Evidence for iNOS-dependent peroxynitrite production in diabetic platelets.
OBJECTIVE
The aim of the present study was twofold. Firstly, to determine whether diabetic platelets produce more peroxynitrite than normal platelets and secondly to correlate the peroxynitrite production with the intraplatelet induction of the inducible isoform of nitric oxide-synthase.


METHODS
Intraplatelet peroxynitrite production was monitored with dichlorofluorescin acetate with a combination of confocal microscopy and steady-state fluorescence. The platelets were probed for the induction of the inducible-nitric oxide-synthase by western immunoblotting.


RESULTS
In the presence of extracellular L-arginine (100 micromol/l), platelets from subjects with Type I (insulin-dependent) diabetes displayed about 5 times higher fluorescence than those from control subjects. To determine whether inducible-nitric oxide-synthase was the source of peroxynitrite, dichlorofluorescein production was quantified as a function of L-arginine as well as nitric oxide-synthase inhibitors, in platelets from control subjects, subjects with Type I diabetes and subjects with Type II (non-insulin-dependent) diabetes mellitus. Platelets from subjects with Type I yielded about sevenfold and those from Type II about threefold larger amounts of L-arginine/nitric oxide-synthase-dependent dichlorofluorescein fluorescence than those from control subjects. The platelets were then immunologically probed for inducible-nitric oxide-synthase, which has previously been implicated in peroxynitrite production and detected in megakaryocytes of subjects with coronary heart disease. Western immunoblots of intraplatelet proteins indicated that the inducible-nitric oxide-synthase was absent in control subjects. Platelets from both Type I and Type II diabetic subjects, however, contained inducible-nitric oxide-synthase.


CONCLUSIONS
Inducible-nitric oxide-synthase-derived peroxynitrite is a source of platelet damage in diabetes.
Tannous
M
M
School of Physical Sciences, Chemistry and Biochemistry, University of Windsor, Ontario, Canada.
Rabini
R A
RA
Vignini
A
A
Moretti
N
N
Fumelli
P
P
Zielinski
B
B
Mazzanti
L
L
Mutus
B
B
D000328:Adult; D001120:Arginine / Q000494:pharmacology; D001792:Blood Platelets / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D015153:Blotting, Western; D003922:Diabetes Mellitus, Type 1 / Q000097:blood*; D003924:Diabetes Mellitus, Type 2 / Q000097:blood*; D005260:Female; D005456:Fluorescent Dyes; D006801:Humans; D008297:Male; D018613:Microscopy, Confocal; D008875:Middle Aged; D009566:Nitrates / Q000097:blood*; D019001:Nitric Oxide Synthase / Q000097:blood*; D052247:Nitric Oxide Synthase Type II; D013050:Spectrometry, Fluorescence
D005456:Fluorescent Dyes; D009566:Nitrates; C039642:peroxynitric acid; D001120:Arginine; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II