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PubMed1996

Role of the thrombin receptor in development and evidence for a second receptor.

Thrombin, a coagulation protease generated at sites of vascular injury, activates platelets, endothelial cells, leukocytes and mesenchymal cells. A G-protein-coupled receptor that is proteolytically activated by thrombin is a target for drug development aimed at blocking thrombosis, inflammation and proliferation. Here we show that although disruption of the thrombin receptor (tr) gene in mice causes about half of the tr-/- embryos to die at embryonic day 9-10, half survive to become grossly normal adult mice with no bleeding diathesis. Strikingly, tr-/- platelets respond strongly to thrombin, whereas tr-/- fibroblasts lose their ability to respond to thrombin. We conclude that the thrombin receptor plays an unexpected role in embryonic development, suggesting a possible new function for the 'coagulation' proteases themselves. Moreover, a second platelet thrombin receptor exists, and different thrombin receptors have tissue-specific roles. This may allow development of therapeutics that will selectively block thrombin's different cellular actions.

Adenosine Triphosphate↗

PubMed1997

Thrombin has a bimodal effect on glioma cell growth.

Using rat glioma C6 cells as a model, we have found a bimodal effect of alpha-thrombin on cell growth. In C6 cells treated with alpha-thrombin at concentrations from 0.02 nM to 1.0 nM, inhibition of cell proliferation was noted. Because the thrombin receptor agonist peptide TRAP-6 also induced inhibition of cell proliferation and the thrombin receptor antagonist peptide T1 prevented the inhibitory effect of alpha-thrombin on C6 glioma cell growth, thrombin receptor involvement in antiproliferative action of alpha-thrombin in C6 glioma cells is highly likely. However, stimulation of cell proliferation observed when C6 cells were treated with alpha-thrombin at higher doses (> 1.0 nM) seems to be mediated by as yet undefined thrombin receptor-independent biochemical mechanisms.

Animals↗

PubMed1999

Pro- and anti-inflammatory actions of thrombin: a distinct role for proteinase-activated receptor-1 (PAR1).

1. Thrombin has well characterized pro-inflammatory actions that have recently been suggested to occur via activation of its receptor, proteinase-activated receptor-1 (PAR1). 2. In the present study, we have compared the effects of thrombin to those of two peptides that selectively activate the PAR1 receptor, in a rat hindpaw oedema model. We have also examined whether or not thrombin can exert anti-inflammatory activity in this model. 3. Both thrombin and the two PAR1 activating peptides induced significant oedema in the rat hindpaw following subplantar injection. 4. The oedema induced by thrombin was abolished by pre-incubation with hirudin, and was markedly reduced in rats in which mast cells were depleted through treatment with compound 48/80 and in rats pretreated with indomethacin. In contrast, administration of the PAR1 activating peptides produced an oedema response that was not reduced by indomethacin and was only slightly reduced in rats pretreated with compound 48/80. 5. Co-administration of thrombin together with a PAR1 activating receptor resulted in a significantly smaller oedema response than that seen with the PAR1 activating peptide alone. This anti-inflammatory effect of thrombin was abolished by pre-incubation with hirudin. 6. These results demonstrate that the pro-inflammatory effects of thrombin occur through a mast-cell dependent mechanism that is, at least in part, independent of activation of the PAR1 receptor. Moreover, thrombin is able to exert anti-inflammatory effects that are also unrelated to the activation of PAR1.

Animals↗

PubMed2001

Activation of pro-(matrix metalloproteinase-2) (pro-MMP-2) by thrombin is membrane-type-MMP-dependent in human umbilical vein endothelial cells and generates a distinct 63 kDa active species.

