaged CNS cells, such as neurons and oligodendrocytes.
Goals for therapy include shortening acute exacerbations, decreasing frequency of exacerbations, and relieving symptoms; maintaining the patient's ability to walk is particularly important. Acute exacerbations may be treated with brief courses of corticosteroids. However, although they may shorten acute attacks and perhaps slow progression, corticosteroids have not been shown to affect long-term outcome.
Immunomodulatory therapy decreases frequency of acute exacerbations and delays eventual disability. Immunomodulatory drugs include interferons (IFNs), such as IFN-β1b and IFN-β1a. Glatiramer acetate may also be used. Other potential therapies include the immunosuppressant methotrexate and Natalizumab, an anti-α4 integrin antibody that inhibits passage of leukocytes across the blood-brain barrier. Immunosuppressants such as mycophenolate and cyclophosphamide have been used for more severe, progressive MS but are controversial.
In addition to suppressing the pathological immune response it is important to protect CNS cells from further damage and to induce repair of injured cells since some cells such as neurons have few progenitors in the adult mammalian brain and are thus limiting.
Limited therapeutic benefit achieved with the above-mentioned immunotherapies may relate to the apparent pathogenetic and clinical heterogeneity of MS. An improved understanding of the pathologic processes involved may allow therapies to be targeted to subgroups of MS patients that are most likely to respond. Clearly, in order to tailor therapy for each patient, classification of pathogenetic mechanism, preferably by using noninvasive methods, will be necessary.
In recent years, a “systems biology” approach using large-scale analysis of proteins and gene transcripts has illuminated new aspects of pathogenesis for complex disease networks including malignancies, neurodegenerative disorders and infections. Similarly, large-scale transcriptional profiling of MS lesions has identified involvement of novel molecules and pathways such as osteopontin and Notch/Jagged signalling, respectively. However, transcriptomic analysis fails to provide a comprehensive understanding of effector molecules involved in MS pathogenesis due to the susceptibility of mRNA to degradation and the discrepancy between mRNA and protein expression levels. Transcriptomic analysis also overlooks signaling molecules from serum, hormones and neurotransmitters.
The present invention provides an alternative approach, where characterization of MS lesions utilized focused proteomic analysis, enriched by laser-capture microdissection (LCM) and analyzed by sensitive tools such as mass spectrometry, provides functional insights into MS pathogenesis.
SUMMARY OF THE INVENTION
The invention provides methods for treating neurological inflammatory diseases, which may be demyelinating autoimmune diseases, such as multiple sclerosis, etc. It is shown herein that MS lesions are associated with the presence of proteins specific to that lesion type, and that reversing the physiological effects of these proteins can ameliorate disease. Proteins of interest include the proteins set forth in any one of Table 3, 4 and 5. In some embodiments the targeted protein is protein C inhibitor. In other embodiments the targeted protein is tissue factor.
In some embodiments of the invention, the neurological inflammatory disease is multiple sclerosis, which may be of the chronic active plaque (CAP) type. In one aspect, the invention is directed to a method to ameliorate the clinical effects of disease in a patient having one or more CAP-type lesions, comprising the steps of administering an agent that reverses the physiological effects of protein C inhibitor in a dose effective to reduce the severity of the disease. In some embodiments the agent is activated protein C (aPC), which is a well-defined anti-coagulant enzyme. The enzyme may be recombinantly produced human aPC. The enzyme may also be a variant aPC with reduced anticoagulant activity.
In certain embodiments, the methods of the invention comprise identifying the presence of one or more CAP-type lesions in the patient, administering to the patient an agent that reverses the physiological effects of protein C inhibitor in a dose effective to reduce the severity of the disease. The administering may be systemic or local administration that provides for a prolonged localized concentration, which may utilize sustained release implants, viscous solutions, or other topical formulation, are of particular interest.
In other embodiments, methods are provided for proteomic analysis of MS lesions, the method comprising staining a section of tissue suspected of containing one or more MS lesions, isolating lesions by laser microscopy, and performing sequencing of proteins present in the lesions by mass spectroscopy.
As provided herein, MS lesions were stained and classified by histological types: acute plaque (AP), chronic active plaque (CAP), and chronic plaque (CP). The lesions were isolated by LCM and saturated sequencing performed by mass spectrometry. The polypeptides thus identified can be analyzed by various means for specificity, utility as a therapeutic and/or diagnostic target, and the like. The markers are useful in screening, diagnostic and therapeutic applications.



BRIEF DESCRIPTION OF THE DRAWINGS
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
FIG. 1: Histopathology of MS brain lesions. (a,d,g=H&E; e,h=LFB; b,c,f,i=IH). a, b, c) Active plaque (AP). a) Marked inflammation, vacuolation (arrowheads) and edema. b) Patchy demyelination (normal myelin=brown), anti-PLP200-219. c) Perivenous and parenchymal (arrows) inflammatory cells; anti-CD45. d, e, f Chronic active plaque (CAP). d, e) Well-demarcated lesion edge (arrows) with recent inflammation (e) (blocked arrow). f) Macrophages, on-going demyelination in CAP; anti-CD68. g, h, i) Chronic plaque (CP). g) Hypocellular fibrotic CP. h) Well-demarcated edge. i) Astrogliosis, anti-GFAP. Scale bars; d (a,d,e,f=50 μM, =25 μM (c,f,i), g (b,g,h)=50 μM.
FIG. 2: a) Schematic procedure of the proteomic analysis of MS lesions. b) Representative tandem mass spectra of peptide(s) identified from b) TF or c) PCI from CAP samples. Sequences of identified peptides are shown above the mass spectra and underlined in the protein sequence; bn or yn denotes the ion generated by cleavage of the peptide bond after the nth amino acid from the amino terminus or the carboxyl terminus; identified b or y ions are shown in boldface letters; and values of m/z (mass/charge) for ions are indicated in the table.
FIG. 3: Thrombin inhibition suppresses inflammation in EAE. a) Mean clinical scores±SEM of EAE mice treated with PBS (black) or recombinant hirudin (white) (10 mg/kg) (n=10/group) at the peak of disease (arrow) (p<0.05, Mann-Whitney analysis). b) In vitro proliferation rates of splenocytes and lymph node cells activated with PLP (20 μg/ml) and cytokine production from d) splenocytes and e) lymph node cells of PBS or hirudin-treated mice. Mean±SEM. *p<0.05, **<0.02 (t test) (From triplicate culture wells). c) Quantitation of inflammatory lesions from brain and spinal cord of EAE mice treated with PBS or hirudin (n=5/group). Data represents means±SEM (**p<0.01).
FIG. 4: aPC modulates Th1 and Th17 responses in EAE. a) Mean clinical scores±SEM of EAE mice treated with PBS (black) and aPC (white) at maximal paralysis (arrow)(p<0.05, Mann-Whitney analysis); b) proliferation rates of splenocytes and lymph node cells following activation with PLP peptide in culture and cytokine levels of c) lymph nodes and d) splenocytes from PBS and aPC-treated EAE mice. Means±SEM (pg/ml) (*p<0.05, **p<0.02, t test) e) Quantitation of inflammatory foci from paraffin-embedded sections from brain and spinal cord of EAE mice treated with PBS or aPC. Data represent mean±SEM, (p<0.05, t test).
FIG. 5: Molecular mechanism of aPC during EAE. SJL/J mice with established EAE (n=7/group) were treated with (arrow) PBS or aPC-WT (a), aPC-L8W (b) or aPC-K193E (c)(0.46 mg/kg). Mean clinical scores SEM (p*<0.05, Mann-Whitney analysis). d-h) Macrophages (d, e), primary astrocytes (f, g) or purified T cells (h) were pre-treated with recombinant murine aPC-WT (30 nM) and activated with LPS (100 ng/ml) (d-g) or CD3/CD28 (5 μg/ml) (h) and cytokine levels were measured from culture supernatant. Means±SE (pg/ml) (p<0.05, t test). i) Immunoblot of total cell lysate (50 pg) from purified T cells treated with aPC (30 minutes time point) probed with anti-IκB-α.



