Thrombin Receptor Peptide Research Articles

Homocysteine levels and platelet reactivity in coronary artery disease patients treated with ticagrelor

Background and aimRecurrent atherothrombotic events have been reported in certain higher risk subsets of patients even with ticagrelor, a potent first-line antiplatelet agent for the management of patients with acute coronary syndrome (ACS). Hyperhomocysteinemia is a known determinant of platelet function abnormalities. Therefore, the aim of our study was to evaluate the impact of homocysteine (Hcy) levels on platelet reactivity in patients receiving Ticagrelor. Methods and resultsPatients with ACS undergoing percutaneous coronary revascularization and on dual antiplatelet therapy with ASA + Ticagrelor (90mg/twice a day) were scheduled for platelet function assessment 30–90 days post-discharge. Aggregation tests were performed by Multiple Electrode Aggregometry (MEA). Suboptimal platelet inhibition HRPR-high residual platelet reactivity was defined if above the lower limit of normality (417 AU*min).We included 432 patients, divided according to Hcy tertiles. Higher Hcy levels were associated with age, renal failure, creatinine levels and use diuretics (p < 0.001).Patients with higher Hcy levels displayed a higher platelet reactivity at COL test (p = 0.002), and ADP test (p = 0.04), with a linear relationship between Hcy and platelet aggregation after stimulation with collagen (r = 0.202, p < 0.001), thrombin receptor peptide (r = 0.104, p = 0.05) and ADP (r = 0.145, p = 0.006).However, Hcy levels did not significantly affect the rate of HRPR with Ticagrelor (9.9% vs 13.7% vs 10.7%, p = 0.89; adjusted OR [95% CI] = [0.616–1.51], p = 0.99). ConclusionsAmong patients with ACS, despite the elevated platelet reactivity associated to hyperhomocysteinemia, DAPT with ticagrelor could overcome such phenomenon, achieving an adequate platelet inhibition in the majority of the patients.

A novel P2Y12-Independent Signaling Pathway Mediating Akt Phosphorylation In Response to Thrombin Receptors

AbstractAbstract 3191The serine-threonine kinase Akt plays an important role in regulating platelet activation. Stimulation of platelets with various agonists results in Akt activation as indicated by Akt phosphorylation. However, the mechanisms of Akt phosphorylation in platelets are not completely understood. It has been previously shown that Akt phosphorylation in response to thrombin receptors is dependent on Gi signaling activated primarily by secreted ADP through its receptor P2Y12. By using P2Y12 knockout mice combined with recombinant CHO models, we demonstrate a Gi-independent pathway for Akt phosphorylation in response to thrombin. Although Akt phosphorylation in response to low dose thrombin or the PAR4 thrombin receptor peptide AYPGKF was abolished in P2Y12 deficient platelets, at high concentrations, they stimulated substantial phosphorylation of both Akt residues Thr308 and Ser473 in P2Y12 deficient platelets, demonstrating that there are P2Y12-dependent and -independent pathways contributing to Akt phosphorylation in response to thrombin receptors. Thrombin or AYPGKF repressed forskolin-induced cAMP production in the wild type mouse platelets but not in the P2Y12 deficient platelets, suggesting that Gi activation by thrombin or AYPGKF is dependent on ADP receptor P2Y12. Therefore, thrombin- or AYPGKF-induced Akt phosphorylation in P2Y12 deficient platelets is Gi independent. AYPGKF-induced Akt phosphorylation was enhanced by expression of recombinant PAR4 cDNA in CHO cells, demonstrating that stimulation of thrombin receptor PAR4 is able to elicit Akt phosphorylation in the absence of platelet secretion. It is unlikely that PAR4-induced Akt phosphorylation in CHO cells involves the P2Y12 pathway, because CHO cells apparently do not express functional P2Y12. This conclusion is supported by the observation that ADP failed to stimulate Akt phosphorylation and inhibit forskolin-induced cAMP production in CHO cells. Unlike thrombin, U46619-induced Akt phosphorylation was dramatically decreased by P2Y12 deficiency, suggesting that TXA2-induced Akt phosphorylation is largely P2Y12 dependent. In addition, P2Y12-independent Akt phosphorylation was not inhibited by the integrin inhibitor peptide RGDS or integrin β3 deficiency, demonstrating that integrin αIIbβ3 outside-in signaling is not required for thrombin-induced, P2Y12-independent, Akt phosphorylation. Furthermore, Akt phosphorylation in response to thrombin or AYPGKF in P2Y12 deficient platelets was inhibited by the calcium chelator dimethyl-BAPTA, the Src family kinase inhibitor PP2, or PI3K inhibitors, but was not affected by PKC inhibitors. Thus, our results reveal a novel P2Y12-independent signaling pathway mediating Akt phosphorylation in response to thrombin receptors. Disclosures:No relevant conflicts of interest to declare.

