Original Paper Acta Haematol 2017;137:44–50 DOI: 10.1159/000450783 Received: June 27, 2016 Accepted after revision: September 8, 2016 Published online: December 7, 2016 Integrin αIIbβ3-Dependent ERK Signaling Is Regulated by Src and Rho Kinases in Both Leu33 and Pro33 Polymorphic Isoforms Khon C Huynh a, c Thi-Hiep Nguyen a Dinh Chuong Pham b Huong T.T Nguyen c Toi Van Vo a Marianna Gyenes c Volker R Stoldt c a Biomedical Engineering Department, International University, Vietnam National University, Ho Chi Minh City, Vietnam; b Faculty of Applied Sciences, Ton Duc Thang University, Ho Chi Minh City, Vietnam; c Department of Hemostasis, Hemotherapy, and Transfusion Medicine, Heinrich Heine University Medical Center, Düsseldorf, Germany Abstract Platelet integrin αIIbβ3 possesses a Leu/Pro polymorphism at residue 33 (Leu33/HPA-1a or Pro33/HPA-1b) The Pro33 isoform has been suggested to exhibit prothrombotic features αIIbβ3-expressing CHO (Chinese hamster ovary) cells on immobilized fibrinogen show activation of the MAP kinase family member ERK2, with an enhanced ERK2 activity in Pro33 cells compared to Leu33 cells In our present work, we examined how the Leu/Pro polymorphism modulates the ERK2 activation stimulated by differently triggered outside-in signalings We either treated the CHO cells with Mn2+ or allowed them to adhere to fibrinogen Moreover, we studied which signaling cascades are involved in ERK2 activation In contrast to immobilized fibrinogen, Mn2+ did not significantly increase ERK2 activation However, Mn2+ had a synergistic effect on ERK2 phosphorylation when combined with immobilized fibrinogen Pro33 cells adherent to fibrinogen exhibited a significantly greater ERK2 activity than Leu33 cells in the presence of Mn2+, which peaked after 10 of © 2016 S Karger AG, Basel E-Mail karger@karger.com www.karger.com/aha adhesion Our data showed that Src family and rho kinases play a crucial role in the integrin αIIbβ3-dependent outsidein signaling to ERK2 © 2016 S Karger AG, Basel Introduction The major platelet integrin, the fibrinogen receptor αIIbβ3, interacts with numerous plasma and extracellular matrix proteins and thus plays an important role in platelet adhesion and aggregation during hemostasis and thrombosis Upon ADP or thrombin activation of the platelets, integrin αIIbβ3 becomes activated (inside-out signaling), and it can bind soluble fibrinogen, which in turn induces the activation of various cellular responses such as spreading and aggregation (outside-in signaling) [1–3] The β3 subunit of αIIbβ3 is polymorphic at residue 33, and these alleles encode either Leu (HPA-1a) or Pro (HPA-1b) Platelets expressing the Pro33 phenotype show an increased αIIbβ3 function, e.g., enhanced aggregation, shorter bleeding times, and a greater affinity on immobilized fibrinogen [4–7] The possible clinical aspects of this polymorphism have been published in several studies Khon C Huynh Biomedical Engineering Department International University, Vietnam National University Ho Chi Minh City (Vietnam) E-Mail hckhon @ hcmiu.edu.vn Downloaded by: Rutgers University Alexander Library 128.6.218.72 - 2/20/2017 12:55:45 PM Keywords αIIbβ3 · ERK signaling · Leu33 · Pro33 · Polymorphisms · Rho kinase · Src signaling Materials and Methods Antibodies and Reagents Anti-Src pY418 was from Invitrogen (Darmstadt, Germany), anti-v-Src from Calbiochem (Darmstadt, Germany), phosphorylated ERK1/2 from Cell Signaling Technology (Danvers, MA, USA), and anti-ERK1/2 from Promega (Mannheim, Germany) Nonconjugated IgG mouse was from Sigma (Taufkirchen, Germany), secondary antibody rabbit HRG and mouse HRG from GE Healthcare (Munich, Germany), FITC-conjugated clone P2 antibody and clone SZ21 antibody from Immunotech (Krefeld, Germany), and FITC-conjugated nonspecific mouse IgG from Becton-Dickinson (Heidelberg, Germany) Alfazyme was from PAA αIIbβ3-Dependent ERK Signaling Is Regulated by Src and Rho