The autosomal dominantly inherited east Texas bleeding disorder is linked to an A2440G variant in exon 13 of the F5 gene. Affected individuals have normal levels of coagulation factor V (FV) activity, but demonstrate inhibition of global coagulation tests. We demonstrated that the A2440G mutation causes upregulation of an alternatively spliced F5 transcript that results in an in-frame deletion of 702 amino acids of the large activation fragment, the B domain. The approximately 250-kDa FV isoform (FV-short), which can be fully activated by thrombin, is present in all A2440G carriers' plasma (n = 16). FV-short inhibits coagulation through an indirect mechanism by forming a complex with tissue factor pathway inhibitor-α (TFPIα), resulting in an approximately 10-fold increase in plasma TFPIα, suggesting that the TFPIα:FV-short complexes are retained in circulation. The TFPIα:FV-short complexes efficiently inhibit thrombin generation of both intrinsic and extrinsic coagulation pathways. These data demonstrate that the east Texas bleeding disorder-associated F5 A2440G leads to the formation of the TFPIα:FV-short complex, which inhibits activation and propagation of coagulation. IntroductionCoagulation factor V (FV) is a cofactor protein that can both promote and inhibit coagulation (1, 2). Located on 1q24-25, the F5 gene is composed of 25 exons that transcribe a 6.8-kb mRNA (3-7). The translated 330-kDa glycoprotein precursor contains 2,196 amino acids organized into the domain structure A1-A2-B-A3-C1-C2 and is highly homologous to factor VIII (FVIII), sharing 35%-40% identity in the A and C domains (4, 5, 8). Human FV is primarily produced by hepatocytes and circulates in plasma as an intact 330-kDa precursor at a concentration of about 20 nM (7 μg/ml) (9-12). Approximately 20% of the total human FV found in whole blood is stored in platelet α granules in a partially proteolyzed form in conjunction with the binding protein multimerin. This platelet FV derives from the plasma FV pool and is secreted upon platelet activation to create a high, localized concentration of the cofactor at sites of injury (12)(13)(14)(15).The procoagulant cofactor function of FV is primarily dictated by its interaction and cleavage by thrombin and active factor X (FXa). Its cleavage by thrombin is deemed the most biologically important early event in the blood clot formation process (16)(17)(18)(19). As an intact single-chain precursor, FV expresses less than 1% of its potential procoagulant cofactor activity (20). Upon sequential cleavage of Arg-709, Arg-1018, and Arg-1545 by thrombin, the large connecting B domain is removed from the intact FV molecule to produce the heavy chain (A1-A2) and the light chain (A3-C1-C2) that have M r s of 105,000 and 74,000, respectively. The heavy chain and the light chain noncovalently associate in the presence of calcium to produce an active procoagulant cofactor (FVa). FVa and FXa assemble on negatively charged phospholipid (PL) membranes to form the prothrombinase (PTase) complex. The presence of FVa in...

Coagulation factor V (FV) deficiency is characterised by variable bleeding phenotypes and heterogeneous mutations. To add new insights into the FV genotype-phenotype relationship, we characterised the R1698W change in the A3 domain, at the poorly investigated interface with the A2 domain. The FV R1698W mutation was responsible for a markedly reduced expression level (10% of FV-WT) and specific activity in thrombin generation (0.39). Interestingly, the FVa1698W showed rapid activity decay upon activation due to increased dissociation rate between the heavy and light chains. The importance of the size and charge of the residue at position 1698 was investigated by three additional recombinant mutants, FVR1698A, FVR1698Q, and FVR1698E. FVR1698A and FVR1698Q expression (30 and 45% of FV-WT), specific activity (both 0.57) and stability were all reduced. Noticeably, FVR1698E showed normal activity and stability despite poor expression (10% of FV-WT). These data indicate the essential role of R1698 for normal biosynthetic process and support local flexibility for positively or negatively charged residues to produce stable and functional A3-A2 domain interactions. Their experimental alteration produces a gradient of FV defects, which help to interpret the wide spectrum of phenotypes in FV-deficient patients.