UBC Theses and Dissertations
Functional regulation of anticoagulant protein C and clotting factor Va by fibrinolytic plasmin Lee, Frank Man Hon
Thrombosis is the leading cause of death and disability. Clots (i.e. thrombi) that obstruct blood flow from delivering oxygen and nutrients to tissues require rapid removal. The main treatment is with tissue plasminogen activator (tPA), which activates the enzyme plasmin from its circulating precursor plasminogen. Plasmin works by degrading fibrin, a meshwork of fibers that form the structural scaffold of the clot. Although these thrombolytic drugs have saved countless lives, their use is associated with life-threatening complications including increased bleeding risk and cellular toxicity. By greatly elevating plasmin concentrations with intravenous tPA, the effect of plasmin on its other substrates apart from fibrin may play an important role in dysregulating hemostasis, giving insight into physiological mechanisms that are impacted by thrombolysis. This thesis focuses on the modulatory effects of plasmin on protein C (PC) and activated clotting factor (F) V (FVa). Fragmentation of PC by plasmin assessed in conjunction with activated PC (APC)-specific chromogenic substrate cleavage showed increased amidolytic activity correlating with proteolysis of the PC heavy chain (PC-Hc). Plasmin-cleaved PC inhibited inactivation of FVa and had no appreciable effect on prolonging plasma clotting times in PC-deficient plasma compared with APC. N-terminal sequencing showed that plasmin cleaves PC-Hc within the autolysis loop and at the canonical thrombin activation site. Our findings so far are consistent with natural and engineered APC variants, affected within or adjacent to the autolysis loop, that have reduced anticoagulant function. Interestingly, these mutants still signal through protease-activated receptor 1, suggesting a novel role for plasmin-modulated (A)PC in conferring cytoprotection. For FVa, plasmin-specific cleavage of chromogenic substrate and protein staining were used to confirm the requirement for proteolysis by plasmin in converting FVa into an accelerator of tPA. Free and lipid-binding fragments both enhanced tPA activity. 125I-radiolabeled plasminogen binding indicated an interaction with an unbound fragment derived from FVa-Hc that is C-terminal lysine dependent. The tPA-accelerating fragment of FVa-Lc is derived from the C1 and C2 lipid-binding domains and could non-enzymatically enhance fibrinolysis specifically at the site of vascular injury. Understanding the functional regulation of PC and FVa by plasmin offers novel insights toward improving thrombolysis therapy.
Item Citations and Data
Attribution-NonCommercial-NoDerivatives 4.0 International