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Background: Ximelagatran has been approved in Europe for VTE prophylaxis in orthopedic surgery at fixed doses and without laboratory monitoring. Aim of the study was to evaluate safety and efficacy of ximelagatran in a meta-analysis of prophylaxis and/or treatment randomized controlled trials., Methods: Absolute risk of events for ximelagatran and OR for its comparison with LMWH and coumarins were calculated. Subgroup analysis was performed for ximelagatran regimen, comparator agent, type of surgery, starting time of prophylaxis., Results: Twelve studies and 16,992 patients were meta-analysed. Ximelagatran showed an absolute risk of major VTE of 4.04% and 1.69% and of major bleedings of 1.68% and 1.03% in prophylaxis and treatment trials, respectively. In prophylaxis trials, a significant excess mortality (OR: 2.5; 95% CI: 1.02 - 6.13) and an excess in major bleedings (OR: 1.41; 95% CI: 0.93 - 2.14) was found in the whole ximelagatran group. No evidence of treatment effect for major VTE was seen in the comparison with LMWH (OR: 1.01; 95% CI: 0.52 - 1.97). The cohort of patients treated with 24 mg b.i.d. showed similar results. An increase in the absolute risk of bleeding (from 1.04% to 3.03%) was found between post and preoperative administration of ximelagatran. Major VTE risk was increased when ximelagatran was compared to b.i.d. LMWH., Conclusions: Ximelagatran can be considered for its potential advantages for prevention and treatment of VTE. Future efforts are needed by researchers to prospectively investigate the best postoperatively starting time and by clinicians to monitor overall mortality in prophylactic use.

Background: Patients with kidney failure present endothelial dysfunction, which was shown to be partly corrected by hemodialysis. No data exist on the effects of hemodialysis on endothelial dysfunction in kidney failure patients with associated vascular risk factors. The aim of this study was to evaluate the acute effects of hemodialysis on endothelial dysfunction in patients with kidney failure and associated vascular risk factors and to assess the role of endothelium-toxic substances., Methods: We assessed endothelial dysfunction in 13 patients with chronic renal failure and other vascular risk factors before and after hemodialysis and in 13 healthy controls and simultaneously measured nitric oxide (NO) synthesis and activity. Endothelial dysfunction was studied using an echographic method as flow-mediated dilation (FMD) of the brachial artery; plasma NO2- and NO3-, cyclic guanosine-5-monophosphate (cGMP), plasma homocysteine levels and low molecular mass-advanced glycation end-products (LMM-AGEs) were simultaneously measured., Results: As compared with healthy controls, patients with renal failure showed a reduced FMD (2.89 +/- 1.43 vs. 7.81 +/- 1.54%, p < 0.01) which was not corrected by dialysis (after dialysis 2.40 +/- 1.65%, p = NS vs. pre). Plasma NO2- and NO3- were normal or slightly increased and remained unchanged after dialysis. Plasma cGMP levels were reduced and remained unchanged after dialysis. Homocysteine and LMM-AGE plasma levels were raised and, although significantly reduced by dialysis, remained higher than in controls., Conclusions: Patients with kidney failure and associated vascular risk factors show an endothelial dysfunction related to defective NO activity, which is not corrected by hemodialysis despite the reduction, though not to normal, in homocysteine and LMM-AGE levels. Endothelial dysfunction may contribute to the progression of atherosclerosis in patients with kidney failure and vascular risk factors.

Dermatan sulphate (DS) is a glycosaminoglycan which selectively catalyzes the inactivation of thrombin by Heparin Cofactor II without interacting with Antithrombin III. DS does not interact with other coagulation factors and, unlike heparin, is able to inactivate thrombin bound to fibrin or to the surface of an injured vessel. Efficacy and safety of DS have been validated in studies on thromboprophylaxis and on the anticoagulation for hemodialysis. Studies on thromboprophylaxis have been performed in "medical" patients as well as in general, orthopedic and oncological surgery. In this last setting, DS proved to be more efficacious than heparin, in the absence of excess bleeding. No statistically significant differences were observed between DS and heparin in hemodialysis. A low-molecular-weight DS,which shows a higher bioavailability after s.c. administration, has been tested in pilot studies on the treatment of venous thromboembolism with encouraging results. Two DS-containing compounds, sulodexide and, particularly, mesoglycan, have been clinically studied in a number of trials and found to be effective in the treatment of venous and arterial leg diseases.

