Our laboratory reported that protein S inhibits prothrombin activation by competition for phospholipid binding sites.2 The paper by Seré et al1 concludes that our hypothesis was based on experiments performed with protein S preparations that contained multimeric forms of protein S. This conclusion was based upon studies performed with their preparations of protein S, using which they observed that protein S was polymerized when analyzed in the absence of Ca2+.3 Their data, in fact, confirmed a previous publication from our laboratory4 that examined the polymerization of protein S and the Ca2+ dependence of this process. In the latter paper, we showed that protein S, both intact and thrombin cleaved, polymerizes in the absence of Ca2+ but is totally depolymerized by 0.8 mM calcium with a Ca2+ dissociation constant of 0.42 mM. Sedimentation equilibrium studies conducted in the presence of Ca2+ revealed that protein S was homogeneous with a molecular weight of 75 800 ± 4200 (S vedberg units = 4.2). In the presence of ethylenediaminetetraacetic acid (EDTA), polymerized forms of the protein were observed both by sedimentation equilibrium and sedimentation velocity (S vedberg units = 7.2). We cannot comment on the qualities of the protein S prepared by Seré and colleagues.
The experiments performed by our laboratory on prothrombin activation reported that protein S slowed the rate of prothrombin activation in experiments conducted using solutions equilibrated with Ca2+ (2 mM) prior to mixing in the reaction system. We also showed that phospholipids overcame the inhibition that was observed, consistent with a phospholipid-dependent process, and that the inhibition occurred in systems with platelets and factors II, VIIa, IX, X, VIII, and V at their mean physiologic concentration, with reactions initiated with tissue factor.
In the report by Seré et al,1 there are fundamental defects in the experimental protocol that overcome the ability to interpret the experimental results. Their experiments are conducted in diluted citrate plasma with initiation of the reactions by the addition of Ca2+. Thus, at the start of each experiment protein S is polymerized, and depolymerization is occurring during the course of the reaction itself. The investigators, in fact, make a point of this and indicate that the preaddition of CaCl2 to the experimental system caused a “decrease in lag time”1(p3626) consistent with the rate processes required for the conformational changes of procoagulant vitamin-K-dependent proteins as well as depolymerization of the inhibitor protein S. These conflicting processes render the experimental results uninterpretable. In addition, in the endogenous thrombin potential (ETP) measurements used by the investigators, a substrate for thrombin is included. This addition, however, confuses the results of ETP interpretations by providing a competitive substrate for thrombin, thus influencing thrombin concentration throughout the measurements.
To conclude, the authors state that they “quantified the APC-independent anticoagulant activity of protein S in its natural environment [emphasis added].”1(p3629) This is hardly the case. The natural environment for protein S is blood that, in addition to the components of plasma, contains cells and platelets, but does not include dilution, citrate, or a synthetic thrombin inhibitor.