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PubMed1983

Thrombin-platelet interactions: an assessment of the roles of saturable and nonsaturable binding in platelet activation.

Thrombin binds to platelets and induces platelet activation, but the relationship of binding to activation is not clear. To better define this relationship, we have analyzed parameters of binding and activation by alpha-thrombin and by three analogous proteases that activate platelets somewhat differently. The proteases were nitro-alpha-thrombin, a derivative with nitrated tyrosine, gamma-thrombin, a product of partial proteolysis of alpha-thrombin, and trypsin, a homologous protease. Nitro-alpha-thrombin and native alpha-thrombin activated platelets similarly, whereas gamma-thrombin and trypsin activated to a slightly lesser extent than alpha-thrombin and only after a distinctive delay. alpha-Thrombin and nitro-alpha-thrombin bound to platelets to about the same extent, but only alpha-thrombin showed evidence of saturable binding. Hirudin, a thrombin inhibitor, blocked both platelet activation and saturable binding by alpha-thrombin. With nitro-alpha-thrombin, hirudin blocked platelet activation, but it had no effect on binding. gamma-Thrombin and trypsin bound less than alpha-thrombin and with no evidence of saturable binding. There were identical relationships between the total amount bound and the extent of platelet activation for the four proteases (some show no saturable binding) but distinct differences in the relationships of total amount bound and the rate of activation; similar rates of activation required the binding of three to five times more gamma-thrombin or trypsin than alpha-thrombin. That is, without saturable binding, activation was slower. These data thus show a correlation between total amount bound and extent of activation but no correlation between amount saturably bound and the extent of platelet activation. Conversely, the rate of activation is more closely correlated with saturable binding than with total binding. We conclude that high-affinity saturable binding is not essential for thrombin-induced platelet activation but that it may accelerate the reaction.

Binding Sites↗
PubMed2003

Perioperative topical bovine thrombin exposure is not associated with hemodialysis graft thrombosis.

Arteriovenous (AV) graft use as hemodialysis access remains highly prevalent, with a consequent high thrombosis rate. The magnitude of this problem requires that all potentially modifiable risk factors for graft thrombosis be thoroughly investigated. During graft surgery, topical bovine thrombin is often administered, which can lead to the development of antibovine thrombin antibodies and subsequent hemostatic changes. A recent study correlated the presence of plasma antibovine thrombin antibodies with graft thrombosis in hemodialysis patients. We therefore hypothesized that perioperative topical bovine thrombin exposure would lead to the development of antibovine thrombin antibodies and graft thrombosis. We screened 314 hemodialysis patients and identified 73 patients who had 74 grafts placed for whom complete data on perioperative topical bovine thrombin exposure and subsequent graft outcomes was available. Sixty-one of these patients were available for retrospective measurement of antibovine thrombin antibodies, antihuman thrombin antibodies, and the thrombin activation markers thrombin activatible fibrinolysis inhibitor (TAFI) and thrombin precursor protein (TpP). In these grafts, there was no significant association between topical bovine thrombin exposure and primary assisted patency (P = 0.37). The presence of antibovine thrombin antibodies (P = 0.13), antihuman thrombin antibodies (P = 0.10), and increased TAFI (P = 0.18) were associated with trends toward reduced primary assisted patency which did not reach significance. There was a correlation between antibovine thrombin antibodies and antihuman thrombin antibodies (r = 0.30, P < 0.0001) and between TAFI and TpP trade mark (r = 0.30, P < 0.0001), but no significant correlation between topical bovine thrombin exposure and elevated levels of antibovine thrombin antibodies, antihuman thrombin antibodies, TAFI or TpP trade mark. We conclude that perioperative topical bovine thrombin exposure is not associated with subsequent graft thrombosis.

Aged↗
PubMed2003

Bound thrombin-induced upregulation of myosin heavy chain isoform, SMemb messenger RNA expression in cultured rabbit vascular smooth muscle cells.

To investigate whether bound thrombin can induce modulation of SMemb expression in vascular smooth muscle (VSM) cells, messenger RNA (mRNA) expression was measured by in situ hybridization (ISH) and reverse transcription-polymerase chain reaction (RT-PCR) in cultured rabbit aortic VSM cells. To test the concentration- and time-dependent effect of bound thrombin on the expression of SMemb, confluent VSM cells were incubated for 48 h in 10% FBS-DMEM containing 0, 3, 10 and 30 units/ml of bound thrombin. In addition, the confluent VSM cells were incubated for 6, 12, 24 and 48 h in 10% FBS-DMEM containing 10 units/ml of bound thrombin. Consequently, bound thrombin significantly increased SMemb mRNA in a concentration- and time-dependent manner. When compared with the effect of rabbit fibrinogen (10 microg/ml) and native thrombin (10 units/ml), SMemb mRNA was significantly increased by bound thrombin and was slightly increased by native thrombin, but not by fibrinogen. Other myosin heavy chain (MHC) isoform (SM1 and SM2) mRNA expressions were not changed by fibrinogen, native thrombin or bound thrombin. ISH revealed that there was no significant difference in the expression of MHC mRNAs among fibrinogen, native thrombin or bound thrombin. Western blot analysis demonstrated that the SMemb protein level was significantly increased by 2.5-fold by bound thrombin. When the clot-forming activities in cultured medium containing native thrombin or bound thrombin were measured from 0.5 to 48 h, the activity of bound thrombin declined more slowly than that of native thrombin. In conclusion, bound thrombin could upregulate the expression of SMemb mRNA and protein in cultured VSM cells and the activity of bound thrombin was maintained for longer than that of native thrombin in culture medium.