Objective: To assess the effect of treatment with mesoglycan, a sulphated polysaccharide compound, on the walking capacity of patients with stage II peripheral arterial disease., Methods: Non-diabetic outpatients with intermittent claudication, duplex ultrasound evidence of peripheral atherosclerosis, ankle/arm index <0.80, systolic ankle pressure >50 mmHg, and absolute walking distance (AWD) between 100 and 300 m (standardised treadmill test) were eligible. After a 5-week run-in on single-blind placebo, patients were randomised to double-blind treatment with mesoglycan, 30 mg/day intramuscularly for 3 weeks followed by 100 mg/day orally for 20 weeks, or matching placebo. All patients received low-dose aspirin and lifestyle instructions. Clinical response was defined as an AWD increase at Week 23 >50% over baseline. Health-related quality of life and ischaemic events were assessed as secondary efficacy variables., Results: 242 patients were randomised and 237 were assessed for clinical response. Patients achieving clinical response were 59/118 with mesoglycan (50.0%) and 31/119 with placebo (26.1%; p <0.001). Geometric mean AWD increased from 192 to 298 m with mesoglycan, and from 192 to 238 m with placebo (p <0.001). Pain-free walking distance showed a non-significant increase with mesoglycan (p = 0.057). Changes in quality of life scores were in favour of mesoglycan. The rate of ischaemic events was 1/120 on mesoglycan and 6/122 on placebo (p = 0.053). The rate of non-ischaemic adverse events leading to treatment discontinuation was 7/120 and 4/122, respectively., Conclusion: Treatment with mesoglycan improves the walking capacity of patients with intermittent claudication, and might confer additional antithrombotic protection over that of aspirin.

Background and Objectives: Thrombin-induced thromboembolism in mice is a model in which the feed-back clotting activation produced by the injected enzyme greatly contributes to fibrin accumulation in lungs and to mortality. Using this model we have previously shown that activated human protein C (aPC), by interrupting endogenous clotting activation at a high level (factors Va and VIIIa), prevents mortality inducing only a minor hemostatic impairment. With the same model we have now compared the antithrombotic and prohemorrhagic effects of two low molecular weight heparins (LMWHs), reviparin and tinzaparin, which are expected to inhibit preferentially the positive feed-back triggered by thrombin (anti Xa activity), with those of unfractionated heparin (UFH) and PEG-hirudin, which inhibit mainly or exclusively thrombin activity (anti IIa activity)., Design and Methods: Pulmonary thromboembolism was induced in mice by i.v. injection of bovine thrombin (1,000U/kg). Drugs (from 0.12 to 1.2 mg/kg) were given as bolus injection 2 min prior to thrombin challenge and mortality was assessed within 15 min. The bleeding time was assessed by a tail tip transection model. Activated partial thromboplastin time (aPTT), thrombin clotting time (TcT), fibrinogen assay and anti Xa activity determination were performed in citrated plasma from saline- or drug-treated animals., Results: All drugs protected mice from thrombin-induced mortality in a dose-dependent way. At comparable antithrombotic dosages, the anti IIa activity generated in plasma (assessed by TcT) was highest with UFH, intermediate with tinzaparin and very low with reviparin. Accordingly, the fibrinogen drop, which is caused mainly by the injected thrombin, was prevented by the heparins to an extent that was fairly well related to their anti IIa activity. aPTT and bleeding time, used as measures of hemorrhagic risk, were markedly more prolonged by UFH than by reviparin. Tinzaparin, instead, had an intermediate effect. Interestingly, PEG-hirudin, at equipotent antithrombotic dosages, caused a prolongation of bleeding time comparable to that observed with UFH., Interpretations and Conclusions: Our data show that, in our model, drugs acting at a high level of the blood clotting cascade, like LMWHs with a high anti Xa/anti IIa ratio, display a better antithrombotic/prohemorrhagic profile than drugs acting prevalently on thrombin.

