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PubMed1993

alpha-Thrombin within fibrin clots: inactivation of thrombin by antithrombin-III.

To demonstrate that human alpha-thrombin is effectively inactivated by human antithrombin III (AT) during the production of a fibrin clot we measured the amount of alpha-thrombin activity which can be recovered from a clot generated from purified human proteins. We discovered that 0.05-0.07% of the original alpha-thrombin activity is recovered from a fibrin clot produced from a reaction mixture where the initial concentrations of AT and alpha-thrombin were chosen at a ratio (17.5) to allow complete conversion of fibrinogen to fibrin. These results indicated that alpha-thrombin is successfully inactivated by AT during the production of a fibrin clot. Further, when an amount of alpha-thrombin equal to that recovered from a fibrin clot is introduced into a solution of fibrinogen and AT identical to that utilized to produce the clot only 4% of the fibrinogen is converted to fibrin. These results suggest that i) when a fibrin clot is dissolved during fibrinolytic therapy little active alpha-thrombin should be released from the clot and ii) this amount of thrombin is insufficient to catalyze rethrombosis without proposing de novo production of thrombin. The action on factors XI, VIII, and V of the small amount of thrombin released upon thrombolysis, however, may provide the stimulus for de novo production of sufficient thrombin to catalyze rethrombosis.

Antithrombin III↗

PubMed1998

Ligand binding to thrombin exosite II induces dissociation of the thrombin-heparin cofactor II(L444R) complex.

Heparin cofactor II (HCII) inhibits thrombin rapidly in the presence of heparin or dermatan sulfate. The product of the inhibition reaction is a kinetically stable, 1:1 complex between the two proteins. We recently observed that heparin induces dissociation of complexes containing thrombin and the reactive site mutant HCII(L444R) to yield active thrombin and cleaved inhibitor (Han, J. -H., Van Deerlin, V. M. D., and Tollefsen, D. M. (1997) J. Biol. Chem. 272, 8243-8249). In the current study, we have investigated the mechanism by which heparin induces dissociation of the thrombin-HCII(L444R) complex. Heparin oligosaccharides >/=6 sugars in length induce dissociation, which suggests that dissociation does not depend on binding of a heparin molecule simultaneously to both proteins in the complex. Binding of heparin to HCII(L444R) in the complex also does not appear to be required, since the heparin dose response is unaltered for complexes containing the double mutant HCII(L444R/K173Q), which has decreased affinity for heparin. By contrast, binding of heparin to thrombin appears to be necessary and sufficient to induce dissociation. First, heparin fails to induce dissociation of complexes that contain thrombin(K236E), a variant with decreased heparin affinity. Second, a monoclonal IgG that interacts with the heparin-binding site of thrombin mimicks heparin in its ability to induce dissociation of the thrombin-HCII(L444R) complex. Finally, the complex of HCII(L444R) with thrombin(desPPW), which binds normally to heparin but lacks Pro60BPro60CTrp60D in an insertion loop ("60-loop") between the heparin-binding site and the catalytic site, does not dissociate in the presence of heparin. These results suggest that binding of heparin to thrombin induces an allosteric effect causing destabilization of the thrombin-HCII(L444R) complex and that the allosteric effect may be mediated by the 60-loop.

Arginine↗

PubMed1998

Role of thrombin anion-binding exosite-I in the formation of thrombin-serpin complexes.

