Lipopolysaccharide (LPS) induced pulmonary neutrophil recruitment and platelet activation is mediated via the P2Y1 and P2Y14 receptors in mice

Abstract

Platelet activation occurs during host defence and in various inflammatory disorders. In animal models of infection and inflammation, experimental depletion of platelets leads to significantly reduced leukocyte recruitment and impaired clearance of pathogens from the lung. It is now appreciated that purinergic receptor activation is required for leukocyte activation, motility and adhesion, and platelet interactions with leukocytes can be modulated by purinergic stimulation of platelets. Here, we have investigated the role of platelet P2Y_1, P2Y₁₂, P2Y₁₄, and P2X₁ receptors on leukocyte recruitment and chemotaxis. Mice were administered either vehicle controls or selective P2Y_1, P2Y₁₂, P2Y₁₄, or P2X₁ antagonists intravenously before intranasal administration of lipopolysaccharide (LPS) to investigate the effect of these drugs on pulmonary leukocyte recruitment, peripheral platelet counts, bleeding times, and ex vivo platelet aggregation. Separately, platelets were incubated with P2Y_1, P2Y₁₂, P2X₁ antagonists, or P2Y₁₄ agonists to assess effects on platelet-induced neutrophil chemotaxis in vitro. Pulmonary neutrophil recruitment induced by intranasal LPS administration was inhibited in mice administered either with P2Y₁ or P2Y₁₄ antagonists, but not with P2Y₁₂ or P2X₁ antagonists. Furthermore, the administration of either a P2Y₁ or a P2Y₁₄ antagonist reversed the incidence of peripheral thrombocytopaenia associated with LPS exposure. Bleeding times were significantly increased in mice administered P2Y_1, P2Y₁₂, or P2X₁ antagonists, whilst ex vivo platelet aggregation to ADP was significantly reduced. These haemostatic responses remained unaltered following antagonism of P2Y₁₄. In vitro chemotaxis assays revealed direct antagonism of platelet P2Y_1, but not P2Y₁₂ or P2X₁ receptors suppressed platelet-dependent neutrophil motility towards Macrophage derived chemokine (MDC, CCL22). Furthermore, the stimulation of platelets with selective P2Y₁₄ agonists (UDP-glucose, MRS2690) resulted in significant platelet-dependent neutrophil chemotaxis. These results reveal a role for P2Y₁ and P2Y₁₄ activation of platelets following exposure to LPS, whilst haemostatic indices were unaffected by inhibition of platelet function with the P2Y₁₄ antagonist in response to LPS.