bluuebunny/crossref_metadata_2025 huggingface dataset:
PRODUCTION OF A TISSUE INHIBITOR OF METALLOPROTEINASES BY BOVINE VASCULAR CELLS
Authors: DeClerck Y A , Bock R , Laug W E
Published: August 2018

Tissue Inhibitor of Metalloproteinases (TIMP) plays an important role in collagen turnover in tissue due to its ability to irreversibly inhibit mammalian collagenases. We have investigated the production of such an inhibitor by various cells of bovine vessels including endothelial cells of arterial, venous and capillary origin and arterial smooth muscle cells. While large amounts of collagenase inhibitor (800 mU/106 cells/24 hr) were produced by vascular smooth muscle cells, smaller amounts were detected in* the medium conditioned by either arterial, capillary or venous endothelial cells (90, 1.7 and 1.1 mU/106 cells/24 hr respectively). An inhibitor with a Mr of 28,500 was purified from serum free medium conditioned by bovine smooth muscle cells using molecular sieve followed by heparin sepharose and carboxy-methylcellulose chromatography. It inhibited several vertebrate collagenases but was inactive against bacterial collagenase. This inhibitor was resistant to treatment with acid and heat but sensitive to trypsin and reduction alkylation. It formed with vertebrate collagenase an enzyme-inhibitor complex resistant to organomercurials or trypsin. This inhibitor, therefore, is similar to a collagenase inhibitor produced by human fibroblasts and a tissue inhibitor of metalloproteinases extracted from human amniotic fluid and rabbit bone.

The production of TIMP by bovine vascular smooth muscle cells markedly increased during cell proliferation. In addition, when endothelial cells were grown on a preformed layer of smooth muscle cells, the production of TIMP was more than additive suggesting an enhancing effect of endothelial cells on vascular smooth muscle cells.

These data suggest that the large amount of TIMP produced by vascular muscle cells may be responsible for the accumulation of collagen characteristically observed in conjunction with smooth muscle cells hyperplasia in atherosclerotic plaques.
STEREOGEOMETRY OP THE ACTIVE SITES OF SERINE ENZYMES GATHERED FROM SYNTHETIC THROMBIN-INHIBITORS
Authors: Hijikata-Okunomiya A , Okamoto S , Kikumoto R , Tamao Y
Published: August 2018

MD-805 is a potent thrombin-inhibitor having the structure of tri-pods; Arg skeletone, N-terminal side and C-terminal side. MD-805 showed weaker inhibitory activity to other enzymes than thrombin. In this report, to gather more detailed informations about the structural features of serine enzymes concerning the specificity, we experimentally examined the structure-activity relationship (SAR) of a number of arginine derivatives including MD-805 and theoretically generated a MD-805-trypsin complex model using the results of X-ray crystallography of MD-805 and BPTI-trypsin complex by calculation in principle to minimize van der Waals contacts, and thus we discussed to interpret SAR from the molecular level. SAR of C-terminal side of arginine derivatives was obtained with the inhibitory activity to trypsin, plasmin, and glandular kallikrein and compared with the previous results of thrombin, the followings being indicated: (1) The hydrophobic binding pocket (HBP), which was reported by us to be at least partly similar in stereogeometry between trypsin and thrombin, had the depth corresponded to the length of ethylpiperidine, (2) concerning the site (termed the P site) next to HBP, there were large differences in stereogeometry between trypsin and thrombin; the P site of trypsin could accept propyl and phenyl group attached to 4-position of piperidine, while that of thrombin was unable to accept them and (3) the P sites of plasmin and glandular kallikrein resembled that of trypsin in being able to accept phenyl group. MD-805-trypsin complex model supported the reasonable understanding that the stereogeometrical similarity in HBP between thrombin and trypsin was attributable to the high homology in amino acid sequences in Ser-195 loop and that the dissimilarity in the P sites between thrombin and the others was attributable to 9 amino acids insertion found only in thrombin (Loop B). Furthermore, many dansylarginine derivatives showed very strong inhibition for pseudocholinesterase, however, SAR for C-terminal side of these derivatives revealed the similarity and dissimilarity in HBP and the P site between pseudocholinesterase and the proteases described above.
NUCLEOTIDE SEQUENCE OF THE STAPHYLOCOAGULASE GENE FROM STAPHYLOCOCCUS AUREUS STRAIN BB
Authors: Kaida S , Miyata T , Kawabata S , Morita T , Yoshizawa Y , Igarashi H , Iwanaga S
Published: August 2018

Staphylocoagulase (SC) is a secretary protein produced by several strains of Staphylococcus aureus (S. aureus). This protein forms a molecular complex ("staphylothrombin") with human prothrombin in a molar ratio of 1:1. The complex displays the ability to clot fibrinogen and to hydrolyze the synthetic tripeptide substrates for α-thrombin. The formation of staphylothrombin does not require proteolytic cleavage of the prothrombin molecule, and this mechanism differs markedly from the activation process by either blood-clotting factor Xa or snake venom procoagulant.

In the present studies, a pAT153 library containing partial Mbo I-digested DNA prepared from aureus strain BB has been screened with a fibrin gel formation method. The identity of these clones with SC was confirmed by DNA sequence analysis and by comparison of the derived amino acid sequence with that determined for the purified SC protein. One of the positive colonies was isolated and 3.1 Kb of the insert DNA was determined by the dideoxy chain termination method. The results indicated that the insert DNA consists of 148 bp 5' flanking region, protein coding region of 715 amino acids and 746 bp 3' flanking region, and that SC from strain BB is synthesized as a precursor with a signal peptide of 26 amino acids. Thus, the mature form was composed of 689 amino acids with a molecular weight of 77,337- The NH2-terminal sequence (324 amino acids) of SC isolated from S. aureus strain 213 (S. Kawabata et al. (1986): J. Biol. Chem. 261, 527-531) was compared with that of SC derived from strain BB. The sequence homology between them was found to show 57 %. It was also found that SC derived from strain BB was composed of 8 tandem repeats (27 amino acid residues in length) in the COOH-terminal region, although their functions are not known.
EXPRESSION IN ONTOGENESIS OF HUMAN BLOOD COAGULATION FACTORS
Authors: Hassan H J , Leonardi A , Chelucci C , Guerriero R , Mannucci P M , Peschle C
Published: August 2018

We have analyzed the expression of several blood coagulation factors (IX, VIII, X, fibrinogen chains) and inhibitors (antithrombin III, protein C) in human embryonic and fetal livers, obtained from legal abortions at 6-11 week post-conception. The age was established by morphologic staging and particularly crown-rump lenght measurement.

