British Journal of Haematology, 2001, 115, 687±691

Marked elevation of thrombin generation in patients with

elevated FVIII:C and venous thromboembolism

James O'Donnell,1 Andrew D. Mumford,2 Richard A. Manning1 and Michael A. Laffan1 1Department of

Haematology, Imperial College School of Medicine, Hammersmith Hospital, and 2MRC Haemostasis Research Group,

London, UK

Received 16 February 2001, accepted 4 July 2001

Summary. Elevated plasma factor VIII coagulant activity(FVIII:C, . 150 IU/dl) is a risk factor for venous throm-boembolism (VTE). We hypothesized that increased FVIII:Cmay exert a prothrombotic effect by increasing basalthrombin generation. To test this hypothesis we havemeasured prothrombin fragment 1 1 2 (F1 1 2) andthrombin±antithrombin complex (TAT) in three groups: (i)patients with objectively confirmed VTE and elevatedFVIII:C; (ii) patients with VTE and no detectable thrombo-philia; and (iii) healthy age- and sex-matched controlsubjets. In the group of patients with elevated FVIII:C,TAT and F1 1 2 levels were increased in 85% and 78% ofindividuals respectively. This frequency of coagulationactivation is dramatically higher than that reported forother recognized constitutional thrombophilias. In the

group of patients with VTE but no proven thrombophilia,increased thrombin generation was present in 30% ofindividuals. Basal thrombin generation was significantlyhigher in patients with elevated FVIII:C compared withindividuals with VTE but no documented thrombophilia(median TAT � 8´65 mg/l versus 2´95 mg/l, medianF1 1 2 � 1´5 nmol/l versus 0´87 nmol/l; P , 0´0001,P , 0´001). Overall FVIII:C levels were strongly correlatedwith levels of thrombin generation (r� 0´5, P , 0001).The clinical significance of such markedly increased F1 1 2and TAT levels in patients with high FVIII:C levels remainsunclear.

Keywords: elevated FVIII:C, thrombin generation, venousthrombosis.

An association between high levels of plasma factor VIIIcoagulant activity (FVIII:C) and arterial disease wassuggested many years ago and its role in coronarythrombosis was demonstrated in the Northwick ParkHeart Study (Meade et al, 1994). More recently, it hasemerged as a powerful and highly prevalent risk factor forvenous thrombosis (Koster et al, 1995; Kraaijenhagen et al,2000). Three independent groups have each reported thatelevated plasma FVIII is found in < 25% of patients withvenous thrombosis (Koster et al, 1995; O'Donnell et al,1997; Kraaijenhagen et al, 2000). Using multivariateanalysis of the Leiden Thrombophilia study, an adjustedodds ratio of 4´8 was determined for first deep veinthrombosis (DVT) in those individuals with FVIII:C levels. 150 IU/dl compared with those with FVIII:C , 100 IU/dl(Koster et al, 1995). In this study and in the prospectivestudy of post-operative DVT (Lowe et al, 1999a) the risk wasfound to be associated with FVIII:C but not von Willebrandfactor. The increased FVIII:C levels observed in these

patients is persistent and independent of any post-thrombo-tic acute phase reaction (O'Donnell et al, 2000). Early datasuggest that unlike many other risk factors for first DVT,high levels of FVIII (. 234 IU/dl) also identify patients athigh risk of recurrence (Kyrle et al, 2000). Interestingly,although the Leiden study reported a dose±responserelationship between FVIII and risk of thrombosis, Kyrleet al (2000) observed a threshold FVIII:C level below whichrisk of recurrence was not increased. Neither study was ableto determine whether the risk of thrombosis continues torise with ever higher levels of FVIII, or whether the effectreaches a plateau.