A G(i) -independent mechanism mediating Akt phosphorylation in platelets.

The serine-threonine kinase Akt plays an important role in regulating platelet activation. Stimulation of platelets with various agonists results in Akt activation as indicated by Akt phosphorylation. However, the mechanisms of Akt phosphorylation in platelets are not completely understood. We used P2Y₁ knockout mice to address the role of P2Y₁₂ in Akt phosphorylation in response to thrombin receptors in platelets. Thrombin or the PAR4 thrombin receptor peptide AYPGKF at high concentrations stimulated substantial phosphorylation of Akt residues Thr³⁰⁸ and Ser⁴⁷³ in P2Y₁₂-deficient platelets. AYPGKF-induced Akt phosphorylation is enhanced by expression of recombinant human PAR4 cDNA in Chinese hamster ovary (CHO) cells. P2Y₁₂ -independent Akt phosphorylation was not inhibited by integrin inhibitor peptide RGDS or integrin β₃ deficiency. Akt phosphorylation induced by thrombin or AYPGKF in P2Y₁₂-deficient platelets was inhibited by the calcium chelator dimethyl-BAPTA, the Src family kinase inhibitor PP2, and PI3K inhibitors, respectively. Our results reveal a novel P2Y₁₂-independent signaling pathway mediating Akt phosphorylation in response to thrombin receptors.

PAR1 antagonist mediated antithrombotic activity in extracorporeal arterio-venous shunt in the rat

The activation of platelets by thrombin is mediated at least in part by cleavage of protease-activated receptors (PARs) [ [1] Vu T.K. Hung D.T. Wheaton V.I. Coughlin S.R. Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation. Cell. 1991; 64: 1057-1068 Abstract Full Text PDF PubMed Scopus (2641) Google Scholar ]. Four distinct PARs have been cloned to date, with PAR1, PAR3 and PAR4 being activated by thrombin, whereas PAR2 is sensitive to trypsin [ [2] Ossovskaya V.S. Bunnett N.W. Protease-activated receptors: contribution to physiology and disease. Physiol Rev. 2004; 84: 579-621 Crossref PubMed Scopus (908) Google Scholar ]. Human platelets express PAR1 and PAR4 and activation of either is sufficient to trigger platelet aggregation and secretion [ 1 Vu T.K. Hung D.T. Wheaton V.I. Coughlin S.R. Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation. Cell. 1991; 64: 1057-1068 Abstract Full Text PDF PubMed Scopus (2641) Google Scholar , 3 Kahn M.L. Nakanishi-Matsui M. Shapiro M.J. Ishihara H. Coughlin S.R. Protease-activated receptors 1 and 4 mediate activation of human platelets by thrombin. J Clin Invest. 1999; 103: 879-887 Crossref PubMed Scopus (680) Google Scholar ]. Studies with selective neutralizing antibodies have indicated that PAR1 mediates activation of human platelets at low thrombin concentrations and that, in the absence of PAR1 function, PAR4 can mediate platelet activation but only at high thrombin concentrations [ [4] Coughlin S.R. Thrombin signalling and protease-activated receptors. Nature. 2000; 407: 258-264 Crossref PubMed Scopus (2083) Google Scholar ]. Curiously, PAR1 appears not to play a role in rat platelets. PAR1-activating peptides do not activate rat platelet aggregation [ 5 Kinlough-Rathbone R.L. Rand M.L. Packham M.A. Rabbit and rat platelets do not respond to thrombin receptor peptides that activate human platelets. Blood. 1993; 82: 103-106 PubMed Google Scholar , 6 Derian C.K. Santulli R.J. Tomko K.A. Haertlein B.J. Andrande-Gordon P. Species differences in platelet responses to thrombin and SFLLRN receptor-mediated calcium mobilization and aggregation, and regulation by protein kinases. Thromb Res. 1995; 78: 505-519 Abstract Full Text PDF PubMed Scopus (65) Google Scholar ]. In contrast to this, PAR3 is not expressed by human or guinea-pig platelets, but by mouse and rat platelets [ [7] Hishihara H. Connolly A.J. Zeng D. Kahn M.L. Zheng Y.W. Timmons C. et al. Protease-activated receptor 3 is a second thrombin receptor in humans. Nature. 1997; 386: 502-506 Crossref PubMed Scopus (794) Google Scholar ]. Recently, Leger et al. [ [8] Leger A.J. Jacques S.L. Badar J. Kaneider N.C. Derian C.K. Andrade-Gordon P. et al. Blocking the protease-activated receptor 1-4 heterodimer in platelet-mediated thrombosis. Circulation. 2006; 113: 1244-1254 Crossref PubMed Scopus (187) Google Scholar ] elegantly showed that in human platelets PAR1 and PAR4 form a stable heterodimer that enables thrombin to act as a bivalent functional agonist. Collectively, all these studies reveal the complexity of the PAR-mediated platelet activation in different animal species. Thus, the aim of the present study was to investigate the putative in vivo role of PAR1 in a thrombosis model conducted in anesthetized rats. We have investigated the effects of selective PAR4 antagonist, YD-3 [ [9] Wu C.C. Hwang T.L. Liao C.H. Kuo S.C. Lee F.Y. Lee C.Y. et al. Selective inhibition of protease-activated receptor 4-dependent platelet activation by YD-3. Thromb Haemost. 2002; 87: 1026-1033 PubMed Google Scholar ] but also relatively selective PAR1 antagonists ER-121958 and SCH-203099 [ 10 Ahn H.S. Foster C. Boykow G. Stamford A. Manna M. Graziano M. Inhibition of cellular action of thrombin by N3-cyclopropyl-7-[[4-(1-methylethyl)phenyl]methyl]-7H-pyrrolo[3,2-f]quinazoline-1,3-diamine (SCH 79797), a nonpeptide thrombin receptor antagonist. Biochem Pharmacol. 2000; 60: 1425-1434 Crossref PubMed Scopus (138) Google Scholar , 11 Ahn H.S. Chackalamannil S. Nonpeptide thrombin receptor antagonists. Drugs Future. 2001; 26: 1065-1085 Crossref Scopus (13) Google Scholar , 12 Chackalamannil S. Xia Y. Thrombin receptor (PAR-1) antagonists as novel antithrombotic agents. Expert Opin Ther Patents. 2006; 16: 493-505 Crossref PubMed Scopus (27) Google Scholar ] and conventional antithrombotic agents on an extracorporeal arteriovenous shunt model in the rat.

