Regulation of prothrombinase activity by protein S

The independent effect of protein S as prothrombinase inhibitor has been pr oposed to depend on binding to both coagulation factors Va and factor Xa or on the binding to phospholipid thereby limiting the phospholipid available for prothrombinase activity. In this study we show that plasma concentrati ons of protein S (300 nM) equilibrated with the prothrombinase components ( factor Va, factor Xa, phospholipid) cause a profound inhibition at low phos pholipid concentrations (similar to 0.2 mu M). This inhibition by protein S of prothrombinase activity is abrogated with increasing phospholipid conce ntrations. Modeling of the effect of protein S on prothrombinase based only on the reported affinity of protein S for phospholipids (K-d similar to 10 (-8) M) in an equilibrium model (Clotspeed), predicted the experimentally o btained thrombin generation rates at low phospholipid in the presence of pr otein S based on the diminished available phospholipid binding sites for th e prothrombinase components. Consistently, initial rates of prothrombinase activity are. already maximally inhibited when protein S is preincubated wi th the phospholipid prior to the addition of factor Xa, factor Va and proth rombin. The results indicate that the order of addition of prothrombinase c omponents and the availability of phospholipid may have a profound influenc e on observed effects of protein S on prothrombinase activity. All prothrom binase components (factor Xa, factor Va, phospholipid) become available dur ing the course of the physiological thrombin generation. The effect of prot ein S was therefore studied on tissue factor-induced, platelet-dependent th rombin generation. Protein S delayed and inhibited the rate of thrombin gen eration of tissue factor-induced thrombin formation when surface is provide d at physiologic concentrations using isolated platelets (2 X 10(8)/ml). In contrast, protein S hardly affected thrombin generation in this model when platelets were pre-activated with collagen. Furthermore, the observed effe cts of addition of protein C and thrombomodulin in the absence or presence of protein S on tissue factor-induced, platelet-dependent thrombin generati on, indicate that protein S and protein C may cooperate in the regulation o f prothrombinase activity through independent mechanisms.