Total cellular RNA was isolated from partially purified hepatocytes or total liver homogenate using the guanidine isothiocyanate method. Poly(A)+ RNA was selected by oligodT cellulose chromatography. The size and the number of the embryonic and fetal transcripts are equivalent to those observed in adult liver, as evaluated by Northern blot analysis of total or poly(A)+ RNA hybridized to human cDNA probes.

The level of coagulation factor transcripts in embryonic and fetal liver was evaluated by dot hybridization of total RNA (0.5-10 ug), as compared to RNA extracted from normal adult liver biopsies. The expression of blood coagulation factors in embryos is generally reduced for all factors, but at a different degree. In 5-11 wk liver, the level of factor IX is 5-10% of that observed in adults, while fibrinogen, protein C, antithrombin III RNA level rises from 25 to 50% and factor X is expressed at a level comparable to that observed in adult liver.

We conclude that during these stages of development blood coagulation factors are expressed according to three different time, curves, possibly due to the effect of different types of regulatory mechanisms.
THE ACTIVITY OF A SINGLE CHAIN rt-PA MUTANT IN A PRIMATES AND RABBITS
Authors: Hotchkiss A , Knitter G , DeGuzman L , Rsfino C
Published: August 2018

Recombinant tissue-type plasminogen activator(rt-PA) contains a plasmin sensitive peptide bond domain between amino acids (AAs) 275 and 276. Since the single chain form of rt-PA is rapidly clipped to the two chain form during thrombolysis, one way to determine the relative activities of single chain and two chain rt-PA is to produce a mutant which no longer has this clip site; therefore, a single AA substitution was made at position 275, replacing Glu for Arg (Tate, et al., Biochemistry 1987, in Press). The thrombolytic activity of this mutant (Glu275) was compared to the activity of the wild type rt-PA (Arg275) in a new primate thrombolysis model.

An arterio-venous shunt model (Hotchkiss et al, this meeting) was used. Various doses of the rt-PAs were infused over a 90 min period with 10% of the total dose administered as an initial bolus. The rate and extent of thrombolysis were quantitated by the measurement of radioactivity remaining in the thrombus at 15, 30, 60 and 90 min, using an external probe. Data were collected from 5 cyncmolgus monkeys (male 5 to 6 kg). The dose response curves were used to determine the ED40 (dose for 40% clot lysis). The ED40 for Arg275 and Glu275 wire 870 and 680 (ugAg)r respectively. The plasna concentrations of rt-PA were measured at plateau for various doses and the regression of the plasma concentrations (Y,ng/ml) versus the dose rates (X, ug/kg/min) was calculated. The regression equations were; Y=28+51X and Y=39+113X for Arg275 and Glu275, respectively. This indicates that the clearance rate of Glu275 is 2.2 fold slower than Arg275.

These experiments were also performed in rabbits. The ED40 for Arg275 and Glu275 were 740 and 660 (ug/kg) respectively. The Glu275 was cleared more slowly, having a 1.7 fold slower clearance rate.

These results support the conclusion, that the "one chain" Glu275 rt-PA is still active in vivo and that it is slightly more potent than the wild type in these animal models. Some of this increase in potency is apparently due to a slower clearance rate for the Glu275 rt-PA.
EXPRESSION OF A RECOMBINANT ANTIBODY-TARGETED THROMBOLYTIC MOLECULE
Authors: Quertermous T , Schnee J M , Runge M S , Matsueda G R , Hudson N W , Seidman J G , Haber E
Published: August 2018

We have recently shown that targeting tissue-type plasminogen activator (t-PA) by covalent linkage to a fibrin-specific monoclonal antibody (59D8) produces a more potent thrombolytic agent which also induces less fibrinogenolysis. A recombinant molecule encoding a t-PA-59D8 fusion protein was constructed to provide a ready source of this agent for further study, and to allow tailoring of the active moities for maximal activity. DNA sequence coding for the 59D8 heavy chain (HC) antigen combining site was cloned from a lambdaphage library by selection with a joining region probe. Gene segments coding for this cloned HC rearrangement, the amino portion of the mouse gamma 2b HC constant region, and the catalytic B chain of t-PA were joined in the pSV2-gpt expression vector. The desired coding sequence was confirmed by nucleotide sequence analysis. The construct was transfected by electroporation into 59D8 hybridoma HC loss variants. Transfectants were screened for antifibrin antibody activity. Positive clones were shown to produce mRNA which hybridized to the human t-PA gene in Northern blot analysis. Supernatants from 5 of these clones were subjected to affinity chromatography on a synthetic fibrin-like peptide-Sepharose column followed by a benzamidine-Sepharose column. Western blots of SDS polyacrylamide gels run under reducing conditions revealed binding to a 60 kd band by a monoclonal antihuman t-PA antibody, consistent with a 59D8 HC-t-PA fusion protein. Also, binding to a 25 kd band by goat anti-mouse Fab indicated the presence of 59D8 light chain. Affinity purified protein was shown to have amidolytic activity of similar potency to t-PA in a chromogenic substrate assay utilizing S-2288. Bifunctionality of the purified protein was demonstrated first by an assay which requires the protein to bind to immobilized fibrin and simultaneously exhibit activity in the S2288 assay, and second by simultaneous fibrin and iodinated anti-t-PA binding.
IN VITRO AND IN VIVO EFFECT OF VITAMIN E ON NORMAL HUMAN PLATELETS. AGGREGATION AND ARACHIDONIC ACID (AA) METABOLISM
Authors: Kockmann V , Véricel E , Croset M , Lagarde M
Published: August 2018