DETAILED DESCRIPTION OF THE EMBODIMENTS
Before the present methods are described, it is to be understood that this invention is not limited to particular methods described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.
Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, subject to any specifically excluded limit in the stated range. As used herein and in the appended claims, the singular forms “a”, “and”, and “the” include plural referents unless the context clearly dictates otherwise.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.
The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed.
DEFINITIONS
“Diagnosis” as used herein generally includes determination of a subject's susceptibility to a disease or disorder, determination as to whether a subject is presently affected by a disease or disorder, prognosis of a subject affected by a disease or disorder (e.g., identification of disease states, stages of MS, or responsiveness of MS to therapy), and use of therametrics (e.g., monitoring a subject's condition to provide information as to the effect or efficacy of therapy).
The term “biological sample” encompasses a variety of sample types obtained from an organism and can be used in a diagnostic or monitoring assay. The term encompasses blood, cerebral spinal fluid, and other liquid samples of biological origin, solid tissue samples, such as a biopsy specimen or tissue cultures or cells derived therefrom and the progeny thereof. The term encompasses samples that have been manipulated in any way after their procurement, such as by treatment with reagents, solubilization, or enrichment for certain components. The term encompasses a clinical sample, and also includes cells in cell culture, cell supernatants, cell lysates, serum, plasma, biological fluids, and tissue samples.
The terms “treatment”, “treating”, “treat” and the like are used herein to generally refer to obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete stabilization or cure for a disease and/or adverse effect attributable to the disease. “Treatment” as used herein covers any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the disease or symptom from occurring in a subject which may be predisposed to the disease or symptom but has not yet been diagnosed as having it; (b) inhibiting the disease symptom, i.e., arresting its development; or (c) relieving the disease symptom, i.e., causing regression of the disease or symptom.
The terms “individual,” “subject,” “host,” and “patient,” used interchangeably herein and refer to any mammalian subject for whom diagnosis, treatment, or therapy is desired, particularly humans.
A “host cell”, as used herein, refers to a microorganism or a eukaryotic cell or cell line cultured as a unicellular entity which can be, or has been, used as a recipient for a recombinant vector or other transfer polynucleotides, and include the progeny of the original cell which has been transfected. It is understood that the progeny of a single cell may not necessarily be completely identical in morphology or in genomic or total DNA complement as the original parent, due to natural, accidental, or deliberate mutation.
“Comparable cell” shall mean a cell whose type is identical to that of another cell to which it is compared. Examples of comparable cells are cells from the same cell line.
“Inhibiting” the onset of a disorder shall mean either lessening the likelihood of the disorder's onset, or preventing the onset of the disorder entirely. In the preferred embodiment, inhibiting the onset of a disorder means preventing its onset entirely. As used herein, onset may refer to a relapse in a patient that has ongoing relapsing remitting disease. The methods of the invention are specifically applied to patients that have been diagnosed with an autoimmune disease. Treatment is aimed at the treatment or prevention of relapses, which are an exacerbation of a pre-existing condition.
“Inhibiting” the expression of a gene in a cell shall mean either lessening the degree to which the gene is expressed, or preventing such expression entirely.
“Specifically hybridize” to a nucleic acid shall mean, with respect to a first nucleic acid, that the first nucleic acid hybridizes to a second nucleic acid with greater affinity than to any other nucleic acid.
“Specifically inhibit” the expression of a protein shall mean to inhibit that protein's expression (a) more than the expression of any other protein, or (b) more than the expression of all but 10 or fewer other proteins.
“Subject” or “patient” shall mean any animal, such as a human, non-human primate, mouse, rat, guinea pig or rabbit.
“Suitable conditions” shall have a meaning dependent on the context in which this term is used. That is, when used in connection with an antibody, the term shall mean conditions that permit an antibody to bind to its corresponding antigen. When this term is used in connection with nucleic acid hybridization, the term shall mean conditions that permit a nucleic acid of at least 15 nucleotides in length to hybridize to a nucleic acid having a sequence complementary thereto. When used in connection with contacting an agent to a cell, this term shall mean conditions that permit an agent capable of doing so to enter a cell and perform its intended function. In one embodiment, the term “suitable conditions” as used herein means physiological conditions.
The term “inflammatory” response is the development of a humoral (antibody mediated) and/or a cellular (mediated by antigen-specific T cells or their secretion products) response. An “immunogen” is capable of inducing an immunological response against itself on administration to a mammal or due to autoimmune disease.
Unless otherwise apparent from the context, all elements, steps or features of the invention can be used in any combination with other elements, steps or features.
General methods in molecular and cellular biochemistry can be found in such standard textbooks as Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., Harbor Laboratory Press 2001); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); Nonviral Vectors for Gene Therapy (Wagner et al. eds., Academic Press 1999); Viral Vectors (Kaplift & Loewy eds., Academic Press 1995); Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998). Reagents, cloning vectors, and kits for genetic manipulation referred to in this disclosure are available from commercial vendors such as BioRad, Stratagene, Invitrogen, Sigma-Aldrich, and ClonTech.
The present invention has been described in terms of particular embodiments found or proposed by the present inventor to comprise preferred modes for the practice of the invention. It will be appreciated by those of skill in the art that, in light of the present disclosure, numerous modifications and changes can be made in the particular embodiments exemplified without departing from the intended scope of the invention. For example, due to codon redundancy, changes can be made in the underlying DNA sequence without affecting the protein sequence. Moreover, due to biological functional equivalency considerations, changes can be made in protein structure without affecting the biological action in kind or amount. All such modifications are intended to be included within the scope of the appended claims.
The subject methods are used for prophylactic or therapeutic purposes. As used herein, the term “treating” is used to refer to both prevention of relapses, and treatment of pre-existing conditions. For example, the prevention of autoimmune disease may be accomplished by administration of the agent prior to development of a relapse. The treatment of ongoing disease, where the treatment stabilizes or improves the clinical symptoms of the patient, is of particular interest.
Methods of the Invention
The invention provides methods for treating neurological inflammatory diseases, which may be a demyelinating autoimmune disease, such as multiple sclerosis. The methods of the invention comprise administering to the subject an effective amount of an agent that reverses the physiological effects of proteins demonstrated herein to be specifically associated with neurological lesions. Proteins of interest include the proteins set forth in any one of Table 3, 4 and 5. Of particular interest is the administration of an agent that provides protein C activity, particularly the administration of activated protein C (aPC), in a dose effective to decrease the severity of ongoing disease, where the aPC may be recombinantly produced human aPC; variant human aPC with reduced anticoagulant activity; and the like.
In this invention, administering the instant compositions can be effected or performed using any of the various methods and delivery systems known to those skilled in the art. The administering can be performed, for example, intravenously, orally, via implant, transmucosally, transdermally, intramuscularly, intrathecally, and subcutaneously. The delivery systems employ a number of routinely used pharmaceutical carriers.
Inflammatory neurological diseases include multiple sclerosis (MS), which is characterized by various symptoms and signs of CNS dysfunction, with remissions and recurring exacerbations. The most common presenting symptoms are paresthesias in one or more extremities, in the trunk, or on one side of the face; weakness or clumsiness of a leg or hand; or visual disturbances, e.g. partial blindness and pain in one eye (retrobulbar optic neuritis), dimness of vision, or scotomas. Other common early symptoms are ocular palsy resulting in double vision (diplopia), transient weakness of one or more extremities, slight stiffness or unusual fatigability of a limb, minor gait disturbances, difficulty with bladder control, vertigo, and mild emotional disturbances; all indicate scattered CNS involvement and often occur months or years before the disease is recognized. Excess heat may accentuate symptoms and signs.
The course is highly varied, unpredictable, and, in most patients, remittent. At first, months or years of remission may separate episodes, especially when the disease begins with retrobulbar optic neuritis. However, some patients have frequent attacks and are rapidly incapacitated; for a few the course can be rapidly progressive (primary progressive MS, PPMS). Relapsing remitting MS (RR MS) is characterized clinically by relapses and remissions that occur over months to years, with partial or full recovery of neurological deficits between attacks. Such patients manifest approximately 1 attack, or relapse, per year. Over 10 to 20 years, approximately 50% of RR MS patients develop secondary progressive MS (SP MS) which is characterized by incomplete recovery between attacks and accumulation of neurologic deficits resulting in increasing disability.
Diagnosis is indirect, by deduction from clinical, radiographic (brain plaques on magnetic resonance [MR] scan), and to a lesser extent laboratory (oligoclonal bands on CSF analysis) features. Typical cases can usually be diagnosed confidently on clinical grounds. The diagnosis can be suspected after a first attack. Later, a history of remissions and exacerbations and clinical evidence of CNS lesions disseminated in more than one area are highly suggestive.
MRI, the most sensitive diagnostic imaging technique, may show plaques. It may also detect treatable nondemyelinating lesions at the junction of the spinal cord and medulla (e.g., subarachnoid cyst, foramen magnum tumors) that occasionally cause a variable and fluctuating spectrum of motor and sensory symptoms, mimicking MS. Gadolinium-contrast enhancement can distinguish areas of active inflammation from older brain plaques. MS lesions may also be visible on contrast-enhanced CT scans; sensitivity may be increased by giving twice the iodine dose and delaying scanning (double-dose delayed CT scan).
Conventional treatments for MS include interferon β (Avonex, Betaseron, Rebif), Copaxone (Glatiramer acetate), and anti-VLA4 (Tysabri, natalizumab), any of which may be combined with the agents utilized in the present invention. MS is also treated with immunosuppressive agents including methylprednisolone, other steroids, methotrexate, cladribine and cyclophosphamide. Many biological agents, such as anti-IFNγ antibody, CTLA4-Ig (Abetacept), anti-CD20 (Rituxan), and other anti-cytokine agents are in clinical development for MS.
In some embodiments of the invention, a tissue section suspected of comprising a lesion is characterized by staining, e.g. with Hematoxylin and Eosin (H&E), Luxol Fast Blue (LFB), etc. Alternatively plaques may be characterized according to expression of the markers as described herein. The treatment with an agent may be selected on the basis of the lesion characterization. For example, a patient with a CAP type lesion may be selected for treatment with aPC.
Lesions can be characterized as acute plaque (AP) having florid parenchymal and perivascular inflammatory cell infiltration, abundant astroglial hypertrophy, myelin fragmentation, edema and ongoing demyelination with indistinct margins. CAP lesions have chronic demyelination, sharply defined margins and recent areas of inflammatory demyelination at the edges including pronounced immunoglobulin and complement reactivity associated with degenerating myelin at the active plaque edge and with myelin degradation products within macrophages. CP lesions have areas of demyelination with well-demarcated borders and abundant astrogliosis but few or no inflammatory cells. These latter two types of lesions show sharply demarcated perivenular demyelination with loss of all myelin proteins apparently occurring simultaneously. Sparing of oligodendrocytes in active plaques and repopulation of inactive plaques with high numbers of oligodendrocytes was observed.
A more complete description of the plaques is publicly available, for example as described, inter alia, by Kuhlmann et al. (2008) Acta Neuropathol.; Lassmann et al. (2007) Brain Pathol. 17(3):325; Lassmann et al. (2004) Ann Neurol. 56(2):308; Lassmann et al. (2001) Trends Mol Med 7:115-2; each of which are specifically incorporated by references for teachings related to plaque classification.
The present invention identifies polypeptides that are differentially expressed in MS plaques. Methods are provided in which these polypeptides, which may be collectively referred to as plaque-specific markers, are used for assessing and treating disease. Methods may use one or a combination of markers, where a combination may include 2, 3 or more markers.
In some embodiments, the markers are expressed as a level at least 2× the expression level of a cell free of disease, e.g. a human oligodendrocyte or neural cell, or a differently classified plaque, where expression may be determined as the level of transcription, mRNA accumulation, and/or protein accumulation. In other embodiments the markers are expressed as a level at least 3×, at least 4×, at least 5×, at least 10×, at least 20× or greater, than the expression level of a counterpart non-diseased cell.
The present invention provides methods of using the markers described herein in diagnosis of MS, classification and treatment of MS and related conditions according to expression profiles. The methods are useful for characterizing plaques, facilitating diagnosis of MS and the severity and nature of the disease in a subject, facilitating a determination of the prognosis of a subject, and assessing the responsiveness of the subject to therapy. The detection methods of the invention can be conducted in vitro or in vivo, on isolated cells, or in whole tissues, e.g. needle biopsy samples, and the like.
A polynucleotide or polypeptide sequence that corresponds to, or represents a marker means that at least a portion of a sequence of the polynucleotide or polypeptide is present. A sequence may also be “identified” by a polynucleotide or polypeptide if the polynucleotide or polypeptide corresponds to or represents the marker. An “identifying sequence” is a minimal fragment of a sequence of contiguous nucleotides or amino acids that uniquely identifies or defines a polynucleotide or polypeptide sequence. Sequences of interest include those set forth in Tables 3-5, which are differentially expressed in the specified MS plaques.
Methods are also provided for optimizing therapy, by first classification, and based on that information, selecting the appropriate therapy, dose, treatment modality, etc. which optimizes the differential between delivery of a treatment to the undesirable target cells, while minimizing undesirable toxicity. The treatment is optimized by selection for a treatment that minimizes undesirable toxicity, while providing for effective anti-proliferative activity.
Therapeutic Agents
In one embodiment of the invention, agents that provide activated protein c activity, e.g. aPC polypeptides, nucleic acids encoding aPC, and the like are used in the treatment of inflammatory disease, including demyelinating autoimmune disease, such as MS.
“Activated protein C” shall mean the activated form of the human protein encoded by the mRNA sequence set forth in GenBank Accession No. NM—000312 and as described by Beckmann et al. (1985) Nucleic Acids Res. 13 (14), 5233-5247; and Foster et al. (1985) Proc. Nati. Acad. Sci. U.S.A. 82 (14), 46734677, all biologically active variants and homologues thereof. Protein C is a vitamin K-dependent serine protease zymogen. Purified human activated protein C selectively destroys factors Va and VIII:C in human plasma and thus has an important anticoagulant role. The protein is activated by thrombin.
An FDA approved recombinantly produced form of activated protein C is available as Drotrecogin alpha (activated), from Eli Lilly under the trade name XIGRIS. Drotrecogin alfa (activated) is a glycoprotein of approximately 55 kilodalton molecular weight, consisting of a heavy chain and a light chain linked by a disulfide bond. Drotrecogin alfa (activated) and human plasma-derived Activated Protein C have the same sites of glycosylation, although some differences in the glycosylation structures exist.
Variants of protein C have been described, e.g. see Mosnier et al. (2004) Blood 104:1740; Griffin et al. (2007) J Thromb Haemost. 2007 July; 5 Suppl 1:73-80; Preston et al. (2006) J Biol Chem. 281(39):28850-7; Gale et al. (2006) J Thromb Haemost. 4(6):1315-22; and Preston et al. (2005) FEBS J. 272(1):97-108; each of which is herein specifically incorporated by reference for teachings of protein C variants and their use.
Activated protein c polypeptides, which can be used in the methods of the invention, comprise at least about 50 amino acids, usually at least about 100 amino acids, at least about 150 amino acids, at least about 160 amino acids, at least about 170 amino acids, and which may include up to 175 amino acids of an activated protein c protein, or modifications thereof, and may further include fusion polypeptides as known in the art in addition to the provided sequences. The activated protein c sequence may be from any mammalian or avian species, e.g. primate sp., particularly humans; rodents, including mice, rats and hamsters; rabbits; equines, bovines, canines, felines; etc. Of particular interest are the human proteins.
In some embodiments of the invention, the aPC protein, or a functional fragment or variant thereof is administered to a patient. A “variant” polypeptide means a biologically active polypeptide as defined below having less than 100% sequence identity with a native sequence polypeptide. Such variants include polypeptides wherein one or more amino acid residues are added at the N- or C-terminus of, or within, the native sequence; from about one to forty amino acid residues are deleted, and optionally substituted by one or more amino acid residues; and derivatives of the above polypeptides, wherein an amino acid residue has been covalently modified so that the resulting product has a non-naturally occurring amino acid. Ordinarily, a biologically active variant will have an amino acid sequence having at least about 90% amino acid sequence identity with a native sequence polypeptide, preferably at least about 95%, more preferably at least about 99%.
