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PubMed1992

PPACK-thrombin is a noncompetitive inhibitor of alpha-thrombin binding to human platelets.

Recent studies from our laboratory indicate that purified kininogens are noncompetitive inhibitors of human alpha-thrombin but not PPACK-thrombin, binding to human washed platelets. In order to understand the mechanism by which the kininogens inhibit alpha-thrombin binding, investigations were initiated to determine if alpha-thrombin and PPACK-thrombin bound to the same site on human platelets. Initial investigations reveal that alpha-thrombin is a more potent inhibitor of 125I-PPACK-thrombin binding than PPACK-thrombin. Further studies show that PPACK-thrombin is a noncompetitive inhibitor of 125I-alpha-thrombin binding to platelets. These studies suggest that human alpha-thrombin binds on the platelet surface to a different site or binds differently to the same site from PPACK-thrombin. These data indicate that the ability of the kininogens to block alpha-thrombin binding to platelets but not PPACK-thrombin binding results from these thrombins having either two different binding sites or one binding site on the platelet surface which they interact with differently.

Amino Acid Chloromethyl Ketones↗
PubMed1989

Conformational lability of vitronectin: induction of an antigenic change by alpha-thrombin-serpin complexes and by proteolytically modified thrombin.

We previously showed that the alpha-thrombin-antithrombin III complex causes antigenic change in vitronectin as monitored by the monoclonal anti-vitronectin antibody 8E6 (Tomasini & Mosher, 1988). We have extended these studies to other protease-serpin complexes and to gamma-thrombin, a proteolytic derivative of alpha-thrombin. In the presence of heparin, recognition of vitronectin by 8E6 was increased 64- or 52-fold by interaction with the complex of alpha-thrombin and heparin cofactor II or the Pittsburgh mutant (Met358----Arg) of alpha 1-protease inhibitor, respectively. This was comparable to the value obtained with the alpha-thrombin-antithrombin III complex. Factor Xa-serpin complexes were approximately 4-fold less effective than the corresponding thrombin complexes. alpha-Thrombin-serpin complexes but not Xa-serpin complexes formed disulfide-bonded complexes with vitronectin. Antigenic changes and disulfide-bonded complexes were not detected when trypsin- or chymotrypsin-serpin complexes were incubated with vitronectin. gamma-Thrombin caused 7- and 34-fold increases in recognition of vitronectin by MaVN 8E6 in the absence and presence of heparin, respectively. In contrast, alpha-thrombin by itself had no effect. The antigenic change induced by gamma-thrombin was maximal when gamma-thrombin and vitronectin were equimolar, was not dependent on cleavage of vitronectin, and was abolished by inhibition of gamma-thrombin with Phe-Pro-Arg-chloromethyl ketone but not with diisopropyl fluorophosphate. These data indicate that alpha-thrombin is the component in alpha-thrombin-serpin complexes that induces the antigenic change in vitronectin, probably via a region that is preferentially exposed in gamma-thrombin.

Animals↗
PubMed1981

Conformational differences between high clotting human alpha-thrombin and nonclotting gamma-thrombin.

The conformations of human alpha-thrombin and gamma-thrombin have been compared by circular dichroism, solvent perturbation different spectroscopy, and chemical modification. Circular dichroism studies indicate that proteolytic conversion of alpha-thrombin to gamma-thrombin is accompanied by considerable conformational changes which include a decrease in alpha-helical content from 5-7% to 0-1%. Solvent perturbation at pH 6.0 obtained with 20% ethylene glycol, 20% glycerol, and 20% dimethyl sulfoxide indicates an apparent exposure of 3.5 +2- 0.2 tryptophan and 7.8 +/- 0.1 tyrosine residues in alpha-thrombin and 4.6 +/- 0.2 tryptophan and 9.2 +/0 0.3 tyrosine residues in gamma-thrombin. This increased exposure is substantiated by the greater reactivity of tryptophan residues in gamma-thrombin toward dimethyl (2-hydroxy-5-nitrobenzyl) sulfonium bromide. It suggests that gamma-thrombin is a less compact molecule than the parent alpha-thrombin. Solvent perturbation studies of alpha-thrombin and gamma=thrombin inhibited by phenyl-methanesulfonyl fluoride showed that 0.3 +/- tryptophan and 0.9 +/- 0.3 tyrosine residues in alpha-thrombin and 0.6 +/- 0.3 tryptophan and 1.3 +/- 0.4 tyrosine residues in gamma-thrombin were blocked by the inhibitor. These subtle differences in the extent of blocking of tyrosine and tryptophan suggest a tighter conformation in the catalytic site of gamma-thrombin compared to that of alpha-thrombin.

