HUMAN NEUTROPHIL ELASTASE ACTIVATES HUMAN FACTOR-V BUT INACTIVATES THROMBIN-ACTIVATED HUMAN FACTOR-V

The effect of human neutrophil elastase (HNE) on human factor V (F.V) or alpha-thrombin-activated human factor V (F.Va) was studied in vitro by prothrombinase assays, sodium dodecyl sulfate-polyacrylamide gel e lectrophoresis (SDS-PAGE), and NH2-terminal sequence analysis. Incubat ion of F.V (600 nmol/L) with HNE (2 nmol/L) in the presence of Ca2+ re sulted in a time-dependent increase in its cofactor activity. In contr ast, treatment of F.Va (600 nmol/L) with HNE (60 nmol/L) in the presen ce of Ca2+ resulted only in a time-dependent decrease in its cofactor activity. Under the conditions of these experiments, the maximum exten t of F.V activation accomplished by incubation with HNE was approximat ely 65% to 70% of that observed with alpha-thrombin in presence of Ca2 +. The extent of both the HNE-dependent enhancement in F.V cofactor ac tivity and the HNE-dependent decrease in F.Va cofactor activity was no t influenced by the addition of phosphatidylcholine/phosphatidylserine (PCPS) vesicles (50 mu mol/L). The HNE-derived cleavage products of F .V, which correlated with increased cofactor activity, as demonstrated by SDS-PAGE under reducing conditions, were different from those gene rated using alpha-thrombin. Treatment of F.V (600 nmol/L) with HNE (2 nmol/L) in the presence of Ca2+ resulted in the production of three cl osely spaced doublets of: 99/97, 89/87, and 76/74 kD whose appearance over time correlated well with the increased cofactor activity as judg ed by densitometry. Treatment of F.Va (600 nmol/L) with HNE (60 nmol/L ) in the presence of Ca2+ resulted in the cleavage of both the 96 kD h eavy chain and the 74/72 kD light chain into products of: 56, 53, 35, 28, 22, and 12 kD. Although densitometry indicated that both the heavy and light chains of F.Va were hydrolyzed by HNE, cleavage of the 96 k D heavy chain was more extensive during the time period (10 to 30 minu tes) of the greatest loss of F.Va cofactor activity. NH2-terminal sequ ence analysis of F.V treated with HNE indicated cleavage at Ile(819) a nd Ile(1484) under conditions during which the procofactor expressed e nhanced cofactor activity in the prothrombinase complex. MH2-terminal sequence analysis of F.Va treated with HNE indicated cleavage at Ala(3 41), Ile(508), and Thr(1767) under conditions, which the cofactor beca me inactivated, as measured by prothrombinase activity. The activation and inactivation cleavage sites are close to those cleaved by the phy siological activator and inactivator of F.V and F.Va, namely alpha-thr ombin (Arg(709) and Arg(1545)) and Activated Protein C (APC) (Arg(306) and Arg(506)), respectively. These results indicate that HNE can gene rate proteolytic products of F.V, which initially express significantl y enhanced procoagulant cofactor activity similar to that observed fol lowing activation with alpha-thrombin. In contrast, HNE treatment of F .Va resulted only in the loss of its cofactor activity, but again, thi s is similar to that observed following inactivation by APC. (C) 1997 by The American Society of Hematology.