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PubMed1983

Adhesion and aggregation of thrombin prestimulated human platelets on surface-bound fibrinogen: evidence for involvement of ADP and arachidonic acid pathways.

The purpose of this study was to examine the influence of substances released from human platelets upon their accumulation on human fibrinogen-coated glass tubes. After prestimulation with thrombin for one minute or in the absence of prestimulation, washed human platelets suspended in Eagle's medium with RBC were drawn through the tubes at 1 ml/min, 80 s-1, for 1, 2 or 6 min. Thrombin prestimulation (0.02, 0.05 or 0.25 U/ml) was followed by inactivation with hirudin (0.1, 0.25 or 1.25 U/ml) before flow. Singly adherent platelets were observed in the absence of thrombin or with thrombin for exposure times of 1 and 2 min. At 6 min after at least 0.05 U/ml of thrombin, surface-bound aggregates were observed. The initial rate of adhesion increased with the amount of thrombin used for prestimulation. For adhesion to fibrinogen in the absence of prestimulation, platelet-derived ADP was a stimulator. Adhesion was shown to be independent of the ADP and arachidonic acid pathways in response to prestimulation with a low level of thrombin, 0.02 U/ml. For adhesion and cohesion, aggregation, in the presence of sufficient thrombin for prestimulation, 0.05 U/ml, ADP, serotonin and substances from arachidonic acid metabolism acted jointly to stimulate platelets.

Adenosine Diphosphate↗
PubMed1980

Localizaton of fibrin converted by thrombin from released platelet fibrinogen.

Treatment of platelets (10(9) cells/ml) with thrombin (1 U/ml) resulted in rapid disappearance of fibrinogen from the system as measured by the tanned red cell hemagglutination inhibition immunoassay (TRCHII). Plasmin digestion of individual pellet and supernatant fractions that had been previously separated from thrombin-treated platelet suspensions by centrifugation resulted in recovery of TRCHII-detectable material in platelet pellets. To elucidate the specific association of fibrin to platelet membranes, control and thrombin-treated platelets were homogenized by a modified glycerol-loading and nitrogen decompression technique. Ultracentrifugation of homogenates through 27% sucrose cushions yielded three subcellular fractions: supernatant, small membrane vesicles, and a particulate fraction for controls; and supernatant membrane vesicles, and aggregated membrane "ghosts" for thrombin preparations. Ultrastructurally identifiable fibrin was noted only in the thrombin fraction containing membrane ghosts. Fibrinogen recovered from 3 thrombin fractions was markedly decreased (3% of the control). Plasmin digestion produced 23% and 46-fold increase in TRCHII-detectable material from 3 subcellular fractions of control and thrombin preparations, respectively. More than 97% of TRCHII material recovered from thrombin preparations was in the fraction containing aggregated membrane fractions. Results suggest that platelet plasma membranes function as surfaces for fibrin deposition.

Blood Platelets↗
PubMed1984

[Kininogenase activity of alpha and beta/gamma forms of bovine thrombin].

The kininogenase activity of alpha- and beta/gamma-forms of bovine thrombin with respect to the high molecular weight (HMW) and low molecular weight (LMW) human kininogens was studied. It was shown that both forms of the enzyme split of bradykinin from these kininogens. The kininogenase activity of alpha-thrombin is completely blocked by the highly specific thrombin inhibitor Nalpha-dansyl-L-arginine-p-ethylpiperidineamide, but not by the soya bean trypsin inhibitor. The alpha- and beta/gamma-forms of thrombin hydrolyze HMW (Km(app) = 4.5 and 3.3 microM, respectively) and LMW (Km(app) = 10.1 and 4.7 microM, respectively). The specific constants (kcat/Km(app) ) for thrombin with respect to the substrates differ about 7-fold, predominantly due to the high catalytic rates of HMW as compared to LMW; the kcat values are 0.18 and 0.06 min-1, respectively. alpha-Thrombin upon a long-term (over 1 hour) exposure to HMW, besides bradykinin, splits off the product inhibiting the kininogenase activity of thrombin. No differences in the specificity of the beta/gamma-form of thrombin with resect to HMW and LMW were detected.

