Abstract
Blood coagulation cascade ensures the arrest of bleeding and restoration of vascular integrity after physical damage and therefore is essential to normal physiology. However, many pathological conditions can disturb the tightly regulated haemostatic balance. Inflammation or malignancies may promote a hypercoagulable phenotype, while autoimmune diseases may lead to either thrombosis
... read more
or bleeding depending on the antigen. In this thesis we evaluated the role of plasma proteins β2-glycoprotein I (β2GPI), factor VII activating protease (FSAP) and protein S in coagulation pathophysiology. Thrombin generation assay as a global coagulation assay was instrumental in this thesis.
β2GPI is a highly abundant plasma protein which in the antiphospholipid syndrome (APS) in complex with anti-β2GPI autoantibodies may create a prothrombotic phenotype. From the B-cell repertoire of two APS patients with the help of phage display we constructed four β2GPI-binding monoclonal antibodies which recognized domain I of β2GPI with or without domain II. Various coagulation assays revealed that their lupus anticoagulant potency is dependent on the affinity for and presence of β2GPI. These antibodies may serve as useful tools in further research on the pathogenesis of APS. Self-antigen recognition and presentation is a crucial step in autoimmune disease, therefore the uptake and the subsequent presentation of β2GPI by dendritic cells was explored. Despite efficient internalization by immature dendritic cells, only a limited number of β2GPI peptides were identified to be presented on MHC class II molecules after dendritic cell maturation. Finding of these peptides will promote further research on T-cell recognition implicated in immune response associated with APS.
Since the discovery of FSAP, numerous functions have been proposed including interactions with the haemostatic system. To contest the initial reported name-giving activity of FSAP, we used recombinant thermolysin-activated FSAP-R313Q mutant to find that FSAP was a poor activator of FVII while retaining its reactivity towards other known substrates of plasma-derived FSAP. Therefore FSAP does not mediate haemostasis through activation of FVII. Instead, thrombin generation experiments revealed that FSAP had a procoagulant effect in plasma which was restricted to low tissue factor concentrations and involved inhibition of tissue factor pathway inhibitor (TFPI). Furthermore, the Marburg-I mutant of FSAP had a decreased interaction with TFPI and a diminished procoagulant effect in thrombin generation. Our data show that FSAP but not its Marburg-I variant may amplify thrombin generation by inhibiting TFPI at the onset of coagulation.
While the role of protein S as a natural anticoagulant is beyond doubt, regulation of its anticoagulant activity by cleavage in the thrombin sensitive region is less recognized. We showed that only intact but not cleaved protein S has anticoagulant properties in plasma. In individuals with pathological conditions characterised by extreme low and high platelet counts, as occurred in patients with haematological malignancies or chemotherapy-induced thrombocytopenia, we were able to correlate the extent of in vivo protein S cleavage with platelet counts and thus pinpoint the source of the cleaving protease to activated platelets. The modulation of protein S cleavage by platelets may have a potentiating effect on the bleeding phenotype associated with chemotherapy-induced thrombocytopenia or thrombotic phenotype in malignant platelet proliferation.
show less
