Flow dynamics and haemostasis

Fluid-dynamic conditions that are compatible with tensile stress on the bonds between platelet glycoprotein Ibalpha and immobilized Von Willebrand factor A1 domain (VWF-A1) led to Ca++ release from intracellular stores (type alpha/beta peaks), which preceded stationary platelet adhesion. Raised levels of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate inhibited these [Ca++]i oscillations and prevented stable adhesion. Once adhesion was established through the integrin alphaIIbbeta3, new [Ca++]i oscillations (type gamma) of greater amplitude and duration, and involving a transmembrane ion flux, developed in association with the recruitment of additional platelets into aggregates. We have defined the distinct roles that the two ADP receptors, P2Y1 and P2Y12, play in the early events that follow the initial platelet interaction with immobilized VWF-A1 under high flow conditions. We have examined the consequences of specific pharmacologic inhibition of P2 receptors and our findings demonstrate a differential role of P2Y1 and P2Y12, respectively, in platelet adhesion and aggregation onto immobilized VWF under elevated shear stress, and highlight the distinct contribution of signaling pathways dependent on Src family kinases, PLC, and phosphoinositide 3-kinase (PI 3-K) to these processes. Results have been achieved through original experiments under flow, thoroughly characterized by ad hoc image analysis techniques and quantitative kinetic analysis.