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PubMed1991

Effect of the hirudin carboxy-terminal peptide 54-65 on the interaction of thrombin with platelets.

The carboxy-terminal region of hirudin (residues 54-65) has previously been shown to inhibit thrombin clotting activity without binding to the catalytic site of the enzyme. In the present study, the effect of hirudin 54-65 on thrombin interaction with specified platelet proteins has been investigated. Hirudin 54-65 was found to inhibit thrombin-induced platelet aggregation and secretion in a dose-dependent manner. Substitution of either Phe56, Glu57, Ile59, Pro60 or Leu64 showed that these residues were critical for inhibition of thrombin-induced platelet activation whereas sulfation of Tyr63 increased the inhibitory potency of the peptide. Hydrolysis of glycoprotein V, a platelet membrane substrate for thrombin, was only partially inhibited by hirudin 54-65. Although hirudin 54-65 did not decrease the amount of thrombin bound to platelets during cross-linking experiments, it was found to inhibit the specific binding of thrombin to platelet glycoprotein Ib. Since the carboxy-terminal region of hirudin has previously been reported to bind near the trypsin-catalyzed beta cleavage site, we have analyzed the consequences of alpha to beta-thrombin conversion on both thrombin-hirudin 54-65 interaction and thrombin activity toward platelets. The beta cleavage induced a decrease in the affinity of thrombin for both glycoprotein Ib and hirudin 54-65. Altogether, our results indicate that thrombin recognition sites for hirudin 54-65 and platelet membrane glycoprotein Ib share common structures located near the beta cleavage site at Arg 73 on the thrombin B chain.

Amino Acid Sequence↗
PubMed1983

Studies on the mechanism of thrombin. Interaction with fibrin.

Fibrin monomer Sepharose was used to investigate the interactions of thrombin with fibrin. Thrombin binding was found to be reversible and saturable and to depend on the thrombin: fibrin ratio. Scatchard analysis indicated a single class of binding sites with K alpha = 4.9 X 10(5) M-1. Ca2+ ions caused rapid desorption and elution of thrombin from fibrin monomer, and the Ca2+ concentration needed for maximal desorption depended on the fibrin:thrombin ratio. Mg2+, Mn2+, and Sr2+ also released thrombin from fibrin monomer but not as efficiently as Ca2+. These results indicate that divalent metal ions induce a physical change in fibrin monomer which results in desorption of thrombin. Thrombin binding to fibrin in a gel was compared to binding to fibrin monomer. These studies showed that as fibrin monomers polymerize to form the gel network, thrombin is released. Under static conditions the released thrombin remains associated with the gel because diffusion is limited by the gel. However, the thrombin can be readily removed when buffer is allowed to flow through the gel. These results lead to the possibility that thrombin binding to fibrin monomer and its subsequent release, either by Ca2+ or by polymerization, may have important consequences for regulating the effective thrombin concentration in vivo.

Calcium↗
PubMed1980

[Ultraviolet difference spectroscopy study of alpha- and beta gamma-thrombin binding to heparin].

The interaction of alpha- and beta gamma-thrombin with heparin was studied by ultraviolet difference spectroscopy within the wavelength range of 230-300 nm. The absorption difference spectrum of the thrombin-heparin complex was negative and had two maxima at 255 nm (5300 M-1 cm-1) and 282 nm (4700 M-1 cm-1) for alpha-thrombin and at 240 nm (4900 M-1 cm-1) and 282 nm (4100 M-1 cm-1) for beta gamma-thrombin. It is assumed that the conformational changes induced by heparin in the enzyme molecule involve the transfer of some tryptophan and tyrosine residues from the interior of the protein to the surface. The absorption changes during alpha-thrombin--heparin interaction at physiological ionic strength suggest binding of some alpha-thrombin molecules to a heparin molecule at the ligand-enzyme molar ratio lower than 1. Under the same conditions beta gamma-thrombin forms an equimolar complex with heparin with the dissociation constant equal to 7,0.10(-9) M. The ionic strength increase up to 0,217 M NaCl results in some disturbances in beta gamma-thrombin-heparin interaction and prevents the binding of additional alpha-thrombin molecules to an equimolar complex of alpha-thrombin with heparin. Therefore the kinetics of the two enzyme forms interaction with heparin are similar, the alpha-thrombin affinity for heparin being a little higher. The data obtained suggest that alpha-thrombin binding to heparin is essential for biological inactivation of thrombin.

