Enhanced thrombin generation in patients with cirrhosis‐induced coagulopathy

Summary. Background: Prothrombin time (PT) and the international normalized ratio (INR) are still routinely measured in patients with liver cirrhosis to ‘assess’ their bleeding risk despite the lack of correlation with the two. Thrombin generation (TG) assays are global assays of coagulation that are showing promise in assessing bleeding and thrombosis risks. Aim: To study the relationship between the INR and TG profiles in cirrhosis‐induced coagulopathy. Methods: Seventy‐three patients with cirrhosis were studied. All TG parameters were compared with those from a normal control group. Contact activation was prevented using corn trypsin inhibitor. TG was also assayed in the presence of Protac^®. The endogenous thrombin potential (ETP) ratio was derived by dividing the ETP with Protac® by the ETP without Protac®. Results: The INR (mean 1.7) did not correlate with the ETP and the velocity of TG (P > 0.05). There was no difference between the lag time and ETP of the two groups (P > 0.05). The velocity of TG was increased in cirrhosis (67.95 ± 34.8 vs. 45.05 ± 25.9 nM min^?1; P = 0.016) especially in patients with INRs between 1.21 and 2.0. Both the ETP with Protac^® and the ETP ratio were increased in cirrhosis (mean 1074 ± 461.4 vs. 818 ± 357.9 nM min, P = 0.004 and 0.80 ± 0.21 vs. 0.44 ± 0.15, P ≤ 0.0001, respectively). Conclusion: Despite a raised INR, TG parameters are consistent with a hypercoagulable profile in cirrhosis‐related coagulopathy. This confirms that the PT or INR should not be used to assess bleeding risk in these patients, and other parameters, such as TG, need to be explored as clinical markers of coagulopathy.     

Fixed dosage of low-molecular-weight heparins causes large individual variation in coagulability, only partly correlated to body weight

Summary. Backgrounds: Low‐molecular‐weight heparins (LMWHs) are routinely given without the control of their effect on coagulation. The endogenous thrombin potential (ETP) is a sensitive detector of the heparin effect. Question: What is the interindividual variation in TG after a fixed dose of LMWH in normal volunteers, is it explained by variation in weight? Methods: Subcutaneous (s.c.) injection, in 12 healthy volunteers, of 9000 aXa‐units of unfractionated heparin (UFH) and of three heparins with narrow MW distribution around 10.5, 6.0 and 4.5 kD. Measurement of anti‐thrombin (aIIa) and antifactor Xa (aXa)‐activities and ETP at 11 time points over 24 h. Results: The coefficient of variation (CV) of the AUCs of aXa‐ and aIIa‐activities is 50% for UFH and 22–37% for LMWHs. Because of the hyperbolic form of the dose–response curve, the CV of the inhibition of the ETP is lower: 32% for UFH and 13–21% for the LMWHs. Fixed dosage of LMWH caused under‐dosage in 10–13% of the samples and over‐dosage in 5–11%. High or low response is an individual property independent of the type of heparin injected and only partially explained by variation in body weight. Conclusion: Optimized individual dosage of LMWH is possible through recognition of high and low responders, which requires one measurement of the heparin concentration or, preferably, the heparin effect on the ETP, 2–5 h after a first injection.     

Treating hepatic encephalopathy in cirrhotic patients admitted to ICU with sodium phenylbutyrate: a preliminary study

Hepatic encephalopathy (HE) influences short‐term and long‐term prognoses. Recently, glycerol phenylbutyrate (PB), that lowers ammonia by providing an alternate pathway to urea for waste nitrogen excretion, has shown that it was effective in preventing the occurrence of HE in RCT. The aim was to assess the benefits of sodium PB in cirrhotic patients admitted to ICU for overt HE, in terms of ammonia levels decrease, neurological improvement, and survival. Cirrhotic patients who presented with overt HE, ammonia levels >100 μmol/L, and did not display any contra‐indication were included. Sodium PB was administered at 200 mg/kg/day. Control group included historical controls treated by standard therapy, matched for age, sex, MELD score, and severity of HE. Eighteen patients were included and treated with sodium PB (age: 59 [45–68], male gender: 15 [83%], Child–Pugh B: 8 [44%], Child–Pugh C: 10 [56%], and MELD score: 16 [13–23]). Ammonia levels significantly decreased in the PB as compared to the control group from inclusion to 12 h and from inclusion to 48 h (P = 0.0201 and P = 0.0230, respectively). The proportion of patients displaying neurological improvement was only higher in the PB‐treated group as compared to controls at ICU discharge (15 [83%] vs. 9 [50%], P = 0.0339). ICU discharge survival was significantly higher in patients treated with PB (17 [94%] vs. 9 [50%], P = 0.0017). In cirrhotic patients with overt HE, sodium PB could be effective in reducing ammonia levels and might be effective in improving neurological status and ICU discharge survival. More extensive data, especially a RCT, are mandatory.     

