Platelet activation and tissue factor release in hemolytic uremic syndrome

University dissertation from Department of Pediatrics, Lund University

Abstract: Hemolytic uremic syndrome (HUS) is a clinical syndrome characterized by microangiopathic hemolytic anemia, thrombocytopenia and acute renal failure. There are two subtypes: typical HUS associated with enterohemorrhagic E. coli (EHEC) and atypical HUS associated with uninhibited activation of the alternative pathway of complement. EHEC produce virulence factors such as lipopolysaccharide (LPS) and Shiga toxin. Atypical HUS is associated with mutations in complement regulators, mainly in factor H. During HUS platelets are activated and consumed leading to thrombocytopenia. The overall aim of this thesis was to investigate how platelet activation occurs and if tissue factor is released during typical and atypical HUS, as pathogenetic mechanisms explaining the formation of thrombotic microangiopathy. LPS was shown to bind to platelets through a novel receptor complex composed of TLR4/CD62. Binding led to platelet activation and aggregation. Shiga toxin bound to activated platelets and monocytes. In whole blood LPS and Shiga toxin induced platelet-leukocyte complex formation, generation of tissue factor-expressing platelet microparticles and release of functional tissue factor into plasma. Patients with typical HUS were shown to have LPS and Shiga toxin bound to their platelets and increased levels of platelet-leukocyte complexes, tissue factor-expressing platelet microparticles and circulating tissue factor were demonstrated. Mutated factor H was incapable of protecting platelets from complement activation. This was demonstrated using serum from patients with atypical HUS and defined mutations in factor H. Purified mutated factor H exhibited decreased binding to the platelet surface and allowed complement activation to occur. Complement activation on platelets led to their activation and release of tissue factor-expressing microparticles. Clusterin is one of the regulators of the terminal complement pathway. A novel mutation in clusterin (Q433P) was found in a child with atypical HUS. This mutated protein could not bind C5b-7 and serum from the patient induced complement activation on normal platelets. The results presented in this thesis show that LPS and complement deposition on platelets can activate platelets, release tissue factor and ultimately result in a prothrombotic state.

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