ENZYMES THAT DEPHOSPHORYLATE INOSITOL PHOSPHATES IN HUMAN PLATELETS

Mapping Intimacies ◽

10.1055/s-0038-1644522 ◽

1987 ◽

Author(s):

Luis Molina ◽

y Vedia ◽

Eduardo G Lapetina

Keyword(s):

Molecular Weight ◽

Protein Kinase C ◽

Protein Kinase ◽

Membrane Fraction ◽

Inositol Phosphates ◽

Human Platelets ◽

Particulate Fraction ◽

Cytosolic Fraction ◽

Specific Marker ◽

Kinase C

Inositol trisphosphate (IP3) is now recognized as a second messenger molecule that mobilizes Ca2+ from intracellular stores to the cytosol. The persistence of the action of IP3 depends on the specific phosphatase that converts IP3 to inositol bisphosphate (IP2). The activation of IP3 phosphatase is important in terminating the Ca2+ signal in stimulated cells. In platelets it has previously been shown that this enzyme is regulated by protein kinase C since it is stimulated by phorbol esters and 1,2-diacylglycerol (Molina y Vedia, L., and Lapetina, E.G. J. Biol Chem. 261, 10493-10495, 1986) and the cytosolic platelet enzyme is phosphorylated by brain protein kinase C, resulting in a 4-fold increase in IP3 phosphatase activity (Connolly, T. M., Lawing, W.J., Jr., and Majerus, P.W., Cell, 46, 951-958, 1986). We have studied the subcellular distribution of the phosphatases that hydrolyze IP3, IP2 and inositol monophosphate (IP) in human platelets. Three subcellular fractions were obtained from human platelets lysed by freezing and thawing: a cytosolic fraction, a membrane fraction and a mixed particulate fraction containing granules, mitochondria and membranes. These fractions have been characterized by specific marker enzymes. The highest specific activity of IP3 -phosphatase is associated with the membrane fraction and accounts for about 10-15% of the total activity. The mixed particulate fraction has 35-40% of the activity while about 50% is cytosolic. The Km of the membrane fraction enzyme is 100 μM. This enzyme is extracted by 1M NaCl and hydrodynamic studies revealed a molecular weight of 50 kDa. The NaCl extracted-enzyme has been further purified by hydrophobic and gel filtration chromatographies. This activity does not hydrolyses IP but hydrolyse IP2 at a lower rate. The enzyme that hydrolyses IP to inositol is confined to the cytosolic fraction, has a Km of 130 μM, is inhibited by Li+, and hydrodynamic studies show an apparent molecular weight of 91 kDa.

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The action of the protein kinase C inhibitor, staurosporine, on human platelets. Evidence against a regulatory role for protein kinase C in the formation of inositol trisphosphate by thrombin

Biochemical Journal ◽

10.1042/bj2490345 ◽

1988 ◽

Vol 249 (2) ◽

pp. 345-350 ◽

Cited By ~ 149

Author(s):

S P Watson ◽

J McNally ◽

L J Shipman ◽

P P Godfrey

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Phosphatidic Acid ◽

Phorbol Esters ◽

Inositol Phosphates ◽

Protein A ◽

Polymyxin B ◽

Human Platelets ◽

Phosphoinositide Metabolism ◽

Kinase C

The ability of several putative inhibitors of protein kinase C (PKC) to block dioctanoylglycerol (DC8)-induced phosphorylation of a 47 kDa protein (a recognized substrate for PKC) in human platelets was investigated. Staurosporine (1 microM) caused complete inhibition of phosphorylation, whereas the other reagents were either inactive (polymyxin B) or gave only partial inhibition (C-1, H-7, tamoxifen). Staurosporine (1 microM) fully inhibited the phosphorylation of the 47 kDa protein in platelets challenged with thrombin, but also inhibited the phosphorylation of a 20 kDa protein which is a substrate for myosin light-chain kinase. The inhibition of both kinases by staurosporine was associated with the inhibition of thrombin-induced secretion of ATP and 5-hydroxytryptamine and a slowing of the aggregation response; staurosporine, however, had no effect on the formation of phosphatidic acid and inositol phosphates induced by thrombin. Staurosporine also reversed the inhibitory action of phorbol esters on thrombin-induced formation of phosphatidic acid. These data are consistent with a role for these two kinases in secretion and aggregation (although there must be additional control signals, since aggregation was only slowed, not inhibited), but suggest that neither kinase is involved in the regulation of phosphoinositide metabolism. This latter conclusion contradicts previous observations that the activation of PKC by phorbol esters or membrane-permeable diacylglycerols alters the apparent activity of both phospholipase C and inositol trisphosphatase. Possible explanations for this discrepancy are discussed.

