Biomedical subjects

K M Lerea

Publications and source records attributed to K M Lerea.

7 recordsLinked to original sources

PubMed1992

Inhibitors of protein phosphatase type 1 and 2A attenuate phosphatidylinositol metabolism and Ca(2+)-transients in human platelets. Role of a cdc2-related protein kinase.

The addition of either okadaic acid or calyculin A desensitizes human platelets to thrombin. One objective of this study was to determine which step(s) leading to secretion reactions may be affected by these protein phosphatase inhibitors. In a dose-dependent manner, okadaic acid or calyculin A inhibits phosphatidylinositol metabolism and Ca(2+)-transients. In all cases, calyculin A was approximately 10-fold more potent than okadaic acid, and it had maximal effects at a concentration of 1 microM. Although thrombin-induced rises in [Ca2+]i were diminished, an increase in the phosphorylation state of myosin light chains (MLC) was still observed. Changes in this phosphorylation were diminished, however, following the addition of thrombin to calyculin A-treated platelets that were loaded with dimethyl-BAPTA. These data demonstrate that calyculin A and okadaic acid lower agonist-induced Ca(2+)-transients, which in turn prevents responses such as secretion reactions. Calyculin A/okadaic acid-induced phosphorylation events were not diminished in BAPTA-loaded platelets, suggesting that these phosphorylations are Ca(2+)-insensitive. Thus, a second objective of this study was to identify the protein kinase(s) that was(were) responsible for the calyculin A-induced phosphorylations. In a platelet lysate system, calyculin A caused an increase in the incorporation of [32P]phosphate into p50. This phosphorylation event was identical to that observed in the intact platelet and was not mimicked by cAMP, cGMP, Ca2+, or a Ca2+/phospholipid/diacylglycerol mixture. Kinase activity was removed after the lysate was incubated with p13suc1-Sepharose. This suggests that a p13suc1-sensitive protein kinase, e.g., a cell cycle-dependent protein kinase, is responsible for the calyculin A-sensitive phosphorylation events.(ABSTRACT TRUNCATED AT 250 WORDS)

Blood Platelets↗

PubMed1991

Thrombin-induced effects are selectively inhibited following treatment of intact human platelets with okadaic acid.

The involvement of protein phosphatases in regulating platelet activation was studied. The major portion of the phosphorylase phosphatase activity found in platelet lysates appears to be of the type 1 variety. The identification of this enzyme was based on the finding that greater than 80% of protein phosphatase activity was inhibited by the heat-stable inhibitor protein inhibitor 2 and, while only 20% of the phosphorylase phosphatase activity in platelet extracts was inhibited by 2 nM okadaic acid, greater than 95% of the activity was inhibited in the presence of 1 microM okadaic acid. Increases in protein phosphorylations occurred and thrombin-induced release of serotonin was prevented as a result of artificially inhibiting the enzyme with okadaic acid in intact platelets. This implies either that the regulation of okadaic acid sensitive protein phosphatases is necessary for some agonist-induced effects or that okadaic acid sensitive phosphatases are required for maintaining platelets in a responsive state.

Blood Platelets↗

PubMed1989

Vanadate and molybdate increase tyrosine phosphorylation in a 50-kilodalton protein and stimulate secretion in electropermeabilized platelets.

Addition of vanadate and molybdate to electropermeabilized human platelets caused a time- and dose-dependent increase in the phosphotyrosyl content of 50- and 38-kDa proteins. This effect can most likely be attributed to an inhibition of protein-tyrosine-phosphatase activity because vanadate and molybdate inhibited this activity in platelet extracts by greater than 97% while causing an increase in tyrosyl phosphorylation of artificial substrates that had been added to the same extracts. The addition of vanadate and molybdate to the electropermeabilized platelets also induced an increase in serotonin and PDGF secretion. Interestingly, the secretion of these components tightly correlated in a time- and dose-dependent fashion with the phosphorylation of the 50-kDa protein on tyrosyl residues. This suggests that the tyrosine phosphorylation of this protein may be closely linked to the platelet activation cascade.

Blood Platelets↗

PubMed1988

Phosphorylation results in activation of a cAMP phosphodiesterase in human platelets.

