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PubMed1994

Effects of thrombin and thrombin peptide activating receptor (SFLLRN) on proteoglycan synthesis and distribution in human endothelial cells.

In the present study we compared the effect of alpha-thrombin and the active receptor peptide (SFLLRN) on proteoglycan (PG) release from human endothelial cells (HUVEC) exposed for 24 h in the presence of 35S-sulfate and 3H-glucosamine. The effect of thrombin (5U/ml) and receptor peptide (100 microM) was evaluated on the distribution of PGs in the conditioned medium, cellular extract and extracellular matrix. In the conditioned medium, thrombin increased the amount of PGs. In extracellular matrix thrombin decreased radioactive PGs measured by both isotopes; no difference was observed in cell extracts. SFLLRN peptide showed a consistent increase in PG content in the medium. At variance with thrombin, in cell extracts the peptide increased the amount of PGs. No relevant differences were observed in PG content of extracellular matrix. These results indicate that thrombin and the peptide act differently on PG distribution and underline the different role of the serine protease and of the receptor binding activities of thrombin.

Amino Acid Sequence↗
PubMed1996

Differential mobilization of tyrosine kinases in human platelets stimulated with thrombin or thrombin receptor agonist peptide.

Both thrombin and thrombin receptor agonist peptide (TRAP) activated p72syk and p60c-src with similar magnitudes. Both thrombin and TRAP induced translocation of p60c-src and p54/58lyn to cytoskeleton in an aggregation-dependent manner. Thrombin also induced cytoskeletal association of p72syk, but independent of platelet aggregation. Furthermore, p72syk associated with cytoskeleton underwent marked proteolysis, which was partially dependent upon calpain activation. In contrast, TRAP, even at concentrations as high as 100 mu M, did not induce p72syk translocation to cytoskeleton. Our findings suggest that cytoskeletal translocation of p72syk induced by thrombin is governed by a mechanism distinct from those of p60c-src and p54/58lyn translocation. It is also suggested that p72syk translocation induced by thrombin requires additional signal(s) other than that mediated by the recently-cloned thrombin receptor that couples with GTP-binding proteins and interacts with TRAP.

Blood Platelets↗
PubMed1994

Contractile effects of thrombin in porcine pulmonary arteries and the influence of thrombin inhibitors.

Thrombin catalyzes not only the conversion of fibrinogen to fibrin but also activates several receptor-mediated cell responses. In ring segments of porcine pulmonary arteries the contractile effect of thrombin was studied in the presence and absence of endothelium. The integrity of endothelium was assessed by the bradykinin-induced relaxation of PGF2 alpha (3 mumol/l)-precontracted vessels which was absent after mechanical removal of endothelium. Thrombin at 0.1 to 10 U/ml (i.e. about 1-100 nmol/l) caused a sustained contraction in endothelium-denuded arteries with a maximum at 20-30 min. In vessels with intact endothelium a significant increase in tension up to 1 U/ml was observed preceded by a transient relaxant response. The contractile effect in vessels with intact endothelium was comparatively weaker. This is probably due to the release of EDRF from endothelial cells since blockade of EDRF synthesis by NG-nitro-L-arginine augmented the thrombin-induced contractions in arteries with intact endothelium. Indomethacin did not alter the contractile effect. However, in vessels with endothelium and in endothelium-denuded vessels the contractions were reduced when extracellular calcium was omitted. Verapamil (10 mumol/l) significantly diminished the contractile effect only in endothelium-denuded vessels. On preincubation of endothelium-denuded arterial ring segments with myo-[2-3H]inositol the addition of thrombin (10 U/ml) caused an accumulation of [3H]inositol-1,4,5-triphosphate (IP3). A maximum was observed after 2 min preceding the maximum increase in contraction. Measurement of thrombin-induced endogenous IP3 generation by radioreceptor assay yielded the same results. The thrombin-induced contractile effect requires the proteolytic activity of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Glycosylation of high-affinity thrombin receptors appears necessary for thrombin binding.

