M. McLaren

Dark chocolate inhibits platelet aggregation in healthy volunteers

Cardiovascular disease is a leading cause of death in the UK. The flavonoids found in cocoa may produce a cardio-protective role for chocolate with a high cocoa content. Thirty healthy volunteers were randomised to receive 100 g of white, milk or dark chocolate, and assessments of platelet function were undertaken on venous blood samples before and after chocolate consumption. White and milk chocolate had no significant effect on platelets. However dark chocolate inhibited collagen-induced platelet aggregation in platelet rich plasma. In the future dark chocolate may have a role in prevention of cardiovascular and thromboembolic diseases.

The White Blood Cell Adhesion Molecule E-Selectin Predicts Restenosis in Patients With Intermittent Claudication Undergoing Percutaneous Transluminal Angioplasty

BACKGROUND Experimental studies have shown that endothelial dysfunction is an early event preceding restenosis. Monocytes and neutrophils have been shown to bind to damaged endothelium via the cell adhesion molecules (CAMs). The selectins are involved in capturing the leukocytes and tethering them to the endothelium. E-selectin is a CAM that is only expressed on activated endothelial cells. Its ligands are expressed on monocytes and neutrophils and it has been found to exist in a soluble form. This soluble form may represent a marker for endothelial damage and may be a precursor of smooth muscle proliferation. METHODS AND RESULTS Fifty-four patients who were undergoing peripheral arterial balloon angioplasty had blood sampled before angioplasty. E-selectin was measured in plasma with the use of an ELISA. At follow-up angiogram, 30% (n=14) of the patients had restenosed at 1 year. There was a significant difference in baseline E-selectin levels in patients who restenosed compared with those who did not (65.3 ng/mL [58.25 to 78.05] versus 52.3 [34.2 to 62.1], Mann-Whitney U, P<.007). Endothelial activation with subsequent adherence of white blood cells is an important step in restenosis. CONCLUSIONS We have shown an increased level of shed E-selectin in patients destined for restenosis and suggest that this work further supports a role for white blood cell/endothelial interaction in restenosis after angioplasty.

Abnormal markers of endothelial cell activation and oxidative stress in children, adolescents and young adults with type 1 diabetes with no clinical vascular disease.

Endothelial cell dysfunction is an early feature of vascular disease and oxidative stress may be involved in its pathogenesis.

The Relationship of Oxidative Stress to Thrombotic Tendency in Type 1 Diabetic Patients with Retinopathy

Increased free radical activity may contribute to thrombosis via effects on platelet aggregation and the prostanoid balance. To investigate this further we studied 15 Type 1 diabetic patients with retinopathy, matched with uncomplicated Type 1 patients for age, duration of diabetes and HbA1, together with matched healthy non‐diabetic control subjects. The oxidative effects of free radicals as total diene conjugates and lipid peroxides were measured, together with redox status extracellularly as plasma albumin‐thiols and intracellularly as erythrocyte superoxide dismutase activity. Platelet count, aggregation of platelets in whole blood to collagen, thromboxane B2, and prostacyclin stimulating factor (PGI2SF) were also assessed. Free radicals measured as lipid peroxides were significantly higher (9.6 (8.1–11.6) μmol 1‐1 (median and interquartile range) in diabetic patients with retinopathy than in control subjects (8.1 (7.4–9.2) μmol l‐1; p < 0.05). There were also significant reductions in redox status both extracellularly as plasma albumin thiols (408 (383–473) vs 490 (456–517) μmol l‐1, p < 0.001) and intracellularly as erythrocyte superoxide dismutase activity (34 (27–41) vs 44 (36–51) g l‐1, p < 0.05) between patients with retinopathy and control subjects. Platelet counts were increased in diabetic patients with retinopathy (p < 0.05), as was collagen‐induced platelet aggregation (p < 0.01). Prostacyclin stimulating factor was reduced in patients with retinopathy (p < 0.05) and correlated within the plasma with lipid peroxides (r = – 0.53, p < 0.04) and albumin thiols (r = 0.64, p < 0.01). The results suggest that diabetic patients, particularly with retinopathy, are under oxidative stress and have an increased thrombotic tendency with increased platelet reactivity and a reduction in prostacyclin stimulating factor.