Thrombin, a critical enzyme in the coagulation cascade, has also been associated with angiogenesis and activation of the zymogen form of matrix metalloproteinase-2 (MMP-2 or gelatinase-A). We show that thrombin activated pro-MMP-2 in a dose- and time-dependent manner in cultured human umbilical-vein endothelial cells (HUVECs) to generate a catalytically active 63 kDa protein that accumulated as the predominant form in the conditioned medium. This 63 kDa thrombin-activated MMP-2 is distinct from the 62 kDa species found following concanavalin A or PMA stimulated pro-MMP-2 activation. Hirudin and leupeptin blocked thrombin-induced pro-MMP-2 activation, demonstrating that the proteolytic activity of thrombin is essential. However, activation was also dependent upon membrane-type-MMP (MT-MMP) action, since it was blocked by EDTA, o-phenanthroline, hydroxamate metalloproteinase inhibitors, tissue inhibitor of metalloproteinase-2 (TIMP-2) and TIMP-4, but not TIMP-1. Thrombin inefficiently cleaved recombinant 72 kDa pro-MMP-2, but efficiently cleaved the 64 kDa MT-MMP-processed intermediate form in the presence of cells. Thrombin also rapidly (within 1 h) increased cellular MT-MMP activity, and at longer time points (>6 h) it increased expression of MT1-MMP mRNA and protein. Thus signalling via proteinase-activated receptors (PARs) may play a role in thrombin-induced MMP-2 activation, though this does not appear to involve PAR1, PAR2, or PAR4 in HUVECs. These results indicate that in HUVECs the activation of pro-MMP-2 by thrombin involves increased MT-MMP activity and preferential cleavage of the MT-MMP-processed 64 kDa MMP-2 form in the presence of cells. The integration of these proteinase systems in the vascular endothelium may be important during thrombogenesis and tissue remodelling associated with neovascularization.

Cells, Cultured↗

PubMed1994

Plasmin modulates the thrombin-evoked calcium response in C6 glioma cells.

Extracellular proteinases may be selectively targeted to cell surfaces by specific receptors or binding sites. In previous studies, we have characterized cellular binding sites for plasminogen and plasmin on rat C6 glioma cells. In this investigation, we studied the response of C6 cells to alpha-thrombin and plasmin by measuring the rapid kinetics of free intracellular Ca2+ concentrations ([Ca2+]i). Thrombin produced a strong, concentration-dependent rise in [Ca2+]i with an onset within 3 s and peak levels achieved in less than 10 s. A similar response was also evoked by an SFLLRN-containing thrombin-agonist peptide. C6 cells did not respond to plasmin (25 nM-1.5 microM). By contrast, pretreatment of C6 cells with 100 nM plasmin significantly inhibited the [Ca2+]i response to thrombin and the thrombin-agonist peptide. The peak [Ca2+]i response to thrombin, in cells pretreated with plasmin, was reduced by approx. 50%. The effect of plasmin on the cellular response to thrombin was selective, as pretreatment of the cells with plasmin did not affect the [Ca2+]i response to platelet-activating factor. Di-isopropylphosphorylplasmin and plasminogen did not inhibit the cellular response to thrombin, indicating that plasmin activity is required and that occupancy of cellular plasmin(ogen)-binding sites alone is insufficient. These studies demonstrate that plasmin does not directly induce a response in C6 cells, but may affect cellular function by specifically modulating the thrombin response.

Amino Acid Sequence↗

PubMed1999

Acceleration of Ca2+ ionophore-induced arachidonic acid liberation by thrombin without the proteolytic action toward the receptor in human platelets.

We investigated the regulation of arachidonic acid liberation catalyzed by group-IV cytosolic phospholipase A2 (cPLA2) in human platelets upon stimulation with thrombin through interaction with protease-activated receptor-1 (PAR-1) or glycoprotein Ib. Leupeptin, a protease inhibitor, completely inhibited thrombin-induced arachidonic acid liberation and Ca2+ mobilization, with inhibition of its protease activity. However, preincubation with thrombin in the presence of leupeptin potentiated Ca2+ ionophore-induced arachidonic acid liberation. The preincubation did not affect the intracellular Ca2+ level or cPLA2 activity in response to ionomycin. Human leukocyte elastase, which cleaves glycoprotein Ib, did not inhibit the enhancement of arachidonic acid liberation by thrombin in the presence of leupeptin. However, the effect of thrombin with leupeptin was abolished by a peptide corresponding to residues 54-65 of hirudin (hirudin peptide), which impairs the binding of thrombin to PAR-1. Furthermore, Phe-Pro-Arg chloromethyl ketone (PPACK)-thrombin, which binds to platelets but has no protease activity, also enhanced Ca2+ ionophore-induced arachidonic acid liberation. In contrast, trypsin with leupeptin did not mimic the effect of thrombin with leupeptin, and furthermore trypsin-induced arachidonic acid liberation was insensitive to hirudin peptide. On the basis of the present results, we suggest that thrombin may accelerate cPLA2-catalyzed arachidonic acid liberation through non-proteolytic action toward PAR-1 but not toward glycoprotein Ib in co-operation with the proteolytic action leading to Ca2+ mobilization.

