Factor V Leiden pseudo‐homozygotes have a more pronounced hypercoagulable state than factor V Leiden homozygotes

Abstract

The factor V (FV) R506Q mutation (FV Leiden) [1Bertina R.M. Koeleman B.P. Koster T. Rosendaal F.R. Dirven R.J. de Ronde H. van der Velden P.A. Reitsma P.H. Mutation in blood coagulation factor V associated with resistance to activated protein C.Nature. 1994; 369: 64-7Crossref PubMed Scopus (3807) Google Scholar], which is present in approximately 5% of Caucasians, is associated with activated protein C (APC) resistance [2Dahlbäck B. Carlsson M. Svensson P.J. Familial thrombophilia due to a previously unrecognized mechanism characterized by poor anticoagulant response to activated protein C: prediction of a cofactor to activated protein C.Proc Natl Acad Sci U S A. 1993; 90: 1004-8Crossref PubMed Scopus (2003) Google Scholar] and increases the risk of venous thrombosis seven‐fold in heterozygotes and 80‐fold in homozygotes [3Rosendaal F.R. Koster T. Vandenbroucke J.P. Reitsma P.H. High risk of thrombosis in patients homozygous for factor V Leiden (activated protein C resistance).Blood. 1995; 85: 1504-8Crossref PubMed Google Scholar]. 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% [4Simioni P. Scudeller A. Radossi P. Gavasso S. Girolami B. Tormene D. Girolami A. ‘Pseudo homozygous’ activated protein C resistance due to double heterozygous factor V defects (factor V Leiden mutation and type I quantitative factor V defect) associated with thrombosis: report of two cases belonging to two unrelated kindreds.Thromb Haemost. 1996; 75: 422-6Crossref PubMed Scopus (82) Google Scholar]. Based on APC resistance measurements, FV Leiden pseudo‐homozygotes are generally considered to have a hypercoagulable state similar to that of FV Leiden homozygotes [4Simioni P. Scudeller A. Radossi P. Gavasso S. Girolami B. Tormene D. Girolami A. ‘Pseudo homozygous’ activated protein C resistance due to double heterozygous factor V defects (factor V Leiden mutation and type I quantitative factor V defect) associated with thrombosis: report of two cases belonging to two unrelated kindreds.Thromb Haemost. 1996; 75: 422-6Crossref PubMed Scopus (82) Google Scholar, 5Brugge J.M. Simioni P. Bernardi F. Tormene D. Lunghi B. Tans G. Pagnan A. Rosing J. Castoldi E. Expression of the normal factor V allele modulates the APC resistance phenotype in heterozygous carriers of the factor V Leiden mutation.J Thromb Haemost. 2005; 3: 2695-702Crossref PubMed Scopus (0) Google Scholar, 6Simioni P. Castoldi E. Lunghi B. Tormene D. Rosing J. Bernardi F. An underestimated combination of opposites resulting in enhanced thrombotic tendency.Blood. 2005; 106: 2363-5Crossref PubMed Scopus (0) Google Scholar]. 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 [7Castaman G. Tosetto A. Ruggeri M. Rodeghiero F. Pseudohomozygosity for activated protein C resistance is a risk factor for venous thrombosis.Br J Haematol. 1999; 106: 232-6Crossref PubMed Scopus (13) Google Scholar] and to that of homozygotes in another study [6Simioni P. Castoldi E. Lunghi B. Tormene D. Rosing J. Bernardi F. An underestimated combination of opposites resulting in enhanced thrombotic tendency.Blood. 2005; 106: 2363-5Crossref PubMed Scopus (0) Google Scholar]. 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 [8Duckers C. Simioni P. Spiezia L. Radu C. Gavasso S. Rosing J. Castoldi E. Low plasma levels of tissue factor pathway inhibitor in patients with congenital factor V deficiency.Blood. 2008; 112: 3615-23Crossref PubMed Scopus (111) Google Scholar]. 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 [9Dahm A. van Hylckama Vlieg A. Bendz B. Rosendaal F. Bertina R.M. Sandset P.M. Low levels of tissue factor pathway inhibitor (TFPI) increase the risk of venous thrombosis.Blood. 2003; 101: 4387-92Crossref PubMed Scopus (214) Google Scholar]. 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 [8Duckers C. Simioni P. Spiezia L. Radu C. Gavasso S. Rosing J. Castoldi E. Low plasma levels of tissue factor pathway inhibitor in patients with congenital factor V deficiency.Blood. 