Monosaccharide binding competition, lectin affinity chromatography, and glycosylation inhibitors have been used to determine if glycosylation plays a role in thrombin-receptor interactions. Mannose appeared to specifically inhibit thrombin binding to mouse embryo (ME) and hamster fibroblasts. Concanavalin A bound to antibody-purified receptor fractions, and was used as an affinity ligand to purify receptor fractions that retained thrombin binding activity. Cells treated with tunicamycin (6.25 ng/ml) for 24 h lost approximately 35% of their high-affinity thrombin binding sites, yet binding of receptor monoclonal antibody TR-9 was not affected, indicating that the receptor was present in the membrane, but unable to bind thrombin. Thus thrombin receptor glycosylation may be directly involved in thrombin binding.

Animals↗
PubMed1988

Platelet activation by thrombin in the absence of the high-affinity thrombin receptor.

The receptor status of the moderate-affinity platelet binding site for alpha-thrombin has been established by treating platelets with Serratia marcescens protease under conditions causing cleavage of 95-97% glycoprotein Ib (2.5 micrograms for 30 min). High-affinity binding was lost under these conditions, but the platelets continued to show moderate-affinity binding (Kd1 = 16 +/- 5 nM; 930 +/- 300 sites/platelet) and low-affinity binding (Kd2 = 4.6 +/- 3 microM; 170,000 +/- 90,000 sites/platelet), in good agreement with the values previously obtained for moderate- and low-affinity binding in intact platelets [Harmon, J.T., & Jamieson, G.A. (1986) J. Biol. Chem. 261, 15928-15933]. Platelets treated with Serratia protease under these conditions were about 4-fold less sensitive to activation by alpha-thrombin, as measured by serotonin secretion. Crossover studies with analogues showed that binding of alpha-thrombin was compatible by both D-phenyl-alanyl-L-prolyl-L-arginine chloromethyl ketone treated thrombin and N alpha-p-tosyl-L-lysine chloromethyl ketone treated thrombin, and both analogues were capable of inhibiting activation of Serratia-proteolyzed platelets by alpha-thrombin. These studies establish that the moderate-affinity platelet binding site for alpha-thrombin is a receptor, occupancy of which is required for platelet activation in the absence of the high-affinity receptor.

Blood Platelets↗
PubMed1994

Evidence for a selective inhibitory effect of thrombin on megakaryocyte progenitor growth mediated by the thrombin receptor.

An efficient method for the culture of human megakaryocyte precursors in serum-free medium has been developed facilitating study of the effect of regulators of megakaryocyte growth and maturation without interference by serum-derived factors. We have investigated how megakaryocytes and their precursors respond to the procoagulant enzyme, thrombin. In addition to its already documented agonist effect on mature megakaryocytes, thrombin was found to have a marked inhibitory effect on the growth of megakaryocyte colonies from CD34+ bone marrow cells stimulated by IL3. This inhibitory effect, not previously reported, was selective for megakaryocytic cells. The growth of granulomonocytic and erythroid colonies was not affected. A monoclonal antibody which neutralized the effect of exogenous transforming growth factor beta (TGF beta) was unable to fully neutralize the inhibitory effect of thrombin. With the use of a synthetic peptide, corresponding to the tethered thrombin receptor ligand, and of a recombinant inactive form of thrombin, we provide direct evidence that both the inhibitory effect of thrombin on megakaryocyte proliferation and its agonist effect on mature megakaryocytes are mediated by a receptor analogous to the recently cloned platelet thrombin receptor.

Antigens, CD↗
PubMed2000

Extracellular mutations of protease-activated receptor-1 result in differential activation by thrombin and thrombin receptor agonist peptide.

