Investigation of new anti-clotting approaches

In normal haemostasis, key receptors on human blood platelets, glycoprotein (GP)Ibα of the GPIb-IX-V complex that binds von Willebrand factor (VWF) and GPVI that binds collagen, initiate rapid platelet activation and secretion of agonists such as ADP, leading to activation of the platelet integrin αIIbβ3 (GPIIb-IIIa) that mediates platelet aggregation (thrombus formation). The haemostatic response is critical for blood loss following injury, however defects in this process can increase bleeding risk, or lead to thrombotic disease such as myocardial infarction (MI) or stroke. It is currently poorly understood how changes in expression levels of these platelet-specific receptors, GPIbα and GPVI, on human platelets control platelet function and/or are related to bleeding/thrombotic risk in disease. The overall aim of studies described in this thesis is to develop reliable quantitative methods for analysing human platelet receptor expression and function in platelet-rich plasma or whole blood. Importantly, these flow cytometry-based methods use relatively small volumes of blood and can be used for analysis both in clinical samples at normal to low platelet count or normal to high platelet size, and in experimental systems including microfluidic analysis of thrombus formation using confocal imaging. The specific aims are (i) to analysis surface expression of platelet GPIbα, GPVI, αIIb (of the integrin αIIbβ3) and a tetraspanin CD9 (control) using phycoerythrin (PE)-conjugated mouse monoclonal antibodies in platelet-rich plasma from healthy donors with normal platelet size; (ii) to establish a new 2-colour flow cytometry-based assays for analysis of platelet GPIbα/GPVI expression in platelet-rich plasma and in whole blood from individuals with abnormally large platelets (macrothrombocytopenia), using PE-conjugated antibodies against GPIbα and GPVI together with an Alexa 488-conjugated anti-αIIbβ3 antibody (Abciximab) to overcome the difficulty of identifying oversized platelets in whole blood; and (iii) to evaluate the functional consequences of different expression levels of platelet GPIbα and GPVI on the rate and extent of thrombus formation on a collagen-coated surface under flow conditions using whole blood anticoagulated with the thrombin inhibitor, hirudin. The major results include establishing the normal range of expression levels of GPIbα, GPVI, αIIbβ3 and CD9 in >100 healthy donors. The 2-colour assay was validated using platelet-rich plasma and whole blood from healthy donors, and in a small number of whole blood samples with macrothrombocytopenia, demonstrating significantly lower GPIbα expression in 5 individuals with the rare inherited defect of GPIb-IX, Bernard-Soulier syndrome (BSS). Finally, the consequences of altered surface expression of platelet receptors was analysed by flowing blood at arterial shear rates over a collagen-coated surface ex vivo, where platelet adhesion is dependent on the GPIbα-VWF interaction. Analysis of healthy donors (n = 22) showed a significant correlation between surface expression levels of GPIbα and thrombus surface coverage and volume on a capillary coated with collagen type I at an input shear rate of 1800 s-1 under steady flow conditions, whereas a correlation of both GPIbα and GPVI and thrombus volume was observed at the same input shear rate (1800 s-1) but under pulsatile flow conditions (n = 8). For this analysis, thrombi of platelets stained with the membrane dye, DiOC6, were analysed over 3-15 min using confocal imaging of 0.5-micron z-sections under conditions of steady or pulsatile flow. Together, these studies are significant because they provide an approach for profiling receptor expression on human platelets in small samples of blood, including at low platelet count or where analysis may be complicated by increased platelet size, and relating changes in expression of these receptors to platelet thrombus formation under flow conditions. In future, these studies can increase understanding of potential anti-platelet targets to attenuate shear-dependent thrombosis, and provide new diagnostic approaches to evaluate bleeding or thrombotic risk and use of antiplatelet agents in individuals. Nvestigation of new anti-clotting approaches