The sequence of activated protein c peptides as described above may be altered in various ways known in the art to generate targeted changes in sequence. The sequence changes may be substitutions, insertions or deletions. Such alterations may be used to alter properties of the protein, by affecting the stability, specificity, etc. Techniques for in vitro mutagenesis of cloned genes are known. Examples of protocols for scanning mutations may be found in Gustin et al., Biotechniques 14:22 (1993); Barany, Gene 37:111-23 (1985); Colicelli et al., Mol Gen Genet 199:537-9 (1985); and Prentki et al., Gene 29:303-13 (1984). Methods for site specific mutagenesis can be found in Sambrook et al., Molecular Cloning: A Laboratory Manual, CSH Press 1989, pp. 15.3-15.108; Weiner et al., Gene 126:3541 (1993); Sayers et al., Biotechniques 13:592-6 (1992); Jones and Winistorfer, Biotechniques 12:528-30 (1992); Barton et al., Nucleic Acids Res 18:7349-55 (1990); Marofti and Tomich, Gene Anal Tech 6:67-70 (1989); and Zhu Anal Biochem 177:1204 (1989).
The activated protein c for use in the subject methods may be produced from eukaryotic or prokaryotic cells, or may be synthesized in vitro. Where the protein is produced by prokaryotic cells, it may be further processed by unfolding, e.g. heat denaturation, DTT reduction, etc. and may be further refolded, using methods known in the art.
Modifications of interest that do not alter primary sequence include chemical derivatization of polypeptides, e.g., acylation, acetylation, carboxylation, amidation, etc. Also included are modifications of glycosylation, e.g. those made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing or in further processing steps; e.g. by exposing the polypeptide to enzymes which affect glycosylation, such as mammalian glycosylating or deglycosylating enzymes. Also embraced are sequences that have phosphorylated amino acid residues, e.g. phosphotyrosine, phosphoserine, or phosphothreonine.
Also included in the subject invention are polypeptides that have been modified using ordinary molecular biological techniques and synthetic chemistry so as to improve their resistance to proteolytic degradation or to optimize solubility properties or to render them more suitable as a therapeutic agent. Analogs of such polypeptides include those containing residues other than naturally occurring L-amino acids, e.g. D-amino acids or non-naturally occurring synthetic amino acids. D-amino acids may be substituted for some or all of the amino acid residues.
The aPC polypeptides may be prepared by in vitro synthesis, using conventional methods as known in the art. Various commercial synthetic apparatuses are available, for example, automated synthesizers by Applied Biosystems, Inc., Foster City, Calif., Beckman, etc. By using synthesizers, naturally occurring amino acids may be substituted with unnatural amino acids. The particular sequence and the manner of preparation will be determined by convenience, economics, purity required, and the like.
The polypeptides may also be isolated and purified in accordance with conventional methods of recombinant synthesis. A lysate may be prepared of the expression host and the lysate purified using HPLC, exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification technique. For the most part, the compositions which are used will comprise at least 20% by weight of the desired product, more usually at least about 75% by weight, preferably at least about 95% by weight, and for therapeutic purposes, usually at least about 99.5% by weight, in relation to contaminants related to the method of preparation of the product and its purification. Usually, the percentages will be based upon total protein.
Active polypeptides or polynucleotides can serve as the active ingredient in pharmaceutical compositions formulated for the treatment of various disorders as described above. The active ingredient is present in a therapeutically effective amount, i.e., an amount sufficient when administered to substantially modulate the effect of the targeted protein or polypeptide to treat a disease or medical condition mediated thereby. The compositions can also include various other agents to enhance delivery and efficacy, e.g. to enhance delivery and stability of the active ingredients.
Thus, for example, the compositions can also include, depending on the formulation desired, pharmaceutically-acceptable, non-toxic carriers or diluents, which are defined as vehicles commonly used to formulate pharmaceutical compositions for animal or human administration. The diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, buffered water, physiological saline, PBS, Ringer's solution, dextrose solution, and Hank's solution. In addition, the pharmaceutical composition or formulation can include other carriers, adjuvants, or non-toxic, nontherapeutic, nonimmunogenic stabilizers, excipients and the like. The compositions can also include additional substances to approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents, wetting agents and detergents. The composition can also include any of a variety of stabilizing agents, such as an antioxidant.
Further guidance regarding formulations that are suitable for various types of administration can be found in Remington's Pharmaceutical Sciences, Mace Publishing Company, Philadelphia, Pa., 17th ed. (1985). For a brief review of methods for drug delivery, see, Langer, Science 249:1527-1533 (1990).
The aPC compositions may be administered in a single dose, or in multiple doses, usually multiple doses over a period of time, e.g. daily, every-other day, weekly, semi-weekly, monthly etc. for a period of time sufficient to reduce severity of the disease, which may comprise 1, 2, 3, 4, 6, 10, or more doses.
Determining a therapeutically or prophylactically effective amount an agent that provides aPC activity can be done based on animal data using routine computational methods. In one embodiment, the therapeutically or prophylactically effective amount contains between about 0.1 mg and about 1 g of protein. In another embodiment, the effective amount contains between about 1 mg and about 100 mg of protein, as applicable. The effective dose will depend at least in part on the route of administration. The agents may be administered orally, in an aerosol spray; by injection, e.g. i.m., s.c., i.p., i.v., etc. The dose may be from about 0.1 μg/kg patient weight; about 1 μg/kg; about 10 μg/kg; to about 100 μg/kg.
Treating, treatment, or therapy of a disease or disorder shall mean lessening the severity of adverse clinical symptoms by administration of an aPC composition. As used herein, ameliorating a disease and treating a disease are equivalent.
The method also provide for combination therapy, where the combination may provide for additive or synergistic benefits. Combinations of activated protein c may be obtained with a second agent selected from one or more of the general classes of drugs commonly used in the non-antigen specific treatment of autoimmune disease, which include corticosteroids and disease modifying drugs; or from an antigen-specific agent. Corticosteroids have a short onset of action, but many disease modifying drugs take several weeks or months to demonstrate a clinical effect. These agents include methotrexate, leflunomide (AravaTM), etanercept (EnbrelTM), infliximab (RemicadeTM), adalimumab (HumiraTM), anakinra (KineretTM), rituximab (RituxanTM), CTLA4-Ig (abatacept), antimalarials, gold salts, sulfasalazine, d-penicillamine, cyclosporin A, cyclophosphamide azathioprine; and the like.
Corticosteroids, e.g. prednisone, methylpredisone, prednisolone, solumedrol, etc. have both anti-inflammatory and immunoregulatory activity. They can be given systemically or can be injected locally. Corticosteroids are useful in early disease as temporary adjunctive therapy while waiting for disease modifying agents to exert their effects. Corticosteroids are also useful as chronic adjunctive therapy in patients with severe disease.
Disease modifying anti-rheumatoid drugs, or DMARDs have been shown to alter the disease course and improve radiographic outcomes in RA. It will be understood by those of skill in the art that these drugs are also used in the treatment of other autoimmune diseases.
Methotrexate (MTX) is a frequent first-line agent because of its early onset of action (4-6 weeks), good efficacy, favorable toxicity profile, ease of administration, and low cost. MTX is the only conventional DMARD agent in which the majority of patients continue on therapy after 5 years. MTX is effective in reducing the signs and symptoms of RA, as well as slowing or halting radiographic damage. Although the immunosuppressive and cytotoxic effects of MTX are in part due to the inhibition of dihydrofolate reductase, the anti-inflammatory effects in rheumatoid arthritis appear to be related at least in part to interruption of adenosine and TNF pathways. The onset of action is 4 to 6 weeks, with 70% of patients having some response. A trial of 3 to 6 months is suggested.
Antigen specific therapeutic methods include administration of an antigen or epitope specific therapeutic agent. One method to induce immune tolerance is tolerizing DNA vaccines (Garren et al. (2001) Immunity, 15:15-22; Robinson et al. (2003) Nature Biotechnology 21:1033-9). Tolerizing DNA vaccines are DNA plasmids containing the regulatory regions necessary for expression of the encoded cDNA in mammalian cells, and would be engineered to contain cDNA sequence encoding all or a portion of activated protein c in order to induce immune tolerance to the encoded epitopes. To enhance the ability of such plasmids to induce immune tolerance, the immunostimulatory CpG sequences (Krieg et al. (1998) Trends Microbiol. 6:23-27) can be reduced in number or completely removed from the plasmid vector. Additionally, immunoinhibitory GpG sequences can be added to the vector (see Ho et al. (2005) J. Immunology, 175:6226-34).
As an alternative, or in addition to DNA tolerization, specific peptides, altered peptides, or proteins may be administered therapeutically to induce antigen-specific tolerance to treat autoimmunity. Native peptides targeted by the autoimmune response can be delivered to induce antigen-specific tolerance (Science 258:1491-4). Native peptides have been delivered intravenously to induce immune tolerance (J Neurol Sci. 152:31-8). Delivery of peptides that are altered from the native peptide, is also known in the art. Alteration of native peptides with selective changes of crucial residues (altered peptide ligands or “APL”) can induce unresponsiveness or change the responsiveness of antigen-specific autoreactive T cells. In another embodiment, whole protein antigens targeted by the autoimmune response can be delivered to restore immune tolerance to treat autoimmunity (Science 263:1139).
Lesion Specific Markers
The invention provides polypeptides and polynucleotides that encode them that represent proteins that are differentially present in specific classes of MS lesions. These polynucleotides, polypeptides and fragments thereof have uses that include, but are not limited to, diagnostic probes, and primers as starting materials for probes and primers, as immunogens for antibodies useful in MS diagnosis and therapy, and the like as discussed herein.
In general, the term “polypeptide” as used herein refers to both the full length polypeptide, as well as portions or fragments thereof. “Polypeptides” also includes variants of the naturally occurring proteins, where such variants are homologous or substantially similar to the naturally occurring protein, and can be of an origin of the same or different species as the naturally occurring protein. In general, variant polypeptides have a sequence that has at least about 80%, usually at least about 90%, and more usually at least about 98% sequence identity with a differentially expressed polypeptide described herein. The variant polypeptides can be naturally or non-naturally glycosylated, i.e., the polypeptide has a glycosylation pattern that differs from the glycosylation pattern found in the corresponding naturally occurring protein.
Fragments of the polypeptides disclosed herein, particularly biologically active fragments and/or fragments corresponding to functional domains, are of interest. Fragments of interest will typically be at least about 10 aa to at least about 15 aa in length, usually at least about 50 aa in length, and can be as long as 300 aa in length or longer, but will usually not exceed about 1000 aa in length, where the fragment will have a stretch of amino acids that is identical to a polypeptide encoded by a polynucleotide having a sequence of any one of the polynucleotide sequences provided herein, or a homolog thereof. A fragment “at least 20 aa in length,” for example, is intended to include 20 or more contiguous amino acids. In this context “about” includes the particularly recited value or a value larger or smaller by several (5, 4, 3, 2, or 1) amino acids.
A polypeptide marker can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography (“HPLC”) is employed for purification.
Polypeptides can also be recovered from: products purified from natural sources, including bodily fluids, tissues and cells, whether directly isolated or cultured; products of chemical synthetic procedures; and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast higher plant, insect, and mammalian cells.
Polypeptides can be prepared and used for raising antibodies for experimental, diagnostic, and therapeutic purposes. Antibodies may be used to identify MS lesions or subtypes of lesions. These antibodies are specific to an epitope on the polypeptide, and can precipitate or bind to the corresponding native protein in a cell or tissue preparation or in a cell-free extract of an in vitro expression system.
The antibodies may be utilized for immunophenotyping of cells and biological samples, e.g. a needle biopsy. Monoclonal antibodies directed against a specific epitope, or combination of epitopes, will allow for the screening of cell populations potentially containing the marker. Various techniques can be utilized using monoclonal antibodies to screen for cellular populations expressing the marker(s), and include RIA, ELISA, immunohistochemistry, magnetic separation using antibody-coated magnetic beads, “panning” with antibody attached to a solid matrix (i.e., plate), and flow cytometry (See, e.g., U.S. Pat. No. 5,985,660; and Morrison et al. Cell, 96:737-49 (1999)). These techniques allow for the screening of particular populations of cells; in immunohistochemistry of biopsy samples; in detecting the presence of markers shed into biologic fluids, and the like.
In many embodiments, the level of a subject polypeptide is measured. By measured is meant qualitatively or quantitatively estimating the level of the polypeptide in a first biological sample either directly (e.g. by determining or estimating absolute levels of polypeptide) or relatively by comparing the levels to a second control biological sample. In many embodiments the second control biological sample is obtained from an individual not having MS. As will be appreciated in the art, once a standard control level of gene expression is known, it can be used repeatedly as a standard for comparison.
Each publication cited in this specification is hereby incorporated by reference in its entirety for all purposes.
It is to be understood that this invention is not limited to the particular methodology, protocols, cell lines, animal species or genera, and reagents described, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which will be limited only by the appended claims.
As used herein the singular forms “a”, “and”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a cell” includes a plurality of such cells and reference to “the culture” includes reference to one or more cultures and equivalents thereof known to those skilled in the art, and so forth. All technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs unless clearly indicated otherwise.
Experimental
Proteomic Analysis of Active Multiple Sclerosis Lesions Reveals Therapeutic Targets
Laser-capture microdissection (LCM) and proteomics were used to identify proteins unique to three major types of multiple sclerosis (MS) lesions: acute plaque (AP), chronic active plaque (CAP) and chronic plaque (CP). Comparative proteomic profiles identified tissue factor (TF) and protein C inhibitor (PCI) within CAP samples, suggesting dysregulation of molecules associated with coagulation. In vivo administration of the thrombin inhibitor hirudin or recombinant activated protein C (aPC) reduced disease severity in experimental autoimmune encephalomyelitis (EAE) and suppressed Th1 and Th17 cytokines in astrocytes and immune cells. Administration of mutant forms of recombinant aPC showed that both its anticoagulant and its signaling functions were essential for optimal amelioration of EAE. A proteomic approach illuminated potential therapeutic targets selective for specific pathological stages of MS and implicated participation of the coagulation cascade.
Here, we classified MS brain lesions into distinct histological types: acute plaque (AP), chronic active plaque (CAP) and chronic plaque (CP). We then isolated the lesions by LCM and performed saturated sequencing by mass spectrometry. We selected two candidate proteins, tissue factor (TF) and protein C inhibitor (PCI), via an analysis using a computer-guided system. We then validated their potential therapeutic roles in experimental autoimmune encephalomyelitis (EAE). We also studied the cellular and molecular mechanism of how activated protein C (aPC), an intrinsic inhibitor of PCI, ameliorates EAE. These findings emphasize how lesion-specific proteomic profiling of diseased tissue from MS patients can identify potential therapeutic targets. In addition, we reveal the extensive interface between the coagulation system and brain inflammation.
Results
Histological characterization of MS lesions. MS brain autopsy samples from patients with different clinical subtypes (Table 1) were evaluated by staining with Hematoxylin and Eosin (H&E), Luxol Fast Blue (LFB) and immunohistochemistry (IH). Lesions with florid parenchymal and perivascular inflammatory cell infiltration, abundant astroglial hypertrophy, myelin fragmentation, edema and ongoing demyelination with indistinct margins were classified as AP (FIG. 1a-c). CAP lesions had chronic demyelination, sharply defined margins and recent areas of inflammatory demyelination at the edges (FIG. 1d-f). CP lesions had areas of demyelination with well-demarcated borders and abundant astrogliosis but few or no inflammatory cells (FIG. 1g-i). Age-matched control brain samples were analyzed similarly and were devoid of CNS abnormalities.
Proteomic profiling of MS lesions. We compared the different histological stages of MS lesions by proteomics analysis to determine their global protein expression profiles. LCM enabled selective isolation of MS lesions from the adjacent white matter from the same tissue blocks evaluated for histological characterization. Samples isolated by LCM were separately analyzed by nano-liquid chromatography and tandem mass spectrometry (FIG. 2a). To ascertain reliable protein identification, we used the criteria of stringent mass tolerance and eliminated false positive proteins by searching against a forward and reverse human protein database. Furthermore, to enhance maximal protein detection coverage, MS samples were analyzed repeatedly by mass spectrometric analysis (4-7 times) until a saturation point was reached. Analysis of control, AP, CAP and CP samples yielded a total of 2,574 proteins with high confidence. Among these, 2,302 proteins were related to MS samples (three types of lesions combined), and 1,492 proteins belonged to control samples (see Table 3 for a complete listing). For individual MS lesion types we identified 1,082, 1,728 and 1,514 proteins for AP, CAP and CP samples respectively (Table 2). To our knowledge this is the largest and the most comprehensive proteome of MS brain lesions characterized to date (Tables 3 and 5).