The protease-activated thrombin receptor-1 (PAR-1) can be activated by both the tethered ligand exposed by thrombin cleavage and a synthetic peptide having the tethered ligand sequence (thrombin receptor agonist peptide or TRAP). We conducted a mutational analysis of extracellular residues of the receptor potentially involved in interaction with both the tethered ligand and the soluble peptide agonist. Agonist-stimulated calcium efflux in X. laevis oocytes or inositol phosphate accumulation in COS-7 cells was used to assess receptor activation. We have also examined the binding of a radiolabeled TRAP for the wild-type and mutant PAR-1 receptors. Our results indicated that most of the mutations strongly affected TRAP-induced responses without significantly altering thrombin-induced responses or TRAP binding. Several point mutations and deletion of extracellular domains (DeltaEC3, DeltaNH3) drastically altered the ability of mutant receptors to respond to TRAP, but not to thrombin, and did not affect the affinity for the radiolabeled TRAP by these mutant receptors. Only mutations that disrupted the putative disulfide bond or substitution of multiple acidic residues in the second extracellular loop by alanine had a significant effect on both ligand binding and thrombin activation. These results suggest that although both agonists can activate PAR-1, there are profound differences in the ability of thrombin and TRAP to activate PAR-1. In addition, we have found PAR-1 mutants with the ability to dissociate receptor-specific binding from functional activity.

Amino Acid Sequence↗
PubMed1983

Influence of thrombin inhibitors on the thrombin-induced activation of human blood platelets.

The influence of thrombin inhibitors, which differ both in their chemical structure and type of inhibition, on the thrombin-platelet reaction was studied. The inhibitory effects of the competitive inhibitors tested on the thrombin-induced platelet reaction correlated well with their effects on the thrombin-catalyzed fibrinogen conversion and on the thrombin-catalyzed hydrolysis of synthetic substrates. Heparin and antithrombin III inhibited the thrombin-induced platelet reaction. However, heparin did not potentiate the inhibition by antithrombin III of the thrombin-platelet reaction.

Antithrombin III↗
PubMed1992

The function of calcium in protein C activation by thrombin and the thrombin-thrombomodulin complex can be distinguished by mutational analysis of protein C derivatives.

Protein C activation is catalyzed on endothelium by a complex between thrombin and thrombomodulin. Ca2+ stimulates protein C activation in the presence, and inhibits in the absence, of thrombomodulin. Protein C has Asp residues at the P3 and P3' positions relative to the scissile bond at Arg169-Leu. To determine the contribution of these residues to the Ca2+ effect on activation, we have expressed human 4-carboxyglutamic acid (Gla)-domainless protein C and 3 mutants with Asp-->Gly substitutions at P3, P3', and both positions. Ca2+ interaction with the protein C derivatives was monitored by changes in intrinsic fluorescence, and the Ca2+ dependence of activation by thrombin and a complex of thrombin-thrombomodulin with a soluble thrombomodulin derivative (the fourth through sixth epidermal growth factor domains). The affinity for Ca2+ of the mutants was reduced 3-6-fold, which was reflected by a comparable change in the Ca2+ concentration required for the half-maximal rate of activation by the thrombin-thrombomodulin complex. However, Ca2+ no longer effectively inhibited activation of the mutants by thrombin alone. We conclude that 1) the Asp residues play a specific role in the Ca(2+)-dependent inhibition of protein C activation by thrombin; 2) these mutations alter the affinity of Ca2+ for the high affinity binding site; and 3) the Asp residues in the P3 and P3' sites do not contribute in a positive fashion to rapid activation by the thrombin-thrombomodulin complex.

Animals↗
PubMed1995

Human thrombin receptors are insensitive to thrombin-like snake venom enzymes.

Thrombin-like snake venoms enzymes, flavoxobin, and okinaxobin I isolated from Trimeresurus flavoviridis and Trimeresurus okinavensis, respectively, were examined in SH-EP cells and evaluated whether or not they can activate human thrombin receptors. Flavoxobin was almost completely inactive in both assays for phosphoinositide turnover and DNA synthesis. In contrast, okinaxobin I stimulated phosphoinositide turnover in a dose dependent manner, but considerably weakly. The EC50 value was about 100 nM, which was 4,000 times larger than that of alpha-thrombin. This stimulation was not inhibited by hirudin, an effective inhibitor of alpha-thrombin. Okinaxobin I also induced a very weak stimulation of DNA synthesis. These results suggest that thrombin-like snake venom enzymes interact with human thrombin receptors in inefficient ways. Weak interactions of the enzymes with thrombin receptor and inhibitor were ascribed to the incomplete formation of a lysine-cation cluster necessary for electrostatic molecular recognition.