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A diacylglycerol kinase inhibitor, R59022, potentiates secretion by and aggregation of thrombin-stimulated human platelets

Biochemical Journal ◽

10.1042/bj2430809 ◽

1987 ◽

Vol 243 (3) ◽

pp. 809-813 ◽

Cited By ~ 58

Author(s):

D L Nunn ◽

S P Watson

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Phosphatidic Acid ◽

Kinase Inhibitor ◽

Inositol Phosphates ◽

Protein A ◽

Diacylglycerol Kinase ◽

Human Platelets ◽

Kinase C ◽

Kinase Phosphorylation

The diacylglycerol kinase inhibitor R59022 (10 microM) potentiates secretion and aggregation responses in human platelets challenged with sub-maximal concentrations of thrombin. Potentiation correlates closely with increased formation of diacylglycerol, increased phosphorylation of a 40 kDa protein, a known substrate for protein kinase C, and with decreased formation of phosphatidic acid, the product of diacylglycerol kinase. Phosphorylation of myosin light chains, formation of inositol phosphates and the mobilization of Ca2+ by thrombin are not affected by R59022 (10 microM). These data support a role for protein kinase C in platelet aggregation and secretion, and provide further evidence that endogenous diacylglycerols bring about the activation of this enzyme. These data also add further argument against a role for phosphatidic acid in platelet activation.

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Activation of human platelets by peroxovanadate is associated with tyrosine phosphorylation of phospholipase Cγ and formation of inositol phosphates

Biochemical Journal ◽

10.1042/bj2900471 ◽

1993 ◽

Vol 290 (2) ◽

pp. 471-475 ◽

Cited By ~ 44

Author(s):

R A Blake ◽

T R Walker ◽

S P Watson

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Phospholipase C ◽

Tyrosine Phosphorylation ◽

Platelet Activation ◽

Inositol Phosphates ◽

Human Platelets ◽

Functional Responses ◽

Phosphorylation Of Proteins ◽

Kinase C

Vanadate ions in the presence of H2O2 (peroxovanadate) induce a marked increase in the degree of tyrosine phosphorylation of proteins in human platelets. This increase preceded the onset of platelet shape change and aggregation, and is associated with activation of phospholipase C and increased [32P]phosphorylation of proteins of 47 kDa, a substrate for protein kinase C, and 20 kDa, a substrate for both myosin light-chain kinase and protein kinase C. The non-selective inhibitor of protein kinases, staurosporine, inhibits the increase in tyrosine phosphorylation of nearly all proteins and inhibits completely all other functional responses, suggesting that these events may be linked. In support of this, peroxovanadate stimulates tyrosine phosphorylation of phospholipase C gamma 1, suggesting that this may underlie its mechanism of platelet activation. Staurosporine also inhibited activation of phospholipase C by collagen, suggesting that tyrosine phosphorylation has an important role in the early stages of collagen-induced platelet activation.

