Coagulation factor VA2440G causes east Texas bleeding disorder via TFPIα

Abstract

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...

Factor V‐short and protein S as synergistic tissue factor pathway inhibitor (TFPIα) cofactors

“…The recent elucidation of the molecular mechanisms of the East Texas Bleeding disorder revealed that FV‐Short, a splice variant of FV lacking 702 amino acid residues of the activation peptide B domain, is an important carrier of TFPIα in circulation 23. In this disease, a point mutation in the FV gene activates a normally existing but inefficient splice donor site in the codon for S756 and as a result affected individuals have greatly increased levels of circulating FV‐Short.…”

“…The FV‐Short binds TFPIα with high affinity and through mechanisms that are yet to be elucidated, the concentration of FV‐Short/TFPIα complexes in circulation are more than 10‐fold increased as compared to unaffected individuals. In individuals with the wild‐type allele, the concentration of the circulating FV‐Short is not yet elucidated but can be estimated to be in the sub‐nM range (≈0.1‐0.2 nmol L −1 ), approximately 100‐fold lower than the concentration of full length FV 23. We now demonstrate that FV‐Short not only is an important carrier for TFPIα in circulation but also functions as an efficient synergistic TFPIα cofactor together with protein S in the inhibition of FXa.…”

“…Thrombin generation was performed as previously described with slight variations 23. Either 40 uL pooled normal citrated (50 ug mL −1 CTI) or FV‐deficient plasma were mixed with different combinations of FV‐Short (before and after activation with 1.6 nmol L −1 thrombin (0.5 units mL −1 ) for 10 minutes and then inhibition with 1 unit/ml hirudin), anti‐bodies against protein S or TFPI (100 ug mL −1 final concentration), TFPIα (40 uL) and 30 uL PL (10 umol L −1 ; 10/20/70 of PS/PE/PC)/TF (1.4 pmol L −1 ) mixture as indicated in each experiment.…”

“…Family members carrying the mutation have high plasma concentrations of FV‐Short (≈2‐5 nmol L −1 ). The C‐terminus of the truncated B domain of FV‐Short exposes the negatively charged cluster to which TFPIα binds with high affinity 12, 23. As a result, the TFPIα concentration in circulation increases 10‐fold and causes a moderately severe bleeding phenotype.…”

“…As a result, the TFPIα concentration in circulation increases 10‐fold and causes a moderately severe bleeding phenotype. Under normal conditions, the FV‐Short is present in plasma at sub‐nM concentrations circulating in complex with TFPIα23. FV‐Short is thus an important carrier of TFPIα in the normal circulation.…”