Amino Acid Sequence↗
PubMed1996

Crystal structure of two new bifunctional nonsubstrate type thrombin inhibitors complexed with human alpha-thrombin.

The crystal structures of two new thrombin inhibitors, P498 and P500, complexed with human alpha-thrombin have been determined at 2.0 A resolution and refined to crystallographic R-factors of 0.170 and 0.169, respectively. These compounds, with picomolar binding constants, belong to a family of potent bifunctional inhibitors that bind thrombin at two remote sites: the active site and the fibrinogen recognition exosite (FRE). The inhibitors incorporate a nonsubstrate type active site binding fragment: Dansyl-Arg-(D)Pipecolic acid (Dns-Arg-(D)Pip), reminiscent of the active-site directed inhibitors MD-805 and MQPA, rendering them resistant to thrombin-induced hydrolysis. The FRE binding fragment of these inhibitors corresponds to the hirudin55-65 sequence. They differ in the chemical nature of the nonpeptidyl linker bridging these two functional activities. In both cases, the active site binding fragment is well defined in the electron density. The DnsH1, ArgH2, and (D)PipH3 groups occupy the S3, S1, and S2 subsites of thrombin, respectively, in a way similar to that observed in the thrombin-MQPA complexes. Binding in the active site of thrombin is characterized by numerous van der Waals contacts and ring-ring system interactions. Unlike in the substrate-like inhibitors, ArgH2 enters the S1 specificity pocket from the P2 position and adopts a bent conformation to make an hydrogen bond to the carboxylate of Asp189. In this noncanonical position, its carbonyl points away from the oxyanion hole, which is now occupied by well-ordered solvent molecules. The linkers fit in the groove extending from the active site to the FRE. The C-terminal fragments of both inhibitors bind in the same way as analogous FRE binding elements in previously described complexes.

Amino Acid Sequence↗
PubMed1987

Binding of heparin or dermatan sulfate to thrombin is essential for the sulfated polysaccharide-accelerated inhibition of thrombin by heparin cofactor II.

Heparin cofactor II (HC II) and thrombin were chemically modified with pyridoxal 5'-phosphate, and their effects on the inhibition of thrombin by HC II in the presence of heparin or dermatan sulfate were studied. The inhibition of thrombin by HC II was enhanced about 7000-fold in the presence of heparin or dermatan sulfate. However, this enhancement by heparin dwindled to 110- and 9.6-fold when the modified HC II and the modified thrombin, respectively, were substituted for native proteins. Essentially identical results were obtained from the experiments using dermatan sulfate. These results indicate that the binding of heparin or dermatan sulfate to both thrombin and HC II is required for the sulfated polysaccharide-dependent acceleration of the thrombin inhibition by HC II, and the binding to thrombin is more essential for the reaction.

Antithrombins↗
PubMed1984

Monoclonal antibodies directed against human alpha-thrombin and the thrombin-antithrombin III complex.

Human alpha-thrombin was poorly immunogenic in Balb/c mice. Nevertheless, following fusion of spleen cells from a responding mouse with NS-1 cells, 8 mouse monoclonal antibodies against alpha-thrombin were isolated, and 6 were characterised. Five of these were isotype IgG2a, and one was IgG1. One, EST 1, bound thrombin only minimally, and was directed against a neoantigen on the thrombin-ATIII (T-AT) complex. This antibody also recognised a site on prothrombin, though with much lower affinity. Its binding was markedly temperature-dependent, indicating a requirement for molecular mobility. A second antibody, EST 4, would not bind the T-AT complex. It inhibited both the clotting and amidase activities of thrombin, and modification of the active site histidine, but not the active site serine, reduced the affinity constant of binding to EST 4. This antibody appears to be directed against an epitope in the vicinity of the enzyme active site. The epitopes for EST 1 and EST 4 were both remote from those of the other monoclonal antibodies, EST 2, 6, 7 and 8. These four competed with each other for binding to thrombin, and all inhibited clotting but not amidase activity. Thrombin binding was not affected by modification of the active site, though formation of the T-AT complex reduced the affinity of binding to EST 6 and EST 8. These monoclonals recognise epitopes in the region of the fibrinogen binding site.

