Effect of FV-Leiden Mutation and Antiphospholipid Syndrome on Thethrombin GenerationTest.

Introduction: The incidence of Factor V Leiden mutation (Arg 506 Gln) is significantly high in patients with venous and arterial thrombosis. In patients with antiphospholipid syndrome (APS), the FVL mutation may play a major role in the occurrence of thrombosis. The aim of this study was to demonstrate that an increased Endogenous Thrombin Potential (ETP) may be an important mechanism for the thrombotic risk associated with FVL and / or APS and to know if the FVL mutation would increase the risk of thrombosis in patients with APS. Additionally, we tested if fluorometric determination of ETP( area under the curve), thrombin generation velocity (PEAK) and TIME TO PEAK (time from starting to reaching the peak), is suitable to determine changes in haemostatic parameters.

Methods and patients: We measured the thrombin generation in 120 patients (67F, 53M) with a median age of 40 year categorized in 3 groups of 40 patients each:(1) either heterozygous or homozygous FVL mutation (14F, 26M of whom 29 were heterozygous and 11 were homozygous) (2) presence of antiphospholipid antibodies (aPL) (20F, 20M) and (3) a combination of both (33F,7M). Three characterizing thrombin generation parameters were measured: ETP, PEAK and TIME TO PEAK. Platelet poor plasma samples (PPP) were derived from citrated blood. In a microtiter plate, we used different concentrations of TF with phospholipids after adding PRP(platelet poor plasma) and buffer. The reaction was started after adding substrate solution. Fluorometer was the Fluoroskan Ascent Type 374.

Results: Using four concentrations (Innovin dilutions 1:600, 1:6.000, 1:50.000 and 1:500.000) of TF, the following results (ETP units = relative fluorescence units or RFU; PEAK units: RFU/min; TIME TO PEAK units = min) were obtained for the 3 groups:

• For all patients TF 1:600 median ETP, PEAK and TIME TO PEAK were taken as 100 % because less dilution gives no further increase resp. decrease in these parameters

• Patients with aPL: median ETP decreased from 100 % to 36,6 % (lowest TF concentration), PEAK to 34,4 % and TIME TO PEAK increased to 218 %.

• Patients with FVL median ETP decreased from 100 % to 2,64 % (lowest TF concentration), PEAK to 9 % and TIME TO PEAK increased to 282 %.

• Patients with aPl and FVL => median ETP decreased from 100 % to 33 % (lowest TF concentration), PEAK to 30,8 % and TIME TO PEAK increased to 143 %.

While there was no detectable thrombin generation in aPL patients in low concentration of TF, at the two highest concentrations the thrombin generation was comparable to same concentrations in the haemostatically normal control samples(data not shown). For those affected by FVL and aPL, the threshold of detectable thrombin generation was even shifted to lower concentrations of TF, regarding a “residual activity” of about 33 % for ETP and PEAK, as for patients with FVL. In patients with APS, TF 1:50.000 caused a thrombin generation comparable to TF 1:6.000 in haemostatically normal controls, showing a shift in coagulation factor reactability. In a physiological environment this could indicate a stronger haemostatic reaction to minor vascular lesions in patients affected by factor V Leiden mutation than in those without it.

Conclusion: A thrombin generation assay utilizing lower concentrations of TF as coagulation initiating agent could be usefully performed to assess thrombophilic states due to coagulation factor mutations and /or presence of antiphospholipid antibody.