Binding Sites↗
PubMed1994

Intercellular gap formation induced by thrombin in confluent cultured bovine retinal pigment epithelial cells.

PURPOSE: Thrombin is formed at the site of intraocular hemorrhage and may be important in the development of progressive retinal damage. The authors observed that thrombin-treated bovine retinal pigment epithelial (RPE) cell cultures develop intercellular gaps and initiated this study to examine in detail the effects of thrombin on RPE cell morphology, adhesion, and cytoskeleton. METHODS: Confluent cultures of bovine RPE cells were incubated for various times (0 to 24 hours) with alpha-thrombin (0.1 to 100 U/ml) or enzymatically inactive thrombin. Intercellular gaps were quantitated by light microscopy in ten representative fields (magnification X400) as number of gaps per field (gaps/f). RPE cytoskeleton was studied using immunofluorescent staining for vinculin and F-actin. The mechanism of thrombin-induced RPE cell gap formation was studied by preincubation with specific drugs, including a protein kinase inhibitor (staurosporine), protein kinase C inhibitors (H-7 and calphostin C), cyclic adenosine monophosphate (cAMP) inducer (forskolin), and cytoskeleton-disrupting agents (cytochalasin B or colchicine). RESULTS: Intercellular gaps (20 to 80 microns in diameter) were markedly increased in number in thrombin-treated cultures in a dose-dependent and time-dependent manner and were associated with an alteration in the distribution of F-actin and vinculin. Whereas control cultures showed 3.3 +/- 2.4 gaps/f, incubation with 8 U/ml of alpha-thrombin for 3 hours resulted in 44.8 +/- 15.3 gaps/f. These changes were most prominent shortly after the 3-hour coincubation, but the cultures did return to their original confluent state within 24 hours. Cultures treated with an enzymatically inactive thrombin showed fewer intercellular gaps than those treated with enzymatically active thrombin but had significantly more intercellular gaps than control cultures. Thrombin-induced intercellular gap formation was blocked by preincubation with forskolin (14.6 +/- 7.1 gaps/f), staurosporine (10.2 +/- 5.0 gaps/f), or H-7 (24.5 +/- 9.8 gaps/f). CONCLUSIONS: Exposure to an enzymatically active thrombin results in formation of intercellular gaps between cultured RPE cells. Inhibition of this phenomenon by protein kinase inhibitors and by a cAMP inducer suggests that this effect is mediated, at least in part, through protein kinase C- and cAMP-dependent pathways. Thrombin generation associated with intraocular hemorrhage may thus result in direct damage to the RPE monolayer, possibly via the same pathway(s).

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1993

Evidence for thrombin binding to dermatan sulphate sites in the rabbit aorta subendothelium in vitro.

The proposal that thrombin binds to dermatan sulphate chains of extracellular proteoglycans has been examined directly using the subendothelium of the rabbit aorta. Freshly excised aortas were de-endothelialized by balloon catheter in vitro and then incubated with 125I-thrombin to allow adsorption of 20-30 fmol of thrombin/cm2. Pretreatment of the subendothelium with FPR-thrombin or chondroitinase ABC partially inhibited thrombin binding, each by approximately 40-45%. The addition of dermatan sulphate inhibited, competitively, up to 50% of thrombin from binding to the subendothelium whereas chondroitin-4 or -6 sulphates had little or no effect. By contrast, protamine inhibited 90% of FPR-thrombin binding. Of subendothelium-bound thrombin, chondroitinase ABC released only a small proportion (3-12%) of bound thrombin but up to 44% of bound FPR-thrombin. It is concluded that, when 125I-thrombin is bound in vitro at a concentration of < 30 fmol/cm2 of aorta intima-media, approximately 50% of subendothelial 125I-thrombin is bound to dermatan sulphate chains of proteoglycan in the extracellular matrix. The possibility is discussed that dermatan sulphate chains may function as thrombin-binding loci to control or augment thrombin activity in the ECM of the injured vascular wall in vivo.

Animals↗
PubMed1998

Thrombomodulin modulates the mitogenic response to thrombin of human umbilical vein endothelial cells.