Animals↗
PubMed1981

Antigenic changes produced by complex formation between thrombin and antithrombin-III.

Immunization of goats and mules with human thrombin resulted in an antiserum that reacted only weakly with the parent molecule, prothrombin. Some of the antibodies in this antiserum showed a greater affinity for thrombin complexed to its naturally occurring inhibitor, antithrombin-III, than for active thrombin. An antiserum against the human thrombin inhibitor, antithrombin-III, produced 2 precipitin lines against human serum but only 1 against plasma. The 2nd line in serum was shown to represent precipitation of a complex of thrombin with antithrombin-III. The neoantigens appearing in antithrombin-III after complex formation were also present in complexes prepared with purified clotting factor Xa and antithrombin-III. Since purified host (mule) thrombin was also capable of causing formation of the neoantigenic sites when complexed to human antithrombin-III, it seems likely that these determinants result from interaction in the host between the immunogens (either human thrombin or antithrombin) and the appropriate interacting host protein (mule antithrombin-III or thrombin, respectively). Studies by radioimmunoassay showed that the antibodies formed are not completely specific for the neoantigens since they also react to a lesser extent with the free proteins.

Animals↗
PubMed1982

The use of the synthetic peptide, Gly-Pro-Arg-Pro, in the preparation of thrombin-degranulated rabbit platelets.

The method for preparing thrombin-degranulated platelets has been modified to avoid the use of plasmin or successive treatments with small amounts of thrombin, while still achieving more than 90% release of platelet amine storage granule contents. It was necessary to prevent the fibrinogen released from the platelets during thrombin treatment from forming an insoluble fibrin mesh that could trap the platelets and hinder their deaggregation. To accomplish this we have treated rabbit platelets with 0.73 U/ml of thrombin for 1 min in the presence of the synthetic peptide, Gly-Pro-Arg-Pro, which prevents the polymerization of fibrin molecules. We have demonstrated that it also prevents 125I, initially added as 125I-fibrinogen, from associating with the platelets in a form that was not removed by centrifuging and washing during the preparation of thrombin-degranulated platelets, and we infer that products formed from the fibrinogen released from the platelets would also be prevented from associating with them. Thrombin-degranulated platelets prepared by this method have lost 92% of their granule contents and they can be washed and resuspended. These platelets aggregate normally upon stimulation with thrombin, adenosine diphosphate (ADP), or arachidonate. Thus, Gly-Pro-Arg-Pro is useful in preparing thrombin-degranulated platelets for studying platelet reactions without the complicating effects of released materials such a ADP and fibrinogen.

Animals↗
PubMed1982

Effect of thrombin on isolated canine blood vessels.

The aim of the present study was to assess whether or not thrombin affects vascular smooth muscle activity by a direct action on the blood vessel wall. In isolated saphenous veins and femoral and pulmonary arteries thrombin did not affect basal tension, but in pulmonary and portal mesenteric veins it caused contraction. In saphenous and femoral veins and in femoral and pulmonary arteries the enzyme depressed contractions evoked by alpha-adrenergic stimulation. In the saphenous vein thrombin also depressed contractions induced with acetylcholine or potassium ions. In this blood vessel the relaxation caused by thrombin was abolished by antithrombin III and the tripeptide D-phenylalanine-proline-arginine-CH2Cl, a specific inhibitor of the enzyme center of thrombin. In pulmonary and portal mesenteric veins, thrombin potentiated the contractile response to norepinephrine. These contractile effects of thrombin were also abolished by D-phenylalanine-proline-arginine-CH2Cl. Thus, thrombin causes relaxation or contraction of blood vessels through a direct action on the blood vessel wall. These effects are due to its proteolytic activity.

Acetylcholine↗
PubMed1982

Effect of membrane cholesterol on phospholipid metabolism in thrombin-stimulated platelets. Enhanced activation of platelet phospholipase(s) for liberation of arachidonic acid.

