Platelets stimulated with ADP bind fibr specifically, and this ability correlates well with their ability to aggregate when they are shaken with ADP and fibr. Others found that platelets incubated with chymotrypsin (CT) bind and aggregate with fibr without stimulation by ADP; as with ADP-induced stimulation, these responses are inhibited by excess EDTA. Phillips et al. showed that thrombin activation produced a large Triton-insoluble cytoskeleton in the presence of either EDTA or CaCl2, but

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... 125I-labeled glycoprotein(s) IIb-III were incorporated into the cytoskeleton only when CaCl2 was present and aggregation occurred. This system cannot be used to assess the role of fibr in membrane glycoprotein incorporation, as exogenous fibr is not required for thrombin-induced aggregation. We therefore used CT-treated platelets labeled with 125I. They were suspended in calcium-poor Hepes-buffered Tyrode's solution at 22°C for 30 min with 2.5 mM EDTA or 1 mM CaCl2 with or without 100 ug/ml fibr. Only samples with CaCl2 + fibr aggregated, and similar samples prepared with CT-treated platelets which had first been incubated with EDTA at 37°C for 15 min failed to aggregate despite recalcification. Cytoskeletons were prepared by adding an equal vol of 2% Triton X-100-5 mM EGTA-0.1 M Tris pH 7.4 and centrifuging 1 hr later. Cytoskeletal protein was 10-25% of total and did not vary with aggregability. CT-treated platelets showed more 125I bands than untreated labeled platelets. The two bands with highest Mr on SDS-PAGE were present only when the platelets aggregated during incubation, i.e., under conditions in which fibr is bound. Possibly the dimeric fibr molecule must link two surface molecules before they can attach to the cytoskeleton.