Giuseppe G. Nenci

Diabetes Mellitus, Hypercholesterolemia, and Hypertension but Not Vascular Disease Per Se Are Associated With Persistent Platelet Activation In Vivo Evidence Derived From the Study of Peripheral Arterial Disease

BACKGROUNDnPrevious studies relating increased thromboxane (TX) biosynthesis to cardiovascular risk factors do not answer the question whether platelet activation is merely a consequence of more prevalent atherosclerotic lesions or reflects the influence of metabolic and hemodynamic disturbances on platelet biochemistry and function.nnnMETHODS AND RESULTSnWe examined 64 patients with large-vessel peripheral arterial disease and 64 age- and sex-matched control subjects. TXA2 biosynthesis was investigated in relation to cardiovascular risk factors by repeated measurements of the urinary excretion of its major enzymatic metabolite, 11-dehydro-TXB2, by radioimmunoassay. Urinary 11-dehydro-TXB2 was significantly (P = .0001) higher in patients with peripheral arterial disease (57 +/- 26 ng/h) than in control subjects (26 +/- 7 ng/h). Seventy percent of patients had metabolite excretion > 2 SD above the normal mean. However, 11-dehydro-TXB2 excretion was enhanced only in association with cardiovascular risk factors. Multivariate analysis showed that diabetes, hypercholesterolemia, and hypertension were independently related to 11-dehydro-TXB2 excretion. During a median follow-up of 48 months, 8 patients experienced major vascular events. These patients had significantly (P = .001) higher 11-dehydro-TXB2 excretion at baseline than patients who remained event free.nnnCONCLUSIONSnThe occurrence of large-vessel peripheral arterial disease per se is not a trigger of platelet activation in vivo. Rather, the rate of TXA2 biosynthesis appears to reflect the influence of coexisting disorders such as diabetes mellitus, hypercholesterolemia, and hypertension on platelet biochemistry and function. Enhanced TXA2 biosynthesis may represent a common link between such diverse risk factors and the thrombotic complications of peripheral arterial disease.

Thromboxane synthase inhibitors, thromboxane receptor antagonists and dual blockers in thrombotic disorders

Thromboxane A2 (TXA2) plays a pivotal role in platelet activation and is involved in the development of thrombosis. Thromboxane synthase inhibitors suppress TXA2 formation and increase the synthesis of the antiaggregatory prostaglandins PGI2 and PGD2; however, accumulated PGH2 may interact with the platelet and vessel wall TXA2 receptor, thus reducing the antiplatelet effects of this class of drug. TXA2 receptor antagonists block the activity of both TXA2 and PGH2 on platelets and the vessel wall. Very recently, drugs possessing both thromboxane synthase-inhibitory and thromboxane receptor-antagonist properties have been developed. Paolo Gresele and colleagues explain here why these drugs can be more efficacious than traditional antiplatelet agents and review the available experimental studies involving these drugs.

Evidence for a Storage Pool Defect in Platelets From Cirrhotic Patients With Defective Aggregation

The mechanisms underlying the defective platelet function in cirrhotic patients were investigated. Eleven cirrhotic patients with mild disease (group 1), 20 patients with severe cirrhosis (group 2), and 31 controls were studied. Platelet aggregation was significantly reduced in cirrhotics compared with controls. Compared with controls, cirrhotic patients in group 2 showed a significant reduction in the total content of adenosine triphosphate (57.8 +/- 7.8 vs. 26.1 +/- 6.3 mumol/10(11) platelets; P less than 0.05), 5-hydroxytryptamine (285 +/- 26 vs. 104 +/- 38 nmol/10(11) platelets; P less than 0.05), beta-thromboglobulin (2129 +/- 120 vs. 1223 +/- 161 ng/10(8) platelets; P less than 0.01), and platelet factor 4 (1389 +/- 108 vs. 805 +/- 176 ng/10(8) platelets; P less than 0.05). In patients with severe disease, an increase in plasma beta-thromboglobulin-platelet factor 4 ratio, an index of in vivo platelet activation, was observed (controls, 3.50 +/- 0.50; group 1, 4.02 +/- 0.80; and group 2, 6.59 +/- 1.15). Our data indicate the existence of a platelet storage pool defect, which may favor the bleeding tendency of cirrhotic patients.

Activated human protein C prevents thrombin-induced thromboembolism in mice. Evidence that activated protein c reduces intravascular fibrin accumulation through the inhibition of additional thrombin generation.

