Neuroprotective effects of ultra‐low‐molecular‐weight heparin in vitro and vivo models of ischemic injury

This study was conducted to demonstrate ultra‐low‐molecular‐weight heparin’s neuroprotective effects on ischemic injury both in vivo and in vitro studies. In vitro, the effect of ultra‐low‐molecular‐weight heparin was tested in cultured PC12 cells exposed to Earle’s solution containing sodium dithionite, to identify its neuroprotection to PC12 cells damaged by oxygen‐glucose deprivation (OGD). The cell injury was detected by the tetrazolium salt 3‐(4,5‐dimethyl‐2‐thiazolyl)‐2,5 diphenyl‐2H tetrazolium bromide (MTT) assay. In vivo, male Wistar rats with middle cerebral artery occlusion were evaluated for infarct volume followed by the treatment with ultra‐low‐molecular‐weight heparin. The results in vitro showed that ultra‐low‐molecular‐weight heparin significantly inhibited PC12 cells damage induced by OGD. Results in vivo showed that vein injection of Ultra‐Low‐molecular‐weight heparin at doses of 0.5 and 1.0 mg/kg exerted significant neuroprotective effects on rats with focal cerebral ischemic injury by significantly reducing the infarct volume compared with the injury group. All the findings suggest that ultra‐low‐molecular‐weight heparin might act as a neuroprotective agent useful in the treatment of cerebral ischemia.     

Fatal evolution of a huge right atrial free‐floating thrombus

Right atrial thrombus is a rare medical emergency that should be suspected in all cases of pulmonary embolism, and rapid action should be taken to ensure a timely, proper management.     

Aggregated low‐density lipoprotein induce impairment of the cytoskeleton dynamics through urokinase‐type plasminogen activator/urokinase‐type plasminogen activator receptor in human vascular smooth muscle cell

Summary. Background: Urokinase‐type plasminogen activator (UPA) regulates vascular smooth muscle cell (VSMC) functions relevant in vascular remodeling by facilitating proteolysis at the cell surface and inducing cell signaling pathways. Our previous results demonstrated that aggregated low‐density lipoprotein (agLDL) impair cytoskeleton dynamics, a key event contributing to VSMC behavior during progression of atherosclerotic plaques. Objectives: To investigate whether mechanisms underlying inhibition of cytoskeleton dynamics in lipid‐loaded VSMC occurs through a UPA‐mediated process. Methods: Adhesion assay was performed in lipid‐loaded human VSMC after 16‐h exposition to agLDL (100 μg mL^?1). Protein subcellular localization and actin‐fiber formation were assessed by confocal microscopy. For analysis of protein expression western blots were carried out. Co‐immunoprecipitates of UPAR were examined by one‐dimensional‐ or two‐dimensional electrophoresis (1‐DE or 2‐DE), mass spectrometry MALDI‐TOF and western blot. Results: agLDL induced UPA subcellular delocalization and significantly decreased UPA levels during attachment of VSMC. UPA (enhanced endogenous‐expression or exogenous added) acting as a urokinase‐type plasminogen activator receptor (UPAR)‐ligand restored actin‐cytoskeleton organization and adhesion capacity of lipid‐loaded cells to control levels. UPAR co‐immunoprecipitated with the unphosphorylated form of myosin regulatory light chain (MRLC) in lipid‐loaded cells. The detrimental effects of agLDL on MRLC phosphorylation were reversed by high levels of UPA. The UPA effects on VSMC exposed to agLDL involved FAK phosphorylation. Conclusions: The detrimental effects of atherogenic LDL on VSMC are mediated by a decrease and delocalization of the UPA–UPAR interaction that result in an impairment of cytoskeleton dynamics and adhesion capacity affecting cell phenotype and function.     

