Platelets and vascular risk: an option for treatment.

Epidemiological studies have linked platelet hyperactivity with an increased risk of vascular events. Even more convincing is the evidence from appropriately designed clinical trials showing that antiplatelet agents decrease the risk of vascular events (e.g. myocardial infarction, MI and stroke). These findings are compatible with the known thrombotic action of platelets. A considerable limitation in platelet research is the absence of a reliable, universally accepted marker of platelet activity. Therefore, it is difficult to reliably identify the 'high risk patient' and/or evaluate the efficacy of any administered treatment other than by calculating event rates over a period of time. This review will focus on the preventive aspects of antiplatelet intervention while also briefly considering the assessment of platelet hyperactivity and the mechanisms involved in platelet-induced thrombosis.

Does meta-chlorophenylpiperazine (mCPP) activate human platelets?

mCPP (meta-chlorophenylpiperazine), an agonist at serotonin (5-hydroxytryptamine, 5-HT) 5-HT2 receptors, has been used as a probe of serotonergic function. We assessed its effect on platelet activation by measuring median platelet volume (MPV), the Sonoclot (SCT) pattern and plasma and intraplatelet serotonin. (a) In vitro study: MPV was measured (n = 7) using a high-resolution channelyzer: Saline (median and range (5.23 fl; 5.10-6.18) vs. mCPP (5.36; 5.10-6.44) P = 0.03; ADP (5.42; 5.29-6.44) vs. ADP + mCPP (5.67; 5.42-6.63) P = 0.02; mCPP (5.36; 5.10-6.44) vs. ADP + mCPP (5.67; 5.42-6.63) P = 0.02. Therefore, mCPP increases the MPV and enhances the effect of ADP. (b) In vivo study: The SCT time to inflection (TI) and time to peak (TP) were measured following the oral administration of mCPP (0.5 mg/kg) or aspirin (300 mg) (n = 10). Ingestion of mCPP significantly shortened TI and TP indicating platelet activation. TI: 0 h (mean +/- SD: 10.2 +/- 2.0 min) vs. 6 h (9.3 +/- 1.5) P = 0.03; TP: 0 h (31.9 +/- 7.6) vs. 6 h (23.1 +/- 2.9) P = 0.01. Aspirin had no effect on TI or TP. There were no significant changes in plasma and intraplatelet 5-HT. It is concluded that mCPP activates human platelets via 5-HT receptors.

The effects of transdermal estradiol alone or with cyclical dydrogesterone on markers of cardiovascular disease risk in postmenopausal women with type 2 diabetes: a pilot study.

The objective of this open, longitudinal, controlled study was to assess the effect of transdermal estradiol alone or combined with cyclical dydrogesterone on the markers of cardiovascular disease (CVD) risk in postmenopausal women with type 2 diabetes. The control group consisted of postmenopausal diabetic women who declined menopausal hormone replacement therapy (HRT). Twenty-eight postmenopausal women (19 on HRT and 9 controls) with type 2 diabetes were followed up for 12 months. From the active treatment group 14 women with a uterus in situ had 80 microg/24 hr transdermal estradiol (Fematrix 80; Solvay Healthcare Ltd, Southampton, UK) and oral dydrogesterone 10 mg daily for the first 12 days of the calendar month, whereas 5 women with previous hysterectomy had 80 microg/24 hr transdermal estradiol (Fematrix 80) alone. CVD risk markers were measured before and at regular intervals after starting HRT. The main outcome measures were weight, systolic and diastolic blood pressure, fasting plasma glucose, glycated hemoglobin (HbA1c), glucose/insulin ratio, total cholesterol, high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, triglycerides, lipoprotein (a), high-sensitivity C-reactive protein (hs-CRP), fibrinogen, and endothelin-1. Transdermal estradiol with or without dydrogesterone in women with type 2 diabetes did not adversely affect any of the measured markers of cardiovascular risk. There was a significant decrease in HbA1c, total cholesterol, and LDL cholesterol at 6 months in women receiving HRT. Some of the cardiovascular disease risk markers may improve in postmenopausal women with type 2 diabetes with transdermal estradiol. This effect may have important clinical implications and it deserves further investigation in appropriately designed trials.

