Thrombin-reactive polypeptides of platelets may regulate inhibition of thrombin by antithrombin

The central enzyme involved in blood coagulation and activation of platelets is the serine proteinase thrombin. The principal inhibitor of this proteinase in plasma is antithrombin. The mechanism of regulation of the thrombin-antithrombin reaction remains unknown. Two polypeptides of 74 and 55 kDa present on the platelet surface and in plasma are known to specifically enhance the activity of thrombin on different substrates. This study was undertaken to assess the effects of these platelet proteins on thrombin-antithrombin interaction. Direct measurements of residual thrombin activity in mixtures of thrombin and antithrombin, in the presence or absence of the platelet proteins, were made utilizing a specific chromogenic substrate. Under these conditions, when 60% of thrombin activity was inhibited by antithrombin in controls, 100% of enzyme activity was retained in the presence of the platelet proteins. When heparin was used in these assays, the rate of inhibition of thrombin by antithrombin was much more rapid and 62% of thrombin activity remained after 1 min. Under these conditions, the platelet proteins continued to protect thrombin from inactivation with 98% activity remaining at 1 min and 85% activity at 5 min. In contrast, the inhibition of trypsin by antithrombin was not affected by the platelet proteins. Additional studies in platelet aggregation showed that the platelet polypeptides have two effects on thrombin: (i) protection of the enzyme inhibition by antithrombin and (ii) stabilization of thrombin from loss of activity due to aging. The results suggest a novel role for the platelet proteins in hemostasis - regulation of the inhibition of thrombin by antithrombin.     

Isolation and quaternary structure of the photosynthetic core of the marine purple sulfur bacterium Chromatium purpuratum

Abstract Porphyromonas gingivalis produces a trypsin-like enzyme, Protease I, which is thought to be an important virulence determinant of the organism in adult periodontal disease. Protease I is transiently inhibited by physiological inhibitors of human thrombin. The aim of the present work was to establish whether Protease I was able to mimic thrombin by activation of the thrombin receptor on human platelets. Protease I caused true platelet activation at concentrations comparable to thrombin as measured by aggregometry, morphology and fluorescence flow cytometric analysis of CD63 expression. The effect was blocked by protease inhibitors but not by anti-thrombin receptor antibodies which, by contrast, blocked platelet activation by thrombin. We conclude that the activation of platelets by P. gingivalis Protease I involves proteolysis, but not scission of the thrombin cleavage site of the thrombin receptor.     

Localization of the single-stranded DNA binding site in the thrombin anion-binding exosite.

Single-stranded DNA molecules containing a 15-nucleotide consensus sequence have been reported to inhibit thrombin activity. The mechanism of the inhibition was studied using a consensus 15-mer oligonucleotide and two recombinant mutant thrombins: the anion-binding exosite mutant thrombin R70E, and thrombin K154A, in which the mutation was located in a surface loop outside of the exosite. The consensus 15-mer oligonucleotide inhibited both fibrinogen-clotting and platelet-activation activities of plasma-derived thrombin, recombinant wild type thrombin, and mutant thrombin K154A in a sequence-specific and dose-dependent manner, whereas it did not inhibit either activity of mutant thrombin R70E. The 15-mer oligonucleotide also inhibited thrombomodulin-dependent protein C activation by plasma-derived thrombin. In competition equilibrium binding experiments, binding of 125I-labeled diisopropyl phosphoryl-thrombin to thrombomodulin was completely inhibited by the consensus 15-mer oligonucleotide with a Kd value of 2.68 +/- 0.16 nM. These results suggest that Arg-70 in the anion-binding exosite of thrombin is a key determinant for interaction with specific single-stranded DNA molecules, and that binding of single-stranded DNA molecules to the exosite prevents the interaction of thrombin with fibrinogen, the platelet thrombin receptor, and thrombomodulin.     

