Activation of the small GTPase Rap2B in agonist-stimulated human platelets.
The activation of the small GTPase Rap2B in resting and agonist-stimulated human platelets was investigated. Both thrombin, that stimulates heterotrimeric G-protein-coupled receptors, and the GPVI ligand convulxin, that activates a tyrosine-kinase based signaling pathway, were able to induced the rapid and sustained binding of GTP to Rap2B. Similarly, a number of other agonists tested, previously known to activate the highly related protein Rap1B, were also able to stimulate Rap2B. In contrast, platelet antagonists that increase the intracellular concentration of cAMP did not signal to Rap2B. Thrombin- and convulxin-induced activation of Rap2B was not dependent on thromboxane A2, did not require the interaction of the protein with the cytoskeleton, and was not regulated by integrin alphaIIbbeta3-dependent outside-in signaling. When secreted ADP was neutralized, activation of Rap2B induced by thrombin, but not by convulxin, was significantly reduced. ADP itself was found to induce the rapid and sustained binding of GTP to Rap2B, and this effect was predominantly mediated by stimulation of the Gi-coupled P2Y12 receptor. Activation of Rap2B promoted by both thrombin and convulxin was regulated by intracellular Ca2+, while protein kinase C was found to be involved in convulxin- but not in thrombin-induced activation of Rap2B. Moreover, Rap2B activation induced by thrombin, but not by convulxin, was totally dependent on phosphatidylinositol 3-kinase activity. These results demonstrate that the small GTPase Rap2B is involved in platelet activation, and outline some important differences between the regulation of highly related GTPases Rap2B and Rap1B in human platelets.
Greco
F
F
Center of Excellence for Applied Biology, Department of Biochemistry, University of Pavia, Pavia, Italy.
Sinigaglia
F
F
Balduini
C
C
Torti
M
M
D000244:Adenosine Diphosphate / Q000378:metabolism; D001792:Blood Platelets / Q000201:enzymology*; D002118:Calcium / Q000378:metabolism; D002850:Chromatography, Gel; D003435:Crotalid Venoms / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D037181:Lectins, C-Type; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D015539:Platelet Activation; D011401:Promoter Regions, Genetic; D011493:Protein Kinase C / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D015398:Signal Transduction; D013917:Thrombin / Q000378:metabolism; D013997:Time Factors; D020668:rap GTP-Binding Proteins / Q000378:metabolism*
D003435:Crotalid Venoms; D037181:Lectins, C-Type; C028890:convulxin; D000244:Adenosine Diphosphate; D000242:Cyclic AMP; D005982:Glutathione Transferase; D019869:Phosphatidylinositol 3-Kinases; D011505:Protein-Tyrosine Kinases; D011493:Protein Kinase C; D013917:Thrombin; C065562:RAP2B protein, human; D020668:rap GTP-Binding Proteins; D002118:Calcium