Plasmin-mediated proteolysis in microvascular thrombosis

This thesis investigates the role of plasmin-mediated proteolysis in the breakdown of VWF-rich microthrombi. Thrombi in the microvasculature differ from those in large vessels because they contain a relatively large contribution of von Willebrand factor (VWF) and comparatively little fibrin. Consequently, fibrin-based markers such as D-dimer provide limited information about the formation and breakdown of these occlusions.

VWF is regulated under normal conditions by the metalloprotease ADAMTS13, which cleaves ultra-large VWF multimers and limits their platelet-binding capacity. In thrombotic thrombocytopenic purpura (TTP), severe ADAMTS13 deficiency allows highly adhesive VWF multimers to accumulate and form platelet-rich microthrombi. Earlier work suggested that plasmin may provide an auxiliary mechanism for VWF degradation when ADAMTS13 activity is insufficient. The aim of this thesis was therefore to investigate whether plasmin-mediated VWF proteolysis generates a measurable cleavage product associated with microthrombus breakdown.

Chapter 2 describes the development of a bioassay for detecting plasmin-cleaved VWF (cVWF) in plasma. The assay recognizes a plasmin-generated cleavage product that is biochemically distinct from intact VWF, ADAMTS13-cleaved VWF, and fragments generated by several other proteases. In vitro, cVWF is released during plasmin-mediated degradation of platelet-VWF complexes, both under static conditions and under flow. cVWF was detected during acute TTP attacks, but not during remission or in healthy controls. In a mouse model of TTP, therapeutic stimulation of plasminogen activation increased cVWF formation and coincided with VWF clearance. These findings demonstrate that the assay can be used to study plasmin-mediated VWF proteolysis in experimental and disease settings.

Chapter 3 examined cVWF in an independent cohort of Japanese patients with acute TTP. cVWF levels were elevated compared with healthy controls and showed a moderate association with LDH, reflecting hemolysis and tissue injury. However, cVWF levels did not predict one-year TTP-related mortality. These findings confirm that cVWF formation is a reproducible feature of acute TTP in an independent patient population.

Chapter 4 investigated plasmin-mediated VWF cleavage in patients with acute and chronic liver disease and in patients with sepsis. cVWF was detected in patients with advanced or acute liver disease and in patients with sepsis, but not in healthy controls or patients with stable cirrhosis. Its levels increased with disease severity and correlated with D-dimer, suggesting an association with severe disease and fibrinolytic activity. These findings are compatible with a role for cVWF in studying disease-related microvascular processes, although its precise biological significance remains to be established.

Chapter 5 examined how plasmin disrupts platelet-VWF complexes. Plasmin-cleaved VWF retained its platelet-binding capacity, whereas plasmin-mediated cleavage and shedding of the platelet receptor GPIbα reduced the ability of platelets to re-form aggregates. VWF binding enhanced plasminogen recruitment to the platelet surface and increased GPIbα shedding. This mechanism was most evident in vitro, while its contribution to platelet-VWF complex breakdown in vivo remains to be determined.

Overall, this thesis establishes an experimental approach for measuring a specific plasmin-generated cleavage product of VWF, and advances our understanding of the actions of plasmin on both VWF and GPIbα.