Summary

Recombinant HPA-1a antibodies with Fc, mutated to removedestructive effector functions, have been developed as a potentialtherapy for fetomaternal alloimmune thrombocytopenia(FMAIT), via blockade of binding of human HPA-1a polyclonalantibodies to fetal HPA-1a1b platelets. We have assessed theeffect of the IgG1 HPA-1a antibody B2G1 and two mutatedderivatives in various functional assays in resting and agonist-stimulatedplatelets of the three HPA-1 genotypes. WithHPA-1a1b platelets (fetal genotype), the antibodies did notactivate signalling or CD62P expression in resting platelets, didnot change in vitro bleeding time (IVBT), and did not inhibitplatelet adhesion to collagen in flowing blood. Adhesion ofHPA-1a1b platelets to fibrinogen was reduced by 20%,and aggregation induced by ADP by 50%, but collagen-related peptide (CRP-XL)-induced aggregation was normal. WithHPA-1a1a platelets, aggregation to both ADP and CRP-XL wasinhibited, with total blockade of adhesion to fibrinogen and ofIVBT responses. Interestingly, a monovalent antibody fragmentwith identical specificity had no inhibitory effect on aggregation.In static adhesion assays using human αIibβ3 or αVβ3 transfectantsof HPA-1a (Leu ³³) phenotype, attachment to fibrinogen ofthe latter but not of the former was completely blocked by theHPA-1a antibodies. These observations are best explained byantibody-mediated blockade of the RGD binding site on β3by a mechanism of steric hindrance. As the effect on plateletfunction is modest with HPA-1a1b (fetal type) platelets, themutated HPA-1a antibodies described here could be developedfurther for FMAIT therapy.