ADP Binding Proteins in Thrombasthenic Platelet Membranes

Thrombasthenia is a severe congenital platelet disorder characterized by absence of ADP-induced platelet aggregation, diminished clot retraction and abnormalities of platelet membrane glycoproteins. The finding of normal ADP-induced shape change suggests that initial ADP binding might be normal. We have previously shown that the synthetic ADP analogue 5′-p-fluorosul-fonylbenzoyladenosine (5′FSBA) inhibits ADP-induced platelet shape change and covalently binds to specific proteins in normal platelet membranes. This affinity label was thus used to identify ADP binding proteins in platelet membranes from a patient with thrombasthenia. Isolated platelet membranes were incubated with 3H-5′-FSBA (0.1μM) at 37°C for 1 hr. then dissolved in 19% SDS, 8M urea, and 0.2M dithiothreitol, and subjected to SDS disc gel electrophoresis. The radiochromatograms of disc gels from 16 normal individuals demonstrated consistent labelling of polypeptides of 200, 120, 100, and 43 χ 103 daltons. The covalent binding was almost completely prevented by simultaneous incubation with a 100-fold excess of ADP or ATP, only partially prevented by AMP, adenosine and GDP, and was unaffected by UDP, thrombin, and epinephrine. When 5′FSBA was incubated with membranes from thrombasthenic platelets, the incorporation was identical to that found in normal membranes. Further the SDS-disc gel pattern showed the same 4 radiolabelled polypeptides despite the characteristic thrombasthenic membrane protein abnormalities. These experiments demonstrate that the ADP binding proteins of thrombasthenic platelet membranes are similar to those of normals and support the concept that the defect in thrombasthenia occurs subsequent to initial ADP binding.