Kinases Laboratories GmbH (Pasching, Germany), PP1 from Biomol (Hamburg, Germany), PP3 from Merck (Darmstadt, Germany), and the staining kit from Bio-Rad (Munich, Germany) Protease and phosphatase inhibitors, apyrase, PGE1, human fibrinogen, and all other reagents were from Sigma Flow Cytometry Two CHO cell clones stably expressing αIIbβ3 isoforms Leu33 and Pro33 were obtained from the Department of Hemostasis, Hemotherapy, and Transfusion Medicine, Heinrich Heine University Medical Center, Düsseldorf, Germany [22] To check the expression of αIIbβ3 isoforms, cells were resuspended in PBS, incubated with either FITC-conjugated CD-41 clone P2 antibody (1: 10) or FITC-conjugated HPA-1a-specific antibody (CD61 clone SZ21, 1:10) in 50 μL end volume for 15 at room temperature As a control, nonspecific mouse IgG-FITC was used The labeled cells were analyzed on a FACScalibur flow cytometer (Becton Dickinson) Cell Adhesion to Immobilized Fibrinogen CHO cells were grown to 70–80% confluence, detached by Alfazyme for at 37 ° C, collected by centrifugation at 300 g for at room temperature, and resuspended in Tyrode’s buffer (137 mM NaCl, 2.8 mM KCl, 12 mM NaHCO3, 0.4 mM NaH2PO4, mM MgCl2, mM CaCl2, and 5.5 mM glucose, pH 7.4) Six-well tissue plates were coated with 500 μL (100 μg/mL) fibrinogen or 1% heat-denaturated BSA in PBS Approximately 1.5 × 105 cells were added to each well and incubated for the indicated time periods at 37 ° C in a 5% CO2 incubator For Western blotting, adherent cells were lysed on the plates with ice-cold lysis buffer (10 mM Tris, 100 mM NaCl, mM EDTA, mM EGTA, mM NaF, 20 mM Na4P2O7, mM Na3VO4, 1% Triton X-100, 10% glycerol, and 0.5% sodium deoxycholate; pH 7.4) supplied with 250 μg/mL AEBSF, 15 μg/mL pepstatin, chymostatin, antipain, 55 μg/mL leupeptin, and a phosphatase inhibitor mixture For immunoprecipitation, we used a lysis buffer containing 1% Triton X-100, 20 mM Tris, 136 mM NaCl, mM Na3VO4, mM NaF, mM EDTA, and 20 mM Na4P2O7 (pH 7.4) supplied with protease and phosphatase inhibitors A nonadherent cell suspension was added to ice-cold 2× lysis buffer The lysates were chilled for 30 on ice and clarified by centrifugation at 13,200 rpm for 30 at ° C in a microcentrifuge Protein concentration was determined by the Bradford method Gel Electrophoresis and Western Blotting Equal amounts of protein were subjected to electrophoresis, and all samples were electrophoresed either in 8% (for Src) or 10% (for ERK) acrylamide gel for SDS-PAGE, transferred onto PVDF membranes, and subjected to immunoreaction The signals were densitometrically visualized with a chemiluminescence ECL (Amersham Biosciences) system and quantified using an Azure c300 Imaging System (Azure Biosystems) Results Expression of αIIbβ3 Isoforms in CHO Cells We obtained αIIbβ3-transfected CHO cells with the appropriate αIIbβ3 isoforms (Leu33 or Pro33) [22] The Acta Haematol 2017;137:44–50 DOI: 10.1159/000450783 45 Downloaded by: Rutgers University Alexander Library 128.6.218.72 - 2/20/2017 12:55:45 PM demonstrating a potential association between these symptoms, acute coronary syndromes [8], and a premature myocardial infarction by patients with coronary artery disease who are carriers of HPA-1b/1b [9] Although the activation of integrin αIIbβ3 mostly occurs via inside-out signaling, adhesive ligand occupation (i.e., immobilized fibrinogen/fibronectin) to the integrin can also generate the active conformation of the integrin leading to outside-in signaling [2] Divalent Mn2+ cations have also been reported to induce an active conformational state of αIIbβ3 [10] and to generate a subsequent outside-in signaling [11, 12] Src tyrosine kinase is associated with the cytoplasmic tail of the β3 subunit and has been reported to play a crucial role in the integrin-mediated outside-in signaling [13, 14] A number of other signal molecules and pathways have also been identified to participate in the integrin-mediated