Vitamin K-dependent protein S - studies of synthesis, structure and function

Sammanfattning: This thesis is based on four projects with the overall aim to investigate vitamin K-dependent protein S functions in relation to its structure. The anticoagulant functions of protein S can be divided in three: 1) cofactor for activated protein C (APC), 2) cofactor for tissue factor pathway inhibitor (TFPI) and 3) APC and TFPI-independent anticoagulant functions. Microparticles might be shed from any cell type, and elevated levels of circulating microparticles are associated with several diseases ranging from thrombotic disorders, cancer, diabetes, autoimmune conditions and sickle cell disease. Platelet-derived microparticles are part of normal haemostasis, assisting in efficient formation of a blood clot at the site of vascular injury. However in circulation, the procoagulant microparticles are potential initiators of undesirable thrombus formation. Protein S has been shown to bind microparticles, and we therefore studied the function of the anticoagulant APC-system on the surface of platelet- and erythrocyte-derived microparticles. We could confirm binding of protein S to the microparticles. The bound protein S supported APC in regulation of coagulation, both in purified systems and in plasma, suggesting the APC-mediated regulation of coagulation to be highly effective on the surface of microparticles. The high concentration of protein S in circulation might therefore counteract the procoagulant properties of microparticles. The TFPI-cofactor function of protein S was quite recently described, protein S stimulating TFPI-mediated regulation of the initiation of coagulation, in a manner that is dependent on direct interaction between protein S and TFPI. In an attempt to localize the interaction site on protein S, we created four protein S variants and tested them for TFPI-cofactor function. We found two variants with impaired function and could therefore suggest a region in the most C-terminal domain of protein S to be of importance. Furthermore, in silico experiments were performed and possible poses of interaction between protein S and TFPI were proposed. Only 30-40% of the circulating protein S has full anticoagulant function, the rest is bound to the complement regulator C4b-binding protein (C4BP). Protein S interacts with C4BP in an affinity so high that all C4BP in circulation carry a protein S molecule. In a previous study, it was shown that patients with protein S deficiency, undergoing treatment that further decreases the protein S production, also had decreased concentration of C4BP. By the use of recombinant protein expression we could see that protein S and C4BP need to be produced in the same cell, at the same time for efficient secretion of C4BP. Our results could therefore explain the low concentration of C4BP in protein S deficient patients.