The action of factor Xa, thrombin and trypsin on human factor II

The proteolytic action of human and bovine Factor Xa, bovine thrombin and bovine pancreatic trypsin Factor II at pH 7.5 and 25°C was monitored by sodium dodecylsulfate gel electrophoresis and thrombin assays. Purified human and bovine Factor Xa, and trypsin, were found to activate Factor II to thrombin. The conversion of Factor II to thrombin by either Factor Xa or trypsin was found to proceed through two thrombogenic intermediates. The reaction pathway appears to be sequential in that the Factor II (75 000 daltons) is first cleaved to a 55 000-dalton thrombogenic product (Intermediate 1) and a 25 000-dalton non-thrombogenic product (Fragment 1). Intermediate 1 is subsequently converted to an inactive 37 000-dalton thrombogenic protein (Intermediate 2) and a 16 000-dalton protein (Fragment 2). Intermediate 2 is finally converted to an active 37 000-dalton thrombin (α-thrombin). Purified bovine thrombin readily converted Factor II to Intermediate 1 and Fragment 1, but possessed little capacity to catalyze subsequent cleavages to produce active thrombin. The ability of thrombin to cleave Factor II was entirely obviated in the presence of hirudin. Under the conditions of the incubation, the maximum thrombin yield obtainable by Factor Xa or trypsin activation was 50% when compared to the two-stage potential thrombin.     

Effect of rabbit thrombomodulin on the inhibition of thrombin by the antithrombin-heparin complex: role of the acid domain of thrombomodulin

Acidic and non-acidic forms of rabbit thrombomodulin were studied with regard to their effects on the inhibition of thrombin by antithrombin in the presence of exogenous heparin. The non acidic form was obtained by proteolytic cleavage of a polyanionic component (presumably a sulfated polysaccharide) from the parent acidic form of thrombomodulin, and purified by ion-exchange chromatography. It was previously found that the acidic form of thrombomodulin increases the rate of thrombin inactivation by antithrombin. The present study showed that thrombin bound to acidic thrombomodulin was inactivated at a lower rate by antithrombin in the presence of exogenous heparin than was free thrombin or thrombin bound to the non-acidic form of thrombomodulin. The data suggest that the acidic component of thrombomodulin is primarily responsible for the retardation of thrombin-antithrombin complex formation in the presence of exogenous heparin. It is proposed that the polyanionic component of thrombomodulin blocks a site on thrombin required for heparin binding, thus rendering the antithrombin-heparin complex ineffective.     

Identification of anti-plasmin antibodies in the antiphospholipid syndrome that inhibit degradation of fibrin

The combined presence of anti-phospholipid Ab (aPL) and thrombosis is recognized as the antiphospholipid syndrome (APS). The aPL represent a heterogeneous group of Ab that recognize various phospholipids (PL), PL-binding plasma proteins, and/or PL-protein complexes. Recently, we found the presence of antithrombin Ab in some APS patients and that some of these anti-thrombin Ab could inhibit thrombin inactivation by antithrombin. Considering that thrombin is homologous to plasmin, which dissolves fibrin, we hypothesize that some APS patients may have Ab that react with plasmin, and that some anti-plasmin Ab may interfere with the plasmin-mediated lysis of fibrin clots. To test this hypothesis, we searched for anti-plasmin Ab in APS patients and then studied those found for their effects on the fibrinolytic pathway. The results revealed that seven of 25 (28%) APS patients have IgG anti-plasmin Ab (using the mean OD plus 3 SD of 20 normal controls as the cutoff) and that six of six patient-derived IgG anti-thrombin mAb bind to plasmin with relative K(d) values ranging from 5.6 x 10(-8) to 1 x 10(-6) M. These K(d) values probably represent affinities in the higher ranges known for human IgG autoantibodies against protein autoantigens. Of these mAb, one could reduce the plasmin-mediated lysis of fibrin clots. These findings suggest that plasmin may be an important driving Ag for some aPL B cells in APS patients, and that the induced anti-plasmin Ab may act either directly, by binding to plasmin and inhibiting its fibrinolytic activity, or indirectly, by cross-reacting with other homologous proteins in the coagulation cascade to promote thrombosis.     

