Consensus meeting on "Relevance of parenteral vitamin C in acute endothelial dependent pathophysiological conditions (EDPC)".

The 22 supersetnd Hohenheim Consensus Workshop took place in at the University of Stuttgart-Hohenheim. The subject of this conference was vitamin C and its role in the treatment of endothelial dysfunction. Scientists, who had published and reviewed scientific and regulatory papers on that topic were invited, among them basic researchers, toxicologists, clinicians and nutritionists. The participants were presented with eleven questions, which were discussed and answered at the workshop, with the aim of summarising the current state of knowledge. The explicatory text accompanying the short answers was produced and agreed on after the conference and was backed up by corresponding references. The therapeutic relevance of administration of the physiological antioxidant vitamin C in high parenteral doses in Endothelial Dependent Pathophysiological Conditions (EDPC) was discussed. Endothelial dysfunction is defined as including disturbed endothelial dependant relaxation of resistance vessels, breakdown of the microvascular endothelial barrier and/or loss of anti-adhesive function. It occurs in severe burn injury, intoxications, acute hyperglycemia, sepsis, trauma, and ischemic-reperfusion tissue injury and is induced by oxidative stress. Reduced plasma ascorbate levels are a hallmark of oxidative stress and occur in severe burns, sepsis, severe trauma, intoxication, chemotherapy/radiotherapy and organ transplantation. Vitamin C directly enhances the activity of nitric oxide synthase, the acyl CoA oxidase system and inhibits the actions of proinflammatory lipids. There is experimental evidence that parenteral high-dose vitamin C restores endothelial function in sepsis. In vitro, supraphysiological concentrations (> 1mM) of ascorbate restore nitric oxide bioavailability and endothelial function. Only parenterally, can enough vitamin C be administered to combat oxidative stress. There is no evidence that parenteral vitamin C exerts prooxidant effects in humans. Theoretical concerns in relation to competitive interactions between vitamin C and glucose cellular uptake are probably only relevant for oxidised vitamin C (dehydroascorbate).

Large platelets continue to circulate in an activated state after myocardial infarction.

This study was intended to investigate the actual platelet activation status after an acute coronary event. The activation status of circulating platelets was assayed directly by measuring the membrane activation markers CD62 and CD63 with the Düsseldorf III flow cytometry test in 22 patients with the diagnosis of acute myocardial infarction during the 48-h observation period following the acute event. The number of activated, marker-positive sample platelets was significantly increased in the post-MI patients: CD62: 5.8% x 2.25 +/- 1 vs. 3.5% x 2.32 +/- 1, P < or = 0.05; CD63: J8.7% x 1.77 +/- 1 vs. 4.6% x 2.16 +/- 1, P < or = 0.00.1. The platelet volume and count were concomitantly increased (12.1 +/- 2.4 fl/ 236 +/- 90 x 10(3) microliters-1 compared to 8.3 +/- 1.6 fl/ 187 +/- 42 x 10(3) microliters-1) in the control group. Particularly large platelets were identified as being activated documented by the exponential increase in the difference in CD63-binding sites per sample platelet above the 90%-percentile and below the 10%-percentile of the volume distribution: delta + 1341 +/- 903 (MI patients) vs. delta + 276 +/- 126 (controls), P < or = 0.00.1. Significant creatine kinase elevation and decrease in platelet count was found in the non-survivor subset (n = 5). We conclude that predominantly large platelets continue to circulate in an activated state after MI. This study provides direct evidence that the assumption of an increased thrombotic potential becomes operative in vivo in MI patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Increased GPIIB/IIIA expression and altered DNA-ploidy pattern in megakaryocytes of diabetic BB-rats.

