The Role Of Membrane-Bound Tubulin In Platelet Functions
Evidence has been presented which suggests the existence of tubulin, subunit protein of microtubules, as an integral part of plasma membrane of certain cells. We have investigated whether tubulin is also a constituent of platelet plasma membrane or not, and, if so, what the functional significance is? Platelet membranes isolated by glycerol lysis technique according to the method of Barber and Jamieson retained colchicine-binding activity, 6.2 ± 1.4 n mol colchicine per 100 mg platelet membranes. Colchicine-binding activity of platelet membranes was not decreased after membranes were washed 3 times, indicating that colchicinebinding activity of membranes is not due to contamination of loosely bound cytoplasmic soluble tubulin. On SDS-poly- acrylamide gel electrophoresis, platelet membranes revealed Coomassie blue stained band of molecular weight 55,000, which comigrated with purified cytoplasmic tubulin isolated from human platelets by two successive cycles of temperature -dependent polymerization depolymerization as described previously(Ikeda & Steiner, J. Biol. Chem. 251:6135, 1976). Monospecific antibody against platelet tubulin was prepared in rabbits by injecting soluble tubulin at weekly intervals for 4 weeks. Platelets preincubated with anti-tubulin F(ab’)2 fragment showed reduced platelet aggregation and shape change induced by collagen, but not by ADP or epinephrine. Collagen-induced release of 14C-serotonin was also inhibited by anti-tubulin F(ab’)2 fragment while ADP- or epinephrine-induced serotonin release was not inhibited(collagen 2μg/ml:45.6% of control, ADP 10μM:92.0% of control, epinephrine 4μg/ml: 98.0% of control).Our results suggest that membrane-associated tubulin may play important roles in collagen-platelet interactions.