The early effect of lipid-lowering treatment on carotid and femoral intima media thickness (IMT).

OBJECTIVES: an increased intima media thickness (IMT) is an early indicator of the atherosclerotic process. We investigated the early effect of atorvastatin on the common carotid artery (CCA) and common femoral artery (CFA) IMT. METHODS: the IMT was measured in the CCA and the CFA of hyperlipidaemic patients referred with peripheral vascular disease. The measurements were performed using an automated radio frequency IMT technique pre-treatment and at 4 and 8 weeks post-treatment with 20 mg/day atorvastatin. RESULTS: patients (14 men; 11 women), median age 69 years (range: 48-81) had a CCA-IMT mean (SD) of 0.79 (0.21) mm pre-treatment, 0.75 (0.22) mm after 4 weeks, and 0.64 (0.15) mm after 8 weeks. The ANOVA test was significant (p=0.024) for the CCA-IMT trend. The corresponding CFA-IMT readings were 0.83 (0.13) mm, 0.80 (0.09) mm and 0.69 (0.14) mm (p=0.0003). After 8 weeks of treatment there was a significant reduction in total cholesterol 6.0 (0.3) to 4.3 (0.8) mmol/l, p=0.0004 and low-density lipoprotein cholesterol 3.7 (0.2) to 2.2 (0.5), p=0.0001. There was a significant decrease in median serum creatinine levels after 8 weeks treatment: 87 micromol/l (range 67-114) to 84 micromol/l (range: 64-112), p=0.007. CONCLUSIONS: cholesterol-lowering with atorvastatin 20 mg/day leads to a decrease in CCA-IMT and CFA-IMT. This difference achieved significance after 8 weeks of treatment, but a trend was visible at 4 weeks. These rapid changes in IMT may be attributable to an anti-inflammatory effect. IMT measurement may be a useful tool to rapidly assess the effect of drug treatment on the atherosclerotic process.

Lipoprotein (a) does not participate in the early acute phase response to training or extreme physical activity and is unlikely to enhance any associated immediate cardiovascular risk.

AIMS: To investigate the proposal that lipoprotein (a) (Lp(a)) contributes to the acute phase response and thus possibly to the acute cardiac risks associated with major physical effort. METHODS/RESULTS: Fit, healthy, British army recruits were reviewed at the beginning and the end of a 10 week programme of basic training concluding with an intense 48 hour military exercise. Final recruit assessment was staggered over the last week of training, giving rise to six recruit groups, with determination of Lp(a), C reactive protein (CRP), fibrinogen, albumin, and total creatine kinase values from 12 hours to five days after the final exercise. A clear acute phase response was seen following the final exercise, marked by a significant increase in circulating concentrations of fibrinogen and a reduction of albumin, and a trend with non-significant increases in CRP. CONCLUSION: Lp(a) did not behave as an early marker of the acute response. Previous reports may have been confounded by concurrent disease in older subjects and by late sampling. Lp(a) determination for cardiovascular risk profiling is not confounded by associated physical effort. It is also unlikely that the acute risks of major physical effort are enhanced by any process involving Lp(a).

Fibrinogen, lipoprotein (a) and lipids in patients with erectile dysfunction. A preliminary study.