Amino Acid Chloromethyl Ketones↗

PubMed1996

Thrombin attenuates neuronal cell death and modulates astrocyte reactivity induced by beta-amyloid in vitro.

beta-Amyloid protein has been implicated as a potential causative agent in the neuropathology associated with Alzheimer's disease. This possibility is supported by observations that beta-amyloid induces neuronal degeneration and astrocyte reactivity in vitro by as yet undefined mechanism(s). In this report, we present data demonstrating that the pathological effects of beta-amyloid on cultured cells are modulated by activation of the thrombin receptor. At concentrations between 50 and 500 nM, thrombin pretreatment significantly attenuates neurotoxicity mediated by fibrillar aggregates of beta 1-42 and beta 25-35 peptides. In cultured astrocytes, the stellate morphology induced by beta 1-42 and beta 25-35 aggregates can be prevented and reversed by thrombin exposures between 10 pM and 1 microM. In contrast, thrombin potentiates rather than attenuates the beta-amyloid-induced increased expression of basic fibroblast growth factor, suggesting that thrombin differentially modulates the effects of beta-amyloid on astrocytes. Thrombin's effects on both neurons and astrocytes are mimicked by thrombin receptor-activating peptide and inhibited by two potent thrombin inhibitors, hirudin and protease nexin-1. These data provide both new insight into the signaling pathways underlying the cellular effects of beta-amyloid and additional support for the role of thrombin as an important mediator of neuropathological events.

Amyloid beta-Peptides↗

PubMed1997

Endothelial cell thrombin receptors and PAR-2. Two protease-activated receptors located in a single cellular environment.

Human endothelial cells express thrombin receptors and PAR-2, the two known members of the family of protease-activated G protein-coupled receptors. Because previous studies have shown that the biology of the human thrombin receptor varies according to the cell in which it is expressed, we have taken advantage of the presence of both receptors in endothelial cells to examine the enabling and disabling interactions with candidate proteases likely to be encountered in and around the vascular space to compare the responses elicited by the two receptors when they are present in the same cell and to compare the mechanisms of thrombin receptor and PAR-2 clearance and replacement in a common cellular environment. Of the proteases that were tested, only trypsin activated both receptors. Cathepsin G, which disables thrombin receptors, had no effect on PAR-2, while urokinase, kallikrein, and coagulation factors IXa, Xa, XIa, and XIIa neither substantially activated nor noticeably disabled either receptor. Like thrombin receptors, activation of PAR-2 caused pertussis toxin-sensitive phospholipase C activation as well as activation of phospholipase A2, leading to the release of PGI2. Concurrent activation of both receptors caused a greater response than activation of either alone. It also abolished a subsequent response to the PAR-2 agonist peptide, SLIGRL, while only partially inhibiting the response to the agonist peptide, SFLLRN, which activates both receptors. After proteolytic or nonproteolytic activation, PAR-2, like thrombin receptors, was cleared from the endothelial cell surface and then rapidly replaced with new receptors by a process that does not require protein synthesis. Selective activation of either receptor had no effect on the clearance of the other. These results suggest that the expression of both thrombin receptors and PAR-2 on endothelial cells serves more to extend the range of proteases to which the cells can respond than it does to extend the range of potential responses. The results also show that proteases that can disable these receptors can distinguish between them, just as do most of the proteases that activate them. Finally, the residual response to SFLLRN after activation of thrombin receptors and PAR-2 raises the possibility that a third, as yet unidentified member of this family is expressed on endothelial cells, one that is activated by neither thrombin nor trypsin.

Endopeptidases↗

PubMed1997

Thrombin causes a marked delay in skeletal myogenesis that correlates with the delayed expression of myogenin and p21CIP1/WAF1.

Thrombin is a multifunctional serine protease whose activity is regulated in the extravasculature by an extracellular inhibitor, protease nexin-1. Because protease nexin-1 expression has been shown to be regulated during skeletal muscle cell differentiation, we reasoned that thrombin inactivation may be an important requirement for this developmental process. To test this hypothesis, we examined the effects of thrombin on differentiating C2C12 myoblasts. We report here that myogenesis, as scored by myotube formation, is considerably delayed by thrombin. This regulation correlated with delayed expression of myogenin and p21(CIP1/WAF1), both considered critical components of the skeletal muscle cell differentiation program. Regulation occurred at the RNA level, indicating that the effect of thrombin is either transcriptional or post-transcriptional. Furthermore, we present evidence suggesting that this regulation is mediated by the thrombin receptor. Although thrombin is mitogenic for certain cell types, we found that delay of myogenesis in C2C12 cells did not involve a mitogenic signal. Taken together, these results imply that inhibition of the serine protease thrombin may be required for proper progression through the myogenic differentiation program. The data point to potentially important roles that thrombin and protease nexin-1 may play during skeletal muscle development.

