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The Pro33 Isoform of Integrin β3 Enhances Outside-in Signaling in Human Platelets by Regulating the Activation of Serine/Threonine Phosphatases

2005; Elsevier BV; Volume: 280; Issue: 23 Linguagem: Inglês

10.1074/jbc.m500872200

ISSN

1083-351X

Autores

K. Vinod Vijayan, Yan Liu, Wensheng Sun, Masaaki Ito, Paul F. Bray,

Tópico(s)

Blood properties and coagulation

Resumo

Integrin β3 is polymorphic at residue 33 (Leu33 or Pro33), and the Pro33-positive platelets display enhanced aggregation, P-selectin secretion, and shorter bleeding times. Because outside-in signaling is critical for platelet function, we hypothesized that the Pro33 variant provides a more efficient signaling than the Leu33 isoform. When compared with Pro33-negative platelets, Pro33-positive platelets demonstrated significantly greater serine/threonine phosphorylation of extracellular signal-regulated kinase (ERK2) and myosin light chain (MLC) but not cytoplasmic phospholipase A2 upon thrombin-induced aggregation. Tyrosine phosphorylation of integrin β3 and the adaptor protein Shc was no different in the fibrinogen-engaged platelets from both genotypes. The addition of Integrilin (αIIbβ3-fibrinogen blocker) or okadaic acid (serine/threonine phosphatase inhibitor) dramatically enhanced ERK2 and MLC phosphorylation in the Pro33-negative platelets when compared with Pro33-positive platelets, suggesting that integrin engagement during platelet aggregation activates serine/threonine phosphatases. The phosphatase activity of myosin phosphatase (MP) that dephosphorylates MLC is inactivated by phosphorylation of the myosin binding subunit of MP at Thr696, and aggregating Pro33-positive platelets exhibited an increased Thr696 phosphorylation of MP. These studies highlight a role for the dephosphorylation events via the serine/threonine phosphatases during the integrin outside-in signaling mechanism, and the Leu33 → Pro polymorphism regulates this process. Furthermore, these findings support a mechanism whereby the reported enhanced α granule secretion in the Pro33-positive platelets could be mediated by an increased phosphorylation of MLC, which in turn is caused by an increased phosphorylation and subsequent inactivation of myosin phosphatase. Integrin β3 is polymorphic at residue 33 (Leu33 or Pro33), and the Pro33-positive platelets display enhanced aggregation, P-selectin secretion, and shorter bleeding times. Because outside-in signaling is critical for platelet function, we hypothesized that the Pro33 variant provides a more efficient signaling than the Leu33 isoform. When compared with Pro33-negative platelets, Pro33-positive platelets demonstrated significantly greater serine/threonine phosphorylation of extracellular signal-regulated kinase (ERK2) and myosin light chain (MLC) but not cytoplasmic phospholipase A2 upon thrombin-induced aggregation. Tyrosine phosphorylation of integrin β3 and the adaptor protein Shc was no different in the fibrinogen-engaged platelets from both genotypes. The addition of Integrilin (αIIbβ3-fibrinogen blocker) or okadaic acid (serine/threonine phosphatase inhibitor) dramatically enhanced ERK2 and MLC phosphorylation in the Pro33-negative platelets when compared with Pro33-positive platelets, suggesting that integrin engagement during platelet aggregation activates serine/threonine phosphatases. The phosphatase activity of myosin phosphatase (MP) that dephosphorylates MLC is inactivated by phosphorylation of the myosin binding subunit of MP at Thr696, and aggregating Pro33-positive platelets exhibited an increased Thr696 phosphorylation of MP. These studies highlight a role for the dephosphorylation events via the serine/threonine phosphatases during the integrin outside-in signaling mechanism, and the Leu33 → Pro polymorphism regulates this process. Furthermore, these findings support a mechanism whereby the reported enhanced α granule secretion in the Pro33-positive platelets could be mediated by an increased phosphorylation of MLC, which in turn is caused by an increased phosphorylation and subsequent inactivation of myosin phosphatase. Integrin αIIbβ3 mediates platelet adhesion and aggregation at the site of vascular injury and plays a pivotal role in hemostasis and thrombosis. The binding of platelet integrin αIIbβ3 to ligands such as fibrinogen and von Willebrand factor during aggregation/adhesion transmits information into the platelets called outside-in signaling. These signals are critical for platelet physiology and contribute to platelet thrombus formation by promoting the secretion of internal granules, the secondary wave of aggregation, and the formation of membrane vesicles with procoagulant activities (1Phillips D.R. Nannizzi-Alaimo L. Prasad K.S. Thromb. Haemostasis. 