Rap1B and Rap2B Translocation to the Cytoskeleton by von Willebrand Factor Involves FcγII Receptor-mediated Protein Tyrosine Phosphorylation

Abstract

Stimulation of human platelets with von Willebrand factor (vWF) induced the translocation of the small GTPases Rap1B and Rap2B to the cytoskeleton. This effect was specifically prevented by an anti-glycoprotein Ib monoclonal antibody or by the omission of stirring, but was not affected by the peptide RGDS, which antagonizes binding of adhesive proteins to platelet integrins. Association of Rap2B with the cytoskeleton was very rapid, while translocation of Rap1B occurred in a later phase of platelet activation and was totally inhibited by cytochalasin D. vWF also induced the rapid tyrosine phosphorylation of several proteins that was prevented by the tyrosine kinases inhibitor genistein and by cAMP-increasing agents. Under these conditions, also the association of Rap1B and Rap2B with the cytoskeleton was prevented. Translocation of Rap proteins to the cytoskeleton induced by vWF, but not by thrombin, was inhibited by a monoclonal antibody against the Fc␥II receptor. The same antibody inhibited vWF-induced tyrosine phosphorylation of selected substrates with molecular masses of about 75, 95, and 150 kDa. Three of these substrates were identified as the tyrosine kinase pp72 syk , the phospholipase C␥2, and the inositol 5-phosphatase SHIP. Our results indicate that translocation of Rap1B and Rap2B to the cytoskeleton is regulated by tyrosine kinases and suggest a novel role for the Fc␥II receptor in the mechanism of platelet activation by vWF.Rap proteins are low molecular weight GTP-binding proteins that share about 50% sequence homology with the product of the ras protooncogene. Human platelets express two members of the Rap family of proteins, Rap1B and Rap2B, which are located at the membrane as a consequence of post-translational modifications, including isoprenylation, proteolysis, and carboxylmethylation (1). The role of Rap1B and Rap2B in platelet function is still poorly understood. Rap1B is phosphorylated by the cAMP-dependent protein kinase A (PKA) 1 (2) and is rapidly activated upon platelet stimulation with extracellular agonists (3, 4). By contrast, Rap2B is not a substrate for PKA (5), and its activation upon cell stimulation has not been reported. In thrombin-treated platelets both Rap1B and Rap2B translocate to the cytoskeleton (6, 7). This actin-based structure is not only responsible for the morphological changes of activated platelets, but also represents a network connecting several molecules involved in signal transduction processes, including protein-tyrosine kinases, lipid metabolizing enzymes, and membrane glycoproteins (8). Translocation of Rap2B to the cytoskeleton in thrombin-stimulated platelets requires cell aggregation and is promoted by secondary signals generated by binding of fibrinogen to the membrane glycoprotein IIb-IIIa (GP IIb-IIIa) (9). Similarly, fibrinogen binding to GP IIb-IIIa strongly supports Rap1B translocation to the cytoskeleton, although a small amount of this protein associates with the actin-based structures, even in activated cells in the absence of aggregation a...

Platelet adhesion signalling and the regulation of thrombus formation

“…Binding of VWF to GPIb-V-IX upregulates integrin αIIbβ3 affinity (Asazuma et al, 1997;Kasirer-Friede et al, 2004;Milner et al, 1998;Munday et al, 2000;Nesbitt et al, 2002;Torti et al, 1999;Yuan et al, 1999). The integrin can then bind to VWF, thereby enhancing adhesion, and contributing to thrombus formation by binding to fibrinogen.…”

“…The binding of VWF to GPIb under shear stress can stimulate calcium mobilization (Kroll et al, 1991;Mazzucato et al, 2002;Milner et al, 1998;Nesbitt et al, 2002;Yap et al, 2000), protein kinase C (PKC), protein kinase G (PKG) (Li et al, 2003;Yap et al, 2000), phosphoinositide 3-kinase (PI3K) (Jackson et al, 1994;Munday et al, 2000;Yap et al, 2000) and cytoskeletal rearrangements (Torti et al, 1999;Yuan et al, 1999). VWF binding also upregulates integrin αIIbβ3 affinity indirectly through the stimulation of ADP secretion (Moake et al, 1988).…”

“…2). GPIb might also activate platelets by triggering Src-family-kinase-mediated phosphorylation of FcRγ and FcγRIIA, receptors with which GPIb physically associates (Canobbio et al, 2001;Falati et al, 1999;Sullam et al, 1998;Torti et al, 1999;Wu et al, 2001).…”

“…GPIb binding is associated with the stimulation of tyrosine kinase signalling (Asazuma et al, 1997;Ozaki et al, 1995;Razdan et al, 1994). Principal players in this pathway include the non-receptor tyrosine kinases Src, Fyn, Lyn and Syk, phospholipase Cγ2 (PLCγ2), and adaptor proteins such as Shc, linker for activation of T cells (LAT) and SLP-76 (Asazuma et al, 1997;Falati et al, 1999;Jackson et al, 1994;Marshall et al, 2002;Torti et al, 1999;Wu et al, 2001). Exactly how these components cooperate in GPIb-V-IX signalling is not known, but there is good evidence that it might be similar to GPVI signalling in platelets (described in detail below; Fig.…”

Structure, functional regulation and signaling properties of Rap2B

“…In platelets, the GPIb-IX-V complex, a member of the leucine-rich GP gene family, is the main receptor for vWF (58). In 1999, Torti et al (59) revealed that the translocation of Rap1B and Rap2B to the cytoskeleton was induced by vWF, following binding of vWF to the GPIb-IX-V complex through a GP IIb/IIIa-independent mechanism. In a previous study, Rap2B was prevented from associating with the cytoskeleton by cytochalasin D, which did not inhibit platelet aggregation (60).…”