Severe factor V (FV) deficiency is associated with mild to severe bleeding diathesis, but many patients with FV levels lower than 1% bleed less than anticipated. We used calibrated automated thrombography to screen patients with severe FV deficiency for protective procoagulant defects. Thrombin generation in FV-deficient plasma was only measurable at high tissue factor concentrations. Upon reconstitution of FV-deficient plasma with purified FV, thrombin generation increased steeply with FV concentration, reaching a plateau at approximately 10% FV. FV-deficient plasma reconstituted with 100% FV generated severalfold more thrombin than normal plasma, especially at low tissue factor concentrations (1.36 pM) or in the presence of activated protein C, suggesting reduced tissue factor pathway inhibitor (TFPI) levels in FV-deficient plasma. Plasma TFPI antigen and activity levels were indeed lower (P < .001) in FV-deficient patients (n ‫؍‬ 11; 4.0 ؎ 1.0 ng/mL free TFPI) than in controls (n ‫؍‬ 20; 11.5 ؎ 4.8 ng/mL), while persons with partial FV deficiency had intermediate levels (n ‫؍‬ 16; 7.9 ؎ 2.5 ng/mL). FV immunodepletion experiments in normal plasma and surface plasmon resonance analysis provided evidence for the existence of a FV/TFPI complex, possibly affecting TFPI stability/clearance in vivo. Low TFPI levels decreased the FV requirement for minimal thrombin generation in FV-deficient plasma to less than 1% and might therefore protect FV-deficient patients from severe bleeding. (Blood. 2008;112:3615-3623) IntroductionCoagulation factor V (FV) is a large multidomain glycoprotein structurally and functionally homologous to factor VIII (FVIII). 1 After biosynthesis in the liver, FV is released in the bloodstream, where it is found in both plasma (80%; concentration of 21-25 nM) and platelets (20%). The activated form of FV (FVa) acts as an essential cofactor of activated factor X (FXa) in prothrombin (PT) activation, thereby enhancing thrombin formation by several orders of magnitude. 2 The generation of thrombin is physiologically down-regulated by several anticoagulant mechanisms, including the protein C pathway 3 and the tissue factor pathway inhibitor (TFPI) system. 4 Activated protein C (APC) is a vitamin K-dependent serine protease which, in concert with its nonenzymatic cofactor protein S, inactivates FVa and FVIIIa by limited proteolysis. A poor anticoagulant response of plasma to exogenous APC (APC resistance 5 ) is the most common risk factor for venous thrombosis. Conversely, TFPI is a Kunitz-type protease inhibitor that binds and inhibits both FXa and the tissue factor (TF)/FVIIa complex in a 2-step reaction, 6 the first step being stimulated by protein S. 7,8 TFPI is synthesized primarily by the vascular endothelium, and most of it (approximately 80%) is associated with the endothelial surface as a full-length protein, the form that most effectively inhibits FXa. 9 Another 2% of all TFPI is stored in platelets. 10,11 The remainder circulates in plasma at a concentration of 2.0 to 2.5 nM, of which app...

Coagulation factor V (FV), present in plasma and platelets, is indispensable to thrombin formation, yet patients with undetectable plasma FV seldom experience major bleeding. We used thrombin generation assays to explore the role of platelet

SummaryCoagulation factor V (FV), present in plasma and platelets, is an indispensable clotting factor, as demonstrated by the uniform lethality of FV knock-out mice. Surprisingly, however, severe FV deficiency is rarely fatal in humans. In fact, although several cases of life-threatening intracranial haemorrhage have been reported in FV-deficient newborns, many patients with undetectable FV levels experience only mild to moderate bleeding and do not require routine prophylaxis. While the reasons for this variable phenotypic expression are largely unknown, several observations from different laboratories indicate platelets as crucial players in FV deficiency. Moreover, we have recently shown that plasma levels of tissue factor pathway inhibitor are considerably reduced in FV-deficient plasma, which results in enhanced thrombin generation especially at very low FV levels (<2%). The present review discusses and integrates these findings in the context of the biology of FV and the clinical features of FV deficiency.

To cite this article: Castoldi E, Duckers C, Radu C, Spiezia L, Rossetto V, Tagariello G, Rosing J, Simioni P. Homozygous F5 deep-intronic splicing mutation resulting in severe factor V deficiency and undetectable thrombin generation in platelet-rich plasma. J Thromb Haemost 2011; 9: 959-68. Summary. Background: Coagulation factor (F) V deficiency is associated with a bleeding tendency of variable severity, but phenotype determinants are largely unknown. Recently, we have shown that three patients with undetectable plasma FV and mild bleeding symptoms had sufficient residual platelet FV to support thrombin generation in platelet-rich plasma (PRP). Therefore, we hypothesized that FV-deficient patients with severe bleeding manifestations may lack platelet FV. Objectives: To characterize a FV-deficient patient with a severe bleeding diathesis. Patients/Methods: We performed FV mutation screening and functional studies in a 31-year-old male (FV:C < 1%) with umbilical bleeding at birth, recurrent hemarthrosis and muscle hematomas, and a recent intracranial hemorrhage. Results: The proband was homozygous for a deep-intronic mutation (F5 IVS8 +268A fi G) causing the inclusion of a pseudo-exon with an in-frame stop codon in the mature F5 mRNA. Although platelet FV antigen was detectable by immunoprecipitation followed by Western blotting, no FV activity could be demonstrated in the probandÕs plasma or platelets with a prothrombinase-based assay. Moreover, no thrombin generation was observed in PRP triggered with 1-50 pM tissue factor (even in the presence of platelet agonists), whereas an acquired FV inhibitor was excluded. Clot formation in the probandÕs whole blood, as assessed by thromboelastom-etry, was markedly delayed but not abolished. Conclusions: This is the first report of a pathogenic deep-intronic mutation in the F5 gene. Our findings indicate that the minimal FV requirement for viability is extremely low and suggest that thrombin generation in PRP may predict bleeding tendency in patients with undetectable plasma FV.

