PB0758 - An αIIbβ3 Ligand-Mimetic mAb Shows Platelet Activation (via Engaging FcγIIA Receptor) and Inhibition (via blocking αIIbβ3 Ligand Binding) and Serves as a Model of Disorders Involving FcγRIIA

Background: LB and JW contributed equally.
Platelet integrin receptor αIIbβ3 is required for platelet aggregation and clot retraction.

Aims: To produce an αIIbβ3 murine monoclonal antibody (mAb) that inhibits clot retraction.

Methods: BALB/cAnN mice were immunized with human platelets and hybridoma supernatants were tested for their ability to bind to αIIbβ3 and inhibit clot retraction.

Results: The IgG1 mAb R6H8 inhibited clot retraction for up to 3 hours at 20 µg/ml (Figure 1). Paradoxically, after 20 min of incubation at room temperature, R6H8 at 5 µg/ml initiated platelet aggregation immediately upon initiation of stirring; it also induced P-selectin expression, fibrinogen binding, and PAC-1 binding. At 20 µg/ml, however, R6H8 completely inhibited aggregation induced by 25 µg/ml PAR-1 activating peptide SFLLRN (T6). R6H8 at 20 µg/m also dramatically inhibited T6-induced fibrinogen and PAC-1 binding to platelets.
The platelet aggregation induced by 5 µg/ml R6H8 could be inhibited by mAb IV.3, which blocks the FcγIIA receptor and the Fab fragment of R6H8 did not induce platelet aggregation, supporting a model in which R6H8 binds to both αIIbβ3 and FcγRIIA.
Cryo-EM analysis of the R6H8 Fab–αIIbβ3 structure (Figure 2) revealed that R6H8: 1) Binds to the αIIbβ3 RGD binding pocket via an RYD sequence in its heavy chain CDR3, 2) Interacts with β3 Asp126 via an adjacent CDR3 Arg, leading to the reorientation of Asp126 and loss of the ADMIDAS Ca2+, and 3) Initiates swingout of the β3 hybrid domain away from the MIDAS.

Conclusion(s): We conclude that R6H8 is an αIIbβ3 ligand-mimetic mAb that activates platelets via FcγRIIA at low concentrations and potently inhibits platelet aggregation and clot retraction at high concentrations. R6H8 simulates the actions of a number of pathological antibodies, including a subpopulation of platelet-activating antibodies developed after therapy with glycoprotein IIb/IIIa (αIIbβ3) inhibitors and platelet-activating antibodies in heparin-induced thrombocytopenia and vaccine-induced immune thrombotic thrombocytopenia