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PubMed1994

Effect of thrombin, the thrombin receptor activation peptide, and other mitogens on vascular smooth muscle cell urokinase receptor mRNA levels.

Bovine vascular smooth muscle cells (SMC) express the urokinase-type plasminogen activator receptor (u-PAR) claimed to be important in cell invasion. Receptor numbers and affinity are regulated by thrombin and several other mitogens involved in SMC proliferation. We investigated the effects of these mitogens on u-PAR mRNA levels. On continuous thrombin stimulation the u-PAR message in SMC was 10 +/- 2.3-fold elevated reaching a maximum between 6 and 9 hours and declining to control values within 48 hours. Thrombin present for 30 minutes on the cell surface produced similar effects. Stimulation with the thrombin receptor activation peptide S-F-L-L-R-N representing the NH2-terminus of the tethered ligand also increased u-PAR mRNA levels with an identical time course. D-Phe-Pro-Arg-chloromethyl ketone (PPACK) active site blocked thrombin and the catalytically inactive thrombin mutant S205A did not affect u-PAR mRNA levels. Thrombin stimulation also resulted in a 2 +/- 0.2-fold transient increase in thrombin receptor mRNA preceding the rise in u-PAR message. Transforming growth factor beta 1 (TGF beta 1) and platelet-derived growth factor (PDGF) showed similar time courses for the elevation of u-PAR mRNA levels with a maximal 5.5 +/- 0.9 and 12 +/- 2.5-fold increase, respectively. Basic fibroblast growth factor (bFGF) and phorbol myristate acetate (PMA) showed a more prolonged effect increasing u-PAR mRNA levels 8 +/- 2.0-fold and 12.3 +/- 2.5-fold, respectively, within 6 hours but remaining 5 to 10-fold elevated at 48 hours. In order to decide if the u-PAR mRNA increase was due to message stabilization or a consequence of transcriptional activation we used the RNA polymerase II inhibitor 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole (DRB) during the stimulation experiments. u-PAR mRNA levels on TGF beta 1 stimulation of SMC decayed after the addition of DRB indicating that enhancement of transcriptional activity was involved in the induction. In contrast, the time course of u-PAR mRNA elevation on thrombin, bFGF, and PMA stimulation was not significantly altered in the presence of DRB suggesting that in these latter cases u-PAR mRNA message accumulation was at least in part due to mRNA stabilization. Increased transcriptional activity, mRNA stabilization and expression of u-PAR protein on the SMC surface in response to growth factors may facilitate enhanced cell surface protease activity, cell migration, and development of atheromatous lesions.

Amino Acid Sequence↗
PubMed1997

Release of the thrombin receptor (PAR-1) N-terminus from the surface of human platelets activated by thrombin.

Platelet activation by thrombin is at least partially mediated by a G-protein-coupled receptor whose extended N-terminus is cleaved by thrombin. Theoretically, this should release a small fragment containing the original receptor N-terminus. However, the fate of this fragment is unknown, as is its biological role, if any. To begin to examine these issues, we have prepared monoclonal anti-receptor antibodies whose epitopes lie entirely N-terminal to the thrombin cleavage site. By flow cytometry and fluorescence microscopy of human platelets and megakaryoblastic CHRF-288 cells, these antibodies were found to recognize intact receptors and receptors activated by the agonist peptide, SFLLRN, but not receptors whose N-terminus had been cleaved by thrombin or cathepsin G. Incubating CHRF-288 cells with thrombin released pre-bound antibody from the cell surface. An assay based upon the antibodies was able to detect a fragment containing the original receptor N-terminus in the supernate of thrombin-treated human platelets. The concentration of the fragment obtained with platelets from 15 normal donors was 4.8 +/- 0.9 pmol per 10(9) platelets (mean +/- sem), which is similar to the value expected if all of the thrombin receptors present on human platelets have been cleaved. Taken together, these results demonstrate that 1) following receptor cleavage a fragment containing the original N-terminus of the receptor is released from the platelet surface, 2) based upon epitope mapping, this fragment is at least 15-20 residues long, 3) it is possible to quantitate the receptor fragment in the supernates of cells exposed to thrombin, and 4) the results of the quantitation suggest that on platelets all of the receptors have been cleaved and 100% of the fragment is present in the cell supernate. Depending on it survival time, measurements of the receptor fragment in blood or urine samples may eventually prove to be a useful marker for thrombin receptor activation in vivo.

