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.