Factor VIII's C Domains Bind to Platelets.

The C domains of factor VIII contain the primary binding site for the cofactor, activated factor VIII, to interact with the phospholipid membranes, including those on the platelet surface. Isolated C2 domain has been shown to bind to phosphotidyl-L-serine-rich lipids and platelets; under flow cytometry, binding to activated platelets was confirmed. For comparison, C1C2, expressed in E.coli, was prepared with up to mg quantities isolated. Fresh, gel-filtered platelets were then studied in a flow cytometer either with or without activation by the thrombin receptor peptide, SFLLRN-amide. Depending upon the conditions, up to 80% of the platelets could be stained with a monoclonal antibody to C2 (ESH8) that is known not to compete with lipid or von Willebrand factor binding. The results were confirmed using a S2296C mutant C1C2 where the free suflhydryl group was either biotinylated and detected by fluorescein labeled streptavidin or directly labeled with fluorescein. As shown in the figure, essentially all platelets bound directly fluorescein labeled C1C2. Using standardized, labeled microbeads, it was estimated that there are 7000–10,000 binding sites per platelet. After platelet activation, the number of platelets binding C1C2 increased with all three detecting systems but only by 15–30%. In contrast, binding of isolated C2, as determined either by ESH8 or as a C2296 biotinylated species, was much lower when the same molar amounts were added, and was primarily detectable following platelet activation. C1C2 binding appeared independent of von Willebrand factor as platelets from two unrelated subjects with severe, type 3 von Willebrand disease gave the same patterns on flow cytometry as seen in platelets from normal subjects. ESH4, a monoclonal antibody known to inhibit binding of C2 to lipid membranes effectively competed C1C2 binding to platelets. Although an indirect alteration the C2 domain conformation cannot be excluded, results support a direct role of C1 in enhancing platelet binding. [Display omitted]

Role of the C2 Domain of Factor VIIIa in the Assembly of Factor-X Activating Complex on the Platelet Membrane

Optimal rates of factor X (FX) activation require binding of factor IXa (FIXa), factor VIII(a) [FVIII(a)], and FX to activated platelet receptors. To define the FVIIIa domains that mediate platelet interactions, albumin density gradient washed, gel-filtered platelets (3.5 x 10(8)/mL) activated by the thrombin receptor peptide, SFLLRN (25 microM), were incubated with 125I-labeled FVIII C2 domain, or 125I-FVIIIa, or 125I-FVIII((LC)), or peptides from the C2 domain region, with or without anti-C2 domain monoclonal antibodies (MoAb), ESH4 or ESH8. FVIIIa (Kd approximately 1.7 nM), FVIII((LC)) (Kd approximately 3 nM), and the C2 domain (Kd approximately 16 nM) all interacted with approximately 700-800 binding sites/platelet. Unlike FVIIIa, the C2 domain did not respond to the presence of excess EGR-FIXa (45 nM) and FX (1.5 microM) with enhanced binding stoichiometry and affinity. Both the MoAb ESH4 and a synthetic peptide corresponding to FVIII residues 2303-2332 (epitope for FVIII MoAb, ESH4) inhibited FVIIIa binding to platelets, whereas MoAb ESH8 and a C2 domain peptide corresponding to residues 2248-2285 (epitope for the FVIII MoAb, ESH8) failed to inhibit FVIIIa binding. Thus, a major platelet-binding site resides within residues 2303-2332 in the C2 domain of FVIIIa, and an additional site within residues 2248-2285 increases the stoichiometry and affinity of FVIIIa binding to activated platelets only in the presence of FIXa and FX but does not directly mediate FVIIIa binding to the platelet surface.