Circular Dichroism↗
PubMed1989

Anti-thrombin activities of heparin. Effect of saccharide chain length on thrombin inhibition by heparin cofactor II and by antithrombin.

The interactions of two proteinase inhibitors, heparin cofactor II and antithrombin, with thrombin are potentiated by heparin. Using two methods, we have studied the potentiating effects of a series of heparin (poly)saccharides with high affinity for antithrombin and mean Mr ranging from approx. 1700 to 18,800. First, catalytic amounts of heparin (poly)saccharide were added to purified systems containing thrombin and either heparin cofactor II or antithrombin. Residual thrombin activity was determined with a chromogenic substrate. It was found that only the higher-Mr polysaccharides (Mr greater than 8000) efficiently catalysed thrombin inhibition by heparin cofactor II, there being a progressive catalytic effect with increasing Mr of the polysaccharide. Weak accelerating effects were noted with low-Mr saccharides (Mr less than 8000). This contrasted with the well-characterized interaction of heparin with antithrombin and thrombin, where heparin oligosaccharides of Mr less than 5400 had absolutely no ability to accelerate the reaction, while (poly)saccharides of Mr exceeding 5400 showed rapidly increasing catalytic activity with increasing Mr. Secondly, these and other heparin preparations were added in a wide concentration range to plasma with which 125I-labelled thrombin was then incubated for 30 s. Inhibited thrombin was determined from the distribution of labelled thrombin amongst inhibitor-thrombin complexes, predominantly antithrombin-thrombin and heparin cofactor II-thrombin complexes. In this situation, where the inhibitors competed for thrombin and for the (poly)saccharides, it was found that, provided the latter were of high affinity for antithrombin and exceeded a Mr of 5400, thrombin inhibition in plasma was mediated largely through antithrombin. Polysaccharides of Mr exceeding 8000 that were of low affinity for antithrombin accelerated thrombin inhibition in plasma through their interaction with heparin cofactor II. High concentrations of saccharides of Mr 1700-5400 exhibited a size-dependent acceleration of thrombin inhibition, not through their interaction with antithrombin, but through their interaction with heparin cofactor II.

Antithrombins↗
PubMed1994

Identification of a region in protein C involved in thrombomodulin-stimulated activation by thrombin: potential repulsion at anion-binding site I in thrombin.

During coagulation human protein C is activated by thrombin; however, this cleavage reaction is slow unless thrombin is complexed with a cofactor, thrombomodulin. Near the thrombin cleavage site in protein C is a cluster of basic residues, at positions P5' (Lys-174), P8' (Arg-177) and P9' (Arg-178). We have explored the role of this basic cluster in the activation of protein C by thrombin, and by thrombin-thrombomodulin complex, by substitution of glutamic acid at each position to generate the acidic protein C derivative P'-EEE. The activation rate of P'-EEE by free alpha-thrombin was approx. 12-fold faster than that observed for wild-type (wt) human protein C zymogen (HPC) in the presence of calcium, but unchanged in the absence of calcium. While the thrombin-catalysed activation of wt-HPC was stimulated approx. 300-fold by thrombomodulin, we observed no effect of thrombomodulin on thrombin-catalysed activation of the P'-EEE derivative. Using synthetic peptides that bind to anion-binding site I of thrombin (thrombin-receptor sequence 52-66 and hirudin sequence 54-65 SO4 Tyr), we found that the rate of thrombin-catalysed activation of wt-HPC in the presence of calcium could be increased severalfold in a dose-dependent manner. However, the enhanced rate of thrombin-catalysed activation of P'-EEE could be progressively reduced to wt-HPC levels with increasing concentrations of both synthetic peptides. Our data suggest that the P' basic cluster in protein C reduces interaction with free alpha-thrombin through electrostatic repulsion with anion-binding site I, a site that is masked when thrombomodulin binds thrombin. Further, the lack of thrombomodulin cofactor activity with thrombin-catalysed activation of P'-EEE suggests that the basic cluster in protein C forms a contact site with thrombomodulin.

