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Factor VIIa levels in prothrombotic conditions

University of British Columbia

The coagulation cascade is continuously in a state of preparedness to rapidly respond to vascular damage at sites of injury. However, an overactivity of the hemostasis system resulting in a hypercoagulable state can lead to thrombotic disorders such as coronary heart disease (CHD) and stroke. Recent prospective studies have implicated elevated factor VII (FVII) activity as an independent risk factor in incidences of coronary heart disease. Indeed, factor VII and its cofactor, tissue factor (TF), are generally thought to play the principle role in the physiological initiation of clotting which may help explain the association between FVII and the risk of CHD. Recent evidence has indicated that trace levels of the activated form of FVII, called factor Vila (FVlIa), circulates in the blood. This active form is unique among the clotting factors in that FVlIa has a long half life of 2 to 2.5 hours, is highly resistant to inhibitors, and can auto-activate zymogen FVII. However, conventional assays for FVII activity employing tliromboplastin were not able to separate FVIIa from its zymogen precursor, FVII. This has resulted in a great deal of controversy over whether plasma FVIIa levels per se or total FVII (zymogen and FVIIa) is more closely associated with the risk of CHD. In the present study, a specific assay for factor Vila was developed using a recombinant soluble form of tissue factor (sTF), deleted of the membrane binding region, that is deficient in promoting the conversion of factor VII to factor Vila. However, this soluble tissue factor, in the presence of phospholipid vesicles, retains its ability to clot plasma using the pre-activated factor Vila in the plasma sample. The sTF was engineered by using site-directed mutagenesis to delete the transmembrane domain of the TF gene and the truncated protein was expressed in a eucaryotic expression system employing baby hamster kidney (BHK) cells. Using the unique properties of sTF, an automated clot-based assay was developed and optimized to quantitatively determine the concentration of FVIIa in plasma samples. The assay was employed to measure FVIIa levels in a normal, healthy population and in a group of patients in the late stages of complicated pregnancies The FIIa assay developed in the present study had a sensitivity range of 20 pg/ml to 1000 ng/ml FVIIa and required only 5 ul of plasma per assay. The within-run and between-run coefficients of variation were 3.9% and 4.9%, respectively, indicating high reproducibility. The assay also showed excellent correlation (r = 0.998) with a commercial FVIIa kit produced by Diagnostica Stago in a patient comparison study over a range of 0.1 ng/ml to 30 ng/ml FVIIa. The present study was standardized using an international FVIIa standard and therefore FVIIa levels could be measured in concentration (ng/ml) or international activity units (mU/ml). Results of the initial population study on FVIIa confirmed the existence of trace amounts of FVIIa in normal healthy subjects. The normal range in the present study was 0.7 to 4.8 ng/ml (11 to 100 mU/ml) and the mean plasma FVIIa was 2.08 ± 0.90 ng/ml (42 ± 15 mU/ml). There was a significant correlation between FVIIa levels and increasing age but no significant difference between FVIIa levels in men and women. Factor Vila levels in complicated pregnancy patients were significantly higher than in an age-matched group of healthy women. The mean FVIIa in the patients was 4.1 ± 2.2 ng/ml (85 ± 42 mU/ml) compared to 1.86 ng/ml (37 mU/ml) in healthy women. However, more extensive clinical studies on FVIIa and FVH in other groups at risk of CHD are required to better establish the importance of FVIIa levels in prothrombotic conditions.

Thesis/Dissertation

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2009-03-24

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http://hdl.handle.net/2429/6444

Biochemistry and Molecular Biology

University of British Columbia

UBCV

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