Lipid Raft Association of a Shared Factor X/Prothrombin Binding Site on Human Platelets Is Mediated by the Gla Domain.

Factor X (FX) initially binds to a high-capacity, low-affinity platelet binding site shared with prothrombin (FII) which then presents FX to a specific, high-affinity site consisting of FVIIIa bound to a high-affinity, low-capacity receptor on activated platelets. We have demonstrated the localization of FX in lipid rafts and shown that FX-raft association requires Ca2+ and is enhanced by saturating concentrations of FVIIIa. Here we investigate FII-raft association and define the domains through which shared FX and FII sites are mediated on the surface of human platelets. Activated (thrombin receptor peptide, SFLLRN, 25 μM) gel-filtered platelets (3.5 x 108/ml) were incubated with 125I-FII or 125I-FX to determine direct platelet binding and then they were lysed with Triton-X100 (0.025-0.25%) followed by sucrose density gradient centrifugation. FII was localized to lipid rafts in SFLLRN stimulated (~25% total binding) but not to unactivated platelets. The optimal associations of FX and FII with lipid rafts required Ca2+ and were not affected by the presence of EGR-FIXa or FIX (45 nM). The association of FII with lipid rafts was completely abolished in the presence of FX (1.5 μM) whereas, Gla (des) FX was unable to compete with raft associated FII. Similarly, 125I-FII fragment 1 association with lipid rafts was inhibited by FX but not by Gla (des) FX. Prothrombin and FII fragment 1 (residues 1-155) were equipotent inhibitors of FX-raft association. FVIIIa (20 nM) had no effect on FII-raft association but significantly increased (~2-fold to ~45%) FX-raft association. In contrast, the presence of FVa (20 nM) had no effect on FX-raft association but significantly (~2-fold to ~45%) increased FII-raft association. The structural integrity of lipid rafts was completely disrupted by 10 mM methyl-β-cyclodextrin (MβCD), a known cholesterol depleting drug, which completely prevented FII or FX association with lipid rafts, and this removal was reversed by cholesterol repletion. Furthermore, MβCD (up to 40 mM) had no effect on the amount of FII or FX bound to activated platelets, thus suggesting that neither platelet activation by SFLLRN nor the exposure of FII receptors was affected by MβCD treatment. These experiments demonstrate the localization of a shared FX/FII site in lipid rafts and support the hypothesis that these interactions are mediated by the Gla-domains of FX and FII and are specific and essential for the assembly of F-X activating complex on the activated platelet membrane.

Intracellular signaling triggered by antiphospholipid antibodies in platelets and endothelial cells: a pathway to targeted therapies

Understanding the intracellular events triggered by antiphospholipid (aPL) antibodies in platelets and endothelial cells (ECs) is important in designing new modalities of targeted therapies for the treatment of thrombosis in Antiphospholipid Syndrome (APS). A recent study showed a significant increase in the expression of GPIIb/IIIa on platelets treated with aPL antibodies and a thrombin receptor peptide agonist (TRAP), and these effects were abrogated by hydroxychloroquine (HQ). Hydroxychloroquine has also been shown to reduce in vivo aPL-induced thrombus formation. Furthermore, aPL-enhanced thrombosis in vivo can be abrogated by infusions of a GPIIb/IIIa antagonist (1B5) monoclonal antibody, and aPL-mediated thrombophilia is not observed in GPIIb/IIIa-deficient mice. Treatment of platelets with aPL antibodies has resulted in a significant increase in p38 mitogen-activated protein kinase (p38MAPK) phosphorylation and aPL-induced platelet aggregation and thromboxane B2 (TXB2) production was abrogated by SB203580 (a p38MAPK inhibitor). aPL antibodies induce increased expression, function and transcription of tissue factor (TF) on EC. Activation of ECs and thrombogenicity of aPL in vivo can be reversed by treatment of the animals with statins. Upregulation of TF on ECs can also be abrogated by treatment of the cells with fluvastatin. There is also indication of activation of nuclear factor kappa B (NFκB), increase in phosphorylation of p38MAPK in ECs by aPL antibodies that can be reversed by specific inhibitors MG132 and SB203580, respectively. The data open the possibility to new treatment modalities that may include the use of hydroxychloroquine, statins, specific antagonists of GPIIb/IIIa (such as abciximab or equivalent) and specific p38MAPK inhibitors, after the completion of well-designed clinical studies.