On the assumption that antithrombotic drugs are able to reduce the incidence of any vascular event independently of where it first occurs, they are used for the secondary prevention of arterial vascular disease in different locations. The Antiplatelet Trialists' Collaboration meta-analysis has shown that the net benefit of antiplatelet drugs in the prevention of stroke, acute myocardial infarction and vascular death is about the same for patients with prior stroke or prior myocardial infarction. Most of the trials included in the Antiplatelet Trialists' Collaboration meta-analysis used aspirin, which was shown to lower the risk of stroke, myocardial infarction, and vascular death in patients with a history of transient ischemic attack or stroke. Aspirin should be given to patients operated on for symptomatic carotid stenosis and should be considered for asymptomatic patients. In a comparative study ticlopidine (500 mg) vs aspirin (650 mg), ticlopidine reduced the relative risk of vascular events by 9% and of recurrent stroke by 21%. When clopidogrel (75 mg) was compared with aspirin (325 mg), a 7.3% relative risk reduction was seen in the stroke group (6431 patients) of the CAPRIE study; a reduction in hemorrhagic events, especially in gastrointestinal bleeding, was also seen. At variance with previous studies, the ESPS-2 showed an advantage when dipyridamole (400 mg) was added to aspirin (50 mg). Oral anticoagulants are more effective than aspirin in the prevention of cardioembolic stroke in patients with atrial fibrillation. The higher efficacy of indobufen with respect to aspirin in this particular setting needs confirmation. Inhibition of thrombosis may be one of the mechanisms explaining the effect of statins in reducing both stroke and cardiac events in high-risk patients.

Defibrotide is an antithrombotic drug which enhances prostacyclin production and activates fibrinolytic system. The aim of this study was to investigate the improvement of walking distance in patients with intermittent claudication treated with defibrotide. DICLIS was a double blind, placebo-controlled study which included patients with walking distance autonomy at a standardized treadmill test < or =350 > or =100 meters. A total of 310 patients were randomly allocated to placebo (n = 101), defibrotide 800 mg/day (n = 104) or defibrotide 1200 mg/day (n = 105). During a one year follow-up, the Absolute Walking Distance (AWD) was measured six times (0, 30, 60, 90, 180, 360 days). Similar improvement in walking distance was found in the three groups until the 90th day; thereafter placebo group showed no further increase, while AWD continued to increase in the defibrotide groups. Between the 180th and 360th day visits, AWD was significantly higher (P <0.01) in patients given defibrotide than in patients given placebo. No difference in efficacy was observed between the two dosages of defibrotide. No differences in side effects were observed among the three groups. The results of the present trial suggest that long-term administration of defibrotide improves walking distance in patients with intermittent claudication.

Excessive oxidative stress due to hyperglycemia and glycoxidation leads to an increased production of F2-isoprostanes, one of which, 8-iso-PGF2 alpha, reaches high concentrations in plasma and urine in both insulin-dependent and non-insulin-dependent diabetics. This is associated with an increase in platelet activation, reflected by an increased urinary excretion of platelet-derived TxB2. Improved metabolic control or vitamin E supplementation reduces urinary 8-iso-PGF2 alpha and TxB2, whereas aspirin or indobufen reduces TxB2 but not 8-iso-PGF2 alpha. Since TxB2 in the urine seems to represent the common link between diabetes (as well as other risk factors) and the thrombotic complications of vascular disease, platelet activation due to lipid-glycoxidation is an important aspect in the pathogenesis of vascular complications of diabetes mellitus. Among the various plasma coagulation and fibrinolysis factors that are found to be altered in diabetes, the increased level of plasminogen activator inhibitor (PAI-1) in the plasma and in the vessel wall is of the utmost importance. Indeed, it is suspected that the atherosclerotic plaques formed in the presence of high concentrations of PAI-1 are more prone to rupture and ensuing thrombosis. The thrombosis-oriented modifications of blood platelets, coagulation and fibrinolysis are an important cause behind the high prevalence of vascular events in diabetes.

Platelets contain, besides alpha- and delta-granules, lysosomes which store glycohydrolases able to degrade glycoproteins, glycolipids and glycosaminoglycans. While several studies have shown that alpha- and delta-granule secretion takes place "in vivo" in humans upon platelet activation, no data are available on the "in vivo" release of lysosomes. We have studied the release of platelet lysosomal contents "in vivo" in healthy volunteers at a localized site of platelet activation by measuring markers of lysosomal secretion in the blood oozing from a skin wound inflicted for the measurement of the bleeding-time. The levels of beta-N-acetylhexosaminidase (Hex) were 13.1 +/- 0.85 mU/ml in bleeding-time blood and 10.2 +/- 0.66 mU/ml in plasma (p <0.001). Hex in serum was 16.4 +/- 0.72 mU/ml. The levels of beta-galactosidase were also higher in bleeding-time blood than in plasma (0.85 +/- 0.07 mU/ml vs 0.4 +/- 0.05 mU/ml, p <0.001). In bleeding-time blood collected at one minute intervals, Hex rose progressively consistent with ongoing platelet activation and flow-cytometry showed a progressive increase of the expression of LIMP and LAMP-2, two lysosomal associated proteins. In conclusion, our data demonstrate that platelet lysosomal glycohydrolases are released "in vivo" in humans upon platelet activation.

