Synergistic effect of signaling from receptors of soluble platelet agonists and outside-in signaling in formation of a stable fibrinogen-integrin αIIbβ3-actin cytoskeleton complex

• Formation of stable aggregate requires platelet activation for more than 1 min.
• Maximal αIIbβ3–actin association requires at least 5 min of platelet activation.
• αIIbβ3–actin association requires fibrinogen mediated platelet-to-platelet bridging.
• Direct αIIbβ3 activation is not sufficient for cytoskeleton complex formation.

IntroductionThrombus formation in the injured vessel wall is a highly complex process involving various blood-born components that go through specific temporal and spatial changes as observed by intravital videomicroscopy. Platelets bind transiently to the developing thrombus and may either become stably incorporated into or disengage from the thrombus. The aim of the present study was to reveal the processes involved in the formation of a stable thrombus.MethodsPlatelet-rich plasma and washed platelets were studied by the aggregometer. The aggregate stability was challenged by eptifibatide. Platelet Triton-insoluble fraction was prepared and the actin and αIIb content in the cytoskeleton was analyzed by western blot.ResultsMaximal actin polymerization is achieved 1 min after platelet activation while maximal αIIbβ3–actin cytoskeleton association requires 5 to 10 min of activation and fibrinogen-mediated platelet-to-platelet bridging. Thus, actin polymerization is dependent on platelet activation and requires neither αIIbβ3 integrin occupation nor platelet aggregation. Formation of a stable aggregate requires platelet activation for more than 1 min, complete increase in actin cytoskeleton fraction and partial association of αIIbβ3 with the actin cytoskeleton. However, direct αIIbβ3 activation is not sufficient for cytoskeleton complex formation. Thus, stable αIIbβ3–fibrinogen interaction, representing stable aggregate, is achieved after more than 1 min agonist activation, involving inside-out and outside-in signaling but not after direct integrin activation, involving only outside-in signaling.ConclusionsFormation of a stable fibrinogen–αIIbβ3–actin cytoskeleton complex is the result of the combined effect of platelet stimulation by soluble agonists, activation of αIIbβ3, fibrinogen binding and platelet-to-platelet bridging.