EXTRACELLULAR CALCIUM AND PLATELET GLYCOPROTEINS

Platelet membrane glycoproteins lib and Ilia form Ca⁺⁺-dependent heterodimer complexes that contain binding sites for fibrinogen and therefore are relevant to the ability of platelets to aggregate together. In this study we investigated the effects of extracellular Ca⁺⁺ on the stability and expression of IIb-IIIa complexes using a IIb-IIIa complex-specific monoclonal antibody M148. Its specificity was examined using crossed immunoelectrophoresis: the antibody reacted only with intact IIb-IIIa complexes and not with either glycoprotein alone.

SDS-polyacrylamide gel electrophoresis of immunoprecipitates of soluble glycoproteins that interacted with Ml48 showed that lib and Ilia were present as complexes in Ca⁺⁺-depleted media at 25°C, pH7.4. However, Ca⁺⁺-depletion at 37°C, pH7.4 or 37°C, pH8.7 or 25°C, pH8.7 caused dissociation of the complex

The effect of extracellular Ca⁺⁺ on the expression of IIb-Illa complexes on the surface of intact platelets was studied by a technique which is based upon indirect binding of M148 using a fluorescent- labelled second antibody (FITC-RAM) and measuring the fluorescence per platelet using the FACS IV cytofluorometer. Intact platelets were incubated in Ca⁺⁺-depleted media at 25°C, pH7.4 or 37°C, pH7.4 either (i) prior to or (ii) after adding M148. At 25°C increased M148-binding was observed, compared to the value prior to Ca⁺⁺-depletion. This increased binding could be reversed by adding Ca⁺⁺ back to the preparation. Under condition (i) at 37°C a marked decrease in M148 binding was observed, which could not be reversed by restoring Ca⁺⁺, while under condition (ii) at 37°C the results were the same as at 25°C.

Our studies demonstrate that (a) Ca⁺⁺-depletion at 37°C and/or alkaline pH causes dissociation of the Ilb-IIIa complex (b) Ca⁺⁺ depletion at 25°C possibly alters distribution of the complexes thereby increasing their availability to the antibody and (c) M148 prevents the dissociation of complexes in Ca⁺⁺-depleted media at 37°C, possibly by holding lib and Ilia together