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Coagulation factor V : pathology and biochemistry

University of British Columbia

ROLE OF GLU96, ASP1O2 AND ASP111 IN FACTOR V Coagulation factor V activity is unmasked by thrombin-mediated excision of the central B domain resulting in a noncovalent heterodimer, factor Va. To understand the role of individual amino acids in maintaining the Ca²⁺-depenadent subunit interaction, G1u96 (E96A), Asp1O2 (D1O2A) or Asp111 (D111A) were mutated because of known effects on chelator sensitivity. The primary clotting activity of each mutant was reduced by “40%. Demonstrating at least two distinct inhibition mechanisms, only D111A was further inhibited by thrombin pre-treatment consistent with spontaneous subunit dissociation and severely inhibited Ca²⁺ binding. The parental factor V construct used here has a truncated B domain that does not require excision for activity. Therefore inhibition of D111A by thrombin-cleavage reveals a new B domain function that maintains factor V in a factor Va-like configuration independent of Ca²⁺ binding. In addition to Ca²⁺, factor V binds Cu²⁺, but with unknown function. Unexpectedly, D111A also lost detectable Cu²⁺. Finding that a single amino acid substitution simultaneously alters Ca²⁺ and Cu²⁺ suggests an interdependent metal ion binding site. Unlike D111A, the thrombin-mediated factor Va derived from E96A and D1O2A was stable, had only moderately faster subunit dissociation upon chelation and had normal metal ion binding. Thus, the current study defines the highly conserved acidic segment spanning G1u96-Asp112 in factor V as multifunctional. Of the three amino acids I evaluated, Asp111 is essential and likely functions through direct and indirect metal ion interactions. G1u96 and Asp102 individually influence factor V/Va function by more subtle effects at the metal ion-dependent subunit interface. FACTOR V-DEFICIENT PATIENT A factor V-deficient patient due to Y1702C mutation has been studied. The patient however did not suffer from severe bleeding despite of undetectable levels of plasma and platelet factor V. A close inspection of the patient’s blood coagulation cascade showed that the lack of available factor V was compensated by other factors that influence the intrinsic pathway. This finding suggests that the commonly observed phenotypic differences shown among factor V-deficient patients with the same genotypes may be due to existing hypercoagulant factors that influence the outcome of the disease.

Thesis/Dissertation

application/pdf

2009-03-05

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/5595

Pathology

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/