Platelet activation induces the formation of a stable gelsolin-actin complex from monomeric gelsolin.
We have studied the interactions between gelsolin and actin in crude extracts from activated and unactivated platelets and in mixtures of purified platelet gelsolin and muscle actin. Extracts were prepared using 10 mM EGTA from human platelets treated either with 100 microM aspirin and 2.5 mM tetracaine to retard activation or with the calcium ionophore A23187 to effect activation. The extracts were fractionated by gel filtration on Sephadex G-150 or by sedimentation on sucrose gradients and then analyzed using anti-gelsolin immunoblots and actin filament nucleation assays. The nucleation activity in both extracts was associated with gelsolin. The activity in the extracts from unactivated platelets sedimented with an S value of 5.2 and had an Mr = 90,000. The activity in the extracts prepared with EGTA from activated platelets sedimented at 6.8 S and had an Mr = 130,000. We have shown previously that the Mr = 130,000 species is an EGTA-stable binary complex of one actin and one gelsolin. Transient exposure of the extracts from unactivated platelets to 100 microM Ca2+ and subsequent fractionation in EGTA-containing buffers demonstrated that the formation of the binary complex occurs in the presence of Ca2+. Fractionation in the presence of 100 microM Ca2+ demonstrated higher order complexes including a ternary complex with a sedimentation constant of 8.2 S and an Mr = 165,000. Sedimentation and gel filtration experiments using purified platelet gelsolin and rabbit skeletal muscle actin demonstrated that formation of the EGTA-stable binary complex required Ca2+. At least one additional actin is bound to the binary complex in the presence of Ca2+, but is not sufficiently stable to be purified when EGTA is added. The results suggest that gelsolin exists either as a monomer or perhaps as a weak complex with actin in unactivated platelets but complexes tightly with actin during the transient Ca2+ rise that occurs during activation.
D000199:Actins / Q000097:blood*; D000244:Adenosine Diphosphate / Q000494:pharmacology; D001241:Aspirin / Q000494:pharmacology; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000097:blood; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D004533:Egtazic Acid / Q000494:pharmacology; D018260:Gelsolin; D006801:Humans; D008970:Molecular Weight; D013748:Tetracaine / Q000494:pharmacology; D013917:Thrombin / Q000494:pharmacology
D000199:Actins; D002135:Calcium-Binding Proteins; D018260:Gelsolin; D013748:Tetracaine; D000001:Calcimycin; D004533:Egtazic Acid; D000244:Adenosine Diphosphate; D013917:Thrombin; D001241:Aspirin; D002118:Calcium