Effect of phospholipid membranes on α-thrombin activity

Abstract α-Thrombin is generated from its inactive precursor prothrombin by a cascade of zymogen activation reactions at a membrane/solution interface. Since thrombin is a water-soluble protein, it can dissociate from the membrane where it was activated and subsequently act in solution. However, since most thrombin substrates are membrane bound, α-thrombin acts in a membrane rich environment and thus the possibility that thrombin recognizes its substrates on the membrane itself cannot be excluded. We had previously obtained evidence, by a.c. polarography, that thrombin binds and penetrates model membranes containing phosphatidylserine (PS) and phosphatidylcholine (PC). Therefore, we thought it important to asses whether thrombin activity on a synthetic chromogenic substrate (S 2238) is affected by the presence of phospholipids. In the present study, we found a dissociation constant of 3.2 × 10 −7 M for thrombin interacting with monolayers containing 25% PS–75% PC, the same order of magnitude as for prothrombin in the absence of Ca 2+ . Amidolytic thrombin activity on vesicles was measured and the effect of phospholipids on the kinetic parameters was studied. In the presence of either 25% PS–75% PC or of 100% PS, the limiting maximum rate of enzyme activity V m was halved and the Michaelis constant K m was not significantly changed. Thus, we found that membrane-bound thrombin remains active, to some extent, and we hypothesize that in this state it could be protected from the inhibitors.