Cleavage and inactivation of Factor IX by granulocyte elastase.

Abstract

Radioiodinated Factor IX was cleaved by a crude sonicate from leukocytes. In the absence of calcium, fragments of less than 15,000 mol wt were seen from reduced samples on gel electrophoresis. After digestion in 2 mM calcium, however, electrophoresis of reduced samples showed, in addition to low molecular weight fragments, protein bands corresponding in size to heavy and light chains of Factor XIa-activated Factor IX. The cleaving activity in leukocyte sonicates was inhibited by soybean trypsin inhibitor, but only to a small extent by aprotinin. Granulocyte elastase was isolated from purified polymorphonuclear leukocyte granules by affinity chromatography on soybean trypsin inhibitor-agarose and further chromatography on carboxymethyl cellulose. The purified fraction contained two isozymes on acidic gels which cleaved both an ester sensitive to elastase and radiolabeled Factor IX. These two activities were inhibited by elastase-specific chloromethyl ketone. The isolated protease fraction rapidly inactivated apparent Factor IX activity in a coagulant assay system. The degree of inactivation correlated with the amount of intact, radiolabeled Factor IX cleaved. As with the crude sonicate, generation of the larger heavy and light chain-sized fragments was dependent upon calcium. To assess directly the effect of elastase on Factor IX, an immunospecific, active site-directed assay was developed. In this assay, the sample was incubated with solid-phase antibody to Factor IX and the amount of activated product was detected as that which had complexed with radioiodinated antithrombin III. In this system, exposure of Factor IX to Factor XIa showed progressive increase in the ability to bind antithrombin III, whereas after elastase, Factor XIa was unable to generate antithrombin III binding. The elastase-degraded Factor IX did not inhibit activation of additional Factor IX in clotting assays. When Factor IXa was incubated with elastase, binding of antithrombin III was decreased, corresponding to appearance of low molecular weight fragments on parallel samples that were reduced and electrophoresed. These data are consistent with elastase inactivating Factor IX by cleaving bonds near, but distinct from, bonds cleaved by Factor XIa.