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Regulation of platelet receptors in health and disease
thesisposted on 2017-02-06, 02:22 authored by Qiao, Jianlin
Platelet-specific receptors glycoprotein (GP)VI that binds collagen, and GPIbα, a subunit of GPIb-IX-V complex that binds von Willebrand factor (VWF), initiate platelet adhesion, activation and aggregation in response to exposed collagen/VWF or elevated fluid-shear stress in haemostasis or arterial thrombosis. The overall hypothesis underlying studies in this thesis is that quantitative assessment of platelet receptor expression, function and proteolytic status could increase understanding of mechanisms underlying platelet receptor shedding, and enable more detailed analysis of thrombotic/bleeding risk and/or platelet production/clearance abnormalities in a range of human diseases. New analyses focusing on platelet-specific GPIbα, GPVI and associated receptors in healthy individuals (Chapter II) revealed that: 1, surface expression of GPIbα, GPVI, CD9 or αIIbβ3 are independent of platelet count in platelet-rich plasma (PRP); 2, surface expression of GPVI, CD9 or αIIbβ3 are independent of mean platelet volume (MPV); 3, surface expression of GPIbα directly correlates with MPV, supporting a role for GPIbα in regulating platelet size; 4, surface expression of GPVI strongly correlates with shed soluble GPVI (sGPVI) levels in human plasma; 5, immature platelet fraction (IPF%) is independent of platelet count, but directly correlates with MPV, consistent with younger platelets being larger. This methodology was applicable to the analysis of samples from patients with platelet-related disorders. First, in a patient with myelodysplastic syndrome (MDS) associated with a selective GPVI-dependent defect (Chapter III), platelet signaling pathways initiated by collagen or collagen-related peptide (CRP) were distinct from signaling by convulxin (which activates platelets via GPIbα), and defective GPVI-dependent platelet reactivity was associated with aberrant proteolysis of Syk. Second, in patients with lymphoproliferative disease before or during treatment with ABT-263 (an apoptosis drug) (Chapter IV), surface expression of GPVI and GPIbα, as well as CD9, was significantly lower in all patients with lymphoproliferative disease, prior to treatment with ABT-263, following treatment in the same individuals, and in additional patients with steady-state ABT-263 treatment. This drug, therefore, does not significantly affect platelet receptor expression at doses used in patients with lymphoproliferative disease. Further, it was shown using a novel two-colour flow cytometry-based method for quantitation of GPIbα proteolytic status, that patients with immune thrombocytopenia (ITP) (Chapter V) exhibited significantly lower expression of GPVI and GPIbα, likely due to increased shedding, as indicated by higher plasma sGPVI levels and lower GPIbα intact:total ratio. These results imply that analysis of surface receptor expression/shedding is a potentially useful marker for diagnosis or evaluating response to drug treatment in thrombocytopenia. Another new ELISA assay designed to measure soluble FcγRIIa levels in human plasma (Chapter VI) revealed that soluble FcγRIIa in human plasma may be derived from human platelets by mechanism(s) other than metalloproteinase-mediated ectodomain shedding. It was additionally shown that the FcγRIIa genotype at position 27 and 131 influences FcγRIIa stability in healthy individuals. Together, these combined results test the potential utility of systematic analysis of platelet-specific receptors GPIbα and GPVI, for investigating regulatory mechanisms underlying their expression/shedding, as well as revealing diagnostic potential for discriminating different causes of immune or non-immune thrombocytopenia due to production or destruction defects.