In the search for a more acceptable route of heparin administration that can also be used for long-term treatment, we evaluated the bioavailability and antithrombotic activity of intraduodenal administration of unfractioned heparin (UFH) in rats. A radioiodinate derivative of UFH was administered intraduodenally in rats in conjunction with unlabeled UFH. We found that radioactivity increases very rapidly in plasma, as well as on the surface of aortic and caval segments, so that peak radioactivity was already achieved within 5 min of drug administration. Subsequently, plasma radioactivity declined rapidly, although 1.5-2.5% of the total radioactivity administered was still circulating 3 h after drug administration. The plasma anti-Xa activity was much lower and longer lasting than expected from the radioactivity counts in both peripheral and portal blood, and its level was very similar in the two circulatory districts, never exceeding 023 U/ml. This suggests that extensive degradation of the drug already occurs during its gastrointestinal absorption. Nevertheless, in a stasis-induced venous thrombosis model, intraduodenal UFH prevented thrombus formation in a dose-dependent way (ED50, 2000 IU/kg). The maximum antithrombotic effect was observed when the drug was administered 30-60 min before ligature of the vena cava, and a 40% reduction of thrombus weight was still present at 180 min. Since antithrombotic kinetics does not match the kinetics of either the plasma or vessel-bound radioactivity but approaches what is found in anti-Xa activity, the antithrombotic activity of oral heparin may be dependent on the release of unlabeled endogenous glycosaminoglycans deposited in the vessels.

The main aim of medical treatment for intermittent claudication (IC) is the reduction of mortality and morbidity from ischemic cardiovascular disease. However, symptomatic treatment with the aim of improving exercise performance and the overall quality of life may also be an important target of the clinical management of patients with intermittent claudication. Cloricromene, a drug with antithrombotic and anti-ischemic activities, has previously shown some promising results in patients with claudication. We have carried out a clinical trial to assess the effect of cloricromene on the claudication distance and on the quality of life of patients with IC chronically treated with aspirin. A total of 159 patients with IC, Stage II (Fontaine), were enrolled in a double-blind, randomized, prospective, multicenter study comparing cloricromene (100 mg orally b.i.d.) or an identical placebo for 6 months. All patients received 160 mg/day aspirin. The primary end-point was the improvement of initial claudication distance (ICD) at 6 months as measured by a standardized treadmill test. The secondary end-points were the absolute claudication distance (ACD) at 6 months, the percentage of patients defined as responders to treatment (improvement of ICD of at least 40%), changes in the ischemic window (IW), quality of life as assessed by the SF-36 questionnaire, and the occurrence of major cardiovascular events. The ICD increased in both treatment groups, with a non-significant difference at 6 months in favor of cloricromene of +12.3 m. The ACD, percentage of responders to treatment and ischemic window also improved in both groups with a slight, non-significant trend in favor of cloricromene. Pretreatment quality of life scores showed only a slight worsening compared with an age-matched, healthy population and did not change upon treatment. A post hoc subgroup analysis showed a significant benefit from cloricromene in patients with an ICD at enrollment higher than the median of the patient population. In conclusion, treatment with cloricromene for 6 months does not significantly improve claudication in patients with Stage II Fontaine peripheral arteriopathy chronically treated with aspirin. An improvement of 40-60 m in the ICD on a standardized treadmill test does not translate into a self-perceived improvement in the quality of life as assessed by the SF-36 questionnaire.

The use of low molecular weight heparin (LMWH) for the prevention and treatment of venous thromboembolism has been validated by numerous clinical trials and meta-analyses over the past 25 years. More recently, the possibility of extending treatment with LMWH to the arterial disease where thrombosis is a prominent feature has led to the planning of many clinical trials, several of which have been already published. LMWH has been tested in settings such as acute coronary syndromes, including myocardial infarction, surgery or percutaneous revascularization for coronary and peripheral arteries, and stroke. In most indications, LMWH has proved to be superior to or at least as effective as unfractionated heparin and it is also easier to administer.