Animals↗

PubMed2001

Modulation of angiogenesis and progelatinase a by thrombin receptor mimetics and antagonists.

The angiogenic action of thrombin has been shown to be mediated by activation of the thrombin receptor. In this report we studied the effects of SFLLR, an agonist of the activated thrombin receptor and thrombin receptor peptide and non peptide antagonists on angiogenesis in the chick chorioallantoic membrane (CAM) system. As antagonists were used the tripeptide FPR and non-peptide 1,4-disubstituted piperazine derivatives. The pentapeptide SFLLR, like thrombin, caused a marked stimulation of angiogenesis in the CAM. FPR and the piperazine derivatives caused suppression of angiogenesis and in combination with thrombin antagonized its angiogenic effect. Thrombin and SFLLR activated progelatinase A (MMP-2) in the culture medium of human umbilical cord endothelial cells (HUVECs). MMP-2 is involved in the early steps of angiogenesis leading to local dissolution of basement membrane collagen and migration of the activated endothelial cells. FPR and the piperazine derivatives inhibited the activation of this enzyme. They also antagonised the effects of both thrombin and SFLLR on MMP-2 activation. These results suggest that non-thrombogenic agonists or antagonists of the activated thrombin receptor can be used as modulators of angiogenesis.

Animals↗

PubMed1980

Binding of thrombin to cultured human fibroblasts: evidence for receptor modulation.

Previous work from this laboratory has indicated that thrombin's influence on cell growth can be negative as well as positive. Addition of enzyme to actively growing or confluent cultures of human skin fibroblasts produced growth stimulation, whereas cultures receiving thrombin at the time of subculture displayed inhibited DNA synthesis and mitosis. The specific binding of [125I]thrombin to cells under stimulatory and inhibitory conditions has been studied. Fibroblasts receiving enzyme at subculture bound about two times more [125I]thrombin than those processed in the same way several hours later. The apparent dissociation constant for both groups was approximately 1.5 x 10(-8) M. In each case binding was saturable, although cells receiving enzyme at subculture showed a much higher rate of binding. Experiments were conducted in which enzyme was added to cells at various times after subculture. It was found that the ability of these fibroblasts to specifically bind [125I]thrombin decreased progressively over a 2-h period after subculture and then remained constant for at least 24 h. Evidence is also presented indicating that the binding of [125I]thrombin in both experimental groups was inversely dependent upon the culture density. The biological effects of elevated thrombin binding in cells receiving enzyme at subculture were examined. It was found that inhibited DNA synthesis and altered cellular morphology were directly to this parameter. This study suggests that fibroblasts may possess cryptic thrombin receptors that become exposed during subculture or after injury in vivo. These possibilities and the relationship of cell shape to the availability of thrombin receptors are discussed.

Cell Count↗

PubMed1953

Observations on thrombin inactivation by human serum.

The inactivation of thrombin by serum follows a first order kinetics with thrombin concentrations of the order of 20 to 40 units per ml. of serum. With higher thrombin concentrations of 300 to 600 units per ml. of serum, the rate slows down and not the logarithm of the clotting time, but the clotting time itself increases proportionally with the incubation time. The antithrombic factor is stable at +4 degrees C. for a period of 2 weeks, or for 3 months at -15 degrees C. Heating over 58 degrees C. destroys it rapidly. The heat inactivation has an energy of activation of 121,000 cal. With low thrombin concentrations the rate of thrombin inactivation increases linearly with increasing serum concentration. The pH optimum of the inactivation is at 8.5. Increasing the temperature increases the rate. The energy of activation of the thrombin destruction by serum is 14,000 cal. Heparin increases the rate considerably with low thrombin concentrations, but does not affect the rate with high thrombin concentrations. The effect of heparin can be abolished with protamine or toluidine blue. Some other reagents were also tested with respect to their effect upon the rate of inactivation of thrombin by serum.

Heparin↗

PubMed2006

The effect of different anticoagulants on thrombin generation.

