Purinergic control of platelet mediated leukocyte recruitment

Platelet-leukocyte complexes form as a result of the rheological displacement of blood cells. This 'traps' leukocytes into an environment rich in platelets towards the vessel periphery, greatly enhancing the possibility of collisions between platelets and leukocytes. However, unless platelets become activated by inflammatory stimuli, the result of these collisions appears to be unresponsive in terms of adhesion to the vascular endothelium, as recognised by the presence of circulating platelet-leukocyte complexes found in control subjects with no inflammation. It would therefore appear that platelets require additional inflammatory stimuli above that which is required for transitory interactions between platelets and leukocytes before firm adhesion to the endothelium takes place. Three purinergic receptors are expressed on the surface of platelets. The P2X1 cation channel is activated by adenosine tri-phosphate (ATP), whilst two G-protein coupled receptors: P2Y1 and P2Y12 are both activated by ADP. Evidence now suggests that purinergic receptors are involved in platelet mediated inflammatory events. Both P2Y1 and P2Y12 activation leads to the expression of platelet P-selectin and the formation of platelet-leukocyte complexes which can be inhibited by selective antagonists. Indeed, the activation of platelets via chemokines, and low levels of primary agonists such as ADP have been shown to be dependent on the purinergic P2Y1 receptor rather than the P2Y12 receptor, which is believed to have a greater role in sustained thrombus formation. Although the phenomenon of platelet-leukocyte complex formation and subsequent tissue recruitment has not been tested after platelet activation via chemokines or other inflammatory mediators, ADP signalling may contribute to the initial stages of platelet activation in an inflammatory setting. This may resemble a 'bottleneck' in the directing of leukocyte migration into inflamed tissue via the orchestration of multiple chemokines and inflammatory mediators. We propose a programme of research to address the mechanisms involved in ADP induced platelet activation and ensuing leukocyte recruitment to inflamed endothelium, and to explore a possible role for chemokine-induced platelet activation in a murine model of allergic inflammation. Our initial studies will establish the importance of ADP induced activation of platelets on pulmonary leukocyte recruitment in sensitized mice after allergen exposure. We will use a pharmacological approach using selective P2Y receptor antagonists and the role of specific P2Y receptors will be corroborated in mice deficient in the expression of P2Y1 or P2Y12. ADP induced platelet activation is involved in establishing platelet-leukocyte aggregates in circulating blood and platelet adhesion under flow conditions. Using a intravital microscope to visualise the mouse tracheal circulation, we will establish whether P2Y1 or P2Y12 (or both) activation is necessary for platelet induced leukocyte rolling and firm adhesion to endothelium. This technique will be enhanced by visualizing platelets with a fluorescent label (e.g. BCECF, Molecular probes) using a standard fluorescent adaption to the intravital microscope in use in our laboratory. Time lapse recording (Zeiss Axiocam) of the exposed venule will be conducted, of four different parameters: the total number of accumulated cells, the number of rolling cells, the number of firmly arrested cells, and the rolling velocity for the period of analysis comparing leukocytes attached/ not attached to platelets. We will study whether allergen or chemokine activation of platelets induces platelet P-selectin expression using flow cytometric analysis, since platelet P-selectin is important for platelet-leukocyte complex formation. The ability of receptor antagonists for P2Y1 and P2Y12 or the use of platelets taken from P2Y1-/- or P2Y12-/- mice at inhibiting these events will finally be evaluated.