CAP expresses coagulation proteins. Following mass spectrometric protein identification, we utilized the INTERSECT software program to determine proteins specific to each MS lesion type. There were 158, 416 and 236 proteins unique to AP, CAP and CP (Table 4). We then applied the PROTEOME-3D software to assign biological functions and sub-cellular localization to these proteins. The analysis revealed that proteins of unknown function made up more than half of the unique proteins identified in all three MS lesion types. Of the proteins with known function, structural proteins, adhesion molecules, cell surface receptors and components of channels were among the most numerous (6% or greater). They were followed by proteins involved in the cell cycle, in synaptic transmission, in cellular signaling and in the components of the machinery for transcription and translation (2-6%). Least numerous were proteins with functions associated with molecular chaperones and cellular metabolism (<2%). Interestingly, the analysis revealed five proteins involved in coagulation: Tissue Factor (TF), protein C inhibitor (PCI), thrombospondin, Fibronectin and vitronectin (FIG. 2b, c). These coagulation proteins were unique to CAP samples.
Thrombin inhibition attenuates EAE. TF, a coagulation factor, is expressed in monocytes and astrocytes during inflammation and promotes proinflammatory thrombin signaling via Protease Activated Receptor (PAR) family of proteins. PCI is a serum protein that accumulates in the CAP lesions probably secondary to the disruption of the blood-brain barrier during neuroinflammation. PCI inhibits aPC. aPC also signals through PAR-1 and endothelial protein C receptor (EPCR). Despite sharing a common signaling pathway with procoagulant TF, aPC is an anticoagulant with cytoprotective properties. The combined presence of TF and PCI suggests proinflammatory thrombin formation and suppression of protein C (PC) pathway in CAP lesions.
To test the role of thrombin signaling during neuroinflammation, SJL/J mice that had been immunized with myelin proteolipid protein (PLP139-151) peptide were treated daily with either intravenous injection of the thrombin inhibitor hirudin (Refludan, recombinant lepirudin, Berlex), or with phosphate buffered saline (PBS), at the peak of clinical disease. Mice treated with hirudin showed dramatic improvement of disease severity (FIG. 3a). This was accompanied by decreased immune cell proliferation (FIG. 3b) and suppression of cytokines IL-6, tumor necrosis factor (TNF) and IL-17 (FIG. 3d, e). There were no differences in the production of IL4, IL-10, IL-12 and interferon-γ (IFN-γ) cytokines between the vehicle-treated and the hirudin-treated groups. Hirudin had no effect on relapse rates or disease course. The brains and spinal cords of mice treated with hirudin showed fewer inflammatory foci (FIG. 3c). Amelioration of EAE by hirudin treatment was observed only up to day 35, probably secondary to development of autoantibodies against hirudin.
aPC administration ameliorates EAE. aPC has anti-inflammatory and anti-apoptotic functions and its therapeutic benefits have previously been observed in meningococcemia and in SIRS (Systemic Inflammatory Response Syndrome). The presence of PCI in CAP samples and evidence of low serum levels of PC in MS patients suggest suppression of the PC pathway during MS. To determine the effects of aPC during neuroinflammation, we induced EAE in 7-8 week-old SJL/J mice and treated them with either recombinant murine aPC (0.2 mg/kg) or vehicle (PBS) beginning at the peak of disease. During the course of EAE, mice treated with aPC showed significant amelioration of disease severity (FIG. 4a). Treatment had no effect on relapse rates nor did it after the disease course. This effect was accompanied by decreased immune cell proliferation in splenocytes and lymph node cells (FIG. 4b) and inhibition of Th1 and Th17 cytokines in aPC-treated mice (FIG. 4c, d). Additionally, fewer inflammatory foci were observed in the CNS tissue of EAE mice treated with aPC (FIG. 4e).
Molecular mechanism of aPC during EAE. aPC functions both as an anticoagulant and a signaling molecule. Structure-function studies have identified the domain of aPC required for its anticoagulant function as distinct from its signaling function. To determine whether the amelioration of EAE by aPC treatment is mediated through anti-coagulant or signaling functions we induced EAE in SJL/J mice and treated with two recombinant aPC mutants, aPC-L8W and aPC-K193E. One mutant, aPC-L8W, retains anticoagulant properties but lacks PAR-1 signalling due to the defective interaction with its receptor EPCR at L8. The other mutant, aPC-K193E, mainly participates in PAR-1 signalling and lacks anticoagulant activities. The clinical status of the mice treated with aPC mutants were compared against those treated with either vehicle (PBS) or aPC wild type (WT). Mice treated with aPC-L8W and aPC-K193 showed significant amelioration early in the disease course (days 20-25) whereas mice treated with aPC-WT showed improvement in the latter part of disease course (days 25-30)(FIG. 5a-c). These data suggest that both activities of aPC may be required for maintaining an extended effect in this model.
To understand the effects of aPC on CNS and immune cells, we separately isolated peritoneal macrophages, astrocytes and T cells and activated them in vitro with either lipopolysaccharide (LPS) or CD3/CD28 following pre-treatment with recombinant murine aPC. Activated macrophages and astrocytes treated with aPC produced less IL6 and IL17 (FIG. 5d-g). Similarly, low levels of IL17 were detected in T cells exposed to aPC (FIG. 5h)., These data suggest that aPC suppresses inflammation in both the CNS and the periphery.
Since aPC suppressed NF-κB signaling during neuronal injury, we analyzed protein extracts from cultured T cells treated with murine aPC in cell activation assays by Western blot analysis. The results demonstrate less IκB breakdown in cells treated with aPC. This implies inhibition of NF-κB signalling by aPC.
This study provides the first ever information on large-scale protein identification from highly characterized MS brain lesions. Proteomic expression profiling of MS brain lesions has identified several candidate therapeutic targets. Reversing the physiological effects of two of these newly implicated proteins (TF and PCI) ameliorates disease in EAE. A parallel approach in identification of targets in EAE had previously led to development of new therapies in MS as in the case of Natalizumab which targets a critical integrin involved in homing of monocytes to the inflamed brain. Thus, this exercise has precedents in leading ultimately to new and effective therapies in MS.
Brück and Lucchinelti have classified active MS lesions according to their distinct histological and immunocytological characteristics (Lassmann et al. (2001) Trends Mol Med 7:115-21). The proteomic analysis of MS lesions illuminates the dynamic biological events that influence lesion development and pathogenesis. These proteomics techniques have been refined in the present invention to analyze specific areas in MS tissues (e.g. normal appearing white matter, areas of oligodendrocyte destruction) in order to identify proteins unique to these particular regions of interest.
The reversal of neurological deficits in EAE by administration of thrombin inhibitor and aPC demonstrates several new options for MS therapy. Heparin therapy was previously shown to improve symptoms during MS relapses and active EAE (see Maschmeyer et al. (1961) Bulletin of the Los Angeles Neurological Society 165-71; and Lider et al. (1989) J Clin Invest 83, 752-6), but treating MS patients with an anticoagulant such as hirudin is not optimal because of the increased risk of bleeding. Serum of EAE mice-treated with hirudin also showed the presence of anti-hirudin antibodies, which may have interfered with the protective effects of hirudin during EAE.
However, aPC provides a useful drug for therapy in MS, which may be further improved through the use of an aPC variant with reduced bleeding potential (see Mosnier et al. (2007) Blood 109, 3161-72; and Bernard et al. (2001) N Engl J Med 344:699-709). Experiments utilizing function-specific aPC mutants described herein demonstrate that both the anticoagulant and signalling properties of aPC ameliorate EAE, perhaps through different mechanisms. One explanation consistent with our findings centers on PAR-1 activation, such that either sending a cytoprotective signal (through EPCR and PAR-1 via aPC-K193E) or inhibiting the generation of thrombin (via aPC-L8W, and thus suppressing its pro-inflammatory signals through PAR-1) is independently sufficient to improve function in EAE.
We used the approach of systems biology to identify the molecular composition of the proteins in defined MS lesions. The lesion-specific proteome reveals. a “New World” with unique proteins identified in all three MS lesions. Proteins like those of the coagulation cascade are clearly playing new and unexpected pathobiological roles. The intersection of the coagulation cascade and inflammation in MS is the first of many new discoveries emerging from this catalogue of proteins. These proteomes constitute a vocabulary for the biological language whose rules and structures allow understanding of the disease.
Methods
All solvents, high performance liquid chromatography (HPLC) or mass spectrometry grade, reagents for histology were from Fisher (New Jersey, USA). Metal-rim slides and micro centrifuge tube for LCM were from MMI (Knoxville, Tenn., USA). Recombinant murine aPC, recombinant human aPC wild type (aPC-WT) and mutants (aPC-L8W and aPC-K193E) were generously provided by Lilly Research Laboratories. Protease inhibitor cocktail tablets were from Roche Applied Science (Mannheim, Germany). ABC kit, secondary antibodies (biotinylated horseradish peroxidase conjugates) and Diaminobenzidine were from Vector Inc. (Burlingame, Calif. USA). Monoclonal anti-GFAP (Glial fibrillary acidic protein), anti-CD3, anti-CD45 and anti-CD68 were from Dako Cytomation (Cabinteria, Calif.) and anti-CD 28 was from BD Biosciences. Rabbit polyclonal antibodies against IκB-α and β actin were from Cell Signaling and Sigma, respectively. Monoclonal anti-PLP was prepared as described previously.
Human brain samples from MS cases and normal controls. Fresh frozen MS and normal control brain samples were obtained at autopsy under an IRB approved protocol. MS brain samples and accompanying paraffin-embedded sections were kindly provided by Dr. C. S. Raine. Normal control samples were obtained from the University of Washington Alzheimer's disease brain consortium. Samples were harvested, rapidly frozen and stored at −80° C. All samples were obtained from the cerebral hemispheres.
Histopathological characterization and classification of MS lesions. Unfixed, frozen brain tissue from MS and control samples were partially thawed and  ̃1 cm tissue blocks were embedded in OCT compound (Sakura Finetek, USA). Frozen blocks were cut into 6 μM cryosections, then fixed in acetone briefly and analyzed by H&E, LFB and IH staining using antibodies against PLP, GFAP, CD3, CD45 and CD68 as previously described by Chabas et al. (2001) Science 294:1731-5. MS lesions were classified according to the criteria used by Lock et al. (2002) Nat Med 8:500-8. Normal control brain samples were ruled out for obvious CNS pathology.
Isolation of MS plaques by LCM and sample preparation. The LCM microscope and laser system were from MMI Systems (Geneva, Switzerland). MS lesions were isolated from samples (frozen blocks) used for histological characterization. 15 μM sections were cut on MMI membranes, briefly fixed in 75% ethanol and MS lesions were then isolated by LCM as previously described (Bagnato et al. (2007) Molecular & Cellular Proteomics 6, 1088-102). MS lesions from 400 tissue sections were isolated and extracted first with modified RIPA buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% SDS, 1% Triton X100, 1% deoxycholate, 5 mM EDTA, supplemented with protease inhibitor cocktail) and subsequently with 2% SDS buffer (62.5 mM Tris-HCl, pH7.5, 2% SDS) for 15 mins each at 65° C. White matter from the control samples was also isolated in a similar manner.
In-gel trypsin digestion, nano liquid chromatography and tandem mass spectrometry. Protein extracts (100 μg per sample) from MS and control brain samples were resolved by 1-dimensional SDS-PAGE using a 4-12% NuPAGE gel (Invitrogen, San Diego, Calif.) and stained with Coomassie Brilliant Blue G-250. Protein bands (20 per sample) were then digested with trypsin and peptides were then extracted as described (Hwang et al. (2006) Molecular & Cellular Proteomics 5, 1131-45). Tryptic peptides were analyzed using an LTQ linear ion trap mass spectrometer (Thermo Finnigan, San Jose, Calif.) equipped with a commercial nanospray source (Thermo Finnigan). Samples were loaded into an in-house C18 micro column (100-μm inner diameter, 360-μm outer diameter, 10-cm length, 5-μm bead size, 100-Å pore size, Column Engineering Inc., Ontario, Canada) by a microautosampler (Famos-Dionex, Sunnyvale, Calif.) and separated by an Agilent 1,100 high performance binary pump. Peptides were loaded for 20 mins with solvent A (5% acetonitrile, 0.4% acetic acid and 0.005% heptafluorobutyric acid) at a flow rate of  ̃200 nl/min by flow splitting. The solvent gradient of HPLC was linear from 95% solvent A to 30% solvent B (100% acetonitrile, 0.4% acetic acid, and 0.005% heptafluorobutyric acid) for 45 mins. The column was then regenerated by 80% solvent B for 10 mins and 100% solvent A for 10 mins. The eluent was introduced directly into an LTQ mass spectrometer via electrospray ionization. Each full mass spectrometric scan was followed by a five tandem mass spectrometry scan of the most intense ion with data-dependent selection using the dynamic exclusion option (Top 5 method). Dynamic exclusion features were enabled to maximize the fragmentation of low abundance peptide ions. Sample loading, solvent delivery and scan functions were obtained by XCalibur software (Thermo Finnigan). Each sample was analyzed 4-7 times by mass spectrometry.
Database searching and data processing. Data obtained from each gel band generated a .dat file and was searched independently against a non-redundant human protein database (56,709 entries as of Dec. 1, 2004, Advanced Biomedical Computing Center) using the SEQUEST algorithm, resulting in one .html output file. All .html files from each lesion type (AP, CAP, CP) were combined using the INTERACT program. They were then filtered using the following criteria: peptide mass tolerance of 2.0 with differential modification of +16 for oxidized methionine, +80 for phosphorylated serine, threonine and tyrosine, cross-correlation (Xcorr) of 1.9, 2.2, and 3.7 for 1+, 2+, and 3+ charge state peptides respectively and delta correlation (dCn) score greater than or equal to 0.1, excluding single peptide ID. False positive rates were estimated by searching a subset of AP, CAP, CP and control samples against a concatenated forward and reverse human protein database using the formula: False Positive (%)=number of reverse peptide Ids×100/number of forward peptide Ids, based on representative subsets of each category (Table 2).
Protein quantitation. Semi-quantitative protein abundance was estimated by spectral count (SC). SC=the number of tandem mass spectrometric spectra confidently assigned to the protein, as previously defined.
Identification of proteins unique to MS lesions and functional annotation. We used software INTERSECT to determine proteins unique to each lesion type. GO (gene ontology) classification and software PROTEOME-3D were then applied to assign biological functions and sub-cellular localization of these proteins.
EAE induction, treatment with hirudin, aPC (WT and mutants), proliferation assays, cytokine analysis and quantitative histopathology. Mice were maintained in the Research Animal Facility at Stanford University. EAE was induced in 7-8 week old SJL/J female mice by subcutaneous immunization with 100 μg PLP139-151 in emulsion. For hirudin or aPC treatment, EAE mice (n=10/group) were treated with daily intravenous injection of hirudin (10 mg/kg) or mouse recombinant aPC-WT (0.2 mg/kg) at the peak of disease and compared with PBS treated group. Mice were assessed daily for clinical signs of EAE and scored according to: 0, no clinical disease; 1, tail weakness; 2, hindlimb weakness; 3, complete hindlimb paralysis; 4, hindlimb paralysis and some forelimb weakness; 5, moribund or dead.
Recombinant human aPC-WT and mutants (aPC-L8W and aPC-K193E) were prepared as described by Grinnell et al (2007) Critical Care Medicine (suppl). These aPC variants have the following properties on aPTT and PAR-1 signaling function: aPC-WT: aPTT (relative activity)=1, PAR-1 (relative activity)=1, aPC-L8W: aPTT=1, PAR-1=0.02 and aPC-K193E: aPTT=0.03, PAR-1=1. EAE mice (n=7/group) were treated with daily intravenous injection of recombinant human aPC-WT, aPC-L8W, aPC-K193E (0.46 mg/kg) or PBS (control group) at the time of maximal paralysis and assessed daily until day 30.
In vitro immune cell proliferation and cytokine analysis were performed as previously described (Ousman et al. (2007) Nature 448, 474-9). Briefly, splenocytes and lymph node cells harvested from EAE experiments were cultured in flat-bottomed, 96-well plates at a concentration of 0.5×106 cells/well in stimulation media (RPMI 1640 supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mM non-essential amino acids, 100 U/ml2 Penicillin, 0.1 mg/ml2 streptomycin, 0.5 μM 2-mercaptoethanol and 10% fetal calf serum) and activated with PLP139-151 peptide (5-20 μg/ml). To determine proliferation rates, cultures were pulsed with (3H)-Thymidine (1 μCi per well) following 72-hour culture and harvested 18 hours later onto filter paper. The counts per minute (cpm) of incorporated 3H-Thymidine were read using a beta counter. Cytokine levels (IL-2, IL-4, IL-6, IL-10, IL-12p40, IL-17, IFN-γ, TNF) were measured from the supernatant of cultured cells using anti-mouse OPTEIA ELISA kits (BD Pharmigen).
For histopathological analysis, brains and spinal cord of EAE mice were fixed in 10% formaldehyde. 6 μM thick paraffin sections were stained with LFB and H&E and the number of inflammatory foci within the brain and spinal cord were quantified by a neuropathologist who was blinded to the treatment and clinical parameters of the mice.
In vitro Immune cell activation assays and cytokine analysis. T lymphocytes were isolated from pooled splenocytes and lymph node cells from 8 week-old naïve SJL/J mice by negative selection (Pan T cell isolation kit, Miltenyi Biotech). Cells were pre-treated with 30 nM recombinant murine aPC for 15 mins at 37° C. followed by activation with CD3/CD28 (5 μg/ml) coated on 12 well plates. Cells were cultured at 5×106 cells/ml concentration in stimulation media. Culture plates were harvested at different time points (15 min-96 hours) and cytokine levels were measured from culture supernatant by ELISA.
Primary peritoneal macrophages were isolated from naïve 8-9 weeks old SJL/J mice following intraperitoneal injection of thioglycollate (BD Diagnostic Systems) and cultured in complete medium (DMEM supplemented with 10% fetal bovine serum, 1 mM sodium pyruvate, 100 μM penicillin and 0.1 mg/ml streptomycin). Cells (1×106 cells/ml) were treated with 30 nM recombinant murine aPC for 15 minutes, then activated with LPS 100 ng/ml (Sigma), harvested at different time points (15 min-72 hours), and cytokine levels were measured.
Astrocyte culture. Astrocytes were cultured from brain of one-day-old SJL/J pups as described previously. Briefly, the cerebral cortices from pups were minced, cells were disrupted by passing through a filter and cultured in complete DMEM. Purified astrocytes (50-80% confluent) were treated with 30 nM recombinant murine aPC for 15 minutes and activated with 100 ng/ml LPS (Sigma). Cells were harvested at 4, 24, 48 and 72-hours, and supernatant were analyzed by ELISA for cytokine production.
Western blot analysis. For NF-κB activation, total cell lysate from purified T cells treated with aPC were analyzed by SDS-PAGE, transferred to PVDF membrane, probed with antibodies against IκB-α, and β-actin and the signal was visualized by enhanced chemiluminescence.
Statistical analysis. Data are presented as means±SEM. When data were parametric, a t-test (n=2 groups) was used to detect between-group differences. When data were non-parametric, a Mann-Whitney U test was used for comparison between groups (n=2 groups). A p value of <0.05 or lower was considered significant. Error bars in FIG. 3d, e, FIG. 4c, d and FIG. 5d, e are not discernible due to their small size.