Oxygenated metabolism of AA through cyclooxygenase and lipoxygenase pathways involves peroxide species and vitamin E has been extensively studied as an agent that could reduce this lipid peroxidation. In the present study, effects of vitamin E has been investigated upon AA metabolism in normal human platelets with both in vivo and in vitro approaches. Using low doses of vitamin E either in vivo or in vitro, we have succeeded to almost double plasma and platelet a-tocopherol (determinated by HPLC). Despite such an enrichment platelet aggregation induced by agents involved in the AA cascade (collagen, arachidonate and U46619) was not affected. Similarly, the oxygenation of exogenous AA determined by HHT, HETE and TxB2 production was not modified. When the oxygenated products were measured after thrombin stimulation, some variations could be noted, although rarely significant. The tendency was a decrease after in vitro enrichment and an increase when enrichment occured in vivo. Basal oxygenated metabolism of AA in vivo was assessed by measuring 6-Keto-PGFla, 2,3-dinor-6-Keto-PGFla, TxB2 and 2,3-dinor-TxB2 in urine. All of them tended to increase after vitamin E intake, although not significantly. The oxygenation of eicosapentaenoic acid, which is markedly potentiated by AA through its hydroperoxide, 12 HPETE, was not altered after vitamin E treatment, confirming that vitamin E does not alter the specific peroxidation of polyunsaturated fatty acids in normal platelets. We conclude that vitamin E supplement does not affect the AA dependent aggregation neither the oxygenated metabolism of AA in normal human platelets. This does not exclude that it might be however useful in platelets that exhibit a relative deficiency in this vitamin (e.g. diabetes and aging) where it could slow down both AA peroxidation and aggregation.
NEW EICOSANOIDS FORMED DURING PLATELET-NEUTROPHIL INTERACTIONS
Authors: Marcus A J , Safier L B , Ullman H L , Islam N , Broekman M J , Schacky C V
Published: August 2018

In physiologic and pathologic processes such as hemostasis, thrombosis and inflammation, multiple cell types are brought into close proximity - thereby increasing the possibility of metabolic interactions in the microenvironment. Activated platelets synthesize12-hydroxyeicosatetraenoic acid (12-HETE) in the presence or absence of aspirin. During a cell-cell interaction, platelet 12-HETE is metabolized by a cytochrome P-450 enzyme system in unstimulated neutrophils to 12.20-dihydroxyeicosatetraenoic acid (12,20-DiHETE). Recently, we observed time-dependent formation of a new eicosanoid following exposure of neutrophils to 12-HETE. This compound is more polar than the parent eicosanoid 12,20-DiHETE (reversed-phase HPLC). Incubation of purified 12,20-DiHETE with neutrophils resulted in a progressive decrease in the 12,20-DiHETE with increasing formation of the polar metabolite. In the absence of neutrophils, 12.20-DiHETE was quantitatively unchanged. The new metabolite of 12.20-DiHETE has been tentatively identified as 12-hydroxyeicosatetraen-l,20-dioic acid. The UV absorption maximum of the new compound is 237 nm which is identical to that of 12-HETE and 12,20-DiHETE. 20-hydroxy-LTB4 is the omega-hydroxylated derivative of the pro-inflammatory eicosanoid LTB4. When added to neutrophils 15 sec prior to 12,20-DiHETE, equimolar concentrations of 20-hydroxy-LTB4 (2.8uM) inhibited formation of the new metabolite by 28%. A concentration of 8uM 20-hydroxy-LTB4 inhibited the reaction by 49%. These results indicate that the neutrophil enzyme system responsible for conversion of 20-hydroxy-LTB4 to 20-carboxy-LTB4 may also be involved in further metabolism of-12,20-DiHETE. Neutrophil homogenization resulted in loss of the capacity to transform 12.20-DiHETE to the new metabolite despite pretreatment with DFP and addition of NADPH. Our data provide further evidence for the occurrence of transcellular metabolic events during thrombosis and the inflammatory response.
NOVEL GTP-BINDING PROTEINS OF CYTOSOLIC AND MEMBRANE FRACTIONS OF HUMAN PLATELETS
Authors: Lapetina Eduardo G , Reep Bryan R , Vedia Luis Molina Y
Published: August 2018

We have assessed the binding of (α-32P)GTP to platelet proteins from cytosolic and membrane fractions. Proteins were separated by SDS-PAGE and electrophoretically transferred to nitrocellulose. Incubation of the nitrocellulose blots with (α-32p)GTP indicated the presence of specific and distinct GTP-binding proteins in cytosol and membranes. Binding was prevented by 10-100 nM GTP or GTPyS and by 100 nM GDP; binding was unaffected by 1 nM-1 μM ATP. One main GTP-binding protein (29.5 KDa) was detected in the membrane fraction while three others (29, 27, and 21 KDa) were detected in the soluble fraction. Two cytosolic GTP-binding proteins (29 and 27 KDa) were degraded by trypsin; another cytosolic protein (21 KDa) and the membrane-bound protein (29.5 KDa) were resistant to the action of trypsin. Treatment of intact platelets with trypsin or thrombin, followed by lysis and fractionation, did not affect the binding of (α-32P)GTP to the membrane-bound protein. GTPyS still stimulates phospholipase C in permeabilized platelets already preincubated with trypsin. This suggests that trypsin-resistant GTP-binding proteins might regulate phospholipase C stimulated by GTPyS. We have started to purify the membrane-bound, trypsin-resistant, GTP-binding protein. Purification includes 1 M NaCl extraction and the use of an FPLC system with successive phenyl superose and superose 12 columns.
A MONOCLONAL ANTIBODY SOLID PHASE COMPETITIVE RADIOIMMUNOASSAY (RIA) WHICH MEASURES ALL FORMS OF PROTEIN S (PS) IN PLASMA
Authors: Bovill E G , Landesman M , Mann K G , Tracy R P
Published: August 2018

PS is bound to one (C4bp) and possibly two (protein S binding protein) other proteins in plasma, necessitating the validation of any assay, both by standard criteria and for its ability to measure the different forms of PS in plasma. Murine monoclonal antibody PS-2a was adsorbed to solid phase microtiter plate wells at 10 ug/ml, pH 9.6. For the assay, affinity purified PS standard, free of C4bp, or plasma samples were incubated with 125 I labelled PS.

