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Publication

The carboxypeptidase U system in acute ischemic stroke

Book - Dissertation

Subtitle:translation from bench to bedside
Currently, only 20% of acute ischemic stroke (AIS) patients can be successfully treated. New treatment strategies are therefore urgently needed. The antifibrinolytic enzyme carboxypeptidase U (CPU) is an appealing target to improve stroke therapy. CPU circulates in plasma as an inactive zymogen, proCPU, that can be activated by thrombin, the thrombin-thrombomodulin complex, or plasmin. We aimed to assess the CPU system as a potential target to improve ischemic stroke treatment. Activity-based, immunologic and functional assays were optimized and further characterize for application in observational and clinical studies as well as in preclinical animal models of ischemic stroke. Special attention was paid to the pre-analytical phase which is key to accurate CPU measurement. Especially the impact of in vitro hemolysis on the measurement of the CPU system was assessed. Assay-specific cut-off values reflecting maximal allowable oxyhemoglobin levels were determined as significant inhibition of CPU activity was observed. In the preclinical part, a CPU inhibition strategy for the treatment of AIS was tested in a rat model of transient middle cerebral artery occlusion. Clear activation of the CPU system was observed in both saline- and rtPA-treated animals. Administration of the CPU inhibitor AZD9884 resulted in complete inhibition and reduced fibrinogen levels in the brain, which is a parameter of microvascular thrombosis (MT). Two observational studies were performed on AIS patients. CPU activity and CPU+CPUi antigen increased in patients upon arrival in the hospital compared to controls. In AIS patients receiving either rtPA or rtPA with endovascular thrombectomy (EVT), the CPU system was clearly activated. Maximum CPU and CPU+CPUi levels tended to be higher in patients undergoing additional EVT compared to those that received rtPA alone. Some findings point towards a potential role of MT in these patient populations but, large inter-individual variation was observed in CPU and CPU+CPUi kinetics. ProCPU levels were also quantified in the cerebrospinal fluid (CSF) of stroke patients that did not receive thrombolytic treatment and were increased compared to controls Patients with progressive stroke or poor outcome had higher proCPU CSF levels. The proCPU levels were also associated with blood-brain barrier dysfunction, which was found to be the likely cause of the increase. In the last part of this project, we performed the pharmacodynamic assessment of a novel CPU inhibitor in a first-in-man trial. The combined use of the CPU activity assay and the two in vitro clot lysis assays proved to be valuable to confirm the target engagement of the inhibitor.
Number of pages: 306
Publication year:2020
Keywords:Doctoral thesis
Accessibility:Closed