Collagen, Convulxin, and Thrombin Stimulate Aggregation-independent Tyrosine Phosphorylation of CD31 in Platelets: EVIDENCE FOR THE INVOLVEMENT OF Src FAMILY KINASES

Abstract

Platelet endothelial cell adhesion molecule-1 (CD31) is a 130-kDa glycoprotein receptor present on the surface of platelets, neutrophils, monocytes, certain T-lymphocytes, and vascular endothelial cells. CD31 is involved in adhesion and signal transduction and is implicated in the regulation of a number of cellular processes. These include transendothelial migration of leukocytes, integrin regulation, and T-cell function, although its function in platelets remains unclear. In this study, we demonstrate the ability of the platelet agonists collagen, convulxin, and thrombin to induce tyrosine phosphorylation of CD31. Furthermore, we show that this event is independent of platelet aggregation and secretion and is accompanied by an increase in surface expression of CD31. A kinase capable of phosphorylating CD31 was detected in CD31 immunoprecipitates, and its activity was increased following activation of platelets. CD31 tyrosine phosphorylation was reduced or abolished by the Src family kinase inhibitor PP2, suggesting a role for these enzymes. In accordance with this, each of the Src family members expressed in platelets, namely Fyn, Lyn, Src, Yes, and Hck, was shown to co-immunoprecipitate with CD31. The involvement of Src family kinases in this process was confirmed through the study of mouse platelets deficient in Fyn. Platelet endothelial cell adhesion molecule-1 (CD31) is a 130-kDa glycoprotein receptor present on the surface of platelets, neutrophils, monocytes, certain T-lymphocytes, and vascular endothelial cells. CD31 is involved in adhesion and signal transduction and is implicated in the regulation of a number of cellular processes. These include transendothelial migration of leukocytes, integrin regulation, and T-cell function, although its function in platelets remains unclear. In this study, we demonstrate the ability of the platelet agonists collagen, convulxin, and thrombin to induce tyrosine phosphorylation of CD31. Furthermore, we show that this event is independent of platelet aggregation and secretion and is accompanied by an increase in surface expression of CD31. A kinase capable of phosphorylating CD31 was detected in CD31 immunoprecipitates, and its activity was increased following activation of platelets. CD31 tyrosine phosphorylation was reduced or abolished by the Src family kinase inhibitor PP2, suggesting a role for these enzymes. In accordance with this, each of the Src family members expressed in platelets, namely Fyn, Lyn, Src, Yes, and Hck, was shown to co-immunoprecipitate with CD31. The involvement of Src family kinases in this process was confirmed through the study of mouse platelets deficient in Fyn. high affinity receptor for immunoglobin E Fc receptor immunoreceptor tyrosine-based inhibitory motif immunoreceptor tyrosine-based activation motif glycoprotein VI protein phosphatase convulxin Tris-buffered saline polyacrylamide gel electrophoresis 5-hydroxytryptamine Platelet endothelial cell adhesion molecule-1 (CD31) is a membrane-spanning glycoprotein of 130 kDa that is expressed on the surface of platelets, endothelial cells, neutrophils, monocytes, and some T-lymphocyte subsets (1Ohto H. Maeda H. Shibata Y. Chen C.Y. Ozaki Y. Higashihara M. Takeuchi A. Tohyama H. Blood. 1985; 66: 873-881Crossref PubMed Google Scholar, 2Muller W.A. Ratti C.M. McDonnell S.L. Cohn Z.A. J. Exp. Med. 1989; 170: 399-414Crossref PubMed Scopus (292) Google Scholar, 3Albelda S.M. Oliver P.D. Romer L.H. Buck C.A. J. Cell Biol. 1990; 110: 1227-1237Crossref PubMed Scopus (333) Google Scholar, 4Newman P.J. Berndt M.J. Gorski J. White G.C. Lyman S. Paddock C. Muller W.A. Science. 