Date of Award

1995

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Chemistry and Biochemistry

First Advisor

Mutus, Bulent,

Keywords

Chemistry, Biochemistry.

Rights

CC BY-NC-ND 4.0

Abstract

Evidence is presented here for the regulation of platelet activation by intra-platelet levels of glutathione (GSH) and L-arginine. Our data is consistent with the intra-platelet GSH ((GSH) $\sb{\rm ip}$) modulation through the regulation of the thromboxane A$\sb2$ (TxA$\sb2$) biosynthesis. The reduction of (GSH) $\sb{\rm ip}$ by 100 $\mu$M 1-chloro-2,4-dinitrobenzene (CDNB) enhanced thrombin-induced platelet aggregation and ADP-induced platelet aggregation was inhibited by the elevation of (GSH) $\sb{\rm ip}$ through a facilitative GSH-specific transport system. Platelet facilitative GSH uptake was subsequently characterized as being Na$\sp+$-independent, concentration dependent (K$\sb{\rm M}$ and V$\sb{\rm max}$ for GSH uptake in platelet plasma membrane vesicles (PPMV) is $18.2 \pm 3.6\ \mu$M and 178 $\pm$ 27 pmol/min/mg protein, respectively), inhibited by GSH analogs, enhanced by KCl-induced membrane depolarization and sensitive to the intraplatelet thiol redox status since the K$\sb{\rm M}$ and V$\sb{\rm max}$ for GSH uptake in intact platelets changed from 137 $\mu$M and 42.2 pmol/min/10$\sp9$ platelets, respectively, to 31.7 $\mu$M and 31.3 pmol/min/10$\sp9$ platelets, respectively, on reducing intra-platelet GSH with 100 $\mu$M CDNB. Glutathione reductase (GR) was found to be inhibited by physiological levels of GSH with species-dependent differences. With respect to varying GSSG, GSH inhibited GR from human platelets in an apparent uncompetitive manner (K$\sb{\rm i}$ = 6.6 mM), while bovine intestinal mucosa and yeast GRs displayed apparent mixed hyperbolic inhibition (K$\sb{\rm i}$ = 2.9 and 2.4 mM, respectively), and the E. coli enzyme exhibited an apparent, competitive inhibition (K$\sb{\rm i}$ = 12.1 mM). In the course of this study it was observed that 1-(4-chlorophenyl)-4,4-dimethyl-5-diethylamino-1-penten-3-one hydrobromide (CDDP) is an alpha class selective glutathione S-transferase (GST) substrate and that certain analogs of CDDP are GST inhibitors with K$\sb{\rm i}$s ranging from 7.5 to 53.4 $\mu$M. Exogenous L-arginine inhibited ADP-induced platelet aggregation which suggests that platelet-derived NO regulates platelet activation. Platelet L-arginine uptake was via the cationic amino acid transporter, system y$\sp+$, which appears to be regulated by NO. S-nitrosoglutathione (GSNO) was found to be photolyzed by visible light. The release of NO by GSNO photolysis resulted in an enhanced NO-dependent cytotoxicity towards HL-60 cells. GSNO, or related compounds, may therefore find use as photochemotherapeutic agents.Dept. of Chemistry and Biochemistry. Paper copy at Leddy Library: Theses & Major Papers - Basement, West Bldg. / Call Number: Thesis1994 .S49. Source: Dissertation Abstracts International, Volume: 56-11, Section: B, page: 6097. Adviser: Bulent Mutus. Thesis (Ph.D.)--University of Windsor (Canada), 1995.

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