Platelet Microtubules in Clot Structure Formation and Contractile Force Generation: Investigation of a Controversy

SummaryWhether platelet microtubules are involved in clot retraction/ contraction has been controversial. To address this question we have simultaneously measured two clotting parameters, clot structural rigidity and isometric contractile force, using a rheological technique. For recalcified PRP clots these two parameters began rising together at about 15 min after CaCl2 addition. In the concentration range affecting microtubule organization in platelets, colchicine, vinca alkaloids and taxol demonstrated insignificant effects on both clotting parameters of a recalcified PRP clot. For PRP clots induced by adding small amounts of exogenous thrombin, the kinetic curves of clot rigidity were biphasic and without a lag time. The first phase corresponded to a platelet-independent network forming process, while the second phase corresponded to a platelet-dependent process. These PRP clots began generating contractile force at the onset of the second phase. For both rigidity and force parameters, only the second phase of clotting kinetics was retarded by microtubule affecting reagents. When PRP samples were clotted by adding a mixture of CaCl2 and thrombin, the second phase clotting was accelerated and became superimposed on the first phase. The inhibitory effects of micro tubule affecting reagents became less pronounced. Thrombin clotting of a two-component system (washed platelets/ purified fibrinogen) was also biphasic, with the second phase being microtubule-dependent. In conclusion, platelet microtubules are important in PRP clotted with low concentrations of thrombin, during which fibrin network formation precedes platelet-fibrin interactions. On the other hand they are unimportant if a PRP clot is induced by recalcification, during which the fibrin network is constructed in the presence of platelet-fibrin interactions. The latter is likely to be more analogous to physiological processes in vivo.