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.

Sustained MAP kinase activation is required for the expression of cyclin D1, p21Cip1 and a subset of AP-1 proteins in CCL39 cells.

In CCL39 cells thrombin is a potent growth factor which requires sustained activation of mitogen activated protein kinases (MAPKs) to promote DNA synthesis. Some of the effects of thrombin can be mimicked by peptides based on the new amino terminus of the cleaved receptor; however, these thrombin receptor peptides (TRPs) fail to induce sustained activation of MAPK or DNA synthesis. We have used thrombin, TRP-7 and other agonists which elicit sustained or transient MAPK activation to identify immediate-early and delayed-early genes which are only expressed under conditions of sustained MAPK activation focusing on cyclin D1, p21CiP1 and the AP-1 transcription factor. Of the stimuli tested only FBS and thrombin were able to stimulate a sustained activation of MAPK, expression of cyclin D1, p21Cip1 and cell cycle re-entry. The expression of cyclin D1 was strongly, though not completely, inhibited by the MEK1 inhibitor PD098059. Thrombin stimulated a rapid but transient accumulation of c-Fos whereas the expression of Fra-1, Fra-2, c-Jun and JunB was sustained throughout the G1 phase of the cell cycle. We focussed our analysis on c-Fos (typical of AP-1 genes which are expressed rapidly and transiently) and Fra-1 and JunB (typical of AP-1 genes expressed after a delay but in a sustained manner). The expression of c-Fos, Fra-1 and JunB was dependent upon the activation of MAPK since these responses were inhibited by PD098059. However, a comparison of responses to FBS, thrombin, TRPs, LPA and EGF revealed that Fra-1 and JunB expression required sustained activation of MAPK whereas c-Fos expression was strongly induced even by non-mitogenic stimuli which elicited only transient MAPK activation. The expression of c-Fos (in response to thrombin, TRP or LPA) or Fra-1, JunB and cyclin D1 (thrombin only) was also inhibited by pertussis toxin. This suggests that both early and late AP-1 gene expression is regulated by the same Gi-mediated, MEK-dependent MAPK signalling pathway but that expression of late AP-1 genes and cyclin D1 requires that this pathway be persistently activated. The results suggest that the duration of receptor signalling and therefore MAPK activation is a key determinant of qualitative changes in gene expression during cell cycle re-entry.

Tissue-Specific Expression of Functional Platelet Factor XI Is Independent of Plasma Factor XI Expression

AbstractPlatelet factor XI is an alternatively spliced product of the factor XI gene expressed specifically within megakaryocytes and platelets as an approximately 1.9-kb mRNA transcript (compared with ∼2.1 kb in liver cells) lacking exon V. Flow cytometry with an affinity-purified factor XI antibody, with PAC1 antibody (to the GPIIb/IIIa complex on activated platelets), and with S12 antibody (to P-selectin, an α-granule membrane protein expressed on the platelet surface during secretion) on platelets activated with ADP, thrombin, thrombin receptor peptide (SFLLRN amide), or collagen at various concentrations exposed platelet factor XI and PAC1 antibody binding in parallel. Unactivated platelets expressed approximately 40% of total platelet factor XI but no PAC1 binding sites. Enhanced membrane exposure of platelet factor XI is independent of α-granule secretion, because ADP and collagen exposed platelet factor XI but no S12 binding sites. Platelets from four patients with plasma factor XI deficiency (<0.04 U/mL) had normal constitutive and activation-dependent expression of platelet factor XI. Well-washed platelets from normal and from factor XI-deficient donors incubated with low concentrations of thrombin (0.05 to 0.1 U/mL) corrected the clotting defect observed with factor XI-deficient plasma. Thus, functionally active platelet factor XI is differentially expressed on platelet membranes in a tissue-specific manner both constitutively and in a concentration-dependent fashion by various agonists in the absence of detectable plasma factor XI.