Animals↗
PubMed1991

Radioimmunoassay for the detection of active-site specific thrombin inhibitors in biological fluids. II. Heparin affects the binding of hirudin to alpha-thrombin.

A radioimmunoassay for recombinant (r.) hirudin was used to study the effect of heparin and other glycosaminoglycans (GAG's) on interactions between hirudin and thrombin. Binding of 125I-r.hirudin to thrombin in the presence or absence of heparin (and other GAG's) was monitored by immunoprecipitation of thrombin-125I-r.hirudin complexes with a monoclonal antibody to human a-thrombin. Heparin and dextran sulfate substantially reduced hirudin binding to thrombin in human plasma. Inhibition by heparin was restored by addition of increasing amounts of antithrombin (AT) to samples containing constant amounts of heparin, thrombin and 125I-r.hirudin and was not neutralized by protamine sulfate. At very low heparin concentrations competitive displacement of 125I-r.hirudin was observed, while in the presence of heparin-AT complexes some 125I-r.hirudin remained bound to thrombin, suggesting that two or more binding sites for hirudin on the thrombin molecule may be occupied simultaneously.

Binding Sites↗
PubMed1992

Determination of r DNA hirudin and A-human thrombin- hirudin complex in plasma samples: enzyme linked immunosorbent assays for hirudin and complex vs. chromogenic thrombin substrate assay.

rDNA hirudin plasma concentrations in man and rhesus monkeys were determined over a period of 15 and 24 h. The plasma concentration of alpha-human thrombin-hirudin complex was measured after administration of the complex to rhesus monkeys. The complex was also determined after administration of hirudin to man and rhesus monkeys to study a possible formation of a complex with alpha-human thrombin in blood. The determination of hirudin was performed by a sandwich ELISA, using polyclonal and monoclonal antibodies and the chromogenic thrombin substrate assay. The alpha-human thrombin-hirudin complex concentration in the plasma of rhesus monkeys was measured over a period of 48 hours. The results of a sandwich ELISA were compared with those of the chromogenic thrombin substrate assay. A good agreement between the total hirudin concentrations analyzed by the hirudin ELISA and the alpha-human thrombin-hirudin complex ELISA and those of the chromogenic thrombin substrate assay, measuring total hirudin, too, was observed.

Animals↗
PubMed2003

The influence of direct and antithrombin-dependent thrombin inhibitors on the procoagulant and anticoagulant effects of thrombin.

INTRODUCTION: Clinical trials evaluating direct thrombin inhibitors in unstable coronary artery disease (CAD) have been disappointing. The hypothesis tested in the present study was that these agents may inhibit the anticoagulant effect of thrombin to a further extent than the procoagulant effect of thrombin. MATERIALS AND METHODS: We studied both reversible and irreversible thrombin inhibitors and compared the effects of each inhibitor on activated protein C (APC) generation vs. the effect on fibrinopeptide A (FPA) generation. A mixture of protein C, thrombin inhibitor, fibrinogen, fibrin polymerisation blocker and thrombin was incubated with thrombomodulin (TM)-expressing human saphenous vein endothelial cells (HSVECs). The inhibitors investigated were melagatran, inogatran, hirudin, hirugen, D-Phe-D-Pro-D-arginyl chloromethyl ketone (PPACK), and antithrombin (AT) alone or in combination with unfractionated heparin (UFH) or low-molecular-weight heparin (LMWH). RESULTS: All agents, except hirugen, inhibited APC and FPA generation in a dose-dependent manner. FPA inhibition/APC inhibition ratios, based on IC50 for inogatran, melagatran, hirudin, PPACK, AT, AT-UFH and AT-LMWH were 1.73, 0.85, 0.55, 2.1, 0.5, 0.65 and 3.1 respectively. CONCLUSIONS: All agents, except hirugen, inhibited APC and FPA generation approximately to a similar extent. Thus, it can be inferred that the poor efficacy of thrombin inhibitors in recent clinical trials in patients with unstable CAD is unlikely to be a consequence of their effects on the protein C system.