Mean platelet volume is a useful parameter: a reproducible routine method using a modified Coulter Thrombocytometer

The principal physiological function of platelets is to promote haemostasis but they also contribute to thrombosis and atherogenesis. Platelet volume is a marker and possibly a determinant of platelet function in that large platelets are more active than normal sized platelets. Mean platelet volume (MPV), a measure of platelet size, reflects changes in either the level of platelet stimulation or the rate of platelet production. For these reasons, we have developed a sensitive instrument to measure platelet volume, which we believe is more reliable and specific than previously used instruments. It is based on a computer - interfaced Coulter Thrombocytometer and a pulse analyser including a high - speed baseline restorer. We have developed a reproducible method to assay MPV and from the histogram derived the median (MED) and the skewness (SK) values. We have looked at the effects of anticoagulant used and time elapse prior to assay. A normal range has been established for MPV which correlates directly with MED and inversely with SK. The MPV decreases with age but there is no difference between genders. We have demonstrated a negative correlation between whole blood platelet number and MPV and MED, and a direct relationship with the SK of the histogram of the platelet volume.The principal physiological function of platelets is to promote haemostasis but they also contribute to thrombosis and atherogenesis. Platelet volume is a marker and possibly a determinant of platelet function in that large platelets are more active than normal sized platelets. Mean platelet volume (MPV), a measure of platelet size, reflects changes in either the level of platelet stimulation or the rate of platelet production. For these reasons, we have developed a sensitive instrument to measure platelet volume, which we believe is more reliable and specific than previously used instruments. It is based on a computer-interfaced Coulter Thrombocytometer and a pulse analyser including a high-speed baseline restorer. We have developed a reproducible method to assay MPV and from the histogram derived the median (MED) and the skewness (SK) values. We have looked at the effects of anticoagulant used and time elapse prior to assay. A normal range has been established for MPV which correlates directly with MED and inversely with SK. The MPV decreases with age but there is no difference between genders. We have demonstrated a negative correlation between whole blood platelet number and MPV and MED, and a direct relationship with the SK of the histogram of the platelet volume.

The use of Ginkgo biloba in Raynaud's disease: a double- blind placebo-controlled trial

Raynaud’s phenomenon (RP) is a common and painful condition characterized by episodic digital ischaemia produced by emotion and cold. Treatment of RP is notoriously difficult because of the high incidence of side effects. The aim of our study was to investigate the clinical efficacy of a standardized Ginkgo biloba extract (Seredrin) in the treatment of RP in patients with no apparent, associated condition such as systemic sclerosis. A two-week assessment period was done during which patients were asked to record frequency, severity and duration of attacks in diaries. Subjects were then randomized independently of the study centre to receive either active or placebo treatment for 10 weeks, during which time the same data were recorded in their diaries. Patients were seen after two and four weeks of treatment and at the end of the 10-week treatment phase. Blood samples pre- and post-treatment were taken for haemorrheology. Only in the number of attacks per day was there a significant effect of treatment over placebo. The number of attacks per week prior to treatment with Seredrin was 13.2 6 16.5 reducing to 5.8 6 8.3, a reduction of 56%, whereas placebo reduced the number by only 27% (p < 0.00001). There were no significant differences in haemorrheology between the two groups. Ginkgo biloba phytosome may be effective in reducing the number of Raynaud’s attacks per week in patients suffering from Raynaud’s disease.

Biochemical indices of vascular function, glucose metabolism and oxidative stress in horses with equine Cushing's disease

REASONS FOR PERFORMING STUDY The mechanisms underlying the increased risk of laminitis in horses with equine Cushings disease (ECD) are poorly understood. HYPOTHESIS That abnormalities in glucose homeostasis, similar to those which cause microvascular dysfunction in human diabetics, contribute to development of laminitis in horses with ECD. METHODS Thirty-one aged horses were divided into 3 groups based on clinical signs and dexamethasone suppression testing (DST). Group 1 (n = 12) had clinical ECD as evidenced by hirsutism. Group 2 (n = 10) had a positive DST but no hirsutism. Group 3 (n = 9) were controls without ECD, with a negative DST and no clinical evidence of ECD. Biochemical indices of glucose metabolism, vascular function and oxidative stress were determined in single morning blood samples. RESULTS Group 1 had abnormalities in glucose homeostasis, including increased levels of glucose and insulin, compared to Groups 2 and 3. Groups 1 and 2 had significantly lower plasma thiol (PSH) levels and nonsignificantly lower albumin-corrected PSH levels than Group 3, consistent with oxidative stress. CONCLUSIONS AND POTENTIAL RELEVANCE The observed abnormalities in glucose metabolism and oxidative stress could potentially contribute to development of laminitis in horses with ECD, by similar mechanisms to those that cause microvascular dysfunction in human diabetics. The absence of inter-group differences in the biochemical indices of vascular function precludes their use as preclinical diagnostic indicators of vascular dysfunction. The study also highlighted limitations in the premortem diagnosis of ECD.

