title: The effect of recombinant IgG antibodies against the leucine-33 form of the platelet beta3 integrin (HPA-1a) on platelet function.
abstract: Recombinant HPA-1a antibodies with Fc, mutated to remove destructive effector functions, have been developed as a potential therapy for fetomaternal alloimmune thrombocytopenia (FMAIT), via blockade of binding of human HPA-1a polyclonal antibodies to fetal HPA-1a1b platelets. We have assessed the effect of the IgG1 HPA-1a antibody B2G1 and two mutated derivatives in various functional assays in resting and agonist-stimulated platelets of the three HPA-1 genotypes. With HPA-1a1b platelets (fetal genotype), the antibodies did not activate signalling or CD62P expression in resting platelets, did not change in vitro bleeding time (IVBT), and did not inhibit platelet adhesion to collagen in flowing blood. Adhesion of HPA-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. With HPA-1a1a platelets, aggregation to both ADP and CRP-XL was inhibited, with total blockade of adhesion to fibrinogen and of IVBT responses. Interestingly, a monovalent antibody fragment with identical specificity had no inhibitory effect on aggregation. In static adhesion assays using human alphaIIbbeta3 or alphaVbeta3 transfectants of HPA-1a (Leu(33)) phenotype, attachment to fibrinogen of the latter but not of the former was completely blocked by the HPA-1a antibodies. These observations are best explained by antibody-mediated blockade of the RGD binding site on beta3 by a mechanism of steric hindrance. As the effect on platelet function is modest with HPA-1a1b (fetal type) platelets, the mutated HPA-1a antibodies described here could be developed further for FMAIT therapy.
authors.affiliation: University of Cambridge Department of Haematology, National Blood Service Cambridge, Long Road, Cambridge, UK.
authors.forename: Lotta
authors.initials: L
authors.lastname: Joutsi-Korhonen
authors.forename: Sandy
authors.initials: S
authors.lastname: Preston
authors.forename: Peter A
authors.initials: PA
authors.lastname: Smethurst
authors.forename: Martin
authors.initials: M
authors.lastname: Ijsseldijk
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Schaffner-Reckinger
authors.forename: Kathryn L
authors.initials: KL
authors.lastname: Armour
authors.forename: Nicholas A
authors.initials: NA
authors.lastname: Watkins
authors.forename: Michael R
authors.initials: MR
authors.lastname: Clark
authors.forename: Philip G
authors.initials: PG
authors.lastname: de Groot
authors.forename: Richard W
authors.initials: RW
authors.lastname: Farndale
authors.forename: Willem H
authors.initials: WH
authors.lastname: Ouwehand
authors.forename: Lorna M
authors.initials: LM
authors.lastname: Williamson
mesh_terms: D000906:Antibodies / Q000235:genetics / Q000276:immunology / Q000494:pharmacology*; D016824:Antigens, Human Platelet / Q000276:immunology*; D003094:Collagen / Q000378:metabolism; D005260:Female; D005340:Fibrinogen / Q000378:metabolism; D006801:Humans; D007074:Immunoglobulin G / Q000235:genetics / Q000494:pharmacology; D039302:Integrin alphaVbeta3 / Q000378:metabolism; D039661:Integrin beta3 / Q000276:immunology; D007930:Leucine; D015539:Platelet Activation / Q000276:immunology*; D010979:Platelet Function Tests; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000378:metabolism; D020641:Polymorphism, Single Nucleotide / Q000276:immunology*; D011247:Pregnancy; D015202:Protein Engineering; D011994:Recombinant Proteins
chemical_list: D000906:Antibodies; D016824:Antigens, Human Platelet; C061248:ITGB3 protein, human; D007074:Immunoglobulin G; D039302:Integrin alphaVbeta3; D039661:Integrin beta3; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D011994:Recombinant Proteins; D005340:Fibrinogen; D003094:Collagen; D007930:Leucine