Amino Acid Sequence↗
PubMed1991

Interaction of human protein Z with thrombin: evaluation of the species difference in the interaction between bovine and human protein Z and thrombin.

Protein Z is a vitamin K-dependent protein of unknown function present in normal human and bovine plasma. Binding and kinetic studies showed that bovine protein Z interacts with bovine thrombin with a dissociation constant of 0.11 microM in a Ca(2+)-independent fashion and that thrombin becomes associated with phospholipid vesicles in the presence of protein Z but not in its absence (Hogg, P. J. and Stenflo, J. (1991) J. Biol. Chem., in press). In the present study the interaction of human protein Z with human thrombin and the influence of human protein Z on the association of thrombin with phospholipid vesicles was evaluated. In contrast to bovine protein Z, human protein Z bound human DIP-thrombin with a 20-fold weaker affinity at 1.5 mM Ca2+ and in a Ca(2+)-dependent fashion. Human protein Z was also less effective than bovine protein Z in promoting the association of thrombin with phospholipid vesicles. Also, bovine protein Z cleaved by thrombin at Arg-365 bound DIP-thrombin with a 10-fold weaker affinity than did native bovine protein Z. The data suggest that the species difference in the interaction between protein Z and thrombin can be explained by a difference in the COOH-terminal region of bovine protein Z versus human protein Z.

Animals↗
PubMed1982

The action of thrombin on peptide p-nitroanilide substrates: hydrolysis of Tos-Gly-Pro-Arg-pNA and D-Phe-Pip-Arg-pNA by human alpha and gamma and bovine alpha and beta-thrombins.

Human and bovine alpha-thrombins (greater than 90% alpha form) with high fibrinogen clotting activities (approximately 3,000 U.S. units/mg protein) exhibit similar Michaelis menten kinetics with the p-nitroanilide tripeptide substrates Tos-Gly-Pro-arg-pNA (Chromozym-TH) and D-Phe-Pip-Arg-pNA (S-2238). The kinetic parameters at I = 0.11 M, 25 degrees C, pH 7.8 are: (Km = 4.18 +/- 0.22 and 3.61 +/- 0.15 microM; kcat = 127 +/- 8 and 100 +/- 1 s-1) for Chromozym TH and (Km = 1.33 +/- 0.07 and 1.50 +/- 0.10 microM; kcat = 91.4 +/- 1.8 and 98.0 +/- 0.5 s-1) for S-2238 for the human and bovine enzymes, respectively. Unlike the native enzyme forms, their "non-clotting" terminal degradative forms, human gamma-thrombin (approximately 5 units/mg) and bovine beta-thrombin (approximately 200 units/mg), give increased values for these parameters (km = 14.3 +/- 2.4 and 14.4 +/- 2.2 microM; kcat = 160 +/- 9 and 124 +/- 6 s-1) for Chromozym-TH; and (Km = 2.50 +/- 0.36 and 2.99 +/- 0.33 microM; kcat = 106 +/- 3 and 106 +/- 3 s-1) for S-2238. Based on these parameters, 50% degradation of human or bovine alpha-thrombins can be calculated to produce relatively small errors in the kinetic measurement of total thrombin concentrations (maximally 9% and 7% for Chromozym-TH; 7% and 3% for S-2238, respectively) if the kinetic parameters for all alpha forms are erroneously used and assays are at 150 microM substrate. This is in contrast to the large errors inherent in clotting activity measurements on thrombin mixtures. Incorporation of 1 mg/ml of polyethylene glycol 6,000 into assay solutions eliminates systematic errors otherwise caused by thrombin adsorption to surfaces and enables thrombin to be accurately assayed at concentrations less than 0.1 nM or 0.01 clotting unit/ml of alpha-thrombin.