Site-directed mutagenesis was used to investigate the role of basic residues in the thrombin anion-binding exosite-I during formation of thrombin-antithrombin III (ATIII), thrombin-protease nexin 1 (PN1), and thrombin-heparin cofactor II (HCII) inhibitor complexes, in the absence and presence of glycosaminoglycans. In the absence of glycosaminoglycan, association rate constant (kon) values for the inhibition of the mutant thrombins (R35Q, K36Q, R67Q, R73Q, R75Q, R77(a)Q, K81Q, K109Q, K110Q, and K149(e)Q) by ATIII and PN1 were similar to wild-type recombinant thrombin (rIIa), whereas kon values were decreased 2-3-fold for HCII against the majority of the exosite-I mutants. The exosite-I mutants did not have a significant effect on heparin-accelerated inhibition by ATIII with maximal kon values similar to rIIa. A small effect was seen for PN1/heparin inhibition of the exosite-I mutants R35Q, R67Q, R73Q, R75Q, and R77(a)Q, where kon values were decreased 2-4-fold, compared with rIIa. For HCII/heparin, kon values for inhibition of the exosite-I mutants (except R67Q, R73Q, and K149(e)Q) were 2-3-fold lower than rIIa. Larger decreases in kon values for HCII/heparin were found for R67Q and R73Q thrombins with 441- and 14-fold decreases, respectively, whereas K149(e)Q was unchanged. For HCII/dermatan sulfate, R67Q and R73Q had kon values reduced 720- and 48-fold, respectively, whereas the remaining mutants were decreased 3-7-fold relative to rIIa. The results suggest that ATIII has no major interaction with exosite-I of thrombin with or without heparin. PN1 bound to heparin uses exosite-I to some extent, possibly by utilizing the positive electrostatic field of exosite-I to enhance orientation and thrombin complex formation. The larger effects of the thrombin exosite-I mutants for HCII inhibition with heparin and dermatan sulfate indicate its need for exosite-I, presumably through contact of the "hirudin-like" domain of HCII with exosite-I of thrombin.

Amino Acid Sequence↗

PubMed2004

Oxytocic activity of thrombin: modulation of thrombin-induced gravid rat myometrial contractions by 5-hydroxytryptamine receptor antagonists.

BACKGROUND: Thrombin possesses potent oxytocic activity in vitro and in vivo. This activity has been proposed to play a role in post-parturitional uterine contractions and possibly, preterm birth related to intrauterine hemorrhage. Previous workers have demonstrated that cyclo-oxygenase pathway may not play a significant role in oxytocic activity of thrombin. However, the role of 5-hydroxytryptamine (a mediator of some of the biological activities of thrombin) in the oxytocic activity of thrombin is unknown. The present study therefore aimed to examine the possible involvement of 5-hydroxytryptamine in thrombin-induced myometrial contractions. METHODS: Effect of 5-hydroxytryptamine receptor antagonists on thrombin-induced contractions of isolated gravid rat myometrium was studied using isolated tissue bath system. RESULTS: Thrombin-induced myometrial contractions were significantly and concentration-dependently inhibited by ketanserin and methysergide. Furthermore, 12 +/- 2% increase in the force of contractions of gravid rat myometrium pre-contracted with 5-hydroxytryptamine (1 microM) was provoked by 1 U/ml of thrombin. Thrombin-induced augmentation of the uterine stimulating effect of 5-hydroxytryptamine was characterized by pronounced increase in the contractile tone. CONCLUSIONS: 5-hydroxytryptamine may possibly play a role in oxytocic activity of thrombin. Uterine hyperactivity associated with intrauterine hemorrhage could hence involve thrombin-induced 5-hydroxytryptamine production in the uterus.

Animals↗

PubMed2005

Quality of thrombin produced from the patient's own plasma using the TPD, a new Thrombin-processing Device.

Thrombin derived from bovine sources commonly is used to arrest bleeding during surgical procedures. However, complications such as postoperative hemorrhage can occur because of the development of cross-reactive anti-bovine antibodies that inhibit human coagulation factor V. It would thus be advantageous to develop techniques to generate human thrombin. This study evaluated thrombin produced from human plasma using a new Thrombin-Processing Device (TPD). Plasma was introduced into the TPD, mixed with an ethanol/ CaCl2 reagent, incubated for 1 h, and the harvested thrombin was assayed for activity and the ability to activate platelets by in vitro assays. TPD-produced thrombin activity was found to be 51.8 +/- 12.4 IU/mL (n = 145). TPD-produced thrombin also stimulated P-selectin (CD62) expression (83 +/- 13% of the platelet population) and Annexin V binding (10.3 +/- 2% of the platelet population) on platelets in a similar fashion to commercial thrombin (P-selectin expression: 88 +/- 3%; Annexin-V binding: 11.4 +/- 3%). Compared with CaCl2 and batroxobin, TPD-produced thrombin had a significantly greater ability to activate platelets. TPD-produced thrombin from human plasma has consistent activity and significantly activates platelets and, thus, may have attractive applications such as the production of autologous thrombin for surgical patients.

