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Abstract

Heterozygous carriers of factor V (FV) Leiden who also carry FV deficiency often develop venous thromboembolism, but the thrombosis risk associated with this rare condition (pseudohomozygous activated protein C resistance) is still unclear. The thrombosis risk of genetically characterized pseudohomozygotes (n = 6) was compared with that of FV Leiden heterozygotes (n = 683) and homozygotes (n = 50) recruited within a large cohort study on familial thrombophilia. Both thrombin generation and Kaplan-Meier thrombosis-free survival analyses were performed in different FV genotype groups. FV Leiden pseudohomozygotes showed significantly higher thrombosis risk than heterozygotes. The thrombin generation test in pseudohomozygotes showed a pattern similar to homozygotes. Accordingly, early thrombotic manifestations occurred in pseudohomozygotes at a similar rate as in homozygotes. Thus, failure to recognize FV deficiency in FV Leiden heterozygotes may result in an underestimate of the thrombosis risk and inadequate management of affected patients.

Introduction

The coagulation factor V (FV) Arg506→Gln mutation1 (FV Leiden) is a major cause of activated protein C (APC) resistance2 and confers an increased risk of venous thromboembolism (VTE) both in the heterozygous (7-fold) and homozygous (80-fold) condition.3 Interestingly, FV Leiden heterozygotes who carry a null mutation on the counterpart (non-Leiden) FV allele not only have reduced plasma FV levels (∼50%), but also show an APC resistance phenotype comparable to that of FV Leiden homozygotes. This rare condition, known as pseudohomozygous APC resistance,4-5 is attributable to nonexpression of the non-Leiden FV allele and consequent absence of normal FV in plasma.6 Assuming that the overall frequency of FV null alleles in the general population is 0.1%, as estimated from the 1 in 1 000 000 prevalence of severe FV deficiency (“parahemophilia”), approximately 1 of 1000 FV Leiden carriers is predicted to be pseudohomozygous APC-resistant.

Although partial FV deficiency, which is a potential bleeding defect7 (bleeding may occur spontaneously or sometimes during risk situations such as surgical procedures, tooth extractions, and trauma), might counteract the prothrombotic tendency associated with FV Leiden, virtually all reported cases of pseudohomozygous APC resistance are patients with severe thrombotic manifestations. However, since the low prevalence of this condition in the general population has precluded risk quantification via epidemiologic studies, the associated thrombosis risk is still a matter of debate.8 We have compared thrombin generation and thrombosis-free survival curves in FV Leiden pseudohomozygotes, heterozygotes, and homozygotes collected in the context of a large cohort study on familial thrombophilia.

Study design

Study population

Consecutive patients with single or recurrent VTE (deep vein thrombosis, pulmonary embolism, superficial vein thrombosis) referred to the Thrombosis Centre of Padua University between 1994 and 2004 were screened for FV Leiden. All FV Leiden heterozygotes and homozygotes, as well as available family members with and without VTE, were eligible for the study. Seven of 213 probands (3.3%) refused to participate. The whole cohort (n = 1144, including 206 probands) consisted of 50 FV Leiden homozygotes, 689 heterozygotes, and 405 individuals with normal FV genotype. Among the FV Leiden heterozygotes, 6 (5 probands and 1 asymptomatic brother of a proband) were found to have reduced plasma FV levels (∼50%) and marked APC resistance (Figure 1A), and were therefore reclassified as pseudohomozygotes. A detailed medical history was taken by physicians who were unaware of the FV genotype of participating subjects.9 The study was approved by the local ethics committee and informed consent was obtained from all participants.

Figure 1.