Serial dilutions of pooled normal plasma paralleled the standard curve. Competition studies with other vitamin K dependent coagulation proteins, anti-thrombin III, C4bp and bone Gla protein showed no significant crossreactivity. When 5.7 ug of PS was added to 10 normal plasmas the mean recovery was 5.86 ± 0.8 ug/ml. Sensitivity was 0.03 ug/ml at the working plasma dilution (1/64). Normal pooled plasma had a mean of 15 ug/ml with an intra-assay CV of 5.7%. The normal range based on 61 normal plasmas is 9.5-24 ug/ml. The mean of 24 warfarinized plasmas was 8.9 ug/ml (1 SD = 3.5)

Normal pooled plasma was totally depleted of PS using anti-PS-2a coupled to sepharose 4B at 5mg/ml. Depleted and undepleted normal pooled plasmas were then compared to a standard curve of purified PS in a reduced, SDS PAGE Western Blot utilizing a rabbit polyclonal anti PS. This experiment demonstrated that the monoclonal antibody used in the assay totally depleted the pooled plasma of all forms of PS and therefore appears to recognize an epitope on PS not involved in its binding interactions with other plasma proteins.

PS and C4bp were measured following HPLC using a Beckman TSK-3000 column for rapid separation. In pooled plasma 70% of PS appeared bound to C4bp.

(Supported by NIH Grant HL 35058)
INDEPENDENT ISOLATION OF HUMAN PROTEIN S cDNA AND THE ASSIGNMENT OF THE GENE TO CHROMOSOME 3
Authors: Ploos van Amstel J K , van der Zanden A L , Bakker E , Reitsma P H , Bertina R M
Published: August 2018

Protein S is a vitamin K-dependent glycoprotein, that serves as a cofactor of activated protein C. A hereditary deficiency in protein S is associated with an increased risk for the development of venous thrombosis. The deficiency is inherited as an autosomal dominant trait. We isolated a cDNA coding for protein S and assigned its gene to chromosome 3.

A human liver cDNA library in phage xgtll (complexity 1.2 × 106 , D. Stafford, Chapel Hill) was screened by using immuno-purifiedpolyclonal anti-protein S IgG as a probe. Approximately 1.5 x 10 recombinants of the amplified library were screened. Out of eighteen positive clones one clone was found, after nucleotide sequence analysis, to code for a peptide with a high degree of homology with the carboxy terminal region of the already published bovine protein S. This clone pSP84 (450 bp) was used as a probe to screen a human liver cDNA library in plasmid pUC9. From this library we isolated several positive clones. Clone pSUL5 contained the largest insert (2200 base pairs). Dideoxy sequencing revealed that it codes for 330 amino acids of the carboxy terminal part of protein S. Furthermore, it contained a 1200 base pairs 3' untranslated region. The predicted amino acid sequence did not differ from the published sequence of human protein S, although at the nucleotide level some differences could be detected.

Clone pSUL5 was used to localize the protein S gene to its chromosome. The assignment was done by hybridization to Pst I digested DNA from human-hamster c.q. human-mouse somatic cell hybrids. In this way we got strong indication that the protein S gene is located on chromosome 3.
EVIDENCE FOR AN ABNORMAL EXPRESSION OF THE COLLAGEN BINDING DOMAIN IN VON WILLEBRAND'S DISEASE TYPE II
Authors: Ingerslev J , Stenbjerg S , Bukh A , Møller NPH , Zeuthen J
Published: August 2018

A recently developed new series of monoclonal antibodies (MAbs) against the von Willebrand factor (vWf) included antibodies strongly inhibiting ( Mab vWf-41) and partly inhibiting ( Mab vWf-33) the collagen binding of vWf. We also characterized two Mabs with interacting properties against the ristocetin induced platelet aggregation (MAbs vWf-21 and vWf-39). These antibodies were conjugated with horse-radish peroxidase (HRP) and examined in different constructions forming two-site MAb ELISA's for plasma vWf:Ag and compared with polyclonal antibody ELISA. Symmetrical MAb-ELISA ( i.e. same Mab for extraction and detection) gave practical no dose-response in the standard assay, whereas any different combination of Mabs gave favourable dose-response relationships in sensitive ELISA's for vWf:Ag. Two different sandwiches were chosen using MAb vWf-33 and Mab vWf-41 at either side of the ELISA. These two assay models gave results of plasma from normal persons almost identical to those obtained with polyclonal antibody ELISA. Also in type I von Willebrand's disease these three assays performed very uniformly. In subtypes II plasma ( IIA: n=7; IIB: n=3, IIC: n=l, IID: n=i) . the assay using vWf-33 for coating and vWf-41-HRP for detection measured considerably lower than the polyclonal ELISA and the Mab-ELISA based on the opposite combination. We believe, that our results are indicative of a molecular defect in the collagen binding domain of vWf in subtype II plasma.
ON THE FACTOR XIIIa-INDUCED CROSSLINKING OF HUMAN FIBRIN α-CHAINS
Authors: Henschen A , Müller E
Published: August 2018

Factor XIIIa catalysis the formation of isopeptide bonds Between γ-carbamoyl groups of peptide-bound glutamines and ε-amino groups of lysines or lysine analogues. During fibrin crosslinking two such bonds are rapidly formed between the C-termini of two γ-chains in adjacent molecules and then several bonds are more slowly formed between several α-chains. The crosslinking sites in the γ-chain were identified already 15 years ago, those in the α-chain are still only tentatively or partially identified,, However, by determining the incorporation of lysine analogues in the α-chain it could be shown that the glutamines in positions 328, 366 and possibly also 237 may participate in crosslinking reactions. Analyses of cyanogen bromide fragments isolated from crosslinked fibrin indicated the segments 271-776 and 518-587 to contain the primary crosslinking sites.

