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Keywords:

  • venous thromboembolism;
  • factor V Leiden;
  • high factor VIII levels;
  • hypercoagulability;
  • coagulation factors

Abstract

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Factor V Leiden (FVL)-carrying relatives of selected patients with venous thromboembolism (VTE) have much higher venous thrombotic risks than FVL-carrying relatives of unselected consecutive patients with VTE. To find an explanation for this, we explored other risk factors of VTE, in particular the presence of high factor VIII levels, in a retrospective follow-up study. We assessed levels of factor VIII, factor IX, fibrinogen, protein C, protein S, antithrombin, the presence of prothrombin 20210A, and the occurrence of VTE in 61 first-degree relatives of 12 selected thrombophilic families harbouring FVL, and 183 first-degree relatives of 47 unselected families of FVL carriers with a first VTE. In all families, FVL appeared to be an independent risk factor for VTE. Higher thrombosis incidence rates were found in carriers of both FVL and high factor VIII levels (≥ 150 IU/dl), while high levels of factor VIII appeared to be an independent thrombotic risk factor only in selected thrombophilic families. The fraction of individuals with more than one prothrombotic coagulation disorder was 10% higher in selected families. These results and the higher thrombotic risks we found in the thrombophilic families favour the hypothesis that other unknown co-existing genetic defects contribute to thrombophilia.

Numerous acquired and inherited conditions have been identified as risk factors for venous thromboembolism, suggesting that venous thrombosis is a multifactorial disease. Therefore, combinations of (inherited) risk factors will more often be found in selected patients with thrombophilia and their relatives than in unselected patients with venous thrombosis and their relatives.

We recently demonstrated a much higher thrombotic risk for carriers of factor V Leiden in selected thrombophilic families than for carriers in families of unselected patients (Lensen et al, 2000). It is probable that this is caused by the presence of additional known and unknown, genetic and/or acquired risk factors for venous thrombosis in the selected families. Another explanation is purely stochastic: the selected families happened to be those that had the most thrombotic events. The difference is crucial between those two explanations. While an abundance of events would be apparent in retrospect for both situations, we would not predict an increased risk for families that had only coincidentally experienced a large number of thromboses. If, on the other hand, the high incidence of thrombosis in the selected families was caused by the presence of additional risk factors, we would also expect an increased risk in prospect.

Factor V Leiden is known to be the most common genetic risk factor for venous thromboembolism and leads to a sevenfold increased thrombotic risk (Bertina et al, 1994; Ridker et al, 1995; Rosendaal et al, 1995). In Caucasians, factor V Leiden is present in about 5% of healthy individuals, in 20% of unselected consecutive patients with deep vein thrombosis, and in 50% of probands from thrombophilic families (Bertina et al, 1994; Rosendaal et al, 1995).

Several studies have described the effect of combinations of factor V Leiden with other coagulation defects on the risk on venous thrombosis. All these studies demonstrated a higher thrombotic risk for the combination of defects than for the single defect (Koeleman et al, 1994, 1995; Zöller et al, 1995; Beauchamp et al, 1996; van Boven et al, 1996; Martinelli et al, 1998; Mustafa et al, 1998; Tosetto et al, 1998; Salomon et al, 1999). We therefore hypothesized that the higher thrombotic tendency in thrombophilic families is probably explained by the combined presence of two or more (genetic) risk factors. Obviously, more common risk factors such as elevated factor VIII levels, are the most probable candidates. Penick et al (1966) described for the first time an association between thrombophilia and elevated factor VIII levels. Factor VIII levels exceeding 150 IU/dl are present in about 11% of healthy individuals and increase the venous thrombotic risk nearly fivefold compared with levels lower than 100 IU/dl (Koster et al, 1995a; O'Donnell et al, 1997). Ten per cent of asymptomatic individuals have factor VIII levels higher than 175 IU/dl (90th percentile of controls) versus 19% in symptomatic individuals with a single thrombotic event and 33% in symptomatic individuals with recurrences (Kraaijenhagen et al, 2000). Because of this high prevalence and high thrombotic risk, we focused on the influence of high factor VIII levels on the occurrence of venous thrombosis in the families of both selected and unselected patients with factor V Leiden. We hypothesized that if the thrombotic risk differed between the two groups owing to an additional risk factor such as high factor VIII levels, we would observe, first, an excess of individuals with high factor VIII levels in (selected) thrombophilic families compared with unselected families; second, when the two risk factors, i.e. factor V Leiden and high factor VIII levels, are taken into account, the difference in risk between selected and unselected families should largely disappear.