BACKGROUND: Erectile dysfunction is associated with cardiovascular risk factors (e.g. hypertension, smoking, dyslipidemia and diabetes) and is more common in patients with cardiovascular disease. We therefore assessed the prevalence of two predictors of vascular events, fibrinogen and lipoprotein-a, in patients with and without erectile dysfunction. METHODS: Men with erectile dysfunction (48 non-smokers, 48 smokers), aged 45-70 years, were compared with controls (21 non-smokers, 21 smokers) with normal erectile function and no known pathology. RESULTS: Serum total cholesterol was significantly higher in non-smokers with erectile dysfunction compared to both control non-smokers and erectile dysfunction smokers. Men with erectile dysfunction who smoked had a significantly higher plasma fibrinogen level than control smokers. Similarly, men with erectile dysfunction, who did not smoke had higher levels of plasma fibrinogen compared to both smokers and non-smokers without erectile dysfunction. No significant difference in serum lipoprotein-a values was found. CONCLUSIONS: These findings support the concept that cardiovascular risk factors are predictors of erectile dysfunction and that this may be another manifestation of vascular disease.

Doxazosin, an alpha1-adrenoceptor antagonist, inhibits serotonin-induced shape change in human platelets.

Patients with peripheral vascular disease or diabetes mellitus tend to have elevated circulating levels of naturally occurring platelet agonists like serotonin (5 hydroxytryptamine; 5-HT). This bioamine can induce platelet shape change (PSC) an early phase of platelet activation, which is essentially aspirin resistant. In addition, 5-HT exerts other harmful effects (eg stimulating vascular smooth muscle proliferation and inducing vasoconstriction in atheromatous coronary vessels). The aim of this study was to determine whether doxazosin inhibits 5-HT-induced PSC. Doxazosin is a long acting alpha(1)-adrenoceptor antagonist, used in the treatment of essential hypertension and/or benign prostatic hyperplasia (BPH). Platelet rich plasma (PRP) was prepared from healthy volunteers (n = 8; five males and three females with a median age of 32 years, range: 26-57). Agonists (5-HT, 0.06-0.5; ADP, 0.1-0.2 micromol/l or U46619, a TXA(2)analogue, 0.025-0.05 micromol/l) were added to PRP and aliquots were removed at specific time points for median platelet volume (MPV) measurement (using a high-resolution channelyser). The MPV was used as an indicator of PSC. PRP was also incubated with doxazosin (final concentration: 0.33 microM, a concentration similar to therapeutic plasma levels) prior to the addition of each of the above-mentioned agonists. Doxazosin significantly inhibited (P = 0.007 and P = 0.008, at 30 sec and 60 sec, respectively) the 5-HT-induced increase in MPV. Doxazosin did not significantly inhibit ADP- or U46619-induced PSC. The inhibitory effect of doxazosin seems to be specific to platelet 5-HT(2) receptors, since there was no effect on ADP- or U46619-induced PSC. This inhibition of platelet activation may be an additional, clinically relevant, advantage. Future in vivo studies should consider assessing the effect of doxazosin on 5-HT-induced platelet activation.

A potential role for endothelin-1 in peripheral vascular disease.

The distribution of endothelin-1 (ET-1) and its receptors (ET(A)/ET(B)) has been studied in segments of femoral artery obtained from patients undergoing operation for peripheral vascular disease (PVD) using a combination of immunohistochemistry and autoradiography. Both receptor subtypes were located on the tunica media of vessel segments, with ET(A)-receptors predominating. Densitometric analysis showed that there was no difference in receptor binding to proximal/distal arterial segments from PVD patients. High-resolution autoradiography identified ET(B) binding to vascular nerves and vasa vasorum, mainly in distal portions of femoral arteries. Immunoreactive ET-1 was also identified that was associated with the vasa vasorum.

Effect of endothelin-1 on human platelet shape change: reversal of activation by naftidrofuryl.