Activated protein C (APC) is a potent physiologic anticoagulant with profibrinolytic properties, and has been shown to prevent thrombosis in different experimental models. We investigated the effect of human APC on thrombin-induced thromboembolism in mice, a model of acute intravascular fibrin deposition leading to death within minutes. APC given intravenously (i.v.) as a bolus 2 min before thrombin challenge (1,250 U/kg) reduced mortality in a dose-dependent manner despite the lack of thrombin inhibitor activity. Significant inhibition of thrombin-induced death was observed at the dose of 0.05 mg/kg, and maximal protection was obtained with 2 mg/kg (> 85% reduction in mortality rate). Histology of lung tissue revealed that APC treatment (2 mg/kg) reduced significantly vascular occlusion rate (from 89.2 to 46.6%, P < 0.01). The protective effect of APC was due to the inhibition of endogenous thrombin formation as indicated by the fact that (a) the injection of human thrombin caused a marked decrease in the coagulation factors of the intrinsic and common pathways (but not of Factor VII), suggesting the activation of blood clotting via the contact system; (b) APC pretreatment reduced markedly prothrombin consumption; (c) the lethal effect of thrombin was almost abolished when the animals were made deficient in vitamin K-dependent factors by warfarin treatment, and could be restored only by doubling the dose of thrombin, indicating that the generation of endogenous thrombin contributes significantly to death; and (d) APC failed to protect warfarin-treated animals, in which mortality is entirely due to injected thrombin, even after protein S supplementation. Other results suggest that APC protects from thrombin-induced thromboembolism by rendering the formed fibrin more susceptible to plasmin degradation rather than by reducing fibrin formation: in thrombin-treated mice, fibrinogen consumption was not inhibited by APC; and inhibition of endogenous fibrinolysis by epsilon-aminocaproic or tranexamic acid resulted in a significant reduction of the protective effect of APC. Since APC did not enhance plasma fibrinolytic activity, as assessed by the measurement of plasminogen activator (PA) or PA inhibitor (PAI) activities, PAI-1 antigen, or 125I-fibrin degrading activity, we speculate that the inhibition of additional (endogenous) thrombin formation by APC interrupts thrombin-dependent mechanisms that make fibrin clots more resistant to lysis, so that the intravascular deposited fibrin can be removed more rapidly by the endogenous fibrinolytic system.

Altered platelet function associated with the bronchial hyperresponsiveness accompanying nocturnal asthma

BACKGROUNDnNocturnal awakening is a common feature of bronchial asthma, and yet the mechanisms underlying this phenomenon are poorly understood. We investigated whether nocturnal awakening is associated with changes in platelet function with the use of a variety of markers of platelet activation.nnnMETHODSnTen patients with a history of nocturnal asthma and 10 age- and sex-matched healthy control subjects were studied at 10:00 PM, 4:00 AM, and 10:00 AM on 2 consecutive days. The following parameters were tested: forced expiratory volume in 1 second (FEV1), log dose of methacholine inducing a 20% fall in FEV1, platelet count and volume, platelet aggregation induced by collagen or activating factor, and plasma and intraplatelet levels of beta-thromboglobulin and platelet factor 4.nnnRESULTSnWe have demonstrated that altered platelet function and platelet activation occurs at 4:00 AM in patients with nocturnal asthma and is associated with the maximum increases in bronchial reactivity. Such changes were not observed in 10 control subjects. Platelet dysfunction has been detected as a reduced aggregatory response of platelets to collagen and platelet activating factor such that up to 5 times more platelet activating factor and 1.5 times more collagen were required to elicit a threshold aggregatory response in asthmatic subjects when compared with control subjects; this difference was evident at all time points tested. Furthermore, at 4:00 AM there were significantly lower levels of intraplatelet beta-thromboglobulin corresponding to the maximum reduction in peak expiratory flow and to the maximal increase in bronchial responses to inhaled methacholine.nnnCONCLUSIONSnThese results suggest that platelet activation accompanies nocturnal asthma and further suggest that platelets may play a role in this common clinical condition.

Low molecular weight heparins: are they interchangeable? No.

