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Blood, Vol. 92 No. 4 (August 15), 1998:
pp. 1287-1296
Neutrophil Elastase Cleavage of Human Factor IX Generates an
Activated Factor IX-Like Product Devoid of Coagulant Function
John A. Samis,
Eunice Kam,
Michael E. Nesheim, and
Alan R. Giles
From the Departments of Pathology, Biochemistry, and Medicine,
Queen's University, Kingston, Ontario, Canada.
In preliminary studies, the generation of thrombin in vivo was found
to 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 associated with evidence of
increased elastase availability. To study the possibility 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 activated partial
thromboplastin time (aPTT) clotting assay demonstrated that, although
HNE eliminated the potential of F.IX to be activated, it only
marginally reduced the F.IXa activity. Reducing sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) indicated that
HNE treatment of F.IX generated cleavage products of 30 and 20 kD that
could not be distinguished from the respective heavy and light chain
peptides 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 active site probe p-aminobenzamidine; (2) hydrolyze the
synthetic peptide substrate
CH3SO2-Leu-Gly-Arg-p-nitroanilide; and (3)
activate human factor X (F.X). In contrast to
dansyl-Glu-Gly-Arg-chloromethyl ketone (dEGR)-inactivated F.IXa,
HNE-treated F.IX (0.01 to 10,000 pmol/L) failed to inhibit the clotting
activity of F.IXa (10 pmol/L) in the aPTT. NH2-terminal
sequencing indicated that HNE cleaved human F.IX at Thr140,
Thr144, Ile164, Thr172, and
Val181. The cleavages at
Thr140/Thr144 and at
Thr172/Val181 are both very close to the normal
F.XIa  -(Arg145) and  -(Arg180) cleavage
sites, respectively. 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 improper active site formation. These in vitro
observations support the possibility that increased HNE cleavage of
F.IX in vivo may contribute to the disregulation of hemostasis that
occurs in conditions such as disseminated intravascular coagulation
(DIC).
© 1998 by The American Society of Hematology.
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