2001; 86: 246-258Crossref PubMed Scopus (126) Google Scholar). Not surprisingly, Glanzmann thrombasthenic human platelets and mouse platelets expressing β3 integrin in which the cytoplasmic tyrosines have been replaced with phenylalanines have defective outside-in signaling (2Chen Y.P. O'Toole T.E. Ylanne J. Rosa J.P. Ginsberg M.H. Blood. 1994; 84: 1857-1865Crossref PubMed Google Scholar, 3Wang R. Shattil S.J. Ambruso D.R. Newman P.J. J. Clin. Investig. 1997; 100: 2393-2403Crossref PubMed Scopus (105) Google Scholar, 4Law D.A. DeGuzman F.R. Heiser P. Ministri-Madrid K. Killeen N. Phillips D.R. Nature. 1999; 401: 808-811Crossref PubMed Scopus (278) Google Scholar). All these lines of evidence indicate that hemostasis requires a coordinated interaction between integrin αIIbβ3 and the signaling machinery in platelets. αIIbβ3 outside-in signaling is initiated by integrin engagement via an organized interaction of tyrosine and serine/threonine kinases such as Src, Csk, Syk, and protein kinase C-β with the integrin β3 subunit (5Obergfell A. Eto K. Mocsai A. Buensuceso C. Moores S.L. Brugge J.S. Lowell C.A. Shattil S.J. J. Cell Biol. 2002; 157: 265-275Crossref PubMed Scopus (353) Google Scholar, 6Buensuceso C.S. Obergfell A. Soriani A. Eto K. Kiosses W.B. Arias-Salgado E.G. Kawakami T. Shattil S.J. J. Biol. Chem. 2005; 280: 644-653Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar) and calcium and integrin-binding protein to the α subunit (7Naik U.P. Naik M.U. Blood. 2003; 102: 1355-1362Crossref PubMed Scopus (64) Google Scholar). The net result is the phosphorylation of tyrosine and serine/threonine residues of several proteins including extracellular signal-regulated kinases 2 (ERK 2), 1The abbreviations used are: ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; MEK, MAPK/ERK kinase; MLC, myosin light chain; ppMLC, diphosphorylated MLC; cPLA2, cytoplasmic phospholipase A2; PP1, protein phosphatase 1; PP2A, protein phosphatase 2A; MP, myosin phosphatase; MBS, myosin-binding subunit; CHO, Chinese hamster ovary; BSA, bovine serum albumin. myosin light chain (MLC), and cytoplasmic phospholipase A2 (cPLA2). Phosphorylation of ERK, MLC, and cPLA2 modulates various biological functions. For example, tyrosine/threonine phosphorylation of ERK1/ERK2 is involved in cell growth and proliferation (8Lewis T.S. Shapiro P.S. Ahn N.G. Adv. Cancer Res. 1998; 74: 49-139Crossref PubMed Google Scholar), megakaryocyte differentiation and proplatelet formation (9Whalen A.M. Galasinski S.C. Shapiro P.S. Nahreini T.S. Ahn N.G. Mol. Cell. Biol. 1997; 17: 1947-1958Crossref PubMed Scopus (205) Google Scholar, 10Jiang F. Jia Y. Cohen I. Blood. 2002; 99: 3579-3584Crossref PubMed Scopus (54) Google Scholar), and release of stored Ca2+ in platelets (11Rosado J.A. Sage S.O. J. Biol. Chem. 2000; 275: 9110-9113Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Serine/threonine phosphorylation of MLC is essential for migration, cytoskeletal clustering of integrins (12Kamm K.E. Stull J.T. J. Biol. Chem. 2001; 276: 4527-4530Abstract Full Text Full Text PDF PubMed Scopus (472) Google Scholar), and shape change and secretion in platelets (13Daniel J.L. Molish I.R. Rigmaiden M. Stewart G. J. Biol. Chem. 1984; 259: 9826-9831Abstract Full Text PDF PubMed Google Scholar), whereas serine phosphorylation of cPLA2 participates in the release of potent agonist arachidonic acid (14Lin L.L. Wartmann M. Lin A.Y. Knopf J.L. Seth A. Davis R.J. Cell. 1993; 72: 269-278Abstract Full Text PDF PubMed Scopus (1659) Google Scholar), platelet spreading on fibrinogen, and phosphorylation of PP125 focal adhesion kinase in platelets (15Haimovich B. Ji P. Ginalis E. Kramer R. Greco R. Thromb. Haemostasis. 1999; 81: 618-624Crossref PubMed Scopus (16) Google Scholar). Since the net phosphorylation on any protein is regulated by both kinases and phosphatases, it is likely that phosphatases also play a role in the process of outside-in signaling. Indeed, integrin αIIbβ3 engagement activates tyrosine phosphatases PTP1B by calpain cleavage (16Ezumi Y. Takayama H. Okuma M. J. Biol. Chem. 1995; 270: 11927-11934Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar), and we have recently shown that integrin αIIbβ3 engagement can regulate the activity of αIIb-associated protein phosphatase 1 PP1, a serine/threonine phosphatase (17Vijayan K.V. Liu Y. Li T.T. Bray P.F. J. Biol. Chem. 2004; 279: 33039-33042Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Phospho-ERK is dephosphorylated by dual-specific tyrosine/threonine phosphatases from the mitogen-activated protein kinase phosphatase family members (MKP-3, MKP-4) and serine/threonine phosphatases such as protein phosphatase 2A PP2A (18Camps M. Nichols A. Gillieron C. Antonsson B. Muda M. Chabert C. Boschert U. Arkinstall S. Science. 