1990; 247: 1219-1222Crossref PubMed Scopus (826) Google Scholar). The functions that have been identified for CD31 are diverse and include transendothelial migration of leukocytes (5Muller W.A. Weigl S.A. Deng X. Phillips D.M. J. Exp. Med. 1993; 178: 449-460Crossref PubMed Scopus (989) Google Scholar, 6Vaporciyan A.A. DeLisser H.M. Yan H.-C. Mendiguren I.I. Thom S.R. Jones M.L. Ward P.A. Albelda S.M. Science. 1993; 262: 1580-1582Crossref PubMed Scopus (430) Google Scholar), angiogenesis (3Albelda S.M. Oliver P.D. Romer L.H. Buck C.A. J. Cell Biol. 1990; 110: 1227-1237Crossref PubMed Scopus (333) Google Scholar, 7Matsumura P.W. Wolff K. Petzelbauer P. J. Immunol. 1997; 158: 3408-3416PubMed Google Scholar) vasculogenesis (8Brier G. Brevario F. Caveda L. Berthier R. Schnurch H. Gotsch U. Vestweber D. Risau W. Dejana E. Blood. 1996; 87: 630-641Crossref PubMed Google Scholar), integrin regulation (9Tanaka Y. Albelda S.M. Horgan K.J. van Seventer G.A. Shimizu Y. Newman W. Hallam J. Newman P.J. Buck C.A. Shaw S. J. Exp. Med. 1992; 176: 245-253Crossref PubMed Scopus (341) Google Scholar, 10Piali L. Albelda S.M. Baldwin H.S. Hammel P. Gisler R.H. Imhof B.A. Eur. J. Immunol. 1993; 23: 2464-2471Crossref PubMed Scopus (122) Google Scholar), and T-cell receptor function (11Newton-Nash D.K. Newman P.J. J. Immunol. 1999; 163: 682-688PubMed Google Scholar). The cloning of CD31 was as a consequence of its expression on platelets (4Newman P.J. Berndt M.J. Gorski J. White G.C. Lyman S. Paddock C. Muller W.A. Science. 1990; 247: 1219-1222Crossref PubMed Scopus (826) Google Scholar); however, a clear role for CD31 in platelets remains obscure. CD31 was classified to the family of cellular adhesion molecules on the basis of structural and amino acid sequence. Homophilic ligand binding interactions are believed to underlie the functions of CD31, although heterophilic interactions with integrin αVβ3, CD38, and unidentified proteins have been suggested (12Buckley C.D. Doyonnas R. Newton J.P. Blystone S.D. Brown E.J. Watt S.M. Simmons D.L. J. Cell Sci. 1996; 109: 437-445PubMed Google Scholar, 13Deaglio S. Morra M. Mallone R. Ausiello C.M. Prager E. Garbarino G. Dianzani U. Stockinger H. Malavasi F. J. Immunol. 1998; 160: 395-402PubMed Google Scholar). In recent years, considerable attention has been directed to the ability of CD31 to participate in signal transduction. Numerous studies have shown that CD31 becomes tyrosine-phosphorylated in response to a variety of conditions that include CD31 cross-linking (14Edmead C.E. Crosby D.A. Southcott M. Poole A.W. FEBS Lett. 1999; 459: 27-32Crossref PubMed Scopus (27) Google Scholar), activation of the high affinity receptor for immunoglobulin E (FcεRI)1 (15Sagawa K. Swaim W. Zhang J. Unsworth E. Siraganian R.P. J. Biol. Chem. 1997; 272: 13412-13418Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar), platelet aggregation (16Jackson D.E. Ward C.M. Wang R. Newman P.J. J. Biol. Chem. 1997; 272: 6986-6993Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar), shear (17Osawa M. Masuda M. Harada N. Lopes R.B. Fugiwara K. Eur. J. Cell Biol. 1997; 72: 229-237PubMed Google Scholar), and oxidative stress (18Wang R. Paddock C. Augustine J.A. Newman P.J. Blood. 1998; 92 (abstr.): 548aCrossref Google Scholar). The sites of CD31 tyrosine phosphorylation have been mapped to two residues that fall within a conserved signaling motif known as an immunoreceptor tyrosine-based inhibitory motif (ITIM). ITIMs are found in an increasing list of receptors that regulate immune function and are defined by the consensus sequence (L/I/V/S)XYXX(L/V). Many proteins, including CD31, contain tandem ITIMs separated by a 15–30-amino acid residue sequence (19Vivier E. Daeron M. Immunol. Today. 