Blood Loss, Surgical↗

PubMed1990

Formation of a ternary complex between thrombin, fibrin monomer, and heparin influences the action of thrombin on its substrates.

The consequences of the combined effects of fibrin II monomer (FnIIm) and heparin (H) on the hydrolysis of peptidyl p-nitroanilide substrates by thrombin (IIa), the cleavage of prothrombin by thrombin and the thrombin-catalyzed release of fibrinopeptides from fibrinogen have been studied at pH 7.4 and I 0.15. The effects of fibrin II monomer and heparin on chromogenic substrate hydrolysis can be described by a hyperbolic mixed inhibition model in which substrate can interact with four possible enzyme species (IIa, IIa.H, IIa.FnIIm, and IIa.FnIIm.H) that arise as a result of random formation of a ternary complex among thrombin, fibrin II monomer, and heparin (Hogg, P. J. and Jackson, C. M. (1990) J. Biol. Chem. 265, 241-247). The formation of the ternary IIa.FnIIm.H complex results in an increase in the Km values of 7.03 +/- 1.17-fold (1.37-9.65 microM) and 1.94 +/- 0.60-fold (38.1-73.9 microM) for H-D-Ile-Pro-Arg-pNA and Cbz-Gly-Pro-Arg-pNA hydrolysis, respectively, and a decrease in the kc values of 0.45 +/- 0.08-fold (49.5-22.3 s-1) and 0.52 +/- 0.05-fold (93.1-48.4 s-1). Fibrin II monomer and heparin in combination also decrease the efficiency (kc/Km) with which thrombin cleaves prothrombin to produce Fragment 1 and Prethrombin 1 by 2.3-fold from 607 +/- 30 to 264 +/- 13 M-1 s-1. In contrast to the effects of fibrin II monomer and heparin on thrombin hydrolysis of chromogenic substrates, its proteolysis of prothrombin and its inactivation by antithrombin III (Hogg, P. J., and Jackson, C. M. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 3619-3623), these components have no discernible influence on the ability of thrombin to cleave fibrinogen. These observations indicate that the substrate specificity of thrombin is altered when it is bound in a complex with fibrin II monomer and heparin and suggest that the catalytic efficiency of thrombin for its physiological substrates will be affected differentially by these interactions. Such ternary complex formation involving thrombin, fibrin II monomer, and heparin may provide a mechanism for selectively regulating thrombin action.

Amino Acid Sequence↗

PubMed1981

The activation and inactivation of human factor VIII by thrombin: effect of inhibitors of thrombin.

The activation and inactivation of human factor VIII by thrombin have been investigated by the use of thrombin inhibitors. The addition of inhibitors to nonactivated factor VIII blocks activation by thrombin. In contrast, their addition to factor VIII activated with thrombin does not block inactivation, but causes an initial period of decay that is more rapid than in the absence of inhibitor. This effect was seen only with protease inhibitors that inhibit thrombin. After the initial decay, low levels of factor VIII coagulant activity persist in the presence of inhibitors, but an assay specific for activated factor VIII showed this to be largely a result of the persistence of nonactivated factor VIII. Only in the case of reversible inhibition is activated factor VIII present in this plateau phase. Possible mechanisms that would account for these observations were studied by iterative computer simulation of model reactions. Two classes were considered: (formula: see text). The experimental results are inconsistent with the first mechanism, which predicts that thrombin indicators should stabilize activated factor VIII (VIIIt). Alternative mechanisms were studied where activation is thrombin-dependent, but inactivation is a first-order reaction (mechanism 2). This family of mechanisms includes those where VIIIt is an VIII. thrombin complex. Simulation of the addition of thrombin inhibitors to such model systems shows the initial rapid decay of activity characteristic of the experimental observations and predicts qualitatively the different effects of reversible and irreversible inhibitors that are observed in the plateau phase. These results argue strongly against a two-cleavage model for the activation and inactivation of factor VII by thrombin and support a one-cleavage model in which inactivation is due to first-order decay. In addition, they provide a plausible mechanistic explanation for the fact that serine protease inhibitors appear to inhibit thrombin-activated factor VIII.