TABLE 1







Characteristics of the MS patients and controls in the study















Age/

Disease

Lesion
Cause of
Autopsy



gender
Type of MS
duration
Prior treatment
type
death
interval




















MS1
42/F
Acute
2
wk
None
AP
Respiratory
12
h.









failure


MS2
54/F
Acute
21⁄2
mo
Corticosteroids
AP
Respiratory
12
h.









failure


MS3
31/F
Chronic
11
y
Corticosteroids
CAP
Respiratory
1.5
h









failure


MS4
27/F
Progressive
10
y.
Corticosteroids
CAP
Broncho-
4
h.









pneumonia


MS5
47/M
Secondary
20
y
Corticosteroids
CP
Respiratory
24
h




Progressive




failure


MS6
46/M
Chronic
15
y
Lioresal
CP
Cardiac
4
h




progressive


Compazine

arrest















Control 1
23/F
Fallopian
N/A
N/A

Respiratory
12
h




tube cancer



failure


Control 2
52/F
Ovarian
N/A
N/A

Respiratory
15.5
h




cancer



failure









Two separate samples of brain lesions were obtained from MS1 and 2 and three separate samples were obtained from MS3-6 and normal control samples. None of the MS cases were treated with disease modifying agents. Full CNS autopsies were performed on all cases.







TABLE 2







Summary of the proteomic data












AP
CAP
CP
CTL















Peptide
40,819
64,678
54,339
68,478


Unique peptide
6,321
10,143
8,967
8,991


Protein*
1,082
1,728
1,514
1,492


Reverse peptide
39
34
10
12


Ids


Forward peptide ids
3612
4937
3687
4020


False positive (%)**
1.08
0.69
0.27
0.3





*Identification filtering criteria: Xcorr 1.9 (1+), 2.2(2+), 3.7(3+), dCn > 0.1, excluding trypsin, including keratin, excluding single peptide ID. The files used to compute false positive rate were searched against concatenated forward and reverse human database.


**Formula for the False Positive (%): number of reverse Ids × 100/number of forward Ids, based on representative subset of each category.













TABLE 3







PROTEINS UNIQUE TO AP








Protein ID
Common Name





2A5E_HUMAN
Serine/threonine protein phosphatase 2A


A3B1_HUMAN
Adapter-related protein complex 3 beta 1 subunit


AGRN_HUMAN
Agrin precursor


ANC1_HUMAN
Anaphase promoting complex subunit 1


ASP2_HUMAN
Apoptosis stimulating of p53 protein 2


AT10_HUMAN
ADAMTS-10 precursor Secreted


ATHA_HUMAN
Potassium-transporting ATPase alpha chain 1


ATX7_HUMAN
Ataxin-7; Spinocerebellar ataxia type 7 protein


BAI2_HUMAN
Brain-specific angiogenesis inhibitor 2 precursor


BDH_HUMAN
D-beta-hydroxybutyrate dehydrogenase


CA17_HUMAN
Collagen alpha-1(VII) chain [Precursor] Basement membrane


CA1G_HUMAN
Collagen alpha 1 Type II membrane protein


CACP_HUMAN
Carnitine O-acetyltransferase Endoplasmic reticulum


CDA9_HUMAN
Protocadherin alpha 9 precursor


CHD1_HUMAN
Chromodomain-helicase-DNA-binding protein 1


CHD2_HUMAN
similarity to CHROMODOMAIN HELICASE DNA-BINDING PR


CHD7_HUMAN
Chromodomain-helicase-DNA-binding protein 7


CMGA_HUMAN
Chromogranin A precursor Neuroendocrine and endocrine


DCE2_HUMAN
Glutamate decarboxylase, 65 kDa isoform


DHE4_HUMAN
Glutamate dehydrogenase 2, mitochondrial precursor


DJA4_HUMAN
DnaJ homolog subfamily A member 4 Membrane-bound


E2F4_HUMAN
Transcription factor E2F4 Nuclear.


EFB1_HUMAN
Ephrin-B1 precursor Type I membrane protein


F20C_HUMAN
Protein FAM20C precursor


FGD1_HUMAN
Putative Rho/Rac guanine nucleotide exchange factor


FX30_HUMAN
F-box only protein


FXR1_HUMAN
Fragile X mental retardation syndrome related protein 1


GCP6_HUMAN
Gamma-tubulin complex component 6; GCP-6


GLR3_HUMAN
Glutamate receptor 3 precursor


GP80_HUMAN
Probable G protein-coupled receptor GPR80


GTR3_HUMAN
Solute carrier family 2, facilitated glucose transporter


HB21_HUMAN
HLA class II histocompatibility antigen


HGF_HUMAN
Hepatocyte growth factor precursor


ITB4_HUMAN
Integrin beta-4 precursor Type I membrane protein


ITN1_HUMAN
Intersectin 1; SH3 domain-containing protein 1A


JAK1_HUMAN
Tyrosine-protein kinase JAK1


LRR8_HUMAN
Leucine-rich repeat-containing protein 8 precursor


M3K1_HUMAN
Mitogen-activated protein kinase kinase kinase 1


MINT_HUMAN
Msx2-interacting protein


MY15_HUMAN
Myosin XV


MYBA_HUMAN
Myb-related protein A


MYM1_HUMAN
Myomesin 1 (190 kDa titin-associated protein)


NAC1_HUMAN
Sodium/calcium exchanger 1 precursor


NAF1_HUMAN
Nef-associated factor 1


NFX1_HUMAN
Transcriptional repressor NF-X1


NI2M_HUMAN
NADH-ubiquinone oxidoreductase B22 subunit


NSD1_HUMAN
Nuclear receptor binding SET domain containing protein 1


NU93_HUMAN
Nuclear pore complex protein Nup93


NUKM_HUMAN
NADH-ubiquinone oxidoreductase 20 kDa subunit, mitochondr NO INFO NO



INFO 23564 10


O15065
KIAA0358 protein; C: cytoplasm NO INFO NO INFO 176077 5.75


O15081
KIAA0376 protein NO INFO NO INFO 99803 5.07


O60611
Supervillin


O75042
KIAA0454 protein


O75160
KIAA0672 protein


O75163
KIAA0676 protein


O75183
KIAA0701 protein


O75691
DRIM protein


O94896
KIAA0804 protein


O95204
Metalloprotease 1


O96005
Cleft lip and palate transmembrane protein 1


P78524
P126


PAPG_HUMAN
Poly(A) polymerase gamma; PAP gamma


PAX3_HUMAN
Paired box protein Pax-3; HUP2


PC16_HUMAN
Protocadherin 16 precursor Type I membrane protein


PCP_HUMAN
Lysosomal Pro-X carboxypeptidase precursor


PER3_HUMAN
Period circadian protein 3


PHF6_HUMAN
PHD finger protein 6


PKHD_HUMAN
Polycystic kidney and hepatic disease 1 precursor


Q13535
FRAP-related protein; F: protein kinase activity


Q66M66
Cdc42-associated guanine nucleotide exchange factor


Q68D12
Hypothetical protein DKFZp781C0723


Q68DP5
Hypothetical protein DKFZp686B2031


Q693C2
Nonstructural protein 1; C: virion


Q6IMI5
SULT1C3 splice variant a; F: sulfotransferase activity


Q6MZP7
Hypothetical protein DKFZp686G04165


Q6NY19
FLJ46061 protein


Q6PK04
MGC16597 protein


Q6ZRV8
Hypothetical protein FLJ46051


Q6ZSJ6
Hypothetical protein FLJ45467


Q6ZSS7
Hypothetical protein FLJ45241


Q6ZUD8
Hypothetical protein FLJ43793


Q6ZV52
Hypothetical protein FLJ42981


Q71RH2
FP1188; C: integral to membrane


Q7KWM6
Similar to Homo sapiens


Q7RTR0
NOD6


Q7Z7J6
Actin alpha 1 skeletal muscle protein


Q86V20
Family with sequence similarity 35


Q8IUN3
FLJ10157 protein


Q8IW51
KIAA1458 protein


Q8IWG2
Hypothetical protein LOC284001


Q8IWV7
Ubiquitin ligase E3 alpha-I; F: ligase activity


Q8IXS1
ARHGAP20 protein


Q8IYQ7
Threonine synthase-like 1


Q8IZ48
FBF1 protein


Q8N3R6
Hypothetical protein DKFZp451G165


Q8N442
Hypothetical protein FLJ13220


Q8N5D9
Similar to spindlin; P: gametogenesis


Q8N6P1
TMPIT protein


Q8N6Z5
PTPN23 protein


Q8N970
Hypothetical protein FLJ38285


Q8N9C0
Hypothetical protein FLJ37794


Q8N9H0
Hypothetical protein FLJ37160


Q8NCD6
Hypothetical protein FLJ90323


Q8ND71
hypothetical protein


Q8NDT2
Hypothetical protein DKFZp547N2215


Q8SSN9
Similar to Homo sapiens


Q8TAI1
Similar to LOC147447


Q8TDM9
Amplified in breast cancer 1


Q8TEN9
FLJ00154 protein


Q8TF60
KIAA1941 protein


Q8WWZ8
LZP; Hypothetical protein FLJ39116


Q8WXX0
Ciliary dynein heavy chain 7


Q8WYL5
HSSH-1L


Q8WYN8
Ribosomal protein S27a


Q96CN6
ADCK2 protein


Q96F76
Putative RNA methyltransferase


Q96FZ8
Citrate synthase, mitochondrial [Precursor]


Q96GK7
Fumarylacetoacetate hydrolase domain containing 2A


Q96I57
MYH7B protein


Q96JB1
DNAH8


Q96M34
Hypothetical protein FLJ32859


Q96Q04
KIAA1883 protein


Q96QE4
Hypothetical protein


Q96RD8
GTP binding protein Rab1a


Q96RK0
Capicua protein


Q9BZS0
Kappa B and V(D)J recombination signal sequences binding prNO


Q9C0B7
KIAA1746


Q9HCD6
KIAA1636 protein


Q9NQW1
Secretory pathway component Sec31B-1


Q9P2H0
KIAA1377 protein


Q9P2J0
KIAA1357 protein


Q9ULU2
KIAA1127 protein


Q9UNJ2
Myosin-IXa


Q9Y2I9
KIAA0984 protein


Q9Y4F4
KIAA0423 protein


R39B_HUMAN
small GTP-binding rab protein


RGE4_HUMAN
RAP guanine-nucleotide-exchange factor 4


RH26_HUMAN
Rho-GTPase-activating protein 26


RP2B_HUMAN
Ras-related protein Rap-2b


RRP5_HUMAN
RRP5 protein homolog; Programmed cell death protein 11


RYR2_HUMAN
Ryanodine receptor 2 Integral membrane protein


S6A1_HUMAN
Sodium- and chloride-dependent GABA transporter 1


SGCE_HUMAN
Epsilon-sarcoglycan precursor Type I membrane protein


SON_HUMAN
SON protein; SON3; Negative regulatory element-binding protein


SPCR_HUMAN
Spectrin beta chain, brain 4 Cytoplasmic


SRB1_HUMAN
Signal-regulatory protein beta-1 precursor; SIRP-beta-1


SRB2_HUMAN
Signal-regulatory protein beta-2 precursor; SIRP-beta-2


T172_HUMAN
TBP-associated factor 172; TAF-172


T4AP_HUMAN
Trpc4-associated protein


TOP1_HUMAN
DNA topoisomerase I


TRP4_HUMAN
Short transient receptor potential channel 4


U13A_HUMAN
Unc-13 homolog A


U13B_HUMAN
Unc-13 homolog B; Munc13-2


UB13_HUMAN
Ubiquitin carboxyl-terminal hydrolase 13


UGG2_HUMAN
UDP-glucose:glycoprotein glucosyltransferase 2 precursor


VATL_HUMAN
Vacuolar ATP synthase 16 kDa proteolipid subunit


Y296_HUMAN
Hypothetical zinc finger protein KIAA0296


Z479_HUMAN
Zinc finger protein 479
















TABLE 4







Proteins unique to CAP








Protein ID
Common Name





2A5D_HUMAN
Serine/threonine protein phosphatase


5NTD_HUMAN
5′-nucleotidase, ecto NO


A1M1_HUMAN
Adaptor-related protein complex


ABR_HUMAN
Active breakpoint cluster


AFAM_HUMAN
Afamin precursor; Alpha-albumin;