1997; 18: 286-291Abstract Full Text PDF PubMed Scopus (327) Google Scholar). The phosphorylated ITIM provides a docking site for signaling proteins that bind via SH2 domains. The protein-tyrosine phosphatases SHP-2 and SHP-1 have been shown to associate with tyrosine-phosphorylated CD31 (16Jackson D.E. Ward C.M. Wang R. Newman P.J. J. Biol. Chem. 1997; 272: 6986-6993Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, 20Hua C.T. Gamble J.R. Vadas M.A. Jackson D.E. J. Biol. Chem. 1998; 273: 28332-28340Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar); and for SHP-2, this interaction has been shown in vitro to stimulate its activation (14Edmead C.E. Crosby D.A. Southcott M. Poole A.W. FEBS Lett. 1999; 459: 27-32Crossref PubMed Scopus (27) Google Scholar). As a consequence, ITIM-containing receptors may counter the effects of tyrosine kinases and thereby negatively regulate signaling pathways, although SHP-2 has been shown to positively regulate growth factor receptor signaling. It has been suggested recently that CD31 would be more correctly classified as a member of the Ig ITIM family of proteins (21Newman P.J. J. Clin. Invest. 1999; 103: 5-9Crossref PubMed Scopus (235) Google Scholar). In contrast to the effects of ITIM-containing receptors are the functions of a number of receptors that possess a conserved signaling motif termed the immunoreceptor tyrosine-based activation motif (ITAM). The consensus sequence for this motif is YXXLX (6–8)YXXL (22Reth M. Nature. 1989; 338: 383-384Crossref PubMed Scopus (1166) Google Scholar) and is found on the cytoplasmic tail of several immune receptors, including T- and B-cell antigen receptors and a number of immunoglobulin receptors (23Qian D. Weiss A. Curr. Opin. Cell Biol. 1997; 9: 205-212Crossref PubMed Scopus (286) Google Scholar, 24Kurosaki T. Curr. Opin. Immunol. 1997; 9: 309-318Crossref PubMed Scopus (181) Google Scholar). It is believed that ITAM- and ITIM-bearing receptors may in some contexts act antagonistically when expressed on the same cell. Indeed, a number of closely related receptors, such as FcγRIIa (ITAM) and FcγRIIb (ITIM), are thought to function in this way (25Daeron M. Latour S. Malbec O. Espinosa E. Pina P. Pasmans S. Fridman W.H. Immunity. 1995; 3: 635-644Abstract Full Text PDF PubMed Scopus (388) Google Scholar). We and others have recently identified the collagen receptor that underlies platelet activation. Collagens are principal platelet agonists at sites of vascular damage and are therefore central to the process of hemostasis. This collagen receptor comprises the recently cloned glycoprotein VI (GPVI) (26Gibbins J.M. Okuma M. Farndale R. Barnes M. Watson S.P. FEBS Lett. 1997; 413: 255-259Crossref PubMed Scopus (261) Google Scholar, 27Tsuji M. Ezumi Y. Arai M. Takayama H. J. Biol. Chem. 1997; 272: 23528-23531Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar, 28Clemetson J.M. Polgar J. Magnenat E. Wells T.N.C. Clemetson K.J. J. Biol. Chem. 1999; 274: 29019-29024Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar) and the Fc receptor (FcR) γ-chain (29Gibbins J. Asselin J. Farndale R. Barnes M. Law C.L. Watson S.P. J. Biol. Chem. 1996; 271: 18095-18099Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). The FcR γ-chain contains an ITAM within its cytoplasmic domain that is tyrosine-phosphorylated upon collagen stimulation. The phosphorylated ITAM is responsible for recruiting signaling molecules such as Syk and phosphatidylinositol 3-kinase to the receptors; and thus, multiple signaling pathways are activated, leading to platelet secretion and aggregation (29Gibbins J. Asselin J. Farndale R. Barnes