Decrease in thrombin generation is the key effect in anticoagulation. The aim of the present study was to investigate the effect of anticoagulants on thrombin generation and the relation to platelet count. Plasma samples from 10 healthy volunteers (mean age 43.0 +/- 9 years) were incubated at preset platelet counts with different doses of the anticoagulants lepirudin, fondaparinux and low molecular weight heparins. Thrombin generation was measured in a tissue factor-mediated assay using a fluorometer and a slow-reacting fluorogenic substrate. The endogenous thrombin potential, the lag phase, the maximum reaction velocity (Vmax) and the concentration of a given anticoagulant required for 50% inhibition of thrombin generation (IC50) are presented. All three anticoagulants decreased endogenous thrombin potential and prolonged the lag phase in a dose-dependent manner. Fondaparinux and low molecular weight heparins, but not hirudin, decreased Vmax in a concentration-dependent manner. With increasing platelet count, the IC50 increased but the extent of this increase was not uniform for the three anticoagulants and the three variables investigated. The influence of anticoagulants on thrombin generation is variable, depending on their basic mechanism of action. In defining and comparing their effects, the endogenous thrombin potential, the lag phase and the maximum reaction velocity should be considered together. Platelets have a considerable influence on the magnitude of thrombin generation.

Adult↗

PubMed1994

Calcium inhibition of the activation of protein C by thrombin. Role of the P3 and P3' residues.

Protein C, a precursor to a natural plasma anticoagulant, and the platelet thrombin receptor, involved in cell activation, both require proteolytic cleavage to be activated. In humans, the sequences adjacent to the scissile bond of protein C, DPR/LID and the thrombin receptor DPR/SFL are similar. Previous studies with Asp-->Gly mutants indicated that both the P3 and the P3' Asp residues make either peptides or protein C a poor substrate for free thrombin, but thrombin interaction with thrombomodulin overcomes these inhibitory interactions. Similar mechanisms are probably operative in the thrombin receptor. In rodents, the P3 Asp residue of the human thrombin receptor is replaced by Asn and in protein C, the P3' residue is Asn. To determine the functional significance of these Asp-->Asn substitutions, the Asp in the P3 or P3' position of human protein C was changed to Asn. The resultant mutants were still resistant to activation by thrombin, and still required Ca2+ for activation by thrombin-thrombomodulin complex. We conclude that, unlike activation of the Asp-->Gly mutants by thrombin, activation of the P3 and P3' Asp-->Asn mutants is still potently inhibited by physiological Ca2+. Furthermore, even though the charge has been deleted, thrombomodulin acceleration is retained.

Asparagine↗

PubMed1986

Thrombin-cellular interactions.

It is clear that there are a number of different types of reactions between thrombin and the cell surface (TABLE 6). In one type, thrombin binds to cell-surface receptors resulting in cellular activation. In other types of reactions, there are at least two components to the thrombin-specific pathway of cellular activation: a classical receptor to which thrombin binds, and a protein that is cleaved. In both types of reactions, thrombin binding and/or proteolysis is linked to changes in GTP-binding proteins, protein kinase C, or other pathways. In most cases, the receptor and membrane substrates involved in cellular activation are not well characterized. In another type of reaction, the interaction between thrombin and proteins in the extracellular fluid is regulated by cell-surface receptors. Binding of thrombin to these receptors can result in acceleration or inhibition of the reactions with the soluble proteins. In the fourth type of reaction, thrombin cleaves a cell-membrane protein that is involved in reactions with plasma proteins. Recognition of the different types of interactions between thrombin and the cell surface is necessary for the correct interpretation of experimental observations. Although the term receptor has classically referred to a cell-surface component to which an agonist binds, it is now clear that there are additional membrane components that specifically bind potential agonists not leading to cellular activation.

Binding Sites↗

PubMed1992

Biphasic increase in c-jun mRNA is required for induction of AP-1-mediated gene transcription: differential effects of muscarinic and thrombin receptor activation.