Patients and methods

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Patients and families We included 61 first-degree relatives (siblings, parents and children of the proband) of 12 selected thrombophilic families with factor V Leiden (i.e. referred to our centre for diagnostic work-up for venous thrombophilia because there were at least two symptomatic relatives in addition to the proband). There were 32 men and 29 women, the median age was 47 years (range, 23–70 years). We contrasted these first-degree relatives with 183 first-degree relatives of 47 unselected factor V Leiden carriers from a consecutive series with a first venous thrombosis. In this group were 80 men and 103 women, and the median age was 49 years (range, 16–70 years). Approximately half the relatives in both groups were carriers of factor V Leiden. One citrated blood sample was obtained from all individuals. A standardized history was taken on the occurrence of venous thromboembolism before screening for the factor V Leiden mutation took place. Venous thrombotic events (excluding superficial thrombophlebitis) were those that were diagnosed by physicians (70% were objectively diagnosed: ultrasound, venography, pulmonary angiography or lung ventilation perfusion scan; 30% were diagnosed on clinical observation only). Blood was collected in a 1/10 volume of 0·106 mol/l sodium citrate and centrifuged for 10 min at 2000 g. Plasma was stored at −70°C. All laboratory assays were carried out by technicians unaware of the patient medical history. As expected, some (four) of the 47 unselected families also met the criteria set for the selection of the thrombophilic families. In the analysis, these families (21 relatives) were regarded as part of the panel of unselected families unless stated otherwise.

Detection of coagulation defects The mutation of the factor V gene was detected as described previously (Bertina et al, 1994). Factor VIII:Ag was measured in triplicate using a sandwich-type enzyme-linked immunosorbent assay (ELISA) as previously described, using two monoclonal antibodies directed against different epitopes on the light chain of factor VIII (CLB CAgA and CLB CAg117). These monoclonal antibodies were kindly provided by Dr J. van Mourik (Department of Blood Coagulation, CLB, Sanguin Blood Supply Foundation, Amsterdam, The Netherlands). The intra- and interassay coefficients of variation were 4·3 and 5·6 respectively. Pooled normal plasma, calibrated against the World Health Organization (WHO) standard (91/666) for von Willebrand factor (VWF):Ag, factor VIII:C and factor VIII:Ag, was used as a reference. We used a factor VIII level of 150 IU/dl as the cut-off point; the same cut-off level had been used previously in the analysis of the Leiden Thrombophilia Study (Koster et al, 1995a). Factor IX antigen levels were also determined using ELISA (van Hylckama Vlieg et al, 2000). Fibrinogen concentration was determined according to the method of Clauss using the DadeR thrombin reagent (Baxter, Miami, USA). The prothrombin genes were analysed for the 20210A mutation using polymerase chain reaction and HindIII digestion as described before (Poort et al, 1996). All individuals were screened for deficiencies of protein C, protein S and antithrombin; no deficiencies were found.

Statistics Incidence rates in different groups were calculated by dividing the number of first events by the total of observation-years (follow-up time). Follow-up started at birth and ended at the date of the interview or the date of the first thrombotic event, whichever came first. We constructed thrombosis-free survival curves using the Kaplan–Meier method. We used Cox regression analysis to study the role of the genetic risk factors: heterozygosity for factor V Leiden and the presence of high levels of factor VIII. In a separate Cox regression analysis including all relatives of both groups of families, we also entered the ‘group of family’ (selected thrombophilic families versus families of unselected consecutive patients) to which the relative belonged as a risk factor in the model (excluding propositi). We investigated interaction under an additive model between factor V Leiden and the presence of high levels of factor VIII in the two groups by comparing the incidence rate for carriers of both disorders with the sum of the incidence rates found in carriers of only factor V Leiden or elevated factor VIII levels. We also investigated interaction under a multiplicative model between the two disorders by comparing the relative risk for carriers of both defects with the product of the relative risks found in carriers of one of the two disorders. As main determinants of risk, we investigated factor V Leiden and factor VIII levels. Fibrinogen levels, factor IX levels and the 20210 G to A prothrombin variant were entered as covariates in some of our models.

Results

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Selected thrombophilic families with factor V Leiden

We investigated 61 first-degree family members of the 12 probands from thrombophilic families. The overall incidence rate of venous thrombosis was 0·7%/year (18 events in 2544 person-years). Of the 61 relatives, 32 carried factor V Leiden and 15 (47%) of these carriers had experienced a thrombotic event (incidence rate: 1·2%/year; Table I). Of the 29 non-carriers, three (10%) had experienced a venous thrombosis (incidence rate: 0·2%/year). Therefore, the rate ratio of factor V Leiden for venous thrombosis was 5·1 (95% CI: 1·5–16·4).

