AN EPITOPE OF A MONOCLONAL ANTIBODY (TM83) AGAINST GLYCOPROTEIN lib/Ilia COMPLEX

 

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GPIIb/lIIa is forming a heterodimer complex on the human platelet membrane.Many monoclonal antibodiesagainst GPIIb/IIIa complex obtained elsewhere react with neither GPIIb nor GPIIIa separately when GPIIb/IIIa is blotted to filter after SDS-PAGE. Therefore the epitope of GPIIb/IIIa complex is not identified actually. Our attemption is to clarify the epitope recognized by a monoclonal antibody against GPIIb/IIIa designated TM83. TM83 (30 yg/ml) inhibited collagen-, ADP-, or thrombin-induced aggregation, but it did not inhibit ATP-secretion induced by 0.01 U/ml of thrombin. TM83 also inhibited fibrinogen-binding approximately to 50 % of total binding. The binding of 125 I- TM83 to platelets decreased to b0% of controlwhen platelets were incubated in the presence of 1 mM EDTA at 37°C for 30 min. However the incubation at25°C for 30 min did not change any binding capacity of 125 I-TM83 to platelets. Thus thebinding of TM83to platelets was dependent on both temperature and calcium concentration in surrouding medium, suggesting that TM83 bound to GPIIb/IIIa complex.

If the small amounts of epitope of GPIIb/IIIa complex is not injured during SDS-PAGE and blotting, we may identify clearly the epitope of GPIIb/IIIa complex. For this aim, GPIIb/IIIa complex was extracted carefully in the presence of 1 mM calcium by the phase separationusing Triton X-11U, and was run on SDS-PAGE in the presence of 100 yM calcium. Western-blot of the membrane preparation showed that 125 I-TM83 was incorporated into both GPIIb and GPIIIa on Durapore filter. Further radio-crossed immunoelectrophoresis showed that I-TM83 was incorporated only into immunoprecipitin of GPIIb/IIIa complex in the presenceof 1 mM calcium. While after addition of 25 mM EDTA to the membrane preparation containing 1 mM calcium,125i-tm83 wasmainly incorporated into GPIIb/IIIa complex as well, however very faint radioactivity fromthe immunoprecipitin corresponding to GPIIIa was observed, but the radioactivity from GPIIb was not identified. While there is a discrepancy inour result, it must be further studied whether one monoclonal antibody can recognize or not two kinds of GPs, GPIIb and Ilia, which are formed in complex in physiological condition.