The inactivation of single-chain urokinase-type plasminogen activator by thrombin on cultured human endothelial cells

Single-chain urokinase-type plasminogen activator (scu-PA) is cleaved by thrombin, resulting in an inactive molecule called thrombin-cleaved two-chain urokinase-type plasminogen activator (tcu-PA/T). There is no knowledge about cell-mediated inactivation of scu-PA. We have studied whether scu-PA bound to cultured human umbilical vein endothelial cells (HUVEC) could be inactivated by thrombin. High molecular weight scu-PA was bound to HUVEC and incubated with increasing amounts of thrombin for 30 min at 37 degrees C. Cell-bound urokinase-type plasminogen activator (u-PA) was released and levels of scu-PA, tcu-PA/T and active two-chain u-PA were measured using sensitive bioimmunoassays. Cell-bound scu-PA was efficiently inactivated by thrombin. Fifty percent inactivation of scu-PA occurred at about 0.2 nM thrombin. In the presence of monoclonal anti-urokinase receptor IgG, at least 50% of the binding of scu-PA to HUVEC was inhibited. The relative amount of tcu-PA/T that was generated by thrombin was not affected by the monoclonal antibody. These results indicated that scu-PA bound to HUVEC via the urokinase receptor can be inactivated by thrombin. The efficient inactivation of cell-bound scu-PA suggests that a cofactor for thrombin may be involved, like thrombomodulin or glycosaminoglycans. It is concluded that scu-PA bound to the urokinase receptor on a cell surface can be inactivated by thrombin, which may have profound effects on u-PA-mediated local fibrinolysis and extracellular proteolysis during processes in which thrombin is also involved.     

The effect of prothrombin fragment 2 on the inhibition of thrombin by antithrombin III.

The effect of prothrombin fragment 2 on the inhibition of thrombin by antithrombin III has been studied. Fragment 2 was found to slow the rate of inhibition of thrombin by antithrombin III about 3-fold. The effect of prothrombin fragment 2 on antithrombin III inhibition was examined by comparing its action in the presence of either thrombin or meizothrombin (des fragment 1). The second order rate constants for antithrombin III inhibition of thrombin with saturating fragment 2 and antithrombin III inhibition of meizothrombin (des fragment 1) were the same. Prothrombin fragment 2 had no effect on either antithrombin III inhibition of meizothrombin (des fragment 1) or Factor Xa. The effect of the fragment on the reaction mechanism of thrombin inhibition was evaluated to see if the fragment altered binding of antithrombin III to thrombin or inhibited the formation of the covalent complex. The fragment was found to have no inhibitory effect on the rate of covalent complex formation, indicating that the protective effect of the fragment is by inhibiting binding of antithrombin III to thrombin. These data suggest that prothrombin fragment 2 may be an important factor in controlling the localization of clot formation by regulating the interaction between thrombin and antithrombin III.     

Neurite outgrowth activity of protease nexin-1 on neuroblastoma cells requires thrombin inhibition

Protease nexin-1 (PN-1) is a protein proteinase inhibitor recently shown to be identical with the glial-derived neurite-promoting factor or glial-derived nexin. It has been shown to promote neurite outgrowth in neuroblastoma cells and in sympathetic neurons. The present experiments were designed to further test the hypothesis that this activity on neuroblastoma cells is due to its ability to complex and inhibit thrombin. It has been suggested that PN-1:thrombin complexes might mediate the neurite outgrowth activity of PN-1. However, the present studies showed that such complexes, unlike free PN-1, did not promote neurite outgrowth. The neurite outgrowth activity of PN-1 was only detected in the presence of thrombin or serum (which contains thrombin). PN-1 did not affect the rate or extent of neurite outgrowth that occurred when neuroblastoma cells were placed in serum-free medium. Retraction of neurites by thrombin was indistinguishable in cells whose neurites had been extended in the presence or absence of PN-1. The neurite-promoting activity of PN-1 was inhibited by an anti-PN-1 monoclonal antibody, which blocks its capacity to complex serine proteinases. The plasma thrombin inhibitor, antithrombin III, stimulated neurite outgrowth but only when its thrombin inhibitory activity was accelerated by heparin. The neurite outgrowth activity of both antithrombin III and PN-1 corresponded to their inhibition of thrombin. Together, these observations show that PN-1 promotes neurite outgrowth from neuroblastoma cells by inhibiting thrombin and suggest that this depends on the ability of thrombin to retract neurites.     

Studies on the mechanism of the rate-enhancing effect of heparin on the thrombin-antithrombin III reaction.