Besides the well-known effect of molecular weight (MW) distribution of the heparin chains of the different low molecular weight heparins (LMWHs) on the relative inhibition of thrombin (IIa) and of factor Xa (fXa), several chemical differences can influence the biological activity of the various commercial fractionated heparins. Random cleavage of glycosidic bonds used to prepare LMWHs will inevitably degrade some of the pentasaccharide units during depolymerization of the parent heparin chains with high affinity to antithrombin III. Therefore, LMWHs prepared for clinical use have a lower proportion of high-affinity chains than unfractionated heparin (UFH) [1] and thus a lower specific activity. This adds to decreased activity due to loss of sulfate ester groups during depolymerization. The extent of activity loss may very well depend on the cleavage procedure adopted for the individual commercial product. Thus, even if differences in terminal residues [2] do not influence bioequivalence and if the same is true for the final ion-exchange clean-up applied to dalteparin, the loss of sulfate ester groups or the resulfatation of some LMWHs can alter the activity spectrum of the product [1]. Mean MW varies from 3800 Da for certoparin to 6500 Da for tinzaparin, and the (a-Xa/a-IIa) ratio can range from 2 for ardeparin to 3.3–3.5 for enoxaparin [3]. In addition to these differences, denaturation during depolymerization may add variability to the pharmacological characteristics and the clinical effects of the various LMWHs, and offer an answer to the question of why prophylactic doses of LMWHs result in different anti-Xa anti-IIa activity in blood of patients. UFH 2000 U injected subcutaneously gave a-Xa values of 0.03–0.13 U mL 1 from 2 to 4 h after administration across most of the comparative studies cited in Table 1. Similar a-Xa levels (as well as similar efficacy and safety) were shown after dalteparin 2500 U, parvaparin 7500 U, reviparin 1750 U or tinzaparin 2500–3500 U. nadroparin 2850 U was associated with an a-Xa level of 0.38 U mL 1 (and the same bleeding rate as the placebo); dalteparin 5000 U gave comparable plasma aXa activity (together with an excess of major bleeds with respect to UFH 5000 IU 2), as did 40 mg and 30 mg 2 enoxaparin (with an excess of local hematomas for the latter dosage), 4200 U reviparin, and as much as parvaparin 15 500 coagulometric a-Xa U. In other hands, dalteparin 5000 U resulted in 0.13 U mL 1 a-Xa activity after 2 h and no excess bleeding when compared with UFH. In one of the few comparative trials in 440 hip surgery patients, reviparin 4200 U and enoxaparin 40 mg resulted in equal efficacy and safety, but a-Xa levels were consistently lower in reviparin-treated patients (all the above studies have been reported by Sarret et al. [3]). It is clear that standardization of a-Xa assays is far from perfect. However, most studies show not only that similar plasma aXa levels are obtained with a broad range of dosages for the different LMWHs, but also that clinical efficacy and safety are largely independent of the a-Xa levels attained by the different LMWHs used for thromboprophylaxis. Several meta-analyses have failed to show a superiority of LMWHs on UFH for thromboprophylaxis in general surgery. However, meta-analyses on a drug class may fail to document a clinical effect in terms of efficacy/safety ratio when the different drugs behave differently in the various studies. In effect, when compared to UFH, some LMWHs showed better efficacy but equal safety [4,5], others better safety but equal efficacy [6–8], and still others equal efficacy but lower safety [9]. Halving the dose of the latter LMWH proved safer but less effective [10,11]; incidentally, this half-dosage had been shown to be equally efficacious but safer than UFH [12] Such conflicting results may annihilate one another when pooled in a meta-analysis. Different results on the treatment of acute coronary syndromes (ACS) with LMWHs have been shown by several trials. In contrast to dalteparin and nadroparin studies, ESSENCE and TIMI 11 B independently showed the superiority of enoxaparin over UFH. Interestingly, enoxaparin but not dalteparin was able to suppress the serious rise of von Willebrand factor seen in ACS patients treated with UFH [13]. It is not clear if at least some of differences in the results of the above trials are due to chance or bias. A possible explanation could be the varying lower number of events or differences in the severity of illness, or in the rates of revascularization [14] as well as the pharmacological profile of the different drugs. In any case, for the time being a difference in the clinical results does exist and must be taken into account. Differences in efficacy and safety between LMWHs probably exist also in the treatment of venous thromboembolism (VTE). When the log odds ratio for bleeding is plotted against that for recurrent VTE of an LMWH used for the treatment of a venous thromboembolic event, there appears to be some variations among six currently available LMWHs [15]. For example, dalteparin proves to be associated with a statistically significant difference in efficacy (which was lower) and safety (which was Journal of Thrombosis and Haemostasis, 1: 12–13

Effects of gemfibrozil on insulin sensitivity and on haemostatic variables in hypertriglyceridemic patients.

In order to assess the efficacy of gemfibrozil on lipid and haemostatic parameters in patients with plurimetabolic syndrome, a multicenter double-blind placebo controlled, parallel study was carried out in 56 patients with primary hypertriglyceridemia and glucose intolerance. These patients had elevated PAI activity and antigen and t-PA antigen levels at rest and after venous occlusion. Gemfibrozil reduced plasma triglyceride levels (P<0.001), whereas it increased free fatty acids (P<0.05) and high density lipoprotein cholesterol levels (P<0.05). In those patients reaching normalization of plasma triglyceride levels (triglyceride reduction > or =50%) (n=15), insulin levels (P<0.05) as well as the insulin resistance index were reduced by gemfibrozil treatment, suggesting an improvement of the insulin resistance index in this patient subgroup. Gemfibrozil treatment did not affect plasma fibrinolysis or fibrinogen levels, despite marked reduction of plasma triglycerides and improvement of the insulin sensitivity associated with triglyceride normalization.