Activation of single‐chain urokinase‐type plasminogen activator by platelet‐associated plasminogen: a mechanism for stimulation of fibrinolysis by platelets

Summary. Background and Objective: Platelets are essential for hemostasis, and they cause resistance to fibrinolysis by tissue‐type plasminogen activator. In contrast, platelets enhance fibrinolysis mediated by single‐chain urokinase‐type plasminogen activator (scu‐PA). This study investigated the mechanism behind this profibrinolytic role of platelets. Methods and Results: Platelets enhanced scu‐PA activity, but not urokinase‐type plasminogen activator (u‐PA) activity, in plasma clot lysis and chromogenic assays. We established, using the non‐cleavable scu‐PA mutant (Lys158→Glu) and protease inhibitors, that platelets increased activation to u‐PA by a serine protease. Activation of scu‐PA was platelet‐dependent, even in plasma. It occurred in platelet‐rich but not in platelet‐poor plasma, as assessed by sodium dodecylsulfate polyacrylamide gel electrophoresis and zymography after addition of plasminogen activator inhibitor‐1. Candidate proteases that are known to activate scu‐PA and are present in platelet preparations were investigated. Factor VII activating protease was detected in platelet preparations by western blotting, but its inhibition by antibodies did not inhibit activation of scu‐PA by platelets. Plasmin and plasma kallikrein both mimicked the platelet effect, but were distinguished by their responses to a range of inhibitors. Analysis of platelet‐associated protease activity and the time course of scu‐PA activation pointed towards plasminogen, and the data were consistent with a mechanism of reciprocal activation. The essential role of plasminogen was revealed using platelets from plasminogen‐deficient mice, which could not activate scu‐PA. Local plasminogen on platelet membranes was markedly more effective than solution‐phase plasminogen in activation of scu‐PA. Conclusions: Platelets enhance fibrinolysis by scu‐PA through reciprocal activation of scu‐PA and platelet‐associated plasminogen, a system that is potentially important in the lysis of platelet‐rich thrombi.     

Validation of a clinical algorithm to identify low‐risk patients with pulmonary embolism

Summary. Background: We previously derived a clinical prognostic algorithm to identify patients with pulmonary embolism (PE) who are at low risk of short‐term mortality and who could be safely discharged early or treated entirely in an outpatient setting. Objectives: To externally validate the clinical prognostic algorithm in an independent patient sample. Methods: We validated the algorithm in 983 consecutive patients prospectively diagnosed with PE at an emergency department of a university hospital. Patients with none of the algorithm’s 10 prognostic variables (age ≥ 70 years, cancer, heart failure, chronic lung disease, chronic renal disease, cerebrovascular disease, pulse ≥ 110 min^–1, systolic blood pressure < 100 mmHg, oxygen saturation < 90%, and altered mental status) at baseline were defined as being at low risk. We compared 30‐day overall mortality among low‐risk patients, on the basis of the algorithm, between the validation sample and the original derivation sample. We also assessed the rate of PE‐related and bleeding‐related mortality among low‐risk patients. Results: Overall, the algorithm classified 16.3% of patients with PE as being at low risk. Mortality at 30 days was 1.9% among low‐risk patients, and did not differ between the validation sample and the original derivation sample. Among low‐risk patients, only 0.6% died from definite or possible PE, and 0% died from bleeding. Conclusions: This study validates an easy‐to‐use, clinical prognostic algorithm for PE that accurately identifies patients with PE who are at low risk of short‐term mortality. Patients who are at low risk according to our algorithm are potential candidates for less costly outpatient treatment.     

静脉低剂量重组组织型纤溶酶原激活物治疗短暂性脑缺血发作的临床对照研究

目的研究和分析静脉低剂量重组组织型纤溶酶原激活物(rt-PA)治疗短暂性脑缺血发作(TIA)的临床效果。方法选取86例TIA患者作为研究对象,根据治疗方式将其分为观察组41例(应用低剂量rt-PA静脉溶栓治疗联合阿司匹林抗血小板治疗)和对照组45例(应用阿司匹林抗血小板治疗)。比较2组患者入院时、治疗第1、3、7、14天时组织型纤溶酶原激活物(t-PA)、纤溶酶原激活物抑制物-1(PAI-1)水平。比较2组患者治疗各时段的TIA控制率,对2组患者进行为期1年的随访,比较患者转为急性脑梗死的比例。结果治疗第1、3天,观察组患者的血浆t-PA水平显著高于对照组(P0.05)。在治疗前、后各时点,2组患者的血浆PAI-1水平的差异均无统计学意义(P0.05)。2组患者各时段TIA控制率的差异无统计学意义(P0.05)。观察组和对照组分别有2例和4例患者于随访期内转为急性脑梗死,2组患者转为急性脑梗死比例的差异无统计学意义(P0.05)。结论在阿司匹林抗血小板治疗的基础上采用静脉滴注低剂量rt-PA进行溶栓治疗,能够提高TIA初期患者的血浆t-PA水平,有助于降低血栓形成风险,但对于患者的血浆PAI-1水平、近期疗效和远期预后的作用并不显著。     

