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Thrombin activatable fibrinolysis inhibitor (TAFI) is a human plasma-derived zymogen that is activated through proteolytic cleavage by thrombin, thrombin in complex with thrombomodulin, or plasmin. Active TAFI attenuates fibrinolysis by removing carboxyl-terminal lysine residues from partially degraded fibrin, thereby inhibiting a potent positive feedback loop in the fibrinolytic cascade. In addition to the plasma pool of TAFI arising from expression in the liver, a distinct pool of TAFI has been reported to be present in platelets. While the antifibrinolytic effect of plasma-derived TAFI has been well-documented by in vitro and in vivo clot lysis assays, characterization of the platelet-derived form has been limited. Here, we not only confirm the presence of TAFI in the medium of washed, thrombin-stimulated platelets, but also that platelet-derived TAFI is capable of attenuating platelet-rich thrombus lysis in vitro independently of plasma TAFI using a novel thrombus lysis assay. Fluorescent thrombi were generated by suspending washed human platelets in plasma immunodepleted of TAFI containing fluorescently-labeled human fibrinogen such that the only TAFI present in the system was of platelet origin. Following platelet activation and clot retraction induced by thrombin, t-PA-dependent platelet-rich thrombus lysis was observed by removal of timed aliquots from the medium of retracted thrombi followed by measurement of fluorescence. When supplementary thrombomodulin was added to the thrombus medium, a 2.3-fold reduction in lysis rate was observed, indicating platelet-derived TAFI could attenuate the fibrinolytic cascade in vitro. Furthermore, when supplementary recombinant TAFI (rTAFI) was included in the medium, platelet-derived TAFI and rTAFI were observed to combine for greater inhibition of fibrinolysis. Taken together, these observations indicate that the secretion of platelet-derived TAFI can augment concentrations of TAFI already present in plasma to enhance attenuation of the fibrinolytic cascade. This could be significant at sites of vascular damage or regions of pathological thrombosis, where activated platelets are known to accumulate and secrete the contents of their granules. Finally, we have purified platelet-derived TAFI from platelet releasates for future characterization studies and mass spectrometry. / Thesis (Master, Biochemistry) -- Queen's University, 2009-09-24 14:22:42.5

The fibrinolytic system of normal and leukaemic bone marrow was examined. Normal bone marrow had a very active fibrinolytic system due to abundant free t-PA, with negligible contribution from u-PA. High levels of PAI-2 antigen were observed in addition to PAI-1. Plasminogen, the precursor of plasmin was detected, mainly in complex with α₂-AP, indicating that plasmin had been generated. The balance of the fibrinolytic system in normal bone marrow contrasted with the system in plasma, where plasmin is not normally generated. In bone marrow the t-PA level was greater than that of the inhibitors while in plasma t-PA circulates in complex with PAI-1. t-PA, u-PA, u-PAR, PAI-1 and PAI-2 were localised to cells of the myeloid, monocytic, megakaryocytic and T-lymphoid lineages in normal marrow. In contrast, cells of the B-lymphoid lineage did not possess the antigens of interest. In addition, non-haematopoietic cells in the marrow were examined, and it was observed that osteocytes, endothelial cells, smooth muscle cells and adipocytes contained the antigens of the fibrinolytic system. PAI-1, PAI-2, u-PA, u-PAR and probably t-PA were synthesised by the cells of the marrow, while plasminogen and α₂-AP arose from the general circulation. The activity and antigen levels of the components of the fibrinolytic system differed between normal and leukaemic bone marrow. In leukaemic marrow, u-PA was observed, while t-PA and PAI-2 were decreased compared with normal bone marrow. PAI-1, plasminogen, α₂-AP and plasmin-α₂-AP complex were similar to normal bone marrow. The appearance of u-PA was probably associated with the malignant phenotype and may confer an invasive advantage on the leukaemic cell. In addition, the profile of the fibrinolytic system observed in leukaemic bone marrow may contribute to the haemorrhagic symptoms associated with certain forms of leukaemia.

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