A role for adhesion and degranulation-promoting adapter protein in collagen-induced platelet activation mediated via integrin α2β1

. Background: Collagen-induced platelet activation is a key step in the development of arterial thrombosis via its interaction with the receptors glycoprotein (GP)VI and integrin alpha(2)beta(1). Adhesion and degranulation-promoting adapter protein (ADAP) regulates aIIb beta 3 in platelets and aL beta 2 in T cells, and is phosphorylated in GPVI-deficient platelets activated by collagen. Objectives: To determine whether ADAP plays a role in collagen-induced platelet activation and in the regulation and function of a2 beta 1. Methods: Using ADAP-/- mice and synthetic collagen peptides, we investigated the role of ADAP in platelet aggregation, adhesion, spreading, thromboxane synthesis, and tyrosine phosphorylation. Results and Conclusions: Platelet aggregation and phosphorylation of phospholipase C?2 induced by collagen were attenuated in ADAP-/- platelets. However, aggregation and signaling induced by collagen-related peptide (CRP), a GPVI-selective agonist, were largely unaffected. Platelet adhesion to CRP was also unaffected by ADAP deficiency. Adhesion to the alpha(2)beta(1)-selective ligand GFOGER and to a peptide (III-04), which supports adhesion that is dependent on both GPVI and alpha(2)beta(1), was reduced in ADAP-/- platelets. An impedance-based label-free detection technique, which measures adhesion and spreading of platelets, indicated that, in the absence of ADAP, spreading on GFOGER was also reduced. This was confirmed with non-fluorescent differential-interference contrast microscopy, which revealed reduced filpodia formation in ADAP-/- platelets adherent to GFOGER. This indicates that ADAP plays a role in mediating platelet activation via the collagen-binding integrin alpha(2)beta(1). In addition, we found that ADAP-/- mice, which are mildly thrombocytopenic, have enlarged spleens as compared with wild-type animals. This may reflect increased removal of platelets from the circulation.