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.     

Evidence for an enhanced fibrinolytic capacity in cirrhosis as measured with two different global fibrinolysis tests

Summary. Background and objectives: It has been known for a long time that cirrhosis is associated with hyperfibrinolysis, which might contribute to an increased risk and severity of bleeding. However, recent papers have questioned the presence of a hyperfibrinolytic state in cirrhotic patients and postulated a rebalanced system owing to concomitant changes in both pro‐ and anti‐fibrinolytic factors. Therefore we re‐investigated the fibrinolytic state of cirrhotic patients using two different overall tests including a recently developed test for global fibrinolytic capacity (GFC) using whole blood. Patients and methods: Blood was collected from 30 healthy controls and 75 patients with cirrhosis of varying severity (34 Child–Pugh A, 28 Child–Pugh B and 13 Child–Pugh C). The plasma clot lysis time (CLT), which is inversely correlated with fibrinolysis, was determined as well as the GFC. Results: The mean CLT was 74.5 min in the controls and decreased significantly to 66.9 min in Child–Pugh class A patients, 59.3 min in class B patients and 61.0 min in class C patients, and hyperfibrinolysis existed in 40% of the patients. The median GFC was 1.7 μg mL^?1 in the controls and increased significantly to 4.0 μg mL^?1 in Child–Pugh class A patients, 11.1 μg mL^?1 in class B patients and 22.5 μg mL^?1 in class C patients, and hyperfibrinolysis existed in 43% of the patients. Taken together, 60% of the patients showed hyperfibrinolysis in at least one of the two global assays. Conclusion: A rebalanced fibrinolytic system may occur, but hyperfibrinolysis is found in the majority of patients with cirrhosis.     

Comparison of the anticoagulant intensities of fondaparinux and enoxaparin in the organization to assess strategies in acute ischemic syndromes (OASIS)‐5 trial

Summary. Background: In the OASIS‐5 trial, fondaparinux reduced major bleeding with similar short‐term efficacy as enoxaparin but lowered death and stroke during long‐term follow‐up. The mechanism of lower bleeding and improved efficacy with fondaparinux is uncertain. Methods and Results: We compared the anti‐Xa concentration (reflecting drug levels), Xa clot time (reflecting anticoagulant effect) and endogenous thrombin potential (ETP; a global test of hemostatic function) in plasma samples collected 6, 24 and 72 h after the first dose of the study drug in 48 patients randomly assigned fondaparinux 2.5 mg day^?1 and 42 patients assigned enoxaparin 1 mg kg^?1 twice daily in the OASIS‐5 trial. Patients assigned to fondaparinux compared with enoxaparin had a significantly lower mean anti‐Xa level [0.52 IU mL^?1 (SD 0.22 IU mL^?1) vs. 1.2 IU mL^?1 (SD 0.45 IU mL^?1), P < 0.0001] and Xa clot time [64.9 s (SD 17.7 s) vs. 111.8 s (SD 29.6 s), P < 0.0001], and significantly higher ETP area under the curve (AUC) [386.7 mA (SD 51.5 mA) vs. 206.4 mA (SD 90.6 mA), P < 0.001] at 6 h, and these differences remained evident at 24 and 72 h. There was significantly less variability of the results of anti‐Xa levels, Xa clot time and ETP AUC for fondaparinux compared with enoxaparin at 6 h (P < 0.001 for each comparison). Conclusion: Fondaparinux 2.5 mg day^?1 compared with enoxaparin 1 mg kg^?1 twice daily produces less variable anticoagulant effect and lower mean anticoagulant intensity. These results most likely explain the reduced risk of bleeding seen with fondaparinux compared with enoxaparin in the OASIS‐5 trial and suggest that a lower intensity of anticoagulation than used in the past may be sufficient to prevent recurrent ischemic events and death in patients with ACS who are concurrently treated with aspirin and clopidogrel.     

