Novel function of coagulation factor Xa : conversion into a clot-dissolving cofactor

• Main Author: Vanden Hoek, Amanda Lynne
• Language: English
• Published: University of British Columbia 2011
• Online Access: http://hdl.handle.net/2429/36854

PLASMIN-MEDIATED CONVERSION OF FXa INTO A CLOT-DISSOLVING COFACTOR. Factor Xa (FXa) is an essential blood clotting enzyme. A previously identified FXa derivative, Xa33/13, is generated by two distinct cleavages by the clot-dissolving (fibrinolytic) enzyme, plasmin. FXa is first converted to FXaβ by excision of a small C-terminal peptide and then proteolyzed at Lys330 in the autolysis loop to yield Xa33/13, which cannot participate in clotting. Instead, these cleavages confer novel fibrinolytic function to Xa33/13 as a tissue plasminogen activator (tPA) cofactor, thereby accelerating plasmin generation. To understand the importance of each cleavage and the role of individual residues in this functional conversion of FXa, five mutants were generated by mutation of basic residues to glutamine: Lys330 and four residues in the β-peptide region. Mutation at Lys330 prevented autolysis loop cleavage, and this mutant dissolved purified fibrin clots faster than plasma-derived FXa derivatives. Additionally, no basic residue within the β-peptide was uniquely targeted by plasmin and no single-point mutation in this region prevented subsequent autolysis loop cleavage. FX-DEFICIENT PATIENT. Factor X (FX) can be activated by two separate protein complexes, known as the initiating (extrinsic) and amplifying (intrinsic) tenases, which are assembled during coagulation. I describe a FX-deficient patient with a novel compound heterozygous mutation associated with differential clotting pathway function. Quantification of plasma FX antigen revealed 15 % of normal, which was consistent with extrinsic pathway activity. Intrinsic pathway activity was reduced to 5 % of normal, suggesting an activatable specific activity 3-fold lower than expected for this branch of the clotting pathway. DNA sequence analysis identified two heterozygous mutations: (1) a previously reported mutation that disrupts the splice site between exons I and II; (2) a novel mutation resulting in an Arg386Cys substitution in the protease domain. I propose that alternate disulfide bond formation and protein folding may reduce circulating FX antigen levels. Additionally, Arg386 may be involved in substrate recognition by the intrinsic tenase complex, providing a possible explanation for the differential effect on the two branches of the coagulation cascade. Recombinant FX mutant studies confirmed our findings in patient plasma and provided further support for these hypotheses.