Adsorption↗
PubMed1984

Thrombin-induced platelet reactions: effect of substrates and inhibitors on binding of thrombin and serotonin release.

Binding of 125I-thrombin to platelets and subsequent serotonin release were confirmed. The binding of unaltered thrombin to platelets was also measured by a new technique using a chromogenic substrate (S-2238). As compared to 125I-thrombin, this method gave similar constants for binding to the high affinity binding site, but lower for binding to the low affinity binding site. Furthermore, the results suggest that the platelets have two classes of independent binding sites. Substrates and inhibitors of thrombin inhibited thrombin induced serotonin release, suggesting that the release reaction depends on the proteolytic activity of thrombin. The serotonin release was more inhibited than the binding of thrombin, suggesting that the platelet binding site and the active site of thrombin are located in different parts of the thrombin molecule.

Alprostadil↗
PubMed1993

Bothrojaracin, a new thrombin inhibitor isolated from Bothrops jararaca venom: characterization and mechanism of thrombin inhibition.

A new thrombin inhibitor, bothrojaracin, has been identified and purified to homogeneity from the venom of Bothrops jararaca, the most common venomous snake of South America. Bothrojaracin has an isoelectric point of 4.2 and a molecular mass of 27 kDa and is made of two distinct polypeptide chains of 15 and 13 kDa, linked by disulfide bridges. Purified bothrojaracin is devoid of phospholipase A2, amidolytic, or fibrino (geno)lytic activity. Bothrojaracin forms a noncovalent complex with alpha-thrombin, without changing its catalytic activity on small peptide substrates. Bothrojaracin behaves as a potent and specific antagonist of thrombin-induced platelet aggregation and secretion, characterized by an IC50 ranging from 1 to 20 nM depending on the alpha-thrombin concentration. Bothrojaracin prolongs fibrinogen clotting time, and this effect is related to a competitive inhibition of the binding of alpha-thrombin to fibrin(ogen) (Ki 15 nM). Binding of alpha-thrombin to thrombomodulin is inhibited up to 87% by bothrojaracin, and the rate of protein C activation by alpha-thrombin is also decreased. Bothrojaracin antagonizes the inhibition of thrombin amidolytic activity by hirudin. These results indicate that bothrojaracin acts as a very potent ligand of the exosite of alpha-thrombin.

Amino Acid Sequence↗
PubMed1996

Tryptophan 60-D in the B-insertion loop of thrombin modulates the thrombin-antithrombin reaction.

In a recent study it was demonstrated that thrombin des-PPW reacts with antithrombin (AT) very poorly. In this study it is shown that a Trp to Ala (W60A) mutant of thrombin also reacts with AT at a lower rate than thrombin. The inhibition kinetics were studied by the slow-binding kinetic approach. In both the presence and absence of heparin, the pseudo-first-order rate constant of thrombin inhibition (kobs) increased linearly with AT concentration, indicating that inhibition, in the concentration range covered, conforms to a bimolecular reaction E+I-->K assn E-I. Only the second-order association rate constant (kassn) for thrombin can be estimated [6.8 +/- 2.7) x 10(3) M-1 s-1 in the absence of heparin and (4.1 +/- 1.2) x 10(6) M-1 s-1 in the presence of heparin]. With W60A and des-PPW, the kobs of inhibition increased hyperbolically as a function of AT concentration, indicating that the inhibition is a two-step process according to E+I<==>K init E.I-->k2 E-I. The kinetic constants for W60A were estimated to be Kinit = 13.6 +/- 3.3 microM and k2 = 0.007 +/- 0.001 s-1 in the absence of heparin and K init = 13.6 +/- 3.1 nM and k2 = 0.008 +/- 0.002 s-1 in the presence of heparin. AT inhibited des-PPW very slowly [K assn = (2.9 +/- 0.7) x 10(1) M-1 s-1], but heparin accelerated the reaction approximately 20,000-fold and made it possible to demonstrate a two-step reaction mechanism for des-PPW with K init = 10.4 +/- 2.3 nM and k2 = 0.006 +/- 0.001 s-1. In contrast to thrombin, an active AT-binding pentasaccharide enhanced the inhibition of des-PPW approximately 15-fold. These results indicate that (1) in contrast to thrombin, the heparin-induced conformational change in AT is required for optimal inhibition of des-PPW and (1) Trp60 is essential for normal thrombin-AT reaction. On the basis of these results, a modified model for thrombin-AT interaction is proposed.