Table I.   Data on thrombotic tendencies in relatives of symptomatic propositi, as represented by incidence rates, according to family type and prothrombotic coagulation disorder.*
 Selected familiesUnselected families
 Number (%)Incidence rateNumber (%)Incidence rate
  • *

    Incidence rates are in percentage/year. There was no plasma available in four relatives of selected families and in 13 relative of unselected families.

Factor V Leiden +32 (52%)1·20101 (55%)0·20
Factor V Leiden −29 (48%)0·2082 (45%)0·11
Factor VII≥ 150 IU/dl20 (33%)1·1977 (42%)0·18
Factor VIII < 150 IU/dl41 (67%)0·39106 (58%)0·14

Unselected families with factor V Leiden

We investigated 183 first-degree relatives of 47 unselected consecutive patients factor V Leiden and a first venous thrombosis. The overall incidence rate of venous thrombosis was 0·16%/year (13 events in 8293 person-years), i.e. four times lower than in the thrombophilic families. Of these 183 relatives, 101 carried factor V Leiden and nine (9%) had experienced a thrombotic event (incidence rate: 0·20%/year). Of the 82 non-carriers, four (5%) had experienced a venous thrombosis (incidence rate: 0·11%/year), also lower than in the thrombophilic families. Here, the rate ratio of factor V Leiden for venous thrombosis was 1·8 (95% CI: 0·6–6·0).

High factor VIII levels

In the selected thrombophilic families with factor V Leiden, high factor VIII levels were found in 33% of the relatives. Of these relatives with high factor VIII levels, 59% had experienced a thrombotic event. As Table I shows, high factor VIII levels were associated with a threefold increased risk of thrombosis in these families (in those with factor VIII levels ≥ 150 IU/dl: 1·19%/year versus 0·39%/year in those with factor VIII levels < 150 IU/dl). In families of the unselected patients with factor V Leiden, high factor VIII levels were found in 42% of the relatives. Of these relatives with high factor VIII levels, 11% had experienced a thrombotic event. In these families, the thrombotic risk in individuals with high factor VIII levels was somewhat higher (0·18%/year) than in individuals with factor VIII levels < 150 IU/dl (0·14%/year). So, we found a high prevalence of high factor VIII levels in thrombophilic families and unselected families, but no significant difference in the prevalence of high factor VIII levels between the two groups [after exclusion of the four unselected families who met the criteria set for the selection of thrombophilic families (unselected thrombophilic families); but the thrombotic incidence in individuals with high factor VIII levels was 0·13%/year versus 0·05%/year in individuals with factor VIII levels < 150 IU/dl].

Factor V Leiden combined with the presence of high factor VIII levels

We explored the effect on the thrombotic incidence of high factor VIII levels in relation to factor V Leiden. Table II shows the incidence rates for factor V Leiden and the presence of high factor VIII levels. In both thrombophilic families and unselected families, relatives with the combination of factor V Leiden and high factor VIII levels had an increased incidence rate of venous thrombosis compared with family members with only high factor VIII levels or only factor V Leiden. The thrombotic incidence, whenever one or two of these factors were present, was invariably higher in thrombophilic families than in unselected families. Lowest incidence rates were found in relatives without factor V Leiden but with high factor VIII levels in the unselected families.

Table II.   Incidence rates of venous thrombosis in relatives of symptomatic propositi in the absence or presence of factor V Leiden without and with the high factor VIII levels according to family type.
 Selected familiesUnselected families
Coagulation defectIncidence rateRate ratio (95% CI)Incidence rateRate ratio (95% CI)
  • *

    Reference category.

  • FVL, factor V Leiden; FVIII≥ , high factor VIII levels. Incidence rates are in percentage per years.

FVL– FVIII < 150 IU/dl0·131*0·121#
FVL– FVIII ≥ 150 IU/dl0·382·9 (0·3–31·8)0·090·8 (0·1–5·7)
FVL+ FVIII < 150 IU/dl0·634·8 (0·6–41·3)0·151·3 (0·2–7·1)
FVL+ FVIII ≥ 150 IU/dl2·1116·2 (2·1–127·2)0·272·3 (0·4–11·8)

When we excluded the four unselected thrombophilic families, the thrombotic incidence in unselected families in individuals without factor V Leiden or high factor VIII levels decreased from 0·12%/year (Table II) to 0·06%/year, more than 50% lower than the incidence found in thrombophilic families (0·13%/year).