The cholesterol to phospholipid mole ratio (C/PL) of human platelets was increased 1.3-fold or maintained at a normal value by incubating platelets with sonicated dispersions of cholesterol and phosphatidylcholine (PC) (C/PL = 3 or 1, respectively). Thrombin-induced mobilization of [3H]arachidonic acid from prelabeled phospholipids and subsequent formation of labeled cyclo-oxygenase and lipoxygenase products were increased in cholesterol-enriched platelets as a function of thrombin concentration. Elevated platelet cholesterol content affected thrombin-induced changes in platelet phospholipids: (a) hydrolysis of PC was more sensitive to thrombin and was markedly enhanced over a wide range of thrombin concentrations (0.1-2 units/ml); (b) hydrolysis of phosphatidylinositol (PI) was increased at thrombin concentrations greater than or equal to 0.2 unit/ml. Increased metabolism of [3H]arachidonic acid in stimulated cholesterol-enriched platelets was due to loss of [3H]arachidonate from PC at 0.1 unit/ml of thrombin. At higher thrombin concentrations (0.2-2 units/ml) it reflected enhanced hydrolysis of predominantly PC, but also PI. We conclude that cholesterol, possibly through its effect on platelet lipid organization, influences arachidonic acid metabolism in stimulated plates by promoting enhanced activity of platelet phospholipase(s) for liberation of arachidonic acid.

Arachidonic Acids↗
PubMed1980

Chemically modified bovine prothrombin as a substrate in studies of activation kinetics and fluorescence changes during thrombin formation.

The activation of bovine prothrombin is known to be accompanied in purified systems by proteolytic reactions catalyzed by the product, thrombin. These reactions, which are directed principally towards the prothrombin substrate and the Factor V cofactor, are eliminated if the lysine residues of prothrombin are chemically modified beforehand with methyl acetimidate. Amidinated prothrombin in which the usual lysine content has been reduced by 75% is cleaved completely by Factor Xa to give thrombin which has little or no activity towards fibrinogen, the thrombin-sensitive bond in prothrombin, or Factor V, but with normal activity towards the synthetic chromogenic substrate D-Phe-Pipecolyl-L-Arg-p-nitroanilide. The formation of thrombin could therefore be studied spectrophotometrically by discontinuous assays of thrombin without complication by the proteolytic feedback activity of this enzyme. Such assays showed that the rate of appearance and yield of thrombin is the same whether from native or amidinated prothrombin, in spite of the lack of proteolytic activity in the product from the latter. Native and amidinated prothrombin have identical fluorescence emission spectra (lambda ex = 280 nm) which, upon activation, show a broadening and a red shift of the peak of emission from 330 to 336 nm. This change is different from the quenching known to occur when prothrombin binds Ca2+ and is contingent upon cleavage by Factor Xa. When monitored at the 370 nm band, the shift is seen as an increase in fluroescence intensity which, when the concentrations of Factor Xa and Factor V are adjusted appropriately, has the same general appearance as a progress curve obtained by discontinuous assay of thrombin activity. However, the curves obtained with the native zymogen appear to contain a component due to proteolysis by the accumulating product. in the case of amidinated zymogen, this is not longer so: the curves reflect only proteolysis by Factor Xa. In addition, a comparison of the fluorescence shift with the time course of thrombin appearance shows that the shift results mainly from the cleavage of intact prothrombin by Factor Xa, with little or no contribution from later events in the activation pathway. Modified Stern-Volmer plots for the quenching of fluorescence (lambda ex = 295 nm) of the intact amidinated zymogen and its activation products by sodium iodide allow the conclusion that activation results in the exposure to solvent of tryptophan residues that were previously sheltered. However, there is a variation in the pattern of quenching with the protein concentration, suggesting that these residues may be sheltered in the zymogen by intermolecular rather than intramolecular interactions.

Animals↗
PubMed1993

Kinetics of inactivation of alpha-thrombin by plasminogen activator inhibitor-1. Comparison of the effects of native and urea-treated forms of vitronectin.

Kinetic studies are presented which show that native human vitronectin, but not urea-treated vitronectin, accelerates the inactivation of human alpha-thrombin by human plasminogen activator inhibitor-1 (PAI-1). We demonstrate that although urea-treated vitronectin binds PAI-1 with an affinity greater than that of native vitronectin, it does not accelerate the rate of inactivation of alpha-thrombin by PAI-1. We present evidence to suggest that the inability of urea-treated vitronectin to accelerate the reaction between alpha-thrombin and PAI-1 results at least in part from the inability of urea-treated vitronectin to bind to alpha-thrombin. The accelerated reaction between PAI-1 and alpha-thrombin can be accounted for by the formation of a tight complex between native vitronectin and PAI-1 that reacts in a saturable manner (Kd = 75 nM) with alpha-thrombin. The second-order rate constant (kI/Kd) for the reaction of the native vitronectin-PAI-1 complex with alpha-thrombin (1.64 x 10(5) M(-)-1 s-1) is 270-fold greater than the second-order rate constant for the reaction in the absence of vitronectin (610 m-1 s-1). The increase in the second-order rate constant is largely due to an increase in the affinity of alpha-thrombin for the native vitronectin-PAI-1 complex, as reflected by a greater than 25-fold reduction in the dissociation constant (Kd) observed for the vitronectin-PAI-1 complex relative to that of uncomplexed PAI-1.

Blood Proteins↗
PubMed1995

Limited proteolysis of human alpha-thrombin by urokinase yields a non-clotting enzyme.

Limited proteolysis of human alpha-thrombin by various proteases has been efficiently used to demonstrate the importance of two insertion loops located on the surface of this molecule. In the present study, we demonstrate that two-chain urokinase (tcu-PA) specifically cleaves the B chain of alpha-thrombin giving rise to a transient derivative, consisting of two non-covalently linked subunits. Although the thrombin derivative conserves its activity towards the synthetic substrate S-2238 (Km = 8.4 microM and kcat = 145 s-1 versus respectively 4.5 microM and 149 s-1 for alpha-thrombin), most of its coagulant activity is lost (140 NIH u/mg versus 3000 NIH u/mg) and its ability to activate platelets is considerably reduced (threshold for full platelet aggregation 2.5 nM versus 0.25 nM). The thrombin fragments were separated by HPLC and after reduction and S-carboxyamidemethylation were digested with a lysylendopeptidase; the resulting peptides were separated by HPLC and sequenced. One fragment corresponded to B chain fragment 1-73 and the second to B chain fragment 74-259 covalently linked to the A chain, indicating that tcu-PA cleaves selectively the peptide bond Arg 73-Asn 74 in the B chain. The proteolytic derivative obtained, designated beta u-thrombin, is therefore identical to the transient proteolytic derivative, beta 1-thrombin, produced by trypsin. Prolonged incubation with tcu-PA resulted in further conversion in a derivative analogous to gamma t-thrombin.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

Displacement of fibrin-bound thrombin by r-hirudin precludes the use of 131I-r-hirudin for detecting pulmonary emboli in the rabbit.

Pulmonary emboli are detectable by filling defects in the pulmonary vasculature upon pulmonary angiography. Emboli derived from venous thrombi are rich in fibrin to which thrombin remains bound. Hirudin, a specific thrombin inhibitor, binds to thrombin to yield a 1:1 stoichiometric complex. We examined whether 131I-recombinant hirudin (r-hirudin) could be used to detect pulmonary emboli in rabbits. Clots were formed by re-calcifying rabbit plasma in vitro, and then injected (0.034 ml) into a femoral vein to lodge in the lungs. 131I-r-hirudin (29 +/- 4 microCi/kg) was injected intravenously but emboli could not be detected by gamma camera in real time. Post-mortem analysis of lung tissue showed that 131I-r-hirudin did not associate with emboli prepared with 125I-fibrin. Because of these findings, we used different techniques to look at the binding of hirudin to plasma clots. Clots formed in vitro were incubated with 131I-r-hirudin in the presence of equimolar amounts of 125I-albumin; specific binding of 131I-r-hirudin was not observed. Experiments with immobilized fibrin(ogen) showed that 125I-r-hirudin did not bind to and remain with fibrin-bound 131I-thrombin but did lead to the inactivation and displacement of up to 70% of bound thrombin as r-hirudin-thrombin complex; residual thrombin bound to fibrin remained active. Thus, released r-hirudin-thrombin complex is probably cleared rapidly from the region of the embolus in vivo; radioiodinated r-hirudin may not, therefore, be useful as a marker for detecting emboli.

Animals↗
PubMedDate not supplied

[The vascular effects of thrombin on canine and human arteries; their independence from the metabolism of arachidonic acid].

Independently of it's effects on the coagulation cascade, thrombin can interact with the endothelium and release vasodilatory mediators as prostacyclin, endothelium dependent relaxing factor and potentiate the vascular changes induced by vasoconstrictors like endothelin or cathecolamines. Therefore, in the present study we tested the effect of thrombin in the pulmonary and femoral canine arteries and compared it with the effects on human umbilical artery; we also explore the possible mechanism of action of thrombin-induced changes in vascular tone by using specific inhibitors. Thrombin induced a concentration-dependent and endothelium-dependent relaxation on canine arteries (pulmonary or femoral) and endothelium-independent contraction of human umbilical arteries, neither the relaxation nor the contraction were significantly affected by incubation of the vessels with: a cyclooxygenase inhibitor (indomethacin), lypooxygenase inhibitor (BW 755C) or a soup of antagonists (atropine, metysergyde, propanolol, meperamine or phenoribenzamine) to block muscarinic, histaminic, serotoninergic or adrenergic receptors. However, incubation of the vessels with heparin or a calcium channel blocker did prevented the vasoconstrictor effect of thrombin in human umbilical veins. This results suggests that thrombin can elicit changes in vascular tone and the effect is dependent of the vessel stimulated, and the presence of the endothelium. Thus, thrombin-dependent change in vascular tone is not mediated by arachidonic acid metabolites, sympathetic or parasympathetic neurotransmitters, histamine or serotonine receptors. Thrombin effects may be mediated by interaction with an specific receptor coupled with a calcium signal.

Animals↗
PubMed1993

Fibrinogen Mitaka II: a hereditary dysfibrinogen with defective thrombin binding caused by an A alpha Glu-11 to Gly substitution.

A new type of A alpha Glu-11 to Gly substitution has been identified in a congenitally abnormal fibrinogen, fibrinogen Mitaka II, derived from a 14-year-old female suffering from easy bruising since childhood. Plasma of the patient and fibrinogen purified therefrom were found to clot slowly by thrombin but in a normal fashion by ancrod, a thrombin-like snake venom enzyme. The ancrod-clotted fibrin gels were normally solid and turbid, whereas the thrombin-clotted gels were initially fragile and transparent but became gradually normalized during further incubation. On reverse-phase high-performance liquid chromatography, there was an additional peptide group eluted distinctly later than the corresponding normal fibrinopeptide A in the clot-liquor of the patient's samples. Sequence analysis of these aberrant peptides and isolated A alpha chains of the patient's fibrinogen showed that Glu at position 11 of the abnormal A alpha chain had been replaced by Gly. Studies using 125I-labeled thrombin showed that the binding with thrombin was evidently reduced for her fibrinogen and the aberrant fibrinopeptide A as compared with that for the normal controls, indicating that A alpha Glu-11 may be critical for the fibrinogen-thrombin interaction. Indeed, A alpha Glu-11 of fibrinogen has recently been proposed to stabilize the local conformation, including the beta-turn, and to form a salt bridge between its side-chain carboxyl group and the guanidino group of Arg-173 of thrombin based on crystallographic analyses using analogs of fibrinopeptide A complexed with thrombin (Stubb et al, Eur J Biochem 206:187, 1992 and Martin et al, J Biol Chem 267:7911, 1992).

