Activated platelets adherent to an intact endothelial cell monolayer bind flowing neutrophils and enable them to transfer to the endothelial surface

We have investigated the role that platelets may play in promoting adhesion of neutrophils to morphologically intact endothelium. Immortalized human microvascular endothelial cells (HMEC-1) were grown to confluence in a glass capillary (microslide) and incorporated in a flow-based assay that allowed video-microscopic quantitation of adhesive interactions of perfused, isolated neutrophils (wall shear rate 140 s(-1)). Platelets (with or without stimulation with thrombin) were first sedimented onto the HMEC-1 cells and formed discrete attachments covering <1% of the surface area. When neutrophils were perfused over the platelet-treated HMEC-1 cells, many short-lived adhesive interactions were seen (lasting approximately 0.3 seconds), whereas none were seen for monolayers without platelets. Few of these interactions converted to stationary adhesion, and only small numbers of neutrophils remained attached after a period of washout unless they were pre-stimulated with formyl peptide (fMLP; 10(-7) mol/L). Then about 30% of adhesive interactions by activated neutrophils were seen to transform to a stationary adhesion, and numerous adherent cells remained after a period of washout. Studies with function-blocking antibodies showed that capture of the neutrophils was dependent on P-selectin exposed on platelets. Initial immobilization was mediated predominantly by the beta2-integrin CD11b/CD18 expressed by neutrophils, but CD11a/CD18 also appeared to play a role in prolonged attachment. Visually, adhesion first occurred at sites occupied by platelets, but some activated neutrophils migrated onto the endothelial cells. These studies indicate that even small numbers of platelets that have adhered to morphologically intact endothelium have the potential to capture flowing neutrophils and facilitate their immobilization at the vessel wall and so promote inflammatory and thrombotic intercellular interactions.