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 F5A2440G leads to the formation of the TFPIα:FV-short complex, which inhibits activation and propagation of coagulation.
Lisa M. Vincent, Sinh Tran, Ruzica Livaja, Tracy A. Bensend, Dianna M. Milewicz, Björn Dahlbäck
Western blot analysis of plasma (equivalent to 0.5 μl) from affected and unaffected family members with FV being detected using the monoclonal AHV-5146 against the heavy chain in combination with HRP-conjugated goat anti-mouse antiserum. Blots were developed with Supersignal West Dura Extended Duration Chemiluminescence Substrate. The SDS-PAGE (4%–15%) was run under nonreducing conditions. Lanes marked with plus signs represent affected individuals carrying the A2440G mutation, whereas those marked with minus signs represent unaffected family members. The arrows point at full-length FV (FV-FL) and FV-short. The 2 lanes in the upper row that are marked with asterisks represent pools of unaffected (–*) and affected (+*) family members. Note the weak FV-short band in the seventh sample from the left in the upper row, the sample being derived from individual IV:17 in family pedigree (Supplemental Figure 1).