1998; 280: 1262-1265Crossref PubMed Scopus (438) Google Scholar, 19Chung H. Brautigan D.L. Cell. Signal. 1999; 11: 575-580Crossref PubMed Scopus (46) Google Scholar), whereas phospho-MLC is dephosphorylated by a serine/threonine phosphatase from the PP1 family such as myosin phosphatase (MP) (20Hartshorne D.J. Acta Physiol. Scand. 1998; 164: 483-493Crossref PubMed Scopus (86) Google Scholar). Platelet MP is composed of three subunits, a 36-kDa catalytic subunit of the type 1 protein phosphatase δ, a 130-kDa regulatory subunit called myosin-binding subunit (MBS) or myosin-targeting subunit, and a small 20-kDa subunit (20Hartshorne D.J. Acta Physiol. Scand. 1998; 164: 483-493Crossref PubMed Scopus (86) Google Scholar). Integrin β3 is polymorphic at residue 33 (Leu33 or Pro33, also known as PlA1 or PlA2, respectively). This polymorphism is not rare, and 25% of individuals of northern European descent express Pro33 isoforms on their platelets (21Williams M.S. Bray P.F. Exp. Biol. Med. (Maywood). 2001; 226: 409-419Crossref PubMed Scopus (47) Google Scholar). Platelets expressing the Pro33 isoform have shortened bleeding times and exhibit enhanced activation, α granule secretion, and aggregation (22Michelson A.D. Furman M.I. Goldschmidt-Clermont P. Mascelli M.A. Hendrix C. Coleman L. Hamlington J. Barnard M.R. Kickler T. Christie D.J. Kundu S. Bray P.F. Circulation. 2000; 101: 1013-1018Crossref PubMed Scopus (337) Google Scholar, 23Feng D. Lindpaintner K. Larson M.G. Rao V.S. O'Donnell C.J. Lipinska I. Schmitz C. Sutherland P.A. Silbershatz H. D'Agostino R.B. Muller J.E. Myers R.H. Levy D. Tofler G.H. Arterioscler. Thromb. Vasc. Biol. 1999; 19: 1142-1147Crossref PubMed Scopus (264) Google Scholar) and are associated with acute coronary syndromes in some studies (21Williams M.S. Bray P.F. Exp. Biol. Med. (Maywood). 2001; 226: 409-419Crossref PubMed Scopus (47) Google Scholar). Furthermore, CHO and 293 cells expressing the Pro33 isoform demonstrate enhanced adhesion and migration (24Vijayan K.V. Goldschmidt-Clermont P.J. Roos C. Bray P.F. J. Clin. Investig. 2000; 105: 793-802Crossref PubMed Scopus (153) Google Scholar, 25Sajid M. Vijayan K.V. Souza S. Bray P.F. Arterioscler. Thromb. Vasc. Biol. 2002; 22: 1984-1989Crossref PubMed Scopus (33) Google Scholar). However, an underlying mechanism for the increased platelet reactivity observed in some but not all functional assays with the Pro33-positive platelets is still elusive. Although our previous study demonstrated an increased phosphorylation of ERK2 and MLC in CHO and 293 cells expressing the Pro33 isoform, outside-in signaling in human platelets expressing the Pro33 isoform has not been investigated. Because outside-in signaling is critical for platelet function, we hypothesized that the Pro33 variant provides a more efficient signaling than the Leu33 isoform. We show here that when compared with the platelets lacking the Pro33 isoform, Pro33-positive platelets demonstrate enhanced phosphorylation of the ERK2-MLC axis pathway. More importantly, this enhanced signaling in the Pro33-positive platelets is in part due to an inefficient activation of a serine/threonine phosphatases following integrin αIIbβ3 ligation and is independent of integrin β3 tyrosine phosphorylation. Reagents—Human fibrinogen was from Enzyme Research Laboratories Inc; (South Bend, IN). Okadaic acid, bovine serum albumin (BSA), and phosphatase inhibitor mixture were from Sigma. Antibodies specific for the phosphorylated ERK1/2, phosphorylated cPLA2, and total cPLA2 were obtained from Cell Signaling Technology (Beverly, MA), whereas anti-ERK1/2 antibody was obtained from Promega (Madison, WI). Antibody specific for the diphosphorylated myosin light chain (ppMLC) was a generous gift from Dr. James Staddon, (Eisai London Research, London, UK). Anti-MLC and -Shc antibodies were from Santa Cruz Biotechnology, Inc; (Santa Cruz, CA). Antibodies that recognize tyrosine-phosphorylated integrin β3 were purchased from BIOSOURCE International, Inc. (Camarillo, CA). Thrombin and eptifibatide were generous gifts from Drs. John Fenton (New York State Department of Health, Wadsworth Center, Albany, NY) and D. Phillips (Portala pharmaceuticals Inc., San Francisco, CA), respectively. Platelet Aggregation and Adhesion—Blood was obtained in acidcitrate-dextrose anticoagulant from normal, healthy, and fasting donors of known PlA genotype. In this study, we used