Amino Acid Sequence↗
PubMed2002

In vivo imaging of thrombin activity in experimental thrombi with thrombin-sensitive near-infrared molecular probe.

OBJECTIVE: Thrombin, a serine protease, plays an important role in thrombosis as well as other cellular and developmental processes. In this study, we investigated the ability of a novel thrombin-activatable molecular probe to provide in vivo images of thrombin activity in experimental thrombi. METHODS AND RESULTS: The thrombin probe consists of a near-infrared (NIR) fluorochrome attached to a delivery vehicle via a thrombin-specific oligopeptide substrate. In human blood, endogenous thrombin activated the thrombin probe and increased the fluorescence signal by 18-fold (P=0.008). Hirudin, a specific thrombin inhibitor, suppressed probe activation by 82% (P=0.007). Imaging of in vivo thrombin activity was then investigated in acute experimental murine thrombosis models up to 12 hours. After systemic thrombin probe injection, focal NIR fluorescence signal enhancement was rapidly detected within acute and subacute thrombi. In contrast, no thrombosis signal enhancement was seen in similar experiments with a control NIR fluorochrome. CONCLUSIONS: Thrombin activity can be imaged in vivo by using a novel thrombin-activatable and thrombin-specific NIR molecular probe. The thrombin probe could enhance the understanding of the role of thrombin in thrombogenesis and other homeostatic and pathological conditions.

Adult↗
PubMed1994

Persistent thrombin generation in humans during specific thrombin inhibition with hirudin.

BACKGROUND: The degree to which antithrombotic drugs suppress thrombin generation is unknown. Because hirudin, unlike antithrombin III, binds intravascular thrombin rapidly and selectively to yield a circulating inactive complex of 3- to 4-hour half-life, we used intravenous hirudin in humans to investigate the course of thrombin generation during and early after anticoagulation with this potent, direct antithrombin. METHODS AND RESULTS: Intravascular thrombin was measured with an ELISA for the thrombin-hirudin complex formed during and for 18 hours after stopping a 6-hour infusion of hirudin at 0.1, 0.2, and 0.3 mg.kg-1.h-1 in three groups of six patients each. With free hirudin in 20- to 10,000-fold molar excess of thrombin and peak activated partial thromboplastin times of 2.3 to 3.0 times baseline, mean plasma thrombin-hirudin complex increased from 794 +/- 85 pg/mL (mean +/- SEM) 15 minutes after the start of the infusion to 1617 +/- 151 pg/mL at 6 hours of infusion to 2667 +/- 654 pg/mL at 24 hours. During the 24-hour observation period, plasma concentration of fragment 1.2 (the peptide released during conversion of prothrombin to thrombin) never fell below baseline but rather increased transiently during the hirudin infusion. Plasma concentrations of thrombin-antithrombin III complex (in ng/mL) decreased from 4.34 +/- 0.40 at baseline to 1.64 +/- 0.13 at 6 hours (P < .001) and gradually increased after stopping the infusion to 5.7 +/- 0.87 at 24 hours (nonsignificant compared with baseline). CONCLUSIONS: Measurement of thrombin-hirudin complex may be used as a marker of thrombin generation in humans. Persistent accumulation of thrombin-hirudin complex and generation of fragment 1.2 during and after completion of potent anticoagulation with hirudin suggest thrombin generation is not blocked by high-affinity thrombin inhibition. The persistent formation of thrombin during declining plasma levels of hirudin may contribute to the pathogenesis of rethrombosis early after antithrombin therapy or during inadequate anticoagulation.

