A novel mutation in the F5 gene (factor V Amsterdam) associated with bleeding independent of factor V procoagulant function

Abstract

Key Points• A novel gain-of-function mutation in factor V leading to increased levels of TFPI and bleeding was identified by whole exome sequencing.• Factor V Amsterdam (F5 C2588G) resembles the mutation (F5 A2350G) leading to East Texas bleeding disorder.We investigated a small Dutch family with a bleeding diathesis, prolonged prothrombin, and activated partial thromboplastin times, in whom no classifying diagnosis was made. The 2 affected relatives had severely decreased in vitro thrombin generation, and levels of tissue factor pathway inhibitor (TFPI) were strongly increased. To identify the genetic cause of the bleeding diathesis, we performed whole exome sequencing analysis of all living relatives. We found a novel gain-of-function mutation in the F5 gene (c.C2588G), which leads to an aberrant splicing of F5 and ultimately to a short factor V protein (missing 623 amino acids from the B domain), which we called factor V Amsterdam. Factor V Amsterdam binds to TFPI, prolonging its half-life and concentration. This is the second report of an association between a shorter form of factor V and increased TFPI levels, resulting in severely reduced thrombin generation and a bleeding tendency. (Blood. 2015; 125(11):1822-1825 IntroductionIn clinical practice, we occasionally encounter patients with a bleeding tendency and prolonged coagulation screening tests, in whom a classifying diagnosis cannot be made. Here, we describe our search toward elucidating the etiology of a bleeding tendency in a mother and her son. Study design Subjects and samplesThe pedigree of the family is depicted in Figure 1A. Detailed clinical features of the family are provided in supplemental Table 1, available on the Blood Web site. We collected blood from the mother (II:2) and son (III:1) with clinical manifestations of bleeding and 4 unaffected relatives. All participants provided written informed consent. Coagulation testing and exome sequencingCoagulation parameters were determined by standard methods. Thrombin generation was performed with calibrated automated thrombography. Genomic DNA was isolated from peripheral blood mononuclear cells. Direct exome sequencing of the TFPI and PROS1 genes was performed in the 2 patients and 1 unaffected relative. Whole exome sequencing data were generated for all relatives, and candidate mutations were confirmed by Sanger sequencing. Details of methods are available in supplemental Materials. Results and discussionThrombin generation was measured in plasma from individuals II:1 (unaffected husband), II:2 (proband), and III:1 (affected son). Thrombin generation initiated with 1 pM tissue factor was severely affected in the proband and son ( Figure 1B); this also occurred at higher concentrations of tissue factor (5 pM, Figure 1C; 20 pM, data not shown). Mixing patient plasma with normal plasma did not normalize thrombin generation, suggesting the presence of a circulating inhibitor (data not shown). Because lupus anticoagulant and deficiencies of antithrombin and protein C had been excluded in both pat...

TFPIα interacts with FVa and FXa to inhibit prothrombinase during the initiation of coagulation

“…Multiple lines of evidence support this assertion: (1) in vitro studies have shown that the TFPIα basic C-terminus binds to forms of FVa containing the acidic region with high affinity ( K d , ~90 pM) but does not bind to forms of FVa lacking the acidic region 12,15 ; (2) 2 FV mutations producing a truncated B-domain that lacks the basic region but retains the acidic region have been identified 16,35 ; these forms of FV circulate bound to TFPIα; and (3) plasma concentrations of TFPIα (~1 nM) do not inhibit thrombin generation by prothrombinase when the TFPIα basic region or the FVa acidic region is absent. 12 In addition, there is evidence suggesting the TFPIα basic region may bind FVa outside of the B-domain acidic region.…”

Reduced prothrombinase inhibition by tissue factor pathway inhibitor contributes to the factor V Leiden hypercoagulable state

“…45–47 FV-deficient patients also have TFPIα deficiency, and immunodepletion of FV from plasma also removes TFPIα. 46 Moreover, two FV mutants have been identified that bind to TFPIα and cause its concentration to increase 10- to 20-fold.…”

“…46 Moreover, two FV mutants have been identified that bind to TFPIα and cause its concentration to increase 10- to 20-fold. 45,47 These two mutations suggest the importance of prothrombinase inhibition by TFPIα, as they are associated with a moderately severe bleeding phenotype. Importantly, however, the FVL patients had normal total TFPI and TFPIα plasma concentrations, indicating the reduced anticoagulant activity of TFPI in FVL plasma was not caused by reduced TFPI concentration.…”

Correlates of plasma and platelet tissue factor pathway inhibitor, factor V, and Protein S

“…Patients producing the East Texas and Amsterdam variants of fV, 3,4 both of which tightly associate with plasma TFPIα, highlight the clinical importance of TFPI. These patients have 10-to 20-fold increased plasma TFPIα, an amount sufficient to cause a moderate bleeding diathesis.…”