M. Law C.L. Watson S.P. J. Biol. Chem. 1996; 271: 18095-18099Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 30Poole A. Gibbins J.M. Turner M. van Vugt M.J. van de Winkel J.G.J. Saito T. Tybulewicz V.L.J. Watson S.P. EMBO J. 1997; 16: 2333-2341Crossref PubMed Scopus (396) Google Scholar, 31Gibbins J.M. Briddon S. Shutes A. van Vugt M.J. van de Winkel J.G. Saito T. Watson S.P. J. Biol. Chem. 1998; 273: 34437-34443Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). The role of the collagen receptor GPVI in CD31 signaling in platelets is particularly interesting since this receptor signals through an ITAM. In this study, we have investigated the role of this collagen receptor in the stimulation of CD31 tyrosine phosphorylation. We demonstrate that activation of this receptor and also platelet thrombin receptors results in tyrosine phosphorylation of CD31 and that this is not dependent on integrin function or platelet aggregation. We show that platelet activation by GPVI and thrombin receptors leads to an increase in the platelet surface expression of CD31 and present data suggesting that Src family kinases are responsible for CD31 tyrosine phosphorylation in platelets. Horm-Chemie collagen (collagen fibers from equine tendons) was purchased from Nycomed (Munich, Germany). Convulxin was purified from the venom of the rattlesnake (Crotalus durissus terrificus) as described previously (32Francischetti I.M.B. Saliou B. Leduc M. Carlini C.R. Hatmi M. Randon J. Faili A. Bon C. Toxicon. 1997; 35: 1217-1228Crossref PubMed Scopus (96) Google Scholar). Anti-phosphotyrosine monoclonal antibody 4G10 was from Upstate Biotechnology, Inc. (Buckinghamshire, UK). Anti-CD31 monoclonal antibody HC1/6 was from Serotec (Oxford, UK); anti-CD31 polyclonal (C-20 and M-20) and monoclonal (AB468F and control antibody AB600F) antibodies were from AutogenBioclear Ltd. (Wiltshire, UK). Anti-Fyn, anti-Lyn, anti-Src, anti-Yes, and anti-Hck polyclonal antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and anti-Fyn (clone 25) and anti-Lyn (clone 42) monoclonal antibodies were from Transduction Laboratories (Lexington, KY). Horseradish peroxidase-conjugated secondary antibodies and the enhanced chemiluminescence detection system was purchased from Amersham Pharmacia Biotech (Buckinghamshire). The Src family kinase inhibitor PP2 and negative control PP3 were purchased from Calbiochem-Novabiochem (Nottingham, UK). Fyn-deficient mice were generated as described elsewhere (33Stein P.L. Lee H.M. Rich S. Soriano P. Cell. 1992; 70: 741-750Abstract Full Text PDF PubMed Scopus (500) Google Scholar) and kindly provided by Dr. Steve Watson (University Department of Pharmacology, Oxford). All other reagents were from previously described sources (29Gibbins J. Asselin J. Farndale R. Barnes M. Law C.L. Watson S.P. J. Biol. Chem. 1996; 271: 18095-18099Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 31Gibbins J.M. Briddon S. Shutes A. van Vugt M.J. van de Winkel J.G. Saito T. Watson S.P. J. Biol. Chem. 1998; 273: 34437-34443Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). Human platelets from drug-free volunteers were prepared on the day of the experiment by differential centrifugation as described previously (31Gibbins J.M. Briddon S. Shutes A. van Vugt M.J. van de Winkel J.G. Saito T. Watson S.P. J. Biol. Chem. 1998; 273: 34437-34443Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar) and suspended in modified Tyrode's/Hepes buffer (134 mm NaCl, 0.34 mm Na2HPO4, 2.9 mm KCl, 12 mm NaHCO3, 20 mm Hepes, 5 mm glucose, and 1 mm MgCl2, pH 7.3) to a density of 8 × 108 cells/ml. Stimulation of platelets (450 μl) with collagen, convulxin (Cvx), and thrombin (delivered in 50 μl) was performed at 37 °C in an