In the present study factor XHI-containing fibrinogen was incubated over night with thrombin in presence of calcium ions and cysteine or, as a control, in presence of EDTA. The fibrin material was cleaved with cyanogen bromide, mercaptolysed, pyri-dylethylated and then subjected to Sephacryl S-300 chromatography. The early protein fractions were tested by reversed-phase high-performance liquid chromatography (HPLC) using fibrinogen fragments as reference. In the control sample Aa-chain fragment 271-776 eluted first but in the crosslinked sample it was missing and instead a heterogeneous mixture of higher-molecular weight components was observed. N-Terminal sequence analysis showed the mixture to contain not only the expected fragments 241-476 and 518-584,but in fact all glutamine- or lysine-containing Aα-chain fragments between positions 208 and 611. In the corresponding 6 fragments a total of 6 glutamines and 21 lysines as potential crosslinking sites are present. Two fragments contain only one each of these residues which therefore must be true crosslinking sites. Remaining sites and the actual linkages were identified after reversed-phase HPLC of the tryptic peptide mixture by N-terminal sequence and total amino acid analyses.

The linkage pattern will provide information about the localisation and conformation of the C-terminal part of the α-chain and its contribution to the fibrin polymer structure.
EXPRESSION OF FACTOR XIII SUBUNIT A IN DIFFERENT TYPES OF HUMAN MACROPHAGES
Authors: Adany R , Kiss A , Kappelmayer J , Ablin R J , Muszbek L
Published: August 2018

In addition to plasma the presence of subunit a of blood coagulation Factor XIII (FXIIl) has been verified in platelets and megakariocytes. Most recently, we demonstrated that human peripheral blood monocytes also contain FXIII subunit a. The present study was designed 1/ to determine the stage in the maturation sequence of bone marrow monocytopoesis in which FXIII appears 2/ to establish if FXIII is retained during differentiation into macrophages 3/ to assess how general is the presence of FXIII subunit a in different types of macrophages. FXIII subunit a was immunomorphologically detected in bone marrow smears, in cytospin preparations of cells from serous cavities (pleural, peritoneal, pericardial and synovial spaces), and paraformaldehyde-fixed paraffin-embedded or frozen sections of different organs where classical types of macrophages have been described earlier (liver, lung, thymus, skin, connective tissue, prostate and developing bone) . Cells containing FXIII subunit a were intensively characterized by immunofluorescent and enzymecytochemical techniques in double and treble labeling systems. Its presence was clearly demonstrated in promonocytes of bone marrow, and in all probability, it is present in monoblasts, as well. FXIII was also found in macrophages from different serous cavities and in embryonic osteoclasts. Cells containing FXIII subunit a of connective tissue were found to be tissue histiocytes, and not fibroblasts as previously thought. Kupffer cells of the liver and Langerhans cells of the epidermis were negative supporting theories that these cells are not members of monocyte-derived macrophage cell population. Immunomorphological detection of FXIII subunit a seems to be a useful marker for labeling the continuum of monocyte/macrophage cell line from the earliest ftrais in the bone marrow to the mature forms of macrophages and might be a valuable tool in the cytological diagnosis of malignant disorders of this cell line.
THE OPPOSING OF BASIC FIBROBLAST GROWTH FACTOR AND TRANSFORMING GROWTH FACTOR BETA ON THE REGULATION OF PLASMINOGEN ACTIVATOR ACTIVITY IN CAPILLARY ENDOTHELIAL CELLS
Authors: Saksela O , Moscatelli D , Rifkin D B
Published: August 2018

Basic fibroblast growth factor (bFGF), a potent inducer of angio-genesis in vivo, stimulates the production of both the cell-associated and the secreted forms of urokinase-and tissue-type plasminogen activators (PA) in cultured bovine capillary endothelial cells. This stimulation was counteracted by picogram amounts of transforming growth factor beta The stimulatory effect of bFGF was not completely abolished by increasing the amount of TGFb However, the inhibition by TGFb was greatly enhanced if the cells were pretreated for 1-3 hours with TGFb before addition of bFGF, and the inhibition was almost total, if the' preincubationtime with TGFb was 6 hours.Sequential chanqes of serum-containing medium prior to addition ofbFGF also blocked the PA stimulatory effect of bFGF. This inhibitory activity of serum was reduced by incubation of the serum with anti-TGFb-IgG. After pro-longed incubation of cultures treated simultaneously with bFGF' and TGFb, the inhibitory effect of the added bFGF dominated as assayed by PAlevels. TGFbdid not alter the receptor binding of labeled bFGF, nor did a 6 hour pretreatment with TGFb reducethe amount of bound bFGF. The major difference between effects by bFGF and TGFb was thatwhile bFGF effectively enhanced PA-activi-ty expressed by the cells, TGF decreased the amounts of both cell-associated and secreted PA activity by decreasing enzyme production and proenzyme activation. Both bFGF and TGFb increased the secretion of the endothelial type 1 plasminogen activatorinhibitor (PAI 1). The highest concentration of TGFb is found in platelets, and it is known to be released during clot formation. The suppression of PA production by theendothelium by the release of TGFb shouldresult in a decrease in the fibrinolytic activity and promote clot maintenance. In addition, the rapid stimulation of high levels of PAI 1 secretion from the surrounding capillarycells by platelet released TGFb may further suppress fibrinolysis'. The reversabil it.y of theTGFb effect and domination of bFGF stimulation may be important in relation to the subsequentonset of clot lysis or angiogenesis leadino to thrombus reorganization and wound healing.
IDENTIFICATION OF LYSINES IN HUMAN THROMBIN ESSENTIAL FOR HEPARIN BINDING AND CLOTTING ACTIVITY
Authors: Meade J B , Noyes C M , Church F C
Published: August 2018