PlA1,A1 homozygous platelets (designated Pro33-negative platelets or Leu33/Leu33) and PlA1,A2 heterozygous or PlA2,A2 homozygous platelets (designated Pro33-positive platelets). Washed platelets from all three genotypes were prepared (26Vijayan K.V. Liu Y. Dong J.F. Bray P.F. J. Biol. Chem. 2003; 278: 3860-3867Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar), suspended in Tyrode's buffer (138 mm NaCl, 5.3 mm KCl, 0.33 mm Na2HPO4, 0.44 mm KH2PO4, 5.5 mm glucose, pH 7.4), and allowed to recover for ∼2 h at 37 °C. Aggregation was performed using varying concentrations of thrombin for 2 min using 2 × 108 platelets in 225 μl of Tyrode's in a BIO-DATA 4-channel platelet aggregometer. In some experiments, 25 μm Integrilin or 250 nm okadaic acid or control Me2SO (0.1%) were added 3 min prior to initiating aggregation. After 2 min, the reaction was stopped by solubilization with 25 μl of 10× SDS sample buffer. For adhesion studies, 12.5 μg/ml fibrinogen or heat-treated BSA was immobilized in a 100-mm tissue culture plate. 3 ml containing 3 × 108 platelets were added to each plate and incubated for 45 min at 37 °C in 5% CO2. The fibrinogen bound platelets and the non-adherent platelets from the BSA-coated plate were lysed in 1 ml of ice-cold Triton X-100 lysis buffer, and the protein content was determined. Immunoblotting—For these studies, 50 μg of protein obtained from the Triton X-100 lysates or 90 μl of SDS protein lysates described above were separated by 10% reducing SDS-PAGE, transferred to nitrocellulose membrane, and blocked with 5% nonfat milk in Tris-buffered saline (20 mm Tris-HCl, pH 7.6, 150 mm NaCl) containing 1% Tween 20 for 1 h at 22 °C. The blots were incubated overnight with the following antibodies: anti-phospho ERK1/2 (Thr183, Tyr185), anti-ppMLC (Thr18, Ser19), anti-phospho cPLA2 (Ser502), anti-phospho integrin β3 (Tyr747) and (Tyr759), anti-phospho Shc (Tyr 317), and anti-phospho MBS (Thr695) at 4°C. The blots were washed with Tris-buffered saline and then incubated with horseradish peroxidase-conjugated anti-rabbit, anti-mouse, or anti-goat antibodies for 2 h, and the immunoreactive bands were visualized using an ECL (Amersham Biosciences) system. To confirm equal loading of proteins, the membrane was stripped in buffer containing 62.5 mm Tris-HCl, pH 6.8, 2% SDS, and 100 mm β-mercaptoethanol for 30 min at 50°C and blocked with 5% nonfat milk. The blots were reprobed with antibodies to ERK1/2, MLC, cPLA2, integrin β3, Shc, and MBS as described above. The signals were scanned using PhotoShop 6 software, and the densitometric quantitation was performed using NIH Image software from beta 4.0.2 of Scion Image, Scion Corp., Frederick, MD. Enhanced Phosphorylation of ERK2 in Aggregating Pro33-positive platelets—To determine whether the PlA2 polymorphism of integrin β3 regulates outside-in signaling to ERK2 in human platelets, we studied thrombin-induced aggregation using the Pro33-positive and Pro33-negative platelets. Significantly (p = 0.02) greater levels of phosphorylated ERK2 were detected in the Pro33-positive platelets when compared with Pro33-negative platelets at varying concentrations of thrombin (Fig. 1A). By densitometry, and when compared with the Pro33-negative platelets, Pro33-positive platelets exhibited a maximum of ∼2.4-fold increase in ERK2 phosphorylation at 0.5 units/ml thrombin (Fig. 1B). The level of total ERK1/2 in each lane was equivalent and could not account for the signaling differences (Fig. 1A). Essentially similar results were obtained with the Pro33 homozygous platelets (Fig. 1C). To address whether the ERK2 phosphorylation was a result of direct thrombin signaling or αIIbβ3-dependent platelet aggregation, we analyzed ERK2 phosphorylation in the absence of platelet aggregation. Inhibition of integrin-fibrinogen engagement with Integrilin blocked platelet aggregation and dramatically enhanced ERK2 phosphorylation in platelets lacking the Pro33 form. (Fig. 1D). These results are consistent with a previous study that showed increased phosphorylation of ERK2 upon blocking integrin-fibrinogen interaction using RGDS peptide or AP-2 antibody (27Nadal F. Levy-Toledano S. Grelac F. Caen J.P. Rosa J.P. Bryckaert M. J. Biol. Chem. 1997; 272: 22381-22384Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). These studies indicate that ERK2 phosphorylation was caused by thrombin signaling, possibly via activation of protease-activated receptors, whereas integrin engagement with fibrinogen during aggregation (αIIbβ3 outside-in signaling) results in a negative regulation (dephosphorylation) of ERK2. Under similar conditions, Integrilin blocked the aggregation of the Pro33-positive platelets and only modestly enhanced ERK2 phosphorylation (Fig. 1D). At a concentration of 0.5 units/ml thrombin, the addition of Integrilin enhanced a maximum of ∼7-fold in ERK2 phosphorylation in the Pro33-negative platelets and only ∼2-fold ERK2 phosphorylation in the Pro33-positive platelets. These studies indicated that the engagement of integrin with fibrinogen during aggregation resulted in the dephosphorylation of ERK2 in a more efficient manner in the Pro33-negative platelets than in the Pro33-positive platelets. Thrombin signaling alone cannot account for the Leu33 → Pro differences in ERK2 signaling since ERK2 phosphorylation was not different in the Integrilin-treated Pro33-positive and Pro33-negative platelets. These studies suggest that a difference in the extent of an ERK2 dephosphorylation event upon integrin engagement in the Pro33-positive platelets, when compared with the Pro33-negative platelets, is likely responsible for the enhanced ERK2 phosphorylation in the Pro33-positive platelets. Enhanced Phosphorylation of MLC in Aggregating Pro33-positive Platelets—We have previously reported an ∼3-fold increase in α granule (P-selectin) secretion in response to thrombin in the Pro33-positive platelets when compared with the Pro33-negative platelets (26Vijayan K.V. Liu Y. Dong J.F. Bray P.F. J. Biol. Chem. 2003; 278: 3860-3867Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Since Thr18/Ser19 phosphorylation of MLC is critical for platelet secretion (13Daniel J.L. Molish I.R. Rigmaiden M. Stewart G. J. Biol. Chem. 1984; 259: 9826-9831Abstract Full Text PDF PubMed Google Scholar), we examined the phosphorylation status of MLC in platelets based on the Leu33 → Pro genotype. Substantially greater levels of diphosphorylated MLC were detected in the Pro33-positive platelets when compared with the Pro33-negative platelets at varying concentrations of thrombin (Fig. 2A). When compared with the platelets lacking the Pro33 form, Pro33-positive platelets exhibited a maximum of ∼2.5-fold increase in phosphorylated MLC at 0.5 units/ml thrombin concentrations (Fig. 2B). This signaling difference was not due to the difference in total MLC loaded (Fig. 2A). In a manner similar to the ERK2 studies, inhibition of platelet aggregation enhanced MLC diphosphorylation by ∼5-fold in Pro33-negative platelets and only by ∼2.0-fold in Pro33-fold in Pro-positive platelets at 0.5 units/ml thrombin concentrations (Fig. 2C). These studies indicate that a difference in the extent of MLC dephosphorylation in the Pro33-positive platelets, when compared with the Pro33-negative platelets, is likely responsible for an enhanced Pro33-mediated MLC signaling. Under similar conditions, Ser505 phosphorylation of cPLA2, a substrate for ERK2 and P38 (29Kramer R.M. Roberts E.F. Um S.L. Borsch-Haubold A.G. Watson S.P. Fisher M.J. Jakubowski J.A. J. Biol. Chem. 1996; 271: 27723-27729Abstract Full Text Full Text PDF PubMed Scopus (437) Google Scholar), was no different in thrombin-induced aggregating Pro33-positive and Pro33-negative platelets (Fig. 2D), indicating that the Pro33-mediated enhanced signaling is specific to ERK2-MLC pathway. Enhanced Signaling in the Pro33-positive Platelets Is Independent of Tyrosine Phosphorylation of Integrin β3 and Adaptor Protein Shc—Previous studies have shown an essential role for tyrosine phosphorylation of β3 in the αIIbβ3 outside-in signaling process (4Law D.A. DeGuzman F.R. Heiser P. Ministri-Madrid K. Killeen N. Phillips D.R. Nature. 1999; 401: 808-811Crossref PubMed Scopus (278) Google Scholar). To understand better whether the enhanced ERK2-MLC signaling observed in Pro33-positive platelets was initiated from the integrin β3 itself, we examined the tyrosine phosphorylation of β3 from the Pro33-positive and Pro33-negative platelets either suspended over BSA substrate or adhered to fibrinogen. When compared with the platelets in suspension, platelets adhering to fibrinogen demonstrated an increased phosphorylation of Tyr747 and Tyr759 of integrin β3 (Fig. 3A, left panel). However, the level of Tyr747 and Tyr759 phosphorylation of integrin β3 as determined by densitometry was no different between the fibrinogen adhered Pro33-positive and the Pro33-negative platelets (Fig. 3A, right panel). The adaptor protein Shc is tyrosine-phosphorylated during the process of αIIbβ3 outside-in signaling (30Cowan K.J. Law D.A. Phillips D.R. J. Biol. Chem. 2000; 275: 36423-36429Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar), and since phosphorylation at Tyr317 is essential for ERK2 activation in other cell types, we examined the status of Tyr317 phosphorylation in platelets based on