AFP2_HUMAN
Arfaptin 2; ADP-ribosylation


AKC1_HUMAN
Aldo-keto reductase family


ANC5_HUMAN
Anaphase promoting complex


ANRY_HUMAN
Ankyrin repeat domain


APC_HUMAN
Adenomatous polyposis coli


APXL_HUMAN
Apical-like protein; APXL


AR1A_HUMAN
Actin-related protein 2/3


ARH2_HUMAN
rhoVrac guanine nucleotide


ASC_HUMAN
Apoptosis-associated speck-like protein


ASPH_HUMAN
Aspartyl/asparaginyl beta-hydroxylase Type


ATCY_HUMAN
Caytaxin; Ataxia Cayman


ATHL_HUMAN
Potassium-transporting ATPase alpha


ATND_HUMAN
Sodium/potassium-transporting ATPase beta-3


ATPR_HUMAN
ATP synthase coupling


ATS6_HUMAN
ADAMTS-6 precursor; A


BAG3_HUMAN
BAG-family molecular chaperone


BC046405_1
Hps5 protein [Mus


BGAL_HUMAN
Beta-galactosidase precursor Lysosomal.


BM02_HUMAN
UPF0185 protein BM-002


BOR4_HUMAN
Cdc42 effector protein


BPEB_HUMAN
Bullous pemphigoid antigen


C10_HUMAN
Putative C10 protein


C3L1_HUMAN
Chitinase-3 like protein


CA11_HUMAN
Collagen alpha 1


CA21_HUMAN
Collagen alpha 2


CA25_HUMAN
Collagen alpha 2


CA26_HUMAN
Collagen alpha 2


CA36_HUMAN
Collagen alpha 3


CADJ_HUMAN
Cadherin-19 precursor; UNQ478/PRO941;


CAFA_HUMAN
Chromatin assembly factor


CALD_HUMAN
hypothetical protein with


CALU_HUMAN
Calumenin precursor; Crocalbin;


CARC_HUMAN
Caspase recruitment domain


CCDA_HUMAN
Coiled-coil domain containing


CD82_HUMAN
CD82 antigen (Inducible


CDK5_HUMAN
Cell division protein


CDS2_HUMAN
Phosphatidate cytidylyltransferase 2;


CEBP_HUMAN
Cyclin-E binding protein


CEG3_HUMAN
Centaurin gamma 3


CHP1_HUMAN
Calcium-binding protein p22;


CLP2_HUMAN
Calponin-2 (Calponin H2,


CLP3_HUMAN
Calponin-3 (Calponin, acidic


CN2A_HUMAN
cGMP-dependent 3', 5' -cyclic phosphodiesterase;


CN3B_HUMAN
cGMP-inhibited 3', 5' -cyclic phosphodiesterase


CN4A_HUMAN
cAMP-specific 3′,5′-cyclic phosphodiesterase


CN4B_HUMAN
cAMP-specific 3′,5′-cyclic phosphodiesterase


CNB3_HUMAN
Cyclic-nucleotide-gated cation channel


CNC3_HUMAN
Protein C14orf123; HSPC134;


COA2_HUMAN
Acetyl-CoA carboxylase 2


COPD_HUMAN
Coatomer delta subunit


COPE_HUMAN
Coatomer epsilon subunit;


COPP_HUMAN
COATOMER BETA PRIME


COPZ_HUMAN
Coatomer zeta-1 subunit


COXJ_HUMAN
Cytochrome c oxidase


CP27_HUMAN
Cytochrome P450 27,


CSN6_HUMAN
COP9 signalosome complex


CT53_HUMAN
Hypothetical protein C20orf53


CXA1_HUMAN
Gap junction alpha-1


CYA5_HUMAN
Adenylate cyclase, type


DAB2_HUMAN
Disabled homolog 2


DCD_HUMAN
Dermcidin precursor Secreted.


DD17_HUMAN
Probable RNA-dependent helicase


DESP_HUMAN
Desmoplakin (DP) Innermost


DHS1_HUMAN
Dehydrogenase/reductase SDR family


DJB6_HUMAN
DnaJ homolog subfamily


DLG5_HUMAN
Discs, large homolog


DPM1_HUMAN
Dolichol-phosphate mannosyltransferase; Dolichol-phosphate


DTNA_HUMAN
Dystrobrevin alpha Cytoplasmic.


DTX2_HUMAN
Deltex protein 2;


DUT_HUMAN
Deoxyuridine 5′-triphosphate nucleotidohydrolase,


EDD_HUMAN
Ubiquitin--protein ligase EDD


ENAH_HUMAN
Enabled protein homolog


ENH_HUMAN
Enigma homolog; Enigma-like


ENP1_HUMAN
Ectonucleoside triphosphate diphosphohydrolase


EWS_HUMAN
RNA-binding protein EWS


EXC7_HUMAN
Exocyst complex component


FABE_HUMAN
Fatty acid-binding protein,


FAK2_HUMAN
Protein tyrosine kinase


FBL2_HUMAN
Fibulin-2 precursor; C: extracellular


FINC_HUMAN
Fibronectin precursor Secreted;


FLR2_HUMAN
Leucine-rich repeat transmembrane


FOG1_HUMAN
Zinc finger protein


G128_HUMAN
Probable G protein-coupled


GBF1_HUMAN
Golgi-specific brefeldin A-resistance


GBGC_HUMAN
Guanine nucleotide-binding protein


GBP1_HUMAN
Interferon-induced guanylate-binding protein


GCP3_HUMAN
Gamma-tubulin complex component


GCST_HUMAN
Aminomethyltransferase, mitochondrial precursor


GDL1_HUMAN
Ganglioside-induced differentiation-associated protein


GL6S_HUMAN
N-acetylglucosamine-6-sulfatase precursor Lysosomal.


GLR2_HUMAN
Glutamate receptor 2


GUAA_HUMAN
GMP synthase [glutamine-hydrolyzing]


HE47_HUMAN
Spliceosome RNA helicase


HEMZ_HUMAN
Ferrochelatase, mitochondrial precursor


HP28_HUMAN
28 kDa heat-


HS47_HUMAN
47 kDa heat


HXA4_HUMAN
Homeobox protein Hox-A4


ICAL_HUMAN
Calpain inhibitor NO


IDHG_HUMAN
Isocitrate dehydrogenase [NAD]


IF2A_HUMAN
Eukaryotic translation initiation


IF31_HUMAN
Eukaryotic translation initiation


IKKA_HUMAN
Inhibitor of nuclear


IM44_HUMAN
Import inner membrane


IP3K_HUMAN
Inositol-trisphosphate 3-kinase A;


IPO9_HUMAN
Importin 9; Imp9;


IPPD_HUMAN
Dopamine- and cAMP-regulated


IPSP_HUMAN
Plasma serine protease


IRF6_HUMAN
Interferon regulatory factor


IRS2_HUMAN
Insulin receptor substrate-2;


IVD_HUMAN
Isovaleryl-CoA dehydrogenase, mitochondrial


JMJ_HUMAN
Jumonji protein; Jumonji/ARID


K2C3_HUMAN
Keratin, type II


KCRM_HUMAN
Creatine kinase, M


KCRS_HUMAN
Creatine kinase, sarcomeric


KF11_HUMAN
Kinesin-like protein KIF11


KF3A_HUMAN
Kinesin-like protein KIF3A


KF4A_HUMAN
Chromosome-associated kinesin KIF4A


KFP3_HUMAN
Kinesin-associated protein 3;


KHL2_HUMAN
Kelch-like protein 2;


KMLS_HUMAN
Myosin light chain


KPCB_HUMAN
Protein kinase C,


KPCE_HUMAN
Protein kinase C,


KPCO_HUMAN
Protein kinase C,


KPR2_HUMAN
Ribose-phosphate pyrophosphokinase II


KV1A_HUMAN
Ig kappa chain


LAP1_HUMAN
Leucine-rich repeat-containing protein


LCF3_HUMAN
Long-chain-fatty-acid--CoA ligase 3;


LD6B_HUMAN
L-lactate dehydrogenase A-like


LGMN_HUMAN
Legumain precursor; Asparaginyl


LMA1_HUMAN
Laminin alpha-1 chain


LPA3_HUMAN
Liprin-alpha 3 Cytoplasmic.


LUM_HUMAN
Lumican precursor Secreted;


LY6H_HUMAN
Lymphocyte antigen Ly-6H


MER1_HUMAN
Mammalian ependymin related


MIME_HUMAN
Mimecan precursor NO


MLEG_HUMAN
Myosin light chain


MLEY_HUMAN
Myosin light chain


MLRA_HUMAN
Myosin regulatory light


MMAA_HUMAN
Methylmalonic aciduria type


MPK4_HUMAN
Dual specificity mitogen-activated


MSAP_HUMAN
MIR-interacting saposin-like protein


MSLN_HUMAN
Mesothelin precursor Attached


MTR1_HUMAN
Myotubularin-related protein 1


MTR3_HUMAN
Myotubularin-related protein 3;


MTRR_HUMAN
Methionine synthase reductase,


MU5B_HUMAN
Mucin 5B precursor;


MY1C_HUMAN
Myosin Ic (Myosin


MY1F_HUMAN
Myosin If; Myosin-IE;


MYG_HUMAN
Myoglobin. NO INFO


MYH2_HUMAN
Myosin heavy chain,


MYH7_HUMAN
Myosin heavy chain,


MYHB_HUMAN
Myosin heavy chain,


MYL4_HUMAN
Myosin light polypeptide


MYO6_HUMAN
Myosin VI. NO


MYPC_HUMAN
Myosin-binding protein C,


N214_HUMAN
Nuclear pore complex


NCB1_HUMAN
Nucleobindin 1 precursor


NDR3_HUMAN
NDRG3 protein; C: cytoplasm


NEB1_HUMAN
Neurabin-I (Neural tissue-specific


NEBL_HUMAN
Nebulette (Actin-binding Z-disk


NEST_HUMAN
Nestin; C: intermediate filament


NGF_HUMAN
Beta-nerve growth factor


NHR2_HUMAN
Na(+)/H(+) exchange regulatory


NIBL_HUMAN
Niban-like protein NO


NIN_HUMAN
Ninein (hNinein) Component


NMZ1_HUMAN
Glutamate [NMDA] receptor


NNP1_HUMAN
NNP-1 protein; Novel


NOL3_HUMAN
Nucleolar protein 3;


NP14_HUMAN
Nucleolar phosphoprotein p130


NUKS_HUMAN
Nuclear ubiquitous casein


NX1A_HUMAN
Neurexin 1-alpha precursor


O00114
Hypothetical human serine-threonine


O00145
SH2 containing inositol-5-phosphatase;


O14827
Ras-GRF2; F: guanyl-nucleotide exchange


O14964
hypothetical protein P1408


O15059
KIAA0351 protein; F: guanyl-nucleotide


O43302
KIAA0418 protein NO


O60293
KIAA0546 protein; C: intracellular


O60316
KIAA0570 protein; F: cysteine-type


O60466
TGF beta receptor


O60526
so09f03.y1 Gm-c1035 Glycine


O60831
JM4 [Homo sapiens]


O75058
KIAA0470 protein NO


O75061
KIAA0473 protein; F: phosphoprotein


O75112
KIAA0613 protein; F: protein


O75815
Breast cancer antiestrogen


O75921
RNA polymerase II


O76045
Pro alpha 1(I)


O95035
WUGSC: H_RG054D04 NO INFO


O96028
Putative WHSC1 protein;


ORP2_HUMAN
Oxysterol binding protein-related


OSB1_HUMAN
Oxysterol-binding protein 1;


OST4_HUMAN
Dolichyl-diphosphooligosaccharide--protein



glycosyltransferaseType I


OSTP_HUMAN
Osteopontin precursor; Bone


OTOF_HUMAN
Otoferlin (Fer-1 like


P2X2_HUMAN
P2X purinoceptor 2;


P4H1_HUMAN
Prolyl 4-hydroxylase alpha-1


P5CS_HUMAN
Delta 1-pyrroline-5-carboxylate synthetase;


PAPA_HUMAN
Pregnancy-associated plasma protein-A


PARA_HUMAN
Alpha-parvin (Calponin-like integrin-linked


PCD5_HUMAN
Programmed cell death


PCFB_HUMAN
Pre-mRNA cleavage complex


PCS1_HUMAN
Phosphofurin acidic cluster


PCTL_HUMAN
PCTP-like protein; PCTP-L;


PDK3_HUMAN
[Pyruvate dehydrogenase [lipoamide]]


PEC1_HUMAN
Platelet endothelial cell


PFD2_HUMAN
Prefoldin subunit 2


PFD4_HUMAN
Prefoldin subunit 4


PGBM_HUMAN
Basement membrane-specific heparan


PGS1_HUMAN
Biglycan precursor Secreted;


PHYI_HUMAN
Phytanoyl-CoA hydroxylase interacting


PKC1_HUMAN
Pleckstrin homology domain


PKL2_HUMAN
Protein kinase C-like


PKP4_HUMAN
Plakophilin 4; p0071;


