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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.
Insights
Human blood platelets maintain their discoid shape due to a stable microtubule (MT) ring. Platelet activation by agonists like ADP or thrombin does not affect the number of MT rings, indicating their resistance to disassembly.
Area of Science:
- Cell Biology
- Hematology
- Biochemistry
Background:
- Human blood platelets possess a discoid shape maintained by a circumferential microtubule (MT) system.
- Previous research demonstrated the isolation of MT rings from resting platelets using detergent and fixative.
Purpose of the Study:
- To investigate the stability of platelet MT rings following activation by agonists.
- To quantify the number of MT rings in activated platelets compared to resting controls.
Main Methods:
- Platelets were activated with adenosine diphosphate (ADP), thrombin, and calcium ionophore A23187.
- Isolated MT rings were quantified after treatment with detergent and fixative.
- Immunofluorescence microscopy using anti-tubulin antibody examined MT stability post-activation.
Main Results:
- Platelet activation by various agonists did not alter the frequency of isolated MT rings.
- The number of MT coils remained consistent or increased slightly in activated platelets up to 120 seconds post-stimulation.
- Statistical analysis showed no significant difference in MT ring counts between control and activated platelets.
Conclusions:
- Platelet microtubules are resistant to disassembly during the early stages of activation.
- The structural integrity of the MT cytoskeleton is maintained despite shape change and internal transformations upon stimulation.