title: Non-enzymatic modifications of prostaglandin H synthase 1 affect bifunctional enzyme activity - Implications for the sensitivity of blood platelets to acetylsalicylic acid.
abstract: Due to its ability to inhibit the blood platelet PGHS-1, acetylsalicylic acid (ASA, Aspirin(®)) is widely used as a preventive agent in atherothrombotic diseases. However, its beneficial effects seem to be lower in diabetic patients, suggesting that protein glycation may impair effective ASA-mediated acetylation process. On the other hand, it is proposed that ASA can prevent some of the late complications of diabetes by lowering the extent of glycation at protein free amino groups. The aim of this work was to evaluate the extents of non-enzymatic N-glycosylation (glycation) and acetylation of blood platelet PGHS-1 (COX-1) and the competition between glycation and acetylation was investigated in order to demonstrate how these two reactions may compete against platelet PGHS-1. When PGHS-1 was incubated with glycating/acetylating agents (glucose, Glu; 1,6-bisphosphofructose, 1,6-BPF; methylglyoxal, MGO, acetylsalicylic acid, ASA), the enzyme was modified in 13.4 ± 1.6, 5.3 ± 0.5, 10.7 ± 1.2 and 6.4 ± 1.1 mol/mol protein, respectively, and its activity was significantly reduced. The prior glycation/carbonylation of PGHS-1 with Glu, 1,6-BPF or MGO decreased the extent of acetylation from 6.4 ± 1.1 down to 2.5 ± 0.2, 3.6 ± 0.3 and 5.2 ± 0.2 mol/mol protein, respectively, but the enzyme still remained susceptible to the subsequent inhibition of its activity with ASA. When PGHS-1 was first acetylated with ASA and then incubated with glycating/carbonylating agents, we observed the following reductions in the enzyme modifications: from 13.4 ± 1.6 to 8.7 ± 0.6 mol/mol protein for Glu, from 5.3 ± 0.5 to 3.9 ± 0.3 mol/mol protein for 1,6-BPF and from 10.7 ± 1.2 to 7.5 ± 0.5 mol/mol protein for MGO, however subsequent glycation/carbonylation did not significantly affect PGHS-1 function. Overall, our outcomes allow to better understand the structural aspects of the chemical competition between glycation and acetylation of PGHS-1.
authors.affiliation: Department of Haemostasis and Haemostatic Disorders, Chair of Biomedical Sciences, Medical University of Lodz, 6/8 Mazowiecka str., 92-215, Lodz, Poland.
authors.forename: Hassan
authors.initials: H
authors.lastname: Kassassir
authors.affiliation: Department of Haemostasis and Haemostatic Disorders, Chair of Biomedical Sciences, Medical University of Lodz, 6/8 Mazowiecka str., 92-215, Lodz, Poland.
authors.forename: Karolina
authors.initials: K
authors.lastname: Siewiera
authors.affiliation: Department of Haemostasis and Haemostatic Disorders, Chair of Biomedical Sciences, Medical University of Lodz, 6/8 Mazowiecka str., 92-215, Lodz, Poland.
authors.forename: Marcin
authors.initials: M
authors.lastname: Talar
authors.affiliation: Central Scientific Laboratory, Medical University of Lodz, 6/8 Mazowiecka str., 92-215, Lodz, Poland.
authors.forename: Emilia
authors.initials: E
authors.lastname: Stec-Martyna
authors.affiliation: Central Scientific Laboratory, Medical University of Lodz, 6/8 Mazowiecka str., 92-215, Lodz, Poland.
authors.forename: Zofia
authors.initials: Z
authors.lastname: Pawlowska
authors.affiliation: Department of Haemostasis and Haemostatic Disorders, Chair of Biomedical Sciences, Medical University of Lodz, 6/8 Mazowiecka str., 92-215, Lodz, Poland. Electronic address: cezary.watala@umed.lodz.pl.
authors.forename: Cezary
authors.initials: C
authors.lastname: Watala
mesh_terms: D000107:Acetylation / Q000187:drug effects; D001241:Aspirin / Q000494:pharmacology*; D001792:Blood Platelets / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D051545:Cyclooxygenase 1 / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation / Q000187:drug effects; D006801:Humans; D007553:Isotope Labeling; D010455:Peptides / Q000032:analysis; D011499:Protein Processing, Post-Translational / Q000187:drug effects*; D053719:Tandem Mass Spectrometry
chemical_list: D010455:Peptides; D051545:Cyclooxygenase 1; D001241:Aspirin
keywords: Acetylation; Acetylsalicylic acid; Diabetes mellitus; Glycation; Non-enzymatic modifications; PGHS-1 activity