Animals↗
PubMed1990

Thrombin immobilized to extracellular matrix is a potent mitogen for vascular smooth muscle cells: nonenzymatic mode of action.

Esterolytically inactive diisopropyl fluorophosphate-conjugated thrombin (DIP-alpha-thrombin) stimulated 3H-thymidine incorporation and proliferation of growth-arrested vascular smooth muscle cells (SMCs), similar to native alpha-thrombin. Half-maximal mitogenic response of SMCs was obtained at 1 nM thrombin and was specifically blocked by the leech-derived, high-affinity thrombin inhibitor, hirudin. Native thrombin and a variety of thrombin species that were chemically modified to alter thrombin procoagulant or esterolytic functions were found to induce 3H-thymidine incorporation to a similar extent. Exposure of SMCs to DIP-alpha-thrombin caused a rapid and transient expression of the c-fos protooncogene, determined by Northern blot analysis. These results indicate that thrombin is a potent mitogen for SMCs through a distinct non-enzymatic domain. Binding of 125I-alpha-thrombin to SMC cultures revealed an apparent dissociation constant of 6 nM and an estimated 5.4 x 10(5) binding sites per cell. This binding was inhibited to the same extent by native thrombin and by its nonenzymatic form, DIP-alpha-thrombin. Moreover, the chemotactic fragment of thrombin (CB67-129), which failed to elicit a mitogenic response, competed for 125I-alpha-thrombin binding to SMCs. Cross-linking analysis of 125I-alpha-thrombin to SMCs revealed a specific cell-surface binding site 55 kDa in size. Finally, thrombin immobilized to a naturally produced extracellular matrix retained potent mitogenic activity toward SMCs. These observations lend support to the possibility that in vivo, subendothelial basement membranes sequester thrombin (as well as other bioactive molecules), which may stimulate localized and persistent growth of arterial SMCs. Thrombin may thus be involved directly in progression of atherosclerotic plaque formation.

Animals↗
PubMed2004

Role of protease-activated and ADP receptor subtypes in thrombin generation on human platelets.

The activated platelet surface serves as an integral part of the prothrombinase complex upon activation by potent platelet agonists such as thrombin and collagen. We determined the receptor specificity through which thrombin was enhancing collagen-induced thrombin generation. Whereas SFLLRN or AYPGKF alone produced minimal thrombin generation or phosphatidylserine exposure through protease activated receptor (PAR) stimulation, they caused a leftward shift in the collagen-induced thrombin generation dose-response curve. Although SFLLRN or AYPGKF potentiated collagen-induced thrombin generation, neither of them potentiated to the same extent as thrombin. However, SFLLRN and AYPGKF together potentiated collagen-induced thrombin generation to the same extent as thrombin. We conclude that thrombin mediates its procoagulant activity through activation of both PAR1 and PAR4 receptors. Similarly, neither PAR1 nor PAR4 stimulation alone mimicked the annexin V-binding response caused by thrombin stimulation. The combination of PAR activating peptides caused minimal increases in annexin V binding, but caused significant thrombin generation, suggesting that events other than phosphatidylserine exposure may play a role in platelet prothrombinase complex formation. We also investigated the ability of ADP to potentiate agonist-induced thrombin generation. Whereas P2Y(1) antagonism did not affect collagen or thrombin-induced thrombin generation, P2Y(12) antagonism did decrease both collagen- and thrombin-induced thrombin generation, suggesting that ADP potentiates thrombin generation primarily through the P2Y(12) receptor. Collectively, these results suggest that stimulation of both the PAR1 and PAR4 receptors are necessary for thrombin-induced procoagulant activity, and that the P2Y(12) receptor, but not the P2Y(1) receptor, is responsible for the potentiation of agonist-induced platelet procoagulant activity.

Annexin A5↗
PubMed1992

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.

Alkaloids↗
PubMed1994

alpha-Thrombin-induced human platelet activation results solely from formation of a specific enzyme-substrate complex.

