Abstract 1937: Generation of a Novel Anti-Human GPIb-Alpha Inhibitor
HIb-1 is a human single chain antibody (ScFv) originally obtained from the Griffin.1 library that recognizes an epitope in the 45 kDa amino terminal region of GPIb-alpha. The present studies were undertaken to explore the ability of both of HIb-1 ScFv and an IgG form to inhibit platelet aggregation in transgenic mice in which the human GPIb-alpha chain has functionally replaced the corresponding murine protein. Surface plasmon resonance analysis was used to establish dissociation constants (KD) between HIb-1and either the extracellular domain of GPIb (GC) derived from human platelets or the CHO-cell secreted GPIb recombinant protein (WT). Lumi-aggregometry was performed using murine platelet rich plasma (PRP) obtained from either normal or transgenic mice following incubation with our antibody. Ristocetin was then added, followed by the addition of human plasma. With GC immobilized on dextran sulfate, the KD for binding by HIb-1 was 600nM. With the WT, the KD was only slightly higher at 900 nM. We additionally studied a double mutant increase-of-function GPIb containing G to V and M to V substitutions. The KD of HIb-1 binding to the double mutant was virtually identical to that of the WT. Addition of ristocetin to the murine PRP resulted in no aggregation in the absence of added human plasma, upon which aggregation then ensued. At concentrations up to 10 ug/mL, both the the ScFv and the IgG forms of HIb-1 reduced the initial rate of aggregation by approximately half; in the case of the IgG form, total extent of aggregation was also reduced by a similar amount. The murine mab C-34, in contrast, produced complete inhibition of ristocetin-induced aggregation; subsequent addition of ADP to these platelets, however, demonstrated a fully normal responsiveness to this agonist. HIb-1 shows strong binding to GPIb, comparable in intensity to that seen with the GPIb mab SZ-2. Transgenic mice with a human GPIb-alpha chain can be aggregated by ristocetin, as long as a source of human vWF is provided. Both murine mabs and mabs derived from human ScFv are capable of inhibiting such aggregation in vitro. These studies provide the necessary foundation for exploring the potential in vivo anti-thrombotic properties of such antibodies in this model system.
This research has received full or partial funding support from the American Heart Association, AHA National Center.