Diagnosis of deep vein thrombosis in asymptomatic high-risk patients

Postoperative deep vein thrombosis is usually asymptomatic so that pulmonary embolism is often the first clinical manifestation of venous thromboembolism. The diagnostic accuracy of impedance plethysmography, 125I-fibrinogen leg scanning and real-time B-mode ultrasonography has been extensively evaluated in patients with asymptomatic deep vein thrombosis. Impedance plethysmography has been evaluated in a number of studies and its sensitivity has been invariably found to be approximately 20% or less. These results seem to be due to the high prevalence in asymptomatic patients of distal, small and nonocclusive thrombi, unable to produce a critical obstruction of the venous flow. The accuracy of 125I-fibrinogen leg scanning has been assessed in a number of studies and found to be significantly different in the initial and more recent studies. This difference has been found to be due to the quality of the study design: the better the methodology, the lower the diagnostic accuracy of 125I-fibrinogen leg scanning. The association of impedance plethysmography and 125I-fibrinogen leg scanning do not result in an improvement of the results obtained by the single diagnostic method. Study methodology strongly influenced the results obtained with real-time B-mode ultrasonography with a reported sensitivity of approximately 50% in the studies performed adopting an appropriate methodology. In conclusion, noninvasive diagnostic methods are inaccurate in the diagnosis of asymptomatic deep vein thrombosis. Thus, venography remains the only accurate diagnostic method for the diagnosis of asymptomatic deep vein thrombosis.

Prolonged Antithrombin Activity of Low-Molecular-Weight Heparins Clinical Implications for the Treatment of Thromboembolic Diseases

BACKGROUNDnThe mechanism for the efficacy of once- or twice-daily subcutaneous injections of low-molecular-weight heparins (LMWHs) for the treatment of venous thromboembolism has been difficult to explain. The confusion exists because the observation from experimental studies that the antithrombin activity of LMWHs is necessary for their antithrombotic effect is inconsistent with the reported short half-life of the antithrombin activity of LMWHs. Previous pharmacokinetic studies were performed with lower doses of LMWHs than have been used in contemporary trials, and antithrombin activity was assessed with the barely sensitive chromogenic assay.nnnMETHODS AND RESULTSnWe performed a pharmacokinetic study to compare the relative half-lives of prophylactic and therapeutic doses of LMWHs assessing antithrombin activity with both the chromogenic and a more sensitive assay (plasma thrombin neutralization assay). An eight-way cross-over randomized study in healthy volunteers was performed. Enoxaparin (20 and 40 mg and 1 and 2 mg/kg) and nadroparin (7500 and 10,000 ICU and 225 and 450 ICU/kg) were administered subcutaneously. The maximal peak activity for aPTT ratio was 1.7. A dose-dependent peak activity was found for both antifactor Xa and antithrombin activities. Disappearance time of these activities after the highest dose of both LMWHs was longer than 16 hours. Overall mean antifactor Xa activity half-life was 4.6 hours. Overall mean antithrombin activity half-life was longer than 4 hours.nnnCONCLUSIONSnOur results provide an explanation for the effectiveness of LMWHs administered either once or twice daily. High and sustained plasma antithrombin activity is achieved when LMWHs are administered in therapeutic doses used in contemporary trials with only a moderate prolongation of the aPTT.

Effects of Desmopressin on Hemostasis in Patients with Liver Cirrhosis

The aim of this double-blind, placebo-controlled crossover study was to investigate the effect of 1-deamino-8-D-arginine vasopressin (dDAVP) on hemostasis in patients with chronic liver disease. Nine consecutive patients with biopsy-proven liver cirrhosis and related coagulation abnormalities received in a random order dDAVP, 0.5 microgram/kg, or saline intravenously. Blood samples were taken before dDAVP infusion and 30, 60 and 180 min after its end. dDAVP infusion induced a statistically significant shortening of the bleeding time from 9 min (range 6.5-15.5) to 6 min (range 4.5-9.5) at 1 h after the infusion. The activated partial thromboplastin time was significantly shortened at 30 and 60 min after dDAVP infusion. Plasma levels of factor VIII, XI and XII coagulant activities were significantly increased at all sampling times after dDAVP infusion. The maximum increase over basal values in plasma levels of factor VIII, XI and XII was 63, 22 and 40%, respectively. dDAVP did not induce any significant changes of prothrombin time, thrombin clotting time, fibrinogen, plasma levels of factor II, V, VII, IX, X, factor XII antigen, protein C (activity and antigen), antithrombin III, plasminogen and alpha 2-antiplasmin. Placebo infusion did not produce any significant changes in the evaluated parameters. We conclude that dDAVP can positively influence the hemostatic system in patients with liver cirrhosis. The clinical relevance of this hemostatic improvement deserves further evaluation.