PENETRATION OF FACTOR Xa INTO PHOSPHOLIPID LAYERS

Negatively charged phospholipids (PL) at the platelet surface play a crucial role in the conversion of prothrombin into thrombin in the prothrombinase complex. Interactions of prothrombin and factor Xa (FXa) with PL are generally thought to occur through ionic interactions via Ca⁺⁺ bridges. However we have shown by various independent approaches that prothrom bin (as well as FV and FVa) actually penetrates into the PL layer. We show that this is also the case, in the presence of Ca⁺⁺ , for the other vitamin K-dependent, Gla-containing protein of the complex, FXa.

Electrochemical surface measurements indicate a reversible increase in the capacitance of condensed PL monolayers (containing various concentrations of phosphatidylserine (PS)), upon interaction with bovine FXa (kindly provided by Dr. C.M. Jackson). Reduction of the FXa disulfide bridges at the contact of the electrode further demonstrates penetration Parallely, radioactive surface measurements allow to follow the adsorption of FXa which is quite reversible on PS gono-layers and to determine the association constant, 6.10⁶ 1/mol, very similar to the Ka of FX, obtained by various different techniques.

Finally, penetration of FXa with PS containing PL vesicles was tested using the radiolabelled apolar probe (INA) which binds selectively to the lipid embedded domains of protein. FXa was radioactively labelled and the label preferentially found on the active site containing FXa heavy chain Moreover, interaction of FXa with PS vesicles decreases its K_m for S 2222.

Supported by grant INSERM n° 845016