肝硬化患者凝血功能、血小板参数、网织红细胞参数的变化与Child-Pugh分级的关系

目的了解肝硬化患者凝血功能、血小板参数、网织红细胞参数的变化与Child-Pugh肝功能分级的关系。方法分别采用全自动血液分析仪和全自动血凝仪测定163例肝硬化患者和50例健康对照者的凝血功能指标(PT、APTT、Fig、TT),血小板参数(PLT、MPV、PDW、PCT)和网织红细胞参数(RET#、RET%、IRF)。比较肝硬化组与对照组、肝硬化Child-Pugh分级后组间凝血功能、血小板参数和网织红细胞参数的变化。结果与对照组比较,肝硬化患者Fig降低,PT、APTT、TT延长,PLT、PCT下降,MPV、PDW升高,RET计数、RET%、IRF升高,差异有统计学意义(P0.05);Fig随着Child-Pugh等级上升逐渐降低,PT、APTT、TT随着Child-Pugh等级上升逐渐延长,PLT、PCT随着Child-Pugh等级上升逐渐下降,MPV、PDW随着ChildPugh等级上升逐渐升高,RET计数、RET%、IRF随着Child-Pugh等级上升逐渐升高(P0.05)。结论肝硬化患者存在凝血功能、血小板参数及网织红细胞参数异常,其凝血功能,血小板参数及网织红细胞参数的变化与Child-Pugh肝功能分级有密切相关,是判定肝脏损害程度、出血倾向及骨髓造血功能的重要指标。     

Serum level of interleukin-6 (IL-6) and N-terminal propeptide of procollagen type I (PINP) in patients with liver diseases

Introduction: The aim of this study was to evaluate the concentration of interleukin-6 and N-terminal propeptide of procollagen type I and their relationship in liver diseases of different etiologies.

Material and methods: Serum samples were obtained from 30 healthy volunteers and patients suffering from alcoholic cirrhosis (AC) – 31, non-alcoholic cirrhosis (NAC) – 28 and toxic hepatitis (HT) – 23 patients. Cirrhotic patients were classified according to Child–Pugh score. IL-6 and PINP concentrations were determined according to the electrochemiluminescence immunoassay.

Results: The mean serum IL-6 concentration was significantly higher in AC (mean?±?SD:21.52?± 15.01?pg/mL), NAC (20.07?±?32.12?pg/mL) and HT (15.14?±?17.18?pg/mL) when compared to the control group (C) (1.67?±?0.42?pg/mL) (Mann–Whitney U test: p?p?p?=?.020 and p?p?p?=?.022, respectively). IL-6 and PINP concentrations appeared to vary depending on the severity of liver damage (p?p?p?Conclusions: We conclude that serum concentrations of IL-6 and PINP change in liver diseases, and those changes reflect the severity of liver disease.     

No accumulation of the peak anti‐factor Xa activity of tinzaparin in elderly patients with moderate‐to‐severe renal impairment: the IRIS substudy

Summary.  Background: In the elderly, concerns have been raised regarding the risk of accumulation of low molecular weight heparins, owing to their renal elimination. Although data exist for tinzaparin, they are observational. Objectives: To assess whether: (i) there was an accumulation of anti‐factor Xa activity; and (ii) there was any relationship between anti‐FXa activity and age, weight, creatinine clearance or clinical outcomes in patients with moderate‐to‐severe renal impairment receiving tinzaparin (175 IU kg^?1 per 24 h) for acute venous thromboembolism. Methods: In 38 centers participating in the Innohep in Renal Insufficiency Study (IRIS), peak plasma anti‐FXa activity was measured on day 2/day 3 and on day 5 or at visit S (VS) (end of tinzaparin treatment). There was considered to be absence of accumulation if the 90% confidence interval (CI) of the (anti‐FXa day 5/VS)/(anti‐FXa day 2/3) ratio did not exceed the predefined limit of 1.25. Results: Eighty‐seven patients, with a mean age of 83 ± 5 years (range: 75–99 years) and a mean creatinine clearance (CrCl) of 40.8 mL min^?1, 24.1% of whom had severe renal impairment, were included. The mean duration of tinzaparin treatment was 8.4 days. No significant accumulation was detected: the mean accumulation ratio was 1.06 (90% CI 1.01–1.11). There was no correlation between the accumulation ratio and age, weight, or CrCl. The mean anti‐FXa activity did not differ significantly between the eight patients who experienced clinically relevant bleeding and those who did not. Conclusions: No accumulation of anti‐FXa activity was observed in elderly patients with renal impairment receiving therapeutic doses of tinzaparin, suggesting that there is no need for systematic anti‐FXa monitoring in these patients. The high proportion of high molecular weight moieties in tinzaparin may account for its reduced dependence on renal elimination.     

