Thrombin stimulates neutrophil adherence by an endothelial cell-dependent mechanism: characterization of the response and relationship to platelet-activating factor synthesis

Abstract

Thrombin, a serine coagulation protease that is generated at sites of tissue injury and inflammation, stimulates the adherence of PMNs and neutrophils to EC. We found that thrombin enhanced the adhesion of neutrophils to primary monolayers of human umbilical vein EC when assayed by the binding of 111Indium-labeled PMNs to the EC, the recovery of unlabeled PMNs after incubation with thrombin-treated EC, and by phase contrast and scanning electron microscopy (SEM). SEM demonstrated that thrombin caused PMNs to intimately adhere to the EC plasma membrane and under some conditions to become polarized. The thrombin-stimulated adherence was a rapid, time-dependent response with an onset within 1 minute of addition of thrombin, a peak at 5-10 minutes, and a decline thereafter. The response was concentration-dependent over the range 0.01-2 U/ml thrombin, and required active thrombin. Prothrombin, factor Xa, and fibrinogen were not effective. Thrombin-stimulated PMN adherence was dependent on the EC, because thrombin did not significantly stimulate neutrophils to adhere to albumin-coated petri dishes, subendothelial matrices, or primary cultures of smooth muscle cells. Human EC, when treated with thrombin, also produce platelet-activating factor (PAF; 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine). The concentration-response relationships and time courses for thrombin-stimulated PMN adherence and PAF production were tightly correlated. Furthermore, PAF itself stimulated the adherence of PMNs to EC, and pretreatment of PMNs with PAF selectively inhibited their adherence response to thrombin. These findings demonstrate two novel biologic activities of thrombin, the stimulation of EC-dependent adherence of PMNs and the production of PAF by EC, and suggest that they are functionally related. In addition, they suggest that thrombin may act as a plasma-derived humoral mediator of inflammation under some conditions.