Signaling role of CD36 in platelet activation and thrombus formation on immobilized thrombospondin or oxidized low‐density lipoprotein

Summary. Background and Objective: Platelets abundantly express glycoprotein CD36 with thrombospondin‐1 (TSP1) and oxidized low‐density lipoprotein (oxLDL) as proposed ligands. How these agents promote platelet activation is still poorly understood. Methods and Results: Both TSP1 and oxLDL caused limited activation of platelets in suspension. However, immobilized TSP1 and oxLDL, but not LDL, strongly supported platelet adhesion and spreading with a major role of CD36. Platelet spreading was accompanied by potent Ca²⁺ rises, and resulted in exposure of P‐selectin and integrin activation, all in a CD36‐dependent manner with additional contributions of α_IIbβ₃ and ADP receptor stimulation. Signaling responses via CD36 involved activation of the protein tyrosine kinase Syk. In whole blood perfusion, co‐coating of TSP1 or oxLDL with collagen enhanced thrombus formation at high‐shear flow conditions, with increased expression on platelets of activated α_IIbβ₃, P‐selectin and phosphatidylserine, again in a CD36‐dependent way. Conclusions: Immobilized TSP1 and oxLDL activate platelets partly via CD36 through a Syk kinase‐dependent Ca²⁺ signaling mechanism, which enhances collagen‐dependent thrombus formation under flow. These findings provide novel insight into the role of CD36 in hemostasis.     

Chronic oral administration of low‐dose combination of fenofibrate and rosuvastatin protects the rat heart against experimentally induced acute myocardial infarction

Fenofibrate and rosuvastatin at low doses might have experimental pleiotropic benefits. This study investigated the combined effect of low doses of fenofibrate and rosuvastatin in isoproterenol‐induced experimental myocardial infarction. Rats administered isoproterenol (85 mg/kg/day, s.c.) for 2 days (day 29 and day 30) of 30 days experimental protocol developed significant myocardial infarction that was accompanied with high myocardial oxidative stress and lipid peroxidation, elevated serum markers of cardiac injury, lipid abnormalities, and elevated circulatory levels of C‐reactive protein. Pretreatment with low doses of fenofibrate (30 mg/kg/day p.o., 30 days) and rosuvastatin (2 mg/kg/day p.o., 30 days) both alone or in combination markedly prevented isoproterenol‐induced myocardial infarction and associated abnormalities while the low‐dose combination of fenofibrate and rosuvastatin was more effective. Histopathological study in isoproterenol control rat heart showed necrosis with edema and acute inflammation at the margins of necrotic area. The rat heart from low‐dose fenofibrate and rosuvastatin pretreated group showed scanty inflammation and no ischemia. In conclusion, fenofibrate and rosuvastatin pretreatment in low doses might have a therapeutic potential to prevent the pathogenesis of myocardial infarction. Moreover, their combined treatment option might offer superior therapeutic benefits via a marked reduction in myocardial infarct size and oxidative stress, suggesting a possibility of their pleiotropic cardioprotective action at low doses.     

Increased anticoagulant response to low‐molecular‐weight heparin in plasma from patients with advanced cirrhosis