outside-in signaling, among others the mitogen-activated protein kinase (MAPK) family member ERK2, whose Tyr/Thr phosphorylation regulates various cellular processes, including the release of stored Ca2+ in platelets [15], cell adhesion, and spreading [16] Via its substrate, the myosin light chain kinase (MLCK), ERK2 modulates the myosin function and thereby the cytoskeletal clustering of integrins, shape changes, and secretion in platelets [17–19] Previously, it has been reported that the substitution of Leu to Pro at residue 33 enhances signaling to ERK2, MLCK, and the extent of the phosphorylated state of the regulatory subunit in the myosin phosphatase [3, 20, 21] As these signal proteins are essential for cytoskeletal rearrangement, adhesion, and spreading, these results correlate well with the increased αIIbβ3 activity observed in the HPA-1b/1b isoform In our work, we examined how the Leu33Pro polymorphism modulates ERK2 activation in outside-in signaling In addition, we studied which signaling pathways are involved in αIIbβ3-mediated ERK activation 46 Acta Haematol 2017;137:44–50 DOI: 10.1159/000450783 Counts HPA-1a (Leu33) HPA-1b (Pro33) 100 101 a 102 103 104 Fluorescence intensity 20 HPA-1b (Pro33) HPA-1a (Leu33) b 100 101 102 103 Fluorescence intensity 104 Fig Characterization of stable αIIbβ3 expression of a CHO cell line; expression levels in HPA-1a (Leu33) and HPA-1b (Pro33) CHO cells were determined by flow-cytometric analysis with the FITC conjugated αIIbβ3-specific P2 antibody (a) To identify the HPA-1a variant, antibody SZ21, a HPA-1a-specific antibody, was utilized (b) Src Family Kinases and Rho Kinases in ERK Signaling ERK2 activation is mediated by dual phosphorylation on threonine 185 and tyrosine 187 residues [23] Therefore, Src tyrosine kinase and Rho kinase (ROCK) are suggested to be involved in ERK activation Following fibrinogen engagement, Src pY418 activity was enhanced in both Leu33 and Pro33 cells (Fig. 4a) To examine the role of Src tyrosine kinases in ERK2 signaling, we incubated Leu33 and Pro33 cells with the selective Src family kinase inhibitor PP1 and subsequently allowed them to adhere to 100 μg/mL fibrinogen Cell suspensions over BSA surfaces were used as a control As shown in Figure 4, PP1 completely blocked the ERK2 phosphorylation in both isoforms indicating an Src kinase-dependent ERK2 actiHuynh/Nguyen/Pham/Nguyen/Van Vo/ Gyenes/Stoldt Downloaded by: Rutgers University Alexander Library 128.6.218.72 - 2/20/2017 12:55:45 PM pERK2 Activity in αIIbβ3-Transfected CHO Cells To study the modulation of the polymorphism onto the integrin-mediated outside-in signaling, we investigated the activation of ERK in αIIbβ3-expressing CHO cells on immobilized fibrinogen After placing cells onto 100 μg/mL immobilized fibrinogen, we allowed them to adhere for 10 followed by ERK activation analysis Pro33 cells exhibit higher ERK activation than Leu33 cells (Fig. 2) Mn2+ is also known to induce integrin activation via shifting the receptor conformation from an inactive to an active state [4] In the next part of our work, we studied the influence of Mn2+ on the ERK2 activation in fibrinogen-adherent CHO cells Moreover, we assessed how the Leu33/Pro33 polymorphism modulates this effect To analyze whether Mn2+ alone induces outside-in signaling to ERK2, we examined the effect of 0.5 mM Mn2+ on ERK2 activation in both Leu33 and Pro33 CHO cell suspensions over BSA surface As shown in Figure 2, Mn2+ slightly stimulated ERK2 activation, but the extent of the stimulation was considerably less than in cells adhering to 100 μg/mL immobilized fibrinogen Higher concentrations of Mn2+ (1 or mM) exhibited a similar effect as 0.5 mM Mn2+ (data not shown) The combination of Mn2+ and immobilized fibrinogen resulted in a synergism of ERK activation Mn2+ concentrations of 0.5 and mM induced significantly greater ERK2 phosphorylation in Pro33 cells than in Leu33 cells