Preparation of monospecific antibody against human platelet tubulin and its effect on platelet aggregation

Abstract

Monospecific antibody against human platelet tubulin was prepared. Human platelet tubulin was isolated by three successive cycles of polymerization using 5×1011 platelets obtained from healthy volunteers by Haemonetics Model 30 blood processor. Densitometric quantification of purified tubulin subjected to SDS-polyacrylamide gel electrophoresis revealed purity of 96%. Purified tubulin 400μg was given to rabbits with Freunds' adjuvant once a week four weeks. The rabbit anti-tubulin serum gave a single precipitin line on double immunodiffusion against platelet tubulin and the high speed supernatant of a platelet sonicate (platelet extract). Anti-tubulin F(ab′)2 fragment was prepared from anti-tubulin rabbit serum. Platelets preincubated with anti-tubulin F(ab′)2 showed decreased aggregation by collagen, but not by ADP or epinephrine.These results may suggest that tubulin, possibly membrane-associated one, plays important roles in the mechanism of platelet aggregation induced by collagen.