Isolation and characterization of actin and actin-binding protein from human platelets.

Abstract

Human blood platelets, which are highly motile cells essential for the maintenance of hemostasis, contain large quantities of actin and other contractile proteins . We have previously introduced a method (Lucas, R. C., T. C. Detwiler, and A . Stracher, J. Cell Biol ., 1976, 70(2, Pt . 2) :259 a) for the quantitative recovery of the platelets' cytoskeleton using a solution containing 1% Triton X-100 and 10 mM EGTA . This cytoskeleton contains most of the platelets' actin, actin-binding protein (ABP, subunit molecular weight = 260,000), and a 105,000-dalton protein. Negative staining of this Triton-insoluble residue on an EM grid shows it to consist of branched cables of actin filaments aligned in parallel .When this cytoskeletal structure is dissolved in high-salt solutions, the actin and ABP dissociate and can subsequently be separated . Here we will present simple and rapid methods for the individual purifications of platelet actin and platelet ABP.When purified actin and ABP are recombined in vitro, they are shown to be both necessary and sufficient for the reformation of the cytoskeletal complex. The reformed structure is visualized as a complex array of fibers, which at the EM level are seen to be bundles of actin filaments. The reformation of the cytoskeleton requires only that the actin be in the filamentous form-no accessory proteins, chelating agents, divalent cations, or energy sources are necessary.In vivo, however, the state of assembly of the platelets' cytoskeleton appears to be under the control of the intracellular concentration of free calcium . Under conditions where proteolysis is inhibited and EGTA is omitted from the Triton -solubiIization step, no cytoskeleton can be isolated . The ability of Ca 12 to control the assembly and disassembly of the platelets' cytoskeleton provides a mechanism for cytoskeletal involvement in shape change and pseudopod formation during platelet activation .

Human platelets contain gelsolin. A regulator of actin filament length.

“…We found previously that rabbit macrophage gelsolin changes the gel point of crosslinked actin networks, and this change correlates with a change in the length of the actin filaments (10). Rabbit skeletal muscle actin was crosslinked with rabbit macrophage actin-binding protein (ABP)', a protein also present in human platelets (4). The critical crosslinker concentration of a 23-,MM F-actin solution was 55 nM ABP.…”

Cytoskeletal domains in the activated platelet

“…3). Glycoprotein IB (150 kD) which binds to actin via filamin [34,38] is detected in blots (Fig. 3) but is not readily visualized by Coomassie staining of protein gels and thus appears to be retained at low levels.…”

“…Proteolysis was consistent, producing the same effect each time. Since extracts were prepared in the presence of high levels of six different proteases and EDTA, this proteolysis is likely to occur during activation, as has been described [34][35][36]. Since this is an intracellular proteolysis that occurs during shape change, it is unlikely to cause the loss of a significant actin binding protein involved in that shape change.…”

Clostridium perfringens iota toxin ADP‐ribosylates skeletal muscle actin in Arg‐177

“…Human platelet cytoskeletal actin was purified according to [13] with an additional gel filtration step as in [ 121. Rabbit skeletal muscle was purified as described [14].…”