Thrombin interacts with its receptor and thrombomodulin on endothelial cells. We evaluated the respective roles of these two proteins on human umbilical vein endothelial cell (HUVEC) growth by comparing thrombin, S195A (a mutant thrombin in which the serine of the charge stabilizing system had been replaced by alanine), and the receptor activating peptide (TRAP). Thrombin and TRAP induced DNA synthesis (half maximal cell proliferation with 5 nM and 25 microM, respectively), whereas S195A thrombin was inactive, inferring that growth is mediated through the thrombin receptor. Surprisingly, cells stimulated by TRAP exhibited a maximal proliferation twice greater than that obtained with thrombin. Combination of thrombin and TRAP resulted in a mitogenic response higher than by thrombin alone, but lower than by TRAP alone. The role of thrombomodulin was evaluated by adding an anti-thrombomodulin antibody, which prevents formation of the thrombin-thrombomodulin complex. Antibody did not interfere with cell proliferation induced by TRAP, but enhanced that induced by thrombin. We conclude that formation of the thrombin-thrombomodulin complex restrains HUVEC proliferation mediated through the thrombin receptor.

Antibodies, Monoclonal↗
PubMed1998

[Thrombin and its pharmacologic regulation].

THROMBUS FORMATION: Thrombin plays a crucial role in thrombus formation. It transforms fibrinogen into fibrin and activates platelets. Thrombin has both stimulating and inhibiting effects controlling the feedback regulation of thrombus formation. Thrombin also stimulates circulating cells other than platelets and vascular cells and thus participates in inflammation and wound repair accompanying hemostasis and thrombosis. Thrombin's interaction with cells is mediated by proteolysis activated receptors (PAR), mainly PAR-1 and probably by the recently cloned PAR-3. MECHANISM OF ACTION: Thrombin is an ellipsoid-shaped serine protease whose active site is located at the bottom of a deep groove. One end of the groove bearing the active site carries exosite 1 which links with the C-terminal end of hirudine, PAR-1 and PAR-3, fibrinogen and fibrin, thrombomoduline and platelet GPIb. Exosite 2 is carried on the top side of the molecule opposite the active site. It binds to certain glycoaminoglycanes such as heparin. Thrombin's high substrate specificity results from multiple interactions which occur between different functional domains and complementary domains on the substrate. ACTION OF HEPARIN: The antithrombotic effect of heparin results from its catalytic effect on antithrombin III (AT) inhibition of thrombin. This effect is however limited because heparin is dependent on the concentration of AT and on the of inactivating proteins such as platelet factor 4. In addition, the inhibitory effect of the AT-heparin complex is limited for thrombin bound to the thrombus. Finally, heparin can produce a rare but severe complication, heparin-induced thrombocytopenia. Because of these different drawbacks, research has been focused on other thrombin inhibitors. DIRECT THROMBIN INHIBITORS: The target of these inhibitors is the active site of thrombin. Many compounds are under study, including several which can be administrated orally. The most advanced clinical trials have been conducted with hirudin. Hirudin binds to thrombin at several sites, blocking all the known functions of thrombin. The problem of choosing an agent to maintain antithombotic therapy in patients with heparin-induced thrombocytopenia remains unresolved. Hirudin would be a likely choice as it has no cross reactivity with heparin or heparinoids. Promising early results have been reported.

Amino Acid Sequence↗
PubMed1991

Thrombin increases proliferation and decreases fibrinolytic activity of kidney glomerular epithelial cells.

Human glomerular epithelial cells (GECs) in culture synthesize single-chain, urokinase-type plasminogen activator (SC-uPA), tissue-type plasminogen activator (t-PA), and plasminogen activator inhibitor 1 (PAI-1) and possess specific membrane-binding sites for u-PA. Using purified 125I-alpha thrombin, we demonstrate here the presence of two populations of specific binding sites for thrombin on GECs (1.Kd = 4.3 +/- 1.0 x 10(-10) M, 5.4 +/- 1.4 x 10(4) M sites per cell, 2. Kd = 1.6 +/- 0.5 x 10(-8) M, 7.9 +/- 1.8 x 10(5) sites per cell). Purified human alpha thrombin promoted the proliferation of GECs and induced a time- and dose-dependent increase of SC-uPA, t-PA, and PAI-1 antigens released by GECs. Thrombin-mediated increase in antigen was paralleled by an increase in the levels of corresponding u-PA and PAI-1 messenger RNA. In contrast, thrombin decreased u-PA activity in conditioned medium. This discrepancy between u-PA antigen and u-PA activity was explained by a limited proteolysis of SC-uPA by thrombin, leading to a two-chain form detected by immunoblotting and that could not be activated by plasmin. Thrombin also decreased the number of u-PA binding sites on GECs (p less than 0.05) without changing receptor affinity. Hirudin inhibited the binding and the cellular effects of thrombin, whereas thrombin inactivated by diisopropylfluorophosphate had no effect, indicating that both membrane binding and catalytic activity of thrombin were required. We conclude that thrombin, through specific membrane receptors, stimulates proliferation of GECs and decreases the fibrinolytic activity of GECs both at the cell surface and in the conditioned medium. These results suggest that thrombin could be involved in the pathogenesis of extracapillary proliferation and persistency of fibrin deposits in crescentic glomerulonephritis.