Aged↗
PubMed1992

Thrombin interaction with platelet glycoprotein Ib: effect of glycocalicin on thrombin specificity.

We describe here the alteration of thrombin specificity induced by its interaction with glycocalicin. Glycocalicin is the external part of platelet glycoprotein Ib alpha (GPIb alpha) and contains binding sites for von Willebrand factor and thrombin. Taking advantage of its solubility, we have used glycocalicin in competition assays on various thrombin activities. Glycocalicin did not inhibit chromogenic substrate hydrolysis nor diisopropylfluorophosphate iPr2 (PF) incorporation, indicating that thrombin binding to GPIb does not alter access to or the conformation of the thrombin catalytic site. Glycocalicin competitively inhibited thrombin binding to fibrin (Ki = 0.1 mumol/L) and blocked fibrinogen clotting activity of thrombin. Glycocalicin also inhibited thrombin binding to thrombomodulin in a competitive manner (Ki = 3 to 5 mumol/L), but failed to prevent thrombin interaction with protein C in the absence of thrombomodulin. Previous results have indicated that GPIb binds to thrombin within the anion binding exosite masked by the carboxy-terminal hirudin peptide 54-65. The present results confirm the implication of the anion binding exosite in GPIb recognition, and further indicate that the thrombin binding site for GPIb overlaps with the thrombin binding sites for fibrin and thrombomodulin, whereas it is distinct from the thrombin binding site for protein C. Some of the structural requirements for thrombin binding to GPIb appear to be very similar to those reported for binding to its platelet receptor. However, thrombin-GPIb interaction does not appear to compete with receptor hydrolysis but rather increases the sensitivity and the rate of platelet responses elicited by the receptor.

Amino Acid Sequence↗
PubMed1984

Thrombin Metz: characterization of the dysfunctional thrombin derived from a variant of human prothrombin.

Thrombin Metz and normal thrombin, resulting from activation of the respective prothrombins by factor Xa in the presence of calcium, phospholipid, and factor Va, were purified by chromatography on sulfopropyl Sephadex. By physicochemical criteria, thrombin Metz is identical to normal thrombin. Its functional properties were investigated in some reactions in which thrombin is classically involved. Thrombin Metz exhibits less than 4% of fibrinogen clotting activity. Both Km and Kcat, determined on S2238, are abnormal. Titration with the high-affinity competitive inhibitor of thrombin, DAPA, shows that fluorescence enhancement of the probe is only 34% in binding to thrombin Metz when compared to that observed in binding to normal thrombin. High-performance liquid chromatography has been used to measure the simultaneous rate of release of fibrinopeptides A and B. A decreased release rate for both fibrinopeptides, more marked for fibrinopeptide B, results in a slow fibrin polymerization, as followed by absorbance at 450 nm. Thrombin Metz is less than 5% as effective as normal thrombin in inducing platelet aggregation. Interaction with antithrombin III is slower than normal when followed by SDS gel electrophoresis and inhibition of the amidolytic activity of thrombin on S2238. This abnormality is not observed in the presence of heparin. However, thrombin Metz binds less tightly to a heparin-Sepharose column, and the direct inhibition of heparin on its activity on S2238 is weaker. From these results, we can predict that the defect in thrombin Metz affects the catalytic site or its vicinity and, jointly or consequently, the region of interaction of thrombin with antithrombin III and heparin.

Antithrombin III↗
PubMed1994

Thrombin-receptor agonist peptides, in contrast to thrombin itself, are not full agonists for activation and signal transduction in human platelets in the absence of platelet-derived secondary mediators.