The rate of the reaction between thrombin and antithrombin III is greatly increased in the presence of heparin. Several mechanisms for this effect are possible. To study the problems commercial heparin was fractionated into one fraction of high anticogulant activity and one of low anticoagulant activity by affinity chromatography on matrix-bound antithrombin III. The strength of the binding of the two heparin fractions to antithrombin III and thrombin, respectively, was determined by a crossed immunoelectrophoresis technique. As was to be expected, the high activity fraction was strongly bound to antithrombin III while the low activity fraction was weakly bound. In contrast, thrombin showed equal binding affinity for both heparin fractions. The ability of the two heparin fractions to catalyse the inhibition of thrombin by antithrombin III was determined and was found to be much greater for the high activity heparin fraction. A mechanism for the reaction between thrombin and antithrombin III in the presence of small amounts of heparin is suggested, whereby antithrombin III first binds heparin and this complex then inhibits thrombin by interaction with both the bound heparin and the antithrombin III.     

Formation of a stable complex of thrombin and the secreted platelet protein glycoprotein G (thrombin-sensitive protein, thrombospondin) by thiol-disulfide exchange

When 125I-labeled thrombin was incubated with washed human platelets or with the supernatant solution of activated platelets, it formed a NaDodSO4-stable complex of apparent mass greater than 450 000 daltons. Formation of the complex was temperature dependent; with 20 nM thrombin incubated with the supernatant solution of ionophore-activated platelets, the initial rate of formation of the stable complex was 1 nM thrombin/min at 37 degrees C, 50 times the rate at 22 degrees C. Thrombin with all free amino groups methylated was still reactive. Active-site-blocked thrombin formed the complex only slowly. The complex that formed with active thrombin was not dissociated by hydroxylamine in urea. Reduction with 2-mercaptoethanol dissociated the complex, and its formation was blocked by the sulfhydryl-blocking agents iodoacetamide and 4,4'-dithiodipyridine. The complex was thus unlike those of thrombin and alpha 2-macroglobulin or antithrombin III, but it had characteristics of a disulfide-linked complex. Of the secreted proteins, albumin and glycoprotein G adhered to an activated thiol-Sepharose column, indicating that they contained free thiol groups. Purified glycoprotein G and thrombin formed a complex similar to the complex formed when thrombin was incubated with the supernatant solution of activated platelets. The purified glycoprotein bound 2.6 mol of radioactive N-ethylmaleimide/mol of protein, indicating three sulfhydryl groups per mole. After reacting with purified glycoprotein G, thrombin developed a new sulfhydryl group. It is concluded that glycoprotein G (thrombin-sensitive protein, thrombospondin) and thrombin form a dissociable complex that leads to a covalent complex by thiol-disulfide exchange of a thiol group on glycoprotein G and a disulfide on thrombin.     

Platelet glycoprotein Ib alpha binds to thrombin anion-binding exosite II inducing allosteric changes in the activity of thrombin

The glycoprotein (GP) Ib-IX complex is a platelet surface receptor that binds thrombin as one of its ligands, although the biological significance of thrombin interaction remains unclear. In this study we have used several approaches to investigate the GPIb alpha-thrombin interaction in more detail and to study its effect on the thrombin-induced elaboration of fibrin. We found that both glycocalicin and the amino-terminal fragment of GPIb alpha reduced the release of fibrinopeptide A from fibrinogen by about 50% by a noncompetitive allosteric mechanism. Similarly, GPIb alpha caused in thrombin an allosteric reduction in the rate of turnover of the small peptide substrate d-Phe-Pro-Arg-pNA. The K(d) for the glycocalicin-thrombin interaction was 1 microm at physiological ionic strength but was highly salt-dependent, decreasing to 0.19 microm at 100 mm NaCl (Gamma(salt) = -4.2). The salt dependence was characteristic of other thrombin ligands that bind to exosite II of this enzyme, and we confirmed this as the GPIb alpha-binding site on thrombin by using thrombin mutants and by competition binding studies. R68E or R70E mutations in exosite I of thrombin had little effect on its interaction with GPIb alpha. Both the allosteric inhibition of fibrinogen turnover caused by GPIb alpha binding to these mutants, and the K(d) values for their interactions with GPIb alpha were similar to those of wild-type thrombin. In contrast, R89E and K248E mutations in exosite II of thrombin markedly increased the K(d) values for the interactions of these thrombin mutants with GPIb alpha by 10- and 25-fold, respectively. Finally, we demonstrated that low molecular weight heparin (which binds to thrombin exosite II) but not hirugen (residues 54-65 of hirudin, which binds to exosite I of thrombin) inhibited thrombin binding to GPIb alpha. These data demonstrate that GPIb alpha binds to thrombin exosite II and in so doing causes a conformational change in the active site of thrombin by an allosteric mechanism that alters the accessibility of both its natural substrate, fibrinogen, and the small peptidyl substrate d-Phe-Pro-Arg-pNA.     

