A model describing the inactivation of factor Va by APC: bond cleavage, fragment dissociation, and product inhibition.
The inactivation of factor Va is a complex process which includes bond cleavage (at three sites) and dissociation of the A2N.A2C peptides, with intermediate activity in each species. Quantitation of the functional consequences of each step in the reaction has allowed for understanding of the presentation of disease in individuals possessing the factor V polymorphism factor VLEIDEN. APC cleavage of membrane-bound bovine factor Va (Arg306, Arg505, Arg662) leads to the dissociation of fragments of the A2 domain, residues 307-713 (A2N.A2C + A2C-peptide), leaving behind the membrane-bound A1.LC species. Evaluation of the dissociation process by light scattering yields invariant mass loss estimates as a function of APC concentration. The rate constant for A2 fragment dissociation varies with [APC], reaching a maximal value of k = 0.028 s-1, the unimolecular rate constant for A2 domain fragment dissociation. The APC binding site resides in the factor Va light chain (LC) (Kd = 7 nM), suggesting that the membrane-bound LC.A1 product would act to sequester APC. This inhibitory interaction (LC.A1.APC) is demonstrated to exist with either purified factor Va LC or the products of factor Va inactivation. Utilizing these experimental data and the reported rates of bond cleavage, binding constants, and product activity values for factor Va partial inactivation products, a model is developed which describes factor Va inactivation and accounts for the defect in factor VLEIDEN. The model accurately predicts the rates of inactivation of factor Va and factor VaLEIDEN, and the effect of product inhibition. Modeled reaction progress diagrams and activity profiles (from either factor Va or factor VaLEIDEN) are coincident with experimentally derived data, providing a mechanistic and kinetic explanation for all steps in the inactivation of normal factor Va and the pathology associated with factor VLEIDEN.
Hockin
M F
MF
Department of Biochemistry, The University of Vermont, College of Medicine, Burlington, Vermont 05405, USA.
Cawthern
K M
KM
Kalafatis
M
M
Mann
K G
KG
D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives / Q000378:metabolism; D002417:Cattle; D003619:Dansyl Compounds / Q000378:metabolism; D015943:Factor Va / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008027:Light; D008081:Liposomes / Q000737:chemistry; D008956:Models, Chemical; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism; D010713:Phosphatidylcholines / Q000737:chemistry; D010718:Phosphatidylserines / Q000737:chemistry; D011486:Protein C / Q000737:chemistry* / Q000494:pharmacology*; D011516:Prothrombin / Q000378:metabolism; D012542:Scattering, Radiation
D003619:Dansyl Compounds; D008081:Liposomes; D010446:Peptide Fragments; D010713:Phosphatidylcholines; D010718:Phosphatidylserines; D011486:Protein C; C019162:dansylarginine N-(3-ethyl-1,5-pentanediyl)amide; D015943:Factor Va; C042511:1,2-dioleoylphosphatidylserine; D011516:Prothrombin; D001120:Arginine; C017251:1,2-oleoylphosphatidylcholine