Role of Monocytes and Neutrophils in Thrombosis
Arterial thrombosis is the major trigger of myocardial infarction and ischaemic stroke. Although platelets are the most abundant cell in arterial thrombi, platelets interact closely with leukocytes such as monocytes and neutrophils and modify their activity. This thesis aims to identify novel mechanisms through which leukocytes and platelet-leukocyte interactions might contribute to thrombosis. The thesis focused on rapid interactions between these cells (on the order of minutes) as these interactions might contribute to initiation of thrombosis. Experimental methods used include microplate-based assays and flow cytometry. Firstly, physical interactions between platelets and leukocytes were studied via flow cytometry following whole blood stimulation with multiple platelet agonists. This identified monocytes and neutrophils as the major leukocyte subtypes to which activated platelets rapidly adhere regardless of the agonist used. Secondly, it was found that activated platelets adhering to monocytes caused the monocytes to rapidly expose on their surface tissue factor, a potent activator of the blood coagulation cascade. Previously reported to take hours, it was instead found that this process could occur on a timescale as short as five minutes, which, if active, might lead to rapid thrombus growth. Whole blood as well as isolated platelets and monocytes were used to study the mechanisms involved and the interaction between platelet P-selectin and its receptor PSGL-1 found on monocytes was identified as both necessary and sufficient to induce this event. The process was successfully inhibited with P-selectin and PSGL-1 blocking antibodies and could be induced in isolated monocytes by the addition of P-selectin-coated beads. Finally, the involvement of monocytes and neutrophils in the generation of the potent procoagulant enzyme thrombin was studied. Using a modified thrombin generation assay, it was found that monocytes do not contribute to the activation of thrombin in human plasma under these conditions, whereas neutrophils do. Furthermore, partial platelet depletion of human plasma revealed that neutrophils were able to maintain normal thrombin activity and support coagulation at platelet concentrations corresponding to mild thrombocytopenia (low platelet count). Neutrophils by themselves resulted in a significant level of thrombin generation following activation with tissue factor or with a protease-activated receptor 2 (PAR2)-specific agonist, even in the complete absence of platelets. Various inhibitors of neutrophil functions and neutrophil granule enzymes were tested to identify the mechanisms involved. Platelets are by far the most well studied cell type involved in thrombosis, whereas the role of leukocytes is less clear. The direct involvement of neutrophils in thrombin generation described in this thesis provides a novel mechanism that could lead to thrombus initiation.
platelets, monocytes, neutrophils, thrombosis
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