Preferential Binding Of Prothrombin Fragments On Monolayers Containing Phosphatidylserine

Phospholipids from platelet membranes play a crucial role in the last step of the blood coagulation cascade, as a catalytic surface for the conversion of prothrombin into thrombin by the prothrombinase complex. It provides a favora ble configuration of the required factors ( factor Xa, prothrombin, factor Va ), concentrated on it’s surface. The maximal biological activity is observed with mixtures of phospholipids containing phosphatidylserine, in presence of Ca⁺⁺.

In order to understand at the molecular level, which part of prothrombin interact with phospholipids, we studied the correlation between the number of prothrombin molecules or fragments of it adsorbed ( by counting the surface radioactivity of ³H-protein ) and their ability to penetrate into the phospholipid monolayer ( measured by the increase of the lipid layer capacitance ), by two recent complementary methods of direct surface measurement ( radioactivity and electrochemistry ), in function of Ca⁺⁺ concentration and phospholipid composition.

We could detect parallely the number of cystine residues ( by electrochemical reduction ) of the penetrating parts of the adsorbed protein.lt occurs that adsorbed protein don’t penetrate. The number of S-S groups reduced per adsorbed protein molecule can be deduced and its variation indicate different orientation of the molecules on the phospholipid surface. Indeed, it is the case at different Ca⁺⁺ concentration,for prothrombin and fragment 1, which contain γ-carboxyglutamic groups.

We could also observe that penetration is specific of phospholipid composition for fragment 2 but not for thrombin and is not specific for both in function of the Ca⁺⁺ concentration.

Results indicate that the ratio of penetration to the total number of adsorbed prothrombin molecules and fragment 1 is important for an optimal catalysis.