Blood Coagulation↗

PubMed1993

Continuous registration of thrombin generation in plasma, its use for the determination of the thrombin potential.

A method is described by which the time-course of thrombin generation in plasma can be obtained from a continuous optical density recording of p-nitroaniline (pNA) production in a 2:3 diluted plasma. A chromogenic substrate, methylmalonyl-methylanalyl-arginyl-pNA (SQ 68), is used that is specifically split by thrombin but at a low rate. The thrombin that appears and disappears in the plasma does not split more than 5% of the substrate added, so the rate of substrate conversion is in good approximation proportional to the amidolytic activity in the plasma over the entire period of thrombin generation. The course of the enzyme concentration can be calculated from the amidolytic activity curve. It is shown that the thrombin generation curves obtained in this way are essentially identical to those obtained via the classical subsampling method. The presence of SQ 68 influences the amount of free thrombin that appears in plasma because it competitively inhibits the inactivation of thrombin by AT III and alpha 2 macroglobulin. The inhibition of the thrombin peak by heparin, relative to an uninhibited control, remains unaltered by the presence of the substrate. From the course of thrombin activity and the prevailing decay constants, the course of prothrombin conversion velocity can be calculated. Prothrombin conversion was seen to be inhibited at high (> 500 microM) substrate concentrations only, and experimental conditions are found under which the inhibition of the clotting process by the substrate is negligible. The amidolytic activity is the sum of the activities of free thrombin and of the alpha 2 macroglobulin-thrombin complex formed.(ABSTRACT TRUNCATED AT 250 WORDS)

Chromogenic Compounds↗

PubMed1996

Human umbilical vein smooth muscle cells as a model to study thrombin generation and function: effect of thrombin inhibitors.

The aim of the present work was to study how human umbilical vein smooth muscle cells (HUVSMC) can initiate the coagulation process and to investigate the responses of these cells to thrombin. Exposure of HUVSMC to recalcified human plasma led to a time-dependent production of thrombin, measured both as amidolytic activity and as release of fibrinopeptide A. Thrombin activity was dose-dependently reduced by an anti-human tissue factor antibody (76 +/- 3% at 10 micrograms/ml) and by inhibitors like heparin, rec-hirudin, hirulog 1, Napap and hirunorm, a novel hirudin-like thrombin inhibitor (IC50 = 2 +/- 0.4, 8 +/- 1, 130 +/- 22, 199 +/- 29 and 68 +/- 8 nM, respectively). The release of fibrinopeptide A was similarly prevented (IC50 = 14 +/- 1, 132 +/- 25 and 50 +/- 8 nM for rec-hirudin, Napap and hirunorm, respectively). Exogenously added thrombin increased thymidine incorporation into HUVSMC to 240 +/- 30% of basal (EC50 = 0.49 +/- 0.09 nM) and thrombin inhibitors blocked this effect (IC50 = 10 +/- 3, 37 +/- 17, 343 +/- 165 and 1402 +/- 758 nM for rec-hirudin, hirunorm, Napap and hirulog-1, respectively). Also recalcified human plasma was mitogenic for HUVSMC and its effect was mainly due to endogenously generated thrombin, as shown by the use of thrombin inhibitors. In conclusion, HUVSMC are capable of initiating the extrinsic coagulation cascade, leading to the formation of thrombin which promotes clotting and stimulates DNA synthesis. Thrombin inhibitors prevent both coagulative and cellular effects of thrombin.