We reported that protein kinase C (PKC) inhibitors increase the release of arachidonic acid induced by fluoroaluminate (AlF4-), an unspecific G-protein activator, in intact human platelets. Now we demonstrate that this effect is independent of the extracellular Ca2+ concentration and that AlF4(-)-induced release of AA is abolished by BAPTA, an intracellular Ca2+ chelator, even in the presence of GF 109203X, a specific and potent PKC inhibitor. This compound also blocks the liberation of the secretory phospholipase A2 in the extracellular medium, indicating that this enzyme is not involved in the potentiation of arachidonic acid by PKC inhibitors. On the other hand, the latter effect is completely abolished by treatment of platelets with AACOCF3, a specific inhibitor of cytosolic phospholipase A2 (cPLA2). These observations indicate that cPLA2 is responsible for the AlF4(-)-induced release of arachidonic acid by a mechanism that is down-regulated by PKC.

Background: Dialysis access occlusion is the most common cause of hospitalization and a frequent indirect cause of mortality in patients on chronic hemodialysis. The clinical assessment of an arteriovenous shunt is presently the most widely adopted method for the diagnosis of vascular access occlusion in hemodialysis patients, but no studies have yet investigated objectively its sensitivity and positive predictive value (PPV). Continuous-wave (CW) Doppler ultrasound is a simple, inexpensive, and noninvasive technique for the assessment of arterial blood flow. We have carried out a prospective evaluation of the PPV of CW Doppler for the diagnosis of vascular access occlusion in hemodialysis patients and compared it with clinical investigation., Methods: Fourty-one hemodialysis patients with clinical diagnosis of occlusion of their fistula were studied, and in 23 of them the diagnosis of occlusion was objectively validated., Results: CW Doppler in the patients in whom occlusion was objectively validated showed PPV of 86 and 83% under basal conditions and after fistula compression, respectively, with sensitivities of 95 and 100%, respectively. Clinical diagnosis, under the same conditions, showed a PPV of 83% and a sensitivity of 100%., Conclusions: CW Doppler and clinical examination have a similar high sensitivity for the diagnosis of occlusion of the dialysis access; thus, there is no need to use routinely Doppler CW examination, unless objective documentation is required.

Patients with thromboembolic diseases who develop heparin-induced thrombocytopenia (HIT) type II require an alternative anticoagulation strategy. Dermatan sulphate (DS) was administered to five patients with thromboembolic diseases who developed HIT type II and showed an in vitro cross-reactivity with low molecular weight heparins. The platelet count and the extension of thrombosis were monitored during DS administration. In four of the five patients the platelet count rapidly increased after heparin was discontinued and DS started. A low platelet count persisted in the single patient with cross-reactivity to DS, up to 4 d after its discontinuation. None of the patients experienced thrombus extension, haemorrhagic side-effects or other adverse events.

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.

Peripheral vascular disease (PVD) is an indicator of diffuse atherosclerosis and is associated with a greatly increased incidence of coronary heart and cerebrovascular disease. Although several studies have assessed whether in vivo platelet activation takes place in patients with PVD, no data are available comparing different platelet function tests in this patient population. We have compared prospectively four tests for the measurement of in vivo platelet activation (plasma betaTG, plasma PF4, intraplatelet betaTG and urinary excretion of 11-dehydro-TXB2) and one in vitro platelet function test (ADP-induced platelet aggregation) in 63 well-characterized patients with intermittent claudication and in 18 age- and sex-matched healthy volunteers. No statistically significant difference was found between patients and controls for plasma betaTG (20.0 +/- 11.8 vs. 18.8 +/- 9.0 ng/ml, respectively), plasma PF4 (5.2 +/- 2.9 vs. 6.3 +/- 3.5 ng/ml), betaTG/PF4 ratio (4.0 +/- 2.9 vs. 3.6 +/- 1.8), intraplatelet betaTG (4503 +/- 1482 vs. 4059 +/- 1065 ng/ml), and threshold aggregatory concentration of ADP (1.7 +/- 0.72 vs. 1.45 +/- 0.56 microM). Urinary 11-dehydro-TXB2 was instead significantly higher in the PVD group (55.4 +/- 27.5 vs. 26.7 +/- 7.0 ng/h, p <0.001). Our study shows that urinary 11-dehydro-TXB2 is a more sensitive index of in vivo platelet activation than the measurement of either platelet specific proteins or of in vitro platelet aggregation in patients with PVD.