Tissue-Specific Expression of Functional Platelet Factor XI Is Independent of Plasma Factor XI Expression

Platelet factor XI is an alternatively spliced product of the factor XI gene expressed specifically within megakaryocytes and platelets as an approximately 1.9-kb mRNA transcript (compared with approximately 2.1 kb in liver cells) lacking exon V. Flow cytometry with an affinity-purified factor XI antibody, with PAC1 antibody (to the GPIIb/IIIa complex on activated platelets), and with S12 antibody (to P-selectin, an alpha-granule membrane protein expressed on the platelet surface during secretion) on platelets activated with ADP, thrombin, thrombin receptor peptide (SFLLRN amide), or collagen at various concentrations exposed platelet factor XI and PAC1 antibody binding in parallel. Unactivated platelets expressed approximately 40% of total platelet factor XI but no PAC1 binding sites. Enhanced membrane exposure of platelet factor XI is independent of alpha-granule secretion, because ADP and collagen exposed platelet factor XI but no S12 binding sites. Platelets from four patients with plasma factor XI deficiency (<0.04 U/mL) had normal constitutive and activation-dependent expression of platelet factor XI. Well-washed platelets from normal and from factor XI-deficient donors incubated with low concentrations of thrombin (0. 05 to 0.1 U/mL) corrected the clotting defect observed with factor XI-deficient plasma. Thus, functionally active platelet factor XI is differentially expressed on platelet membranes in a tissue-specific manner both constitutively and in a concentration-dependent fashion by various agonists in the absence of detectable plasma factor XI.

Sol Sherry lecture in thrombosis: how thrombin 'talks' to cells: molecular mechanisms and roles in vivo.

This article is a summary of the Sol Sherry Lecture of the Council on Arteriosclerosis, Thrombosis, and Vascular Biology, which was presented at the 70th Scientific Sessions of the American Heart Association in November 1997. It highlights work from our laboratory addressing the molecular mechanisms by which the coagulation protease thrombin elicits cellular responses, notes some of the novel issues that protease signaling raises, and cites recent work on the role of thrombin signaling in vivo. Thrombin is a multifunctional serine protease. In adult animals, active thrombin is generated in the context of vascular injury when activation of the coagulation cascade triggers conversion of the circulating zymogen prothrombin to active protease. Thrombin generation may also be important in other contexts, as will be shown below. Several of thrombin’s functions involve cleavage of circulating protein substrates, eg, conversion of fibrinogen to fibrin monomer or activation of protein C. However, thrombin also has important actions on cells. It is the most potent activator of platelets.1 It causes endothelial cells to deliver the leukocyte adhesion molecule P-selectin to their surfaces,2 to secrete von Willebrand factor,2 and to elaborate growth factors and cytokines.3 4 It is also a mitogen for fibroblasts and vascular smooth muscle cells.5 Such cellular actions of thrombin raised several important questions. How does thrombin, a protease, act like a hormone to control cellular behaviors? And what are the roles of thrombin-regulated cellular events in vivo? Thrombin’s actions on platelets are of particular interest. Arterial thrombosis underlies most cases of unstable angina and myocardial infarction. Studies in animal models and clinical trials suggest that these events are both platelet dependent and thrombin dependent, but the relative contributions of and interactions between thrombin-induced platelet activation and fibrin formation in acute coronary syndromes are not known. By elucidating …

Requirement for Rho-mediated Myosin Light Chain Phosphorylation in Thrombin-stimulated Cell Rounding and Its Dissociation from Mitogenesis

Thrombin treatment causes a dose-dependent rounding of 1321N1 astrocytoma cells. This cytoskeletal response is rapid, peaking 2 h after thrombin stimulation, and reverses by 50% after 24 h. The thrombin receptor peptide SFLLRNP also induces cell rounding, whereas other G protein-linked receptor agonists such as carbachol, lysophosphatidic acid, or bradykinin fail to do so. Results of studies using pharmacological inhibitors do not support a requirement for phosphatidylinositol 3-kinase, mitogen-activated protein kinase, or Ca2+ mobilization in this response. Inhibition of protein kinase C or tyrosine kinase produces minimal blockade. Pertussis toxin treatment is also without effect. However, thrombin-induced rounding is fully blocked by the C3 toxin from Clostridium botulinum, which specifically ADP-ribosylates and inactivates the small G protein Rho. Thrombin also leads to a rapid, 2.4-fold increase in 32P incorporation into myosin light chain while carbachol does not. Myosin phosphorylation, like cell rounding is inhibited by inactivation of Rho with C3 exoenzyme, suggesting that myosin phosphorylation is necessary for this cytoskeletal response. This is supported by the observation that thrombin-induced rounding is also blocked by the myosin light chain kinase inhibitor KT5926. However, treatment with KT5926 fails to inhibit mitogenesis. Thus, cell rounding is not prerequisite to thrombin-induced DNA synthesis. We conclude that stimulation of the heterotrimeric G protein-coupled thrombin receptor in 1321N1 cells activates Rho-dependent pathways for both DNA synthesis and cell rounding, the cytoskeletal response being mediated in part through increases in myosin phosphorylation.