2008; 112: 3615-23Crossref PubMed Scopus (111) Google Scholar]. 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 [8Duckers C. Simioni P. Spiezia L. Radu C. Gavasso S. Rosing J. Castoldi E. Low plasma levels of tissue factor pathway inhibitor in patients with congenital factor V deficiency.Blood. 2008; 112: 3615-23Crossref PubMed Scopus (111) Google Scholar]. Plasma levels of prothrombin, FV, total protein S and free TFPI were measured as described before [8Duckers C. Simioni P. Spiezia L. Radu C. Gavasso S. Rosing J. Castoldi E. Low plasma levels of tissue factor pathway inhibitor in patients with congenital factor V deficiency.Blood. 2008; 112: 3615-23Crossref PubMed Scopus (111) Google Scholar]. 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 [10Hemker H.C. Giesen P. AlDieri R. Regnault V. de Smed E. Wagenvoord R. Lecompte T. Béguin S. The calibrated automated thrombogram (CAT): a universal routine test for hyper‐ and hypocoagulability.Pathophysiol Haemost Thromb. 2002; 32: 249-53Crossref PubMed Scopus (0) Google Scholar] 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 inhibitory anti‐TFPI antibodies (an equimolar mixture of anti‐TFPI Kunitz‐1, anti‐TFPI Kunitz‐2, anti‐TFPI Kunitz‐3 and anti‐TFPI C‐terminus monoclonal antibodies from Sanquin Reagents, Amsterdam, the Netherlands). Moreover, 32 μg mL−1 corn trypsin inhibitor (Haematologic Technologies, Essex Junction VT, USA) was added to inhibit contact activation. Measurements at high TF were performed in the absence and presence of APC (Innovative Research, Novi, MI, USA). As the low APC concentration (approximately 5 nm) normally used in the thrombin generation‐based APC resistance test has relatively little effect on thrombin generation in FV Leiden homozygous plasma, precluding discrimination between homozygotes and pseudo‐homozygotes [5Brugge J.M. Simioni P. Bernardi F. Tormene D. Lunghi B. Tans G. Pagnan A. Rosing J. Castoldi E. Expression of the normal factor V allele modulates the APC resistance phenotype in heterozygous carriers of the factor V Leiden mutation.J Thromb Haemost. 2005; 3: 2695-702Crossref PubMed Scopus (0) Google Scholar, 6Simioni P. Castoldi E. Lunghi B. Tormene D. Rosing J. Bernardi F. An underestimated combination of opposites resulting in enhanced thrombotic tendency.Blood. 2005; 106: 2363-5Crossref PubMed Scopus (0) Google Scholar], a higher APC concentration (16 nm) was used in the present study. This APC concentration completely abolished thrombin generation in pooled normal plasma and reduced the endogenous thrombin potential (ETP) of a FV Leiden homozygous plasma pool (prepared by pooling plasma from five FV Leiden homozygotes: one male and four females, mean age 37.8 years) to approximately 30% of the ETP measured in the absence of APC. This FV Leiden homozygous plasma pool was used instead of pooled normal plasma to normalize the APC sensitivity ratio (APCsr). The normalized (n)APCsr was therefore defined as the ratio of the ETPs determined in the presence and absence of APC in sample plasma divided by the ETP ratio in the FV Leiden homozygous plasma pool. All data are presented as mean ± standard deviation. Differences in factor levels and thrombin generation parameters between FV Leiden pseudo‐homozygotes and homozygotes were evaluated using the Student’s t‐test and corrected for age and gender by multiple linear regression analysis. Statistical analyses were performed with IBM SPSS Statistics 18.0 (Somers, NY, USA). The demographic characteristics and the plasma levels of coagulation factors and inhibitors of the 18 FV Leiden homozygotes and the 9 FV Leiden pseudo‐homozygotes under study are presented in Fig. 1A. FV Leiden pseudo‐homozygotes had reduced FV and TFPI levels as compared with homozygotes (FV 62.9% ± 8.5% vs. 104.1% ± 19.3%, P < 0.001; TFPI 6.5 ± 2.0 vs. 10.5 ± 3.4 ng mL−1, P = 0.004). These differences persisted after correction for age and gender. The levels of prothrombin and protein S, two other major determinants of thrombin generation and APC resistance [11de Visser M.C. van Hylckama Vlieg A. Tans G. Rosing J. Dahm A.E. Sandset P.M. Rosendaal F.R. Bertina R.M. Determinants of the APTT‐ and ETP‐based APC sensitivity tests.J Thromb Haemost. 