aggregometer with continuous stirring (1200 rpm). Mouse platelets were prepared as described previously (30Poole A. Gibbins J.M. Turner M. van Vugt M.J. van de Winkel J.G.J. Saito T. Tybulewicz V.L.J. Watson S.P. EMBO J. 1997; 16: 2333-2341Crossref PubMed Scopus (396) Google Scholar) and suspended in modified Tyrode's/Hepes buffer to a density of 1.3 × 108cells/ml. 90 μl of suspension was used for each assay with collagen, Cvx, or thrombin, added in a volume of 10 μl. For protein precipitation experiments, platelets were resuspended in buffer containing 1 mm EGTA to prevent aggregation. In certain experiments, platelets were preincubated prior to stimulation with the tetrapeptide RGDS (0.5 mm) (16Jackson D.E. Ward C.M. Wang R. Newman P.J. J. Biol. Chem. 1997; 272: 6986-6993Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar) or the fibrinogen γ-peptide DVHHLGGAKQAG (125 μm) (34Farrell D.H. Thiagarajan P. Chung D.W. Davie E.W. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10729-10732Crossref PubMed Scopus (301) Google Scholar). Where required, inhibitors and other reagents were added such that the final solvent concentration was 0.2% (v/v) and incubated at 37 °C for 5 min before stimulation. Apyrase was prepared in modified Tyrode's/Hepes buffer, and indomethacin, PP2, and PP3 were dissolved in Me2SO. Platelet stimulation was terminated by the addition of an equal volume of ice-cold lysis buffer (2% (v/v) Nonidet P-40 or 2% (v/v) Brij 96, 20 mm Tris, 300 mm NaCl, 10 mm EDTA, 1 mmphenylmethylsulfonyl fluoride, 2 mmNa3VO4, 10 μg/ml leupeptin, 10 μg/ml aprotinin, and 1 μg/ml pepstatin A, pH 7.3). Detergent-insoluble debris was removed, and the lysates were precleared by mixing with protein A-Sepharose for 1 h at 4 °C (20 μl of a 50% (w/v) suspension of protein A-Sepharose in Tris-buffered saline/Tween (TBS/T; 20 mm Tris, 137 mm NaCl, and 0.1% (v/v) Tween 20, pH 7.6)). Protein A-Sepharose was removed from the lysates before the addition of relevant antibodies (1 or 2.5 μg of anti-CD31 or 1 μg of anti-Lyn, anti-Fyn, anti-Src, anti-Yes, or anti-Hck). Following rotation at 4 °C for 1 h, 0.5 μl of secondary antiserum was added where required (CD31 (human platelets), Lyn, and Fyn: rabbit anti-mouse IgG; and CD31 (mouse platelets) and Src: sheep anti-goat IgG) and mixed for a further 30 min. 25 μl of protein A-Sepharose suspension was added to each sample, and mixing was continued for 1 h before washing the Sepharose pellet in lysis buffer, followed by a second wash in TBS/T and the addition of Laemmli sample treatment buffer. Proteins were separated by SDS-PAGE using 10% gels and transferred to polyvinylidene difluoride membranes by semidry Western blotting. Following Western blotting, polyvinylidene difluoride membranes were blocked by incubation in 10% (w/v) bovine serum albumin dissolved in TBS/T. Primary and secondary antibodies were diluted in TBS/T containing 2% (w/v) bovine serum albumin and incubated with polyvinylidene difluoride membranes for 1 h at room temperature. Blots were washed for 2 h in TBS/T following each incubation with antibodies for 1 h at room temperature and then developed using the enhanced chemiluminescence detection system. Primary antibodies were used at a concentration of 1 μg/ml (anti-phosphotyrosine, anti-CD31, anti-Lyn, anti-Fyn, anti-Src, anti-Yes, and anti-Hck). Horseradish peroxidase-conjugated secondary antibodies were diluted 1:10,000. Platelets were loaded with 5-[3H]HT by incubation with 0.5 μCi/ml platelet-rich plasma for 1 h at 37 °C. Platelets were prepared from the platelet-rich plasma as described above. Stimulation of platelets was terminated by the addition of an equal volume of 6% glutaraldehyde and microcentrifugation, and the level of 5-[3H]HT released into the supernatant was determined by scintillation spectrometry. 5-[3H]HT release is expressed as a percentage of the total tissue content following subtraction of release under basal conditions. Human platelets were stimulated at a density of 8 × 108 cells/ml with Cvx (62.5 or 125 ng/ml) or thrombin (0.1 or 1 unit/ml) in the presence of EGTA (1 mm), indomethacin (10 μm), and apyrase (2 units/ml). Stimulation was terminated by dilution to 1 × 108 cells/ml by the addition of ice-cold buffer (modified Tyrode's/Hepes buffer, pH 7.3, containing 1% (w/v) bovine serum albumin, 1 mm EGTA, and 0.04% (w/v) sodium azide). Platelet suspensions were then incubated in the dark at room temperature for 1 h with a fluorescein isothiocyanate-conjugated anti-CD31 monoclonal antibody (AB468F) diluted to 1:200. Nonspecific antibody binding was determined using a fluorescein isothiocyanate-conjugated isotype-matched IgG (AB600F). Data were collected using a FACScanTM flow cytometer (Becton Dickinson) and analyzed using CELLQuestTM software (Becton Dickinson). CD31 was immunoprecipitated as described above and assayed for kinase activity while immobilized on protein-A Sepharose following the final wash step. The supernatant was removed and replaced with assay buffer (105 mm NaCl, 20 mm Hepes, 5 mm MnCl2, 5 mm MgCl2, 10 μm ATP, and 5 μCi of [γ-32P]ATP, pH 7.4). Samples were incubated at room temperature for 20 min, whereupon reactions were terminated by the addition of reducing Laemmli SDS-PAGE sample treatment buffer. Samples were boiled, and proteins were separated by SDS-PAGE on 10% slab gels and Western-blotted onto polyvinylidene difluoride membranes. Blots were exposed to storage phosphor screens and developed on a PhosphorImager SITM (Amersham Pharmacia Biotech) to detect the incorporation of 32P into proteins present in the immunoprecipitate. Densitometry was performed using ImageQuant software (Amersham Pharmacia Biotech). The identity of 32P-labeled proteins was confirmed by immunoblotting. At sites of vascular damage, platelets may be activated in response to a number of factors, among which the extracellular matrix protein collagen and the enzyme thrombin are considered to be the most thrombogenic. Collagen-stimulated activation is mediated through a tyrosine kinase-dependent pathway via the receptor GPVI, whereas thrombin stimulates an alternative signaling pathway via activation of G protein-coupled thrombin receptors. Since the physiological significance of CD31 in platelet function has not been determined, we sought to characterize the effect of collagen- and thrombin receptor-mediated platelet activation on the tyrosine phosphorylation state of CD31. In addition to use of collagen and thrombin, platelets were also stimulated with Cvx, a protein purified from the venom of the rattlesnake (C. durissus terrificus) that is a selective GPVI agonist (31Gibbins J.M. Briddon S. Shutes A. van Vugt M.J. van de Winkel J.G. Saito T. Watson S.P. J. Biol. Chem. 1998; 273: 34437-34443Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 32Francischetti I.M.B. Saliou B. Leduc M. Carlini C.R. Hatmi M. Randon J. Faili A. Bon C. Toxicon. 1997; 35: 1217-1228Crossref PubMed Scopus (96) Google Scholar). Platelets were stimulated with collagen (10–100 μg/ml), Cvx (62.5–500 ng/ml), or thrombin (0.01–0.5 units/ml) for 90 s, and CD31 was immunoprecipitated from cell lysates. Proteins were separated by SDS-PAGE and immunoblotted to detect tyrosine phosphorylation. Each of the platelet agonists induced robust tyrosine phosphorylation of CD31 in a dose-dependent manner (Fig.1 A). With collagen, tyrosine phosphorylation was detectable at a concentration of 10 μg/ml and reached maximal phosphorylation at ∼100 μg/ml. Cvx is a very potent GPVI agonist (32Francischetti I.M.B. Saliou B. Leduc M. Carlini C.R. Hatmi M. Randon J. Faili A. Bon C. Toxicon. 1997; 35: 1217-1228Crossref PubMed Scopus (96) Google Scholar) and stimulated maximal tyrosine phosphorylation at 62.5 ng/ml. The level of CD31 tyrosine phosphorylation upon thrombin stimulation peaked at 0.1 unit/ml; however, at higher concentrations, additional tyrosine-phosphorylated proteins were co-immunoprecipitated. These unidentified proteins were also detected upon stimulation of platelets with supramaximal concentrations of collagen and convulxin (data not shown). Src family kinases have been implicated in the phosphorylation of CD31. Therefore, the possibility that the smaller tyrosine-phosphorylated protein that co-immunoprecipitated with CD31 (Fig. 1 A) was a Src family kinase was investigated. The protein did not comigrate precisely with any of five Src family kinase members that are expressed in platelets. We report below that such kinases do associate with CD31 in platelets; however, this is not dependent on platelet activation. The larger tyrosine-phosphorylated co-immunoprecipitated protein has an apparent molecular mass of 68 kDa (determined using QuantityOne® software, Bio-Rad) and is likely to be SHP-2, whose association with tyrosine-phosphorylated CD31 is well documented. The kinetics of CD31 tyrosine phosphorylation stimulated with these agonists were examined and are shown in Fig. 1 B. Collagen (100 μg/ml)-stimulated tyrosine phosphorylation was detectable at 20 s of stimulation, and the level peaked at 90 s. This kinetic profile is very similar to that previously reported for the tyrosine phosphorylation of the FcR γ-chain upon stimulation of platelets with collagen (29Gibbins J. Asselin J. Farndale R. Barnes M. Law C.L. Watson S.P. J. Biol. Chem. 1996; 271: 18095-18099Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar) and is consistent with this being an early signaling event. Cvx-stimulated tyrosine phosphorylation was rapid and reached maximal levels after 20 s of stimulation. Stimulation with thrombin also resulted in rapid tyrosine phosphorylation of CD31 that was detectable at 20 s; however, levels continued to rise over 300 s. The stimulation of CD31 tyrosine phosphorylation in platelets following incubation with thrombin-related activatory peptide has been reported to be dependent on aggregation and integrin function. The results discussed above were therefore surprising since the stimulations were performed in the presence of EGTA (1 mm), which prevented platelet aggregation by chelating extracellular calcium that is required for stabilization and function of the fibrinogen receptor integrin αIIbβ3. To confirm this observation, platelets were stimulated with Cvx (62.5 ng/ml) or thrombin (0.1 unit/ml) under alternative conditions that prevent fibrinogen binding to integrin αIIbβ3 and thereby block aggregation (Fig.2 A). Tyrosine phosphorylation was induced by both agonists in the presence of EGTA, the tetrapeptide RGDS (0.5 mm), or the fibrinogen γ-chain peptide (125 μm). The treatments alone did not affect the basal levels of CD31 tyrosine phosphorylation (data not shown), and the differences in the levels of CD31 tyrosine phosphorylation in resting platelets are a consequence of variability between donors and platelet preparations. Aggregation assays were performed on the samples and confirmed complete inhibition of aggregation (data not shown). Platelet activation leads to degranulation and the release or secretion of various factors that further enhance platelet activation. These include thromboxane A2 formed from liberated arachidonic acid via the cyclooxygenase pathway and ADP, which is secreted from dense granules. To determine whether collagen-, Cvx-, or thrombin-stimulated CD31 tyrosine phosphorylation was dependent on the release of thromboxane A2, platelets were stimulated in the presence or absence of indomethacin (10 μm), an inhibitor of cyclooxygenase. Fig. 2 B shows that the level of CD31 tyrosine phosphorylation was not altered in the presence of indomethacin. Furthermore, the inclusion of apyrase (2 units/ml), which catalyzes the conversion of ADP to AMP, had no effect on the level of CD31 tyrosine phosphorylation stimulated by these agonists (Fig.2 B). Slightly different levels of CD31 tyrosine phosphorylation in unstimulated cells in the two experiments shown in Fig. 2 are due to donor and platelet preparation variability. Platelet degranulation following stimulation may be assessed through measurement of 5-HT secretion. Fig. 2 C(panel i) shows the results of 5-HT secretion assays performed on cells stimulated in the absence or presence of EGTA (1 mm), indomethacin (10 μm), and apyrase (2 units/ml). High levels of 5-HT secretion were stimulated with Cvx (62.5 ng/ml) and thrombin (0.1 unit/ml), and this was almost abolished in the presence of EGTA, indomethacin, and apyrase (Cvx, reduced from 79 ± 1.7 to 6.2 ± 3.0%; and thrombin, reduced from 61.8 ± 6.5 to 5.6 ± 4.7% (mean ± S.E., n = 3)). However, Cvx- and thrombin-stimulated tyrosine phosphorylation of CD31 was maintained under these conditions (Fig. 2 C, panel ii). These data support the notion that tyrosine phosphorylation of CD31 following stimulation of the GPVI collagen receptor or the thrombin receptor is an early signaling event that is not dependent on the activity of factors released by the platelets. All subsequent experiments were performed in the presence of EGTA (1 mm), indomethacin (10 μm), and apyrase (2 units/ml). CD31 has been reported to participate in both homophilic and heterophilic binding, and CD31 dimerization or clustering has been shown to stimulate its tyrosine phosphorylation. However, the binding characteristics of this protein on platelets have not been studied. It was not clear from our experiments whether CD31 tyrosine phosphorylation was a direct consequence of intracellular signaling following stimulation of the collagen and thrombin receptors or whether CD31-ligand binding may contribute to the process. The stirring of platelets increases cell-cell collisions and increases the likelihood of intercellular ligand binding of CD31, which may lead to increased tyrosine phosphorylation of the protein. In accordance with this, we observed that stirring platelets induced low-level and time-dependent tyrosine phosphorylation of CD31. This effect is shown in CD31 immunoprecipitates from stirred platelets in Fig. 3 A. The level of tyrosine phosphorylation was considerably lower than when stimulated with collagen, Cvx, or thrombin and continued to rise over a period of 5 min. An increase in CD31 tyrosine phosphorylation upon stimulation with collagen, Cvx, and thrombin may be a direct consequence of activated intracellular signaling mechanisms and/or CD31-ligand binding. If CD31-ligand binding is important for this process, one may expect to observe an increased capacity for such binding in platelets stimulated with Cvx or thrombin. To examine this, the level of cell-surface expression of CD31 in resting platelets and platelets stimulated with Cvx or thrombin was examined by flow cytometry using a fluorescein isothiocyanate-conjugated anti-CD31 antibody. The results shown in Fig. 3 B demonstrate that the cell-surface expression of CD31 was increased following stimulation of platelets with Cvx or thrombin in a dose-dependent manner. The mean fluorescence levels f