One of the aims of research in the area of thrombosis has been to design an effective anticoagulant that would function in a predictable and direct manner. In evaluating the role of coagulation in sepsis we used factor Xa blocked in the active center with [5-(dimethylamino)1- naphthalenesulfonyl]-glutamylglycylarginyl+ ++ chloromethyl ketone (DEGR-Xa). We infused 1 mg/kg of DEGR-Xa together with LD100 concentrations of Escherichia coli (4 x 10(10) organisms/kg) into five baboons. As controls, we infused E coli alone into five baboons. The inflammatory, coagulant, and cell injury responses to E coli of both the treated and control groups were lethal and were similar in every respect except for the complete inhibition of the consumption of fibrinogen in the DEGR-Xa group. The half life of DEGR-Xa was approximately 10 hours and 2 hours, as determined by isotopic and enzyme-linked immunosorbent assays, respectively. These results for the first time demonstrate that, although coagulation occurs in E coli sepsis, fibrin formation per se did not influence the lethal outcome in this model. These results also show the effectiveness of DEGR-Xa as an anticoagulant and raise the possibility that it could serve as an alternative to anticoagulants currently in use.

Previous studies showed that infusion of C4b-binding protein with sublethal Escherichia coli (E. coli) in the primate produced a consumptive coagulopathy followed by microvascular thrombosis and renal failure. The first objective of this study was to characterize the pathophysiology and mechanism of this phenomena following infusion of both these agents with emphasis on defining the role of free protein S. The second objective was to examine the relevance of this model to the hemolytic uremic syndrome. Infusion of C4b-binding protein alone reduced free protein S and decreased platelet concentration to 20% of baseline, whereas infusion of the C4b-binding protein/protein S complex did not. There was no activation of other inflammatory or coagulant factors. Infusion of sublethal E coli alone produced a transient inflammatory response with no reduction of free protein S. However, coinfusion of C4b-binding protein with sublethal E coli reduced free protein S and produced a thrombocytopenia, anemia, and a microvascular thrombotic response, whereas infusion of the C4b-binding protein/protein S complex with sublethal E coli did not. Studies comparing the effects of neutralizing (S-163) and nonneutralizing (S-145) antibodies with protein S coinfused with sublethal E coli produced similar contrasting results. Therefore, we concluded that neutralization of free protein S, and not some other property of C4b-binding protein influenced by protein S, accounted for this microvascular thrombotic response. This response is similar to the hemolytic uremic syndrome characterized by thrombocytopenia, anemia, shistocytosis, and renal glomerular thrombosis with uremia. Comparison of the respective renal histopathologic appearance supports this conclusion. This raises the possibility that inhibition of protein S activity (possibly by one of the forms of C4b-binding proteins) might be one of the factors contributing to microvascular thrombotic disorder, such as the hemolytic uremic syndrome.

This study examines the assumption that both the anticoagulant and fibrinolytic activity that follow the generation of thrombin induced by infusion of factor Xa/PCPS are due to generation of activated protein C. Untreated controls or animals given unrelated antibody were compared with animals pretreated with a specific monoclonal antibody to protein C (HPC4). Compared with untreated controls excess HPC4 substantially reduced the level of protein C activation as observed by protein C immunoblotting and enzyme-linked immunosorbent assay for antitrypsin/activated protein C complexes. Despite this, the anticoagulant activity as reflected by the decline of factors Va and VIIIa levels (as observed by coagulation assays and by factor V immunoblotting) was significantly greater than controls. The fibrinolytic activity (as observed by assays of tissue plasminogen activator, D-Dimer, alpha 2-antiplasmin) also was significantly greater than controls. We conclude that neutralization of the protein C anticoagulant system while resulting in a significantly more intense coagulant response to Xa/PCPS does not preclude inactivation of factors Va and VIIIa and the full expression of the fibrinolytic response. We conclude further that after thrombin generation in vivo, protein C activation is not a prerequisite for the promotion of the fibrinolytic response previously observed, and that the inactivation of factors Va/VIIIa may be mediated by enzymes other than activated protein C. The reduction in alpha 2-antiplasmin levels in association with increased tissue plasminogen activator activity suggests that plasmin is a likely candidate.