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.     

Thrombin causes neurite retraction in neuronal cells through activation of cell surface receptors.

The mechanism by which thrombin induces neurite retraction was studied in NB2a mouse neuroblastoma cells. The rapid effect of thrombin (completed within minutes) appears to involve an interaction between its anion-binding exosite and the thrombin receptor. Structural alterations of this site increase the EC50 for thrombin-mediated retraction, and a hirudin C-terminal peptide that blocks this site inhibits the response. The thrombin effect was mimicked by a 14 amino acid peptide starting with Ser-42, at the proposed cleavage site of the human thrombin receptor. The protein kinase inhibitors staurosporine and H-7 blocked thrombin-induced retraction. It is therefore proposed that thrombin-mediated neurite retraction is caused by cleavage-induced activation of the thrombin receptor and involves stimulation of a protein kinase(s).     

Identification of monoclonal insulin autoantibodies in insulin autoimmune syndrome associated with HLA-DRB1*0401

We have characterized HLA and insulin autoantibodies in a Japanese female patient with insulin autoimmune syndrome. Serological HLA typing demonstrated the patient had HLA-DR4, and DNA typing showed she had HLA-DRB1*0401 which has not been reported in patients with insulin autoimmune syndrome in Japan. A single binding affinity of insulin autoantibodies was demonstrated by Scatchard analysis and immunoglobulin class of insulin autoantibodies was exclusively IgG-kappa. HLA-DRB1*0406 is strikingly associated with patients with insulin autoimmune syndrome who have polyclonal insulin autoantibodies. The present report demonstrated the first Japanese patient with insulin autoimmune syndrome carrying HLA-DRB1*0401 who was revealed to have monoclonal insulin autoantibodies. The present results indicate that HLA molecules are the major determinants of polyclonal insulin autoantibodies and monoclonal insulin autoantibodies in insulin autoimmune syndrome.     

Homologous desensitization of HEL cell thrombin receptors. Distinguishable roles for proteolysis and phosphorylation.

Loss of sensitivity to thrombin following an initial response is characteristic of a number of cell types, including platelets. It has recently been proposed that thrombin receptors resemble other G protein-coupled receptors, but that activation involves a novel mechanism in which thrombin cleaves the receptor, exposing a new N terminus that serves as the ligand for the receptor. Based upon this model, we have examined the mechanism of thrombin receptor desensitization by comparing the effects of thrombin with those of a peptide corresponding to the N-terminal sequence of the receptor following proteolysis by thrombin: SFLLRNPNDKYEPF or TRP42/55. Like thrombin, TRP42/55 stimulated pertussis toxin-sensitive inositol 1,4,5-trisphosphate formation, raised cytosolic Ca2+, and inhibited cAMP formation in the megakaryoblastic HEL cell line. Exposure to either thrombin or TRP42/55 desensitized the cells to both, but not to a third agonist, neuropeptide Y. The rate of recovery after desensitization depended upon the order of agonist addition. Resensitization of the cell to thrombin following a brief exposure to thrombin required up to 24 h and could be inhibited with cycloheximide. Resensitization to TRP42/55 after exposure to thrombin, or to thrombin after exposure to TRP42/55, on the other hand, was detectable within 30 min and could be inhibited by serine/threonine phosphatase inhibitors, but not by cycloheximide. Loss of responsiveness to thrombin and TRP42/55 was also observed following addition of the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA). However, while the protein kinase inhibitor staurosporine completely prevented the desensitization caused by TPA, it had only a limited effect on the desensitization caused by TRP42/55. These results demonstrate that the G protein-mediated effects of thrombin can be reproduced by a receptor-derived peptide and suggest that desensitization occurs by at least two mechanisms. The first, which is seen with thrombin, but not TRP42/55, involves proteolysis and requires protein synthesis for recovery. The second, which occurs with TRP42/55 and TPA, as well as with thrombin, involves phosphorylation, possibly of the receptor itself. Although protien kinase C is activated by thrombin and is presumably responsible for the desensitization caused by TPA, it does not appear to play a major role in receptor desensitization caused by thrombin and TRP42/55. This suggests that other kinases, such as those which inactivate adrenergic receptors and rhodopsin, are involved in the down-regulation of thrombin receptor function.     

