title: Human neutrophil elastase activates human factor V but inactivates thrombin-activated human factor V.
abstract: 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 electrophoresis (SDS-PAGE), and NH2-terminal sequence analysis. Incubation of F.V (600 nmol/L) with HNE (2 nmol/L) in the presence of Ca2+ resulted in a time-dependent increase in its cofactor activity. In contrast, treatment of F.Va (600 nmol/L) with HNE (60 nmol/L) in the presence of Ca2+ resulted only in a time-dependent decrease in its cofactor activity. Under the conditions of these experiments, the maximum extent of F.V activation accomplished by incubation with HNE was approximately 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 activity and the HNE-dependent decrease in F.Va cofactor activity was not influenced by the addition of phosphatidylcholine/phosphatidylserine (PCPS) vesicles (50 micromol/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 generated 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 closely spaced doublets of: 99/97, 89/87, and 76/74 kD whose appearance over time correlated well with the increased cofactor activity as judged 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 heavy 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 kD heavy chain was more extensive during the time period (10 to 30 minutes) of the greatest loss of F.Va cofactor activity. NH2-terminal sequence analysis of F.V treated with HNE indicated cleavage at Ile819 and Ile1484 under conditions during which the procofactor expressed enhanced cofactor activity in the prothrombinase complex. NH2-terminal sequence analysis of F.Va treated with HNE indicated cleavage at Ala341, Ile508, and Thr1767 under conditions, which the cofactor became inactivated, as measured by prothrombinase activity. The activation and inactivation cleavage sites are close to those cleaved by the physiological activator and inactivator of F.V and F.Va, namely alpha-thrombin (Arg709 and Arg1545) and Activated Protein C (APC) (Arg306 and Arg506), respectively. These results indicate that HNE can generate proteolytic products of F.V, which initially express significantly enhanced procoagulant cofactor activity similar to that observed following activation with alpha-thrombin. In contrast, HNE treatment of F.Va resulted only in the loss of its cofactor activity, but again, this is similar to that observed following inactivation by APC.
authors.affiliation: Department of Pathology, Queen's University, Kingston, Ontario, Canada.
authors.forename: J A
authors.initials: JA
authors.lastname: Samis
authors.forename: M
authors.initials: M
authors.lastname: Garrett
authors.forename: R P
authors.initials: RP
authors.lastname: Manuel
authors.forename: M E
authors.initials: ME
authors.lastname: Nesheim
authors.forename: A R
authors.initials: AR
authors.lastname: Giles
mesh_terms: D002118:Calcium / Q000378:metabolism; D004789:Enzyme Activation; D005165:Factor V / Q000737:chemistry / Q000378:metabolism*; D015943:Factor Va / Q000037:antagonists & inhibitors*; D005170:Factor X / Q000378:metabolism; D015951:Factor Xa; D006801:Humans; D019272:Leukocyte Elastase / Q000502:physiology*; D009504:Neutrophils / Q000201:enzymology*; D010446:Peptide Fragments / Q000032:analysis; D013917:Thrombin / Q000502:physiology*
chemical_list: D010446:Peptide Fragments; C022475:prothrombinase complex; D015943:Factor Va; D005165:Factor V; D005170:Factor X; D019272:Leukocyte Elastase; D013917:Thrombin; D015951:Factor Xa; D002118:Calcium