Summary. Introduction: Cirrhotic patients may present thrombotic complications that warrant anticoagulant therapy. However, the efficacy of low‐molecular‐weight heparin (LMWH) in this clinical setting is still unclear. Aims/methods: To evaluate the in vitro effect of LMWH on thrombin generation (TG) in cirrhotic patients at different stages of liver disease. Thirty cirrhotics (10 Child Pugh A, 10 Child Pugh B and 10 Child Pugh C), 10 subjects with inherited type 1 antithrombin (AT) defect and 10 healthy controls were studied. TG was determined at baseline and with anti‐Xa levels after the addition of enoxaparin at 0.35 and 0.7 U anti‐Xa mL. The endogenous thrombin potential (ETP) ratio at 0.35 and 0.7 U anti‐Xa mL was obtained by dividing ETP with LMWH by ETP at baseline. Results: Mean AT levels in all cirrhotic subgroups and in patients with AT deficiency were significantly lower than in controls. The 0.35 ETP ratio was significantly lower in cirrhotic patients than in controls (0.26 ± 0.1 vs. 0.48 ± 0.1, P < 0.001) and the reduction paralleled the severity of liver disease, in spite of the concomitant decrease in AT and anti‐Xa activity. AT‐deficient subjects showed a significantly increased 0.35 ETP ratio compared with both cirrhotic patients and controls (0.69 ± 1 vs. 0.26 ± 0.1, P < 0.001, and vs. 0.48 ± 0.1, P = 0.04 respectively). LMWH at 0.7 U anti‐Xa mL completely inhibited TG in 9/30 cirrhosis patients with more advanced liver disease (Child Pugh B and C), whereas complete TG abolition was seen in only 1/10 controls. Conclusions: Cirrhotic patients show an increased response to LMWH, which correlates with the severity of liver disease, in spite of reduced AT and anti‐Xa activity levels. Thrombin generation may be a useful tool to monitor the response to LMWH in cirrhotic patients.     

小剂量重组组织型纤溶酶原激活物与尿激酶治疗急性心肌梗死的对比研究

目的：评价小剂量重组组织型纤溶酶原激活物（ｒｔＰＡ）静脉溶栓治疗国人急性心肌梗死（ＡＭＩ）的疗效及安全性。方法：６６例ＡＭＩ患者随机分为ｒｔＰＡ组３１例及尿激酶（ＵＫ）组３５例,分别应用ｒｔＰＡ及ＵＫ治疗。结果：心肌梗死相关血管再通率ｒｔＰＡ组８０．６％与ＵＫ组４８．６％比较,Ｐ＜０．０５;４周住院病死率ｒｔＰＡ组０与ＵＫ组８．６％比较,Ｐ＜０．０５;而２组间出血发生率无差异。结论：小剂量ｒｔＰＡ治疗ＡＭＩ血管再通率高,出血并发症少,可降低ＡＭＩ早期病死率,是一种安全有效的溶栓剂     

PRODIGE: a randomized placebo‐controlled trial of dalteparin low‐molecular‐weight heparin thromboprophylaxis in patients with newly diagnosed malignant glioma

Summary. Background and objectives: Venous thromboembolism (VTE) occurs in 20–30% of patients with malignant glioma per year of survival. We tested the efficacy of long‐term dalteparin low‐molecular‐weight heparin (LMWH) for prevention of VTE in these patients. Patients/methods: Adults with newly diagnosed malignant glioma were randomized to receive dalteparin 5000 anti‐Xa units or placebo, both subcutaneously once daily for 6 months starting within 4 weeks of surgery. Treatment continued for up to 12 months. The primary outcome was the cumulative risk of VTE over 6 months. The target sample size was 512 patients. Events were adjudicated by a committee unaware of treatment. Results: The trial began in 2002 and closed in May 2006 because of expiration of study medication. Ninety‐nine patients were randomized to LMWH and 87 to placebo. Twenty‐two patients developed VTE in the first 6 months: nine in the LMWH group and 13 in the placebo group [hazard ratio (HR) = 0.51, 95% confidence interval (CI): 0.19–1.4, P = 0.29]. At 6 months, there were three major bleeds on LMWH and none on placebo; at 12 months, 5 (5.1%) major bleeds on LMWH and 1 (1.2%) on placebo occurred (HR = 4.2, 95% CI: 0.48–36, P = 0.22). All major bleeds were intracranial and occurred while on study medication. The 12‐month mortality rates were 47.8% for LMWH and 45.4% for placebo (HR = 1.2, 95% CI: 0.73–2.0, P = 0.48). Conclusions: Trends suggesting reduced VTE and increased intracranial bleeding were seen in the LMWH thromboprophylaxis group. The role of long‐term anticoagulant thromboprophylaxis in patients with brain tumors remains uncertain.     