Agents such as prostaglandins E1 and I2 which elevate cAMP levels in platelets also increase cAMP phosphodiesterase activity. Since much of the cAMP phosphodiesterase activity in human platelets is due to the cGMP-inhibited isozyme (Macphee, C. H., Harrison, S. A., and Beavo, J. A. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 6600-6663), we examined the regulation of this isozyme by prostaglandins E1 and I2 in intact platelets. Because this isozyme is a minor component of platelet protein, normally requiring several thousand-fold purification to achieve homogeneity, a specific monoclonal antibody (CGI-5) was utilized to identify and isolate the cGMP-inhibited phosphodiesterase activity. Treatment of intact platelets with the prostaglandins promoted an increase in the phosphorylation state of the cGMP-inhibited phosphodiesterase and a corresponding increase in phosphodiesterase activity. The effect on activity and phosphorylation of the cGMP-inhibited phosphodiesterase was observed within 2 min after intact platelets were exposed to the prostaglandins. The half-maximal effective dose for prostaglandin I2 (10 nM) was approximately 10-fold lower than that for prostaglandin E1. The phosphorylated, cGMP-inhibited isozyme migrated as a 110-kDa peptide following sodium dodecyl sulfate gel electrophoresis. Direct in vitro phosphorylation of the platelet cGMP-inhibited phosphodiesterase by the catalytic subunit of cAMP-dependent protein kinase caused a similar increase in phosphodiesterase activity. Treatment with PKI peptide, a specific inhibitor of cAMP-dependent protein kinase, blocked the phosphorylation and the effect on activity. Taken together, the data strongly suggest that the effects of prostaglandins E1 and I2 on platelet phosphodiesterase activity are mediated by a direct cAMP-dependent protein kinase-catalyzed phosphorylation of the cGMP-inhibited phosphodiesterase isozyme.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1987

Agents that elevate cAMP levels in platelets decrease thrombin binding.

The effect of high intracellular levels of cAMP on the ability of rabbit and human platelets to bind and respond to thrombin was examined. Control rabbit platelets differed from human platelets in two interesting respects: they showed thrombin-dependent up-regulation of thrombin binding, but also a 3- to 5-fold lower thrombin-binding capacity. Nevertheless, treatment with prostaglandin E1 + theophylline or with forskolin decreased thrombin binding to both rabbit and human platelets by 60 to 70%. This effect was associated with a marked increase in the level of cAMP and seemed to depend on a decrease in number rather than affinity of thrombin-binding sites. Changes in thrombin binding correlated closely with changes in thrombin-stimulated incorporation of 32Pi into phosphatidic acid and a 40-kDa protein. However, regardless of the amount of thrombin that bound to treated platelets, thrombin-stimulated phosphorylation of a 20-kDa protein and serotonin secretion were severely inhibited. Thus, increased levels of platelet cAMP are associated with a reduced ability to bind and respond to thrombin. However, thrombin binding to platelets correlates more closely with some responses than others, presumably because cAMP inhibits additional platelet reactions.

Alprostadil↗

PubMed1987

Agents that elevate the concentration of cAMP in platelets inhibit the formation of a NaDodSO4-resistant complex between thrombin and a 40-kDa protein.

We studied the influence of prostaglandin E1 and theophylline on the ability of rabbit or human platelets to form NaDodSO4-resistant complexes between 125I-labeled thrombin and a platelet protein of approximately equal to 40 kDa. Control platelets formed two types of these complexes, one that sedimented with the platelets and one that was found in the suspension medium. There were 30-40 sedimentable complexes per platelet and about three times this number of soluble complexes. Pretreatment of rabbit or human platelets with prostaglandin E1 and theophylline decreased the formation of both types of complex by as much as 60-80%. The pretreatment was particularly effective when low doses of thrombin were used. When added to control platelets, such doses of thrombin caused the formation of sedimentable complexes and the concomitant formation of inositol trisphosphate. However, when added to platelets that had been pretreated with prostaglandin E1 and theophylline, low doses of thrombin had little or no effect on either reaction.

Alprostadil↗

PubMed1986

A thiol-sensitive degradative process of liver uncouples autophosphorylation of the insulin receptor from insulin binding.

Insulin receptors derived from highly purified rat liver plasma membranes and Golgi membranes showed differences in insulin-mediated receptor autophosphorylation, even though their insulin-binding characteristics were similar. This difference was related to the generation of a Mr-84,000 fragment of the Mr-90,000 beta subunit of the plasma-membrane receptor, a fragment that was not present in the receptor from Golgi membranes, in the absence of a change in the insulin-binding alpha subunit. When autophosphorylation activity was based on insulin binding, the activity of the plasma-membrane-derived insulin receptor was decreased to 25-30% that of the Golgi-derived receptor. Endoglycosidase F digestion produced changes in the Mr values for both species, but they were not converted into a single subunit, thereby suggesting differences in the protein component of the two subunits. Although the proteinase inhibitors phenylmethanesulphonyl fluoride, ovomucoid and aprotinin failed to block the formation of the Mr-84,000 fragment, the presence of iodoacetamide or EDTA during liver homogenization markedly inhibited fragment generation and allowed the plasma-membrane insulin receptor to retain an autophosphorylation activity comparable with that present in insulin receptors from Golgi membranes. Thus a thiol-sensitive, cation-dependent, degrading activity has been identified that can uncouple the insulin-binding activity of the plasma-membrane insulin receptor from its tyrosine kinase activity.

Animals↗