To determine the contribution to risk of these risk factors (i.e. factor V Leiden and high factor VIII levels) in both settings, we calculated rate ratios for relatives (no propositi) of selected thrombophilic families by application of a Cox regression model (Table IIIA). We included the 20210 G to A prothrombin variant, the presence of fibrinogen levels higher than 4 g/l and the presence of factor IX levels higher than 129 U/dl as additional risk factors for more precise estimations. In Table IIIB, the rate ratios are presented for the unselected families (there were too few individuals to estimate the rate ratio for the 20210 G to A prothrombin variant). For the data shown in Table IIIC, i.e. to study the effect of being a member of a thrombophilic family, we added the four unselected thrombophilic families to the panel of selected thrombophilic families. The strongest risk factor was kinship to thrombophilic families (rate ratio 9:1) followed by the 20210 G to A prothrombin variant (rate ratio 3:9; Table IIIC) and factor V Leiden (rate ratio 3:4).

Table IV shows a summary of the effect of the presence, respectively, of none and one or more coagulation disorders in selected and unselected families. An increase of incidence rate of venous thrombosis with a higher number of coagulation disorders is found in relatives of both selected thrombophilic families and unselected families.

Table IV.   Incidence rates of venous thrombosis in relatives of symptomatic propositi in the absence or presence of one or more prothrombotic coagulation defects according to family type.
 Selected familiesUnselected families
Number of defectNumber of relatives (%)Number of patientsIncidence rateNumber of relatives (%)Number of patientsIncidence rate
  1. Incidence rates are in percentage/year. Relatives from whom one or more defects could not have been detected were excluded (four relatives from the selected families and 13 relatives from unselected families).

012 (20)0025 (14)10·10
119 (31)20·2686 (47)50·13
220 (33)91·0547 (26)40·17
39 (15)61·2419 (10)20·22
41 (1)13·456 (3)10·30
Total61180·71183130·16

Figure 1A and B show the thrombosis-free survival for affected and non-affected relatives with factor V Leiden and/or high factor VIII levels in selected thrombophilic families and unselected families respectively. In selected thrombophilic families, the probability of still being free of venous thrombosis in carriers of both defects was 75% at age 27 years, while in families of unselected propositi this was the case at the age of 55 years.

image

Figure 1.  (A) Thrombosis-free survival curves in non-affected relatives (0), in relatives with only high factor VIII levels (1), in relatives with only factor V Leiden (2) and in relatives with both defects(3), of selected thrombophilia families with factor V Leiden. (B) Thrombosis-free survival curves in non-affected relatives (0), in relatives with only high factor VIII levels (1), in relatives with only factor V Leiden (2) and in relatives with both defects (3), of unselected consecutive patients with factor V Leiden.

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Discussion

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

In order to explain the different venous thrombotic risks in first-degree relatives of probands of selected thrombophilic families and first-degree relatives of unselected patients with thrombosis, we screened for genetic and other common risk factors for thrombosis and analysed their contribution to the thrombotic risk in families of 12 highly selected thrombophilic patients with factor V Leiden and in families of 47 unselected consecutive patients with factor V Leiden. In both groups, factor V Leiden carriership was an independent risk factor, while high factor VIII levels appeared to be an independent risk factor only in selected thrombophilic families (and in unselected families after exclusion of the four unselected thrombophilic families). Highest incidences were found in carriers of both defects in both selected and unselected families. However, individuals with factor V Leiden or high factor VIII levels in thrombophilic families had a fourfold higher thrombotic incidence rate than individuals with the same abnormalities in families of unselected probands. Although this difference between the two types of families remained in multivariate analysis, this difference in thrombotic risk largely disappeared when individuals with neither abnormality were compared. Despite the high incidence rates in the selected families and the wide confidence intervals, interaction between factor V Leiden and high factor VIII levels appeared to be present in selected families (interaction under an additive and multiplicative model). The incidence rate in carriers of both defects in selected families was eight times (95% CI 2·7–22·7) the incidence rate in carriers of both defects in unselected families. The presence of high factor VIII levels appeared to increase the thrombotic incidence three times (95% CI 1·1–9·8) in factor V Leiden carriers of selected families and nearly twofold (95% CI 0·5–6·7) in factor V Leiden carriers of unselected families compared with carriers of factor V Leiden only.

In the most fully adjusted model, ‘family type’ remains the strongest predictor, while in univariate analysis, risks are higher in selected families whenever either factor V Leiden or high factor VIII levels are present (Table II). The only logical explanation seems to be the presence of at least one more genetic risk factor, which has only a mild effect in itself, but that has a synergistic effect with other risk factors such as factor V Leiden and high levels of factor VIII. As deficiencies of protein C, protein S and antithrombin were not present in these families, and as we took into account fibrinogen levels, factor IX levels and the 20210 G to A prothrombin variant, other unknown genetic risk factors are probably responsible for the difference of thrombotic risk in the two differently selected family panels.