Amino Acid Chloromethyl Ketones↗
PubMed1999

Thrombin inhibitors suppress the thrombin-thrombomodulin-mediated generation of activated protein C.

In the treatment of unstable coronary artery disease, direct thrombin inhibitors have shown no or only limited benefit as compared with heparin, despite theoretical advantages. One explanation may be that the direct thrombin inhibitors to a greater extent than heparin have an inhibiting effect on the generation and activity of activated protein C. In the present study, this hypothesis was tested in an in vitro, "purified" system, where human protein C underwent activation to activated protein C by the thrombin-thrombomodulin complex. Direct thrombin inhibitors, inogatran and hirudin, unfractionated heparin+antithrombin, or dalteparin+antithrombin, were added to the system before activation to evaluate their inhibitory effect on the generation of activated protein C. At inhibitor concentrations well below the achieved plasma levels in major clinical trials, the thrombin-thrombomodulin-mediated activation of protein C was inhibited by all the studied inhibitors in a dose-dependent manner, but, contrary to our hypothesis, to a greater extent by unfractionated heparin+antithrombin and dalteparin+antithrombin than by the direct thrombin inhibitors, hirudin and inogatran. Despite difficulties to draw conclusions for the in vivo situation, the in vitro inhibition, by all studied inhibitors, of the generation of activated protein C, found in this study may be a possible explanation for ongoing cardiovascular events despite adequate treatment with thrombin inhibitors, in patients with unstable coronary artery disease. This inhibition of the generation of activated protein C may also contribute to the rebound in cardiovascular events after withdrawal of effective antithrombotic treatment.

Antithrombins↗
PubMed1999

The effect of a low molecular mass thrombin inhibitor, inogatran, and heparin on thrombin generation and fibrin turnover in patients with unstable coronary artery disease.

AIM: This study evaluated a novel specific thrombin inhibitor, inogatran, in comparison with unfractionated heparin, with regard to markers for coagulation activity in patients with unstable coronary artery disease. METHODS AND RESULTS: In the Thrombin Inhibition In Myocardial Ischaemia (TRIM) study patients were randomized to one of three different doses of inogatran or to unfractionated heparin, given intravenously over 72 h. In a subpopulation of 320 patients, markers for coagulation activity were measured at baseline, during and after the study infusion. Prothrombin fragment 1 + 2, indicating thrombin generation, decreased in the low, medium and high dose inogatran groups and in the heparin group during the first 6 h of treatment by 12%, 15%, 21% and 26%, respectively. From 6 h to 72 h after the start of infusion the levels changed by -7%, -6%, -4% and +34%, respectively. The increase in the heparin group continued after the infusion was stopped. Thrombin-antithrombin complex, also indicating thrombin generation, decreased by 0%, 2%, 18% and 22%, respectively, during the first 6 h of treatment. During the same period soluble fibrin, an intermediate in fibrin formation, increased both in the low and medium inogatran group by 9%, while a decrease by 4% and 18%, respectively, was seen in the high dose inogatran group and in the heparin group. Fibrin dissolution, as measured by fibrin D-dimer, decreased during the first 24 h of treatment by 20%, 18%, 18% and 20%, respectively. The first 24 h after discontinuation of infusion, fibrin D-dimer increased by 6%, 23%, 25% and 44%, respectively. After 72 h, at the end of infusion, patients treated with inogatran, to a larger extent than those given heparin, had suffered from death, myocardial infarction or refractory angina pectoris. After 7 days this trend was less marked. CONCLUSION: The more pronounced decrease in thrombin generation and fibrin turnover during the first 6 h of infusion, and the later increase in thrombin generation and fibrin turnover, in the heparin group, as compared to the inogatran groups, may be related to the lower clinical event rate during infusion with heparin compared with inogatran and the recurrence of ischaemic events, early after cessation of heparin infusion.

Aged↗