Amino Acid Sequence↗

PubMed2000

Inhibition of cellular action of thrombin by N3-cyclopropyl-7-[[4-(1-methylethyl)phenyl]methyl]-7H-pyrrolo[3, 2-f]quinazoline-1,3-diamine (SCH 79797), a nonpeptide thrombin receptor antagonist.

A growing body of evidence suggests an important contribution of the cellular actions of thrombin to thrombosis and restenosis following angioplasty. Recently we reported on SCH 79797 (N3-cyclopropyl-7-¿[4-(1-methylethyl)phenyl]methyl¿-7H-pyrrolo[3, 2-f]quinazoline-1,3-diamine) and its analogs as new potent, nonpeptide thrombin receptor antagonists. This study further characterizes the biochemical and pharmacological actions of pyrroloquinazoline inhibitors of protease activated receptor-1 (PAR-1) in human platelets and coronary artery smooth muscle cells (hCASMC). SCH 79797 and its N-methyl analog (SCH 203099) inhibited binding of a high-affinity thrombin receptor-activating peptide ([(3)H]haTRAP, Ala-Phe(p-F)-Arg-ChA-HArg-[(3)H]Tyr-NH(2)) to PAR-1 with IC(50) values of 70 and 45 nM, respectively. SCH 79797 inhibited [(3)H]haTRAP binding in a competitive manner. SCH 79797 and SCH 203099 inhibited alpha-thrombin- and haTRAP-induced aggregation of human platelets, but did not inhibit human platelet aggregation induced by the tethered ligand agonist for protease-activated receptor-4 (PAR-4), gamma-thrombin, ADP, or collagen. SCH 203099 inhibited surface expression of P-selectin induced by haTRAP and thrombin, and it did not increase P-selectin expression or prevent thrombin cleavage of the receptor. Thrombin and TFLLRNPNDK-NH(2) (TK), a PAR-1-selective agonist, produced transient increases in cytosolic free Ca(2+) concentration ([Ca(2+)](i)) in hCASMC. This increase in [Ca(2+)](i) was inhibited effectively by SCH 79797. However, the Ca(2+) transients induced by SLIGKV-NH(2,) a PAR-2-selective agonist, were not inhibited by SCH 79797. Thrombin- and TK-stimulated [(3)H]thymidine incorporation also was inhibited completely by SCH 79797. The results of this study demonstrate that SCH 79797 and SCH 203099 are potent, selective antagonists of PAR-1 in human platelets and hCASMC. These data also suggest that the thrombin stimulation of Ca(2+) transients and mitogenesis in hCASMC is mediated primarily through activation of PAR-1.

Apoptosis Regulatory Proteins↗

PubMed1997

Thrombin induces thrombomodulin mRNA expression via the proteolytically activated thrombin receptor in cultured bovine smooth muscle cells.

Thrombin, an important mitogen governing smooth muscle cell proliferation, binds to cultured bovine aortic smooth muscle cells (BASMCs) via both the proteolytically activated thrombin receptor (PATR) and thrombomodulin (TM). Although TM mRNA expression and functional activity is regulated by thrombin in human endothelial cells and mouse hemangioma cells, it remains unclear in those models whether the increased TM mRNA expression observed upon thrombin stimulation is mediated through the activation of PATR or via TM occupancy. We observed in cultured BASMCs that TM mRNA is increased threefold to sixfold by either thrombin, basic fibroblast growth factor (bFGF), or platelet-derived growth factor (PDGF). The increase in TM mRNA with thrombin is time dependent (maximal at 3 hours), a consequence of increased mRNA stability, and accompanied by increases in cell surface TM functional activity. Thrombin-induced TM mRNA was reproduced by the hexameric thrombin receptor-activating peptide (TRAP6) and augmented by a TM-specific antibody. Together, these data suggest that up-regulation of TM mRNA by thrombin is mediated via the PATR. We speculate that increases in BASMC TM mRNA and activity after thrombin may contribute to the impaired thrombus formation observed after atherosclerotic vascular injury.