Synthetic thrombin receptor peptides (TRPs), comprising the first 6-14 amino acids of the new N-terminus tethered ligand of the thrombin receptor that is generated by thrombin's proteolytic activity, were reported to activate platelets equally with thrombin itself and are considered to be full agonists [Vu et al. (1991) Cell 64, 1057-1068]. Using aspirin plus ADP-scavengers or the ADP-receptor antagonist adenosine 5'-[alpha-thio]triphosphate to prevent the secondary effects of the potent agonists that are normally released from stimulated platelets (i.e. ADP and thromboxane A2), we assessed the direct actions of thrombin and TRPs (i.e. TRP42-47 and TRP42-55). Compared with thrombin, under these conditions, TRPs: (1) failed to aggregate platelets completely; (2) produced less activation of glycoprotein (GP)IIb-IIIa; (3) did not cause association of GPIIb and pp60c-src with the cytoskeleton; and (4) caused less alpha-granule secretion, phosphorylation of cytoplasmic phospholipase A2, arachidonic acid release and phosphatidyl inositol (PtdOH) production. Furthermore, TRPs induced transient increases in protein phosphorylation mediated by protein kinase C and protein tyrosine phosphorylation, whereas these same responses to thrombin were greater and more sustained. Hirudin added after thrombin accelerated protein dephosphorylation, thereby mimicking the rate of spontaneous dephosphorylation seen after stimulation by TRPs. Platelets totally desensitized to very high concentrations of TRPs, by prior exposure to maximally effective concentrations of the peptides, remained responsive to alpha- and gamma-thrombins. Thrombin-stimulated PtdOH production in permeabilized platelets desensitized to TRPs was abolished by guanosine 5'-[beta-thio]diphosphate (GDP[beta S]), as in normal platelets. These results are discussed in terms of the allosteric Ternary Complex Model for G-protein linked receptors [Samama et al. (1993) J. Biol. Chem. 268, 4625-4636]. We conclude that: (1) TRPs are partial agonists for the thrombin receptor and produce incomplete receptor desensitization in keeping with their lower intrinsic activity; (2) thrombin's effects in platelets, even in TRP-desensitized platelets, are entirely mediated through the recently cloned G-protein linked receptor, and (3) thrombin's ability to produce sustained signals, compared with TRPs, may require the continued progressive proteolytic activation of naive thrombin receptors.

Adenosine Diphosphate↗
PubMed1995

Thrombin, phorbol ester, and cAMP regulate thrombin receptor protein and mRNA expression by different pathways.

Human mesangial cells have been used to study the regulation of thrombin receptor protein and mRNA expression during cross-talk between different signal transduction pathways. Persistent activation of thrombin receptor by thrombin led to homologous down-regulation of thrombin receptor protein. However, thrombin receptor mRNA expression was not affected, suggesting that increased receptor degradation is responsible for homologous down-regulation. Chronic activation of protein kinase C by phorbol 12-myristate 13-acetate (PMA) and of adenylylcyclase by prostaglandin E1 (PGE1) resulted in heterologous down-regulation of thrombin receptor protein. In contrast to thrombin, PMA and PGE1 reduced in parallel thrombin receptor mRNA levels to 51% and 24% of control, respectively, indicating that heterologous down-regulation of thrombin receptor protein is, at least in part, due to inhibition of receptor mRNA expression. The mechanisms of heterologous down-regulation of thrombin receptor protein have been studied in detail and compared to homologous down-regulation. PMA-induced down-regulation was completely blocked by GF 109 203 X, an inhibitor of protein kinase C. However, the loss of thrombin receptor induced by thrombin was not prevented by GF 109 203 X, indicating that homologous regulation is not dependent on protein kinase C activation. The heterologous effect of PGE1 was mimicked by 8-bromo-cAMP, isobutylmethylxanthine, and forskolin, suggesting that an increase in intracellular cAMP level is involved in heterologous regulation. Interestingly, heterologous down-regulation induced by PGE1 seems not to require previous internalization of thrombin receptor. These data indicate that thrombin receptor protein and mRNA expression can be regulated in homologous and heterologous ways by different mechanisms.

