Ja. Samis et al., HUMAN NEUTROPHIL ELASTASE ACTIVATES HUMAN FACTOR-V BUT INACTIVATES THROMBIN-ACTIVATED HUMAN FACTOR-V, Blood, 90(3), 1997, pp. 1065-1074
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.