The cause of high FVIII:C levels in these patients remainsunexplained. In particular, it is unclear whether elevatedFVIII:C is a constitutional risk factor, an acquired risk factoror merely a reactive consequence of venous thrombosis.Early family studies reported familial clustering of FVIII:Cplasma concentrations and suggested a contribution froman X-linked locus (Kamphuisen et al, 1998). However, asearch for regulatory polymorphisms in the FVIII genepromoter was unsuccessful (Mansvelt et al, 1998).

The mechanism by which elevated FVIII might increasethe risk of thrombosis also remains unclear. Although it

q 2001 Blackwell Science Ltd 687

Correspondence: Dr James O'Donnell, Department of Haematology,Hammersmith Hospital/ICSM, DuCane Road, East Acton, London

W12 ONN, UK. E-mail: james.o'donnell@virgin.net

may seem obvious that elevation of a coagulation factorshould increase the likelihood of thrombosis, this relation-ship appears to be true for only some (e.g. FVIII, FXI andprothrombin; Koster et al, 1995; Poort et al, 1996; Meijerset al, 2000) and not all factors (FV; Kamphuisen et al,2000). However, mathematical modelling of the coagulationsystem does suggest that plasma FVIII levels can signifi-cantly influence the rate of thrombin generation (Jones &Mann 1994). The effect of varying FVIII:C levels onthrombin generation has also been investigated using anin-vitro coagulation system. Butenas et al (1999) demon-strated that the duration of the initiation phase ofcoagulation was reduced, and maximum thrombin genera-tion increased, when FVIII:C levels were at the upper limit ofthe normal range. However, the effects of FVIII:C levels. 150% on thrombin generation were not reported.

We hypothesized that FVIII elevation may exert aprothrombotic effect by increasing the basal rate ofthrombin generation as has been observed in a proportionof patients with other prothrombotic traits. We havetherefore measured two markers of thrombin formation[plasma thrombin±antithrombin (TAT) complexes andprothrombin fragment 1 1 2 (F1 1 2) levels respectively]in patients with high FVIII:C levels who have had anobjectively confirmed VTE. We expect that if the relationshipbetween FVIII:C and thrombin generation is causal, the rateof thrombin generation should correlate with the FVIII:Clevel. Weak associations have previously been found innormal populations (Lowe et al, 1997). To control for thepossibility that elevated thrombin generation is a feature ofthrombotic patients in general, we measured the sameparameters in a group of patients with venous thrombosisbut who had no detectable thrombophilia trait and hadnormal FVIII:C. A group of age- and sex-matched normalcontrol subjects were recruited to establish normal ranges.

PATIENTS AND METHODS

Patient populationPatients were identified from 249 individuals referred to theHammersmith Hospital for investigation of objectivelyconfirmed venous thrombosis. Seventy-two patients hadsuffered recurrent episodes of deep vein thrombosis (DVT)and/or pulmonary embolism (PE). For each patient, plasmasamples were obtained a minimum of 3 months after theiracute thrombosis and at least 2 weeks after discontinuationof oral anticoagulant therapy. Investigations performedcomprised full blood count, biochemistry screen, C-reactiveprotein, ABO blood group, coagulation screening tests(prothrombin time, activated partial thromboplastin time,thrombin time, fibrinogen), antithrombin, proteins C and S,activated protein C resistance, factor V Leiden genotype,FVIII:C and lupus anticoagulant by dilute Russell's Vipervenom time). Individuals with any thrombophilic trait otherthan elevation of FVIII:C were excluded as these might alsoincrease thrombin generation.

Using the above investigations, we identified a group of 54patients (Group A) in whom the only abnormal finding wasan elevated FVIII:C level (> 150 IU/dl). All these patients

were , 70 years of age with no evidence of underlyingmalignancy, and none of the female patients were pregnantor using oral contraceptives at the time of study. A secondgroup (Group B) consisting of 37 patients was identified inwhom we could not determine any inherited or acquiredthrombophilic trait. A third group (Group C) of 37 healthyvolunteers was used as the normal control group. Theseindividuals were recruited from routine blood donors at theWessex Regional Transfusion Centre, Southampton (UK).The donors selected were age- and sex-matched for thoseindividuals comprising group B. Each donor providedwritten informed consent.

