Activation of αIIbβ3 is a sufficient but also an imperative prerequisite for activation of α2β1 on platelets

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Abstract
Platelet integrins α2β1 and αIIbβ3 play critical roles in platelet adhesion and thrombus formation after vascular injury. On resting platelets, both integrins are in a low-affinity state. However, agonist stimulation results in conformational changes that enable ligand binding that can be detected with conformation dependent monoclonal antibodies (mAbs). By using such conformation-dependent mAbs, we could demonstrate that activation of integrin αIIbβ3 is not only sufficient, but also a prerequisite for α2β1 activation. Compared with platelets in plasma, stimulation of washed platelets resulted in only a minor activation of α2β1, as detected with the activation-sensitive mAb IAC-1. Addition of fibrinogen to stimulated washed platelets greatly potentiated activation of this integrin. Also, treatment of αIIbβ3 with the ligand-mimetic peptide RGDS, resulting in outside-in signaling, led to a powerful α2β1 activation, even in the absence of overall platelet activation, involving tyrosine kinase activity but no protein kinase C activation. The absolute necessity of αIIbβ3 for proper α2β1 activation on platelets was demonstrated by using the αIIbβ3 antagonist aggrastat, which was able to completely abolish α2β1 activation, both under static and flow conditions. In addition, analogous experiments with Glanzmann platelets lacking αIIbβ3 confirmed the indispensability of αIIbβ3 for α2β1 activation.