Isolation of microtubule coils from platelets after exposure to aggregating agents

Abstract

The discoid shape of human blood platelets is supported by a circumferential microtubule (MT) organized in many loops or coils. A recent study reported from the authors' laboratory demonstrated that significant numbers of MT rings could be isolated from resting platelets by simultaneous exposure to detergent and a small amount of fixative. This method has been used in the present investigation to determine the number of MT coils obtained from platelets after activation by ADP, thrombin, and the calcium ionophore, A23187. Concentrations of the agonists that caused shape change and internal transformation in parallel samples did not influence the frequency of MT rings present in activated samples after treatment with fixative and detergent. As many or more MT coils were present 5, 15, 30, 60, 90, and 120 seconds after addition of an agonist as from the control. Statistical analysis revealed no significant difference between the number of isolated coils from controls and activated platelets at any time during early activation. Immunofluorescence microscopic examination of platelets stained with a monoclonal antibody to tubulin at intervals of 5, 15, 30, 60, 90, and 120 seconds after activation on glass surfaces confirmed the suggestion that platelet MTs are resistant to disassembly during the early response to stimulation.