Factor Va enhances thrombin generation by several orders of magnitude. Its function is controlled by activated protein C (APC)-mediated and protein S enhanced proteolysis. The aim of my thesis was to clarify the molecular mechanisms underlying FVa inactivation. For this, all three proteins were recombinantly produced and characterised. As APC-mediated FVa inactivation is phospholipid dependent, assays were optimised to investigate interactions in the presence of phospholipids.
I have shown that protein S and FVa act together to enhance APC association with phospholipid membranes. The presence of protein S is mandatory, as FVa by itself does not increase binding of APC to phospholipids. These findings strongly suggest that APC, protein S and FVa together form an inactivation complex on phospholipid surfaces.
Unlike FVa, FVIIIa does not enhance APC binding to phospholipids, indicating that FVIIIa does not form a similar complex with APC and protein S. C4BP-bound protein S did not efficiently enhance APC-phospholipid binding, indicating that C4BP interfers with complex formation. Results I obtained with protein S variants with impared APC cofactor function, and FV Nara, associated with strong APC resistance, indicate that their mutations essentially abolished their ability to assemble into the tri-molecular complex.
FV-810, with partial B-domain deletion, assembled the complex as efficiently as FVa. Protein S was required for complex enhancement by FV-810. However, results for FV-810 obtained with a FVa inactivation assay indicated that effective inactivation can be achieved in the absence of protein S. These findings suggest that there must be an alternative mechanism involved in APC cofactor function which is not mediated solely by increased binding of APC to phospholipids.
In conclusion, my findings demonstrate that FVa promotes its own APC-mediated degradation by enhancing APC binding to phospholipids together with protein S, but also suggest that more than a single mechanism controls FVa inactivation.