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

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...

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Main Author: Vanden Hoek, Amanda Lynne
Language:English
Published: University of British Columbia 2011
Online Access:http://hdl.handle.net/2429/36854
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spelling ndltd-LACETR-oai-collectionscanada.gc.ca-BVAU.-368542013-06-05T04:19:49ZNovel function of coagulation factor Xa : conversion into a clot-dissolving cofactorVanden Hoek, Amanda LynnePLASMIN-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.University of British Columbia2011-08-22T23:53:39Z2011-08-22T23:53:39Z20112011-08-222011-11Electronic Thesis or Dissertationhttp://hdl.handle.net/2429/36854eng
collection NDLTD
language English
sources NDLTD
description 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.
author Vanden Hoek, Amanda Lynne
spellingShingle Vanden Hoek, Amanda Lynne
Novel function of coagulation factor Xa : conversion into a clot-dissolving cofactor
author_facet Vanden Hoek, Amanda Lynne
author_sort Vanden Hoek, Amanda Lynne
title Novel function of coagulation factor Xa : conversion into a clot-dissolving cofactor
title_short Novel function of coagulation factor Xa : conversion into a clot-dissolving cofactor
title_full Novel function of coagulation factor Xa : conversion into a clot-dissolving cofactor
title_fullStr Novel function of coagulation factor Xa : conversion into a clot-dissolving cofactor
title_full_unstemmed Novel function of coagulation factor Xa : conversion into a clot-dissolving cofactor
title_sort novel function of coagulation factor xa : conversion into a clot-dissolving cofactor
publisher University of British Columbia
publishDate 2011
url http://hdl.handle.net/2429/36854
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