Amino Acid Sequence↗

PubMed2003

The role of thrombin and thrombin receptors in ischemic, hemorrhagic and traumatic brain injury: deleterious or protective?

In the last two decades it has become apparent that thrombin has many extravascular effects that are mediated by a family of protease-activated receptors (PARs). PAR-1, -3 and -4 are activated via cleavage by thrombin. The importance of extravascular thrombin in modulating ischemic, hemorrhagic and traumatic injury in brain has recently become clear. Thus, in vitro, thrombin at low concentration protects neurons and astrocytes from cell death caused by a number of different insults. In vivo, pretreating the brain with a low dose of thrombin (thrombin preconditioning), attenuates the brain injury induced by a large dose of thrombin, an intracerebral hemorrhage or by focal cerebral ischemia. Thrombin may also be an important mediator of ischemic preconditioning. In contrast, high doses of thrombin kill neurons and astrocytes in vitro and cause disruption of the blood-brain barrier, brain edema and seizures in vivo. This review examines the role of thrombin in brain injury and the molecular mechanisms and signaling cascades involved.

Animals↗

PubMed1995

Differential activation of cytosolic phospholipase A2 (cPLA2) by thrombin and thrombin receptor agonist peptide in human platelets. Evidence for activation of cPLA2 independent of the mitogen-activated protein kinases ERK1/2.

The thrombin receptor agonist peptide SFLLRN was less effective than thrombin in eliciting the liberation of arachidonic acid and the generation of thromboxane A2 by human platelets. We found that while SFLLRN evokes an initial transient increase in cystolic free calcium concentration ([Ca2+]i) of similar magnitude as that caused by thrombin, the SFLLRN-induced elevation of [Ca2+]i declines more rapidly to near resting levels than that evoked by thrombin, suggesting that disparate levels of [Ca2+]i may contribute to the attenuated arachidonic acid release. Furthermore, we observed that SFLLRN is less effective than thrombin in mediating the "activating" phosphorylation of cytolic phospholipase A2 (cPLA2). Both thrombin and SFLLRN rapidly and transiently activated kinases that phosphorylate the 21-residue synthetic peptide Thr669 derived from the epidermal growth factor receptor, but the maximal activation of proline-directed kinases by SFLLRN was less pronounced than that by thrombin. MonoQ chromatography and immunoblot analysis of extracts from stimulated platelets revealed that while thrombin induced a prominent activation of the mitogen-activated protein kinases ERK1 and ERK2, SFLLRN completely failed to do so. On the other hand, SFLLRN, like thrombin, stimulated the activity of a proline-directed kinase distinct from ERK1/2, but the activation of this kinase was less pronounced following stimulation of platelets with SFLLRN compared with thrombin. We conclude 1) that the partial activation of cPLA2 and the subsequent attenuated mobilization of arachidonic acid in response to SFLLRN may be the consequence of a less prolonged elevation of [Ca2+]i and insufficient activation of proline-directed kinase(s) by SFLLRN and 2) that the ability of SFLLRN to mediate the activating phosphorylation of cPLA2 in the absence of ERK1/2 stimulation suggest that, at least in human platelets, proline-directed kinases other than ERK1/2 may phosphorylate and activate cPLA2.

Amino Acid Sequence↗

PubMed1997

A strong thrombin-inhibitory prourokinase derivative with sequence elements from hirudin and the human thrombin receptor.

