Abstract
The gamma-carboxyglutamic acid (Gla)-domain region of factor X (residues 1-44 of the light chain) was selectively removed by limited proteolysis with alpha-chymotrypsin. The Gla-domainless factor X was then activated by the factor X coagulant protein of Russell's viper venom. Apparent dissociation constants Kd' values for the interaction of factor Va with either factor Xa or Gla-domainless factor Xa were determined kinetically using prothrombin as the substrate. In the absence of phospholipid, factor Va interacted with Gla-domainless factor Xa with lower affinity (Kd' 4 X 10(-6) M) than with factor Xa (Kd' = 5 X 10(-8) M). At saturating concentrations of factor Va, maximal rates of thrombin formation were similar for either enzyme. The addition of phospholipid increased the affinity of factor Va for factor Xa approximately 75-fold (Kd' = 3.3 X 10(-10) M). In contrast, phospholipid had no effect on the affinity of Gla-domainless factor Xa for factor Va (Kd' = 4 X 10(-6) M). The maximal rate of thrombin formation increased approximately 300-fold with the addition of phospholipid to the factor Xa-factor Va system. Under the same conditions, phospholipid had no effect on the rate of thrombin formation when Gla-domainless factor Xa was the enzymatic moiety. These results demonstrate phospholipid has little or no effect on factor Va function when factor Xa has lost its Gla-mediated Ca2+-binding sites.
Highlights
Phospholipid hadno effect on the affinity and esterolytic properties to those of factor Xa (11).Other of Gla-domainless factor Xa for factor Va
In the presence of the cofactors calcium, phospholipid, and factor Va will prothrombin be activated at a rate rapid enough to support coagulation (1).The complex formed by the association of factor Xa with its cofactors is referred to as the prothrombinase complex and has been extensively
Competition between the active enzyme and increasing concentrations of inactivated enzyme for the factor Xa-binding site on factor Va resulted in a decrease in the rate of thrombin formation as gel-filtered and chromatographed on QAE-Sephadex and was activated by the addition of factor X coagulant protein in the presence of 10 mM Ca2+ (1:100, factor X coagulant protein:Gla-domainless factor X)
Summary
Was removedby dialysis of 4 "C against 0.1M NaCl, 0.02 M Tris-HC1, Gla-domainless factor Xa was prepared as described by Morita and pH 7.5. S2222 assay was sensitive to 0.4 nM factor Xa. Competition between the active enzyme and increasing concentrations of inactivated enzyme for the factor Xa-binding site on factor Va resulted in a decrease in the rate of thrombin formation as gel-filtered and chromatographed on QAE-Sephadex and was activated by the addition of factor X coagulant protein in the presence of 10 mM Ca2+ (1:100, factor X coagulant protein:Gla-domainless factor X). In these experiments, factor Xa or Gla-domainless factor Xa was incubated in the presence of Ca2+and factor Va (with or without phospholipid) such that the factor Va concentration was not saturatedwith respect to theenzyme (90% saturated).
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