FV gene mutations and hemostatic parameters in FV Leiden pseudohomozygotes. (A) Top: F5 gene mutations preventing the expression of the non-Leiden FV allele in pseudohomozygous probands P1-P5. Italics, nucleotide changes; FS, frame shift; X, stop codon. Amino acid changes are indicated with the one-letter code. The large exon 13 is not drawn to scale. Bottom: FV activity (FVc) and antigen (FVag) levels (normal range, 80%-120%) and normalized APC sensitivity ratio (nAPC-sr). APC resistance is defined by an APC-sr less than 0.84; nAPCsr range of FV Leiden homozygotes 0.35 to 0.45. Mutations and patients are aligned. (B) Distribution of the endogenous thrombin potential (ETP) determined in the presence of APC in individuals with normal FV genotype (n = 11), FV Leiden heterozygotes (n = 12), FV Leiden homozygotes (n = 32), and FV Leiden pseudohomozygotes (n = 6). Genotype groups were compared with the Mann-Whitney Wilcoxon test. The horizontal lines indicate the means in the respective genotype groups. FVL stands for Factor V Leiden.

Coagulation laboratory tests

FV antigen and activity levels as well as APC resistance were measured in citrated plasma as previously reported.5

Genetic analysis

The FV Leiden genotype was determined by MnlI-restriction of amplified exon 10. FV mutation screening was performed by polymerase chain reaction (PCR) and direct sequencing, as described.10

Thrombin generation test

Thrombin generation after extrinsic activation of coagulation in the presence of APC was measured as described.11 The endogenous thrombin potential (ETP) was calculated using the Thrombinoscope software (Synapse BV, Maastricht, The Netherlands), and mean values were compared with the Mann-Whitney Wilcoxon test using SPSS 11.5 software. (SPSS, Chicago, IL).

Thrombosis-free survival analysis

Kaplan-Meier thrombosis-free survival curves for each genotype group were constructed and compared using the Statistics software (StatSoft, Tulsa, OK).

Results and discussion

FV mutation screening in the 6 FV Leiden heterozygotes with reduced FV levels yielded 5 different defects (Figure 1A), 3 of which are novel (Q1894X, G2049X) or described in an abstract form (G2112D).12 Three mutations predict premature termination of translation (patients P1, P3, P4, and the asymptomatic brother of P4), and 2 missense mutations (patients P2 and P5) were found to be associated with very low FV levels and severe bleeding in the homozygous condition.12,13 Although a little residual expression has been reported for some frameshift mutations in the F8 gene, and the missense changes could produce FV activity levels below the detection limit, this genetic analysis indicates virtual nonexpression of the non-Leiden FV allele and consequent absence of normal FV in plasma, which defines these patients as true pseudohomozygotes.

To evaluate the hypercoagulable state associated with pseudohomozygous APC resistance, we determined thrombin generation in the presence of APC in plasma from a selection of individuals with different FV genotypes. The 6 FV Leiden pseudohomozygotes were compared to their heterozygous (n = 12) and normal relatives (n = 11) and to unrelated homozygotes (n = 32). Thrombin generation (Figure 1B) was similar between pseudohomozygotes and homozygotes, and significantly higher than in heterozygotes. Since this assay shows a good correlation with the thrombosis risk associated with inherited and acquired thrombophilia,14,15 these findings predict that FV Leiden pseudohomozygotes will show a clinical behavior similar to that of homozygotes.

Figure 2A shows the thrombosis-free survival of the different genotype groups. The median thrombosis-free survival of pseudohomozygotes and homozygotes (27 and 31 years, respectively) was significantly lower than that of heterozygotes (43.5 years) and noncarriers of the FV Leiden mutation (49 years). To avoid overestimating the thrombosis risk in pseudohomozygotes due to selection bias (index-case effect), Kaplan-Meier analysis was also performed in probands only (Figure 2B). The median thrombosis-free survival for probands was 27 years for both pseudohomozygotes and homozygotes and 38 years for FV Leiden heterozygotes (P = .007). Deep vein thrombosis and/or pulmonary embolism were the first thrombotic manifestations in 55% of the heterozygous, 61% of the homozygous, and 40% of the pseudohomozygous probands. Superficial vein thrombosis appeared to be a frequent first thrombotic manifestation in pseudohomozygotes (60%). However, due to the small number of patients, these proportions may not be fully representative of their clinical presentation.