Cell Division↗
PubMed2002

Thrombin enhancement of interleukin-1 expression in mononuclear cells: involvement of proteinase-activated receptor-1.

In addition to its central role in blood coagulation and hemostasis, human alpha-thrombin is considered a pro-inflammatory molecule. We have previously demonstrated that differentiated monocytes express the proteolytically activated receptor for thrombin (PAR-1) and that thrombin enhances the release of interleukin (IL)-6 in human monocytes. In the present study we show that thrombin upregulates the production of both IL-1alpha and IL-1beta in phytohemagglutin (PHA)-activated human peripheral blood mononuclear cells (PBMC). Treating PHA-activated PBMC with the PAR-1 activation peptide, SFLLRN, mimics the effects of thrombin on IL-1alpha and IL-1beta production. Thus, it appears that these pro-inflammatory effects induced by thrombin may be mediated through activation of PAR-1. ELISA and RNase protection assays indicate that thrombin and SFLLRN peptide upregulates IL-1 expression at both protein and mRNA levels. Thrombin directly affects monocyte IL-1 expression, since treatment of differentiated U937 cells with thrombin and SFLLRN enhances IL-1 production. These results may help explain how thrombin can enhance IL-1 expression in normal tissue to initiate tissue repair and why thrombin and thrombin-like enzymes may contribute to inflammatory responses observed in several pathophysiological conditions.

Humans↗
PubMed1997

Thrombin receptor-dependent prostaglandin E2 synthesis in hamster fibroblasts: synergistic interactions with interleukin-1beta.

Cellular responses to alpha-thrombin are mediated through a G-protein-coupled receptor that undergoes proteolytic cleavage, unveiling a tethered peptide ligand with the amino-terminal sequence SFLLRN. The synthetic peptide SFLLRN can mimic many of thrombin's actions via directly stimulating the thrombin receptor. Thrombin has been implicated in several cellular events associated with tissue injury, including fibroblast growth, matrix deposition, and inflammatory responses. The role of the thrombin receptor in fibroblast-dependent release of the inflammatory mediator prostaglandin E2 was evaluated and compared to its well-characterized effect on cell proliferation. Both thrombin and SFLLRN stimulated [3H]thymidine incorporation into DNA but failed to induce prostaglandin E2 release from CCL39 cells. The inflammatory cytokine interleukin-1beta synergized with thrombin and SFLLRN to induce the release of prostaglandin E2, whereas it had no effect on thrombin receptor-mediated DNA synthesis. Interleukin-1beta had no direct effects on thrombin receptor-mediated phosphoinositide hydrolysis, suggesting that its effects were downstream from early signal transduction events. Thrombin and interleukin-1beta together significantly increased the expression of prostaglandin H synthase-2 in accordance with the prostaglandin E2 results. These studies indicate that the fibroblast thrombin receptor differentially couples to intracellular signaling pathways leading to distinct functional responses and that thrombin receptor-effector interactions could be modulated by interleukin-1beta.

Animals↗
PubMed1994

Mechanisms of cholera toxin prevention of thrombin- and PMA-induced endothelial cell barrier dysfunction.