Prior studies using the mutant thrombin, thrombin Quick I, indicate that this protease induces maximum platelet aggregation and intraplatelet [Ca2+] fluxes at agonist concentrations where dissociable, equilibrium platelet binding is undetectable and led to the conclusion that thrombin interaction with its platelet "receptor" is best described kinetically by formation of an enzyme-substrate complex. This conclusion was substantiated further in the present studies by demonstrating that both thrombin Quick I and thrombin mimicked the thrombin receptor agonist peptide in the induction of the platelet activation-dependent events required for functional Prothrombinase assembly and that a rabbit antibody raised against KATNATLDPRSFLLR, a pentadecapeptide which represents amino acids 32-46 in the platelet thrombin receptor/substrate and spans the thrombin cleavage site, inhibited both thrombin- and thrombin Quick I-induced platelet activation responses equivalently. The antipeptide antibody had a more pronounced inhibitory effect on the rate of the thrombin-induced response rather than the magnitude of the response suggesting competition for the cleavage site, consistent with the observation that pretreatment of metabolically-inhibited platelets with thrombin, which was removed by washing, eliminated specific antibody binding due to removal and/or masking of antibody epitopes. Concentrations of the antipeptide antibody that inhibited thrombin- and thrombin Quick I-induced increases in intracellular [Ca2+] flux by as much as 97% did not alter the dissociable equilibrium binding of 125-I-FPR-thrombin to platelets. These combined data indicate that the hydrolytic event initiated by thrombin or thrombin Quick I interaction with the platelet receptor/substrate for thrombin is unrelated to the dissociable equilibrium binding of thrombin to membrane sites described previously by classical receptor-ligand binding analyses.

Amino Acid Sequence↗
PubMed1997

Role of glycoprotein V in the formation of the platelet high-affinity thrombin-binding site.

The glycoprotein (GP) Ib-IX-V complex contains a high-affinity binding site for thrombin on the platelet surface with a poorly defined role in platelet activation by this agonist. Four polypeptides comprise the complex: GP Ib alpha, GP Ib beta, GP IX, and GP V. The site within the complex that binds thrombin has been localized to a 45-kD region at the amino terminus of GP Ib alpha, which also contains the site through which the complex interacts with von Willebrand factor. A GP Ib-IX complex that lacks GP V can be efficiently expressed on the surface of transfected cells. We examined the ability of L cells expressing the GP Ib-IX complex (L2H cells) to bind thrombin at high affinity, and found no increase over the level of thrombin binding to control L cells. Because it is one of the few substrates for thrombin on the platelet surface, GP V has also been implicated as possibly participating in thrombin's actions on the platelet. To examine the role of GP V in forming the high-affinity thrombin-binding site, we compared the binding of thrombin to L2H cells versus cells that express the entire GP Ib-IX-V complex (L2H/V cells). Surface expression of GP Ib alpha was equivalent in these two stable cell lines. Thrombin binding to L2H/V cells was detectable at 0.25 nmol/L thrombin and reached a plateau at 1 nmol/L. No binding to L2H cells was detectable at these concentrations. Comparable results were obtained when thrombin binding to L2H cells transiently expressing GP V was compared with its binding to sham-transfected L2H cells. Again, only cells transiently expressing GP V bound thrombin specifically. As with the platelet polypeptide, thrombin cleaved GP V from the surface of L2H/V cells. To test whether GP V cleavage was required for enhancing thrombin binding to the complex, we tested the binding of enzymatically inactive D-phenylalanyl-L-prolyl-L-arginine chloromethylketone (PPACK)-thrombin to L2H and L2H/V cells. Like native thrombin, PPACK-thrombin at 1 nmol/L bound only to L2H/V cells, indicating that GP V cleavage is not a prerequisite for the formation of the high-affinity thrombin receptor. These data provide the first indication of a physiologic function for GP V, and suggest that formation of the high-affinity thrombin receptor on the platelet surface has complex allosteric requirements.

Allosteric Regulation↗
PubMed1999

Anophelin: kinetics and mechanism of thrombin inhibition.