2005; 3: 1488-94Crossref PubMed Scopus (0) Google Scholar, 12Dielis A.W. Castoldi E. Spronk H.M. van Oerle R. Hamulyák K. Ten Cate H. Rosing J. Coagulation factors and the protein C system as determinants of thrombin generation in a normal population.J Thromb Haemost. 2008; 6: 125-31Crossref PubMed Scopus (175) Google Scholar], did not differ between the two groups. To investigate the effect of reduced plasma TFPI levels in FV Leiden pseudo‐homozygotes, thrombin generation was measured under TFPI‐sensitive conditions. At low TF in the absence of APC (Fig. 1B), the peak height of thrombin generation was 1.6‐fold higher in FV Leiden pseudo‐homozygotes than in homozygotes (85.2 ± 48.3 vs. 53.3 ± 26.8 nm, P = 0.008 after correction for age and gender). This difference disappeared in the presence of anti‐TFPI antibodies (212.9 ± 63.0 vs. 210.5 ± 36.0 nm, P = n.s.), indicating that the low TFPI levels are responsible for the elevated thrombin generation in FV Leiden pseudo‐homozygotes. At high TF in the absence of APC, the ETP was similar in FV Leiden pseudo‐homozygotes and homozygotes (706 ± 276 vs. 692 ± 150 nm min, P = n.s.). However, in the presence of APC the ETP was higher in FV Leiden pseudo‐homozygotes than in homozygotes (312 vs. 213 nm min, P = 0.030 after correction for age and gender), making FV Leiden pseudo‐homozygotes approximately 1.5 times more APC‐resistant than homozygotes (nAPCsr 1.36 vs. 0.91, P = 0.003 after correction for age and gender; nAPCsr normalized against the FV Leiden homozygous plasma pool; Fig. 1C). As TFPI is the major determinant of APC resistance measured with the thrombin generation‐based assay [11de Visser M.C. van Hylckama Vlieg A. Tans G. Rosing J. Dahm A.E. Sandset P.M. Rosendaal F.R. Bertina R.M. Determinants of the APTT‐ and ETP‐based APC sensitivity tests.J Thromb Haemost. 2005; 3: 1488-94Crossref PubMed Scopus (0) Google Scholar, 12Dielis A.W. Castoldi E. Spronk H.M. van Oerle R. Hamulyák K. Ten Cate H. Rosing J. Coagulation factors and the protein C system as determinants of thrombin generation in a normal population.J Thromb Haemost. 2008; 6: 125-31Crossref PubMed Scopus (175) Google Scholar], the observed difference in APC resistance between FV Leiden pseudo‐homozygotes and homozygotes is probably attributable to their difference in plasma TFPI levels. A control experiment performed with pooled plasma from FV Leiden homozygotes and pooled plasma from FV Leiden pseudo‐homozygotes indeed showed that the difference in APC resistance between the two pools could be abolished by the addition of anti‐TFPI antibodies (data not shown). The higher thrombin generation and APC resistance in FV Leiden pseudo‐homozygotes indicate a more pronounced hypercoagulable state than in homozygotes, which may translate in a higher risk of venous thrombosis. The clinical implication of this finding is that protocols for VTE prophylaxis and treatment in FV Leiden pseudo‐homozygotes should be similar to those used in FV Leiden homozygotes (or double heterozygotes for the FV Leiden and prothrombin G20210A mutations) according to the current antithrombotic guidelines. In a previous study we have shown that the lack of normal FV expressing APC‐cofactor activity in factor (F)VIIIa inactivation makes FV Leiden pseudo‐homozygotes more APC‐resistant than FV Leiden heterozygotes and similar to homozygotes [5Brugge J.M. Simioni P. Bernardi F. Tormene D. Lunghi B. Tans G. Pagnan A. Rosing J. Castoldi E. Expression of the normal factor V allele modulates the APC resistance phenotype in heterozygous carriers of the factor V Leiden mutation.J Thromb Haemost. 