the Pro33 genotype. Shc Tyr317 phosphorylation increased during aggregation, reaching a maximum at 20 s after the addition of thrombin, and decreased with further continued stirring (Fig. 3B). Similar patterns of Shc phosphorylation in response to thrombin-induced aggregation were reported previously (30Cowan K.J. Law D.A. Phillips D.R. J. Biol. Chem. 2000; 275: 36423-36429Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). However, the extent of Tyr317 phosphorylation of Shc was no different between the aggregating Pro33-positive and Pro33-negative platelets (Fig. 3B). Nevertheless, the same blot reprobed for ERK2 revealed enhanced ERK2 phosphorylation in the Pro33-positive platelets (not shown), suggesting that the ERK2 phosphorylation status in platelets was not dependent on Shc phosphorylation. Taken together, these results imply that the enhanced ERK2 and MLC signaling in the Pro33-positive platelets are independent of Shc and β3 tyrosine phosphorylation. Differential Activation of Serine/Threonine Phosphatases in the Pro33-positive platelets—Since integrin engagement during platelet aggregation resulted in a dephosphorylation of ERK2 and MLC, we examined a possible role for serine/threonine phosphatases in this process. Because serine/threonine phosphatases PP2A and PP1 participate in dephosphorylating ERK2 and MLC, generic PP2A/PP1 inhibitor was employed. The addition of 250 nm okadaic acid, a concentration that inhibits PP2A and PP1 (31McCluskey A. Sim A.T. Sakoff J.A. J. Med. Chem. 2002; 45: 1151-1175Crossref PubMed Scopus (215) Google Scholar), efficiently enhanced ERK2 and MLC phosphorylation in platelets lacking the Pro33 form (Fig. 4, A and C). In contrast, under similar conditions, okadaic acid only had a modest increase in ERK2 and MLC phosphorylation in the Pro33-positive platelets (Fig. 4, B and D). These results are consistent with the observations in Figs. 1D and Fig. 2C wherein Pro33-negative platelets but not Pro33-positive platelets exhibited an increased phosphorylation of ERK2 and MLC upon treatment with Integrilin. Thus, the phosphorylation of ERK2 and MLC in the Pro33-negative platelets can be increased by blocking integrin-fibrinogen engagement at the platelet surface or by inactivating serine/threonine phosphatases inside the platelet. These studies indicate that integrin-fibrinogen engagement during aggregation negatively regulates ERK2/MLC signaling, possibly through an efficient activation of serine/threonine phosphatases in platelets lacking the Pro33 form. In contrast, integrin engagement in the Pro33-positive platelets only modestly activated these phosphatases. Because okadaic acid failed to appreciably enhance MLC phosphorylation in the Pro33-positive platelets, we considered whether the PP1-myosin phosphatase is rendered inactive in these platelets following integrin engagement. The phosphatase activity of myosin phosphatase is regulated by phosphorylation of its regulatory subunit (MBS), such that phosphorylation at residue Thr696 inactivates the phosphatase (32Ichikawa K. Ito M. Hartshorne D.J. J. Biol. Chem. 1996; 271: 4733-4740Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). We therefore examined the phosphorylation status of MBS at Thr696 in the aggregating platelets based on the Leu33 → Pro genotype. A greater phosphorylation of MBS was detected in the Pro33-positive platelets when compared with the Pro33-negative platelets at 0.5 units/ml thrombin (Fig. 5A). By densitometry and when compared with Pro33-negative platelets, Pro33-positive platelets exhibited an ∼2-fold increase in the level of phosphorylated MBS (Fig. 5B). This result suggests that the enhanced MLC phosphorylation observed in the Pro33-positive platelets upon integrin engagement may in part be due to the increased phosphorylation and thus inactivation of myosin phosphatase. Platelet physiology is influenced by outside-in signals that are generated by the engagement of integrin αIIbβ3. In this study, we use platelet aggregation and adhesion studies to investigate the impact of the Leu33 → Pro polymorphism of integrin β3 on outside-in signaling. Several prior studies have considered a key role for phosphorylation events via kinases in the process of outside-in signaling, but the contribution of the dephosphorylation events via phosphatases during outside-in signaling is poorly understood. The results from our studies identify an enhanced outside-in signaling to ERK2-MLC axis in the Pro33 variant of integrin β3, which is caused in part by an inefficient activation of serine/threonine phosphatase(s) following integrin ligation. Furthermore, this study illustrates a novel role for the Pro33 