Anophelin is a 6.5-kDa peptide isolated from the salivary gland of Anopheles albimanus that behaves as an alpha-thrombin inhibitor. In this paper, kinetic analyses and the study of mechanism of alpha-thrombin inhibition by anophelin were performed. Anophelin was determined to be a reversible, slow, tight-binding inhibitor of alpha-thrombin, displaying a competitive type of inhibition. The binding of anophelin to alpha-thrombin is stoichiometric with a dissociation constant (K(i)) of 5.87 +/- 1.46 pM, a calculated association rate constant (k(1)) of 2.11 +/- 0.06 x 10(8) M(-1) s(-1), and a dissociation rate constant (k(-1)) of 4.05 +/- 0.97 x 10(-4) s(-1). In the presence of 0.15 and 0.4 M NaCl, a 17.6- and 207-fold increase in the K(i) of anophelin-alpha-thrombin complex was observed, respectively, indicating that ionic interactions are important in anophelin-alpha-thrombin complex formation. Incubation of alpha-thrombin with C-terminal hirudin fragment 54-65 that binds to alpha-thrombin anion binding exosite 1 (TABE1) attenuates alpha-thrombin inhibition by anophelin; anophelin also blocks TABE1-dependent trypsin-mediated proteolysis of alpha-thrombin. Using gamma-thrombin, an alpha-thrombin derivative where the anion binding exosite has been disrupted, anophelin behaves as a fast and classical competitive inhibitor of gamma-thrombin hydrolysis of small chromogenic substrate (K(i) = 0. 694 +/- 0.063 nM). In addition, anophelin-gamma-thrombin complex formation is prevented by treatment of the enzyme with D-Phe-Pro-Arg-chloromethyl ketone (PPACK), a reagent that irreversibly blocks the catalytic site of thrombin. It is concluded that anophelin is a potent dual inhibitor of alpha-thrombin because it binds both to TABE1 and to the catalytic site, optimal binding being dependent on the availability of both domains. Finally, anophelin inhibits clot-bound alpha-thrombin with an IC(50) of 45 nM and increases the lag phase that precedes explosive in vitro alpha-thrombin generation after activation of intrinsic pathway of blood coagulation. Because of its unique primary sequence, anophelin may be used as a novel reagent to study the structure and function of alpha-thrombin.

Animals↗
PubMed1996

Heparin enhances the catalytic activity of des-ETW-thrombin.

The thrombin mutant, des-ETW-thrombin, lacking Glu(146), Thr(147), and Trp(148) within a unique insertion loop located at the extreme end of the primary specificity pocket, has been shown previously to exhibit reduced catalytic activity with respect to macromolecular and synthetic thrombin substrates and reduced or enhanced susceptibility to inhibition. Investigation of the hydrolysis of peptidyl p-nitroanilide substrates by des-ETW-thrombin showed increased activity in the presence of heparin and other sulphated glycosaminoglycans. No effect was observed upon the activity of wild-type thrombin. Heparin was found to decrease the K(m) for cleavage of four thrombin-specific substrates by des-ETW-thrombin by 3-4-fold. Similarly, pentosan polysulphate (PPS) decreased the K(m) with these substrates by 8-10-fold. Heparin also increased the rate of inhibition of des-ETW-thrombin by antithrombin III and D-phenylalanyl-prolyl-arginylchloromethane (PPACK). The inhibition of des-ETW-thrombin by a number of thrombin-specific peptide boronic acids also showed significant reduction in the final K(i) in the presence of heparin, due to reduction in the off-rate. A peptide analogue of a sequence of hirudin which binds thrombin tightly to exosite I (fibrinogen recognition site) potentiated the activity of des-ETW-thrombin against peptide p-nitroanilide substrates in a manner similar to heparin. The K(i) for the inhibition of des-ETW-thrombin by p-aminobenzamidine was decreased by these ligands from 9.7 mM to 7.5 mM, 5.1 mM, and 2.5 mM in the presence of heparin, hirudin peptide and PPS respectively, suggesting the increased catalytic activity is due to enhanced access to the primary specificity pocket. The positive influence of these ligands on des-ETW-thrombin was reversed in the presence of ATP or ADP; the latter has previously been shown to inhibit thrombin activity by blocking initial interaction with fibrinogen at exosite 1. Because the effect of heparin and PPS is similar to that of hirudin peptide, it is proposed that the most likely mechanism is that binding at the heparin-binding site (thrombin exosite 2) facilitates interaction at exosite 1 causing a conformational change which partially corrects the defective ground-state binding of the mutant thrombin. Although no effect was observed upon the activity of wild-type thrombin, our findings do provide further evidence of an allosteric property of thrombin which may control the geometry of, and access to, the primary specificity pocket.

Amino Acid Sequence↗
PubMed1996

The cloned thrombin receptor is necessary and sufficient for activation of mitogen-activated protein kinase and mitogenesis in mouse lung fibroblasts. Loss of responses in fibroblasts from receptor knockout mice.

