Thrombin-induced generation of neutrophil activating factors in blood

Abstract

The authors examined the in vitro effects of serum generated by the action of alpha-thrombin on citrated whole blood or plasma. Serum samples were assessed for their ability to induce neutrophil aggregation, neutrophil adherence to cultured endothelial cell monolayer, and superoxide anion generation. The results indicated that both blood- and plasma-derived sera induced similar increases in neutrophil aggregation. Both sera also induced neutrophil adherence to cultured bovine pulmonary artery endothelial monolayer. The adherence increased in a time-dependent manner, producing little change after 5 minutes, and reached maximal response at 60 minutes. Full potency of the neutrophil adherence was evident with serum samples obtained after clotting time of 20 minutes but not at 5 minutes clotting time. The adherence-inducing activity of serum was gradually lost after storage at -20 C and was abolished by 100 C treatment for 2 minutes. In contrast, heating serum to 56 C for 30 minutes did not alter the adherence-inducing activity. Both ether and aqueous fractions obtained after ether extraction of serum possessed adherence-promoting activities, indicating that multiple factors (both lipid and water soluble) were involved. Pretreatment of the endothelium with serum resulted in increased endothelial adhesiveness, but the serum pretreatment failed to induce adhesiveness to subendothelial matrix, indicating that endothelial cells play an active role in acquiring the adhesive property. Serum obtained from thrombin-generated whole blood also resulted in a modest degree of neutrophil superoxide anion generation, but this did not occur with plasma-derived serum. The results indicate that serum factors obtained by clotting blood with thrombin induce neutrophil aggregation and neutrophil adherence to the endothelium as well as superoxide production. These factors appear to be generated in the plasma phase, because the responses were similar in blood- and plasma-derived serum.