Using a concentration of mM Mn2+, both HPA-1 isoforms showed approximately equal ERK activation (Fig. 2) To analyze the kinetics of the ERK2 phosphorylation as a consequence of the immobilized fibrinogen-Mn2+ combination, we allowed Leu33 and Pro33 CHO cells to adhere to fibrinogen surfaces in the presence of 0.5 mM Mn2+ for various periods of time As shown in Figure 3, both Leu33 and Pro33 cells exhibited a maximal ERK2 activity after 10 of incubation with a subsequent decrease after 20 adhesion 64 Counts clones were confirmed for an equivalent expression level of Leu33 and Pro33 isoforms prior to adhesion experiments Flow-cytometric analysis with FITC-conjugated anti-αIIbβ3 antibody P2 demonstrated equivalent receptor expression in the cell lines generated with the Leu33 (HPA-1a)- and Pro33 (HPA-1b)-containing αIIbβ3 isoforms, respectively SZ21, a specific monoclonal antibody to the HPA-1a isoform, presented a substantially lower affinity for the Pro33 cells than for the Leu33 cells (Fig. 1) pERK2 Total ERK1/2 Fig pERK2 activity in αIIbβ3-expressing * 1.0 0.5 + 0.5 mM Mn2+ Fibrinogen vation PP3, an inactive analogue of PP1, did not exhibit an inhibitory effect To examine the potential role of ROCK in the αIIbβ3dependent ERK2 activation, we performed adhesion experiments on fibrinogen in the presence of either Y27632 or HA1077, pharmacologically distinct specific inhibitors of ROCK Both inhibitors completely blocked the ERK activation (Fig. 5) These observations suggest an involvement of the Src family kinases and ROCK in the fibrinogen-mediated αIIbβ3 outside-in signaling to ERK2 + mM Mn2+ BSA + 0.5 mM Mn2+ Total ERK1/2 2.0 * 1.5 0.5 Leu33 Pro33 * * 1.0 The platelet integrin αIIbβ3 plays a crucial role in platelet aggregation and thrombus formation by binding to fibrinogen initiating fibrinogen-dependent plateletcrosslinking [24, 25] The fibrinogen engagement of the integrin activates a great variety of outside-in signals leading to elevated intracellular Ca2+ flux and cytoskeletal rearrangement [2, 10] Several polymorphisms in the integrin β3 subunit have been associated with platelet dysfunction Among them, the Leu33Pro substitution of αIIbβ3 has been reported to exhibit prothrombotic characteristics in several works [4, 7, 20] ERK1 and ERK2 are involved in cell growth, proliferation, and adhesion, megakaryocyte differentiation, proplatelet formation [26, 27], and the release of stored Ca2+ + mM Mn2+ pERK2 Discussion αIIbβ3-Dependent ERK Signaling Is Regulated by Src and Rho Kinases * * 2.5 10 Adhesion time, 20 Fig Activation of ERK2 in αIIbβ3-expressing CHO cells adhering to immobilized fibrinogen in the presence of 0.5 mM Mn2+ CHO cells were incubated in the presence of 0.5 mM Mn2+ for and subsequently allowed to adhere to 100 μg/mL fibrinogen or maintained in suspension over 1% BSA for 2.5, 5, 10, and 20 at 37 ° C After the incubation time, cells were solubilized, and the lysates were analyzed for pERK2 activity The enhanced ERK2 activation in Pro33 compared to Leu33 cells was significant at 5, 10, and 20 Results are representative of experiments * p < 0.05, evaluated by unpaired t test Acta Haematol 2017;137:44–50 DOI: 10.1159/000450783 47 Downloaded by: Rutgers University Alexander Library 128.6.218.72 - 2/20/2017 12:55:45 PM Leu33 Pro33 * 1.5 pERK2/total ERK1/2 2.0 pERK2/total ERK1/2 CHO cells adhering to immobilized fibrinogen in the presence of various concentrations of Mn2+ CHO cells were incubated in the absence or presence of the indicated concentrations of Mn2+ for and subsequently allowed to adhere to 100 μg/mL fibrinogen or maintained in suspension over 1% BSA After a 10-min incubation at 37 ° C, cells were solubilized, and the lysates were processed as described in Materials and Methods The enhanced ERK2 activation in Pro33 compared to Leu33 cells was significant at concentrations of 0, 0.5, and mM Mn2+ Results are representative of experiments * p < 