Animals↗
PubMed1996

Binding of fibrin monomer and heparin to thrombin in a ternary complex alters the environment of the thrombin catalytic site, reduces affinity for hirudin, and inhibits cleavage of fibrinogen.

Interaction of the blood clotting proteinase, thrombin, with fibrin monomer and heparin to form a thrombin.fibrin monomer.heparin ternary complex is accompanied by a change in thrombin catalytic specificity. Equilibrium binding interactions in the assembly of the ternary complex were characterized quantitatively using thrombin labeled at the active site with a fluorescent probe and related to changes in thrombin specificity toward exosite I-dependent binding of hirudin and cleavage of fibrinogen. Changes in the active site environment accompanying binding of heparin or fibrin to thrombin in binary complexes were reported by fluorescence enhancements which contributed additively to the perturbation accompanying formation of the ternary complex. Quantitative analysis of the interactions supports a preferentially ordered path of ternary complex assembly, in which initial binding of heparin to thrombin facilitates binding of fibrin monomer with an approximately 40-fold increased affinity. Binding of fibrin monomer in the ternary complex decreased the affinity of native thrombin for hirudin by >100-fold and inhibited cleavage of fibrinogen, but this inhibition was overcome when fibrin(ogen)-fibrin interactions occurred. These results support a ternary complex model in which heparin binding through exosite II of thrombin facilitates fibrin monomer binding via exosite I, with accompanying changes in thrombin catalytic specificity resulting from perturbations in the active site and reduced accessibility of exosite I to hirudin and fibrinogen.

Anilino Naphthalenesulfonates↗
PubMed1999

Thrombin interacts with thrombomodulin, protein C, and thrombin-activatable fibrinolysis inhibitor via specific and distinct domains.

A collection of 56 purified thrombin mutants, in which 76 charged or polar surface residues on thrombin were mutated to alanine, was used to identify key residues mediating the interactions of thrombin with thrombomodulin (TM), protein C, and thrombin-activatable fibrinolysis inhibitor (TAFI). Comparison of protein C activation in the presence and absence of TM identified 11 residues mediating the thrombin-TM interaction (Lys(21), Gln(24), Arg(62), Lys(65), His(66), Arg(68), Thr(69), Tyr(71), Arg(73), Lys(77), Lys(106)). Three mutants (E25A, D51A, R89A/R93A/E94A) were found to have decreased ability to activate TAFI yet retained normal protein C activation, whereas three other mutants (R178A/R180A/D183A, E229A, R233A) had decreased ability to activate protein C but maintained normal TAFI activation. One mutant (W50A) displayed decreased activation of both substrates. Mapping of these functional residues on thrombin revealed that the 11 residues mediating the thrombin-TM interaction are all located in exosite I. Residues important in TAFI activation are located above the active-site cleft, whereas residues involved in protein C are located below the active-site cleft. In contrast to the extensive overlap of residues mediating TM binding and fibrinogen clotting, these data show that distinct domains in thrombin mediate its interactions with TM, protein C, and TAFI. These studies demonstrate that selective enzymatic properties of thrombin can be dissociated by site-directed mutagenesis.

Animals↗
PubMed2003

Effects of melagatran, the active form of the oral direct thrombin inhibitor ximelagatran, and dalteparin on the endogenous thrombin potential in venous blood from healthy male subjects.