Background: Previous 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., Methods and Results: We 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., Conclusions: The 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.

Five widely used activated partial thromboplastin time (APTT) reagents (Actin-FS, Actin-FSL, Hemolab Silimat, IL-Test APTT Ellagic Acid and Thrombofax Activated) were compared for their sensitivity and precision in measuring the effect of dermatan sulphate on blood coagulation. On each of 4 days, aliquots of the same normal human plasma pool were mixed with dermatan sulphate (MF701) at concentrations ranging from 10 to 100 micrograms/ml, and APTT was measured in duplicate with all reagents by a photo-optical coagulometer. The order of testing between and within reagents was changed every day. The relationship of APTT ratio to dermatan sulphate concentration was linear with all the reagents. There were statistically significant differences between reagents in their sensitivity to DS, as reflected by linear regression slopes. IL-Test was the most sensitive reagent. At dermatan sulphate concentrations of 20, 50, and 80 micrograms/ml APTT ratio ranged from 1.5 to 1.7, 1.9 to 2.3 and 2.3 to 2.9, respectively, according to the reagent. The lambda coefficient and coefficient of variation derived from regression analysis, both reflecting assay precision, ranged from 0.57 to 0.71 and from 4.6 to 5.1%, respectively, with all but the least precise reagent. The best sensitivity/precision balance was displayed by IL-Test. The APTT reagent should therefore be standardized, with special regard to sensitivity to DS, when testing the relationship of dermatan sulphate clinical effects to APTT response.

A high incidence of post-discharge venous thromboembolism in orthopaedic surgery patients has been recently reported drawing further attention to the unresolved issue of the optimal duration of the pharmacological prophylaxis. We performed an overview analysis in order to evaluate the incidence of late occurring clinically overt venous thromboembolism in major orthopaedic surgery patients discharged from the hospital with a negative venography and without further pharmacological prophylaxis. We selected the studies published from January 1974 to December 1995 on the prophylaxis of venous thromboembolism after major orthopaedic surgery fulfilling the following criteria: 1) adoption of pharmacological prophylaxis, 2) performing of a bilateral venography before discharge, 3) interruption of pharmacological prophylaxis at discharge in patients with negative venography, and 4) post-discharge follow-up of the patients for at least four weeks. Out of 31 identified studies, 13 fulfilled the overview criteria. The total number of evaluated patients was 4120. An adequate venography was obtained in 3469 patients (84.1%). In the 2361 patients with negative venography (68.1%), 30 episodes of symptomatic venous thromboembolism after hospital discharge were reported with a resulting cumulative incidence of 1.27% (95% C.I. 0.82-1.72) and a weighted mean incidence of 1.52% (95% C.I. 1.05-1.95). Six cases of pulmonary embolism were reported. Our overview showed a low incidence of clinically overt venous thromboembolism at follow-up in major orthopaedic surgery patients discharged with negative venography. Extending pharmacological prophylaxis in these patients does not appear to be justified. Venous thrombi leading to hospital re-admission are likely to be present but asymptomatic at the time of discharge. Future research should be directed toward improving the accuracy of non invasive diagnostic methods in order to replace venography in the screening of asymptomatic post-operative deep vein thrombosis.