Hyperactive platelets contribute to angiopathic complications in diabetes mellitus. It is unclear whether the increased platelet function is a primary pathogenetic factor in diabetes or follows vascular injury. Increased platelet size and numbers of glycoprotein receptors on diabetic platelets suggest that thrombopoiesis is altered in diabetes mellitus. For further support of this hypothesis we studied whether megakaryocytes are changed with regard to the DNA-ploidy pattern and the GPIIB/IIIA expression in 10 acute diabetic (AD) and 24 insulin treated diabetic (ITD) BB rats in comparison with 22 diabetes resistant (ND) BB rats. In the AD group megakaryocyte size (P = 0.035) and the modal DNA-ploidy distribution dropped (P = 0.0001) concomitant with increased TNF-alpha activity (P = 0.001). GPIIB/IIIA expression and the peripheral platelet status were unchanged. After 4 weeks of insulin substitution metabolic parameters (glucose, cholesterol, triglycerides) were lowered, but remained still elevated. As compared to the AD group the modal DNA-ploidy pattern reversed, but the relative percentage of 64n megakaryocytes increased 2.3-fold and GPIIB/IIIA expression increased 1.6-fold. Simultaneously, the peripheral platelet count and size increased. From these results we conclude that alterations of the megakaryocyte compartment occur at early onset of diabetes. These changes could reflect a response to increased systemic cytokine production during inflammatory islet cell destruction. The peripheral platelet thrombotic potency increased with insulin treatment. This was associated with an increase of 64n-megakaryocytes with upregulated GPIIB/IIIA expression and could reflect a mitogenic effect of insulin upon the endomitotic cycle of the megakaryocytes.

Reduced platelet thromboxane formation after long-term administration of a dihydropyridine calcium channel blocker: a prospective, double-blind, placebo-controlled study with nitrendipine in borderline hypertensive patients with IDDM-type diabetes mellitus.

Twenty-nine IDDM patients with borderline hypertension were randomly allocated to placebo or nitrendipine treatment. Nitrendipine was given orally at a dosage of 20 mg once daily over 4 weeks. Stimulated platelet thromboxane formation at rest and after standardized, non exhausting exercise was measured by standard methods. In addition, plasma levels of platelet factor 4 and aggregation responses to collagen and ADP were determined. In the treatment group thromboxane formation after stimulation with collagen (0.3 and 1.0 micrograms/ml) and 1 mM arachidonic acid (AA) was reduced in the resting state. Exercise induced change of thromboxane synthesis in response to 1.0 micrograms/ml collagen was significantly lower as compared to placebo (p < 0.05). In parallel, PF4 plasma levels were significantly lowered (p < 0.05). Whole blood aggregation after collagen stimulation (1.0 micrograms/ml) was reduced after 4 weeks of nitrendipine treatment, but ADP (5 microM) induced aggregation was not. These effects of nitrendipine were not seen in platelet rich plasma. In conclusion long-term nitrendipine treatment may inhibit collagen dependent platelet activation in the blood of diabetic patients with borderline hypertension.

Flow-cytometric detection of surface membrane alterations and concomitant changes in the cytoskeletal actin status of activated platelets.

Occlusive vascular diseases are promoted by a "prethrombotic state" with increased platelet activity. Polymerization of cytoskeletal proteins and exposure of subcellular structures or rebinding of secreted proteins have been characterized as early reactions after platelet activation preceding adhesion and aggregation. Here, we demonstrate the kinetic increase in specific binding of monoclonal antibodies to thrombospondin (P10) and to platelet membrane activation markers CD63 (GP53, a 53 kD lysosomal protein) and CD62 (GMP140, a 140 kD alpha granule protein) by using a flow-cytometric bio-assay and the related change in the actin status by using the DNase-I inhibition assay after stimulation of normal human platelets with 0.2 U/ml thrombin. F-actin was raised from 41% to 51% of total platelet actin content 30 s after stimulation and remained thereafter constant (50% at 60 s). Simultaneously, the percentage of P10, CD63, and CD62 positive platelets was elevated from 5.4%, 24.4%, and 9.1% to 67.4%, 80.2%, and 82.3% respectively. The mean number of P10, CD63, and CD62 antibody binding sites increased from 3,300, 1,715, and 2,146 to 6,400, 6,800, and 9,016 per platelet. Conclusively, changes in the organization of the cytoskeletal protein "actin" and exposure of subcellular structures indicating platelet secretion can be regarded as markers of early platelet activation. Thus, the parallel response in both analytical systems provides further support for the diagnostic concept of flow-cytometric detection of preactivated platelets in the peripheral blood by using fluochrome staining procedures detecting activation dependent structural alterations directly at the cellular level.