The effect of the oral direct thrombin inhibitor ximelagatran and its active form, melagatran, on thrombin generation was investigated in vitro and ex vivo using a thrombin generation assay. In-vitro thrombin generation was triggered in human platelet-poor plasma by the addition of tissue factor, and the endogenous thrombin potential (ETP) was measured. The ETP IC(50) values for melagatran and the low-molecular-weight heparin dalteparin were 0.44 micromol/l and 0.06 IU/ml, respectively. In contrast to dalteparin, melagatran increased the time-to-thrombin peak in a concentration-dependent manner. ETP was also studied ex vivo in platelet-poor plasma collected from healthy male subjects (n = 54) at pre-dose and 2 h post-dose, with ximelagatran (60 mg) orally, dalteparin (120 IU/kg) subcutaneously, or control (water) orally. After ximelagatran or dalteparin administration, the time-to-thrombin peak was prolonged by 41 and 95%, and the ETP was decreased by 61 and 77%, respectively. Thus, melagatran, the active form of the oral direct thrombin inhibitor ximelagatran, efficiently delays and inhibits the generation of thrombin in plasma both in vitro and ex vivo.

Administration, Oral↗
PubMed2004

Inhibition of thrombin-induced feedback activation of factor V: a potential pathway for inhibition of thrombin generation by melagatran.

The feedback mechanism by which melagatran, the active form of the oral direct thrombin inhibitor ximelagatran, inhibits thrombin generation was investigated in vitro, using an endogenous thrombin potential (ETP) assay. Melagatran decreased ETP in a concentration-dependent manner and increased the time to thrombin peak. FEIBA reversed the melagatran-induced reduction in ETP in a concentration-dependent manner and marginally reduced the prolongation of the time to thrombin peak. Similar results were observed for prothrombin as were seen with FEIBA. Both activated factor V and Russell's Viper Venom-factor V activator reversed the melagatran-induced prolongation in time to thrombin peak in a concentration-dependent manner and partially restored ETP. Prothrombin, in combination with Russell's Viper Venom-factor V or activated factor V, reversed both the melagatran-induced reduction in ETP and the prolongation in time to thrombin peak, in a concentration-dependent manner. These results indicate that inhibition of thrombin-mediated amplification reactions in blood coagulation is an effective way to delay or inhibit thrombin generation.

Anticoagulants↗
PubMed1979

Thrombin receptors of human platelets: thrombin binding and antithrombin properties of glycoprotein I.

Washed human platelets were solubilized and the proteins were separated by preparative gel electrophoresis in the presence of sodium dodecyl sulphate. The gel was cut into slices and the effect of the eluted proteins on the clotting of fibrinogen by thrombin was evaluated. The isolate from only one gel slice strongly inhibited the clotting of fibrinogen. The prolongation of the clotting time was dependent on the concentration of the protein and reached a plateau around 5 microgram. Gel electrophoresis of this isolate showed a prominent glycoprotein with an apparent Mr=150 000. Gel filtration studies with [125I]thrombin showed that the protein isolate bound a significant amount of thrombin which could be displaced with unlabelled thrombin. Another preparation from the same gel or purified gamma-globulin did not bind thrombin or prolong the clotting time of fibrinogen. Glycoprotein I was isolated from human platelets by affinity chromatography on lectin-Sepharose columns. The isolated glycoprotein prolonged the clotting of fibrinogen and bound [125I]thrombin which could be displaced by unlabelled thrombin. It is proposed that the high affinity receptor of thrombin on human platelets is glycoprotein I. In addition, the antithrombin activity of intact platelets is due to binding of thrombin to this glycoprotein.

Antithrombins↗
PubMed1979

Routes of thrombin action in the production of proteolytically modified, secondary forms of antithrombin-thrombin complex.

The reaction between thrombin and antithrombin results in the formation of an inactive, stable, equimolar complex between the two proteins. However, under most reaction conditions several secondary complex forms, which have lower apparent molecular weights in dodecyl sulfate/polyacrylamide gel electrophoresis, appear concomitantly with or immediately following the production of the primary form of the complex. Purification of nascent, intact complex and treatment of this complex form with thrombin demonstrated that these subsidiary forms of antithrombin-thrombin complex may arise by proteolysis of the nascent complex by excess thrombin. Dissociation of such proteolytically modified complex preparations by hydroxylamine, and examination of the dissociation products by dodecyl sulfate/polyacrylamide gel electrophoresis suggested that degradation occurs primarily in the thrombin part of the complex, and only after prolonged proteolysis in its antithrombin moiety also. Incubation of antithrombin with several autolytically modified thrombin preparations showed that formation of subsidiary complex forms can also occur by an alternative route, i.e. between premodified thrombin forms and the inhibitor. In contrast, complex formation between thrombin and active forms of antithrombin, which have been modified by thrombin before complex formation, is unlikely, since no such active forms of antithrombin could be demonstrated.

