VENOUS LEVELS OF SHEAR SUPPORT NEUTROPHIL-PLATELET ADHESION AND NEUTROPHIL AGGREGATION IN BLOOD VIA P-SELECTIN AND BETA(2)-INTEGRIN

Background-After activation, platelets adhere to neutrophils via P-sel ectin and beta(2)-integrin. The molecular mechanisms and adhesion even ts in whole blood exposed to venous levels of hydrodynamic shear in th e absence of exogenous activation remain unknown. Methods and Results- Whole blood was sheared at approximate to 100 s(-1). The kinetics of n eutrophil-platelet adhesion and neutrophil aggregation were measured i n real time by flow cytometry. P-selectin was upregulated to the plate let surface in response to shear and was the primary factor mediating neutrophil-platelet adhesion. The extent of neutrophil aggregation inc reased linearly with platelet adhesion to neutrophils, Blocking either P-selectin, its glycoprotein ligand PSGL-1, or both simultaneously by preincubation with a monoclonal antibody resulted in equivalent inhib ition of neutrophil-platelet adhesion (approximate to 30%) and neutrop hil aggregation (approximate to 70%). The residual amount of neutrophi l adhesion was blocked with anti-CD11b/CD18. Treatment of blood with p rostacyclin analogue ZK36374, which raises cAMP levels in platelets, b locked P-selectin upregulation and neutrophil aggregation to baseline. Complete abrogation of platelet-neutrophil adhesion required both ZK3 6374 and anti-CD18. Electron microscopic observations of fixed blood s pecimens revealed that platelets augmented neutrophil aggregation both by forming bridges between neutrophils and through contact-mediated a ctivation. Conclusions-The results are consistent with a model in whic h venous levels of shear support platelet adherence to neutrophils via P-selectin binding PSGL-1. This interaction alone is sufficient to me diate neutrophil aggregation. Abrogation of platelet adhesion and aggr egation requires blocking Mac-1 in addition to PSGL-1 or P-selectin. T he described mechanisms are likely of key importance in the pathogenes is and progression of thrombotic disorders that are exacerbated by leu kocyte-platelet aggregation.