Joutsi-Korhonen Lotta, Preston Sandy, Smethurst Peter A, Ijsseldijk Martin, Schaffner-Reckinger Elisabeth, Armour Kathryn L, Watkins Nicholas A, Clark Michael R, de Groot Philip G, Farndale Richard W, Ouwehand Willem H, Williamson Lorna M
University of Cambridge Department of Haematology, National Blood Service Cambridge, Long Road, Cambridge, UK.
Thromb Haemost. 2004 Apr;91(4):743-54. doi: 10.1160/TH03-07-0484.
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.
已开发出具有Fc且经过突变以消除破坏性效应功能的重组HPA-1a抗体，通过阻断人HPA-1a多克隆抗体与胎儿HPA-1a1b血小板的结合，将其作为胎儿母体同种免疫性血小板减少症（FMAIT）的一种潜在治疗方法。我们在三种HPA-1基因型的静息和激动剂刺激血小板的各种功能试验中评估了IgG1 HPA-1a抗体B2G1及其两种突变衍生物的效果。对于HPA-1a1b血小板（胎儿基因型），这些抗体在静息血小板中不激活信号传导或CD62P表达，不改变体外出血时间（IVBT），也不抑制血小板在流动血液中与胶原蛋白的粘附。HPA-1a1b血小板与纤维蛋白原的粘附减少了20%，由ADP诱导的聚集减少了50%，但胶原相关肽（CRP-XL）诱导的聚集正常。对于HPA-1a1a血小板，对ADP和CRP-XL的聚集均受到抑制，纤维蛋白原粘附和IVBT反应完全被阻断。有趣的是，具有相同特异性的单价抗体片段对聚集没有抑制作用。在使用HPA-1a（Leu(33)）表型的人αIIbβ3或αVβ3转染体的静态粘附试验中，HPA-1a抗体完全阻断了后者而非前者与纤维蛋白原的附着。这些观察结果最好通过空间位阻机制由抗体介导的对β3上RGD结合位点的阻断来解释。由于对HPA-1a1b（胎儿型）血小板的功能影响较小，本文所述的突变HPA-1a抗体可进一步开发用于FMAIT治疗。