In‐vivo high‐resolution X‐ray microtomography for liver and spleen tumor assessment in mice

The present study sought to validate the use of glycery1‐2‐oley‐1,3‐bis‐[7‐(3‐amino‐2,4,6‐triiodophenyl)‐ heptanoate] (DHOG) contrast agent for mouse spleen tumor and liver metastasis imaging by high‐resolution X‐ray microtomography. Three groups of female nude mice were compared: controls (n = 5), and mice injected with 2.5 × 10⁶ STC1 tumor cells in the spleen, imaged at 15 days (group G15, n = 5) and at 30 days (group G30, n = 5, of which one died before imaging). Micro‐CT scans (X‐ray voltage, 50 kVp; anode current, 200 µA; exposure time, 632 ms; 180 rotational steps resulting in 35 µm isotropic spatial resolution) were acquired at 0, 0.75, 2 and 4 h after i.v. injection of DHOG. CT number (Hounsfield units: HU) and contrast‐to‐noise ratios (CNR) were determined in three organs. Statistical analysis was performed by Mann–Whitney U‐test. Contrast enhancement in normal spleen and liver increased, respectively to 1020 ± 159 and 351 ± 27 HU over baseline at 4 h, and 482 ± 3 and 203 ± 14 HU on day 6 after a single contrast injection. Automated three‐dimensional reconstruction and modeling of the spleen provided accurate and quantifiable images. Spleen tumor and liver metastases did not take up DHOG, making them detectable in contrast to the increased signal in normal tissue. The smallest liver metastasis detected measured 0.3 mm in diameter. High‐resolution X‐ray micro‐CT in living mice using DHOG contrast agent allowed visualization and volume quantification of normal spleen and of spleen tumor and its liver metastases. Copyright © 2007 John Wiley & Sons, Ltd.     

Maintained cerebral metabolic ratio during exercise in patients with β‐adrenergic blockade

Background: Decreased cerebral metabolic ratio (CMR) [molar uptake of O₂ versus molar uptake of (glucose + ½ lactate)] during exercise is attenuated by intravenous administration of the non‐selective β‐adrenergic receptor antagonist propranolol. We evaluated to what extent cirrhotic patients in oral treatment with propranolol are able to mobilize brain non‐oxidative carbohydrate metabolism. Methods: Incremental cycle ergometry to exhaustion (86 ± 4·2 W; mean ± SD) was performed in eight cirrhotic patients instrumented with a catheter in the brachial artery and one retrograde in the right internal jugular vein. Healthy subjects form the control group. Results: In β‐blocked cirrhotic patients arterial lactate increased from 1·5 ± 0·3 to 5·1 ± 0·8 mM (P<0·05) and the arterial–jugular venous difference (a–v diff) from ?0·01 ± 0·03 to 0·30 ± 0·05 mM (P<0·05) at rest and during exercise, respectively. During exercise the glucose a–v diff of 0·46 ± 0·06 mM remained at a level similar to rest (0·54 ± 0·03 mM) and at exhaustion the CMR was not significantly changed (5·8 ± 1·1 versus 6·0 ± 0·6). In controls, CMR decreased from 5·6 ± 0·9 at rest to 3·4 ± 0·7 (P<0·05) during maximal exercise and at a lactate level comparable to that achieved by the patients it was 3·8 ± 0·4. Conclusion: During exhaustive exercise in cirrhotic patients the CMR is maintained and a significant cerebral uptake of lactate is demonstrated. The data suggest that oral treatment with a non‐selective β‐adrenergic receptor antagonist attenuates cerebral non‐oxidative metabolism.     