Mitogenic Responses Mediated Through the Proteinase-Activated Receptor-2 Are Induced by Expressed Forms of Mast Cell α- or β-Tryptases

AbstractThe proteinase-activated receptor-2 (PAR-2) is the second member of a putative larger class of proteolytically activated receptors that mediate cell activation events by receptor cleavage or synthetic peptidomimetics corresponding to the newly generated N-terminus. To further study the previously identified mitogenic effects of PAR-2, we used the interleukin-3 (IL-3)–dependent murine lymphoid cell line, BaF3, for generation of stable cell lines expressing PAR-2 (BaF3/PAR-2) or the noncleavable PAR-2 mutant PAR-2Arg36 → Ala36. Only BaF3 cells expressing either wild-type or mutated receptor exhibited mitogenic responses when grown in IL-3–deficient media supplemented with PAR-2 activating peptide (SLIGRL, PAR39-44). This effect was dose dependent with an EC50 of ∼80 μmol/L, sustained at 24, 48, and 72 hours, and was also demonstrable using thrombin receptor peptide TR42-47. Because tryptase shares ∼70% homology with trypsin (previously shown to activate PAR-2), we studied recombinantly expressed forms of α- and β-tryptases as candidate protease agonists for PAR-2. Hydrolytic activity of the chromogenic substrate tosyl-glycyl-prolyl-argly-4-nitroanilide acetate was present as a sharp peak at Mr ∼130, confirming the presence of secretable and functionally active homotetrameric α- and β-tryptases in transfected COS-1 cells. Dose-dependent proliferative responses were evident using either secreted form of tryptase with maximal responses seen at ∼3 pmol/L (0.1 U/L). Receptor proteolysis was necessary and sufficient for mitogenesis because active site-blocked tryptase failed to induce this response, and proliferative responses were abrogated in BaF3 cells expressing PAR-2Arg36 → Ala36. These results specifically identify both forms of mast cell tryptases as serine protease agonists for PAR-2 and have implications for elucidating molecular mechanisms regulating cellular activation events mediated by proteases generated during inflammatory, fibrinolytic, or hemostatic-regulated pathways.

Functional Interactions Between the Thrombin Receptor and the T-Cell Antigen Receptor in Human T-Cell Lines

AbstractThe proteolytically activated thrombin receptor (TR) is expressed by T lymphocytes, which suggests that thrombin may modulate T-cell activation at sites of hemostatic stress. We examined the relationship between TR function and T-cell activation in the Jurkat human T-cell line and in T-cell lines with defined defects in T-cell antigen receptor (TCR) function. Stimulation with thrombin or the synthetic TR peptide SFLLRN produced intracellular Ca2+ transients in Jurkat cells. As the concentration of TR agonist was increased, peak Ca2+ mobilization increased, but influx of extracellular Ca2+ decreased. TR signaling was enhanced in a TCR-negative Jurkat line and in T-cell lines deficient in the tyrosine kinase lck or the tyrosine phosphatase CD45, both of which are essential for normal TCR function. TCR cross-linking with anti-CD3 IgM desensitized TR signaling in Jurkat cells, but not in CD45-deficient cells. A proteinase-activated receptor (PAR-2)–specific agonist peptide, SLIGKV, produced small Ca2+ transients in both MEG-01 human megakaryocytic cells and Jurkat cells, but was less potent than the TR-specific agonist TFRIFD in both cell types. Like TR signaling, PAR-2 signaling was enhanced in TCR-negative or lck-deficient Jurkat clones. These findings provide evidence for functional cross-talk between proteolytically activated receptors and the TCR.

Suppression of the voltage-gated K + current of human megakaryocytes by thrombin and prostacyclin

We examined the effects of platelet activators and inhibitors of platelet function on the voltage-gated delayed rectifier K + current of human megakaryocytes. We found that both the activators such as thrombin, the thrombin receptor peptide (TRP 42–47) and ADP and the inhibitors such as prostacyclin suppressed the delayed rectifier current through two different mechanisms. The cAMP dependent protein kinase (A-kinase) inhibitor IP 20 blocked the suppression of the delayed rectifier current by prostacyclin and failed to block the suppression by thrombin, TRP 42–47 and ADP. The effects of IP 20 suggest that the action of prostacyclin is mediated by A-kinase and the action of the three activators is not mediated by A-kinase. Pertussis toxin (PTX) an inhibitor of the inhibitory GTP-binding proteins (G i) blocked the suppression of the delayed rectifier current by thrombin, TRP 42–47 and ADP and failed to block the suppression by prostacyclin. The effects of PTX suggests that the action of the three activators is mediated by G i or some other PTX-sensitive GTP-binding protein. We speculate that thrombin and other platelet activators that activate G i may be suppressing the delayed rectifier current via a direct interaction of G i or a subunit of it with the delayed rectifier potassium channel itself.

