Purified protein S contains multimeric forms with increased APC-independent anticoagulant activity.
Protein S, the cofactor of activated protein C (APC), also expresses anticoagulant activity independent of APC by directly inhibiting prothrombin activation via interactions with factor Xa, factor Va, and phospholipids. In different studies, however, large variations in APC-independent anticoagulant activities have been reported for protein S. The investigation presented here shows that within purified protein S preparations different forms of protein S are present, of which a hitherto unrecognized form (<5% of total protein S) binds with high affinity to phospholipid bilayers (K(d) < 1 nM). The remaining protein S (>95%) has a low affinity (K(d) = 250 nM) for phospholipids. Using their different affinities for phospholipids, separation of the forms of protein S was achieved. Native polyacrylamide gel electrophoresis 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-exclusion chromatography revealed that the slower-migrating bands represented multimeric forms of protein S. Multimeric protein S (<5% of total protein S) appeared to have a 100-fold higher APC-independent anticoagulant activity than the abundant form of protein S. Comparison of purified protein S preparations that exhibited a 4-fold difference in APC-independent anticoagulant activity 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.
Seré
K M
KM
Department of Biochemistry, Cardiovascular Research Institute Maastricht, University of Maastricht, Maastricht, The Netherlands.
Janssen
M P
MP
Willems
G M
GM
Tans
G
G
Rosing
J
J
Hackeng
T M
TM
D000327:Adsorption; D000925:Anticoagulants / Q000097:blood / Q000737:chemistry* / Q000378:metabolism*; D015153:Blotting, Western; D002498:Centrifugation; D005591:Chemical Fractionation; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008051:Lipid Bilayers / Q000378:metabolism; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism; D010713:Phosphatidylcholines / Q000378:metabolism; D010718:Phosphatidylserines / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism; D011485:Protein Binding; D011486:Protein C / Q000378:metabolism / Q000502:physiology*; D020033:Protein Isoforms / Q000097:blood / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017293:Protein S / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D011516:Prothrombin / Q000037:antagonists & inhibitors / Q000378:metabolism
D000925:Anticoagulants; D008051:Lipid Bilayers; D008081:Liposomes; D010713:Phosphatidylcholines; D010718:Phosphatidylserines; D010743:Phospholipids; D011486:Protein C; D020033:Protein Isoforms; D017293:Protein S; C042511:1,2-dioleoylphosphatidylserine; D011516:Prothrombin; C017251:1,2-oleoylphosphatidylcholine