One of our initial hypotheses was that the thrombotic tendency in thrombophilic families is higher than in unselected families because of clustering of prothrombotic coagulation defects in thrombophilic families. This hypothesis was partly confirmed: we did not find a higher frequency of prothrombotic coagulation abnormalities in relatives of selected patients with factor V Leiden (80%) than in unselected families with factor V Leiden (86%). On the other hand, we found that combinations of prothrombotic coagulation disorders (49%) were slightly more often found in thrombophilic families than in unselected families (39%). Also, the fraction of families harbouring the 20210 G to A prothrombin variant was higher in thrombophilic families (2/12, 17%) than in unselected families (2/47, 4%). The idea that co-existing coagulation defects cause a higher thrombotic tendency was well illustrated by the increasing incidence rate in individuals with a combination of more coagulation disorders.

It is still unclear whether high factor VIII levels are caused by genetic or environmental mechanisms, apart from the genetic blood group effect. Results from other studies suggest that factor VIII levels are at least in part genetically determined (Kamphuisen et al, 1998). Both in thrombophilic families and unselected families, higher percentages of individuals with high factor VIII levels were found than in the general population.

We compared factor VIII levels ≥ 150 IU/dl with factor VIII levels < 150 IU/dl, while in our previous case–control (Leiden Thrombophilia Study, LETS) study we compared factor VIII levels ≥ 150 IU/dl with factor VIII levels < 100 IU/dl (Koster et al, 1995a). This is responsible for the lower rate ratios in this study (see Table III). When comparing levels ≥ 150 IU/dl with levels < 150 IU/dl in the same LETS data, a lower relative risk of 2·65 (95% CI 1·7–4·1) is also found. The synergistic effect between factor V Leiden and high factor VIII levels previously observed in a case–control study (LETS) was confirmed by the results of the present family studies (Koster et al, 1995b).

Table III.   Adjusted rate ratios (95% CI) for the main potential risk factors on venous thrombotic events.
(A) For relatives (no propositi) of thrombophilic families.*
Potential risk factorRate ratio (95% CI)
Factor V Leiden6·2 (95% CI 1·6–24·5)
High factor VIII levels3·9 (95% CI 1·2–12·9)
20210 G to A prothrombin variant4·8 (95% CI 0·8–29·6)
High fibrinogen levels1·4 (95% CI 0·2–8·0)
High factor IX levels1·0 (95% CI 0·3–3·3)
(B) For relatives (no propositi) of unselected families.
Potential risk factorRate ratio (95% CI)
Factor V Leiden2·2 (95% CI 0·6–7·2)
High factor IX levels2·1 (95% CI 0·7–6·6)
High factor VIII levels1·2 (95% CI 0·4–3·9)
High fibrinogen levels0·3 (95% CI 0·1–2·7)
(C) For all (246) relatives (no propositi) from both family panels.
Potential risk factorRate ratio (95% CI)
  • *

    The rate ratios are the risks of thrombosis in the presence of the risk factor compared with its absence, adjusted for the other factors in the model. All variables are coded as yes/no. For (C), we included the four families from the unselected patients fulfilling the criteria for thrombophilic families among the thrombophilia families.

Relationship to thrombophilia families9·1 (95% CI 3·6–23·2)
Factor V Leiden3·4 (95% CI 1·4–8·2)
High factor VIII levels2·8 (95% CI 1·3–6·4)
20210 G to A prothrombin variant3·9 (95% CI 0·8–18·9)
High factor IX levels1·2 (95% CI 0·5–2·6)
High fibrinogen levels0·9 (95% CI 0·2–3·4)

A limitation of the present study is the fact that one third of the thrombotic events were not objectively diagnosed. This may cause a small overestimation of the rate ratios: after exclusion of the non-objectified thrombotic events, we found slightly lower rate ratios (but with wider confidence intervals).

In conclusion, elevated factor VIII levels are a moderate determinant of thrombotic risk, while the combined presence of factor VIII levels ≥ 150 IU/dl with factor V Leiden appeared to be a strong determinant of thrombotic risk, particularly in selected families with thrombophilia. The higher risks found in thrombophilic families suggest the presence of other unknown genetic risk factors in these families.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

We would like to thank Hans de Ronde and Irma van der Linden for expert technical assistance.

This study was supported by a grant of the Netherlands Heart Association (grant no. 95.26).

References

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
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