The mitogenic activity of thrombin on fibroblasts and smooth muscle cells may contribute to embryonic development and normal wound healing, and it may also play a role in pathological responses to vascular injury. To examine the importance of thrombin signaling in vivo and to define the cloned thrombin receptor's role, we disrupted the thrombin receptor gene (tr) in mice. Platelets from tr-/- mice responded normally to thrombin, but tr-/- fibroblasts showed no thrombin-induced calcium mobilization or phosphoinositide hydrolysis. Thus distinct thrombin receptors act in different tissues. This study focuses on the role of the thrombin receptor in thrombin-induced mitogenesis and mitogen-activated protein (MAP) kinase activation in mesenchymal cells. Thrombin and thrombin receptor agonist peptide both stimulated DNA synthesis and MAP kinase activation in fibroblasts derived from wild-type mice. These responses were selectively lost in fibroblasts from tr-/- mice. Activation of the cloned thrombin receptor is therefore necessary and sufficient for thrombin-induced mitogenesis and MAP kinase activation in mouse lung fibroblasts. The tr-/- mouse thus provides a valuable model for defining the role of thrombin-induced proliferative events in vivo. Because thrombin-induced MAP kinase activation was attributable to a single receptor expressed at natural levels, mouse lung fibroblasts presented an opportunity to define the pathways that normally mediate activation of MAP kinase by the thrombin receptor. Elimination of phorbol-sensitive protein kinase C by prolonged exposure to phorbol ester only partially inhibited MAP kinase activation by thrombin but completely blocked c-Raf kinase activation. Pertussis toxin partially inhibited MAP kinase activation by thrombin but had no significant effect on c-Raf kinase activation. Thus in mouse lung fibroblasts, one thrombin receptor utilizes two pathways for MAP kinase activation: one is protein kinase C- and c-Raf dependent, and a second is Gi-dependent and c-Raf-independent.

Animals↗
PubMed1986

Binding of various thrombin fractions to fibrin and the influence of AT-III on their adsorption.

Human thrombin with high affinity for fibrin was obtained by subjecting purified thrombin to affinity chromatography on Sepharose insolubilized fibrin monomers, after addition of a radioiodinated subsample of thrombin, molar ratio 1:600. As judged by radioprofiling of the electrophoretic distribution of high-affinity thrombin on 10 per cent polyacrylamide gel containing urea/SDS, the preparation consisted of 70 per cent alpha-thrombin, 28 per cent beta-thrombin and only 2 per cent gamma-thrombin. Although alpha-thrombin was bound more strongly to insolubilized fibrin monomers than the other subfractions, complete separation of the individual components could not be achieved. High-affinity thrombin was employed for studies on thrombin adsorption to polymerized fibrin, assuming equal behaviour of labelled and unlabelled thrombin. To avoid passive entrapment of thrombin within the fibrin meshwork at physiological pH, ionic strength and calcium concentration, the optimal fibrinogen concentration was found to be 2.94 umol/l. During such conditions, adsorption of thrombin to polymerized fibrin did not exceed 65 per cent of added thrombin, despite an increasing availability of fibrin-related thrombin binding domains obtained by reducing the thrombin concentration. Adsorption of thrombin to polymerized fibrin increased by 25 per cent when the ionic strength was reduced to 0.05 mol/l. These findings suggest the presence of thrombin subfractions with different affinities for polymerized fibrin. Aggregates of high-affinity thrombin formed during its preparation by affinity chromatography, but were prevented by adding polyethylene glycol (m.w. 6,000, final conc. 6.6 g/l). Such aggregates were not inactivated by AT-III, but could still adsorb to polymerized fibrin.(ABSTRACT TRUNCATED AT 250 WORDS)

Adsorption↗
PubMed1993

Mutagenesis of thrombin selectively modulates inhibition by serpins heparin cofactor II and antithrombin III. Interaction with the anion-binding exosite determines heparin cofactor II specificity.

Thrombin is a multifunctional serine protease that plays a critical role in hemostasis. Thrombin is inhibited by the serpins antithrombin III and heparin cofactor II in a reaction that is dramatically accelerated by glycosaminoglycans. The structural basis of the interaction with these inhibitors was investigated by introducing single amino acid substitutions into the anion-binding exosite (R68E, R70E) and unique insertion loops (K52E, K154A) of thrombin. The rate of inhibition of these recombinant thrombins by antithrombin III and heparin cofactor II was determined in the absence and presence of glycosaminoglycan. The second order rate constant (k2) for inhibition by antithrombin III without heparin was 3.7 x 10(5) M-1 min-1 for wild-type thrombin; rates for the mutant thrombins varied less than 2-fold. For inhibition by antithrombin III with heparin, the rate constant was 4.5 x 10(8) M-1 min-1 for wild-type thrombin with no significant differences between any of the recombinant thrombins. In contrast, the rate constant for inhibition by heparin cofactor II without glycosaminoglycan was 4.3 x 10(4) M-1 min-1 for wild-type thrombin; rates were 10-fold slower for thrombin K52E and 2- to 3-fold slower for thrombins R68E and R70E. The rate constants for inhibition of wild-type thrombin by HCII in the presence of heparin or dermatan sulfate were 9.2 x 10(8) M-1 min-1 and 9.0 x 10(8) M-1 min-1, respectively. Compared to wild-type thrombin, the rate of inhibition by HCII with glycosaminoglycan was 5- to 15-fold slower for thrombins K52E and R70E and 50- to over 100-fold slower for thrombin R68E. Thrombin K154A was inhibited by heparin cofactor II with rates similar to wild-type thrombin in all assays. These results suggest that heparin cofactor II interacts with residue Lys-52 in the proposed S1' subsite and with residues Arg-68 and Arg-70 in the anion-binding exosite of thrombin, and that these interactions contribute to the molecular basis of heparin cofactor II specificity for thrombin.