Thrombin-induced endothelial cell (EC) activation leads to compromise of monolayer barrier function due to cellular retraction/contraction and intercellular gap formation. Cyclic AMP induces relaxation in other contractile cells and promotes barrier function in EC. To investigate mechanisms involved in cAMP protection in thrombin-induced permeability, we pretreated bovine pulmonary arterial EC monolayers with 1 microgram/ml cholera holotoxin which catalyzed ADP ribosylation of Gs and increased synthesis of cAMP. The holotoxin, but not the binding subunit, reduced basal permeability and prevented gap formation and permeability following challenge with 1 microM thrombin, 100 microM thrombin receptor-activating peptide, or 1 microM phorbol myristate acetate (PMA). Furthermore, thrombin-induced gap formation and permeability were reversed by cholera toxin post-treatment. Pretreatment with 5 microM forskolin or 1 mM dibutyryl cAMP, with or without 1 mM isobutyl methylxanthine, but not cGMP analogs, protected against thrombin-induced EC permeability, mimicking the cholera toxin effect. Although downregulation of protein kinase C attenuated both thrombin- and PMA-induced permeability, cholera toxin did not alter either PMA-induced protein kinase C activation or thrombin-induced Ca2+ mobilization. In contrast, cholera toxin attenuated thrombin-induced myosin light chain phosphorylation and largely prevented actin redistribution. These studies suggest that cholera toxin: (1) protects endothelial barrier function and reverses established dysfunction via increased cAMP (2) does not alter thrombin receptor interaction or early signal events such as Ca2+ mobilization and PKC activation, (3) attenuates myosin light chain kinase activation and actomyosin contractile interaction subsequent to thrombin activation, and (4) abrogates contractile processes subsequent to PKC activation, which is also an important mechanism in thrombin-induced permeability but is independent of myosin light chain kinase activation.

Animals↗
PubMed2000

Distribution and potential biologic function of the thrombin receptor PAR-1 on human keratinocytes.

Thrombin has recently been shown not only to exert procoagulant activities, but also to induce mitogenic responses of different cell types involved in wound healing via binding to and cleavage of the thrombin receptor. In order to further explore these aspects of thrombin function, human keratinocytes (HaCaT cell line) were examined for their potential mitogenic responsiveness to thrombin and for the dependency of this process on the expression of the high-affinity thrombin receptor. Quiescent keratinocytes were stimulated in the mitogenic assay with alpha-thrombin and the thrombin receptor activating peptides TRAP42-55 (SFLLRNPNDKYEPY) and TRAP42-46 (SFLLR). A strong induction of cell proliferation was noted with alpha-thrombin, TRAP42-55 and TRAP42-46, but not with the "scrambled" peptide (FSLLR). These findings confirm that keratinocytes express the thrombin receptor and that the sequence of the first two amino acids of the generated neo-N-terminus are important for the activation of the receptor. Using cDNA fragments of the 5' coding sequence of the receptor, Northern blot analysis confirmed that HaCaT keratinocytes express the thrombin receptor. Expression of the receptor was also detected on normal human keratinocytes by immunohistochemistry and in situ hybridization. These data demonstrate the expression and biologic function of the human thrombin receptor on human keratinocytes, suggesting that thrombin, among other mediators, plays an important part in the orchestration of epidermal growth and repair processes.

Blotting, Northern↗
PubMed1997

The thrombin receptor in adrenal medullary microvascular endothelial cells is negatively coupled to adenylyl cyclase through a Gi protein.

The effects of thrombin on adenylyl cyclase activity were examined in rat adrenal medullary microvascular endothelial cells (RAMEC). Confluent RAMEC monolayers were stimulated for 5 min with cAMP-generating agents in the absence and presence of thrombin, and intracellular cAMP was measured with a radioligand binding assay. Thrombin (0.001-0.25 U/ml) dose-dependently inhibited IBMX-, isoproterenol- and forskolin-stimulated cAMP accumulation. A peptide agonist of the thrombin receptor, gamma-thrombin, and the serine proteases trypsin and plasmin, also inhibited agonist-stimulated cAMP levels, while proteolytically inactive PPACK- or DIP-alpha-thrombins were without effect. Moreover, the thrombin inhibitor hirudin abolished the inhibitory effect of thrombin but not of the peptide agonist. These results suggest that the inhibitory action of thrombin on cAMP accumulation is mediated by a proteolytically-activated thrombin receptor. The inhibitor of G(i)-proteins pertussis toxin abolished the inhibitory effect of thrombin on isoproterenol- or IBMX-stimulated cAMP production, while the phorbol ester PMA partly impaired it. The protein kinase C inhibitors staurosporine or H7 and the intracellular Ca2+ chelator BAPTA-AM were without effect. Collectively, our data suggest that the thrombin receptor in RAMEC is negatively coupled to adenylyl cyclase through a pertussis toxin-sensitive G(i)-protein.

Adenylate Cyclase Toxin↗
PubMed1981

Covalent binding of thrombin to specific sites on corneal endothelial cells.

Binding of 125I-labeled human alpha-thrombin to endothelial cells derived from bovine corneas was studied in tissue culture. Specific and saturable binding to the cell surface occurred at 37 degrees C but to a much smaller extent at 4 degrees C. Binding of [125I]thrombin to a specific site on these cells with formation of a 77000-dalton complex was demonstrated by NaDodSO4 (sodium dodecyl sulfate)-polyacrylamide gel electrophoresis. Binding of [125I]thrombin was blocked by a 100-fold excess of unlabeled alpha-thrombin and by the thrombin inhibitor, hirudin. There are approximately 100000 of these thrombin binding sites on the cell surface. Formation of the complex could be detected as early as 15 s, increased rapidly over the next 20-30 min, and then continued at a slower rate for the next 2.5 h. The catalytically active site of the enzyme was required for formation of the NaDodSO4-stable complex as shown by the inability of diisopropyl phosphorofluoride inactivated thrombin to form stable complexes with these cells. The complex was dissociated in NaDodSO4 with 1.0 M hydroxylamine, suggesting an acyl linkage of the enzyme to the cellular binding site. The thrombin-endothelial cell complex was distinct from the thrombin-antithrombin III complex (Mr approximately 90000) on gel electrophoresis, and its formation was not enhanced by heparin. Additional thrombin-cell complexes (Mr less than 77000) were also identified; however, they represent a small fraction of the total thrombin bound to the cells. These observations demonstrate that alpha-thrombin is capable of reacting specifically with corneal endothelial cells to form a NaDod-SO4-stable complex which requires the catalytically active enzyme.

Animals↗
PubMed2000

Enhanced contractile response to thrombin in the pregnant rat myometrium.

Thrombin causes various cellular events by activating protease-activated receptors (PARs). Here, we showed, for the first time, that thrombin induced myometrial contraction. To determine the mechanism of thrombin-induced myometrial contraction, we simultaneously measured intracellular Ca(2+) concentration ([Ca(2+)](i)) and tension of fura-PE3-loaded rat myometrium using front-surface fluorimetry. The expression of thrombin receptor mRNA in the rat myometrium were determined by reverse transcription-polymerase chain reaction analysis (RT - PCR analysis). Thrombin (0.01 - 3 u ml(-1)) caused dose-dependent increase in [Ca(2+)](i) and tension in the rat myometrium, and this effect was greatly enhanced in the pregnant myometrium. PAR1-activating peptide mimicked the effects of thrombin. In Ca(2+)-free PSS, thrombin induced no increase in [Ca(2+)](i) and tension in the pregnant myometrium. Both diltiazem (10 microM) and SK-F 96365 (10 microM) significantly inhibited the thrombin-induced elevations of [Ca(2+)](i) and tension, and their effects were additive. RT - PCR analysis revealed an approximately 10 fold increase in the level of thrombin receptor mRNA in the pregnant myometrium compared to that obtained in the non-pregnant myometrium. In conclusion, the contractile response to thrombin was greatly enhanced in the pregnant myometrium, mainly due to the up-regulation of thrombin receptor. We propose that initiation of a post-parturitional myometrial contraction is one of the most important physiological roles of thrombin receptor.

Animals↗
PubMed1994

Cathepsin G and thrombin: evidence for two different platelet receptors.

Neutrophil cathepsin G and thrombin, the only platelet agonists that are proteases, exhibit a mandatory requirement for catalytic activity to induce platelet aggregation and signal transduction. The thrombin receptor is a G-protein-coupled receptor which undergoes proteolysis to generate a tethered ligand that causes self-activation. Since cathepsin G strongly resembles thrombin in its ability to activate platelets, we have attempted to determine whether cathepsin G and thrombin function through the same or different receptors. Evidence that thrombin and cathepsin G act at different receptors was as follows: (a) an antibody directed against the thrombin receptor blocked thrombin-induced but not cathepsin G-induced platelet responses; (b) human fibroblasts responded to thrombin and to a synthetic thrombin receptor peptide (comprising residues 42-55 of the thrombin receptor) by exhibiting an elevation in cytosolic Ca2+ concentration but did not respond to cathepsin G; and (c) platelets pretreated with neutrophil elastase failed to respond to thrombin but responded when rechallenged by cathepsin G. Thrombin and cathepsin G exhibit heterologous desensitization that is potentiated by okadaic acid and is attenuated by staurosporine, indicating that phosphorylation of serine/threonine residues is important for desensitization and that protein kinase C may be involved. Since catalytic activity of cathepsin G is required for platelet stimulation, it is probable that platelet activation by cathepsin G requires receptor proteolysis and that a tethered ligand mechanism is involved, suggesting that platelets may possess a family of protease receptors.

