NEUTROPHIL ELASTASE CLEAVAGE OF HUMAN FACTOR-IX GENERATES AN ACTIVATED FACTOR IX-LIKE PRODUCT DEVOID OF COAGULANT FUNCTION

In preliminary studies, the generation of thrombin in vivo was found t o induce a 92% loss of functional activity of factor IX (F.IX) despite the detection by Western blotting of a product resembling activated F .IX (F.IXa) and a 25% increase in F.IX antigen levels (Hoogendoorn et al, Thromb Haemost 69:1127, 1993 [abstr]). These changes were associat ed with evidence of increased elastase availability. To study the poss ibility that these two observations were related, a detailed physical and functional characterization of the hydrolysis of purified human F. IX by human neutrophil elastase (HNE) was performed in vitro. An activ ated partial thromboplastin time (aPTT) clotting assay demonstrated th at, although HNE eliminated the potential of F.IX to be activated, it only marginally reduced the F.IXa activity. Reducing sodium dodecyl su lfate-polyacrylamide gel electrophoresis (SDS-PAGE) indicated that HNE treatment of F.IX generated cleavage products of 30 and 20 kD that co uld not be distinguished from the respective heavy and light chain pep tides that were identified in parallel studies when F.IX was activated by activated bovine F.XI (F.XIa), one of its physiological activators . In addition, nonreducing SDS-PAGE demonstrated that HNE-treated F.IX formed no complexes with antithrombin III (ATIII) in the presence of heparin. Furthermore, HNE-treated F.IX was unable to (1) bind the acti ve site probe p-aminobenzamidine; (2) hydrolyze the synthetic peptide substrate CH3SO2-Leu-Gly-Arg-p-nitroanitide; and (3) activate human fa ctor X (F.X). In contrast to dansyl-Glu-Gly-Arg-chloromethyl ketone (d EGR)-inactivated F.IXa, HNE-treated F.IX (0.01 to 10,000 pmol/L) faile d to inhibit the clotting activity of F.IXa (10 pmol/L) in the aPTT NH 2-terminal sequencing indicated that HNE cleaved human F.IX at Thr(140 ), Thr(144), IIe(164), Thr(172) and Val(181). The cleavages at Thr(140 )/Thr(144) and at Thr(172)/Val(181) are both very close to the normal F.XIa alpha-(Arg(145)) and beta-(Arg(180)) cleavage sites, respectivel y. In summary, the results suggest that the activatability of F.IX is eliminated after cleavage by HNE and that the inability of HNE-treated F.IX to support F.IXa-like coagulant function is a consequence of imp roper active site formation. These in vitro observations support the p ossibility that increased HNE cleavage of F.IX in vivo may contribute to the disregulation of hemostasis that occurs in conditions such as d isseminated intravascular coagulation(DIC). (C) 1998 by The American S ociety of Hematology.