Laboratory analysisBlood collection. Blood was collected from the antecubital

vein into Becton-Dickinson (Oxford, UK) Vacutainerw tubescontaining 0´105 ML trisodium citrate. Citrated plasma wasobtained by centrifugation at 2000 g for 20 min and thenaliquotted and stored at 2708C before testing. Samples frompatients with thrombosis were obtained at a routine clinicvisit, and from controls at a normal donor session.

Screening for deficiency of antithrombin, protein C (func-tional) and protein S (free and total), activated protein Cresistance and factor V Leiden were carried out using standardmethods as previously described (Mansvelt et al, 1998).

FVIII:C and factor VIII antigen (FVIII:Ag) assays. FVIII:Cwas measured by the one-stage clotting method, using FVIII-deficient substrate (Immuno, Vienna, Austria): Kaolin/plate-let substitute mixture was from Diagnostic Reagents Ltd.,Thames, England. Appropriate dilutions were performed toensure the test clotting times fell within those of the referencecurve. The inter assay coefficient of variation (CV) was 6% forpooled normal plasma. In group A patients (FVIII:C . 150IU/dl), plasma FVIII:Ag levels were also assayed using acommercial enzyme-linked immunosorbent assay (ELISA) kit(Immunozym, Technoclone, Dorking, UK) according to themanufacturer's recommendations. All samples were mea-sured against a reference plasma calibrated in IU/ml.

F1 1 2 and TAT assays. Plasma F1 1 2 and TAT werealso measured using commercially available ELISA kits(Enzygnost F1 1 2 and Enzygnost TAT, Behring, Marburg,Germany). F1 1 2 and TAT values were normally distrib-uted within the normal control population. The 95thpercentile of the control group was taken as the upperlimit of the normal range. (F1 1 2 , 1´07 nmol/l;TAT , 3´8 mg/ml).

Statistical analysisBecause the TAT and F1 1 2 levels were not normallydistributed in groups A and B, and failed to normalize bytransformation, all results have been expressed as medianand range unless stated otherwise, and non-parametric testsfor significance performed. Statistical significance wasassigned at P , 0´05.

RESULTS

Group A: VTE and elevated FVIII:C . 150 IU/dlWe investigated thrombin generation in 54 patients (36

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women and 18 men) with objectively confirmed VTE andFVIII:C levels . 150 IU/dl. The median age of theseindividuals was 45 years (range 24±69 years). ElevatedFVIII:Ag levels were present in 23/36 patients (64%).

TAT and F1 1 2 levels were increased relative to the

normal range in 46/54 (85%) and 42/54 (75%) ofindividuals respectively. The median TAT and F1 1 2 levelswere 8´65 mg/l and 1´5 nmol/l respectively.

Group B: VTE and no proven thrombophilia (FVIII:C , 150IU/dl)Thirty-seven patients (23 women and 14 men) wereidentified with confirmed VTE but no indication ofthrombophilia. The median age of these patients was38 years (range 17±57 years). TAT and F1 1 2 levelswere increased in 10/37 (27%) and 11/37 (30%) ofindividuals respectively (median TAT and F1 1 2 levelswere 2´95 mg/l and 0´87 nmol/l respectively).

FVIII:C and thrombin generationAs expected, there was a highly significant correlationbetween TAT and F1 1 2 (P , 0´0001; r � 0´76; Spear-man's rank correlation).

TAT and F1 1 2 levels were both significantly higher inpatients with VTE and elevated FVIII:C compared withindividuals with VTE but no documented thrombophilia(P , 0´0001 and P , 0´001 respectively; Mann±Whitney)(Fig 1A and B). Also, TAT and F1 1 2 levels weresignificantly higher in patients with VTE but no identifiedthrombophilia compared with healthy age- and sex-matched controls (P � 0´002 and P � 0´015 respectively;Mann±Whitney) (Fig 1A and B).