Antithrombin III↗
PubMed1996

Influence of thrombin on proliferation and prostaglandin production in cultured bovine endometrial cells.

The control of cell proliferation by thrombin was studied in vitro in cultured epithelial and stromal cells of the endometrium. The effect of thrombin was studied after chronic treatment (72 hr) in medium containing 10% fetal bovine serum (FBS) combined or not with sex steroids. Thrombin inhibited slightly the proliferation (based on DNA measurements) only in epithelial cells (P < 0.05). 17 beta-estradiol (E) and progesterone (P4) had no mitogenic effects. The presence of functional thrombin receptors was estimated by stimulation of second messenger generation in response to increasing doses of thrombin (0-1,500 ng/ml). In confluent cultures of epithelial cells, the addition of thrombin for 10 min stimulated cAMP production by 50% with a maximal response at 500 ng/ml (P < 0.05). Similarly, in stromal cells, thrombin stimulated cAMP production in a dose-dependent manner (P < 0.01). Generation of inositol-phosphates was also stimulated by 50% in epithelial cells (P < 0.03), with a maximal response at 500 ng/ml, and by 45% in stromal cells (P < 0.01), with a maximal response at 50 ng/ml. The effect of thrombin on cell proliferation was investigated by 3H-thymidine incorporation in serum-free medium for 24 hr. Thrombin inhibited incorporation in epithelial cells (P < 0.0001) in a dose-dependent manner. Conversely, thrombin stimulated significantly incorporation of stromal cells (P < 0.05) at 50 ng/ml. The effect of sex steroids was also evaluated and it was found that E had no effect on cell proliferation, while P4 inhibited the incorporation in both epithelial (P < 0.001) and stromal cells (P < 0.001). The effect of a combined treatment with thrombin and E inhibited both epithelial (P < 0.001) and stromal cell (P < 0.001) growth, but a combination of thrombin and P4 had no additional effect on growth compared to P4 alone. Further investigation of the role of thrombin has been carried out by measuring prostaglandin (PG) responses. Addition of thrombin for 24 hr inhibited PGF2 alpha production by epithelial cells (P < 0.0001) but had no effect on PGE2 production by stromal cells. Therefore, functional receptors for thrombin appear to be present in epithelial and stromal cells of the bovine endometrium. The minimal effect of thrombin alone or in combination with sex steroids on endometrial cell proliferation in vitro combined with the evidence of functional thrombin receptor in these cells, suggest that: (1) the effect of sex steroids in cultured endometrial cells is not modulated by the presence of thrombin, and (2) other factors are necessary for the full expression of mitogenic responses to sex steroids in vitro.

Animals↗
PubMed2000

Identification of thrombin receptors in rat brain capillary endothelial cells.

Both thrombin and plasmin induce contraction of brain endothelial cells, which may increase capillary permeability thereby leading to disruption of the blood-brain barrier. Identification of thrombin receptors, as well as the influence of plasmin on their activation, in capillary endothelial cells and astrocytes are therefore essential for understanding injury-related actions of thrombin in the brain. Using the reverse transcriptase-polymerase chain reaction method, the present study shows that primary cultures of rat brain capillary endothelial (RBCE) cells and astrocytes derived from rat brain express two different thrombin receptors. The first is proteolytically activated receptor (PAR)-1, the receptor responsible for the vast majority of the thrombin's cellular activation functions; the second is PAR-3, a receptor described to be essential for normal responsiveness to thrombin in mouse platelets. In addition to these thrombin receptors, the mRNA (messenger RNA) for PAR-2, a possible trypsin receptor, was also identified. Functional significance of thrombin receptors was indicated by changes in [Ca2+]i in response to thrombin, as measured by FURA-2 fluorescence in RBCE cells. Thrombin as low as 4 nmol/L induced an abrupt increase in [Ca2+]i whereas, upon addition of active site-blocked thrombin or plasmin, [Ca2+]i remained unchanged. The [Ca2+]i signal attributable to thrombin was smaller in a low Ca2+-containing medium, indicating that an influx of Ca2+ from the extracellular medium makes a contribution to the overall [Ca2+]i rise. The amplitude of the transient [Ca2+]i signal was dependent on the concentration of thrombin, and repeated application of the enzyme caused an essentially complete and long-term desensitization of the receptor. The PAR-1 agonist peptide SFLLRN also elicited a transient increase in [Ca2+]i. After activation by SFLLRN, cells showed a diminished response to thrombin, but the response was not absent, indicating that PAR-3 might contribute to the generation of the [Ca2+]i signal. Pretreatment of RBCE cells with 100 nmol/L plasmin completely prevented [Ca2+]i rise attributable to thrombin. These data show that RBCE cells and astrocytes express at least two receptors for thrombin, PAR-1 and PAR-3, and probably both receptors are involved in thrombin-induced [Ca2+]i signals. Plasmin itself does not elevate [Ca2+]i but prevents the activation of receptors by thrombin.

