Christopoulos, Constantinos; (1992) Flow cytometric observations in quantitative and qualitative platelet disorders and in humans receiving infusions of a chimaeric monoclonal antibody to glycoprotein IIb-IIIa. Doctoral thesis (M.D.), University College London.

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Abstract

A flow cytometric method was developed for quantitation of platelet surface-bound immunoglobulin G (IgG). A novel approach was used to calibrate the assay; this was based on the creation of reference curves by measuring PSIgO in platelets coated with known amounts of a chimaeric construct (supplied by Centocor Inc.) consisting of human IgG whose hypervariable region (HVR) had been replaced by the HVR of the murine monoclonal antibody (MoAb) 7E3, which binds with high affinity to human platelet glycoprotein (gp) Ilb-IIIa. By using a fluorescein- conjugated polyclonal anti-IgG it was calculated that platelets of healthy subjects (n=71) bear 1463 (SD=927) IgG molecules, whereas PSIgG elevations of up to 39,000 molecules/platelet were often detected in a cohort (n=87) of thrombocytopenic individuals. FC was also used to monitor platelet-bound 7E3, residual 7E3 binding sites and PSIgG instable angina patients receiving infusions of Fab fragments of chimaeric MoAb 7E3 (c7E3-Fab), which is currently under evaluation as an anti-platelet agent. The distribution of c7E3-Fab in the platelet population was unimodal at all time points following the infusion, demonstrating in vivo transfer of antibody to newly released platelets; clearance of platelet-bound antibody followed an exponential model but all circulating platelets were bearing c7E3-Fab at time points beyond the lifespan of the platelets exposed to c7E3-Fab during the infusion. Ex vivo and in vitro mixing experiments suggested that c7E3- Fab transfer between platelets was possible. TurbidometricaUy measured platelet aggregation response to ADP was shown to be a linear function of the logarithm of residual binding sites for 7E3. Infusion of c7E3-Fab was frequently associated with recruitment of IgG onto the platelet surface; in vitro evidence suggested that this was caused by naturally occurring IgG bindable to proteolytically derived Fab fragments of chimaeric but not murine 7E3-IgG. A new type of pseudothrombocytopenia, seen upon EDTA exposure of c7E3-Fab-bearing platelets was observed; this also appeared to be immune-mediated. The physiological, pathophysiological and therapeutic implications of these findings are discussed.

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