We are studying human thrombin (IIa) in order to determine the significance of particular amino acid residues critical for interaction with various substrates and cofactors.Previously, we demonstrated the importance of lysyl residues of Ila during interaction with heparin as well as fibrinogen (Griffith, M.J., (1979) J. Biol. Chem. 254:3401; Church, F.C., et al., (1985) J. Biol. Chem. 260:4936). Identification of these essential lysyl residues in Ila has been approached by chemical modification using the amino group-specific reagent pyridoxal-5'-phosphate (PLP) in the presence and absence of heparin. Ila phosphopyri-doxylated in the absence of heparin (unprotected) showed approximately 2 mols of PLP incorporated per mol of Ila and had greatly reduced inhibition by antithrombin III (ATIII)-heparin as well as reduced clotting activity. Ila phosphopyridoxylated in the presence of heparin (protected) showed approximately 1 mol of PLP incorporated per mol of Ila and had reduced clotting activity but essentially normal inhibition by ATIII-heparin. Both modified thrombins showed normal inhibition by ATIII and heparin cofactor II in the absence of heparin at 25×C and pH 8.0 with apparent second-order rate constant values ranging from 3-5 × 10-5 and 4-6 × 10-4 M-1 min-1, respectively. In contrast to native Ila, neither protected nor unprotected PLP-Ila derivatives bound to a fibrin monomer-agarose column equilibrated at 25×C with 50 mM Tris-HCl, 50 mM NaCl, pH 7.4. Samples of both modified thrombins were reductively denatured, S-carboxymethylated, and hydrolyzed with trypsin at 37×C. The resultant peptide mixtures were separated using reverse-phase high performance liquid chromatography. Peptides showing a high degree of PLP incorporation were sequenced by automated Edman degradation and the modified lysyl residues were identified in the primary sequence of Ila. In unprotected-IIa, lysyl residues 21, 65, 174 and 252 of the B-chain were modified. In heparin-protected-IIa, only Lys 21 and 65 of the B-chain were modified. These results suggest that Lys 174 and 252 of the B-chain of thrombin are essential for binding to heparin and that Lys 21 and/or 65 are essential for clotting activity.
AGGREGATION OF BLOOD PLATELETS BY HUMAN MELANOMA CELL LINES IS MEDIATED BY THE Ilb-IIIa GLYCOPROTEIN COMPLEX: INVESTIGATION PERFORMED USING GLANZMANN THROMBASTHENIC PLATELETS AND AN ANTI-Ilb/IIIa MONOCLONAL ANTIBODY (LYP18)
Authors: Boukerche H , McGregor J L , Berthier Vergnes O , DOré J F , Dechavanne M
Published: August 2018

Human melanoma (M) cell lines with low tumorigenicity (LT) (M1D0., M2Ge.) or high tumorigenicity (HT) (M3Da., M4Be., Mel3Bis) were tested for their ability to aggregate platelets from healthy human donors. HTM cell lines had a potent platelet aggregation activity (on platelets present in heparinized-PRP) compared to the LTM cell lines. Platelet aggregation induced by HTM was inhibited by apyrase. Fab fragments of monoclonal antibody (Mab) LYP18 directed against the Ilb-IIIa glycoprotein (GP) complex completely inhibited platelet aggregation induced by HTM cell lines. Moreover, Mab LYP8 directed against thrombosPondin (TSP) partially blocked this aggregation. Mab AN51 directed against GPIb had no effect on platelet aggregation induced by HTM cell lines. Platelets from two Glanzmann thrombasthenic patients (lacking GPIIb and Ilia) did not aggregate following the addition of HTM cell lines. In immunofluorescence, Mab LYP18 was found to be intensely distributed on the surface of the HTM cell lines. An anti-Illa polyclonal antibody bound to a GP band (having the same Mr as GPIIIa) present on a western blot of SDS-electrophoresed HTM cell lines. Moreover, TSP was present in lysed SDS-treated HTM cell lines (as observed by western blot using a polyclonal anti-TSP) and was secreted by HTM cells iruthe culture medium (using an assay set up by using Mab LYP8). 125I-labelled LYP8 and LYP18 bound specifically to HTM cells. These results suggest that HTM cell-platelet interactions may be mediated by a mechanism involving ADP and the GPIIb-IIIa complex. Moreover, secreted TSP may act as an additional link between HTM cells and stimulatedplatelets through similar membrane receptors.
EFFECT OF ANTICOAGULANT TREATMENT WITH RA-233 (MOPIDAMOLE)ON SURVIVAL IN BRONCHIAL CANCER
Authors: Schneider B , Geser C , Feuerer W
Published: August 2018

RA-233 (mopidamole) is a phosphodiesterase inhibitor that has been -shown previously to limit progression of malignancy in certain experimental animal models and in pilot studies in humans. To test its therapeutic effect a multicenter double blind controlled clinical trial was performed from 1982-1986 with patients suffering from (non small-cell) bronchial cancer. 7 centers participated and the data of 270 patients (147 treated with placebo and 123with RA-233) could be analyzed. Median treatment time was 18 months, median observation time 21 months. With RA-233 treatment survival couldbe increased significantly (Savage-test; p = 0.05 (one sided)) compared to placebo treatment (mean survival time in the RA-233 group 1080 days, in the placebo group 960 days).Cox-analysis showed a significant reduction of the hazard function to 0.6 by RA-233 treatment compared with placebo. As additional significant influence factors of the hazard-function , the postsurgical TNM-class and the histological type could be revealed. Patients from stage T1or T2 and NO survived significantlylonger than patients with other TNM-stages. But there is no interaction between treatment and TNM-class; i.e. RA-233 effect is in all TNM-stages similar. In contrast to survival metastasis-frequency and metastasis-free interval could not be influenced by RA-233.
REGULATION OF PLATELET RECEPTORS FOR FIBRINOGEN AND VON WILLEBRAND FACTOR BY PROTEIN KINASE
Authors: Timmons Sheila , Hawiger Jack
Published: August 2018