1. A phospholipase A2 (PLA2) represents the key enzyme in the remodelling pathway of platelet-activating factor (PAF) synthesis in human polymorphonuclear (PMN) leucocytes. 2. PLA2 activation is also the rate-limiting step for the release of the arachidonic acid utilized for the synthesis of leukotrienes in stimulated leucocytes; however, it is unknown whether the PLA2s involved in the two biosynthetic pathways are identical. 3. Cloricromene (8-monochloro-3-beta-diethylaminoethyl-4-methyl-7-ethoxy- carbonylmethoxy coumarin) is an antithrombotic coumarin derivative which inhibits platelet and leucocyte function and suppresses arachidonic acid liberation by interfering with PLA2 activation. 4. The aim of the present study was to assess whether chloricromene inhibits PAF synthesis by stimulated human polymorphonuclear leucocytes (PMNs). 5. Cloricromene (50-500 microM) inhibited in a concentration-dependent manner the release of PAF, as measured by h.p.l.c. bioassay, from A23187-stimulated PMNs. Significant inhibition (45%) of PAF-release was obtained with 50 microM cloricromene and the IC50 was 85 microM. Mepacrine (500 microM), a non-specific PLA2 inhibitor, strikingly reduced PAF release. 6. The incorporation of [3H]-acetate into [3H]-PAF induced by serum-treated zymosan in human PMNs was also inhibited concentration-dependently by cloricromene, with an IC50 of 105 microM. Mepacrine also suppressed [3H]-acetate incorporation into [3H]-PAF. 7. Cloricromene did not affect the activities of the enzymes involved in PAF-synthesis acetyltransferase or phosphocholine transferase. 8. Our data demonstrate that cloricromene, an inhibitor of PLA2-activation in human leucocytes, reduces the synthesis of PAF by stimulated PMNs. This finding has a twofold implication: the PLA2s (or the mechanisms that regulate their activation) involved in PAF synthesis and arachidonate release in human leucocytes are either identical or else indistinguishable by their sensitivity to cloricromene; the inhibition of PAF release by activated leucocytes may contribute to the antithrombotic and anti-ischaemic activities exerted by cloricromene.

Background: A strong positive correlation exists between the breast cancer tissue content of either urokinase-plasminogen activator (uPA) or plasminogen activator, inhibitor type I (PAI-1), quantified in the tissue extracts by immunoassays, and the survival of patients with breast cancer. Furthermore, several studies assign to the urokinase-type plasminogen activator receptor (uPAR) a pivotal role in triggering the proteolytic activity of the urokinase pathway involved in tumor stroma degradation, tumor spread and metastasis. However, the pattern of distribution of uPAR in normal and cancerous human tissue and the pattern of coexpression of activators and inhibitors that occurs in breast cancer tissues is not completely known., Methods: The immunohistochemical localization of uPAR, uPA, tPA) and PAI-1 was evaluated by using the avidin-biotin immunoperoxidase technique and affinity-purified monoclonal antibodies from American Diagnostica Inc. Studies were performed in formalin fixed, paraffin-embedded tissue prepared from 23 surgically excised non-neoplastic breast tissues and 18 ductal breast carcinomas., Results: While the expression of uPAR protein represents a constant feature of invasive ductal breast cancer, it was also observed in most of the breast tissue samples, including the normal breast tissues. The staining for uPAR was mainly localized on normal or tumoral epithelial cells, even if the co-expression of uPAR in stromal cells was frequently observed in adjacent slides. A semiquantitative analysis of immunohistochemical results showed that uPAR and PAI-1 were overexpressed in invasive breast cancer in comparison with normal and benign breast tissues. In addition, uPA was higher in both invasive breast carcinomas and benign breast lesions with respect to normal breast tissues., Conclusions: We showed that overexpression of uPAR, uPA, and its main inhibitor, PAI-1, is a constant feature of invasive ductal breast carcinomas. However, the expression of the above fibrinolytic reactants is not specific for breast cancer since positive staining for these molecules was frequently observed in benign breast lesions as well as in normal breast tissues. The combined increased expression of uPA and its cellular receptor, uPAR on the surface of tumor epithelial cells may account for the activation of the proteolytic system which occurs in breast cancer.

Washed intact human platelets were prelabelled with [3H]arachidonic acid ([3H]AA) and stimulated with thrombin or with AlF4-, a known unspecific activator of G-proteins. Both stimuli induced the liberation of [3H]AA, the release of beta-thromboglobulin (beta-TG) and platelet aggregation. PMA did not induce liberation of [3H]AA although it induced beta-TG release and aggregation; preincubation with PMA did not modify significantly the amounts of [3H]AA and beta-TG released by thrombin or AlF4-. Different inhibitors of PKC (staurosporine, H-7 and calphostin C) increased the release of [3H]AA and inhibited beta-TG release and aggregation induced by AlF4- but they had no effect when platelets were stimulated with thrombin (0.5 U/ml). Calphostin C was able to release [3H]AA by itself without inducing aggregation of beta-TG release. Okadaic acid (a serine/threonine phosphoprotein phosphatase inhibitor) greatly inhibited the release of [3H]AA, beta-TG and aggregation in AlF4--stimulated platelets. These results indicate the presence of a G-protein mediated mechanism for the activation of a platelet phospholipase A2 which is negatively affected by a protein kinase, sensible to putative inhibitors of protein kinase C, and it is activated by a protein phosphatase, sensible to okadaic acid.