title: Calreticulin Transacetylase mediated activation of human platelet nitric oxide synthase by acetyl group donor compounds.
abstract: Polyphenols have attracted immense interest because of their diverse biological and pharmacological activities. Surprisingly, not much is documented about the biological activities of acetoxy derivatives of polyphenol called polyphenolic acetates (PA). In our previous reports, we have conclusively established the Calreticulin Transacetylase (CRTAase) catalyzed activation of neuronal nitric oxide synthase (nNOS) and tumor necrosis factor-α (TNF-α) induced nitric oxide synthase (iNOS) by PA. In the present work, specificity of CRTAase to various classes of PA was characterized in human platelet. The effect of PA, on platelet NOS and intracellular cyclic guanosine monophosphate (cGMP), and adenosine diphosphate (ADP)-induced platelet aggregation were studied in an elaborated manner. Platelet CRTAase exhibited differential specificities to polyphenolic acetates upon incubation with l-arginine leading to activation of NOS. The intraplatelet generation of NO was studied by flowcytometry using DCFH-DA. The differential specificities of CRTAase to PA were found to positively correlate with increased production of NO upon incubation of PRP with PA and l-arginine. Further, the inhibitory effect of l-NAME on PA induced NO formation in platelets substantiated the CRTAase catalyzed activation of NOS. The real-time RT-PCR profile of NOS isoforms confirmed the preponderance of eNOS over iNOS in human platelets on treatment with PA. Western blot analysis also reiterated the differential pattern of acetylation of eNOS by PA. PA were also found effective in increasing the intraplatelet cGMP levels and inhibiting ADP-induced platelet aggregation. It is worth mentioning that the effects of PA were found to be in tune with the specificities of platelet CRTAase to PA as the substrates.
authors.affiliation: Department of Biochemistry, V.P. Chest Institute, University of Delhi, Delhi, India. ajitvpci@gmail.com
authors.forename: Ajit
authors.initials: A
authors.lastname: Kumar
authors.forename: Anupam
authors.initials: A
authors.lastname: Sushama
authors.forename: Sushma
authors.initials: S
authors.lastname: Manral
authors.forename: Rajesh
authors.initials: R
authors.lastname: Sinha
authors.forename: Rini
authors.initials: R
authors.lastname: Joshi
authors.forename: Usha
authors.initials: U
authors.lastname: Singh
authors.forename: Vishwajeet
authors.initials: V
authors.lastname: Rohil
authors.forename: Ashok K
authors.initials: AK
authors.lastname: Prasad
authors.forename: Virinder S
authors.initials: VS
authors.lastname: Parmar
authors.forename: Hanumantharao G
authors.initials: HG
authors.lastname: Raj
mesh_terms: D000085:Acetates / Q000737:chemistry / Q000494:pharmacology; D000107:Acetylation; D000123:Acetyltransferases / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000494:pharmacology; D001120:Arginine / Q000494:pharmacology; D001792:Blood Platelets / Q000187:drug effects* / Q000378:metabolism*; D002478:Cells, Cultured; D003374:Coumarins / Q000494:pharmacology; D006152:Cyclic GMP / Q000378:metabolism; D003579:Cytochrome Reductases / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005452:Fluoresceins / Q000494:pharmacology; D006801:Humans; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009569:Nitric Oxide / Q000378:metabolism; D020030:Nitric Oxide Donors / Q000494:pharmacology; D052247:Nitric Oxide Synthase Type II / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D009599:Nitroprusside / Q000494:pharmacology; D010974:Platelet Aggregation / Q000187:drug effects; D059808:Polyphenols / Q000737:chemistry / Q000494:pharmacology*; D013329:Structure-Activity Relationship
chemical_list: C114369:7,8-diacetoxy-4-methylcoumarin; D000085:Acetates; D003374:Coumarins; D004791:Enzyme Inhibitors; D005452:Fluoresceins; D020030:Nitric Oxide Donors; D059808:Polyphenols; D009599:Nitroprusside; C029569:diacetyldichlorofluorescein; D009569:Nitric Oxide; D000244:Adenosine Diphosphate; D001120:Arginine; C496319:NOS2 protein, human; C496314:NOS3 protein, human; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; D003579:Cytochrome Reductases; D000123:Acetyltransferases; C539012:calreticulin transacetylase, human; D006152:Cyclic GMP; D019331:NG-Nitroarginine Methyl Ester