Naftidrofuryl (Praxilene; NAF) significantly improves claudication distance in patients with peripheral vascular disease (PVD). Endothelin-1 (ET-1) is a powerful endogenous vasoconstrictor and the circulating levels of ET-1 are elevated in patients with vascular disease. Platelet rich plasma (PRP) was prepared from healthy volunteers. NAF at concentrations similar to therapeutic levels (3.5-14 micromol/l), inhibited (P < 0.02) platelet activation (as indicated by a fall in median platelet volume, MPV) induced by ET-1 (0.4 micromol/l) alone. NAF also inhibited (P <0.0001) shape change (PSC; an early phase of platelet activation, characterised by an increase in MPV) induced by ET-1 (0.4 micromol/l) in combination with ADP (0.05-0.15 micromol/l) or serotonin (0.03-0.13 micromol/ l). We assessed the effect of ET(A) (BQ123, 50 nmol/l) or ET(B) (BQ788, 50 nmol/l) receptor antagonists on PSC induced by ET-1 alone. Both antagonists significantly inhibited PSC. We conclude that ET-1 activates human platelets. Both ET(A) and ET(B) receptors probably contribute to this response by a complex mechanism that requires further elucidation. NAF antagonises the action of ET-1 on human platelets. These actions may contribute to the beneficial effects of NAF in PVD.

Angiotensin II can induce and potentiate shape change in human platelets: effect of losartan.

Platelet shape change (PSC) is an early phase of platelet activation that precedes platelet aggregation. This phase of platelet activation is essentially aspirin resistant. PSC was monitored, by measuring the median platelet volume (MPV) using a high resolution channelyser. Angiotensin (Ang) II, added in vitro, caused a significant (P = 0.004) increase in MPV in platelet rich plasma prepared from healthy subjects (n = 14). This increase in MPV was marked (>0.40 fl) in 57% (n = 8) of these subjects and was significantly inhibited (P<0.008) by losartan (a selective Ang II antagonist) at concentrations similar to those achieved in the circulation during treatment. Ang II also significantly enhanced sub-maximal PSC induced by ADP and serotonin in all subjects tested. Losartan significantly (n = 9; P<0.001) inhibited U46619 (a thromboxane A2 analogue)-induced PSC. These findings suggest that losartan, in addition to its blood pressure lowering action, has antiplatelet activity. This property may be clinically relevant because of the increased risk of vascular events in hypertensive patients.

Shape change in human platelets: measurement with a channelyzer and visualisation by electron microscopy.

The increase in median platelet volume (MPV) that occurs following the addition of various agonists can be recorded by high-resolution channelyzers. However, this increase in MPV may not represent the platelet shape change (PSC), an early phase of platelet activation characterised by the formation of pseudopodia. We therefore compared the MPV after adding various agonists (serotonin, ADP or a thromboxane A2 analogue) with scanning electron micrographs (EM) of the platelets in the same sample. Saline alone did not increase the MPV or alter platelet morphology. In contrast, the selected agonists altered both the MPV and EM. Adrenaline (5.0 mumol/l) alone did not influence either of these indices of platelet activation. However, when a lower concentration of adrenaline (0.2 mumol/l) was added in combination with a dose of serotonin (0.02 mumol/l) that did not affect the MPV on its own, the measured MPV increased significantly and the EM showed pseudopodia formation. Because the channelyzer method is reproducible and suited to serial measurements of the MPV, it may become a useful tool for the investigation of platelet activation.

An investigation of the serotonergic effects of fenfluramine, dexfenfluramine and dexnorfenfluramine using platelets as neuronal models.

The appetite-suppressant, fenfluramine (d,l-F) has been used for several decades to treat obesity. Dexfenfluramine (d-F), the d-enantiomer of d,l-F, was approved in 1996 for long-term administration. Subsequently, these drugs were voluntarily withdrawn due to reports of adverse effects on heart valves. So far, the evidence regarding the serotonergic action of d,l-F and d-F has relied on animal-based experiments. We used human platelets as neuronal models to assess the serotonergic action of both d,l-F, d-F and the main metabolite of d-F, d-norfenfluramine (d-norF). This was evaluated using a sensitive method that assesses platelet shape change (PSC) as expressed by an increase in median platelet volume (MPV). Human platelets increased their MPV in response to d-norF. This action was probably mediated via serotonin (subtype 2) receptors because a specific antagonist blocked it. This is the first demonstration, using human tissue, of the serotonergic action associated with the administration of d-F.