Summary
The independent effect of protein S as prothrombinase inhibitor has been proposed 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 concentrations of protein S (300 nM)
equilibrated with the prothrombinase components (factor Va, factor Xa, phospholipid) cause a profound inhibition at low
phospholipid concentrations (~0.2 μM). This inhibition by protein S of prothrombinase activity is abrogated with increasing phospholipid concentrations. Modeling of the
effect of protein S on prothrombinase based only on the reported affinity of protein S for phospholipids (Kd ~ 10-8 M) in an equilibrium model (Clotspeed), predicted the experimentally obtained thrombin
generation rates at low phospholipid in the presence of protein S based on the diminished
available phospholipid binding sites for the prothrombinase components. Consistently, initial rates of prothrombinase activity are already maximally inhibited when protein S is preincubated with the
phospholipid prior to the addition of factor Xa, factor Va and pro-thrombin. The results
indicate that the order of addition of prothrombinase components and the availability of phospholipid may have a profound influence on
observed effects of protein S on prothrombinase activity. All prothrombinase components (factor Xa, factor Va, phospholipid) become available during the course
of the physiological thrombin generation. The effect of protein S was therefore studied
on tissue factor-induced, platelet-dependent thrombin generation. Protein S delayed
and inhibited the rate of thrombin generation of tissue factor-induced thrombin formation
when surface is provided at physiologic concentrations using isolated platelets (2
× 108/ml). In contrast, protein S hardly affected thrombin generation in this model when
platelets were pre-activated with collagen. Furthermore, the observed effects of addition
of protein C and thrombomodulin in the absence or presence of protein S on tissue
factor-induced, platelet-dependent thrombin generation, indicate that protein S and
protein C may cooperate in the regulation of prothrombinase activity through independent mechanisms.
