Binding of anticoagulant vitamin K-dependent protein S to platelet-derived microparticles.
Vitamin K-dependent protein S is an anticoagulant plasma protein serving as cofactor to activated protein C in degradation of coagulation factors Va and VIIIa on membrane surfaces. In addition, it forms a noncovalent complex with complement regulatory protein C4b-binding protein (C4BP), a reaction which inhibits its anticoagulant function. Both forms of protein S have affinity for negatively charged phospholipids, and the purpose of the present study was to elucidate whether they bind to the surface of activated platelets or to platelet-derived microparticles. Binding of protein S to human platelets stimulated with various agonists was examined with FITC-labeled monoclonal antibodies and fluorescence-gated flow cytometry. Protein S was found to bind to membrane microparticles which formed during platelet activation but not to the remnant activated platelets. Binding to microparticles was saturable and maximum binding was seen at approximately 0.4 microM protein S. It was calcium-dependent and reversed after the addition of EDTA. Inhibition experiments with monoclonal antibodies suggested the gamma-carboxyglutamic acid containing module of protein S to be involved in the binding reaction. An intact thrombin-sensitive region of protein S was not required for binding. The protein S-C4BP complex did not bind to microparticles or activated platelets even though it bound to negatively charged phospholipid vesicles. Intact protein S supported binding of both protein C and activated protein C to microparticles. Protein S-dependent binding of protein C/activated protein C was blocked by those monoclonal antibodies against protein S that inhibited its cofactor function. In conclusion, we have found that free protein S binds to platelet-derived microparticles and stimulates binding of protein C/activated protein C.(ABSTRACT TRUNCATED AT 250 WORDS)
Dahlbäck
B
B
Department of Clinical Chemistry, University of Lund, Malmö General Hospital, Sweden.
Wiedmer
T
T
Sims
P J
PJ
D000911:Antibodies, Monoclonal; D001665:Binding Sites; D001792:Blood Platelets / Q000378:metabolism* / Q000648:ultrastructure; D002118:Calcium / Q000494:pharmacology; D002352:Carrier Proteins / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism*; D003094:Collagen / Q000494:pharmacology; D003169:Complement Inactivator Proteins; D004492:Edetic Acid / Q000494:pharmacology; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate; D005455:Fluorescent Antibody Technique; D005456:Fluorescent Dyes; D006023:Glycoproteins; D006801:Humans; D015539:Platelet Activation / Q000187:drug effects; D011486:Protein C / Q000378:metabolism; D017293:Protein S / Q000378:metabolism* / Q000494:pharmacology; D013917:Thrombin / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D003169:Complement Inactivator Proteins; D005456:Fluorescent Dyes; D006023:Glycoproteins; D011486:Protein C; D017293:Protein S; D003094:Collagen; D004492:Edetic Acid; D013917:Thrombin; D016650:Fluorescein-5-isothiocyanate; D002118:Calcium