Study of bioaccumulation of dalteparin at a therapeutic dose in patients with renal insufficiency

Summary. Background: Low‐molecular‐weight heparins (LMWH) are effective, safe and convenient for anticoagulation. Their use is limited in patients with renal insufficiency (RI) because of bioaccumulation. Objectives: Evaluate pharmacokinetic data of dalteparin at a therapeutic dose in patients with RI. Patients and methods: Prospective observational cohort study. Inpatients were included into three groups according to glomerular filtration rate (GFR): A ≥ 60, B 30–59, C < 30 mL min^?1 1.73 m^?2. Dalteparin was injected subcutaneously (s.c.) twice daily. Peak plasma anti‐factor Xa activity (anti‐Xa) was measured and adjusted to applied dose and body weight after the first dose, on day 2, and every 2nd day afterwards. Bioaccumulation factor R was calculated as quotient of the last and the first adjusted anti‐Xa. Data are shown as median (interquartile range, IQR). Results: Thirty‐two patients (23 men) receiving dalteparin for ≥ 2 days were analyzed. Follow‐up was 6 days (IQR 4–10, range 2–22). Median dose was 90 (73–106) units kg^?1 per 12 h (P = 0.68). After the first dose, adjusted anti‐Xa levels were 3.5 (2.6–5.0), 4.8 (3.3–5.5), 4.5 (3.7–7.5) × 10^?3 for the groups A, B, C; P = 0.26. On the last day, they were 6.1 (3.7–7.3), 7.1 (5.6–8.3), 10.2 (7.8–13.2) × 10^?3; A compared with C, P = 0.002. R was 1.46 (1.15–1.82), 1.36 (1.20–2.16) and 2.28 (1.53–2.93); A compared with C, P = 0.18. Conclusion: Therapeutically dosed dalteparin accumulates in patients with severe RI (group C). Dose adjustments according to anti‐Xa are recommended for dalteparin if used in this patient population. However, no simple dosing scheme can be suggested yet because of wide inter‐individual variation.     

Unfractionated heparin dosing in young infants: clinical outcomes in a cohort monitored with anti‐factor Xa levels

Summary. Background: Unfractionated heparin (UFH) is a widely used anticoagulant. Current American College of Chest Physicians guidelines for infants extrapolated from adults recommend 28 U kg^?1 h^?1 of UFH to achieve an anti‐factor Xa level of 0.35–0.7 IU mL^?1. Objective: To assess the profile of anti‐FXa‐based UFH dosing guidelines in infants. Patients/Methods: We included all infants aged < 6 months treated with per‐protocol intravenous UFH at the Hospital for Sick Children, Toronto, over a 3.5‐year period. Results: Of 100 infants, 11% achieved sustained therapeutic anti‐FXa levels with current dose recommendations. Only 15% achieved target anti‐FXa levels within 24 h with per‐protocol dose escalations. Seventeen per cent of patients never achieved therapeutic anti‐FXa levels, despite up to 60 days of therapy and triple the recommended dose. The median dose needed to achieve therapeutic anti‐FXa levels in the remaining 83 infants was 33 U kg^?1 h^?1 (interquartile range, 30–36). Two in three infants had decreased thrombus size at completion of therapy and no thrombus progression/recurrence, and 11/100 infants suffered major bleeding. Without exclusion of extracorporeal membrane oxygenation patients, an activated partial thromboplastin time (APTT) of > 180 s was detected as a risk factor for major bleeding. Conclusions: UFH monitoring is challenging in infants. Despite their delay in reaching therapeutic anti‐FXa levels, infants monitored with the adult‐based anti‐FXa range have a high thrombus resolution rate, no thrombus progression, but a relatively high bleeding rate. Extreme APTT elevation may contribute to this bleeding risk, particularly in critically ill patients. Current UFH guidelines for young infants may still be inadequate, and laboratory methods with age‐appropriate ranges may be required to further improve clinical outcomes within this population.     