When data from groups A and B were pooled, there was asignificant correlation between FVIII:C and thrombingeneration (P , 0´001; r� 0´52; and P , 0´001;r� 0´51 for TAT and F1 1 2, respectively, Spearman'srank correlation) (Fig 2). However, when group A (highFVIII) was analysed alone, this relationship was no longerapparent (P � 0´4; r� 0´12; and P � 0´13; r� 0´21 forTAT and F1 1 2, respectively, Spearman's rank correlation).

DISCUSSION

Elevated levels of F1 1 2 and TAT have been sought in

Fig 1. (A) Plasma prothrombin fragment 1 1 2 and (B) thrombin±

antithrombin complex levels. Plasma F1 1 2 and TAT levels inpatients with VTE and high FVIII:C levels (X); patients with VTE

and no identified thrombophilia (O); and age-and sex matched

controls (K). The dashed lines represent the upper limit of the

normal ranges at 1´1 nmol/l (F1 1 2) and 3´8 mg/ml (TAT). Thedifferences are significant as shown.

Fig 2. F1 1 2 against FVIII:C levels inpatients with venous thrombosis. Group A:

patients with VTE and high FVIII:C levels

(X); Group B: patients with VTED and no

identified thrombophilia (O). Takentogether there was a significant relationship

between F1 1 2 and FVIII (P , 0´0001;

r� 0´51). However, this correlation was lost

when the two groups were analysed inde-pendently. (P � 0´13; r� 0´21 and

P � 0´37; r� 0´15: Spearman's rank cor-

relation). The dashed lines represent theupper limits of the normal range (FVIII:-

C , 1´5IU/ml and F1 1 2 , 1´1 nmol/l).

Essentially the same results were obtained

with TAT (data not shown) (P � 0´4;r� 0´12 and P � 0´7; r� 0´05: Spear-

man's rank correlation).

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Thrombin Generation in Patients with Elevated FVIII: C 689

many prothrombotic states to detect evidence of increasedthrombin generation. These studies have had limitedsuccess. Approximately one third of individuals withheterozygous protein C or protein S deficiency haveF1 1 2 levels greater than the upper limit of the normalrange (Bauer et al, 1988; Mannucci et al, 1992). Similarly,in patients with the factor V Leiden mutation Martinelli et al(1996) demonstrated high levels of F1 1 2 and TATcomplexes in 32% and 23% respectively. On the otherhand, several studies have shown only a small increase inF1 1 2 levels in antithrombin-deficient patients (Bauer et al,1991; Simioni et al, 1996) and a population-based studyfailed to find an association between F1 1 2 or TAT andfactor V Leiden (Lowe et al, 1999b). Moreover, Kyrle et al(1997a) have shown clearly that elevated levels or F1 1 2and TAT are not predictive of future thrombosis. Thus, therelationship between increased thrombin generation andthe prothrombotic state is unclear.

In this study, we have shown that increased thrombingeneration is present in < 80% of patients with confirmedvenous thrombosis and increased plasma FVIII:C levels. Thisis a much higher prevalence than has been found in theother prothrombotic states. Furthermore, the increase inthrombin generation is also of much greater degree thanpreviously reported. The clinical significance of thisobservation remains unclear, but it may be useful inunderstanding the nature of the prothrombotic state andthe aetiology of elevated FVIII. The mean age of the highFVIII group (A) was higher than the other two groups, andFVIII increases slowly with age but the effect is small(0´05iu/ml per decade) compared with the elevation seen inthese patients. (Kamphuisen et al, 1998).