Cells, Cultured↗
PubMed1985

Interaction of human alpha-thrombin and gamma-thrombin with antithrombin III, protein C and thrombomodulin.

Conversion of human alpha-thrombin to gamma-thrombin by limited proteolysis resulted in a decrease in the inactivation rate of the enzyme by antithrombin III. The second-order rate constants were similar but significantly different: 11 +/- 1.7 X 10(3) and 7 +/- 0.5 X 10(3) M-1 s-1 for alpha- and gamma-thrombin respectively. This difference is probably related to a slight change in reactivity of the catalytic site, rather than to a structural alteration of the recognition site for antithrombin III. The rate of protein C activation, measured in the absence of thrombomodulin, was greatly reduced by conversion of alpha-thrombin to gamma-thrombin. In addition, gamma-thrombin failed to displace alpha-thrombin from its complex with thrombomodulin, as demonstrated by measuring either the rate of protein C activation by thrombin-thrombomodulin, or the fibrinogen clotting activity of thrombin-thrombomodulin, in the presence of competing diisopropylphospho-thrombin. It is concluded that the recognition sites involved in protein-C-thrombin and thrombomodulin-thrombin interactions are both dramatically affected by the loss of peptide material occurring during the conversion of alpha-thrombin to gamma-thrombin and/or by the resulting conformational changes.

Animals↗
PubMed1989

Iatrogenic immunization with bovine thrombin: a mechanism for prolonged thrombin times after surgery.

Unexplained very-prolonged thrombin times (greater than 300 s) were found in plasma from four patients. Other coagulation variables were normal, and there was no history of coagulopathy. Mixing studies suggested the presence of thrombin inhibitors in patient plasma. Substitution of human thrombin for bovine thrombin in performing the thrombin time test resulted in normal clotting times, indicating that the inhibitory activity was directed primarily against bovine thrombin. Each patient had been treated with topical bovine thrombin during previous surgery. In the one patient with a preoperative thrombin time, the initial value was normal and prolongation began 16 days after surgery. An enzyme-linked immunoassay showed elevated levels of IgM or IgG antibodies to bovine thrombin in each patient tested. Affinity-purified antibodies to bovine thrombin from patient serum prolonged the thrombin time of normal plasma. These results suggest that iatrogenic immunization by intraoperative exposure to bovine thrombin is responsible for antibodies to bovine thrombin, which accounts for the prolonged thrombin times found in some patients after surgery.

Adolescent↗
PubMed2005

Stability of human thrombin produced from 11 ml of plasma using the thrombin processing device.

Autologous thrombin can be produced by activating the patient's own plasma. By adding calcium chloride (CaCl2) to the anticoagulated plasma, the coagulation cascade will be initiated, and active thrombin will be produced. However, thrombin obtained by this method degrades very quickly and is not practical for use during surgery. The aim of this study was to investigate the stability of the thrombin produced using the thrombin processing device (TPD; Thermogenesis Corporation). The TPD consists of a tubular chamber containing a negatively charged surface for activation. Plasma (11 ml) and reagent (CaCl2 and ethanol, 3.75 ml) were added to the TPD, and active thrombin was harvested after a 20-minute incubation. The production of thrombin was done at 18 degrees C (64 degrees F), 24 degrees C (75 degrees F), and 27 degrees C (81 degrees F) (n = 4/group). The produced thrombin was stored at the production temperature, 4 degrees C (39 degrees F), and 35 degrees C (95 degrees F). The thrombin activity was assessed by time to clot formation, using a fibrinogen concentrate as substrate, after 2, 4, and 6 hours of storage. Thrombin produced at 18 degrees C had clot times of less than 5 seconds for 2 hours (4.42 +/- 1.3 seconds) when stored at 4 degrees C, but 4 hours (4.1 +/- 1.3 seconds) when stored at 35 degrees C. In contrast, when thrombin was produced at 24 degrees C, the clot times were 4.3 +/- 0.7 and 4.6 +/- 1.6 seconds at 4 degrees C and 35 degrees C, respectively, for up to 6 hours. Similar results were obtained for thrombin produced at 27 degrees C. Active thrombin produced by the TPD is dependent on both the production temperature and the storage temperature. Autologous human thrombin with a stability of up to 6 hours can be obtained using the TPD when produced at 24 degrees C or 27 degrees C and stored at 4 degrees C.