Animals↗
PubMed2005

Impaired inactivation by antithrombin and hirudin and preserved fibrinogen-clotting activity of thrombin in complex with anti-thrombin antibody from a patient with antiphospholipid syndrome.

Immunoglobulin G (IgG) isolated from the blood plasma of a patient with secondary antiphospholipid syndrome (APS) expresses fibrinogen-clotting and amidolytic activity (the thrombin activity in 20 micromole IgG is equivalent to approximately 5 nmole pure thrombin), and activates factor XIII. Hirudin (1 microM) decreases the intrinsic thrombin activity of the APS IgG by only 25%, whereas it inhibits completely pure thrombin with equivalent activity. Under conditions, when antithrombin inactivates 60% of the thrombin activity in the presence of normal IgG, the APS IgG protects almost completely the added thrombin against inactivation by antithrombin. Heparin, however, partially relieves this protective effect and at the same time it facilitates the inhibition of the intrinsic thrombin activity by antithrombin. The APS IgG reduces the thrombin activity in protein C activation assay by 50% compared to the activity in the presence of normal IgG. All described properties are related to the Fab fragment of the antibody. The IgG preserving the fibrin-generating activity of thrombin with concomitant protection against inhibitors unravels a new aspect of the thrombotic mechanism in APS. This condition is probably rare: only one out of 23 examined patients with primary or secondary APS expresses IgG with the described properties.

Adult↗
PubMed1992

Lack of stability of aggregates after thrombin-induced reaggregation of thrombin-degranulated platelets.

The stability of platelet aggregates is influenced by the extent of the release of granule contents; if release is extensive and aggregation is prolonged, deaggregation is difficult to achieve. The relative importance of the contributions of released substances to aggregate stability are not known, although stable thrombin-induced aggregates form in platelet-rich plasma from patients with barely detectable plasma or platelet fibrinogen, and ADP stabilizes thrombin-induced aggregates of platelets from patients with delta storage pool deficiency which otherwise deaggregate more readily than normal platelets. We degranulated platelets with thrombin (0.9 U/ml caused greater than 90% loss of delta and alpha granule contents) and recovered them as individual platelets in fresh medium. The degranulated platelets were reaggregated by thrombin (2 U/ml). To prevent continuing effects of thrombin, FPRCH2Cl was added when thrombin-induced aggregation of thrombin-degranulated platelets reached its maximum. EDTA (5 mM) or EGTA (5 mM) added at maximum aggregation did not deaggregate these platelets, indicating that the stability of these aggregates does not depend on Ca2+ in the medium. Whereas with control platelets a combination of PGE1 (10 microM) and chymotrypsin (10 U/ml) was required for deaggregation, with thrombin-degranulated platelets either PGE1 or chymotrypsin alone caused extensive deaggregation. The rate and extent of deaggregation of thrombin-degranulated platelets by a combination of PGE1 and chymotrypsin was greater than with control platelets.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate↗
PubMed1988

Functional characterization of thrombin Salakta: an abnormal thrombin derived from a human prothrombin variant.