Positive and negative regulation of platelet receptors for adhesive proteins, fibrinogen (F) and von Willebrand Factor (vWF) determines whether binding of these ligands will or will not take place. We have shown previously that ADP stimulates and cyclic AMP inhibits binding of F and vWF to human platelets. Now we show that positive regulation of F and vWF binding to platelets via the glycoprotein 11b/1111a complex is dependent on platelet Protein Kinase C, a calcium- and phospholipid-dependent enzyme. A potent activator of Protein Kinase C, phorbol-12-myristoyl-13-acetate (PMA) induced saturable and specific binding of F and vWF which was inhibited by synthetic peptides, gamma chain .dodecapeptide (gamma 400-411) and RGDS. The phosphorylation of 47kDa protein (P47), a marker of Protein Kinase C activation in platelets, preceded binding of F and vWF induced with PMA as well as with ADP and thrombin. Sphingosine, an inhibitor of Protein Kinase C, blocked binding of F and vWF to platelets stimulated with PMA, ADP, and thrombin. Inhibition of binding was concentration-dependent and it was accompanied by inhibition of platelet aggregation. Thus, stimulation of Protein Kinase C is required for exposure of platelet receptors for adhesive proteins whereas inhibition of Protein Kinase C prevents receptorexposure. Protein Kinase C fulfills the role of an intraplatelet signal transducer, regulating receptors for adhesive proteins, and constitutes a target for pharmacologic modulation of the adhesive interactions of platelets.
THE DEPENDENCE OF PLATELET ATP SECRETION IN RESPONSE TO VARIOUS AGONISTS ON Ca2+ MOBILIZATION AND AGGREGATION IN QUIN 2 LOADED PLATELETS
Authors: Powling M J , Hardisty R M
Published: August 2018

Thrombin (0.O5 and 0.5 u/ml), ADP (10 uM), PAF. (30 nM), vasopressin (vp, 0.018 u/m1 ), arachidonate (AA, 10 uM), U46619 (0.125 ug/ml) and A23187 (2OO nM) induce aggregation ATP secretion, TxB2 generation and a rapid elevation of intracellular calcium (Ca2+i) concentration provided the medium contains 1 mM extracellular Ca2+. Phorbol ester (TPA) induces aggregation and secretion with no alteration in [Ca2+i]. Pre-incubation with Fab fragments of Ml48, a monoclonal antibody against GPIIb/lIIa, completely abolished aggregation in response to all agonists without affecting Ca2+i flux. ADP and A23187-induced secretion was completely abolished by M148 Fab while that induced by VP, PAF, U46619, AA, thrombin and TPA was inhibited by 70%, 70%, 30%, 25%, 25% and 10% respectively.

In the presence of 1 mM EGTA (Ca2+o <100 nM), aggregation was again abolished and the Ca2+i fluxes were about 10-20% of those in the presence of 1 mM Ca2+ o. ADP and A23187 again induced no secretion, but 0.05 u/ml thrombin, PAF, VP, U46619, AA, 0.5 u/ml thrombin and TPA induced respectively 6%, 8%, 12%, 30%, 40%, 57% and 85% of control (1 mM Ca2+ o) levels. Addition of Ml48 Fab and EGTA together had no greater effect than EGTA alone.

At the concentrations used, ADP and A23187 are thus completely dependent on aggregation for the induction of ATP secretion, while TPA and thrombin (0.5 u.ml) can actlargely independently of both aggregation and changes in[Ca2+ i]. Low concentrations of thrombin induce secretion in the absence of aggregation provided the stimulated [Ca2+i] is sufficiently high (ie greater than that seen with <100 nM Ca2+ o). VP and PAF appear to be more dependent on aggregation for their secretory responses, while U46619 and AA are more dependent on stimulated [Ca2+i].
REGULATION OF RECEPTOR-OPERATED Ca2− CHANNEL OPENING IN HUMAN PLATELETS
Authors: Moffat Katrina J , MacIntyre D Euan
Published: August 2018

Agonist-induced elevation of the platelet intracellular free Ca2+ concentration ([Ca2−]i), as monitored using quin2, is not electrically mediated and is attenuated by removal of extracellular Ca2− and by lanthanides (e.g Gd3−).Collectively these data suggest that elevation of [Ca2−]i in platelets derives in part via influx of external Ca2−presumably through a receptor-operated Ca2− channel (ROC). Hal lam & Rink (FEBS Lett. 186: 175: 1985) showed that Mn2−also enters platelets via these ROC. To investigate the possible regulatory mechanisms that govern ROC status, we utilized quin2-labelled human platelets suspended in a Ca2+-free Hepes buffered Tyrodes solution, and monitored agonist-induced Mn2+-mediated quenching of quin2 fluorescence as an index of ROC opening.

Thrombin (Th, 0.01-1 U/ml), Vasopressin (VP, 10-1000 nM) and the TxA2-mitnetic, EP171 (1-100 nM) all induced ROC opening which occurred rapidly (<30s), was maximal within 30-60s and thereafter declined. Gd3+ (≤2 mM) markedly impaired this Mn2ࢤ-mediated quenching of quin2 fluorescence induced by all 3 agonists. The adenylate cyclase stimulant PGD2 (3-3000 nM) and the guanylate cyclase stimulant sodium nitroprusside (0.01-10 μM) impaired ROC opening induced by Th (0.5 U/ml), VP (100 nM) and EP171 (25 nM) whether added to platelets ≤120sbefore or 30s after the agonists. In contrast, agents that selectively antagonize, at the receptor level, the effects of VP (e.g. d(CH2)5Tyr Me AVP, 10 ¼H) or EP171 (e.g.EP092, 250nM), or that inhibit the action of Th(e.g. Hirudin 1 U/ml)only impaired ROC opening when added to platelets simultaneously with or before the agonist.These results indicate that, although initiated by agonist-receptor interaction, maintenance of the open state of ROC in human platelets does not require continued receptor occupancy or activation by agonist. Moreover, besides acting to impair the transduction processes initiated following occupancy by agonist of platelet Vi, TP and Thrombin receptors, cAMP-and cGMP-dependent reactions also can terminate or otherwise limit opening of ROC.
HIV STATUS, T CELL SUBSETS, BLOOD PRODUCT USE, AND HEMATOLOGIC ABNORMALITIES IN CONGENITAL COAGULATION DISORDERS (CCD)
Authors: Lusher J M , Aledort L M , Sarnaik S , Mosley J
Published: August 2018

