Platelet-dependent primary hemostasis promotes selectin- and integrin-mediated neutrophil adhesion to damaged endothelium under flow conditions

Co-localization of blood platelets and granulocytes at sites of hemostasis and inflammation has triggered an intense interest in possible interactions between these cellular processes and induction of vessel wall injury. Leukocyte adhesion to endothelial cells decreases with increasing shear and is dependent on an initial rolling phase mediated by selectins. We hypothesized that flow-dependent platelet adhesion at an injured vessel wall will lead to P-selectin expression by platelets, thus mediating leukocyte co-localization. A perfusion chamber was used in which flowing whole blood induced platelet adhesion to a subendothelial matrix (ECM) of cultured human umbilical vein endothelial cells (HUVEC). We compared neutrophil (polymorphonuclear leukocyte [PMN]) interactions with HUVEC and their ECM with and without adhered platelets. PMNs adhered predominantly to ECM-adhered platelets and not to endothelial cells. ECM alone did not support PMN adhesion under flow conditions. PMN adhesion to unstimulated HUVEC was only substantial at low shear (up to 200 cells/mm² at shear stress 80 mPa). In marked contrast, PMN adhesion to ECM-adhered platelets was dramatically increased, and adhesion was demonstrated at much higher shear stress (up to 640 mPa). Studies with specific antibodies showed that the platelet-dependent neutrophil adhesion was selectin-mediated. Inhibition of P-selectin caused a marked inhibition of adhesion at high shear stress, whereas the role of leukocyte L-selectin was less pronounced. β₂-Integrin-blocking antibodies inhibited static neutrophil adhesion. fMLP induced L-selectin shedding from leukocytes, resulting in decreased leukocyte adhesion. In conclusion, platelet-dependent hemostasis at the ECM appears to be a powerful intermediate in neutrophil-vessel wall interactions at shear stresses that normally do not allow neutrophil adhesion to intact endothelium.