If elevated FVIII:C is responsible for the increased thrombingeneration observed in these individuals, we would expect tofind a correlation between these two variables. Indeed, ahighly significant correlation was apparent when the fullrange of FVIII levels was analysed, supporting this hypoth-esis. This relationship between FVIII and thrombin genera-tion was lost when groups A and B were analysed separately.The lack of correlation may be attributed to smaller numbersand restricted range of FVIII with consequent loss of power inthe subgroup analysis but other explanations are possible.One possible explanation is that at high levels of FVIII,beyond a certain threshold the plasma concentration of FVIIIis no longer a limiting factor in thrombin generation. Asother factors then determine the rate of thrombin generation,the correlation with FVIII levels is lost.

An alternative explanation is that increased thrombingeneration does not result directly from the increased levelsof FVIII:C. Instead, FVIII and thrombin generation may beindependent results of some other unidentified abnormalitythat affects them differentially. A possible link betweenFVIII:C, thrombin generation and thrombosis would be theacute phase reaction. Although we have reported previouslythat elevation of FVIII:C is not associated with elevated Creactive protein (CRP) (O'Donnell et al, 1997), we alsoreviewed CRP measurements in these patient groups. Therewas no relationship between CRP and TAT or F1 1 2 ineither of the thrombotic groups A or B (data not shown).

It might be argued that because the one-stage assay forFVIII:C is susceptible to preactivation, then the elevatedFVIII:C and TAT/F1 1 2 may be measurements of the samephenomenon. We therefore also measured FVIII:Ag in 36individuals with raised FVIII:C levels. Elevation of FVIII:Aglevels was present in 23out of 36 patients. Repeating theanalysis using FVIII:Ag did not significantly alter the results.

Nonetheless it is unlikely that the elevation of FVIII:C issimply an epiphenomenon. The dosage effect seen in theLeiden thrombophilia study, and the effect of ABO bloodgroup suggest that FVIII:C has a direct causal effect on therisk of thrombosis (Koster et al, 1995). This need notnecessarily be via increased thrombin generation. Althoughantithrombin deficiency confers a much higher relative riskfor venous thrombosis than elevated FVIII:C (. 150 IU/dl),it is associated with significantly less basal thrombingeneration (Simioni et al, 1996). Furthermore heterozygousfactor V Leiden, which confers a relative risk for venousthrombosis similar to that of elevated FVIII:C, is associatedwith intermediate levels of thrombin generation (Martinelliet al, 1996; Zoller et al, 1996). These results suggest that thedifferent thrombophilias may be exerting their thromboticeffects via different mechanisms. The models provided byButenas et al (1999) suggest that effects on the initiationand propagation phases of coagulation may also beimportant mechanisms, on which FVIII is known to exertan effect.

In patients with previous venous thrombosis in whom nothrombophilic trait was identified, thrombin generation wasalso increased in approximately 30%. Although thefrequency and intensity of thrombin generation weresignificantly less than that observed among patients withelevated FVIII:C levels (P , 0´001), they were significantlyhigher than age- and sex-matched controls (P , 0´01).These results are similar to those reported by Kyrle et al(1997a) who found that 20% of patients without a definedclotting defect had at least one F1 1 2 level above the upperlimit of the normal range during a 12-month period offollow-up. The mechanism underlying coagulation activa-tion in these patients remains unclear. Clearly a proportionof these individuals may have some undetected underlyingthrombophilic tendency. Hyperhomocysteinaemia, whichwas not included in our routine thrombophilia screen, hasbeen shown recently to increase F1 1 2 levels (Kyrle et al,1997b).

The clinical significance of such markedly increasedF1 1 2 and TAT levels in patients with high FVIII:C levelsis not established. Although, Kyrle et al (1997a) have shownthat F1 1 2 levels cannot be used to identify patients athigher risk of recurrent venous thrombosis, elevated FVIII:Cis clearly associated with a very marked disturbance ofcoagulation homeostasis.

ACKNOWLEDGMENTS

The authors would like to thank Dr Frank Boulton and thestaff of the Wessex Regional Transfusion Centre, South-ampton for their help. James O'Donnell was sponsored by aMedical Research Council Training Fellowship award.

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