Animals↗
PubMed1996

Effects of thrombin on steroid-modulated cultured endometrial stromal cell fibrinolytic potential.

By virtue of their unique chronic expression of tissue factor, the primary initiator of hemostasis, decidualized endometrial stromal cells are capable of significant thrombin generation after vascular disruption. In addition to its potent procoagulant effects, thrombin modifies endothelial and glomerular cell fibrinolytic activity. Therefore, we evaluated whether thrombin affected the expression of endometrial stromal cell urokinase-type (uPA) and tissue-type (tPA) plasminogen activators and their primary inhibitor, type 1 plasminogen activator inhibitor (PAI-1), and whether ovarian steroids modulated putative thrombin effects. Confluent stromal cell cultures were incubated in a defined medium containing vehicle control, 10(-8) mol/L estradiol (E2), 10(-7) mol/L medroxyprogesterone acetate (MPA), or E2 plus MPA for 4 days. The medium was then collected and exchanged for medium containing the corresponding steroids with or without thrombin and the specific thrombin inhibitor, D-phenyl-alanyl-propyl-arginine-chloromethyl ketone, for an additional 24 h. The conditioned medium was then collected and analyzed for immunoreactive (ir) uPA, tPA, and PAI-1 by enzyme-linked immunosorbent assay and for PA activity by chromogenic assay, whereas Northern analysis of the cells was employed to evaluate the expression of thrombin receptor, uPA, tPA, and PAI-1 messenger ribonucleic acid (mRNA) species. The latter studies revealed that confluent cultures incubated in defined medium expressed the 3.45-kilobase thrombin receptor message. Steady state levels of thrombin receptor mRNA were unaffected by exogenous steroids. Thrombin added in the absence of exogenous steroids elevated concentrations of ir tPA, uPA, and PAI-1 compared with control cultures. Conversely, in the absence of added thrombin, MPA added alone or together with E2 inhibited levels of ir tPA and uPA while stimulating PAI-1 levels despite the lack of a response to E2 alone. Interestingly, thrombin counteracted this progestin inhibition of tPA and uPA expression and augmented the progestin-enhanced expression of PAI-1. Northern analysis revealed that steady state levels of tPA and uPA mRNA were also enhanced by thrombin in both control and steroid-containing cultures. Net PA activity reflects the balance between PA and PAI-1. In the absence of thrombin, there is virtually no detectable tPA activity and minimal uPA activity in progestin-exposed cultures. However, thrombin elicited significant increases in tPA and uPA activity in control and E2-treated cultures. Despite the molar excess of PAI-1 in MPA-treated and E2- plus MPA-treated cultures, thrombin reversed progestin inhibition of PA activity. Predictably, the addition of D-phenyl-alanyl-propyl-arginine-chloromethyl ketone, blocked the effects of thrombin on PAI-1, tPA, and uPA protein and mRNA expression and PA activity. In summary, thrombin enhances endometrial stromal cell fibrinolytic and extracellular matrix-degrading protease activity in vitro. Such processes occurring in vivo would probably play a role in menstruation and abnormal uterine bleeding.

Cells, Cultured↗
PubMed2006

[Effects of puerarin on proliferation of vascular smooth muscle cells induced by thrombin].