Figure 2.

Thrombosis-free survival curves of individuals with different FV genotypes. (A) Kaplan-Meier thrombosis-free survival analysis of the whole study cohort (probands and family members). (B) Kaplan-Meier thrombosis-free survival analysis of probands only. □, noncarriers; ○, FV Leiden heterozygotes; ▵, FV Leiden homozygotes; ♦, FV Leiden pseudohomozygotes. Differences between the curves were evaluated with the log-rank test. The apparently solid circles in the curve for FV Leiden heterozygotes are the effect of partial overlapping of open circles.

Taken together, these data indicate that FV Leiden pseudohomozygotes are exposed to the same risk for thrombosis as homozygotes. In fact, although relative risks could not be calculated because we had only one pseudohomozygous family member, the median thrombosis-free survival of FV Leiden pseudohomozygous probands was identical to that of homozygous probands and was significantly lower than that of heterozygous probands.

Interestingly, there were more homozygotes and pseudohomozygotes in the propositi than expected on the basis of the number of FV Leiden heterozygotes in this cohort, which is in accordance with the increased thrombotic risk conferred by these double or combined genetic defects.

Thus, failure to recognize FV deficiency in FV Leiden heterozygotes may result in an underestimate of the thrombosis risk and inadequate prophylaxis and/or treatment of VTE in affected patients. Our findings suggest that pseudohomozygotes might benefit from similar thromboprophylaxis given to homozygous FV Leiden carriers in risk situations for thrombosis, despite the 50% reduction of FV levels in plasma.

The apparent paradox that FV Leiden pseudohomozygotes, whose blood contains only FV Leiden, are exposed to a higher thrombotic risk than heterozygotes, whose blood in addition to the same amount of FV Leiden also contains normal FV, is explained by the observation that FV is not only a procoagulant, but also an anticoagulant protein.16 Evidence has accumulated that FV, but not FV Leiden, stimulates the inactivation of factor VIIIa by APC. In vitro experiments indicate that this anticoagulant activity of FV plays a crucial role in the down-regulation of coagulation by APC.11 Thus, the presence of normal FV protects against thrombosis and explains why heterozygous carriers of the FV Leiden mutation are exposed to a lower risk of venous thrombosis than pseudohomozygotes and homozygotes.

Pseudohomozygous APC resistance teaches us that we need to be careful in considering combinations of altered biologic functions: the combination of a thrombophilic and a slightly prohemorrhagic defect of the same coagulation protein results in increased risk of VTE. Combination of opposites is an ancient philosophic issue. In this case, you might say, “one minus one is 2!”

Footnotes

  • Reprints:
    Paolo Simioni, Department of Medical and Surgical Sciences, Second Chair of Internal Medicine, University of Padua Medical School, Via Ospedale 105; 35100 Padua, Italy; e-mail: paolo.simioni{at}unipd.it.
  • Prepublished online as Blood First Edition Paper, June 16, 2005; DOI 10.1182/blood-2005-04-1461.

  • Supported in part by Ministero della Universitá e della Ricerca Scientifica e Tecnologica (MURST) ex 60%, grant no. 60A07-9773/03 (P.S.) and by Progetto ARTGEA, Comitato dei Sostenitori (F.B.). The funding source had no involvement in the study design, data collection, data analysis, data interpretation, or writing of this report.

  • P.S. and D.T. enrolled the patients, collected clinical information, and performed the Kaplan-Meier analysis; B.L. and F.B. were responsible for the patients' genotyping; E.C. and J.R. performed the thrombin generation experiments; all authors critically contributed to the interpretation of the data and the preparation of the manuscript. All authors had full access to all data in the study and had final responsibility for the decision to submit the manuscript for publication.

  • The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 U.S.C. section 1734.

  • Submitted April 11, 2005.
  • Accepted June 2, 2005.

References

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