polymorphism of integrin β3 in regulating the activation of myosin phosphatases by a mechanism involving a phosphorylation event of the myosin binding subunit. Using platelets of known Leu33 → Pro genotype, we examined the outside-in signaling to ERK2 and MLC during platelet aggregation. When compared with the platelets lacking the Pro33 form, the Pro33-positive platelets exhibited an enhanced phosphorylation of ERK2 and MLC (Figs. 1A and 2A). This observation is consistent with our previous study in which we identified a greater phosphorylation of ERK2 and MLC in Pro33 CHO and 293 cells adhered to fibrinogen (26Vijayan K.V. Liu Y. Dong J.F. Bray P.F. J. Biol. Chem. 2003; 278: 3860-3867Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). ERK2 signaling in platelets is required for the release of stored Ca2+, for GPIb-IX-dependent activation of integrin αIIbβ3 and platelet aggregation to low doses but not to high doses of thrombin, collagen, arachidonic acid, and U46619 (33Li Z. Xi X. Du X. J. Biol. Chem. 2001; 276: 42226-42232Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 34McNicol A. Philpott C.L. Shibou T.S. Israels S.J. Biochem. Pharmacol. 1998; 55: 1759-1767Crossref PubMed Scopus (19) Google Scholar). ERK2 activation promotes cell adhesion and spreading (35Zhu X. Assoian R.K. Mol. Biol. Cell. 1995; 6: 273-282Crossref PubMed Scopus (394) Google Scholar), and dominant negative ERK2 inhibits these processes (36Lai C.F. Chaudhary L. Fausto A. Halstead L.R. Ory D.S. Avioli L.V. Cheng S.L. J. Biol. Chem. 2001; 276: 14443-14450Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar). We observed the Pro33 isoform in CHO cells to enhance actin polymerization, spreading, adhesion, and migration to fibrinogen (24Vijayan K.V. Goldschmidt-Clermont P.J. Roos C. Bray P.F. J. Clin. Investig. 2000; 105: 793-802Crossref PubMed Scopus (153) Google Scholar, 25Sajid M. Vijayan K.V. Souza S. Bray P.F. Arterioscler. Thromb. Vasc. Biol. 2002; 22: 1984-1989Crossref PubMed Scopus (33) Google Scholar). Furthermore, mitogen-activated kinase (MAPK) kinase inhibition abolished the Pro33-mediated enhancement in cell adhesion (26Vijayan K.V. Liu Y. Dong J.F. Bray P.F. J. Biol. Chem. 2003; 278: 3860-3867Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar) and migration (25Sajid M. Vijayan K.V. Souza S. Bray P.F. Arterioscler. Thromb. Vasc. Biol. 2002; 22: 1984-1989Crossref PubMed Scopus (33) Google Scholar) to fibrinogen. On the other hand, MLC phosphorylation promotes myosin ATPase activity and increases actinomyosin contractile responses that are involved in platelet shape change, secretion, and migration (37Klemke R.L. Cai S. Giannini A.L. Gallagher P.J. de Lanerolle P. Cheresh D.A. J. Cell Biol. 1997; 137: 481-492Crossref PubMed Scopus (1103) Google Scholar). Pro33-positive platelets exhibit an ∼3-fold increase in α-granule secretion in response to thrombin (26Vijayan K.V. Liu Y. Dong J.F. Bray P.F. J. Biol. Chem. 2003; 278: 3860-3867Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Moreover, MLC phosphorylation is required for aggregation and secretion in response to ADP (38Hashimoto Y. Sasaki H. Togo M. Tsukamoto K. 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Thus, our findings that the increased outside-in signaling to ERK2 and MLC in the Pro33-positive platelets correlate well with the enhanced functions of these signaling molecules in the physiology of Pro33-positive platelets. Despite the increased phosphorylation of ERK2 in the Pro33-positive platelets, tyrosine phosphorylation of integrin β3 and adaptor protein Shc was not increased in the Pro33-positive platelets (Fig. 3). This observation is in contrast to the classical activation of ERK2 pathway in other cell types that primarily couple Shc to the Ras-Raf-MEK axis (8Lewis T.S. Shapiro P.S. Ahn N.G. Adv. Cancer Res. 1998; 74: 49-139Crossref PubMed Google Scholar). Consistent with our observations, others have reported that activation of Ras (40Tulasne D. Bori T. Watson S.P. Eur. J. Biochem. 