Data are presented on 485 subjects with CCDtreated with blood products at entry into a cooperative study of blood product safety; 376 subjects had hemophilia A, 86 had hemophilia B,and 23 had von Willebrand*is disease (vWD) . Anti-HIV was detected in a total of 323 (66.7%)subjects. Of those treated with pooled product303/397 (76.4%) had anti-HIV; of these, 256/316 (81%) had hemo. A, 44/76 (57.9%) had hemo. B, and 3/5 (60%) had vWD. Of those treated withunpooled products 20/88 (22.7%) had anti-HIV;of these 17/60 (28.3%) had hemo. A; 0 of 10 hadhemo. B, and 3/18 (16.6%) had vWD.The percent of T4 cells in all groups studied were significantly lower in anti-HIV (+) as compared toanti-HIV (-) patients (26% vs 42%) (p=0.0001).T4/T8 ratios demonstrated significant differences in all groups treated (p=0.001) when comparing anti-HIV (+) with anti-HIV (-). However, F VIII concentrate recipients who are anti-HIV(-) have significantly lower T4/T8 when compared to controls (p=0.0001) and single pooled F VIII deficient recipient patients (p=0.0264). Mean platelet counts, WBC, ALC, and Hgb were all significantly lower in anti-HIV ( + ) subjects (p <.001,=.0002,=.002, and =.02) A significantly higher % of anti-HIV (+) subjects had abnormally low WBC, ALC and platelet counts (table). In summary, anti-HIV (+) and lower T4/T8ratios were related to type of blood product used, being seen significantly less frequently in patients receiving only unpooled product. Thrombopenia, leucopenia, and lymphopenia were seen more frequently in anti-HIV (+) patients. _
Demonstration of HIV-encoded Proteins in Cultured and in Uncultured CD 4 Positive Mononuclear Cells from Hemophilia Patients Employing Monoclonal Antibodies against p 15, p 24, GP 41, GP 120, and Reverse Transcriptase
Authors: Wernet P , Scheider E M , Sarin P , Chandra P , Brackmann H H , Kessler M , Egli H
Published: August 2018

In the light of the large percentage of hemophilia patients with antibodies to HIV the identification of a specific virus infection in comparison to HIV antibody negative hemophilia patients has reached crucial importance. The low success rates of direct virus culture techniques together with the as yet low AIDS-di-sease rate observed in these patients separate these patients from the other main risk groups. Within this context, we studied the expression of CD3, CD4, CD8, and HLA class II antigens on fixed cells after PHA stimulation and Interleukin 2 propagation as well as on untreated blood mononuclear cells from healthy individuals and from hemophilia patients by fluorescence activated flow cytometry. Monoclonal antibodies thought to be specific for p 15, p 24, GP 41, GP 120, and for reverse transcriptase revealed a certain number of positive cells on all defined subpopulations analysed. From cell specimen of HIV antibody positive hemophilia patients cells with specific HIV antigens could be enriched by in vitro cultivation. Importantly the expression of virus-encoded antigens preceedes a cytopathic effect for several daVs. Current analyses aim at the prognostic relevance of low amounts of such viral HIV proteins selectively detectable by moAbs.directed to either p 24, GP 41, GP 120, and RT. The reliability, high sensitivity and monoclonal antibody dependent specificity of this newly developed method for the demonstration of HIV specific proteins make it applicable also for longitudinal surveys of hemophilia patients to assess a potential state of viremia or virus antigen processing in their mononuclear cells.
ARACHIDGIIC ACID METABOLISM II PLATELETS STORED FOR FIVE DAYS
Authors: Cascar J M , Kavarro J L
Published: August 2018

Arachidonlc acid (AA) netabolisn has been extensively studied in fresh platelets, but there Is little infomation available for stored platelets . we stored platelets in CLI bags far five days at 22+/-2°C and, on days 0, 3 and 5, six si of platelet concentrate were revved from the container and platelets were labeled with (C-14J-AA. Both incorporation and distribution of radiotracer sere studied in rest and thrcnfcln stivlated platelets.Total uptake of, (C-14)-AA dropped fron day 0 to 5 (p 0.01). Distribution oh day 0 was sinilar to fresh platelets. Incorporation of (C-14)-AA on phosphatidyl inositol (FI) decayed fron 12.4+/-1.5 on day 0, to 7.9+/-0.9 on day 3 (p 0.001), While the percentage attached on phosphatidylserine (PS),increased fron 5.3+/-0.9 to 8.8+/-1.5 (p 0.001). There were not any changes fron day 3 to 5.

On day 0,17.7+/-5.2X of radiactlvity was released fron phospholipids by thronbin. This anount decreased to 7.3+/-2.5X (p 0.01) on day 5. I^xaiment in breakdown of both PI and phosphatidylcholine (PC) was detected. Generation of phosphatidic acid (PA) by thronbin, decreased fron 2.6+/-0.4X of total radiactlvity on day 0 to 1.4+/-0.3X on day 3 (p 0.001) and 0.9+/-0.2X on day 5 (p 0.01). Ve did not find changes in TxB2 and HHT, but HETE decayed fron 7.2+/-2.9X on day 0, to 2.3t/-0.9% on day 5 (p 0.01).

We concluded that both activities of phospholipases A-2 and C are affected by storage.