Amino Acid Sequence↗
PubMed1997

Allosteric modulation of the activity of thrombin.

Substrates containing a P3 aspartic residue are in general cleaved poorly by thrombin. This may be partly due to an unfavourable interaction between the P3 aspartate and Glu192 in the active site of thrombin. In Protein C activation and perhaps also thrombin receptor cleavage, binding of ligands at the anion-binding exosite of thrombin seems to improve the activity of thrombin with substrates containing a P3 aspartate. To investigate the importance of Glu192 and exosite-binding in modulating thrombin's interactions with a P3 aspartate, peptidyl chloromethanes based on the sequence of the thrombin receptor (containing a P3 aspartate) have been synthesized and the kinetics of their inactivation of alpha-thrombin and the mutant Glu192-->Gln determined. The values of the inactivation rate constant (ki) for the chloromethanes containing a P3 aspartate were about two-fold higher with the Glu192-->Gln mutant. A peptide based on the sequence of hirudin (rhir52 65), which binds to the anion-binding exosite of thrombin, was an allosteric modulator of the amidolytic activity of the Glu192-->Gln mutant; a 5-fold decrease in the K(m) value for the substrate D-Phe-pipecolyl-Arg-p-nitroanilide was observed in the presence of saturating concentrations of rhir52-65. This exosite-binding peptide also increased the ki values of chloromethanes containing a P3 aspartate with both alpha-thrombin and the Glu192-->Gln mutant. However, the increases in the ki values were greater with the Glu192-->Gln mutant (5-fold compared with 2-fold for alpha-thrombin). Thus exosite binding does not seem to mitigate putative unfavourable interactions between Glu192 and the P3 aspartate. Moreover, increases in the ki caused by exosite binding were not unique to chloromethanes containing a P3 aspartate; increases of the same magnitude were also observed when the P3 position was occupied by the favourable D-phenylalanine in place of the unfavourable aspartate. The results obtained were consistent with exosite binding's causing changes in the conformation of the S2 and/or S1 site of thrombin.

Allosteric Regulation↗
PubMed2003

Glycoprotein Ib-mediated platelet activation. A signalling pathway triggered by thrombin.

Platelet activation by thrombin plays a major role in the development of haemostasis and thrombosis. Thrombin activates human platelets by cleaving the N-terminal region of G-protein-coupled protease-activated receptors (PARs). On the other hand, the platelet membrane glycoprotein GPIb acts as a thrombin-binding site and promotes platelet activation by low thrombin concentrations. We present here new evidence in favour of a thrombin receptor function for GPIb. We have selected conditions in which thrombin-GPIb interactions were enhanced by thrombin immobilization. Activation was studied independently of PAR cleavage by using active-site-blocked thrombin. We show that immobilized, proteolytically inactive thrombin induces platelet adhesion and spreading, dense granule secretion and integrin alphaIIbbeta3-dependent platelet-platelet interactions. The pathway must be dependent on GPIb because it is deficient in platelets from a patient with Bernard Soulier syndrome and inhibited by a monoclonal antibody to GPIb (SZ2) or by an excess of glycocalicin. Secreted ADP plays a major role in GPIb-dependent thrombin-induced platelet activation which is, in addition, regulated by cAMP concentration. Thrombin-induced GPIb-dependent platelet activation leads to tyrosyl phosphorylation of several proteins. Inhibition of platelet-platelet interactions and protein tyrosine phosphorylations by inhibitors of phosphatidylinositol 3-kinases and protein kinase C implies that activation of the latter are important steps of the GPIb-coupled signalling pathway triggered by thrombin.

Adenosine Diphosphate↗