Date of Award

1994

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

Intraplatelet abnormalities in the platelets from diabetics have been evaluated with a view of understanding diabetes related platelet hyperaggregation. Three of the modulators involved in the platelet regulatory mechanism namely glutathione (GSH), calmodulin (CaM) and nitric oxide (NO) have been assessed in platelets from both normal and diabetic subjects. GSH and NO levels in the platelets from diabetics were lower ($\sim$50% & 57% respectively) when compared to platelets from normal subjects, whereas there was no change in the level of total CaM in platelets from diabetics. Estimates of steady state kinetic parameters (apparent K$\sb{\rm M}$ and apparent V$\sb\max)$ for the platelet enzymes glutathione reductase, $\gamma$-glutamyl transpeptidase, glutathione-S-transferase and glutathione peroxidase have been reported for the first time. The kinetic parameters of platelet glutathione reductase, $\gamma$-glutamyl transpeptidase and glutathione S-transferase were essentially independent of the glycemic state of the subjects. However, the apparent K$\sb{\rm M}$ (t-BuOOH) of glutathione peroxidase was ${\sim}4$ fold higher in platelets from uncontrolled diabetics in comparison to platelets from normal subjects. Further, in vitro and in vivo study established that nonenzymatic glycation of the GSH metabolic enzyme glutathione peroxidase (GSH-Px) resulted in a decrease (4 fold) in affinity for its substrate and thereby a loss in the catalytic efficiency of up to $\sim$33%. The loss in GSH-Px catalytic efficiency and lowered level of GSH in platelets from diabetics provided new evidence for the previously observed elevation in peroxide levels in diabetic platelets. The impairment in GSH-Px activity due to nonenzymatic glycation has been discussed as a potential contributor to the platelet hyperaggregability in diabetics. The study also showed that intraplatelet calmodulin is susceptible to a nonenzymatic glycation reaction and that there is a 3 fold increase in the glycation of CaM in platelets from diabetics (21.6%) when compared to platelets from normal subjects (7.71%). As part of the study an enzyme linked immunosorbent assay system was devised to detect nanogram levels of glycated calmodulin in blood platelets for use in clinical settings as a short time-window index of glycemic status. Subsequently, a distinct isoform of the CaM dependent nitric oxide producing enzyme nitric oxide synthase was purified to homogeneity from human platelets. The native enzyme appears to be dimeric, with an estimated molecular weight of 150 kDa. The platelet nitric oxide synthase exhibited cofactor and kinetic characteristics similar to those from other cells and tissues but most interestingly possessed a distinct subunit molecular weight (${\sim}80$ kDa). The underproduction of NO ($\sim$57%) in platelets from diabetics when compared to the platelets from normal subjects is discussed in terms of a possible contribution to the diabetes related platelet hyperaggregation.Dept. of Chemistry and Biochemistry. Paper copy at Leddy Library: Theses & Major Papers - Basement, West Bldg. / Call Number: Thesis1994 .M87. Source: Dissertation Abstracts International, Volume: 56-11, Section: B, page: 6095. Adviser: Bulent Mutus. Thesis (Ph.D.)--University of Windsor (Canada), 1994.

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