Heterogeneity of autoantibodies in 100 patients with autoimmune myositis: insights into clinical features and outcomes

The objective of this study was to determine the prevalence, mutual associations, clinical manifestations, and diagnoses associated with serum autoantibodies, as detected using recently available immunoassays, in patients with autoimmune myositis (AIM). Sera and clinical data were collected from 100 patients with AIM followed longitudinally. Sera were screened cross-sectionally for 21 autoantibodies by multiplex addressable laser bead immunoassay, line blot immunoassay, immunoprecipitation of in vitro translated recombinant protein, protein A assisted immunoprecipitation, and enzyme-linked immunosorbent assay. Diagnoses were determined using the Bohan and Peter classification as well as recently proposed classifications. Relationships between autoantibodies and clinical manifestations were analyzed by multiple logistic regression. One or more autoantibodies encompassing 19 specificities were present in 80% of the patients. The most common autoantibodies were anti-Ro52 (30% of patients), anti-Ku (23%), anti-synthetases (22%), anti-U1RNP (15%), and anti-fibrillarin (14%). In the presence of autoantibodies to Ku, synthetases, U1RNP, fibrillarin, PM-Scl, or scleroderma autoantigens, at least one more autoantibody was detected in the majority of sera and at least two more autoantibodies in over one-third of sera. The largest number of concurrent autoantibodies was six autoantibodies. Overall, 44 distinct combinations of autoantibodies were counted. Most autoantibodies were unrestricted to any AIM diagnostic category. Distinct clinical syndromes and therapeutic responses were associated with anti-Jo-1, anti-fibrillarin, anti-U1RNP, anti-Ro, anti-Ro52, and autoantibodies to scleroderma autoantigens. We conclude that a significant proportion of AIM patients are characterized by complex associations of autoantibodies. Certain myositis autoantibodies are markers for distinct overlap syndromes and predict therapeutic outcomes. The ultimate clinical features, disease course, and response to therapy in a given AIM patient may be linked to the particular set of associated autoantibodies. These results provide a rationale for patient profiling and its application to therapeutics, because it cannot be assumed that the B-cell response is the same even in the majority of patients in a given diagnostic category.     

Multiple complexes of thrombin and heparin

Fluorescence polarization has been used to study the interaction of thrombin and heparin, and the catalysis by heparin of the combination of thrombin and antithrombin. At low ionic strength (20 mM Tris, pH 7.4), the addition of heparins of known molecular weights to thrombin led to the formation of large complexes (defined as 'complex 1'). Further addition of heparin led to a rearrangement of these large complexes to form smaller complexes (defined as 'complex 2'). The molar ratio of thrombin to heparin in complex 1 increased with increasing heparin molecular weight, and corresponded to one thrombin molecule for every heparin segment of Mr 3000. The stoichiometry of complex 2 was 1 heparin to 1 thrombin, irrespective of the heparin molecular weight. At higher ionic strength (150 mM NaCl) some complex 1 was still formed. However, by reversing the titration and adding thrombin to fluorescein-heparin the dissociation constant for complex 2 was estimated to be 1-3 microM and independent of the heparin molecular weight. The complex formed between thrombin and heparin, to which antithrombin was attached, has a dissociation constant of 1-2 microM, again irrespective of the heparin molecular weight. In the heparin-catalysed thrombin-antithrombin reaction, an increase in the size of heparin leads to a lowering of the observed Km for thrombin. A possible explanation is that thrombin, after initial binding to the heparin, moves rapidly to the site where it combines with antithrombin.     

