Angie S. Brown

Inhibition of platelet activity by S-nitrosoglutathione during coronary angioplasty

Platelet activation is associated with acute vessel occlusion and chronic restenosis after percutaneous transluminal coronary angioplasty (PTCA). Organic nitrates, which act by releasing the vasodilator and anti-platelet agent nitric oxide (NO), have a predominantly vasodilator action and cause hypotension at doses required to inhibit platelet activation. S-nitrosoglutathione (GSNO) is an NO donor with a preferential action on platelets. We investigated platelet activation in patients undergoing PTCA and the effect of GSNO. Blood was sampled from the coronary sinus to measure platelet surface expression of P-selectin and glycoprotein IIb/IIIa as indices of platelet activation. In 7 control patients, PTCA caused a rise in platelet surface expression of P-selectin and glycoprotein IIb/IIIa, which was maximal 5 minutes after PTCA, indicating increased platelet activation despite treatment with aspirin, glyceryl trinitrate, and heparin. 6 patients received an intracoronary infusion of GSNO, starting 10 min before PTCA. GSNO significantly inhibited the PTCA-induced increase in platelet surface expression of P-selectin and glycoprotein IIb/IIIa without altering blood pressure. These findings show that platelets are activated following PTCA and that GSNO can prevent this activation.

Megakaryocyte Ploidy and Platelet Changes in Human Diabetes and Atherosclerosis

Altered platelet morphology and function have been reported in patients with diabetes. They are likely to be associated with the pathological processes and increased risk of vascular disease seen in these patients. Mean platelet volume (MPV), platelet count, and megakaryocyte (MK) ploidy (DNA content) were measured in (1) nondiabetics with normal coronary arteries, (2) nondiabetics with coronary artery atherosclerosis, (3) diabetics without evidence of vascular complications, and (4) diabetics with vascular disease. The platelet count (+/- SD) was increased in all groups but only significantly in the diabetics with vascular disease (236 +/- 65 versus 250 +/- 54 versus 257 +/- 64 versus 295 +/- 90 [P < or = .05] x 10(9)/L, for groups, I, II, II, and IV, respectively). The MPV was significantly increased in patients with atherosclerosis (7.0 +/- 0.4 versus 8.0 +/- 1.2 [P < or = .05] versus 7.2 +/- 0.9 versus 8.1 +/- 0.9 [P < or = .05] IL). Geometric mean MK ploidy was significantly increased in all groups compared with controls (16 +/- 1.5 versus 18.7 +/- 1.8 [P < or = .05] versus 19.8 +/- 1.6 [P < or = .05] versus 20.1 +/- 2.7 [P < or = .05]). Furthermore, some patients with vascular disease and/or diabetes had a modal ploidy shift from 16 (the normal mammalian modal ploidy) to 32, with a concomitant reduction of MKs in the 8 and 16 ploidy classes. This shift was seen particularly in the diabetics with vascular disease (P = .007). Interleukin-6 (IL-6) levels were measured and were elevated in patients with atherosclerosis; the highest levels were found in the diabetic patients (0.7 +/- 0.9 versus 5.3 +/- 5.5 [P < or = .05] versus 2.5 +/- 2.8 versus 6.7 +/- 5.5 [P < or = .05] ng/L). In the diabetic patients with atherosclerosis, fibrinogen levels were also increased (2.85 +/- 0.76 versus 3.34 +/- 1.32 versus 2.43 +/- 1.50 versus 5.59 +/- 1.72 [P < or = .05] g/L). Furthermore, IL-6 levels correlated with MK ploidy (r = .45, P = .009) and fibrinogen levels (r = .5, P = .0001). This study demonstrates that patients with vascular disease, particularly diabetics, have an altered MK ploidy distribution, showing a shift toward higher ploidy in association with an increased platelet mass (count x volume). Changes in platelets in diabetes probably reflect MK changes, which themselves are a response to systemic change.

Nitric oxide-dependent and independent effects on human platelets treated with peroxynitrite

OBJECTIVE Peroxynitrite (ONOO-) is an oxidant formed from the rapid reaction of superoxide and nitric oxide (NO) at sites of inflammation. The literature reports conflicting data on the effects of ONOO- in biological systems, with both NO- and oxidant-dependent effects having been demonstrated. The aim of this study was to investigate these distinct mechanisms through examining molecular aspects of the effects of ONOO- on human platelets, a system in which we have previously shown that ONOO- has both pro- and anti-aggregatory effects. METHODS Platelet function was assessed by measuring platelet P-selectin expression flow cytometrically, intraplatelet Ca2+ concentrations, and by light aggregometry. A colorimetric method was used to measure extracellular platelet membrane thiols. The contribution of NO and cGMP to the pharmacological effects of ONOO- was investigated using an inhibitor of the soluble guanylate cyclase (sGC), 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one (ODQ), and the NO scavenger oxy-haemoglobin. RESULTS Peroxynitrite (50-400 microM) caused a concentration-dependent increase in the number of platelets expressing P-selectin, an increase in intraplatelet Ca2+ concentrations and a decrease in platelet membrane thiols. Peroxynitrite-induced P-selectin expression was augmented by ODQ. In contrast, when P-selectin expression was elicited by collagen, ONOO- acted as an inhibitor of this process, an effect that was further enhanced by the addition of 1% plasma, ODQ or oxy-haemoglobin abolished this inhibitory effect. Finally, low concentrations (50-100 microM) of ONOO- inhibited collagen-induced platelet aggregation, an effect that was reversed by oxy-haemoglobin. CONCLUSIONS Peroxynitrite exerts dual effects on platelets, which are either activating or inhibitory due to the conversion of ONOO- to NO or NO donors. Peroxynitrite-induced platelet activation seems to be due to thiol oxidation and an increase in intracellular Ca2+. It is important to note that inhibitory, NO-dependent effects occur at lower concentrations than the activating effects. These data are then consistent with the conflicting literature, showing both damaging and cytoprotective effects of ONOO- in biological systems. We hypothesize that the conversion of ONOO- to NO is the critical factor determining the outcome of ONOO- exposure in vivo.