2005; 3: 2695-702Crossref PubMed Scopus (0) Google Scholar]. In the present study we show that FV Leiden pseudo‐homozygotes also have reduced plasma levels of TFPI, which further enhances their hypercoagulable state, making them more procoagulant than FV Leiden homozygotes. This difference has been previously overlooked because APC resistance was measured with assays that are rather insensitive to TFPI (e.g. the aPTT‐based assay) and/or because the APC concentration used was too low to discriminate between FV Leiden homozygotes and pseudo‐homozygotes. Our data further suggest that differences in the expression of the normal FV allele in FV Leiden heterozygotes may modulate APC resistance and thrombosis risk not only by affecting the APC‐cofactor activity of FV in factor VIIIa inactivation [5Brugge J.M. Simioni P. Bernardi F. Tormene D. Lunghi B. Tans G. Pagnan A. Rosing J. Castoldi E. Expression of the normal factor V allele modulates the APC resistance phenotype in heterozygous carriers of the factor V Leiden mutation.J Thromb Haemost. 2005; 3: 2695-702Crossref PubMed Scopus (0) Google Scholar], but also by modifying plasma TFPI levels. The relative contributions of these two mechanisms will depend on the determinants of the assay used to measure APC resistance [11de Visser M.C. van Hylckama Vlieg A. Tans G. Rosing J. Dahm A.E. Sandset P.M. Rosendaal F.R. Bertina R.M. Determinants of the APTT‐ and ETP‐based APC sensitivity tests.J Thromb Haemost. 2005; 3: 1488-94Crossref PubMed Scopus (0) Google Scholar]. Similar considerations apply to individuals who are pseudo‐homozygous for other procoagulant FV gene polymorphisms/mutations, including the common R2 haplotype [13Bernardi F. Faioni E.M. Castoldi E. Lunghi B. Castaman G. Sacchi E. Mannucci P.M. A factor V genetic component differing from factor V R506Q contributes to the activated protein C resistance phenotype.Blood. 1997; 90: 1552-7Crossref PubMed Google Scholar] as well as the Cambridge [14Williamson D. Brown K. Luddington R. Baglin C. Baglin T. Factor V Cambridge: a new mutation (Arg306‐‐>Thr) associated with resistance to activated protein C.Blood. 1998; 91: 1140-4Crossref PubMed Google Scholar], Hong Kong [15Chan W.P. Lee C.K. Kwong Y.L. Lam C.K. Liang R. A novel mutation of Arg306 of factor V gene in Hong Kong Chinese.Blood. 1998; 91: 1135-9Crossref PubMed Google Scholar], and Liverpool [16Mumford A.D. McVey J.H. Morse C.V. Gomez K. Steen M. Norstrøm E.A. Tuddenham E.G. Dahlbäck B. Bolton‐Maggs P.H. Factor V I359T: a novel mutation associated with thrombosis and resistance to activated protein C.Br J Haematol. 2003; 123: 496-501Crossref PubMed Scopus (40) Google Scholar] mutations. Moreover, partial FV deficiency might per se increase the risk of venous thrombosis via the associated decrease both in the APC‐cofactor activity of FV and in plasma TFPI levels. Evidence for this has recently been reported in the Japanese population [17Suehisa E. Kawasaki T. Toku M. Hidaka Y. Low level of factor V is associated with development of deep‐vein thrombosis in Japanese patients.Thromb Res. 2010; 125: 128-33Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar], whereas low FV levels were not associated with the risk of venous thrombosis in the Leiden Thrombophilia Study (LETS) [18Kamphuisen P.W. Rosendaal F.R. Eikenboom J.C. Bos R. Bertina R.M. Factor V antigen levels and venous thrombosis: risk profile, interaction with factor V leiden, and relation with factor VIII antigen levels.Arterioscler Thromb Vasc Biol. 2000; 20: 1382-6Crossref PubMed Google Scholar]. This discrepancy might be because of the different patient selection criteria (only idiopathic thromboses in the Japanese study) and/or because of the relatively high prevalence of mild FV deficiencies [17Suehisa E. Kawasaki T. Toku M. Hidaka Y. Low level of factor V is associated with development of deep‐vein thrombosis in Japanese patients.Thromb Res. 2010; 125: 128-33Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar] in the absence of other common thrombophilic defects (FV Leiden and the prothrombin G20210A mutation) in the Japanese population. In either case, further studies are needed to establish whether low FV levels are per se a risk factor for venous thrombosis. The authors state that they have no conflict of interest. This study was supported by a VIDI grant (nr. 917‐76‐312, to E. Castoldi) from the Dutch Organisation for Scientific Research (NWO).