Adolescent↗
PubMedDate not supplied

Design and synthesis of a kininogen-based selective inhibitor of thrombin-induced platelet aggregation.

Thrombin-induced platelet aggregation has been suggested to play an important role in reocclusion following thrombolytic therapy or angioplasty for treatment of myocardial infarction. We previously demonstrated that thrombin-induced platelet aggregation is indirectly mediated by intracellularly activated calpain expressed on the platelet surface through the cleavage of aggregin, a putative ADP-receptor, and that high molecular weight kininogen (HK), a naturally occurring thiol protease inhibitor, modulates thrombin-induced platelet aggregation. Considering the substrate specificity of calpain and the conserved sequence in HK, we studied selective inhibitors of thrombin-induced platelet aggregation by the affinity labeling approach with an S-3-nitro-2-pyridinesulfenyl (Npys) group. H-Phe-Gln-Val-Val-Cys (Npys)-Gly-NH2, which combines chemical and structural features of calpain substrate specificity and the conserved sequence in HK, selectively inhibited thrombin-induced platelet aggregation. It did not inhibit the aggregatory effects of other platelet agonists, and did not inhibit amidolytic activity of thrombin and thrombin-induced platelet shape change. The design and synthesis of such inhibitors could lead to the development of a new class of inhibitors that selectively block thrombin-induced platelet aggregation.

Amino Acid Sequence↗
PubMed1994

Radiolabeled r-hirudin as a measure of thrombin activity at, or within, the rabbit aorta wall in vitro and in vivo.

The behavior of 125I-labeled recombinant hirudin towards the uninjured and de-endothelialized rabbit aorta wall has been studied in vitro and in vivo to determine its usefulness as an indicator of thrombin activity associated with the aorta wall. Thrombin adsorbed to either sulfopropyl-Sephadex or heparin-Sepharose bound > 95% of 125I-r-hirudin and the complex remained bound to the matrix. Binding of 125I-r-hirudin to the exposed aorta subendothelium (intima-media) in vitro was increased substantially if the tissue was pre-treated with thrombin; the quantity of 125I-r-hirudin bound to the de-endothelialized intima-media (i.e. balloon-injured in vitro) correlated positively with the quantity of bound 131I-thrombin (p < 0.01). Aortas balloon-injured in vivo were measured for thrombin release from, and binding of 125I-r-hirudin to, the de-endothelialized intimal surface in vitro; 125I-r-hirudin binding correlated with the amount of active thrombin released (p < 0.001). Uptake of 125I-r-hirudin by the aorta wall in vivo was proportional to the uptake of 131I-fibrinogen (as an indicator of thrombin activity) before and after balloon injury. After 30 min in the circulation, specific 125I-r-hirudin binding to the uninjured and de-endothelialized (at 1.5 h after injury) aorta wall was equivalent to 3.4 (+/- 2.5) and 25.6 (+/- 18.1) fmol of thrombin/cm2 of intima-media, respectively. Possibly, only hirudin-accessible, glycosaminoglycan-bound thrombin is measured in this way.

Animals↗
PubMed1994

Dermatan sulfate is a more potent inhibitor of clot-bound thrombin than unfractionated and low molecular weight heparins.