Clinical Results of Far‐Field R‐Wave Reduction with a Short Tip‐Ring Electrode

Background: Far‐field R‐wave (FFRW) sensing of the atrial lead of AAI or DDD pacemakers causes incorrect mode switches and remains a problem in patients with atrial arrhythmias in whom low voltage sensing is essential. We studied a pacing electrode with a short tip‐ring distance (1.1 mm). We compared our findings with recordings from a conventional electrode with a larger tip‐ring distance (10 mm). Methods: Thirty‐six consecutive patients with an indication for DDD pacing were implanted with the short tip‐ring electrode. Another 23 patients received the conventional electrode. FFRW and P‐wave amplitudes during pacing and intrinsic ventricular depolarization were measured at implantation. Measurements were repeated before hospital discharge and at follow‐up between 10 and 14 days after implantation. Results: P‐wave amplitude was slightly smaller in the short tip‐ring group (2.71 ± 1.04 vs 3.17 ± 1.30 mV in the conventional group, respectively, P = NS). All P‐waves exceeded 1.2 mV. FFRW during pacing was 0.07 ± 0.05 in the short tip‐ring group and 0.54 ± 0.32 mV in the conventional group (P < 0.001). FFRW during intrinsic rhythm was 0.08 ± 0.04 and 0.55 ± 0.31 mV, respectively (P < 0.001). The ratio between P‐wave and FFRW was 48.6 ± 27.2 in the short tip‐ring group and 7.3 ± 4.4 in the conventional group (P < 0.001). FFRW and P‐wave amplitudes did not change at hospital discharge or during follow‐up. Conclusion: FFRW can be suppressed without compromising P‐wave sensing by using a pacing electrode with a short tip‐ring distance. Whether reduced FFRW amplitude results in clinical endpoints remains to be determined.     

Multi‐step binding of ADAMTS‐13 to von Willebrand factor

Summary. Background: ADAMTS‐13 proteolytic activity is controlled by the conformation of its substrate, von Willebrand factor (VWF), and changes in the secondary structure of VWF are essential for efficient cleavage. Substrate recognition is mediated through several non‐catalytic domains in ADAMTS‐13 distant from the active site. Objectives: We hypothesized that not all binding sites for ADAMTS‐13 in VWF are cryptic and analyzed binding of native VWF to ADAMTS‐13. Methods: Immunoprecipiation of VWF–ADAMTS‐13 complexes using anti‐VWF antibodies and magnetic beads was used. Binding was assessed by Western blotting and immunosorbent assays. Results: Co‐immunoprecipitation demonstrated that ADAMTS‐13 binds to native multimeric VWF (K_d of 79 ± 11 nmol L^?1) with no measurable proteolysis. Upon shear‐induced unfolding of VWF, binding increased 3‐fold and VWF was cleaved. Binding to native VWF was saturable, time dependent, reversible and did not vary with ionic strength (I of 50–200). Moreover, results with ADAMTS‐13 deletion mutants indicated that binding to native VWF is mediated through domains distal to the ADAMTS‐13 spacer, probably thrombospondin‐1 repeats. Interestingly, this interaction occurs in normal human plasma with an ADAMTS‐13 to VWF stoichiometry of 0.0040 ± 0.0004 (mean ± SEM, n = 10). Conclusions: ADAMTS‐13 binds to circulating VWF and may therefore be incorporated into a platelet‐rich thrombus, where it can immediately cleave VWF that is unfolded by fluid shear stress.     

Antimetastatic effect of tinzaparin, a low-molecular-weight heparin

Summary. The importance of coagulation activation in cancer patients is suggested by the clinical finding of hypercoagulability, experimental enhancement of metastasis and angiogenesis by coagulation factors such as tissue factor (TF) and thrombin and the possible antitumor effects of anticoagulant agents. Tinzaparin is a low‐molecular‐weight heparin (LMWH) with a relatively high molecular weight distribution and high sulfate to carboxylate ratio. In addition to its ability to inhibit thrombin and factor Xa, tinzaparin is particularly effective at releasing endothelial tissue factor pathway inhibitor (TFPI), the natural inhibitor of both procoagulant and non‐coagulant effects of TF. The present study was undertaken to investigate the effect of tinzaparin on lung metastasis using a B16 melanoma model in experimental mice. Tinzaparin's anticoagulant effect in mice and its ability to release TFPI from human endothelial cells at various time points were demonstrated. Subcutaneous (s.c.) injection of tinzaparin (10 mg kg^?1) 4 h before intravenous administration of melanoma cells (2.0 × 10⁵) markedly (89%) reduced lung tumor formation (3 ± 2) compared with controls (31 ± 23; P < 0.001). In a second group of animals, tinzaparin (10 mg kg^?1, s.c.) administered daily for 14 days following the initial (pretumor cell) dose, before assessment of lung seeding, reduced tumor formation by 96% (P < 0.001). No bleeding problems were observed in any of the tinzaparin‐treated animals, despite a 4‐fold prolongation of the whole blood clotting time after a single s.c. dose of tinzaparin (10 mg kg^?1). Administration of tumor cells (2 × 10⁶) caused a rapid and significant fall in platelet count 15 min after injection (a sensitive marker of intravascular coagulation) in controls (939 ± 37 vs. 498 ± 94 × 10⁶ mL^?1, P < 0.01), but this was prevented by tinzaparin treatment (921 ± 104 × 10⁶ mL^?1). These data provide further experimental evidence to support the potential for LMWH as antimetastatic agents.     