Inactivation of thrombin by murine peritoneal macrophages.

We recently showed that murine peritoneal macrophages cultured in vitro express potent prothrombinase activity (Lindahl, U., Pejler, G., B?gwald, J., and Seljelid, R. (1989) Arch. Biochem. Biophys. 273, 180-188). In the present report, we demonstrate that the macrophages also express anticoagulant activity by inactivating the thrombin that is formed due to the action of the prothrombinase. Addition of exogenous purified thrombin to the macrophage cultures resulted in inactivation of the enzyme at a maximum rate of approximately 5 micrograms/h/10(6) cells. The inactivation appeared to be specific for thrombin, since neither Factor Xa, chymotrypsin, nor trypsin, three serine proteases exhibiting homology with thrombin, were inactivated by the macrophages. Thrombin-inactivating activity was not secreted into the culture medium. Inhibitors of endocytosis did not decrease the rate by which thrombin was inactivated, suggesting that internalization of the coagulation factor was not required. In contrast, the thrombin-inactivating activity was strongly inhibited by the polycation Polybrene. Anion-exchange chromatography of extracts obtained after Triton X-100-solubilization of the macrophages demonstrated that the thrombin-inactivating activity exhibited a high negative charge. Incubation of the thrombin-inactivating activity recovered after anion-exchange chromatography with unlabeled thrombin, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, showed that thrombin was proteolytically cleaved into defined fragments. Similar proteolytic fragments were obtained when 125I-labeled thrombin was added to macrophage cultures. Degradation of thrombin was blocked by phenylmethanesulfonic fluoride, an inhibitor of serine proteases, but not by inhibitors of other classes of proteases. Thrombin that had been chemically modified at its active site was degraded at the same rate by the macrophages as active thrombin. Taken together, these findings indicate that the murine macrophages express surface-bound serine protease activity that specifically inactivates thrombin by proteolytic cleavage. The significance of thrombin-inactivating activity in relation to the involvement of macrophage procoagulant activity in the immune response is discussed.     

Correlation of thrombin-induced glycoprotein V hydrolysis and platelet activation

To assess the possibility that hydrolysis of the platelet surface thrombin substrate, glycoprotein V, is a necessary step in thrombin-induced platelet activation, thrombin-catalyzed hydrolysis of glycoprotein V was correlated with thrombin-induced platelet activation. Hydrolysis of tritium-labeled glycoprotein V on washed human platelets was measured by the appearance of a labeled supernatant fragment, and platelet activation was measured as secretion of ATP. Hydrolysis of glycoprotein V was linear with respect to both thrombin concentration and time of incubation. The extent of platelet activation was correlated with the rate of hydrolysis but not with the amount hydrolyzed. Maximum platelet activation could be obtained with thrombin treatments resulting in hydrolysis of as little as 4% of glycoprotein V per min. Glycoprotein V was partially removed from platelets by pretreatment with either platelet calcium-dependent protease or chymotrypsin. The rate of thrombin-catalyzed hydrolysis of the remaining glycoprotein V from these pretreated platelets was as little as 1.5% the rate from control platelets, but there was no impairment of the extent of platelet activation. Thus, these protease-pretreated platelets compared with control platelets showed a different correlation of glycoprotein V hydrolysis with platelet activation. Glycoprotein V was also partially removed by pretreatment of prostacyclin-inhibited platelets with thrombin. After removal of thrombin and prostacyclin, these platelets were desensitized to subsequent activation by thrombin. Incubation of desensitized platelets with nonsaturating levels of thrombin led to less than 25% of the activation seen with control platelets but to a slightly greater hydrolysis of glycoprotein V. Thus, the desensitization to thrombin was not due to loss of ability of the activating thrombin to hydrolyze glycoprotein V. These results do not exclude a role for glycoprotein V as a component of the platelet thrombin receptor, but they indicate that there is no simple relationship between thrombin-induced hydrolysis of glycoprotein V and platelet activation.