Exercise in Patients with Intermittent Claudication Results in the Generation of Oxygen Derived Free Radicals and Endothelial Damage

Peripheral vascular disease is a major cause of morbidity in Britain. Each year approximately 50,000 patients are admitted to hospital in Britain with a principal diagnosis of peripheral vascular disease and of these over 20,000 have major surgery, including amputations, with an operative mortality of about 10% (Department of Health and Social Security Office of Population Censuses and Surveys 1986). Intermittent claudication is the most common symptom of peripheral vascular disease (Dormandy et al, 1989) and is a symptom of cramp-like muscle pain, brought on by walking, relieved by rest and reproduced by further exercise. It may affect the calf, thigh or buttock muscle groups depending upon the level and degree of vascular obstruction. Intermittent claudication is caused by an inadequate blood supply to the exercising muscles of the lower limb, although the exact pathophysiological mechanism responsible for the symptom remains unknown (Lorentsen, 1973). Five per cent of men over 50 years suffer from intermittent claudication (Dormandy et al,1989). Claudication itself does not cause death, however, the mortality of claudicants is approximately three times that of age and sex matched individuals (Dormandy et al,1989), being about 50% after ten years (Dormandy et al, 1986). Seventy-five per cent of these deaths are due to cardiovascular disease, such as. myocardial infarction, cerebrovascular accidents and aortic aneurysms (Dormandy et al,1989). Paradoxically the disease seems to stabilise symptomatically in seventy-five per cent of patients soon after onset (Dormandy et al,1989) and few claudicants progress to limb threatening ischaemia (Cronenwett et al,1984).

Increased Whole Blood Platelet Aggregation in Patients with Raynaud's Phenomenon with or without Systemic Sclerosis

Platelet activation may have a pathophysiological role in Raynauds phenomenon (RP). However, previous studies have shown conflicting results. This may be related to patient selection and the choice of platelet function assay. In this study, we have assessed platelet function of 30 patients with severe RP with (n = 14) or without (n = 16) systemic sclerosis (SSc), using a whole blood platelet aggregation (PA) assay. Raynauds medication or other drugs which may affect PA were stopped at least 2 weeks previously. Spontaneous whole blood PA and that induced by 0.5 microM adenosine diphosphate and 0.6 and 1 microgram/ml collagen were significantly increased in both groups of patients when compared with controls. There were no significant differences in PA between the 2 groups of patients. Using a more physiological assay, patients with severe RP, whether or not associated with SSc, were shown to have abnormally increased platelet activity. Hyperactive platelets may further impede blood flow in RP.

Soluble Cell Adhesion Molecules - P-selectin and ICAM-1, and Disease Activity in Patients Receiving Sulphasalazine for Active Rheumatoid Arthritis

The aim of this pilot study was to examine soluble cell adhesion molecules before and after sulphasalazine (SSZ) therapy in active RA. Assessment of RA patients (n = 13) was undertaken before and after 3 months of SSZ. sICAM-1, sVCAM-1, sP- and sE-selectin were measured using an ELISA. The mean (+/-SEM) C-reactive protein (CRP) and sP-selectin levels were significantly reduced from 3.9(0.89) to 2.01(0.53) mg/dl and from 332.8 (48.2) to 116.2 (11.1) respectively, after 3 months of SSZ. The sICAM-1 and sP-selectin levels were significantly higher in RA patients at baseline and a reduction occurred of sICAM-1, sVCAM-1 and sE-selectin levels, however this was not significant. The fall in mean (SEM) sICAM-1, from 345.0 (29.8) to 333.5 (30.2), correlated with the change in CRP (r=0.66; p = 0.018), but the fall in sP-selectin did not. SSZ therapy reduced sP-selectin and sICAM-1 levels in active RA, sICAM-1 correlates with disease activity. SSZ may reduce platelet and/or endothelial activity in RA which may be a useful marker of response, however studies of longer duration and more patients are required.