The genetic variant prothrombin Salakta has been described in a patient presenting with a normal level of prothrombin antigen but reduced prothrombin activity. Initial studies indicated that factor Xa-catalyzed cleavages proceed normally but lead to the production of a thrombin molecule with an altered enzymatic activity. To characterize the functional abnormality of thrombin Salakta more precisely, it was purified by chromatography on heparin-Sepharose and diethylaminoethyl-Sephadex. The purified variant does not differ from normal thrombin by size, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and is 93.1% +/- 7.6% active by titration with p-nitrophenyl-p'-guanidinobenzoate. Its activity, however, is altered to various extents toward the following substrates: H-D-phenylalanyl-L-pipecolyl-L-arginine paranitroanilide (S 2238), fibrinogen, factor V, protein C, and antithrombin III. The Michaelis constant (Km) of thrombin Salakta for S 2238 is higher (12.2 +/- 3.3 mumol/L) than normal (2.8 +/- 0.7 mumol/L), whereas the turnover number (Kcat) is normal (84.4 +/- 6.6 s-1 v 85.9 +/- 14.0 s-1 for normal thrombin). The interaction of thrombin Salakta with benzamidine is also altered as evidenced by an increased inhibition constant (Ki = 3.5 mmol/L v 0.28 mmol/L for normal thrombin). The inability of fibrinogen to act as a competitor in the inactivation of thrombin Salakta by diisopropylfluorophosphate clearly indicates that fibrinogen binding to the fibrinopeptide groove is drastically impaired. In contrast, interactions involving sites remote from the active site such as those with fibrin and thrombomodulin are only slightly impaired. These results indicate that thrombin Salakta exhibits a specific pattern of functional alterations different from those reported for other variants. The structural defect seems to affect essentially the primary substrate binding site and to a lesser extent recognition site(s) remote from the catalytic site such as those for fibrin and thrombomodulin.

Catalysis↗
PubMed1986

Interaction of S-protein of complement with thrombin and antithrombin III during coagulation. Protection of thrombin by S-protein from antithrombin III inactivation.

S-protein, the inhibitor in plasma of the membrane attack complex of complement, appears to have a second function in coagulation. S-protein during clotting enters into a trimolecular complex with thrombin and antithrombin III (ATIII). Functionally, S-protein in the presence of low concentrations of heparin, protects thrombin from inactivation by ATIII. Complex formation between S-protein and thrombin, and between S-protein, thrombin, and ATIII, was demonstrated by agarose gel electrophoresis and by two-dimensional immunoelectrophoresis of purified proteins and in recalcified, clotted plasma. Formation of the trimolecular S-thrombin-ATIII complex was strictly dependent on the presence of thrombin. No association was detectable between S-protein and ATIII or between S-protein and prothrombin. Heparin was not required for the formation of the bimolecular S-protein-thrombin complex or the trimolecular S-protein-ATIII complex. The protective effect of S-protein on inactivation of thrombin by ATIII was demonstrated in functional assays with purified proteins and in plasma only in the presence of low concentrations of heparin. Thus, S-protein may mediate its effect by scavenging heparin required for ATIII activation. It is suggested that the protection of thrombin by S-protein from inactivation by ATIII may be of physiological importance.

Antithrombin III↗
PubMed1994

Glycosaminoglycan contributions to both protein C activation and thrombin inhibition involve a common arginine-rich site in thrombin that includes residues arginine 93, 97, and 101.

Proteoglycans play pivotal roles in the regulation of thrombin. Thrombomodulin (TM) binds thrombin through protein-protein contacts and a chondroitin sulfate moiety. The complex activates the anticoagulant zymogen, protein C. Thrombin and a thrombin mutant with Arg93, Arg97, and Arg101 changed to Ala bind soluble TM lacking the chondroitin sulfate with comparable affinities, but the mutant binds TM containing chondroitin sulfate 45-fold weaker than thrombin. A simple hyperbolic relationship describes the Ca2+ dependence of protein C activation with the thrombin mutant-TM complex whether or not the TM contains chondroitin sulfate. A similar Ca2+ dependence is observed with wild type thrombin only when the TM contains chondroitin sulfate. Thus, charge neutralization of Arg93, Arg97, and Arg101 mimics the functional effects of the chondroitin sulfate. The mutant and wild type thrombin are inhibited at comparable rates by antithrombin +/- the pentasaccharide capable of inducing the "active" antithrombin conformation, but heparin acceleration of antithrombin inhibition of the mutant is reduced by more than 95%. Binding studies revealed that the mutant has a > or = 20-fold decrease in heparin affinity. We conclude that heparin and chondroitin sulfate interact with one or more of these Arg residues. These basic residues appear to play a critical role in the regulation of thrombin activity.

Animals↗