Interaction of thrombin with vascular endothelial cells was investigated as a mechanism promoting platelet activation and adherence to endothelial monolayers. We found that pretreatment of endothelium with alpha-thrombin in the absence of platelets results in the attachment of platelets to endothelial cells after the removal of fluid-phase alpha-thrombin. This activity was eliminated by exposure of alpha-thrombin-pretreated endothelial cells to active site inhibitors of alpha-thrombin or by adding alpha-thrombin in the presence of excess diisopropyl fluorophosphate-inhibited thrombin, suggesting retention of active alpha-thrombin by a receptor-mediated mechanism. Morphological data and the results of [14C]serotonin release studies indicate that platelets are activated by alpha-thrombin-pretreated endothelium and that adherence represents aggregates of activated platelets as well as individual platelets. Adherence on alpha-thrombin-pretreated endothelium is dependent on divalent cations. Platelets also adhered to aortic segments pretreated with thrombin. The data of the current studies support the contention that alpha-thrombin can promote adherence of activated platelets to endothelial cells because of the binding and retention of alpha-thrombin to endothelial cells in a manner in which it remains active and available for platelet activation.
We have compared the effects of intracellular pathways initiated by nitric oxide and prostacyclin on thrombin-induced platelet adhesion to endothelial cells. Platelet aggregate adhesion was enhanced when endothelial monolayers were pretreated with NG-monomethyl-L-arginine (L-NMMA), an inhibitor of nitric oxide production. In addition, decreased platelet aggregate adhesion was seen when platelets were pretreated with 8-bromoadenosine 3',5'-cyclic monophosphate (8-bromo-cAMP) or 8-bromoguanosine 3',5'-cyclic monophosphate (8-bromo-cGMP). Single platelet adhesion in isolated perfused lungs under flow conditions in the presence of shear was also assessed. Pretreatment of platelets with either Iloprost, in a dose sufficient to decrease platelet aggregation, or 8-bromo-cAMP did not affect platelet adhesion. However, pretreatment of platelets with 8-bromo-cGMP significantly reduced single platelet adhesion to endothelium. These studies illustrate that nitric oxide inhibits platelet adhesion to endothelium in the presence of shear. They further indicate that prostacyclin is also a regulator of this response but has effects more specifically related to the inhibition of platelet aggregation than platelet-endothelium interactions.
Plasma fibronectin has been shown to increase the binding of fibrin monomer to macrophages in vitro. In the present study we began characterization of the mechanism underlying this fibronectin activity. Fragments of fibronectin containing the amino terminus enhanced macrophage fibrin binding to the same extent as intact fibronectin on an equimolar basis. However, fibronectin fragments containing the gelatin-binding domain or the cell-binding domain, but lacking the amino terminus, had no effect on fibrin binding. Fibronectin enhanced fibrin binding was not affected by the addition of synthetic peptides containing the RGD adhesion sequence. The ability of fibronectin to augment fibrin binding remained after paraformaldehyde fixation of macrophage monolayers. Fixation did not alter the basal levels of fibrin binding by macrophages. Preincubation of macrophages with exogenous fibronectin did not increase the binding of fibrin. Fibronectin enhanced fibrin binding remained unaltered after the removal of endogenous cell surface fibronectin by capping with F(ab')2 fragments of antibodies to fibronectin. These results suggest that the amino terminus of fibronectin supports the attachment of fibrin to macrophages by an initial fluid-phase interaction that precedes cellular binding and does not require a cellular response.
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