Characterization of Anti-Human GPVI Autoantibodies Mediating Persistent Target Immnunodepletion over a 15-Year Period.

Abstract

The first reported patient with platelet GPVI deficiency (Blood 1987; 69:1712) had anti-GPVI antibodies (YA-Abs) in the patient's serum, which contributed to later progress in identification of GPVI as a novel collagen receptor. Although there is now a concept that anti-GPVI Ab induces immunodepletion of GPVI in vivo and may become a promising therapeutic antithrombotic agent, precise nature of anti-human GPVI Ab mediating immunodepletion of GPVI is unknown. Since the patient's platelets are still lacking GPVI and unresponsive to collagen stimulation, we purified YA-Abs from the patient's serum kept in 1988 (YA-Abs-88) and in 2003 (YA-Abs-03) using sequential affinity chromatography on protein A-Sepharose and recombinant human GPVI fused to Fc (rhGPVI-Fc)-Sepharose. The amount of YA-Abs-03 in the serum was estimated to be about 3 μg/mL and twenty times less than that of YA-Abs-88. We also generated 75 mouse monoclonal antibodies (mAbs) against the extracellular part of human GPVI. Among those, several mAbs cross-reacted with YA-Abs for the binding to rhGPVI-Fc, indicating oligoclonal nature of YA-Abs. Two representative mAbs with no cross-reactivity, F1201 and F1232, cross-reacted with YA-Abs-88 and with both YA-Abs-88 and -03, respectively. Previous studies identified two distinct collagen binding sites of human GPVI; the one is a sequence including Lys59 of human GPVI which is recognized by 10B12 Ab, (Blood 2004; 103:903) and the other is a sequence including Gly30, Val34 and Leu36 which involves the binding to 9O12 Ab (J.Biol. Chem. 2004; 279:52293). Point mutated rhGPVI-Fc at Lys59 (K59E) was recognized by YA-Abs-88, -03, and F1232 but not by F1201. Another mutated rhGPVI-Fc in which human GPVI sequence from Pro31 to Leu36 was substituted for mouse GPVI counter sequence was recognized by any of those Abs. YA-Abs-88 and F1201but not F1232 evoked surface P-selectin expression or aggregation of human platelets, while F1232 inhibited collagen-induced aggregation. Since both F1201 and F1232 were found to react with nonhuman primate platelets, either mAb was intravenously injected into monkeys at a dose of 0.3 mg/kg that reached its maximum plasma concentration of 10 μg/mL. Within 1 to 2 days after injection, their platelets showed the lack of both GPVI and collagen-induced aggregation. Thus, GPVI-deficiency of the patient's platelets is now maintained by unexpectedly much decreased amount of specific Abs which contain F1232-like Ab but not F1201-like Ab included in YA-Abs-88. Immunodepletion of GPVI can be safely induced by specific Ab like F1232 recognizing different epitope from previously reported collagen binding sites where their cross-linking causes platelet activation.