0.05, evaluated by unpaired t test BSA Fibrinogen BSA Fibrinogen + 10 μM + 10 μM PP1 PP3 Fibrinogen Src pY418 Total ERK1/2 3.0 2.5 * pERK2/total ERK1/2 Src pY418/total Src BSA pERK2 Total Src 2.0 1.5 1.0 * 0.5 a Fibrinogen + 10 μM + 10 μM PP1 PP3 BSA HPA-1a (Leu33) b HPA-1b (Pro33) * 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 Fig Src pY418 activity (a) and effect of PP1 on ERK2 activity (b) in αIIbβ3-expressing CHO cells adhering to immobilized fibrinogen CHO cells were maintained in suspension over 1% BSA or allowed to adhere to 100 μg/mL fibrinogen To study the effect of PP1 on ERK2 activity, cells were preincubated with 10 μM PP1 for 30 and subsequently allowed to adhere to fibrinogen After 10 * * * HPA-1b (Pro33) HPA-1a (Leu33) of incubation, cells were solubilized, and equal aliquots of samples containing 50 μg of protein were separated by 10% SDSPAGE gel The blots were probed with anti-Src pY418, anti-v-Src, anti-pERK2, or anti-ERK antibodies and quantified by densitometry * p < 0.05, evaluated by unpaired t test Results are representative of experiments 20 μM Fibrinogen 10 μM Y27632 HA1077 BSA Fibrinogen 10 μM Y27632 20 μM HA1077 BSA pERK2 Total ERK1/2 1.2 Fig Effect of Rho kinase inhibition on 48 Acta Haematol 2017;137:44–50 DOI: 10.1159/000450783 0.8 0.6 * 0.4 0.2 HPA-1a (Leu33) HPA-1b (Pro33) Huynh/Nguyen/Pham/Nguyen/Van Vo/ Gyenes/Stoldt Downloaded by: Rutgers University Alexander Library 128.6.218.72 - 2/20/2017 12:55:45 PM * 1.0 pERK2/total ERK1/2 ERK2 activity in αIIbβ3-expressing CHO cells adhering to immobilized fibrinogen CHO cells preincubated either with PBS or with 10 μM Y27632 or 20 μM HA1077 for 30 at 37 ° C and subsequently allowed to adhere to 100 μg/mL fibrinogen After 10 of incubation at 37 ° C, cells were solubilized, and equal aliquots of samples containing 50 μg of protein were separated by 10% SDS-PAGE gel The blots were probed with antiphospho-antibody (pERK2) or anti-ERK antibody and quantified by densitometry (ratio of pERK2 to total ERK in arbitrary units) * p < 0.05, evaluated by unpaired t test Results are representative of experiments in platelets [15] Fibrinogen-adherent Pro33 CHO cells exhibit enhanced αIIbβ3-mediated outside-in signaling to ERK2 and MLC [18], suggesting a role of ERK2 signaling in prothrombotic characteristics of this isoform in platelets Our aim was to further assess which signaling pathways are involved in ERK activation One of the possibilities to activate integrins is triggering an active conformation in their extracellular domains by divalent cations [28, 29] We raised the following question: to what extent Mn2+ ions alone regulate αIIbβ3mediated outside-in signaling in comparison to ligand engagement? To analyze how this distinct manner of activation is reflected in receptor signaling, we examined outside-in signaling induced by immobilized fibrinogen or Mn2+ alone and by a combination of both Furthermore, we analyzed how these processes are modified by the Leu33/Pro33 polymorphism Our observations that Mn2+ cations alone elevate the ERK2 activity only to a small extent when compared to immobilized fibrinogen suggest a less important role of Mn2+ in regulating ERK2 signaling (Fig. 2) Previous studies have shown that Mn2+ increases the binding affinity of αIIbβ3 to ligands, but this activation is not maximal and depends on the integrin isoform type as well as the context [30] Based on previous studies and our data, it is hypothesized that Mn2+ alone cannot induce ERK signaling of αIIbβ3 In contrast, when combined with immobilized fibrinogen, Mn2+ induces a synergistic effect leading to maximal ERK activity after 10 of adhesion In general, upon the whole incubation time, Pro33 cells exhibited a significantly higher ERK activity than Leu33 cells In our work, we showed that the Leu33/Pro33 polymorphism modulates the αIIbβ3-mediated outside-in signaling to Src (Fig. 4a) This tyrosine kinase plays an essential role