2002; 269: 1511-1517Crossref PubMed Scopus (25) Google Scholar) and Raf (41Nadal-Wollbold F. Pawlowski M. Levy-Toledano S. Berrou E. Rosa J.P. Bryckaert M. FEBS Lett. 2002; 531: 475-482Crossref PubMed Scopus (66) Google Scholar) is not sufficient to lead to ERK2 phosphorylation in platelets, although all of these signaling components are expressed in platelets. Outside-in signaling is initiated via several kinases that associate with the β3 tail (5Obergfell A. Eto K. Mocsai A. Buensuceso C. Moores S.L. Brugge J.S. Lowell C.A. Shattil S.J. J. Cell Biol. 2002; 157: 265-275Crossref PubMed Scopus (353) Google Scholar), and phosphorylation of Shc and β3 could favor signaling via kinases. An enhanced signaling in the Pro33-positive platelets without a corresponding increase in β3 and Shc phosphorylation could imply little role for kinases coupled to β3 and Shc in the enhanced ERK2 outside-in signaling. We pursued the possibility that a differential activation of phosphatases (or the extent of dephosphorylation events) could account for the Pro33-mediated increased phosphorylation events. Indeed, inhibition of Pro33-negative platelet aggregation resulted in a 7- and 5-fold enhanced phosphorylation of ERK2 and MLC, respectively, suggesting that αIIbβ3 engagement during aggregation activates serine/threonine phosphatases. A similar ∼10-fold increase in the ERK2 phosphorylation at residue Thr183 upon blocking platelet aggregation with RGDS peptide was reported previously (42Pawlowski M. Ragab A. Rosa J.P. Bryckaert M. FEBS Lett. 2002; 521: 145-151Crossref PubMed Scopus (12) Google Scholar). More importantly, the increase in ERK2/MLC phosphorylation upon blocking integrin-fibrinogen interaction appeared to be lost when we studied the Pro33-positive platelets (Figs. 1D and 2C). These studies suggested that serine/threonine phosphatases are activated by integrin ligation during platelet aggregation, and the Pro33 polymorphism regulated this activation process. This idea is supported by the observations that serine/threonine phosphatase PP1/PP2A inhibitor okadaic acid enhanced ERK2 and MLC phosphorylation more efficiently in the platelets lacking the Pro33 form when compared with the Pro33-positive platelets (Fig. 4). Thus, our studies using platelets from both genotypes suggest that dephosphorylation events are as important as phosphorylation events and that changes in the extent of dephosphorylation events could alter the process of outside-in signaling. How could the engagement of Pro33 isoform of integrin lead to inefficient activation of serine/threonine phosphatases such as MP? Dissociation of MP subunits by arachidonic acid (43Gong M.C. Fuglsang A. Alessi D. Kobayashi S. Cohen P. Somlyo A.V. Somlyo A.P. J. Biol. Chem. 1992; 267: 21492-21498Abstract Full Text PDF PubMed Google Scholar), phosphorylation of MBS at Thr696 by Rho kinases (32Ichikawa K. Ito M. Hartshorne D.J. J. Biol. Chem. 1996; 271: 4733-4740Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar), or phosphorylation of CPI-17, an inhibitory phosphoprotein of MP through protein kinase C (44Watanabe Y. Ito M. Kataoka Y. Wada H. Koyama M. Feng J. Shiku H. Nishikawa M. Blood. 2001; 97: 3798-3805Crossref PubMed Scopus (57) Google Scholar), are different mechanisms that could contribute to the inactivation of MP. Indeed, we observed an ∼2-fold increased phosphorylation of MBS at Thr696 in the Pro33-positive platelets. Thus, increased phosphorylation of regulatory subunits of myosin phosphatase and its subsequent inactivation could account for the increased MLC signaling in the Pro33-positive platelets. Rho kinase is activated by integrin ligation, and its activity regulates the stability of αIIbβ3 adhesion contacts under shear to von Willebrand factor (45Schoenwaelder S.M. Hughan S.C. Boniface K. Fernando S. Holdsworth M. Thompson P.E. Salem H.H. Jackson S.P. J. Biol. Chem. 2002; 277: 14738-14746Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). In addition, we observed an enhanced adhesion of Pro33-CHO cells to von Willebrand factor (28Vijayan K.V. Huang T.C. Liu Y. Bernardo A. Dong J.F. Goldschmidt-Clermont P.J. Alevriadou B.R. Bray P.F. FEBS Lett. 2003; 540: 41-46Crossref PubMed Scopus (27) Google Scholar). It is therefore likely that the Pro33 isoform could induce a greater Rho activation upon integrin ligation, leading to a greater Thr696 phosphorylation of MBS. Our proposed model for an increased signaling in the Pro33-positive platelets is shown in Fig. 6. In conclusion, we have shown that integrin αIIbβ3 engagement during platelet aggregation leads to serine/threonine phosphatase activation (as revealed by the dephosphorylation of ERK2 and MLC) and that the Leu33 → Pro polymorphism regulates the level of the myosin phosphatase activation via Thr696 phosphorylation events. Thus, studies using platelets from both genotypes have 1) shed light on the critical role for the dephosphorylation process in the outside in signaling process and 2) revealed that the Leu33 → Pro polymorphism regulates this dephosphorylation process. These studies provide further insight into the molecular mechanism underlying the increased function (secretion) and increased signaling (MLC phosphorylation) exhibited by a commonly inherited variation of integrin β3.

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