Clot-bound thrombin proteolyses fibrinogen and amplifies the coagulation cascade at its close vicinity, thereby ensuring the growth of fibrin-rich thrombus. The present study compares the ability of various glycosaminoglycans (GAGs) to inhibit these 2 properties. Unfractionated heparin (UH), 3 low molecular weight heparins (LMWHs) with increasing antifactor Xa/antifactor IIa ratio, the synthetic pentasaccharide (PS), devoid of antifactor IIa activity, and dermatan sulfate (DS), a catalyst of thrombin inhibition by heparin cofactor II, were selected on the basis of their different properties. Proteolysis of fibrinogen by clot-bound thrombin was evaluated by measuring fibrinopeptide A (FPA) generation after an incubation of standardized washed clots in plasma for 120 min in absence or in presence of increasing concentrations of heparins or of DS. The results were compared to those obtained when free alpha-thrombin (0.4 nM) was added to plasma in the same experimental conditions. On the basis of equivalent antithrombin units, UH and LMWHs gave identical results. To inhibit by 70% fibrinogen proteolysis induced by clot-bound thrombin (IC 70), 5- to 9-fold higher concentrations of UH or of LMWHs were required in comparison with those required to inhibit free thrombin. For DS, only a 1.3 times higher concentration was required. PS (final concentration 1 anti Xa U.ml-1) was devoid of any inhibitory effect. The amplification of the coagulation cascade induced by clot-bound thrombin was evaluated by measuring the shortening of whole blood clotting time (WBCT) resulting from the incubation of washed clots in native blood. In absence of GAG, clot-bound thrombin reduced WBCT from 18 +/- 2 min to 9 +/- 1 min.(ABSTRACT TRUNCATED AT 250 WORDS)

Dermatan Sulfate↗
PubMed1994

[Regulation of the endothelial function by thrombomodulin and/or thrombin receptor].

Thrombin, the final product of blood coagulation cascade, shows several effect on the vessel-wall cells. However the effects may be regulated by several thrombin receptors on the endothelium. They include thrombomodulin (TM), protease-Nexin, heparin-like molecule-antithrombin III complex. These binding sites do not transduce the signal of thrombin. Especially TM converts thrombin from a procoagulant protease to an anticoagulant. Recently new thrombin receptor was identified on the endothelium and platelets. Through this receptor, thrombin induces activations both on platelet end-endothelium. In brief platelets aggregate and release several factors including serotonin, PDGF, platelet factor4, beta-thromboglobulin on the stimulation by thrombin. The endothelium release t-PA inhibitor; PAI-1, prostacyclin and endothelin. Thus the activations of these cells by thrombin is a key events in hemostasis, wound healing, inflammation, atherosclerosis and restenosis of coronary artery after PTCA.

Blood Platelets↗
PubMed1994

Occupancy of anion binding exosite 2 on thrombin determines Ca2+ dependence of protein C activation.

Thrombomodulin (TM) binds thrombin to form a complex that activates the plasma anticoagulant zymogen protein C. TM is an integral membrane glycoprotein that contains a chondroitin sulfate moiety. Interaction with thrombin involves both the protein component of TM, specifically the growth factor-like repeats 4-6 (TM 4-6), and chondroitin sulfate. Removal of chondroitin sulfate decreases the affinity for thrombin approximately 10-fold and shifts the Ca2+ dependence of protein C activation from simple saturation at > or = 500 microM Ca2+ to a distinct optimum at approximately 100 microM Ca2+. Thrombin possesses two regions of high positive charge, anion binding exosites 1 and 2. Anion binding exosite 1 interacts with the growth factor region of TM while exosite 2 is involved in binding prothrombin activation fragment 2 or heparin. We demonstrate that recombinant TM, truncated at the membrane-spanning domain, or TM 4-6 can bind thrombin when fragment 2 is present either covalently attached (meizothrombin des-fragment 1) or in reversible association. With meizothrombin des-fragment 1, the Ca2+ dependence of protein C activation is independent of the presence of the chondroitin sulfate on TM. At 0.27 mM Ca2+, TM containing chondroitin sulfate binds thrombin (Kd(app) = 0.3 nM) approximately 45 times tighter than meizothrombin des-fragment 1 (Kd(app) = 14 nM). However, the chondroitin-free form binds thrombin (Kd(app) = 2.4 nM) only approximately 4 times tighter than meizothrombin des-fragment 1 (Kd(app) = 9.4 nM). These studies suggest that occupancy of anion binding exosite 2 by either chondroitin sulfate or fragment 2 alters thrombin conformation resulting in the altered Ca2+ dependence of protein C activation.

Amino Acid Sequence↗