A number of inhibitors of thrombin and factor Xa have been described; however, only one inhibitor of factor IXa has been reported. This compound, dansyl-Glu-Gly-Arg chloromethyl ketone (DEGER), inhibits porcine factor IXa with a second-order rate constant of 2.2 X 10(4) M-1 min-1. We now describe the synthesis and characterization of three p-amidinophenyl esters that inhibit human factor IXa with second-order rate constants comparable to those observed with human and bovine factor Xa and alpha-thrombin. These rate constants of inhibition, moreover, are 2-30-fold greater than observed when DEGER is employed to inhibit porcine factor IXa. Additional advantages of these derivatives include their ease of synthesis and low degree of toxicity. The p-amidinophenyl ester of benzoic acid was employed to inhibit human factor IXa, and the plasma clearance of the protein was studied in mice. These experiments demonstrate for the first time that the endothelial binding previously reported with factor IXa is independent of the active site, a finding similar to the behavior observed with factor Xa and alpha-thrombin in this and previous reports.

When factor VIII/von Willebrand factor (FVIII/vWF) is chromatographed on 4% agarose in 0.25 M CaCI2. the procoagulant activity partially separates from the void volume protein peak. If FVIII/vWF is reacted with thrombin prior to chromatography, both the magnitude and resolution of the proe.oagulant activity peak are greatly increased. These observations suggest that activated species of FVIII/vWF are contained in the late-eluting procoagulant activity peak, and therefore, a precursive form of FVIII/vWF may exist. Such a precursor might be expected to: (1) lack procoagulant activity; (2) elute in the void volume from 4% agarose-CaCl2; and (3) be activated by thrombin, after which the procoagulant activity should elute in the inner volume from 4% agarose-CaCI2 columns. The specific procoagulant activities for FVIII/vWF purified in the presence and absence of the protease inhibitors, heparin-bovine pancreatic trypsin inhibitor and diisopropylfluorophosphate, were 27 and 92 U/mg. respectively. For FVIII/vWF isolated in the presence of this mixture of inhibitors. we observed that a peak with very low specific procoagulant activity. but containing at least 80% of the total activity. consistently eluted in the void volume from 4% agarose-O.25 M CaCI2 gel filtration. The FVIII/vWF prepared in the absence of protease inhibitors could be activated 13.8 ± 3.0-fold by thrombin. compared to 6.6 ± 1 .7-fold for FVIII/vWF prepared without protease inhibitors. When FVIII/vWF prepared in the presence of inhibitors is activated by thrombin prior to 4% agarose-0.25 M CaCI2 chromatography, virtually no FVIII procoagulant activity elutes in the void volume; instead, a considerably enhanced symmetrical peak of FVIII procoagulant activity elutes in the inner volume. These data imply that FVIII/vWF circulates in vivo as a precursor with little or no procoagulant activity until activated by thrombin or a thrombin-like enzyme.

The role of endothelium in the inhibition of human factor Xa was studied in a plasma environment. Human factor Xa can bind to and function on bovine aortic endothelium in a manner similar to that of bovine factor Xa. Approximately 70% of the bound factor Xa is subject to inhibition by plasma proteinase inhibitors, and the remaining 30% is irreversibly bound as part of a 125 Kd membrane-associated complex not subject to proteolytic degradation. The proportion reversibly bound and its rate of release do not alter with changes in calcium, citrate, heparin, or active proteinase inhibitor concentrations. The principal plasma proteinase inhibitor of human factor Xa was antithrombin III, which accounted for 60% to 65% of factor Xa released from endothelium, with alpha 1-proteinase inhibitor inactivating 20% to 25% and alpha 2- macroglobulin approximately 15%. All of the reversibly bound factor Xa was identified in complex with one of these three proteinase inhibitors. The thrombin active-site inhibitor hirudin was found to markedly accelerate the displacement of reversibly bound factor Xa from the endothelium and to associate specifically with factor Xa without a loss of activity toward chromogenic substrates, perhaps accounting for a novel mechanism of anticoagulation.

Sera from patients with rheumatoid arthritis containing high titers of anti-streptokinase antibodies were found to contain anti-plasminogen antibodies of the IgG and IgA classes. High titers of anti-plasminogen autoantibodies of the IgA class were also found in sera from patients with systemic lupus erythematosus and Sjögren syndrome. Studies of the immune response to thrombolytic therapy with streptokinase in patients with no prior history of autoimmune disease suggest a strong correlation between streptokinase administration and the appearance of autoantibodies to plasminogen of the IgA class. The IgA anti-plasminogen autoantibody is specific for an epitope in a region of plasminogen which binds streptokinase and the IgG autoantibody reacts with an epitope in the C-terminal region corresponding to the catalytic domain of the plasminogen zymogen. Our findings suggest a different origin for the two classes of anti-plasminogen immunoglobulins in rheumatoid arthritis patients. Since plasminogen binding to rheumatoid synovial fibroblasts is enhanced, the high titers of both classes of anti-plasminogen autoantibodies may add to the localization and perpetuation of the immune response. We suggest that plasminogen may be a target of the immune response in autoimmune disease.