Microtubule inhibitors alter the secretion of β‐glucuronidase by human blood platelets: Involvement of microtubules in release reaction II

Abstract

The effects of the antimicrotubular drugs colchicine and vinblastine on the blood platelet release reaction were studied by measuring release of 14C-5-hydroxytryptamine (14C-5-HT, release I) and P-glucuronidase (release 11) from gel-filtered human platelets. P-glucuronidase release induced by thrombin was significantly inhibited by colchicine (0.01-1 mM) or vinblastine (0.05-0.1 mM). Release of 14C-5-HT, however, was unaffected a t low concentrations of colchicine and only slightly inhibited a t higher concentrations. Inhibition of P-glucuronidase release depended on colchicine or vinblastine concentrations and decreased with longer time intervals (l', 5', 20') after thrombin stimulation. Levels of the cytoplasmic enzyme, lactic acid dehydrogenase, in supernatants of colchicine treated platelets were not significantly different from controls. Colchicine also inhibited P-glucuronidase release, but not I4C-5-HT release, induced by trypsin and sodium arachidonate. Binding of 14C-colchicine by platelets was measured and it was found that platelet aggregation and release of 5-HT induced by adenosine diphosphate, epinephrine and collagen proceeded without any alteration in colchicine binding. However, significant increases in the rate and degree of colchicine binding were observed when platelets were stimulated by thrombin, trypsin and arachidonic acid which induced aggregation, release of both 5-HT and P-glucuronidase. The results suggest that an alteration in platelet microtubules is correlated with the physiologic response resulting in release I1 and that the cellular mechanisms effecting release I and I1 by platelets differ qualitatively in that the microtubules may facilitate release 11.When blood platelets participate in hemostasis and thrombogenesis, they interact with certain physiological substances and undergo a series of cellular reactions which includes aggregation and a release reaction. In the release reaction the contents of intracellular storage organelles are secreted into the extracellular medium (Holmsen e t al., '69). It has been shown that under certain conditions, the platelet release reaction actually proceeds in two, distinct phases (Holmsen and Day, '70). Release I constitutes the discharge of substances, as adenine nucleotides, calcium and 5-hydroxytryptamine (5-HT), stored in the dense bodies of platelets, and release I1 is marked by the extrusion of lysosomal enzymes and other materials, probably sequestered in platelet a-granules (Day and Holmsen, '71; Holmsen, '75).

The protein CD63 is in platelet dense granules, is deficient in a patient with Hermansky-Pudlak syndrome, and appears identical to granulophysin.

“…We obtained sequences on the first 37 amino-terminal amino acids of this protein and compared the predicted sequence with the SWISS-PROT protein library using the FASTA program (32). The sequences were identical to those of stagespecific melanoma antigen ME491 (11) and CD63 /pltgp4o (20,30). The blank position of signals in our sample corresponds to the cysteine residue in those proteins.…”

Effects of antimycin A and 2-deoxyglucose on secretion in human platelets. Differential inhibition of the secretion of acid hydrolases and adenine nucleotides

“…Since phosphorylation of myosin light chain precedes acid hydrolase secretion but not dense-granule secretion (Daniel et al, 1977), ATP-consuming contractile processes may be involved in the former but not in the latter secretory process in platelets. This is further supported by the specific effects on acid hydrolase secretion by cytochalasin B and vinca alkaloids (Kenney & Chao, 1978;Haslam et al, 1975).…”

“…Studies on the subcellular localization and the release kinetics of the granule-stored substances suggest that platelet secretion can be subdivided into a-granule secretion, dense-granule secretion and acid hydrolase secretion (Holmsen, 1978). Acid hydrolase secretion appears to be selectively activated by cytochalasin B (Haslam et al, 1975), inhibited by colchicine and vinblastine (Kenney & Chao, 1978) Vol. 182 and requires greater ATP availability than does dense-granule secretion (Hagen, 1972;Fukami et al, 1976;Holmsen & Robkin, 1977;Holmsen, 1978).…”

Antidote‐Controlled Antithrombotic Therapy Targeting Factor IXa and Von Willebrand Factor

“…Lysosomes contain several acid hydrolases (cathepsins) that digest endocytosed materials, and secretion occurs more slowly than does dense body or α-granule secretion. [19][20][21]…”