Blood Coagulation Tests↗
PubMed1991

"Thrombin" receptor-directed ligand accounts for activation by thrombin of platelet phospholipase C and accumulation of 3-phosphorylated phosphoinositides.

Using three experimental approaches, we have addressed the questions of whether the presence of saturably bound thrombin plays a role in potentiating the activation of platelet phospholipase C (PLC) and/or accumulation of the 3-phosphorylated phosphoinositides (3-PPI), i.e. phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate, and whether the generation of tethered ligand (Vu, T-K.H., Hung, D. T., Wheaton, V. I., and Coughlin, S. R. (1991) Cell 64, 1057-1068) by thrombin can account fully for thrombin's proteolytic effects in activating platelets, as gauged by the above parameters. We have 1) measured PLC activation or 3-PPI after we have exposed platelets to thrombin for various periods and either blocked thrombin's proteolytic activity without interrupting its binding or blocked both binding and proteolytic activity of thrombin; 2) attempted to potentiate 3-PPI accumulation, using combinations of protein kinase C stimulation, Ca2+ elevation, and saturating but proteolytically inactive thrombins; and 3) compared the activation of platelets by thrombin with activation by the "thrombin" receptor-directed peptide, SFLLRNPNDKYEPF (SFLL; a portion of the tethered ligand created by thrombin's proteolytic activity), and examined the effect of thrombin on this latter activation. We conclude that the initial and sustained effects of thrombin in stimulating PLC and the accumulation of 3-PPI are completely attributable to thrombin's proteolytic activity. Further, thrombin's effects in promoting these responses can be accounted for by the actions of SFLL peptide, and by implication, formation of tethered ligand.

Amino Acid Sequence↗
PubMed1988

Effect of thrombin inhibitors on thrombin-induced platelet release and aggregation.

Thrombin-induced platelet activation was interrupted with hirudin or Dansylarginine N-(3-ethyl-1-5-pentanediyl) amide (DAPA) to study the time requirement for receptor occupancy by thrombin in promoting platelet responses at low (0.25 U/ml), intermediate (0.5 U/ml) and high (1 U/ml) thrombin concentrations. Each of these thrombin inhibitors suppressed adenosine triphosphate (ATP) release and aggregation by thrombin when added either before or simultaneously with thrombin or within seconds of the initiation of these responses by thrombin. If the inhibitors were added later, yet before aggregation or release was complete, no effect was present. The period of time for which active thrombin was required in order to promote these reactions had the following characteristics: (i) it is thrombin concentration dependent for a given response; (ii) it is longer for aggregation than for ATP secretion at each thrombin concentration; (iii) it is increased in platelets modified by chymotrypsin or platelets partially inhibited by antimycin A and 2-deoxy-D-glucose, which have prolonged aggregation and ATP release responses. In direct comparison studies, the inhibitory effects of hirudin and DAPA were identical on aggregation and ATP release. Thrombin binding, under similar experimental conditions identical to those used to measure platelet activation, was prevented by hirudin, but not by DAPA. Therefore, the effect of DAPA on thrombin must be at the proteolytic site region and not at the hirudin-inhibitable platelet binding region. It is concluded from these studies that the tight coupling requirements for thrombin to induce platelet dense granule release and aggregation are directly dependent upon both the thrombin concentration and the rate of the individual platelet responses. Catalytic site integrity is required for the duration of this period of receptor occupancy.

Adenosine Triphosphate↗
PubMed1998

Activated human protein C prevents thrombin-induced thromboembolism in mice. Evidence that activated protein c reduces intravascular fibrin accumulation through the inhibition of additional thrombin generation.