PLO1_HUMAN
Procollagen-lysine, 2-oxoglutarate 5-dioxygenase 1


PLO2_HUMAN
Procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2


PLO3_HUMAN
Procollagen-lysine, 2-oxoglutarate 5-dioxygenase 3


POP1_HUMAN
Ribonucleases P/MRP protein


POSN_HUMAN
Periostin precursor; PN;


PP1A_HUMAN
Serine/threonine protein phosphatase


PPCM_HUMAN
Phosphoenolpyruvate carboxykinase, mitochondrial


PRLP_HUMAN
Prolargin precursor Secreted;


PSF1_HUMAN
Proteasome inhibitor PI31


Q01720
FILAGGRIN precursor; PROFILAGGRIN;


Q12869
R kappa B;


Q15042
KIAA0066 protein NO


Q15468
SIL protein; P: cell


Q16630
HPBRII-4 mRNA; HPBRII-7


Q66YK6
Rap1 interacting factor


Q68DE1
Hypothetical protein DKFZp781J2344


Q68DL8
Hypothetical protein DKFZp781L0319


Q69YJ2
Hypothetical protein DKFZp762I0611


Q6AI58
Hypothetical protein DKFZp781A0295


Q6AW92
Hypothetical protein DKFZp686C18106


Q6B0H9
Hypothetical protein NO


Q6DJT3
Organic cation transporter


Q6DV90
Sarcomeric tropomyosin kappa


Q6IA00
FLJ22386 protein NO


Q6IBS0
PTK9L protein; C: intracellular


Q6NSI3
Hypothetical protein NO


Q6NUN7
Hypothetical protein FLJ23554


Q6ZMN5
Hypothetical protein FLJ16798;


Q6ZN20
Hypothetical protein FLJ16514;


Q6ZND8
Hypothetical protein FLJ16191;


Q6ZRT4
Hypothetical protein FLJ46118


Q6ZSQ4
Hypothetical protein FLJ45296


Q6ZTW2
Hypothetical protein FLJ44179


Q6ZV48
Hypothetical protein FLJ42992


Q7Z2Z4
Hypothetical protein DKFZp686I14200;


Q85KX8
Cytochrome oxidase subunit


Q86VN1
CGI-145 protein; OTTHUMP00000040889


Q86XD6
Hypothetical protein FLJ20294


Q8IUI5
Similar to butyrate-induced


Q8IV38
Ankyrin repeat and


Q8IVE9
KIAA2022 protein NO


Q8IWE2
Hypothetical protein LOC92689


Q8IXW0
Hypothetical protein MGC35138;


Q8IYY4
Hypothetical protein FLJ32844;


Q8IZ53
C9orf75 protein NO


Q8IZ83
Hypothetical protein MGC10204;


Q8N1L6
Hypothetical protein FLJ40049


Q8N1N4
Hypothetical protein FLJ39100;


Q8N274
Hypothetical protein FLJ33834


Q8N279
Hypothetical protein FLJ33811;


Q8N3H7
Hypothetical protein DKFZp761G0314


Q8N3X4
FLJ20433 protein NO


Q8N5G2
novel protein [Danio


Q8N5G8
LOC157378 protein NO


Q8N608
Dipeptidylpeptidase 10; C: membrane


Q8N7U5
Hypothetical protein FLJ40332


Q8NBG8
Hypothetical protein FLJ33230


Q8NBS8
Hypothetical protein FLJ90813;


Q8ND87
Hypothetical protein DKFZp434K0835;


Q8NDM2
Hypothetical protein DKFZp727C181


Q8NEY4
V-ATPase C2 subunit;


Q8NEZ3
WD repeat membrane


Q8NHN5
Obscurin NO INFO


Q8TB65
Cytochrome c oxidase


Q8TBP8
Formyltetrahydrofolate dehydrogenase, isoform


Q8TDA3
Guanine nucleotide exchange


Q8TDJ6
Rabconnectin NO INFO


Q8TEC1
Hypothetical protein FLJ23660;


Q8TEP8
FLJ00145 protein NO


Q8WX93
Myoneurin NO INFO


Q8WY20
novel protein [Homo


Q8WZ09
Hypothetical protein NO


Q8WZ35
Hypothetical protein DKFZp434P194


Q96B95
KIAA1799 protein; F: calcium


Q96BR8
LEPRE1 protein; F: oxidoreductase


Q96C32
UBC protein NO


Q96CG5
Hypothetical protein NO


Q96E61
Hypothetical protein NO


Q96EK7
Hypothetical protein KIAA1838;


Q96FJ2
Dynein light chain


Q96GX4
MCM10 protein NO


Q96HU8
Di-Ras2; DIRAS family,


Q96KX7
Thymic dendritic cell-derived


Q96NM0
Hypothetical protein FLJ30596


Q96P57
C6ORF34B NO INFO


Q96QF5
Septin-9; MLL septin-like


Q96SK8
Hypothetical protein FLJ14791;


Q96T23
HBV pX associated


Q9BRG1
Hypothetical protein MGC10540;


Q9BSH5
Haloacid dehalogenase-like hydrolase


Q9BVC6
Hypothetical protein MGC5508


Q9BVZ1
C2orf33 protein NO


Q9GZT6
novel protein [Danio


Q9H009
novel protein similar


Q9H2F7
CTCL tumor antigen


Q9H6E3
Hypothetical protein FLJ22351;


Q9H7C9
Hypothetical protein FLJ21035;


Q9H8C8
Hypothetical protein FLJ13755


Q9HCM2
KIAA1550 protein; C: membrane


Q9HD27
Angiomotin; Hypothetical protein


Q9NPR9
Hypothetical protein; C: integral


Q9P273
KIAA1455 protein NO


Q9UDX0
Oxoglutarate (Alpha-ketoglutarate) dehydrogenase


Q9UHV1
PRO1386 NO INFO


Q9UPN4
KIAA1118 protein; C: muscle


Q9UPV4
KIAA1048 protein; F: ATP


Q9Y5Y9
Sodium channel protein


Q9Y662
Heparan sulfate D-glucosaminyl


R27B_HUMAN
Ras-related protein Rab-27B;


RAC3_HUMAN
Ras-related C3 botulinum


RB31_HUMAN
Ras-related protein Rab-31;


RB35_HUMAN
Ras-related protein Rab-35;


RB3B_HUMAN
Ras-related protein Rab-3B


RCN1_HUMAN
Reticulocalbin 1 precursor;


RDHB_HUMAN
Retinol dehydrogenase 11;


RDHD_HUMAN
Retinol dehydrogenase 13


RGE6_HUMAN
Rap guanine nucleotide


RGSK_HUMAN
Regulator of G-protein


RHG4_HUMAN
Rho-GTPase-activating protein 4


RL2B_HUMAN
60S ribosomal protein


RL35_HUMAN
60S ribosomal protein


RL3_HUMAN
60S ribosomal protein


RLA1_HUMAN
60S acidic ribosomal


RM12_HUMAN
39S ribosomal protein


RN17_HUMAN
RING finger protein


RNT1_HUMAN
Regulator of nonsense


ROCL_HUMAN
novel protein similar


ROG_HUMAN
RNA binding motif


RS26_HUMAN
40S ribosomal protein


RS4Y_HUMAN
40S ribosomal protein


RT36_HUMAN
Mitochondrial 28S ribosomal


RW1_HUMAN
RW1 protein NO


S107_HUMAN
S100 calcium-binding protein


S108_HUMAN
Calgranulin A (Migration


S109_HUMAN
Calgranulin B (Migration


S111_HUMAN
Calgizzarin (S100 calcium-binding


S113_HUMAN
S100 calcium-binding protein


S142_HUMAN
SEC14-like protein 2;


S3B1_HUMAN
Splicing factor 3B


S6AH_HUMAN
Orphan sodium- and


SACS_HUMAN
Sacsin. NO INFO


SAMP_HUMAN
Serum amyloid P-component


SCA1_HUMAN
Secretory carrier-associated membrane


SCA5_HUMAN
Secretory carrier-associated membrane


SEN5_HUMAN
Sentrin-specific protease 5;


SETX_HUMAN
Probable helicase senataxin;


SFD1_HUMAN
Sec1 family domain


SG2_HUMAN
Secretogranin II precursor;


SGTB_HUMAN
Small glutamine-rich tetratricopeptide


SHC3_HUMAN
SHC transforming protein


SJ2B_HUMAN
Synaptojanin 2 binding


SM31_HUMAN
Ubiquitin-like protein SMT3A


SMC3_HUMAN
Structural maintenance of


SN23_HUMAN
Synaptosomal-associated protein 23;


SN29_HUMAN
Synaptosomal-associated protein 29;


SNG2_HUMAN
Synaptogyrin 2 INTEGRAL


SNXP_HUMAN
Sorting nexin 25;


SP20_HUMAN
Spartin; Trans-activated by


SRC_HUMAN
Proto-oncogene tyrosine-protein kinase


SSRA_HUMAN
Translocon-associated protein, alpha


STA1_HUMAN
Signal transducer and


STAU_HUMAN
Double-stranded RNA-binding protein


STOM_HUMAN
Erythrocyte band 7


SX11_HUMAN
Transcription factor SOX-11;


SYTC_HUMAN
Synaptotagmin XII Integral


SZ11_HUMAN
Small inducible cytokine


T2AG_HUMAN
Transcription initiation factor


TBCA_HUMAN
Tubulin-specific chaperone A


TD53_HUMAN
Tumor protein D53;


TES2_HUMAN
Dual specificity testis-specific


TF1A_HUMAN
Transcription intermediary factor


TF_HUMAN
Tissue factor precursor;


TGR3_HUMAN
TGF-beta receptor type


THA2_HUMAN
THAP domain protein


THA_HUMAN
Thyroid hormone receptor


TPCC_HUMAN
Troponin C, slow


TRFM_HUMAN
Melanotransferrin precursor Attached


TRIC_HUMAN
Troponin I, cardiac


TRT2_HUMAN
Troponin T, cardiac


TRY1_HUMAN
Trypsin I precursor;


TSP1_HUMAN
Thrombospondin 1 precursor


TTC1_HUMAN
Tetratricopeptide repeat protein


TYPH_HUMAN
deoA [Mycobacterium tuberculosis


UBQ2_HUMAN
Ubiquilin 2; Protein


UDP2_HUMAN
UTP--glucose-1-phosphate uridylyltransferase 2;


URP1_HUMAN
Unc-112 related protein


VAG1_HUMAN
Vacuolar ATP synthase


VAG2_HUMAN
Vacuolar ATP synthase


VAS1_HUMAN
Vacuolar ATP synthase


VEGP_HUMAN
Von Ebner's gland


VEZA_HUMAN
Vezatin Type III


VP36_HUMAN
Vesicular integral-membrane protein


VP41_HUMAN
Vacuolar assembly protein


VTNC_HUMAN
Vitronectin precursor; Serum


WD37_HUMAN
WD-repeat protein 37


WN3A_HUMAN
Wnt-3a protein precursor


WWP2_HUMAN
Nedd-4-like E3 ubiquitin-protein
















TABLE 5







Proteins unique to CP








Protein ID
Common Name





1A01_HUMAN
HLA class I histocompatibility antigen,


AASS_HUMAN
Alpha-aminoadipic semialdehyde synthase


ABL1_HUMAN
Proto-oncogene tyrosine-protein kinase ABL1; p150;


ACN1_HUMAN
Astrotactin 1; P: cell migration NO


ACY1_HUMAN
Aminoacylase-1; N-acyl-L-amino-acid amidohydrolase


ADAS_HUMAN
Alkyldihydroxyacetonephosphate synthase


ADHX_HUMAN
Alcohol dehydrogenase class III chi


AIP_HUMAN
AH receptor-interacting protein Cytoplasmic. MAY


APL2_HUMAN
Apolipoprotein L2 Cytoplasmic (Probable). May


ARH7_HUMAN
Rho guanine nucleotide exchange factor


ASML_HUMAN
N-acetylserotonin O-methyltransferase-like protein; ASMTL; F:


ATM_HUMAN
Serine-protein kinase ATM PRIMARILY NUCLEAR.


ATPJ_HUMAN
ATP synthase e chain, mitochondrial


BCS1_HUMAN
Mitochondrial chaperone BCS1; BCS1-like protein;


BLMH_HUMAN
Bleomycin hydrolase


CA00_HUMAN
Protein CGI-100 precursor; UNQ397/PRO733


CA16_HUMAN
Collagen alpha 1


CA24_HUMAN
Collagen alpha 2


CADA_HUMAN
Cadherin-10 precursor; T2-cadherin; C: integral to


CALI_HUMAN
Calicin; C: cytoskeleton Calyx; sperm head


CCD6_HUMAN
Coiled-coil domain containing protein 6;


CE29_HUMAN
Centrosomal protein Cep290


CEP2_HUMAN
Centrosomal protein 2


CES6_HUMAN
Cat eye syndrome critical region


CLI5_HUMAN
Chloride intracellular channel protein 5;


CLS1_HUMAN
Calsyntenin-1 precursor Type I membrane


CN39_HUMAN
Hypothetical protein C14orf39


COLI_HUMAN
Corticotropin-lipotropin precursor ACTH


CPSM_HUMAN
Carbamoyl-phosphate synthase [ammonia]


CSN5_HUMAN
COP9 signalosome complex subunit 5;


CTD1_HUMAN
Catenin delta-1 Cytoplasmic and nuclear


CUT2_HUMAN
Homeobox protein Cux-2; Cut-like 2;


CXAR_HUMAN
Coxsackievirus and adenovirus receptor precursor


CYA8_HUMAN
Adenylate cyclase, type VIII Integral


D3D2_HUMAN
3,2-trans-enoyl-CoA isomerase, mitochondrial precursor


DD37_HUMAN
Probable ATP-dependent helicase DHX37; DEAH-box


DJBB_HUMAN
DnaJ homolog subfamily B member


DLP1_HUMAN
Disks large-associated protein 1 Membrane-associated


DMD_HUMAN
Dystrophin. May play


DOC4_HUMAN
Dedicator of cytokinesis protein 4


E15R_HUMAN
Epidermal growth factor receptor substrate


ECH1_HUMAN
Delta3,5-delta2,4-dienoyl-CoA isomerase


EHD2_HUMAN
EH-domain containing protein 2 NO


EMD_HUMAN
Emerin; C: nuclear membrane Nuclear transmembrane


ERC1_HUMAN
ERC protein 1 (ELKS protein)


ERG7_HUMAN
Lanosterol synthase; Oxidosqualene--lanosterol cyclase; 2,3-e


F262_HUMAN
6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 2


F263_HUMAN
6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3


FKB5_HUMAN
FK506-binding protein 5 Nuclear and


FYN_HUMAN
Proto-oncogene tyrosine-protein kinase FYN; P59-FYN;


G6PE_HUMAN
GDH/6PGL endoplasmic bifunctional protein precursor


GAL1_HUMAN
Galactokinase; Galactose kinase; C: cytoplasm NO


GBAP_HUMAN
Gamma-aminobutyric acid receptor associated protein;


GBG4_HUMAN
Guanine nucleotide-binding protein G NO


GBG7_HUMAN
Guanine nucleotide-binding protein G NO


GGA1_HUMAN
ADP-ribosylation factor binding protein GGA1;


HPCA_HUMAN
Neuron specific calcium-binding protein hippocalcin;


HRG_HUMAN
Histidine-rich glycoprotein precursor Secreted. The


HS76_HUMAN
Heat shock 70 kDa protein


IDI1_HUMAN
Isopentenyl-diphosphate delta-isomerase 1 Peroxisomal.