The megakaryocyte platelet system and vascular disease

Abstract. Platelets form a heterogeneous population of cells produced from the uniquely large polyploid cell found in the bone marrow, the megakaryocyte. The platelet megakaryocyte axis forms a dynamic equilibrium varying in normal biology and in disease. Prolonged platelet destruction leads to the production of large platelets from large, high ploidy megakaryocytes. In vivo and ex vivo studies show that such platelets have more haemostatic potential than smaller less dense platelets. The evidence suggesting that prothrombotic changes in the megakaryocyte platelet axis precede coronary artery thrombosis and the importance of platelet reactivity in atherosclerosis will be reviewed.

Immunolocalization of the multi-sarco/endoplasmic reticulum Ca2+ ATPase system in human platelets

We recently identified a multi‐SERCA (sarco/endoplasmic reticulum Ca2+ ATPase) system in haemopoietic cells comprising the SERCA 2b, SERCA 3 and a new monoclonal anti‐Ca2+ ATPase antibody (PL/IM 430) recognizable SERCA isoforms. We have now investigated the subcellular localization of these enzymes in human platelets by Western blotting of subcellular membrane fractions and by immunoelectron microscopy. We precisely defined the recognition specificity of the polyclonal anti‐SERCA 2b, anti‐SERCA 3, anti‐SERCA 1 antibodies as well as of the monoclonal antibody PL/IM 430 by testing their recognition of the tryptic fragments of the SERCA isoforms. The analysis of fragmented membranes enriched in plasma membrane and intracellular membrane components by Western blotting showed that the SERCA 2b and the SERCA 3 isoforms were found in both the plasma membrane and the intracellular membrane fractions, whereas the PL/IM 430 recognizable SERCA isoform was restricted to membranes associated with the plasma membrane fraction. The immunoelectron microscopical study of the SERCA isoforms in resting platelets showed that: (i) the SERCA 2b isoform was expressed in membranes associated with the plasma membrane and open canalicular system, some α‐granules and in unidentified membranes; (ii) the SERCA 3 isoform was found associated with plasma and intracellular membranes; and (iii) the PL/IM 430 recognizable SERCA isoform was observed only in structures associated with the cytoplasmic face of the plasma membranes, as confirmed by flow cytometry. Finally, since the PL/IM 430 antibody was raised against intracellular membranes, we looked for a potential membrane redistribution during the isolation procedure used for the preparation of the immunizing membranes. Neuraminidase treatment indeed induced a translocation of the PL/IM 430 recognizable SERCA isoform from plasma to intracellular membranes.  Thus, the multi‐SERCA system in platelets: (i) is distributed over different platelet membranes, (ii) presents a sub‐compartmental organization with some overlapping, and (iii) is partly associated with motile membranes, reflecting an unrecognized level of complexity of Ca2+ stores in these cells.

Megakaryocytes From Patients With Coronary Atherosclerosis Express the Inducible Nitric Oxide Synthase

Endothelial and platelet generation of nitric oxide (NO) plays an important role in the regulation of hemostasis. Alterations in NO biosynthesis are described in atherosclerosis. We have investigated the NO pathway in megakaryocytes and platelets from patients with atherosclerosis and age-matched control subjects. Megakaryocytes and platelets were isolated from patients with severe coronary atherosclerosis (n = 19) and normal coronary arteries (n = 9) as demonstrated by selective angiography. Constitutive (Ca(2+)-dependent) and inducible (Ca(2+)-independent) NO synthase (cNOS and iNOS, respectively) activities were measured by using the citrulline assay and by immunostaining techniques using an anti-peptide antibody to iNOS. Megakaryocytes from patients with atherosclerosis expressed significantly greater amounts of iNOS (1.28 +/- 0.46 pmol citrulline.mg-1.min-1) than cNOS (0.29 +/- 0.40 pmol.mg-1.min-1). In contrast, megakaryocytes from patients with normal coronary arteries expressed significantly more cNOS (1.48 +/- 0.23 pmol.mg-1.min-1) than iNOS (0.49 +/- 0.40 pmol.mg-1.min-1). Platelets isolated from both groups showed no significant difference in cNOS expression, and no iNOS was seen in either group. Immunostaining confirmed the presence of the iNOS in megakaryocytes. These results suggest there is a link between the expression of iNOS in the megakaryocyte and atherosclerosis.