Activated protein C (APC) is a potent physiologic anticoagulant with profibrinolytic properties, and has been shown to prevent thrombosis in different experimental models. We investigated the effect of human APC on thrombin-induced thromboembolism in mice, a model of acute intravascular fibrin deposition leading to death within minutes. APC given intravenously (i.v.) as a bolus 2 min before thrombin challenge (1,250 U/kg) reduced mortality in a dose-dependent manner despite the lack of thrombin inhibitor activity. Significant inhibition of thrombin-induced death was observed at the dose of 0.05 mg/kg, and maximal protection was obtained with 2 mg/kg (> 85% reduction in mortality rate). Histology of lung tissue revealed that APC treatment (2 mg/kg) reduced significantly vascular occlusion rate (from 89.2 to 46.6%, P < 0.01). The protective effect of APC was due to the inhibition of endogenous thrombin formation as indicated by the fact that (a) the injection of human thrombin caused a marked decrease in the coagulation factors of the intrinsic and common pathways (but not of Factor VII), suggesting the activation of blood clotting via the contact system; (b) APC pretreatment reduced markedly prothrombin consumption; (c) the lethal effect of thrombin was almost abolished when the animals were made deficient in vitamin K-dependent factors by warfarin treatment, and could be restored only by doubling the dose of thrombin, indicating that the generation of endogenous thrombin contributes significantly to death; and (d) APC failed to protect warfarin-treated animals, in which mortality is entirely due to injected thrombin, even after protein S supplementation. Other results suggest that APC protects from thrombin-induced thromboembolism by rendering the formed fibrin more susceptible to plasmin degradation rather than by reducing fibrin formation: in thrombin-treated mice, fibrinogen consumption was not inhibited by APC; and inhibition of endogenous fibrinolysis by epsilon-aminocaproic or tranexamic acid resulted in a significant reduction of the protective effect of APC. Since APC did not enhance plasma fibrinolytic activity, as assessed by the measurement of plasminogen activator (PA) or PA inhibitor (PAI) activities, PAI-1 antigen, or 125I-fibrin degrading activity, we speculate that the inhibition of additional (endogenous) thrombin formation by APC interrupts thrombin-dependent mechanisms that make fibrin clots more resistant to lysis, so that the intravascular deposited fibrin can be removed more rapidly by the endogenous fibrinolytic system.

Animals↗
PubMed2001

Identification of anti-thrombin antibodies in the antiphospholipid syndrome that interfere with the inactivation of thrombin by antithrombin.

The combined presence of anti-phospholipid (PL) Ab, including lupus anticoagulants (LAC) and/or anticardiolipin Ab (aCL), and thrombosis is recognized as the antiphospholipid syndrome (APS). LAC are detected as an inhibitory effect on PL-restricted in vitro blood coagulation tests, and are comprised mainly of Ab against beta(2) glycoprotein I and prothrombin (PT). Recently, anti-PT Ab (aPT) were found to be associated with thrombosis by some investigators, although this is not confirmed by others. Considering that aPT are heterogeneous in patients and that PT is converted into thrombin, we hypothesize that certain aPT in patients may bind to thrombin, and that some of such anti-thrombin Ab may interfere with thrombin-antithrombin (AT) interaction and thus reduce the AT inactivation of thrombin. To test this hypothesis, we searched for anti-thrombin Ab in APS patients and then studied those found for their effects on the AT inactivation of thrombin. The results revealed that most, but not all, aPT-positive patient plasma samples contained anti-thrombin Ab. To study the functional significance of these Ab, we identified six patient-derived mAb that bound to both PT and thrombin. Of these mAb, three could reduce the AT inactivation of thrombin, whereas others had minimal effect. These findings indicate that some aPT in patients react with thrombin, and that some of such anti-thrombin Ab could inhibit feedback regulation of thrombin. Because the latter anti-thrombin Ab are likely to promote clotting, it will be important to develop specific assays for such Ab and study their roles in thrombosis in APS patients.

Adolescent↗