In order to design plasminogen activators with improved thrombolytic properties, bifunctional proteins with both plasminogen-activating and anticoagulative activity were constructed by fusing a thrombin-inhibitory moiety itself comprises four elements: linker 1, a motif directed to thrombin's active site, linker 2 and a fragment of hirudin which binds to the fibrinogen-recognition site of thrombin. In order to improve further the anticoagulative activity, the thrombin-inhibitory domain was modified by substituting linker 2. Introduction of a linker (FLLRNP) from the human thrombin receptor conferred about a 10-fold increase in anticoagulative activity in protein M37 compared with the parent molecule M23 carrying an aliphatic linker. The increase in anticoagulative activity was also reflected in the shift of the Ki value from 159 +/- 20 nM for M23 to 2.0 +/- 0.5 nM for M37. The increased thrombin-inhibitory activity of M37 may be due to the presence of an arginine in the linker from the thrombin receptor which may interact with one of two glutamic acid residues located at the exit of the thrombin substrate binding pocket. This explanation is supported by the observation that another chimera (M35) carrying a linker sequence with two acidic residues has relatively weak thrombin-inhibitory activity. The thrombin-inhibitory activity of M37 may be strong enough to substitute anticoagulative co-medication during fibrinolytic treatment.

Amino Acid Sequence↗

PubMed1992

Enhancement of incisional wound healing and neovascularization in normal rats by thrombin and synthetic thrombin receptor-activating peptides.

To better define thrombin-receptor interactions, we synthesized human thrombin peptides and identified binding-domain peptides that bind thrombin receptors and activate mitogenic signals (Glenn, K.C., G.H. Frost, J.S. Bergmann, and D.H. Carney. 1988. Pept. Res. 1:65-73). Treatment of full dermal dorsal incisions with a single topical application of thrombin receptor-activating peptide (TRAP-508) or human alpha-thrombin in saline enhances 7-d incisional breaking strength in normal rats up to 82% or 55% over saline-treated controls, respectively. Control wounds require approximately 11.5 d to achieve breaking strength equivalent to TRAP-treated wounds at day 7. Thus, a single application of TRAP accelerates healing, shifting the time course forward by up to 4.5 d. Histological comparisons at day 7 show more type I collagen, less evidence of prolonged inflammation, and an increase in number and maturity of capillaries in TRAP- and thrombin-treated incisions. Angiograms also show 50-65% more functional vascularization going across thrombin- and TRAP-treated surgical incisions. Thus, alpha-thrombin and thrombin peptides, such as those released following injury, appear to initiate or enhance signals required for neovascularization and wound healing. The ability to accelerate normal wound healing events with synthetic peptides representing receptor binding domains of human thrombin may offer new options for management of wound healing in man.

Amino Acid Sequence↗

PubMed1999

Inhibition of thrombin-induced neuronal cell death by recombinant thrombomodulin and E5510, a synthetic thrombin receptor signaling inhibitor.

Thrombin, a serine protease generated by the activation of the blood coagulation cascade following vessel injury, converts fibrinogen to fibrin, activates platelets and several coagulation factors, and plays a pivotal role in thrombosis and haemostasis. Thrombin acts as a mitogen and apoptosis inducer in a dose-dependent fashion. We have previously shown that thrombin caused proliferation of vascular smooth muscle cells (VSMCs). Here, we show that a low concentration of thrombin caused proliferation of mouse neuroblastoma (Neuro-2a) and human neuroblastoma (NB-1) cells, while higher concentrations affected cell viability in a time-dependent manner. Similar effects were observed when thrombin receptor agonist peptide (SFLLRNPNDKYEPF, TRAP) was applied. The dying cells showed nuclear condensation and fragmentation, suggesting that cell death occurred by apoptosis. The extent to which thrombin induced cell death was significantly attenuated by recombinant thrombomodulin (rTM), or by a minimum functional domain of TM, termed E456. Furthermore, a synthetic compound that inhibits signaling from the thrombin receptor, 4-cyano-5,5-bis (4-methoxyphenyl)-4-pentanoic acid (E5510), and the antioxidant N-acetyl L-cysteine (NAC), efficiently prevented thrombin-induced Neuro-2a cell death. Thus, thrombin inhibitors and antioxidant appear to neutralize thrombin toxicity.

Acetylcysteine↗

PubMed1995

Contrasting effects of thrombin and the thrombin receptor peptide, SFLLRN, on aggregation and release of 14C-serotonin by human platelets pretreated with chymotrypsin or serratia marcescens protease.

