An underestimated combination of opposites resulting in enhanced thrombotic tendency

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.

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.

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

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 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).

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

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.

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!”