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Ethanol inhibits thrombin-induced secretion by human platelets at a site distinct from phospholipase C or protein kinase C

Biochemical Journal ◽

10.1042/bj2690489 ◽

1990 ◽

Vol 269 (2) ◽

pp. 489-497 ◽

Cited By ~ 32

Author(s):

C Benistant ◽

R Rubin

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Phospholipase C ◽

Phorbol Ester ◽

Inositol Phosphates ◽

Shape Change ◽

Protein A ◽

Human Platelets ◽

Kinase C ◽

A Site

Ethanol is known to inhibit the activation of platelets in response to several physiological agonists, but the mechanism of this action is unclear. The addition of physiologically relevant concentrations of ethanol (25-150 mM) to suspensions of washed human platelets resulted in the inhibition of thrombin-induced secretion of 5-hydroxy[14C]tryptamine. Indomethacin was included in the incubation buffer to prevent feedback amplification by arachidonic acid metabolites. Ethanol had no effect on the activation of phospholipase C by thrombin, as determined by the formation of inositol phosphates and the mobilization of intracellular Ca2+. Moreover, ethanol did not interfere with the thrombin-induced formation of diacylglycerol or phosphatidic acid. Stimulation of platelets with phorbol ester (5-50 nM) resulted in 5-hydroxy[14C]tryptamine release comparable with those with threshold doses of thrombin. However, ethanol did not inhibit phorbol-ester-induced secretion. Ethanol also did not interfere with thrombin- or phorbol-ester-induced phosphorylation of myosin light chain (20 kDa) or a 47 kDa protein, a known substrate for protein kinase C. By electron microscopy, ethanol had no effect on thrombin-induced shape change and pseudopod formation, but prevented granule centralization and fusion. The results indicate that ethanol does not inhibit platelet secretion by interfering with the activation of phosphoinositide-specific phospholipase C or protein kinase C by thrombin. Rather, the data demonstrate an inhibition of a Ca2(+)-mediated event such as granule centralization.

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Regulation of sn-1,2-diacylglycerol second-messenger formation in thrombin-stimulated human platelets. Potentiation by protein kinase C inhibitors

Biochemical Journal ◽

10.1042/bj2690465 ◽

1990 ◽

Vol 269 (2) ◽

pp. 465-473 ◽

Cited By ~ 19

Author(s):

W R Bishop ◽

J August ◽

J M Petrin ◽

J K Pai

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Inositol Phosphates ◽

Strong Support ◽

Human Platelets ◽

Protein Kinase C Inhibitors ◽

Kinase C ◽

Inositol Phospholipids ◽

A Cell ◽

Subsequent Activation

Stimulation of platelets with thrombin leads to rapid degradation of inositol phospholipids, generation of diacylglycerol (DAG) and subsequent activation of protein kinase C (PKC). Previous studies indicated that prior activation of PKC with phorbol myristate acetate (PMA) desensitizes platelets to thrombin stimulation, as indicated by a decreased production of inositol phosphates and decreased Ca2+ mobilization. This suggests that PKC activation generates negative-feedback signals, which limit the phosphoinositide response. To test this hypothesis further, we examined the effects of PKC activators and inhibitors on thrombin-stimulated DAG mass formation in platelets. Pretreatment with PMA abolishes thrombin-stimulated DAG formation (50% inhibition at 60 nM). Pretreatment of platelets with the PKC inhibitors K252a or staurosporine potentiates DAG production in response to thrombin (3-4-fold) when using concentrations required to inhibit platelet PKC (1-10 microM). K252a does not inhibit phosphorylation of endogenous DAG or phosphorylation of a cell-permeant DAG in unstimulated platelets, indicating that DAG over-production is not due to inhibition of DAG kinase. Sphingosine, a PKC inhibitor with a different mechanism of action, also potentiates DAG formation in response to thrombin. Several lines of evidence indicate that DAG formation under the conditions employed occurs predominantly by phosphoinositide (and not phosphatidylcholine) hydrolysis: (1) PMA alone does not elicit DAG formation, but inhibits agonist-stimulated DAG formation; (2) thrombin-stimulated DAG formation is inhibited by neomycin (1-10 mM) but not by the phosphatidate phosphohydrolase inhibitor propranolol; and (3) no metabolism of radiolabelled phosphatidylcholine was observed upon stimulation by thrombin or PMA. These data provide strong support for a role of PKC in limiting the extent of platelet phosphoinositide hydrolysis.

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