Role of P-selectin, β2-integrins, and Src tyrosine kinases in mouse neutrophil-platelet adhesion

Background: The initial interaction of human polymorphonuclear leukocytes (PMN) with activated human platelets is mediated by P-selectin and its leukocyte ligand PSGL-1; subsequently the interaction is strengthened by activation of alphaMbeta2 via protein tyrosine phosphorylation mediated by Src kinases and binding of activated alphaMbeta2 to its platelet counter-receptor(s). Objectives: Because mouse models are being used to define the role of PMN-platelet interactions in thrombosis and the response to vascular injury, we investigated the molecular determinants responsible for the interaction of murine PMNs with activated murine platelets. Methods: Mouse platelets were labeled with the green fluorescent dye BCECF and then activated with thrombin and fixed with 1% paraformaldehyde. Mouse PMNs were labeled with the red fluorescent dye hydroethidine and then stirred with the fixed platelets. After stopping the reaction with paraformaldehyde, formation of mixed cell conjugates was analyzed by flow cytometry. Results: In time course experiments, 90+/-1.9% of PMNs formed mixed conjugates with platelets after 2 min and the mean (+/-SEM) number of platelets per positive PMN was 8.4+/-1.5. A monoclonal antibody to P-selectin reduced the percentage of PMNs with attached platelets to 16+/-2.4% (P=0.001), and only 8+/-5% of PMNs interacted with platelets from P-selectin-/- mice. In contrast, monoclonal antibodies to PSGL-1, beta2-integrin, and alphaIIbbeta3 had much less or no effect on the production of mixed cell aggregates. To. better identify a secondary contribution of beta(2)-integrins, P-selectin interactions were disrupted by briefly adding 5 mM EGTA to already-formed mixed cell aggregates. Brief EGTA treatment alone reduced the percentage of PMNs with attached platelets to 70+/-3.5% (P=0.004 vs. no treatment), but did not modify the number of platelets per positive PMN (9.5+/-1.7). The combination of brief EGTA treatment and a monoclonal antibody to beta(2)-integrin lowered the percentage of PMN with attached platelets to 50+/-7% and reduced the number of platelets attached per positive PMN to 3.6+/-0.7 (P=0.03 vs. brief EGTA treatment only). Brief EGTA treatment did not modify the effect of the other antibodies. When the incubation was stopped with EGTA the Src inhibitors PP1 and PP2 reduced PMN-platelet adhesion, while the inactive analog PP3 was ineffective. Conclusions: These results confirm that P-selectin plays a prominent role in mediating the initial interactions between mouse PMN and platelets, and provide support for additional contributions from beta(2)-integrins and Src family kinases.