Description of the chemical and pharmacological characteristics of a new hemisynthetic ultra-low-molecular-weight heparin, AVE5026

Summary. Background and objectives: AVE5026 is a novel, hemisynthetic, ultra‐low‐molecular‐weight heparin (ULMWH), which is in clinical development for prevention of venous thromboembolism. Its unique structural features result from the highly selective depolymerization of heparin by the phosphazene base that protects the antithrombin (AT)‐binding site from destruction. In the present paper, we describe the chemical and biological characteristics of AVE5026, as well as its effects on experimental thrombosis as compared to those of the low‐molecular‐weight heparin (LMWH) enoxaparin after a single subcutaneous (s.c.) administration in certain animal models. Method and results: AVE5026 has a higher anti‐factor Xa (anti‐FXa) activity (～160 U mg^?1) along with a catalytic anti‐thrombin (anti‐FIIa) activity (～2 U mg^?1) as a result of its structure being strongly enriched in specific AT‐binding oligosaccharides. In human plasma, potent inhibition of thrombin generation by AVE5026 was closely related to its anti‐FXa activity. In a rat venous thrombosis model, AVE5026 showed a dose‐dependent antithrombotic activity comparable to that of enoxaparin (ED50‐AVE5026 = 1.6 mg kg^?1, ED50‐enoxaparin = 2.8 mg kg^?1). Interestingly, non‐occlusive venous thrombosis in rabbits was inhibited by an ED50 of 0.1 mg kg^?1 AVE5026, whereas 0.316 mg kg^?1 enoxaparin was not active. In a canine model, similarly to enoxaparin (ED50 = 1.3 mg kg^?1), AVE5026 dose‐dependently inhibited arterial thrombosis (ED50 = 2.0 mg kg^?1). At equipotent doses, AVE5026 did not affect bleeding parameters, whereas enoxaparin showed increased hemorrhage in rats, rabbits and dogs. Conclusion: These unique structural attributes distinguish AVE5026 from the LMWH class. Based on these data in well‐established arterial and venous thrombosis models, AVE5026 could represent a valuable alternative in thrombosis prevention with an improved benefit‐risk profile as compared to that of enoxaparin.     

AT1 blockade abolishes left ventricular hypertrophy in heterozygous cMyBP‐C null mice: role of FHL1

This research investigated the impact of angiotensin AT1 receptor (Agtr1) blockade on left ventricular (LV) hypertrophy in a mouse model of human hypertrophic cardiomyopathy (HCM), which carries one functional allele of Mybpc3 gene coding cardiac myosin‐binding protein C (cMyBP‐C). Five‐month‐old heterozygous cMyBP‐C knockout (Het‐KO) and wild‐type mice were treated with irbesartan (50 mg/kg/day) or vehicle for 8 weeks. Arterial blood pressure was measured by tail cuff plethysmography. LV dimension and function were accessed by echocardiography. Myocardial gene expression was evaluated using RT‐qPCR. Compared with wild‐type littermates, Het‐KO mice had greater LV/body weight ratio (4.0 ± 0.1 vs. 3.3 ± 0.1 mg/g, P < 0.001), thicker interventricular septal wall (0.70 ± 0.02 vs. 0.65 ± 0.01 mm, P < 0.02), lower Mybpc3 mRNA level (?43%, P < 0.02), higher four‐and‐a‐half LIM domains 1 (Fhl1, +110%, P < 0.01), and angiotensin‐converting enzyme 1 (Ace1, +67%, P < 0.05), but unchanged Agtr1 mRNA levels in the septum. Treatment with irbesartan had no effect in wild‐type mice but abolished septum‐predominant LV hypertrophy and Fhl1 upregulation without changes in Ace1 but with an increased Agtr1 (+42%) in Het‐KO mice. Thus, septum‐predominant LV hypertrophy in Het‐KO mice is combined with higher Fhl1 expression, which can be abolished by AT1 receptor blockade, indicating a role of the renin‐angiotensin system and Fhl1 in cMyBP‐C‐related HCM.     

