Inhibition of factor VII activation by UFH, LMWH and Synthetic Pentasaccharide. Mechanism of action. Preliminary results.

G.T.Gerotziafas^1,2, L. Bara², M.F. Bloch¹, Z. Foka³, P.E. Makris³, M.M. Samama¹.

¹ Service d’Hématologie Biologique, Hôpital Hôtel-Dieu de Paris, France.

² Laboratoire de Thrombose Experimentale Faculté de Medecine, Broussais Hôtel -Dieu, Univ. P. & M. Curie-Paris VI, France.

³Thrombosis & Haemostasis Unit, 1st Prop. Pathologic. Clinic, AHEPA University Hospital, Thessaloniki, Greece.

Factor VII activation is an essential step in the process of blood coagulation and plays an important role in thrombogenesis. Heparins (UFH and LMWH) have a major role in the prevention and treatment of venous and arterial thrombosis. We have recently demonstrated that UFH, LMWHs and the synthetic pentasaccharide (PS) inhibit FVIIa generation during in vitro coagulation of whole blood or PPP. In the present study we investigated some aspects of the mechanism of this activity of heparins and PS on FVIIa generation. Materials & Methods. We measured FVIIa levels in PPP and in the following experimental system : Intrinsic system activation pseudo-serum (ψ -serum) was obtained as follows; PPP was recalcified by adding an equal volume of CaCl₂ (0,025 M). After 60 min incubation in glass tubes at 37^oC, clot was discarded by winding it on a plastic spatula and the supernatant liquid (ψ-serum) was collected. FVIIa levels were measured in ψ-serum prepared by normal pool PPP and factor VII, XII, XI, IX, VIII, X, V and II deficient plasmas. In plasma samples we added either buffer or UFH (0,4 IU/ml), or enoxaparin or PS (0,4 anti-Xa IU/ml) or recombinant Hirudin (2 μg/ml). Normal PPP was incubated with an anti-TFPI monoclonal antibody (moab) (American Diagnostics). After 60 min incubation, we added either buffer or UFH or enoxaparin or PS and ψ-serum was prepared as described. Factor VIIa was measured with a newly developed one stage clotting assay.

Results : UFH and enoxaparin abolished their inhibitory effect on FVIIa generation when ø-sera were prepared from FIX, FXI, or FXII and to lesser degree from FX deficient plasmas. PS inhibited FVIIa generation in every studied plasma. rHirudin did not have any effect on FVIIa generation. Experiments with the anti-TFPI moab demonstrated that in ø-serum treated with the moab, FVIIa levels were 3,6 folds higher than the control. The presence of the anti-TFPI moab did not modify the inhibitory effect of UFH, LMWH and PS which remained important (90-98 % inhibition).

Interpretation : After intrinsic system activation FVII is activated mainly by FIXa. UFH and LMWH intervent in FVIIa generation by inhibiting mainly the activation of FVII by the FIXa and by reducing the generation of FIXa (by inhibiting FXII and FXI). The inhibitory effect of UFH and LMWH on FVIIa generation was partially abolished when FX deficient plasma was used. Thus, the inhibition of FXa by heparins does not seem to play a major role in the inhibition of FVIIa generation in this experimental system. Moreover, the inhibitory effect on FVIIa generation did not pass via the inhibition of FIIa since rHirudin (a pure strong and specific anti-IIa agent) did not have any inhibitory effect on FVIIa generation. PS probably had a direct inhibitory effect on FVIIa generation and/or activity. UFH, LMWH and PS inhibit FVIIa generation independently of the presence of TFPI.