Activation of either muscarinic cholinergic or thrombin receptors increases phosphoinositide turnover, Ca2+ mobilization, and redistribution of protein kinase C and induces rapid transient increases in c-fos mRNA and c-jun mRNA in 1321N1 cells. To determine whether the increases in c-fos and c-jun mRNA induced by carbachol and thrombin are sufficient to stimulate AP-1-mediated transactivation, 1321N1 cells were transfected with a reporter carrying two copies of the tetradecanoyl phorbol acetate response element and the firefly luciferase gene. Thrombin was significantly more effective than carbachol at stimulating AP-1-mediated transactivation. To identify the factors underlying the difference in AP-1 activity induced by carbachol and thrombin, members of the fos and jun families which encode components of AP-1 were examined. Carbachol and thrombin have similar effects on expression of c-fos, fosB, fra-2, junB, and junD, both acutely and over a 24-h time course. However, whereas carbachol leads only to transient induction of c-jun (maximal at 0.5 h), thrombin induces a biphasic increase in c-jun mRNA--an initial peak at 0.5 h and a second, more-prolonged increase at 12 h. Thrombin but not carbachol also induces a late increase in fra-1 mRNA, which peaks at 12 h. The secondary increase in c-jun mRNA is associated with marked increases in c-Jun protein levels and AP-1 DNA-binding activity. The late induction of c-jun and fra-1 mRNA can be prevented by adding the antagonist hirudin 30 min after thrombin, which results in loss of thrombin-stimulated increases in c-Jun protein, AP-1 DNA-binding activity, and AP-1-mediated transactivation. These findings suggest that rapid and transient conduction of c-fos and c-jun mRNA is insufficient to induce prominent changes in gene transcription, while the sustained increase in c-jun mRNA and perhaps the late induction of fra-1 mRNA are required for generation of AP-1 DNA-binding activity and transactivation through AP-1.

Blotting, Northern↗

PubMed1992

Thrombin receptor 14-amino acid peptide binds to endothelial cells and stimulates calcium transients.

We examined the binding characteristics of the recently described thrombin receptor amino-terminal peptide, SFLLRNPNDKYEPF (T. K. H. Vu, D. T. Hung, V. I. Wheaton, and S. R. Coughlin. Cell 64: 1057-1068, 1991), termed TRP-14, and its effect in activating intracellular calcium transients in pulmonary vascular endothelial cells. Binding of 125I-labeled TRP-14 was found to be saturable with a affinity constant of 2 microM and maximum binding of 41 pmol/mg of cell protein. The 125I-labeled TRP-14 also interacted with bovine pulmonary microvessel endothelial cells, human umbilical vein endothelial cells, and porcine pulmonary artery smooth muscle cells. Binding of 125I-labeled diisopropylphosphoryl (DIP)-alpha-thrombin, which is catalytically inactive but binds to thrombin receptors, was not inhibited by TRP-14 or vice versa, indicating that TRP-14 did not compete for the alpha-thrombin binding site(s) on the endothelial cell surface. TRP-14 (> 1 microM) increased the concentration of intracellular calcium ([Ca2+]i) in endothelial cells with kinetics similar to the increase in [Ca2+]i triggered by alpha-thrombin. In contrast, DIP-alpha-thrombin did not increase [Ca2+]i and also did not prevent the rise in [Ca2+]i induced by the subsequent challenge with either TRP-14 or alpha-thrombin. Because the generation of TRP-14 by the proteolytically active forms of thrombin stimulated a rise in endothelial [Ca2+]i, TRP-14 may be the agonist responsible for the activation of the alpha-thrombin receptor in pulmonary vascular endothelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed2003

The role of PAR4 in thrombin-induced thromboxane production in human platelets.

There are two protease-activated receptors (PARs), PAR1 and PAR4, in human platelets. It has been suggested that PAR1 mediates platelet responses to low concentrations of thrombin, whereas PAR4 mediates signaling only at high concentrations. In the present study, we used a selective PAR4 blocker, YD-3, to investigate the role of PAR4 in thrombin-induced thromboxane formation in human platelets. YD-3 completely prevented thromboxane production by either a low concentration of thrombin (0.1 U/ml) or the PAR4 agonist peptide GYPGKF. In contrast, YD-3 did not affect thromboxane production caused by the PAR1 agonist peptide SFLLRN, collagen or arachidonic acid. YD-3 also decreased [(3) H]arachidonic acid release from thrombin-stimulated platelets. Moreover, desensitization of platelets with GYPGKF prevented low thrombin-induced thromboxane formation. The decreased thromboxane production by YD-3 is linked to inhibition of calcium influx in thrombin-stimulated platelets. These results suggest that PAR4 plays an important role in the regulation of thromboxane formation in platelets responding to thrombin through prolonged elevation of [Ca(2+)](i) and activation of phospholipase A(2). These data also indicate that PAR4 can be activated by relatively low concentrations of thrombin in human platelets. The selective inhibition of thrombin-induced thromboxane production by YD-3 may be of therapeutic benefit for thrombotic diseases.

Arachidonic Acid↗