OBJECTIVE: To investigate the effects of puerarin on the proliferation of vascular smooth muscle cells (VSMC) induced by thrombin and the mechanism thereof. METHODS: VSMCs were isolated from the thoracic aorta of a SD rat and cultured, then co-cultured with thrombin of the concentration 0.1, 0.3, 1.0, 3.0, and 10 U/L for 24 h, thrombin of the concentration of 1 U/L for 0, 6, 12, 24, 36, and 48 h respectively, or thrombin of the concentration of 1 U/L combined with puerarin of the concentrations of 1.5 x 10(-5), 1.5 x 10(-4), or 1.5 x 10(-3) mol/L for 24 h. Flow cytometry was used to detect the cell number and cell cycle. Western blotting was used to indicate the protein expression of the oncogenes c-fos and bcl-2 RT-PCR was used to evaluate the thrombin receptor (TR) mRNA expression. RESULTS: he numbers of the groups of VSMCs stimulated by 0.1, 0.3, 1.0, 3.0, and 10 U/L thrombin for 24 hours were 4.82 x 10(4)/ml +/- 0.11 x 10(4)/ml, 6.37 x 10(4)/ml +/- 0.09 x 10(4)/ml, 8.78 x 10(4)/ml +/- 0.08 x 10(4)/ml, 7.37 x 10(4)/ml +/- 0.07 x 10(4)/ml, and 5.28 x 10(4)/ml +/- 0.12 x 10(4)/ml respectively, all significantly higher than that of the control group (4.08 +/- 0.054 x 10(4)/ml, all P < 0.05). The effect of thrombin was in a dose-dependent manner within a concentration range of 0.1 - 1.0 U/L. The suppression rates of VSMC proliferation in the combination groups with puerarin of the concentrations of 1.5 x 10(-5), 1.5 x 10(-4), and 1.5 x 10(-3) mol/L were 10.9% +/- 1.6%, 32.1% +/- 3.3%, and 42.6% +/- 5.2% respectively in comparison with the thrombin group (all P < 0.05). The c-fos protein expression of the VSMCs after thrombin stimulation for 24 h increased by 156.0% +/- 11.3% (P < 0.05), and the bcl-2 protein expression of the VSMCs pretreated with puerarin of the concentrations of 1.5 x 10(-5), 1.5 x 10(-4), and 1.5 x 10(-3) mol/L, and then stimulated by thrombin was significantly lower than that of the VSMCs only stimulated by thrombin with the suppression rates of 20.7% +/- 2.1%, 31.6% +/- 5.2%, and 44.5% +/- 7.5% respectively (all P < 0.05). The bcl-2 protein expression of the VSMCs after thrombin stimulation for 24 h increased by 96.7% +/- 8.3% (P < 0.05), and the bcl-2 protein expression of the VSMCs pretreated with puerarin of the concentrations of 1.5 x 10(-5), 1.5 x 10(-4), and 1.5 x 10(-3) mol/L, and then stimulated by thrombin was significantly lower than that of the VSMCs only stimulated by thrombin with the suppression rates of 7.1% +/- 0.8%, 18.8% +/- 1.2%, and 39.6% +/- 6.4% respectively (all P < 0.05). The stimulation of thrombin increased the TR mRNA expression by 183.9% +/- 9.4%. The puerarin of the concentrations of 1.5 x 10(-5) mol/L and 1.5 x 10(-4) mol/L decreased the increase of TR mRNA expression induced by thrombin, however, without significant differences (both P > 0.05), and puerarin of the concentration of 1.5 x 10(-3) mol/L significantly suppressed the increase of TR mRNA expression induced by thrombin by 17.6% +/- 1.7% (P < 0.05). CONCLUSION: Puerarin suppresses the proliferation and DNA synthesis of VSMC induced by thrombin. The inhibitory effect of puerarin is closely related with the suppression of the protein expression of c-fos and bcl-2n, and partly related with the suppression of the TR mRNA expression.

Animals↗
PubMed1993

Thrombin increases the metastatic potential of tumor cells.

Initial arrest of tumor cells in the microvasculature and their attachment to the endothelium and subendothelial matrix (SEM) are essential prerequisites for metastasis to occur. Factors mediating these interactions are viewed as important determinants of the tumor-cell metastatic phenotype. In this work we have studied the effects of thrombin, its analogs and its precursors on the adhesive properties and metastatic potential of tumor cells. We show that alpha-thrombin, the native form of the key coagulation enzyme, is capable of enhancing tumor-cell adhesion to both the endothelium and SEM components represented by fibronectin. Subclotting, physiological concentrations of alpha-thrombin produced a 2- to 5-fold increase in tumor-cell adhesion. A bell-shaped dose-response curve was observed, with maximal effect at 0.1 U/ml. Maximum effect occurred when cells were exposed to the agonist for 15 min and exposure for up to 4 hr resulted in enhanced tumor-cell adhesion. Prolonged incubation with thrombin resulted in a decline in the thrombin-enhanced adhesion which reached unstimulated control levels by 24 hr. Thrombin precursors and active-site-inhibited thrombin analogs only had minimal adhesion-enhancing activity; nitro- and exosite-alpha-thrombin, which retain a functional active site, mimicked, although to a lesser degree, the action of alpha-thrombin. Tumor-cell incubation with thrombin resulted in an upregulated cell-surface expression of the alpha11b beta 3 integrin, a receptor mediating interactions between tumor cells and endothelial cells, and between tumor cells and SEM. Antibodies against alpha 11b beta 3 integrin effectively inhibited thrombin-enhanced tumor-cell adhesion. Thrombin effects on tumor cells involved the PKC signal transduction pathway as thrombin-enhanced adhesion was inhibited by pre-incubation with PKC inhibitors and a transient PKC translocation from cytosol to membrane was observed following thrombin challenge. In vivo, thrombin-treated tumor cells demonstrated a 2-fold increase in their lung-colonizing ability. In contrast to the adhesion results, the metastasis-enhancing effects of alpha-thrombin were mimicked by a thrombin precursor (prothrombin) and thrombin analogs.

Animals↗