Effect of SanOrg123781A, a synthetic hexadecasaccharide, on clot-bound thrombin and factor Xa in vitro and in vivo

Summary. Factor (F)Xa and thrombin bound to the clot during its formation contribute to the propensity of thrombi to activate the coagulation system. The aim of this work was to study the inhibition of clot‐bound FXa and clot‐bound thrombin by SanOrg123781A, a synthetic hexadecasaccharide that enhances the inhibition of thrombin and FXa by antithrombin (AT). SanOrg123781A, designed to exhibit low non‐specific binding to proteins other than AT, was compared with heparin. In buffer, heparin and SanOrg123781A inhibited FXa and thrombin at similar concentrations [concentration inhibiting 50% (IC₅₀) of Xa and IIa activity were, respectively: heparin 120 ± 7 and 3 ± 1 ng mL^?1; SanOrg123781A 77 ± 5 and 4 ± 1 ng mL^?1]. In human plasma, the activity of both compounds was reduced, although the activity of heparin was much more affected than that of SanOrg123781A (IC₅₀ values for inhibition of FXa and FIIa activity were, respectively: heparin 100 ± 5 and 800 ± 40 ng mL^?1; SanOrg123781A 10 ± 5 and 30 ± 3 ng mL^?1). We demonstrated, in agreement with our previous results, that the procoagulant activity of the clot is essentially due to clot‐bound FXa and to some extent to clot‐bound thrombin. We showed that heparin and SanOrg123781A were able to inhibit fragment F1+2 generation induced by clot‐bound FXa with IC₅₀ values of 2 ± 0.5 µg mL^?1 and 0.6 ± 0.2 µg mL^?1, respectively. Both compounds also inhibited clot‐bound thrombin activity, the IC₅₀ values of heparin and SanOrg123781A being 1 ± 0.01 µg mL^?1 and 0.1 ± 0.1 µg mL^?1, respectively. Moreover, both heparin and SanOrg123781A significantly inhibited fibrinopeptide A generated by the action of clot‐bound thrombin on fibrinogen but also by free thrombin generated from prothrombin by clot‐bound FXa with IC₅₀ values of 4 ± 0.6 and 1 ± 0.1 µg mL^?1, respectively. As with clot‐bound enzymatic activities, SanOrg123781A was three times more active than heparin in vivo on fibrinogen accretion onto a pre‐existing thrombus and as activators of recombinant tissue‐type plasminogen activator‐induced thrombolysis. In conclusion, due to the specific activities of SanOrg123781A, this compound is much more active than heparin in the presence of plasma proteins, on clot‐bound enzymes and in in vivo models of thrombosis/thrombolysis.     

Coagulation status of critically ill patients with and without liver disease assessed using a novel thrombin generation analyzer

The liver synthesizes the majority of pro‐ and anti‐coagulant and fibrinolytic proteins, and during liver dysfunction synthesis of these proteins is reduced. The end point of conventional hemostatic tests, such as the prothrombin time (PT), occurs when only 5% of thrombin generation (TG) has taken place and is not sensitive to the effects of natural anti‐coagulants. The aim of this study was to determine whether TG in the presence of thrombomodulin (TM) provides more useful information about coagulation potential, in comparison to the PT. Analysis was performed on ST Genesia, a novel TG analyzer from Diagnostica Stago. TG was measured using STG‐Thromboscreen, a reagent containing an intermediate concentration of human tissue factor (TF) ± rabbit TM to account for anti‐coagulant protein C (PC) activity. Platelet‐poor plasma (PPP) samples were from the Intensive Care Study of Coagulopathy‐2 (ISOC‐2), which recruited patients admitted to critical care with a prolonged PT (3 seconds above the reference range). Despite a prolonged PT, 48.0% and 60.7% of patients in the liver and non‐liver groups had TG parameters within the normal range. Addition of TM reduced TG by 34.5% and 41.8% in the liver and non‐liver groups, respectively. Interestingly, fresh frozen plasma (FFP) transfusion had no impact on TG. Measurement of TG with addition of TM provides a more informative assessment of coagulation capacity and indicates that hemostasis is balanced in patients with liver disease during critical illness, despite conventional tests suggesting that bleeding risk is increased.