A randomized double‐blind study of low‐molecular‐weight heparin (parnaparin) for superficial vein thrombosis: STEFLUX (Superficial ThromboEmbolism and Fluxum)

Summary. Background: Optimal doses and duration of low‐molecular‐weight heparin (LMWH) for the treatment of superficial vein thrombosis (SVT) are still uncertain. Objectives: To compare the efficacy and safety of different doses and durations of LMWH parnaparin for symptomatic lower limb SVT. Patients and methods: Outpatients with at least a 4‐cm‐long SVT of long or short saphenous veins or their collaterals were randomized to receive parnaparin either 8500 UI once daily ( o.d.) for 10 days followed by placebo for 20 days (group A) or 8500 UI o.d. for 10 days followed by 6400 UI once daily (o.d.) for 20 days (group B) or 4250 UI o.d. for 30 days (group C) in a double‐blind fashion in 16 clinics. Primary outcome was the composite of symptomatic and asymptomatic deep vein thrombosis (DVT), symptomatic pulmonary embolism (PE) and relapse and/or symptomatic or asymptomatic SVT recurrence in the first 33 days with 60 days follow‐up. Results: Among 664 patients, primary outcome occurred in 33/212 (15.6%), 4/219 (1.8%) and 16/217 (7.3%) subjects in groups A, B and C, respectively (B vs. A: absolute risk reduction [ARR]: 13.7%, 95% confidence intervals [CI]: 8–18.9 P < 0.001; B vs. C: ARR: 5.5%; 95% CI: 1.6–9.4 P = 0.011; C vs. A: ARR: 8.2%, 95% CI: 2–14 P = 0.012). During days 0–93, the event rate was higher in group A (22.6%) than either in group B (8.7%; P = 0.001) or C (14.3%, P = 0.034). No major hemorrhages occurred. Conclusions: An intermediate dose of parnaparin for 30 days is superior to either a 30‐day prophylactic dose or a 10‐day intermediate dose for lower limb SVT treatment.     

Complete hematological and major molecular response through treatment with low‐dose Interferon alpha 2a in high‐risk polycythemia vera patient: a case report

Low‐dose interferon‐α 2a treatment may be considered as an alternative to cytoreductive therapy with hydroxyurea or regularly dosed interferon in high‐risk polycythemia vera patients.     

Major bleeding risks of different low‐molecular‐weight heparin agents: a cohort study in 12 934 patients treated for acute venous thrombosis

Essentials

• Low‐molecular‐weight‐heparins (LMWH) kinetics differ which may result in different bleeding risks.
• A cohort of 12 934 venous thrombosis patients on LMWH was followed until major bleeding.
• The absolute major bleeding risk was low among patients registered at the anticoagulation clinic.
• Once‐daily dosing was associated with a lower bleeding risk as compared with twice‐daily.

Summary

Background

Low‐molecular‐weight heparins (LMWHs) are considered members of a class of drugs with similar anticoagulant properties. However, pharmacodynamics and pharmacokinetics between LMWHs differ, which may result in different bleeding risks. As these agents are used by many patients, small differences may lead to a large effect on numbers of major bleeding events.

Objectives

To determine major bleeding risks for different LMWH agents and dosing schedules.

Methods

A cohort of acute venous thrombosis patients from four anticoagulation clinics who used an LMWH and a vitamin K antagonist were followed until they ceased LMWH treatment or until major bleeding. Exposures were classified according to different types of LMWHs and for b.i.d. and o.d. use. Cumulative incidences for major bleeding per 1000 patients and risk ratios were calculated and adjusted for study center.

Results

The study comprised 12 934 patients with a mean age of 59 years; 6218 (48%) were men. The cumulative incidence of major bleeding was 2.5 per 1000 patients (95% confidence interval [CI], 1.7–3.5). Enoxaparin b.i.d. or o.d. was associated with a relative bleeding risk of 1.7 (95% CI, 0.2–17.5) compared with nadroparin o.d. In addition, a nadroparin b.i.d. dosing schedule was associated with a 2.0‐fold increased major bleeding risk (95% CI, 0.8‐5.1) as compared with a nadroparin o.d. dosing schedule.

Conclusions

Absolute major bleeding rates were low for all LMWH agents and dosing schedules in a large unselected cohort. Nevertheless, twice‐daily dosing with nadroparin appeared to be associated with an increased major bleeding risk as compared with once‐daily dosing, as also suggested in a meta‐analysis of controlled clinical trials.