Interactions of Mast Cell Tryptase with Thrombin Receptors and PAR-2

Tryptase is a serine protease secreted by mast cells that is able to activate other cells. In the present studies we have tested whether these responses could be mediated by thrombin receptors or PAR-2, two G-protein-coupled receptors that are activated by proteolysis. When added to a peptide corresponding to the N terminus of PAR-2, tryptase cleaved the peptide at the activating site, but at higher concentrations it also cleaved downstream, as did trypsin, a known activator of PAR-2. Thrombin, factor Xa, plasmin, urokinase, plasma kallikrein, and tissue kallikrein had no effect. Tryptase also cleaved the analogous thrombin receptor peptide at the activating site but less efficiently. When added to COS-1 cells expressing either receptor, tryptase stimulated phosphoinositide hydrolysis. With PAR-2, this response was half-maximal at 1 nM tryptase and could be inhibited by the tryptase inhibitor, APC366, or by antibodies to tryptase and PAR-2. When added to human endothelial cells, which normally express PAR-2 and thrombin receptors, or keratinocytes, which express only PAR-2, tryptase caused an increase in cytosolic Ca2+. However, when added to platelets or CHRF-288 cells, which express thrombin receptors but not PAR-2, tryptase caused neither aggregation nor increased Ca2+. These results show that 1) tryptase has the potential to activate both PAR-2 and thrombin receptors; 2) for PAR-2, this potential is realized, although cleavage at secondary sites may limit activation, particularly at higher tryptase concentrations; and 3) in contrast, although tryptase clearly activates thrombin receptors in COS-1 cells, it does not appear to cleave endogenous thrombin receptors in platelets or CHRF-288 cells. These distinctions correlate with the observed differences in the rate of cleavage of the PAR-2 and thrombin receptor peptides by tryptase. Tryptase is the first protease other than trypsin that has been shown to activate human PAR-2. Its presence within mast cell granules places it in tissues where PAR-2 is expressed but trypsin is unlikely to reach.

Thrombin Interaction with Platelet GPIB: Role of the Heparin Binding Domain

The platelet membrane glycoprotein Ib (GpIb) has a high affinity binding site for alpha-thrombin whose occupancy is thought to positively modulate the thrombin-induced platelet activation. In this study, aimed at further characterizing the thrombin-GpIb interaction, two thrombin anion exosites referred to as "heparin binding site" (HBS) and "fibrinogen recognition site" (FRS) were investigated as the possible domains involved in GpIb binding. The role of thrombin HBS was explored by performing binding measurements of 125I-alpha-thrombin to purified glycocalicin (GC), the extracytoplasmic portion of GpIb, in the presence of heparin as well as after chemical modifications of the thrombin heparin binding site (thrombin-HBS phosphopyridoxylation). These studies showed that a) thrombin binding to GC could be competitively inhibited by heparin and b) the equilibrium association constant for thrombin-GC interaction was reduced up to ten-fold by chemical modifications at the HBS. On the other hand, the role of FRS in the thrombin-GC interaction could be excluded by other experiments showing that GC in solution could not influence the interaction of alpha-thrombin with two substrates which bind to both the catalytic site and the fibrinogen recognition site: 1) the thrombin receptor peptide 38-60 (TR, L38-E60) and 2) the A alpha-chain of fibrinogen. Altogether these results demonstrated that GC interaction with thrombin involves the enzyme heparin binding site, whereas the fibrinogen recognition site does not play a significant role.

Direct evidence for two distinct G proteins coupling with thrombin receptors in human neuroblastoma SH-EP cells

Thrombin receptor-G protein coupling was investigated in the human epithelial neuroblastoma cell line, SH-EP. In these cells, both α-thrombin and thrombin receptor peptides, SFLLRNP (one-letter amino-acid code), which are newly exposed following cleavage by α-thrombin, stimulated GTPase activity about 2-fold over basal activity. Pertussis toxin treatment only partially attenuated α-thrombin-and SFLLRNP-stimulated GTPase activity by 50%, whereas antibody raised against synthetic heptapeptide SFLLRNP blocked α-thrombin-stimulated phosphoinositide hydrolysis more than 80%. Immunoprecipitation studies using this antibody showed that both G i2, a subtype of guanine nucleotide-binding regulatory proteins (G proteins) mediating inhibition of adenylyl cyclase, and G q G 11 , a G protein mediating stimulation of phospholipase C, were activated by α-thrombin. These data suggest that in these cells the thrombin receptor activates pertussis toxin-sensitive and pertussis toxin-insensitive G proteins simultaneously and directly couples to G i2 and G q G 11 , which mediate different signaling pathways.