Signal-transducing mechanisms involved in activation of the platelet collagen receptor integrin alpha(2)beta(1)

Evidence was obtained about the mechanism responsible for platelet integrin alpha(2)beta activation by determining effects of various inhibitors on so luble collagen binding, a parameter to assess integrin alpha(2)beta(1), act ivation, in stimulated platelets, Agonists that can also activate platelet glycoprotein IIb/IIIa are able to activate integrin alpha(2)beta(1),, but t hose operating via glycoprotein Ib cannot. Activation of alpha(2)beta(1), i nduced by low thrombin or collagen-related peptide concentrations was almos t completely inhibited by apyrase, and the inhibitors wortmannin, 4-amino-5 -(chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine bisindolylmaleimide I, and SQ29548 significantly inhibited it. Activation induced by high thrombin or collagen-related peptide concentrations was far less sensitive to these inhibitors. However, only wortmannin markedly inhibited ADP-induced integr in alpha(2)beta(1) activation, and this was not ADP concentration-dependent , These results suggest that at the low agonist concentrations, the release d ADP would be a primary inducer of integrin alpha(2)beta(1), activation, w hile act the high agonist concentrations, there would be several pathways t hrough which integrin alpha(2)beta(1) activation can be induced. Kinetic an alyses revealed that ADP-induced platelets had about the same number of bin ding sites (B-max) as thrombin-induced platelets, but their affinity (K-d) for soluble collagen was 3.7-12.7-fold lower, suggesting that activated int egrin alpha(2)beta(1), induced by ADP is different from that induced by thr ombin, The data are consistent with an activation mechanism involving relea sed ADP and in which there exists two different states Of activated integri n. alpha(2)beta(1); these activated forms of integrin alpha(2)beta(1), woul d have different conformations that determine their ligand affinity.