Recycling of platelet phosphorylation and cytoskeletal assembly.

Abstract

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 .

Platelet activation.

“…107 Phosphorylation is paradoxically also increased by addition of PGI 2 or dibutyryl cyclic AMP; 107 perhaps different sites are involved. Surprisingly, dephosphorylation occurs soon after addition of thrombin only if the platelets have been allowed to aggregate.…”

Platelet Stimulus-Response Coupling and Regulation of Cytoskeletal Assemblies

“…Returning to the oppositional control of these processes, we have recently demon strated that not only was the phosphorylation of myosin light chain completely reversed by PGh or dibutyryl cyclic AMP, but that the extent ofcytoskeletal core assembly and actin polymerization itself was reversed to preacti vation levels [73). The question of the revers ibility of actin-binding protein phosphoryla tion was obscured by its phosphorylation by a cyclic AMP-dependent process.…”

“…Unlike other typi cal platelet activators which can be inhibited or reversed by the oppositional control of cyclic AMP. the actin polymerization, cyto skeletal core assembly, and phosphorylation of platelet proteins induced by PMA is not inhibited by dibutyryl cyclic AMP and cyclic AMP elevating agents [74], It has been re cently suggested that this phorbol ester can directly activate platelet protein kinase C [75], which has been shown to phosphorylate in vitro the 47.000-dalton protein [28]. If the protein kinase C should turn out to be re sponsible for both actin-binding protein and 47,000-dalton protein phosphorylation, and PMA does not initiate myosin light-chain phosphorylation or causes myosin light chains to be phosphorylated at alternate sites, then the selective cytoskeletal formation by PMA could be more readily understood.…”

Erratum: Formation of galaxies and large-scale structure with cold dark matter

“…Reversible light chain phosphorylation also occurs at room temperature using the platelet activator arachidonic acid followed by prostacyclin, which increases cyclic AMP levels 24 • The presence or absence of myosin in the cytoskeletons was correlated with light chain phosphorylation, platelet aggregation and, to a considerable extent, platelet shape. The identification of heavy chain phosphorylation observed here when EDT A and fluoride are present during the lysis and wash steps (Fig.…”