Abstract. To investigate the association of the putative platelet fibrinogen receptor (glycoprotein IIbIIIa) with the cytoskeleton, '25I-surface labeled human platelets washed by gel-filtration were activated under conditions which allow selective assembly of the platelet cytoskeleton. assembly was not altered. The lIla retention correlated with extent of aggregation with maximal retention corresponding to full aggregation. To determine if cytoskeletal development is necessary for the expression of the fibrinogen binding site, binding studies were performed with unlabeled platelets and '25I-fibrinogen. The mean number of binding sites and the mean dissociation constant were not significantly different among the four activation conditions. Although the development of a platelet cytoskeletal core is not required for the expression of the fibrinogen binding site, the retention of the glycoprotein IIb-IIIa complex is dependent on fibrinogensupported aggregation as well as the formation of the pseudopodal cytoskeleton.
The origin and physiological significance of the multiple Mr forms of phosphoinositide-specific phospholipase C in human platelets were investigated. The higher-Mr (400,000 and 270,000) forms of the phospholipase C were converted into the 100,000-Mr form without substantial loss of activity by incubation with a Ca2+-dependent proteinase partially purified from human platelets. These three forms of the phospholipase C were purified approx. 200-500-fold from outdated human platelet supernatants. SDS/polyacrylamide-gel electrophoresis and gel-filtration analysis suggested that the higher-Mr forms of phospholipase C were complexes of 140,000-Mr subunits, whereas the lower-Mr form consisted of a single 95,000-Mr subunit. The substrate specificity of the purified phospholipase C was investigated by using 32P-labelled polyphosphoinositide substrates purified from human platelets by a new method utilizing h.p.l.c. on an amino column. Activity against all three phosphoinositides was detected at micromolar concentrations of Ca2+; this hydrolysis was markedly stimulated by phosphatidylethanolamine and inhibited by phosphatidylcholine. Comparison of the different forms of purified phospholipase C revealed no major differences in Ca2+-sensitivity or substrate specificity. Thus, although the suggestion that the high-Mr forms of human platelet phosphoinositide-specific phospholipase C were converted into a lower-Mr form by a Ca2+-dependent proteinase has been substantiated, the physiological significance of this process remains to be determined.
The shape change and aggregation of washed platelets induced by 10 AM arachidonic acid (AA) can be reversed by 20 ng/ml prostacyclin (PGI2), but these platelets can be reactivated by treatment with 30 AM epinephrine and subsequent addition of 10 AM AA mixture . These events may be modulated by CAMP since 2 mM dibutyryl CAMP also reversed activation without reactivation by epinephrine and AA. We examined protein phosphorylation and formation of cytoskeletal cores resistant to 1 % Triton X-100 extraction of these platelets and correlated these processes with aggregation, fibrinogen binding, and changes in ultrastructure. Unactivated platelet cores contained <15% of the total actin and no detectable myosin or actin-binding protein . AA-induced cytoskeletal cores, which contained 60-80% of the total actin, myosin, and actin-binding protein as the major components, were disassembled back to unactivated levels by PGI2 and then fully reassembled by epinephrine and AA. Phosphorylation of myosin light chain and a 40,000-dalton protein triggered by AA (two-to fivefold) was reversed to basal levels by PG1 2 but was completely restored to peak levels upon addition of the epinephrine and AA mixture . The reversibility of actin-binding protein phosphorylation could not be established clearly because both PGI2 and dibutyryl CAMP caused its phosphorylation independent of activation . With this possible exception, cytoskeletal assembly with associated protein phosphorylation, aggregation, fibrinogen binding, and changes in ultrastructure triggered by activation are readily and concertedly recyclable .
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