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Summary. In the classical concept of platelet integrin activation, it is considered that unidirectional conformational changes of a IIb b 3 and a 2 b 1 regulate the adhesiveness of platelets for fibrin(ogen) and collagen, respectively. Here, we summarize recent evidence that these conformational changes: (i) can also occur in the reverse direction; and (ii) are not independent events. Platelet stimulation through the P2Y 12 receptors provokes only transient a IIb b 3 activation via signaling routes involving phosphoinositide 3-kinases and Rap1b. Furthermore, a IIb b 3 can be secondarily inactivated in platelets with prolonged high Ca 2+ rises, which expose phosphatidylserine and bind coagulation factors. Thus, platelet stimulation with strong agonists (collagen and thrombin) also results in transient integrin activation. Integrin a 2 b 1 is found to be activated by a mechanism that is directly linked to a IIb b 3 activation. Integrin a 2 b 1 can adopt different activation states, depending on the trigger. Conclusively, reversibility and synchrony of platelet integrin activation are newly identified mechanisms to restrict thrombus growth and to allow optimal coagulation factor binding. Back-shifting of activated integrins towards their resting state may be a novel goal of antithrombotic medication.

Platelet integrins ␣ 2 ␤ 1 and ␣ IIb ␤ 3 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 ␣ IIb ␤ 3 is not only sufficient, but also a prerequisite for ␣ 2 ␤ 1 activation. Compared with platelets in plasma, stimulation of washed platelets resulted in only a minor activation of ␣ 2 ␤ 1 , 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 ␣ IIb ␤ 3 with the ligand-mimetic peptide RGDS, resulting in outside-in signaling, led to a powerful ␣ 2 ␤ 1 activation, even in the absence of overall platelet activation, involving tyrosine kinase activity but no protein kinase C activation. The absolute necessity of ␣ IIb ␤ 3 for proper ␣ 2 ␤ 1 activation on platelets was demonstrated by using the ␣ IIb ␤ 3 antagonist aggrastat, which was able to completely abolish ␣ 2 ␤ 1 activation, both under static and flow conditions. In addition, analogous experiments with Glanzmann platelets lacking ␣ IIb ␤ 3 confirmed the indispensability of ␣ IIb IntroductionIntegrins are a large family of heterodimeric transmembrane receptors, each consisting of an ␣ and ␤ subunit, and are key effectors of cell growth, migration, differentiation, and survival. 1,2 Integrins possess the unique ability to signal across the plasma membrane in both directions, and since most integrins are not constitutively active, they are expressed on the cell surface as low-affinity receptors. When cells become activated, cytosolic proteins can bind to the cytoplasmic domains of integrins and as a consequence, the integrins are turned into their high-affinity state ("inside-out" signaling). In a process called "outside-in" signaling, ligand-binding of integrins then again activates intracellular pathways via their cytoplasmic domains, which are connected to the cytoskeleton and are associated with several intracellular signaling molecules. Important signaling modulators necessary for the generation of outside-in signals are members of the Src family protein tyrosine kinases, with c-Src as the initiating molecule due to its constitutive interaction with, for example, ␤ 3 integrins. 3 Such outside-in signals also result in conformational alterations of the integrin (also designated integrin activation), and subsequently these activated integrins can trigger another process of inside-out signaling. 4 This integrin activation upon conformational changes is often defined as an increase in integrin "affinity" for its ligand, and has been the topic of many studies. [4][5][6] In addition, cell activation also promotes clustering of integrins contributing to the "avidity" or "valency" regulation ...