Showing papers by "Alan R. Giles published in 2013"

Thrombin-Activated Human Factor V Human Neutrophil Elastase Activates Human Factor V but Inactivates

Abstract: The effect of human neutrophil elastase (HNE) on human (600 nmol/L) with HNE (60 nmol/L) in the presence of Ca resulted in the cleavage of both the 96 kD heavy chain and factor V (F.V) or a-thrombin–activated human factor V (F.Va) was studied in vitro by prothrombinase assays, sodium dothe 74/72 kD light chain into products of: 56, 53, 35, 28, 22, and 12 kD. Although densitometry indicated that both the decyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE), and NH2-terminal sequence analysis. Incubation of heavy and light chains of F.Va were hydrolyzed by HNE, cleavage of the 96 kD heavy chain was more extensive durF.V (600 nmol/L) with HNE (2 nmol/L) in the presence of Ca resulted in a time-dependent increase in its cofactor ing the time period (10 to 30 minutes) of the greatest loss of F.Va cofactor activity. NH2-terminal sequence analysis of activity. In contrast, treatment of F.Va (600 nmol/L) with HNE (60 nmol/L) in the presence of Ca resulted only in a F.V treated with HNE indicated cleavage at Ile and Ile under conditions during which the procofactor expressed time-dependent decrease in its cofactor activity. Under the conditions of these experiments, the maximum extent of enhanced cofactor activity in the prothrombinase complex. NH2-terminal sequence analysis of F.Va treated with HNE F.V activation accomplished by incubation with HNE was approximately 65% to 70% of that observed with a-thrombin indicated cleavage at Ala, Ile, and Thr under conditions, which the cofactor became inactivated, as measured in presence of Ca. The extent of both the HNE-dependent enhancement in F.V cofactor activity and the HNE-depenby prothrombinase activity. The activation and inactivation cleavage sites are close to those cleaved by the physiological dent decrease in F.Va cofactor activity was not influenced by the addition of phosphatidylcholine/phosphatidylserine activator and inactivator of F.V and F.Va, namely a-thrombin (Arg and Arg) and Activated Protein C (APC) (Arg (PCPS) vesicles (50mmol/L). The HNE-derived cleavage products of F.V, which correlated with increased cofactor activity, and Arg), respectively. These results indicate that HNE can generate proteolytic products of F.V, which initially express as demonstrated by SDS-PAGE under reducing conditions, were different from those generated using a-thrombin. significantly enhanced procoagulant cofactor activity similar to that observed following activation with a-thrombin. In Treatment of F.V (600 nmol/L) with HNE (2 nmol/L) in the presence of Ca resulted in the production of three closely contrast, HNE treatment of F.Va resulted only in the loss of its cofactor activity, but again, this is similar to that observed spaced doublets of: 99/97, 89/87, and 76/74 kD whose appearance over time correlated well with the increased cofacfollowing inactivation by APC. q 1997 by The American Society of Hematology. tor activity as judged by densitometry. Treatment of F.Va