SummaryTAFI (thrombin activatable fibrinolysis inhibitor) down regulates fibrinolysis after activation by relatively high concentrations of thrombin generated during coagulation via thrombin mediated factor XI activation and subsequent activation of the intrinsic pathway. It is this secondary burst of thrombin that is severely diminished in haemophilia A, a deficiency of coagulation factor VIII. We therefore investigated the role of TAFI in haemophilia A by measuring the clot lysis times of tissue factor induced fibrin formation and tPA mediated fibrinolysis. In haemophilia A plasma clot lysis times were normal at relatively high tissue factor concentrations but severely decreased at moderate to low tissue factor concentrations, indicating that the thrombin generation via the extrinsic pathway was insufficient to activate TAFI. Addition of factor VIII, TAFI or thrombomodulin restored the clot lysis times at low tissue factor concentrations. This confirms the hypothesis that the bleeding disorder in haemophilia A is not merely a defect in the initial clot formation but is in fact a triple defect: reduced thrombin formation via the extrinsic pathway at low tissue factor concentrations, a reduced secondary burst of thrombin generation via the intrinsic pathway and a defective down regulation of the fibrinolytic system by the intrinsic pathway.

IntroductionHuman coagulation factor V (FV) is a single-chain glycoprotein that plays an important role in maintaining the hemostatic balance. It circulates in blood as an inactive procoagulant with a M r of 330 kd and a structure consisting of 3 homologous A-type domains and 2 homologous C-type domains connected by a heavily glycosylated B domain in the order A1-A2-B-A3-C1-C2. Proteolytic cleavage by thrombin at R709, R1018, and R1545 (single-letter amino acid codes) results in removal of the B domain and converts the procofactor into the fully active cofactor FVa, which consists of a M r 105-kd heavy chain (A1-A2) and a M r 74-or 71-kd light chain (A3-C1-C2), associated via a single Ca ϩϩ ion. [1][2][3] The difference in molecular weight of the light chain reflects the presence of 2 isoforms of FVa (FVa 1 and FVa 2 ) due to alternative glycosylation of the C2 domain, which leads to different affinities for biologic membranes and subsequent overall procoagulant activity. 4,5 In its active form, FVa forms an essential part of the prothrombinase complex that catalyzes the conversion of prothrombin to thrombin by factor Xa in the presence of calcium and a phospholipid membrane. 1-3 Activated protein C (APC) inactivates FVa through cleavage of the active cofactor at R306, R506, and R679 and requires FV as a cofactor in the APC-mediated inactivation of factor VIIIa (FVIIIa). 6,7 Thus, FV plays an important role in the procoagulant pathway as well as in the protein C anticoagulant pathway. The structure of FV is similar to FVIII (both cofactors share approximately 40% homology in their heavy and light chains) and ceruloplasmin, the copper-binding protein in plasma. 8,9 Recently, the crystal structure of the C2 domain of FV has been established 10 and molecular models for the A and C domains of FV have been proposed. 11,12 The gene for coagulation FV has been mapped to chromosome 1q23 13 and spans more than 80 kilobases (kb). It consists of 25 exons and the messenger RNA (mRNA) encodes a leader peptide of 28 amino acids and a mature protein of 2196 amino acids. Roughly, the heavy chain is encoded by exons 1 to 12 and the light chain by exons 14 to 25. The entire B domain is encoded by exon 13, which contains 2 tandem repeats of 17 amino acids and 31 tandem repeats of 9 amino acids that are absent in the B domain of FVIII. 14,15 Deficiency of FV, or parahemophilia, was first described in 1947 by Owren. 16 It is a rare autosomal recessive bleeding disorder with an estimated frequency of one in one million. The phenotypic expression of FV deficiency is variable; heterozygotes are usually asymptomatic, whereas homozygous patients show mild, moderate, or severe bleeding symptoms. Identifying the molecular basis underlying this disease will help to obtain more insight into the mechanisms involved in this variable clinical expression. The recently published complete nucleotide sequence of the FV gene (GenBank accession number Z99572) has facilitated the molecular characterization underlying FV deficiency and reports have ide...