Doloretta D. Dawicki

The role of microtubules in platelet secretory release

The role of microtubules in platelet aggregation and secretion has been analyzed using platelets permeabilized with digitonin and monoclonal antibodies to alpha (DM1A) and beta (DM1B) subunits of tubulin. Permeabilized platelets were able to undergo aggregation and secretory release. However, threshold doses of agonists capable of eliciting a second wave of aggregation and the platelet release reaction were higher than in control platelets exposed to dimethyl sulfoxide, the solvent for digitonin. Both antibodies to alpha and beta tubulin caused a further increase in the threshold concentration of agonists and inhibited the secretory release of permeabilized platelets, but were ineffective using intact platelets. Neither monoclonal antibody inhibited polymerization or depolymerization of platelet tubulin in vitro. Antibodies to platelet actin and myosin also exhibited an inhibitory activity on platelet aggregation albeit less severe than that observed with the antibodies to alpha and beta tubulin. There was evidence of an interaction between DM1A and DM1B and the antibodies to actin and myosin. The interaction of platelet tubulin and myosin was investigated by two different methods. (1) Coprecipitation of the proteins at low ionic strength at which tubulin by itself did not precipitate and (2) affinity chromatography on columns of immobilized myosin. Tubulin freed of its associated proteins (MAPs) by phosphocellulose chromatography bound to myosin in a molar ratio which approached 2. Platelet actin competed with tubulin for 1 binding site on the myosin molecule. MAPs also reduced the binding stoichiometry of tubulin/myosin. Treatment of microtubule protein with p-chloromercuribenzoate or colchicine did not influence its binding to myosin. DM1A and DM1B inhibited the interaction of tubulin and myosin. This effect could also be demonstrated by reaction of electrophoretic transblots of extracted platelet tubulin with the respective proteins. We interpret these results as evidence for an interference of the two monoclonal antibodies to the tubulin subunits (DM1A and DM1B) with the translocation of microtubule protein from its submembranous site to a more central one during the activation process.

Permeabilization of platelets: an investigation of biochemical, ultrastructural and functional aspects

The biochemical, ultrastructural and functional aspects of digitonin-permeabilized platelets were investigated. Human platelets were permeabilized by exposure to the steroid glycoside digitonin. A 60 microM concentration of this permeabilizer produced a very substantial release of cytosolic enzymes from the platelets. Release from subcellular granules was relatively low and did not inhibit the response of platelets to a series of agonists. Although digitonin-permeabilized platelets required higher threshold concentrations of the usual stimulants, both primary and secondary aggregation as well as the release of nucleotides and enzymes from their respective granules remained intact. Transmission electron micrographs revealed discontinuities in the plasma membrane of digitonin-treated platelets, but scanning electron microscopy showed no difference between control and permeabilized platelets. No substantial loss of structural or membrane proteins could be detected by one- and two-dimensional gel electrophoresis. The pore size produced by digitonin treatment was sufficient to allow entry of 125I-labeled IgG into the platelet cytosolic space.

Studies on the mechanism of short-term regulation of pulmonary artery endothelial cell Na/K pump activity

The Na/K pump is critically important in maintenance of cell homeostasis in the face of injury. Little is known about the regulation of endothelial cell Na/K-pump activity. We previously reported that short-term (30-minute) oxidant-induced endothelial cell perturbation increased Na/K-pump activity in intact monolayers of bovine pulmonary artery endothelial cells (BPAECs). In this study we investigated the mechanism of oxidant-induced increases in endothelial Na/K-pump activity, focusing on short-term modulation of alpha1-pump subunit. By using immunofluorescence microscopy and confocal scanning laser microscopy, we found alpha1 subunit on both apical and basal aspects of BPAECs without polarized distribution. Short-term (30-minute) incubation of PAEC monolayers with H2O2 (1 mmol/L) did not change the relative amounts of alpha1 subunit in membrane fractions, as assessed by immunoblotting. Phosphorylation of the alpha1 subunit also was not affected by H2O2 treatment. Because protein kinases have been reported to alter Na/K-pump activity in several tissues and because H2O2 has been reported to increase PKC activity of endothelial cells, we determined the effects of inhibition and activation of protein kinase C (PKC) on Na/K-pump activity quantitated as ouabain-inhibitable uptake of 86Rb. We also determined the effects of PKC activation and inhibition on H2O2-induced increases in Na/K-pump activity. Inhibitors of PKC increased Na/K-pump activity over a 30-minute period in intact monolayers. Inhibition or depletion of PKC did not prevent H2O2-induced increases in pump activity. These results indicate that PKC is an endogenous regulator of pulmonary artery endothelial cell Na/K-pump activity but that the effects of H2O2 are not mediated by activation of PKC or by changes in the expression or phosphorylation of alpha1 subunit.

Differences in nucleotide effects on intracellular pH, Na+/H+ antiport activity, and ATP-binding proteins in endothelial cells

SummaryBovine (BPAEC) and human (HPAEC) pulmonary artery endothelial cell monolayers were incubated with either ATP, ATP analogues, or UTP, followed by measurement of intracellular pH (pHi) and the rate of recovery from acidosis. ATP increased baseline pHi and the rate of acid recovery in BPAEC. This response was inhibited by the amiloride analogue, methyisobutylamiloride, demonstrating that activation of the Na+/H+ antiport was responsible for the increase in baseline pHi and the recovery from acidosis. This response had the features of both a P2Y and P2U purinergic receptor, based on the responses to a series of ATP analogues and UTP. In contrast, none of the nucleotides had any significant effect on pHi and Na+/H+ antiport activity in HPAEC. This difference in the response to extracellular nucleotides was not due to a difference in ATP metabolism between cell types, since the ectonucleotidase-resistant analogue, ATPγS, also had no effect on HPAEC. Analogues of cAMP had no effect on pHi or acid recovery in either cell type. Incubation of BPAEC and HPAEC with the photoaffinity ligand [32P] 8-AzATP indicated that both BPAEC and HPAEC possess an ATP-binding protein of 48 kDa. However, BPAEC exhibited an additional binding protein of 87 kDa. Thus, the contrasting response to extracellular ATP between bovine and human pulmonary artery endothelial cells may be related to differences in the signal transduction pathway leading to antiport activation, including different ATP-binding sites on the cell membrane.

Agonist-induced changes of platelet tubulin phosphorylation

Changes in the phosphorylation of platelet tubulin were analyzed as a function of platelet activation. Non-activated platelets incubated with [32P]-phosphate showed multiple peaks of radioactivity when solubilized platelet proteins were analyzed by SDS-polyacrylamide gradient gel electrophoresis. Both tubulin monomers were found to be phosphorylated. Agonistic stimulation (thrombin or 1,2-diacylglycerol) resulted in a lowering of the phosphate incorporation into alpha- and beta-tubulin. Such changes we believe are important in the modulation of the reversible polymerization-depolymerization of platelet tubulin that occurs in the course of the agonistic stimulation of platelets.

Time resolved analysis of tubulin phosphorylation during platelet activation

Tubulin phosphorylation was analyzed during the different phases of platelet activation. Platelets preloaded with [32P]-phosphate were stimulated with collagen. Tubulin was immunoprecipitated from serial samples obtained during the activation process. The immunoprecipitates were resolved by SDS-polyacrylamide gel electrophoresis and autoradiographs analyzed by laser densitometry. Agonist induced dephosphorylation of platelets occurred after the onset of shape change at the time of initiation of the secretory release. The dephosphorylation was selective affecting specific peptides.