Effect of various extraction solutions and thrombin activation on the composition of the platelet cytoskeleton.
When human blood platelets were immersed in an ice-cold solution containing 1% Triton X-1200, 40 mM KCl, 10 mM EGTA, 10 mM imidazole-HCl, and 2 mM NaN3 pH 7.0, a flocculent precipitate appeared immediately in the tube. This precipitate was collected at 3,000g and SDS-polyacrylamide gel analysis showed it to consist mainly of actin, alpha-actinin, actin-binding protein (ABP), and varying amounts of myosin. Any modifications of this solution used to isolate the platelets' Triton-insoluble cytoskeleton caused profound changes in the nature of the cytoskeleton isolated. Increasing the KCl concentration resulted in a lower yield of cytoskeletal actin and ABP. Inclusion of EDTA in the solution resulted in an increased amount of myosin associated with the cytoskeleton, whereas including MgATP decreased the myosin yield. Experiments with the purified proteins showed that ABP and myosin can each protect the actin from depolymerizing when dialyzed into the Triton solubilization solution. In addition, it was found that when platelets were stimulated with thrombin for 2 min prior to the addition of the Triton solution, 3-4 times more myosin was associated with the cytoskeletal precipitate. The results suggest, therefore, that any variations in solution conditions used for isolating the cytoskeleton from resting platelets, which results in alterations in the amount of ABP, may have profound effects on the state of actin polymerization. Likewise, in thrombin-activated platelets, it is suggested that the increased association of myosin with the cytoskeleton results in a greater stabilization of the F-actin associated with the cytoskeleton. These factors must be considered when interpreting the results regarding the nature of actin transformations in the resting and activated platelet.
D000185:Actinin / Q000032:analysis; D000199:Actins / Q000032:analysis*; D000255:Adenosine Triphosphate / Q000494:pharmacology; D001704:Biopolymers; D001792:Blood Platelets / Q000032:analysis* / Q000648:ultrastructure; D002352:Carrier Proteins / Q000032:analysis; D003285:Contractile Proteins / Q000032:analysis*; D003599:Cytoskeleton / Q000032:analysis*; D004492:Edetic Acid / Q000494:pharmacology; D018260:Gelsolin; D006801:Humans; D008840:Microfilament Proteins; D009218:Myosins / Q000032:analysis; D013917:Thrombin / Q000494:pharmacology*
D000199:Actins; D001704:Biopolymers; D002352:Carrier Proteins; D003285:Contractile Proteins; D018260:Gelsolin; D008840:Microfilament Proteins; C016953:brevin; D000185:Actinin; D000255:Adenosine Triphosphate; D004492:Edetic Acid; D013917:Thrombin; D009218:Myosins