in integrin signaling and is directly associated with αIIbβ3 integrin [13, 14, 31] Src kinase has been reported to play a central role in the regulation of various pathways, including the MAP kinase cascade [32] On the one hand, it has been shown that in adherent chick embryo fibroblast cells phosphorylated ERK is targeted after integrin engagement or upon v-Src activation to newly forming cell-matrix adhesion [33] On the other hand, in thrombin-activated human platelets, the Src kinase inhibitor PP1 did not block ERK activation [34], indicating Src-independent ERK signaling It seems that integrinmediated ERK activation can occur through several, from each other independent, signaling cascades Therefore, we raised the question whether Src kinases participate in the regulation of ERK2 signaling in fibrinogen-adherent CHO cells Our observation that the Src kinase family in- hibitor PP1 entirely blocked ERK2 activation in both isoforms (Fig. 4) provides evidence that the ERK2 activation in fibrinogen-adhering CHO cells is mediated via Src kinases It has been reported that Src family kinases are also involved in the regulation of the small GTPases [32] These signal proteins are essential for cytoskeleton reorganization, and ROCK is an effector protein of the Rho GTPase with a regulatory function Moreover, ROCK is proposed to be included in MLC phosphorylation [35] As the Thr696 phosphorylation of the PP1-myosine phosphatase regulatory subunit is modulated by the Leu33/Pro33 polymorphism in thrombin-treated platelets [21] and this phosphorylation is regulated by ROCK, we investigated the role of ROCK in the αIIbβ3-mediated outside-in signaling to ERK2 Both Y27632 and HA1077, pharmacologically distinct, specific inhibitors of ROCK, completely blocked ERK2 activation, indicating an essential role of ROCK in αIIbβ3-mediated outside-in signaling to ERK2 (Fig. 5) In conclusion, we provided evidence that the αIIbβ3associated outside-in signaling to ERK is mediated via the Src kinase-ROCK signaling pathway in fibrinogen-adherent CHO cells Although Mn2+ alone only slightly activates ERK, it synergizes the effect of adhesive fibrinogen on ERK activation in both genotypes, showing a significantly higher ERK2 activation in the Pro33 isoform αIIbβ3-Dependent ERK Signaling Is Regulated by Src and Rho Kinases Acta Haematol 2017;137:44–50 DOI: 10.1159/000450783 Acknowledgments We are grateful to Mrs Bianka Maaßen-Weingart und to Mrs Elisabeth Kirchhoff for their excellent technical assistance This work was supported by the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 612, TP B2, and grant No 1161/QĐĐHQG-KHCN of the Vietnam National Universities Ho Chi Minh City Shattil SJ, Newman PJ: Integrins: dynamic scaffolds for adhesion and signaling in platelets Blood 2004;104:1606–1615 Stegner D, Nieswandt B: Platelet receptor signaling in thrombus formation J Mol Med (Berl) 2011;89:109–121 Varga-Szabo D, Pleines I, Nieswandt B: Cell adhesion mechanisms in platelets Arterioscler Thromb Vasc Biol 2008;28:403–412 Michelson AD, Furman MI, GoldschmidtClermont P, Mascelli MA, Hendrix C, Coleman L, Hamlington J, Barnard MR, Kickler T, Christie DJ, Kundu S, Bray PF: Platelet GP IIIa PlA polymorphisms display different sensitivities to agonists Circulation 2000; 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(b) Src Family Kinases and Rho Kinases in ERK Signaling ERK2 activation is mediated by dual phosphorylation on threonine 185 and tyrosine 187 residues [23] Therefore, Src tyrosine kinase and Rho. .. in thrombin-activated human platelets, the Src kinase inhibitor PP1 did not block ERK activation [34], indicating Src- independent ERK signaling It seems that integrinmediated ERK activation can... modulated by the Leu33/ Pro33 polymorphism in thrombin-treated platelets [21] and this phosphorylation is regulated by ROCK, we investigated the role of ROCK in the αIIbβ3-mediated outside -in signaling