Summary. Background: The tissue factor pathway inhibitor (TFPI)/protein S anticoagulant system is a potent inhibitor of blood coagulation. TFPI and protein S are major determinants of thrombin generation (TG) tests determined at low tissue factor (TF) and at high TF concentrations in the presence of activated protein C (APC). Both TFPI and protein S protect against venous thrombosis, but the importance of the TFPI/protein S system in arterial thrombosis remains unclear. Objectives: To investigate the influence of the TFPI/protein S anticoagulant system on the risk of myocardial infarction (MI) in young women. Methods: The RATIO study is a case-control study in women under 50 years of age, including 205 patients and 638 controls. TFPI and protein S were quantified using ELISA. The TFPI/ protein S activity (nTFPIr) and the APC sensitivity ratio (nAPCsr) were determined using TG tests. Odds ratios (ORs) adjusted for putative confounders and corresponding 95% confidence intervals (95% CI) were determined. Results: Women with MI had higher TFPI levels than controls (135.9 ± 40% vs. 124.2 ± 41%), resulting in increased TFPI/protein S activities and increased APC sensitivity. Furthermore, an increased TFPI activity was associated with MI [nTFPIr: adjusted OR Q1 vs. Q4 = 2.1 (95%CI 1.1-4.1)]. Additionally, an increased APC sensitivity was associated with MI [nAPCsr: adjusted OR Q1 vs. Q4 = 1.7 (95% CI 0.9-3.2)] Conclusion: Women with MI had increased TFPI levels compared with controls. Consequently, the TFPI/protein S activity and APC sensitivity are increased in women with MI. Whether this increase in TFPI activity acts as a compensating mechanism for an increased procoagulant state or is a marker of endothelial damage remains to be investigated.

FV (and particularly its FV2 isoform) contributes to the TFPIα-dependent down-regulation of thrombin generation in plasma triggered with low TF.

To cite this article: Duckers C, Simioni P, Tormene D, Carraro S, Rosing J, Castoldi E. Factor V Leiden pseudo-homozygotes have a more pronounced hypercoagulable state than factor V Leiden homozygotes. J Thromb Haemost 2011; 9: 864-7.The factor V (FV) R506Q mutation (FV Leiden) [1], which is present in approximately 5% of Caucasians, is associated with activated protein C (APC) resistance [2] and increases the risk of venous thrombosis seven-fold in heterozygotes and 80-fold in homozygotes [3]. The rare FV Leiden heterozygotes that carry a loss-of-function mutation on the counterpart FV allele (FV Leiden pseudo-homozygotes), express only the FV Leiden allele and have plasma FV levels of approximately 50% [4]. Based on APC resistance measurements, FV Leiden pseudo-homozygotes are generally considered to have a hypercoagulable state similar to that of FV Leiden homozygotes [4][5][6]. Their risk of venous thrombosis is not well established and was found to be similar to that of FV Leiden heterozygotes in a previous study [7] and to that of homozygotes in another study [6]. However, both studies may have been biased by the inclusion of probands in the analysis. Therefore, an intermediate phenotype such as thrombin generation may be more suitable to quantify the thrombotic tendency associated with FV Leiden pseudo-homozygosity.Recently, we have shown that plasma levels of tissue factor pathway inhibitor (TFPI) are markedly reduced in FV deficiency [8]. Since FV Leiden pseudo-homozygotes have low FV levels, we hypothesized that they might have reduced TFPI levels as well. This may exacerbate their hypercoagulable state, as low TFPI levels are also associated with an increased risk of venous thrombosis [9]. To test this hypothesis, we compared plasma TFPI levels and thrombin generation in FV Leiden pseudo-homozygotes and homozygotes.Nine FV Leiden pseudo-homozygotes (five males and four females, mean age 48.4 years), of whom five (55.5%) had experienced venous thrombosis, were included. Eighteen FV Leiden homozygotes (nine males and nine females, mean age 44.8 years), of whom six (33.3%) had experienced venous thrombosis, served as controls. Genotyping for the FV Leiden mutation was performed as described before [8]. All participants gave informed consent to participate in the study, which was carried out in accordance with the Declaration of Helsinki.Platelet-poor plasma was prepared from venous blood as described previously [8]. Plasma levels of prothrombin, FV, total protein S and free TFPI were measured as described before [8]. Pooled normal plasma, prepared by pooling plasma from 15 healthy individuals without FV Leiden (seven males and eight females, mean age 45.1 years), was used as a reference.Thrombin generation was determined using the Calibrated Automated Thrombogram method [10] under conditions that are sensitive to the TFPI anticoagulant pathway, i.e. at low (1.7 pM) tissue factor (TF) and at higher (6.8 pM) TF in the presence of APC. Measurements at low TF were performed in the absence and presence of inh...

People interested in the research are advised to contact the author for the final version of the publication, or visit the DOI to the publisher's website.• The final author version and the galley proof are versions of the publication after peer review.• The final published version features the final layout of the paper including the volume, issue and page numbers. Link to publication General rightsCopyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights.• Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal.If the publication is distributed under the terms of Article 25fa of the Dutch Copyright Act, indicated by the "Taverne" license above, please follow below link for the End User Agreement: