The small GTPase Rap1b regulates the cross talk between platelet integrin α2β1 and integrin αIIbβ3 by Bruno Bernardi, Gianni F. Guidetti, Francesca Campus, Jill R. Crittenden, Ann M. Graybiel, Cesare Balduini, and Mauro Torti Blood Volume 107(7):2728-2735 April 1, 2006 ©2006 by American Society of Hematology

Integrin α2β1-dependent platelet adhesion triggers Rap1b activation. Integrin α2β1-dependent platelet adhesion triggers Rap1b activation. (A) Rap1b activation in platelets adherent to different ligands for integrin α2β1 Human platelets were incubated with immobilized BSA, monomeric collagen, the small proteoglycan decorin, or the collagen-derived peptides CB8(II) and CB11(II), as indicated, for 30 minutes. Active Rap1b (Rap1b-GTP) was precipitated with immobilized GST-tagged RalGDS-RBD and identified by immunoblotting with anti-Rap1 antibody. An identical amount of proteins from each cell lysate was also subjected to immunoblotting analysis with the anti-Rap1 antibody to verify the level of the protein in the different samples (Rap1b TOT). (B) None of the analyzed ligands activate platelet GPVI. The GPVI-associated FcR γ-chain was immunoprecipitated from platelets adherent to decorin, CB8(II), CB11(II), monomeric collagen, and convulxin and analyzed by immunoblotting with antiphosphotyrosine antibody (P-Tyr). The same nitrocellulose membranes were then reprobed with anti-FcR γ-chain (bottom panel). (C) Time course of integrin α2β1-dependent platelet adhesion and Rap1b activation. Human platelets were incubated with the 4 different integrin α2β1 ligands for 15, 30, or 60 minutes, as indicated. The percentage of adherent platelets was determined by a colorimetric assay and is reported above the top panel. The immunoblots show active Rap1b (Rap1-GTP) isolated by the pulldown assay with GST-RalGDS-RBD and the level of total Rap1b (Rap1b TOT) present in the lysates used for the Rap1b activation assays. Bruno Bernardi et al. Blood 2006;107:2728-2735 ©2006 by American Society of Hematology

Integrin α2β1-induced activation of Rap1b does not require platelet-derived secondary agonists. Integrin α2β1-induced activation of Rap1b does not require platelet-derived secondary agonists. Platelet adhesion to monomeric collagen was analyzed using washed platelets preincubated with the ADP scavengers CP/CPK or apyrase or with the ADP receptor antagonists A3P5PS or AR-C69931MX (A) or upon treatment with the cyclooxygenase inhibitors aspirin (asa) and indomethacin (indo) or with the thromboxane A2 receptor antagonist SQ29548 (B), as described in “Materials and methods.” The immunoblots show both active Rap1b (Rap1b-GTP) and total Rap1b (Rap1b TOT) isolated from the same number of adherent platelets. The effect of the different treatments on the extent of platelet adhesion is reported on the top of the panels (adhesion, %), considering as 100% the adhesion of nontreated platelets. Bruno Bernardi et al. Blood 2006;107:2728-2735 ©2006 by American Society of Hematology

Role of PLC-derived second messengers on integrin α2β1-dependent activation of Rap1b. Role of PLC-derived second messengers on integrin α2β1-dependent activation of Rap1b. (A) Washed platelets were left untreated (none) or incubated with the PKC inhibitor Ro31-8220, with the intracellular Ca2+ chelating agent BAPTA-AM, or with both compounds, as indicated in “Materials and methods.” Platelet adhesion to monomeric collagen was evaluated after 30 minutes of incubation and is reported on the top of the panel, considering as 100% the adhesion of untreated platelets. Activated Rap1b in each sample was isolated by the pulldown assay using lysates from an identical number of adherent platelets and identified by immunoblotting (Rap1b-GTP). The bottom panel shows the level of total Rap1b present in identical aliquots of the platelet lysates used for the pulldown assay. (B) Analysis of PLC activation. 32P-labeled platelets were incubated with immobilized monomeric collagen for 30 minutes. Adherent platelets were lysed and total proteins separated by SDS-PAGE on a 5% to 15% acrylamide gradient gel. Phosphorylation of pleckstrin, the main platelet substrate for PKC, was visualized by autoradiography. The migration of phosphorylated pleckstrin is indicated by the arrow on the right, while the migration of molecular mass markers is reported on the left. Some samples of 32P-labeled platelets were preincubated with Ro31-8220, BAPTA-AM, or with the indicated concentrations of U73122, as indicated on the bottom, and analysis of pleckstrin phosphorylation was performed on the same number of adherent platelets. (C) Inhibition of PLC prevents Rap1b activation. Washed platelets were treated with increased concentrations of the PLC inhibitor U73122 or with 10 μM U73343, an inactive related compound, and then incubated with immobilized monomeric collagen. The effect of treatment with U73122 on the extent of platelet adhesion and on the accumulation of active Rap1b in the same number of adherent platelets (Rap1b-GTP), as well as the level of total Rap1b in adherent cells (Rap1b total), are reported. Bruno Bernardi et al. Blood 2006;107:2728-2735 ©2006 by American Society of Hematology