IF5_HUMAN
Eukaryotic translation initiation factor 5


IGB1_HUMAN
Immunoglobulin-binding protein 1; CD79a-binding protein


ITA6_HUMAN
Integrin alpha-6 precursor Type I


JJ2C_HUMAN
Jumonji domain containing protein 2C;


K513_HUMAN
Protein KIAA0513


KHL6_HUMAN
Kelch-like protein 6


KPT3_HUMAN
Serine/threonine-protein kinase PCTAIRE-3; PCTAIRE-motif p


LAP4_HUMAN
LAP4 protein; Scribble homolog protein;


LCF1_HUMAN
Long-chain-fatty-acid--CoA ligase 1; Long-chain acyl-CoA


LMB2_HUMAN
Laminin beta-2 chain precursor Extracellular.


LMG1_HUMAN
Laminin gamma-1 chain precursor Extracellular.


LPB1_HUMAN
Liprin-beta 1; Protein tyrosine phosphatase


LU_HUMAN
Lutheran blood group glycoprotein precursor


MAGM_HUMAN
Mitochondria-associated granulocyte macrophage


MEC2_HUMAN
Methyl-CpG-binding protein 2; MeCP-2 protein;


MPPB_HUMAN
Mitochondrial processing peptidase beta subunit,


MY9B_HUMAN
Myosin IXb; Unconventional myosin-9b; C: actin


NB7M_HUMAN
NADH-ubiquinone oxidoreductase B17


NCR2_HUMAN
Nuclear receptor co-repressor 2 Nuclear.


NGAP_HUMAN
Ras GTPase-activating protein nGAP; RAS


NIAM_HUMAN
NADH-ubiquinone oxidoreductase ASHI subunit,


NID2_HUMAN
Nidogen-2 precursor Secreted; extracellular matCell


NIPM_HUMAN
NADH-ubiquinone oxidoreductase 15 kDa subunit


NR54_HUMAN
Non-POU domain-containing octamer-binding protein


NU14_HUMAN
Uridine diphosphate glucose pyrophosphatase;


NUMA_HUMAN
Nuclear mitotic apparatus protein 1;


O14979
JKTBP2; Heterogeneous nuclear ribonucleoprotein D-like;


O43273
P53 binding protein


O43290
SART-1; Squamous cell carcinoma antigen


O60280
KIAA0528 protein; C: membrane


O60735
dJ69B10.1 (GA17 protein) [Homo sapiens]


O75179
KIAA0697 protein; F: ATP binding


O75339
Cartilage intermediate layer protein; F: phosphoprotein


O75500
tad99c12.y1 Hydra EST Darmstadt I


O95714
HERC2 protein; F: guanyl-nucleotide exchange factor


O95810
Serum deprivation response; F: phospholipid binding


ODBA_HUMAN
2-oxoisovalerate dehydrogenase alpha subunit


P11A_HUMAN
Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit


P285_HUMAN
Peroxisomal proliferator-activated receptor A interacting


P2CA_HUMAN
Protein phosphatase 2C alpha isoform


P2G4_HUMAN
Proliferation-associated protein 2G4


PAC2_HUMAN
Protein kinase C and casein


PASK_HUMAN
PAS domain containing serine/threonine-protein kinase;


PCH7_HUMAN
Protocadherin 7 precursor; Brain-heart protocadherin;


PE5R_HUMAN
PEX5-related protein; Peroxin 5-related protein;


PHR1_HUMAN
Phosphatase and actin regulator 1


PMVK_HUMAN
Phosphomevalonate kinase; PMKase; C: peroxisome Peroxisomal


PSB3_HUMAN
Proteasome subunit beta type 3;


PSB5_HUMAN
Proteasome subunit beta type 5


PSD1_HUMAN
26S proteasome non-ATPase regulatory subunit


PSD6_HUMAN
26S proteasome non-ATPase regulatory subunit


PXF_HUMAN
Peroxisomal farnesylated protein PEROXISOMAL


Q07666
P62; KH domain containing, RNA


Q13446
P60; C: cytosol(ISS); F: protein kinase binding


Q15598
Titin; F: structural constituent of muscle


Q16219
Insulin activator factor; C: insulin control


Q68CR3
Hypothetical protein DKFZp781E17107


Q68DL4
Hypothetical protein DKFZp781J211


Q6BCY4
Cytochrome b5 reductase b5R NO


Q6MZY9
Hypothetical protein DKFZp686M14237


Q6N046
Hypothetical protein DKFZp686H15136


Q6NZI2
Polymerase I and transcript release


Q6P5T6
Hypothetical protein; F: sugar binding NO


Q6PRD1
GPR158-like 1 receptor; C: membrane NO


Q6R7N2
Hemoglobin beta; C: hemoglobin complex NO


Q6U7G8
GTP-GDP dissociation stimulator 1 isoform


Q6UX81
CLCA4; Chloride channel, calcium activated,


Q6XYE4
FP17425


Q6ZML6
FLJ00267 protein


Q6ZRE2
Hypothetical protein FLJ46424


Q6ZUV0
Hypothetical protein FLJ43309; F: hydrolase activity


Q7Z2K9*
homo sapiens (human). dj467I1.1 (kiaa0833)


Q86T29
Hypothetical protein DKFZp686B222; C: nucleus


Q86UF3
Hypothetical protein MGC43026


Q86UQ4
ABC A13; F: ATP binding


Q8IVL1
steerin2 protein [Homo sapiens]


Q8IWY7
novel protein similar to vertebrate


Q8IY03
C9orf77 protein; F: catalytic activity


Q8IYE5
DHX36 protein; F: ATP binding;


Q8IYI6
Exocyst complex 84-kDa subunit; OTTHUMP00000060156


Q8IYS0
DKFZp434C0328 protein


Q8IYU5
SLCO6A1 protein; C: membrane


Q8IYZ3
EFHA2 protein; F: calcium ion binding


Q8IZ16
Hypothetical gene supported by BC031966


Q8IZP7
Heparan sulfate 6-O-sulfotransferase 3; F: transferase


Q8IZQ1
ALFY; F: zinc ion binding


Q8MML2
SYNAPTIC DYNAMIN-ASSOCIATED PROTEIN IIBB


Q8N264
Hypothetical protein FLJ33877


Q8N2F4
Hypothetical protein PSEC0200


Q8N5C0
BAIAP2 protein Secreted


Q8N5E8
PP3856 protein; F: nicotinate phosphoribosyltransferase activity


Q8N5H7
SH2 domain containing 3C; F: guanyl-nucleotide


Q8N6D9
STAM protein; P: intracellular protein transport


Q8N7G2
Hypothetical protein FLJ25675


Q8N883
Hypothetical protein FLJ39837


Q8N8N7
Hypothetical protein FLJ39091; Zinc binding


Q8N987
Hypothetical protein FLJ38214; Neuronal calcium


Q8N9P3
Hypothetical protein FLJ36794


Q8NCZ9
Hypothetical protein DKFZp566B1447


Q8NHP6
Motile sperm domain containing 2;


Q8TDR0
Interleukin 13 receptor alpha 1-binding


Q8TED6
Hypothetical protein FLJ23617; F: hydrolase activity


Q8WXE8
Paraspeckle protein 1 beta isoform


Q969L9
RNA binding protein HQK; RNA


Q96A33
Hypothetical protein FLJ14938; Hypothetical protein


Q96B17
Hypothetical protein; F: protein binding


Q96CH9
Hypothetical protein KIAA1244


Q96EI3
PTD012 protein


Q96GA9
V-crk sarcoma virus CT10 oncogene


Q96H23
FLJ10579 protein


Q96JJ7
KIAA1830 protein; F: calcium ion binding


Q96JN2
KIAA1793 protein


Q96LU3
Hypothetical protein FLJ25064


Q96MG1
Hypothetical protein FLJ32421


Q96S83
Hypothetical protein


Q99442
Translocation protein-1; TLOC1 protein; Sec62;


Q9BRA2
Hypothetical protein TXNL5; Putative 42-9-9


Q9BRF8
Hypothetical protein FLJ11151; CSTP1; F: hydrolase


Q9C0B1
KIAA1752 protein


Q9GZT3
DC50; DC23; Hypothetical protein PD04872;


Q9HAJ2
Hypothetical protein FLJ11539


Q9NQ48
Leucine zipper transcription factor-like 1;


Q9NY47
Calcium channel, alpha 2/delta subunit


Q9UD80
Neuronal voltage-dependent calcium channel alpha


Q9ULH4
KIAA1246 protein


Q9ULM3
KIAA1197 protein


Q9UNU8
Hypothetical protein; C


Q9Y485
X-like 1 protein; F: protein binding


R23A_HUMAN
UV excision repair protein RAD23


RAP1_HUMAN
Rab GTPase binding effector protein


RFL1_HUMAN
Ret finger protein-like 1; RING


RGP1_HUMAN
Ran GTPase-activating protein 1


RGP2_HUMAN
Rap1 GTPase-activating protein 1; Rap1GAP;


RGSC_HUMAN
Regulator of G-protein signaling 12


RL22_HUMAN
60S ribosomal protein L22; Epstein-Barr


RL31_HUMAN
60S ribosomal protein L31


ROH1_HUMAN
heterogeneous nuclear ribonucleoprotein H/F, putative


ROH2_HUMAN
Heterogeneous nuclear ribonucleoprotein H′ Nuclear;


RPGR_HUMAN
X-linked retinitis pigmentosa GTPase regulator


RUV2_HUMAN
RuvB-like 2 (48-kDa TATA box-binding


SC63_HUMAN
Translocation protein SEC63 homolog Integral


SCL2_HUMAN
Sex comb on midleg-like protein


SF45_HUMAN
Splicing factor 45 Nuclear.


SGA2_HUMAN
SLIT-ROBO Rho GTPase activating protein


SHP1_HUMAN
SH3-domain kinase binding protein 1


SM3A_HUMAN
Semaphorin 3A precursor; Semaphorin III;


SMC2_HUMAN
Structural maintenance of chromosome 2-like


SNPH_HUMAN
Syntaphilin; F: syntaxin-1 binding Membrane-associated Inhibits


SP8_HUMAN
Transcription factor Sp8; Specificity protein


SQRD_HUMAN
Sulfide: quinone oxidoreductase, mitochondrial precursor; CGI-


SR14_HUMAN
Signal recognition particle 14 kDa


SRC8_HUMAN
Src substrate cortactin Cytoplasmic. Associated


SUM2_HUMAN
Sulfatase modifying factor 2 precursor;


SYT2_HUMAN
Synaptotagmin-2; Synaptotagmin II; SytII Synaptic


T103_HUMAN
Tetratricopeptide repeat protein KIAA0103 Nuclear


T150_HUMAN
Thyroid hormone receptor-associated protein complex


TAC1_HUMAN
Transforming acidic coiled-coil-containing protein 1


TBBQ_HUMAN
Tubulin beta-4q chain


TGM1_HUMAN
Protein-glutamine gamma-glutamyltransferase K


TIAM_HUMAN
T-lymphoma invasion and metastasis inducing


TIE1_HUMAN
Tyrosine-protein kinase receptor Tie-1 precursor


TLRA_HUMAN
Toll-like receptor 10 precursor Type


TM24_HUMAN
Transmembrane protein 24; DLNB23 protein


TMO1_HUMAN
Tropomodulin 1


TRIA_HUMAN
Thyroid receptor interacting protein 11


TXN4_HUMAN
Thioredoxin domain containing protein 4


TXTP_HUMAN
Tricarboxylate transport protein, mitochondrial precursor;


U84B_HUMAN
Sad1/unc-84-like protein 2; Rab5 interacting


UB37_HUMAN
Ubiquitin carboxyl-terminal hydrolase 37; Ubiquitin


UBQ1_HUMAN
Ubiquilin 1; Protein linking IAP


UBQ4_HUMAN
Ubiquilin 4; Ataxin-1 ubiquitin-like interacting


UN5A_HUMAN
Netrin receptor UNC5A precursor; Unc-5


VATF_HUMAN
Vacuolar ATP synthase subunit F;


WAS2_HUMAN
WAS protein family, member 2


Z217_HUMAN
Zinc finger protein 217 Nuclear


ZYX_HUMAN
Zyxin (Zyxin 2)

## Claims

1. A method for treating multiple sclerosis in a patient, the method comprising:
selecting a patient having chronic active plaque (CAP) type lesions wherein the lesion is typed by determining an increase in expression of at least one protein involved in coagulation, selected from the group consisting of tissue factor (TF) and protein C inhibitor (PCI); and
administering to said patient a therapeutically effective dose of an activated protein C protein or a fragment thereof for a period of time sufficient to decrease the severity of multiple sclerosis.

2. The method of claim 1, wherein the activated protein C protein is Drotrecogin alpha.

3. The method of claim 1, wherein the activated protein C protein is a variant of activated protein C having reduced anticoagulant activity.

4. The method of claim 1, wherein multiple doses of the activated protein C protein or fragments thereof are administered over a period of time.