Purified protein S contains multimeric forms with increased APC-independent anticoagulant activity

Protein S, the cofactor of activated protein C (APC), also expresses antico agulant activity independent of APC by directly inhibiting prothrombin acti vation via interactions with factor Xa, factor Va, and phospholipids. In di fferent studies, however, large variations in APC-independent anticoagulant activities have been reported for protein S. The investigation presented h ere shows that within purified protein S preparations different forms of pr otein S are present, of which a hitherto unrecognized form (<5% of total pr otein S) binds with high affinity to phospholipid bilayers (K-d < 1 nM). Th e remaining protein S (>95%) has a low affinity (K-d = 250 nM) for phosphol ipids. Using their different affinities for phospholipids, separation of th e forms of protein S was achieved. Native polyacrylamide gel electrophoresi s demonstrated that the form of protein S that binds to phospholipids with low affinity migrated as a single band, whereas the high-affinity protein S exhibited several bands that migrated with reduced mobility. Size-exclusio n chromatography revealed that the slower-migrating bands represented multi meric forms of protein S. Multimeric protein S (<5% of total protein S) app eared to have a 100-fold higher APC-independent anticoagulant activity than the abundant form of protein S. Comparison of purified protein S preparati ons that exhibited a 4-fold difference in APC-independent anticoagulant act ivity showed that the ability to inhibit prothrombin activation correlated with the content of multimeric protein S. Multimeric protein S could not be identified in normal human plasma, and it is therefore unlikely that this form of protein S contributes to the APC-independent anticoagulant activity of protein S that is observed in plasma.