Integrin α2β1-dependent activation of integrin αIIbβ3. Integrin α2β1-dependent activation of integrin αIIbβ3. Aspirin-treated human platelets were allowed to adhere to monomeric collagen through integrin α2β1, and specific binding of biotinylated fibrinogen to adherent platelets was measured as described in “Materials and methods.” When indicated, platelets were pretreated with 1 mM RGDS, 3 U/mL apyrase, 10 μM Ro31-8220, 30 μM BAPTA-AM, or 10 μM U73122, alone or in combination, before incubation with immobilized monomeric collagen. Specific binding of fibrinogen has been calculated for the same number of adherent platelets untreated or treated with the different inhibitors. Data are reported considering as 100% the specific fibrinogen binding to control platelets (none) and represent the means ± SD of 3 different experiments performed in duplicate. Bruno Bernardi et al. Blood 2006;107:2728-2735 ©2006 by American Society of Hematology

Analysis of platelet adhesion and integrin α2β1-dependent outside-in signaling in CalDAG-GEFI–deficient murine platelets. Analysis of platelet adhesion and integrin α2β1-dependent outside-in signaling in CalDAG-GEFI–deficient murine platelets. Platelets from wild-type (+/+) and CalDAG-GEFI–deficient (–/–) mice were incubated with immobilized monomeric collagen. Platelet adhesion (A) and spreading (B) were measured using collagen-coated glass coverslips and determined by fluorescence microscopy analysis upon staining of adherent platelets with TRITC-phalloidin. The reported results are the mean ± SD of 3 independent experiments. The statistical significance of the observed differences, calculated by the Student t test, is also shown. PLCγ2 tyrosine phosphorylation, pleckstrin phosphorylation, and Rap1b activation were analyzed in samples containing identical amount of proteins from platelets adherent to collagen-coated 60 mm–diameter dishes as well as from nonadherent platelets, indicated as A (adherent) and NA (nonadherent) on the top of panels C, D, and E. (C) PLCγ2 was immunoprecipitated with specific antibody and then analyzed by immunoblotting with antiphosphotyrosine antibody (P-Tyr), followed by immunoblotting with anti-PLCγ2 antibody, as indicated on the right. (D) Activation of PKC was analyzed on total platelet proteins from adherent and nonadherent platelets by immunoblotting with antiphosphoserine PKC substrates. The position of phosphorylated pleckstrin (47 kDa) detected in adherent but not in nonadherent platelets is indicated by the arrow on the left. On the right, the position of molecular mass markers is reported. (E) Analysis of Rap1b activation (Rap1-GTP) in the same number of adherent and nonadherent platelets, as well as the total amount of Rap1 in the cell lysate used (Rap1b-TOT), are reported. Results from panels C, D, and E are representative of 2 independent experiments. (F) The specific binding of biotinylated fibrinogen to the same number of wild-type (+/+) and CalDAG-GEFI–deficient (–/–) murine platelets adherent to monomeric collagen through integrin α2β1. Fibrinogen binding measured in adherent wild-type platelets is reported as 100%, and the data are the means ± SD of 3 separate experiments. Bruno Bernardi et al. Blood 2006;107:2728-2735 ©2006 by American Society of Hematology