Chymotrypsin cleaves glycoprotein Ib (GPIb) on platelets and reduces their responsiveness to thrombin; platelets from patients with the Bernard-Soulier syndrome, which lack GPIb, are also less responsive to thrombin than platelets from normal donors. However, Bernard-Soulier platelets respond normally to the thrombin receptor peptide SFLLRN (13). We compared responses of 14C-serotonin-labeled, chymotrypsin-treated platelets (and control platelets) to thrombin (0.25-2 U/ml) and SFLLRN (5-40 microM). Chymotrypsin treatment strongly inhibited thrombin-induced aggregation and release of 14C-serotonin when concentrations of thrombin of 0.5 U/ml or lower were used, even though these responses of control platelets remained near the maximum. In contrast, there was little difference between the responses of control and chymotrypsin-treated platelets to SFLLRN, even when the responses of control platelets were less than maximal. Thus, chymotrypsin treatment greatly inhibits the response to thrombin of the seven transmembrane domain thrombin receptor cloned by Coughlin's group (1, 2). Since Serratia marcescens protease also hydrolyses GPIb, but has less effect than chymotrypsin on other glycoproteins, we pretreated platelets with several concentrations of S. marcescens protease. Concentrations that abolished aggregation and release of 14C-serotonin in response to thrombin had little effect on these responses to SFLLRN. One interpretation of these findings would be that by cleaving GPIb, both proteases are affecting an interaction that may be important for activation of the cloned receptor by thrombin, but irrelevant to activation of this receptor by SFLLRN.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1995

Thrombin-bound structures of designed analogs of human fibrinopeptide A determined by quantitative transferred NOE spectroscopy: a new structural basis for thrombin specificity.

The mutation of Gly12 to Val12 in the A alpha chains of human fibrinogen Rouen is associated with a delayed proteolytic release of fibrinopeptide A (FpA or A alpha 1 to 16 of fibrinogen) by thrombin, leading to a bleeding disorder. Analogs of FpA and FpA Rouen have been designed that include a Pro15 to replace Val15 in natural FpA and to mimic the frequent occurrences of a proline residue at equivalent positions of other protein substrates of thrombin. The Pro15 analogs of FpA and FpA Rouen bind specifically to the active site of thrombin as shown by thrombin-induced differential line broadening and transferred nuclear Overhauser effects (transferred NOEs). Pro15 is well tolerated by the thrombin-bound structures of both FpA and FpA Rouen in solution, resulting in enhanced conformational stabilities of the thrombin-FpA complexes. The Val12 mutation in FpA Rouen causes backbone conformational changes in residues Val12 and Gly13 accompanied by an expansion of the hydrophobic cluster of FpA to accommodate the bulky side-chain of Val12. The single turn of helical structure between residues Asp7 and Glu11 is stabilized by hydrogen bonds from the side-chain carboxylate of Asp7 to the exposed backbone NH groups of Ala10 and/or Leu9 (N-capping), and by hydrogen bonds between the exposed backbone carbonyl groups of residues Phe8 and Leu9, and the backbone NH groups of Gly12/Val12 and Gly13 (C-capping). The bound structure of FpA Rouen may be further stabilized by a non-polar (i,i + 4) interaction between the aromatic side-chain of Phe8 and the aliphatic side-chain of Val12. Despite these optimized intrapeptide interactions, the thrombin-peptide interactions are highly dynamic as indicated by the fast rate of dissociation (koff > 100 s-1) of the peptide ligands from the thrombin complexes. Sequence comparison between mammalian fibrinopeptides A and B suggests that the specificity of thrombin is dictated by a four-residue consensus motif, Phe(P4)-Xxx(P3)-Pro(P2)-Arg(P1) or FXPR, when Xxx at P3 can be a charged or a neutral polar residue capable of specific interactions with residues near the active site of thrombin.

Amino Acid Sequence↗