Xa Activity Research Articles

Impact of plasma from patients with severe aortic stenosis on valvular endothelial cells

Abstract Background The natural history of aortic stenosis (AS) encompasses a spectrum of pathophysiological stages, including endothelial dysfunction, inflammation, fibrosis and calcification. Accumulating data underscore the detrimental impact of the haemostatic system on AS, contributing to disease progression and the development of a prothrombotic phenotype within the valve. Understanding these mechanisms is pivotal for the exploration of novel therapeutic avenues, particularly in the context of transcatheter aortic valve replacement (TAVR). Following TAVR, the native diseased aortic valve persists, maintaining its pathological activity and thrombogenic effects on the bioprosthetic valve, potentially influencing its durability and patient outcomes. The precise effects of plasma and its various components on valvular endothelial cells (VECs) dysfunction remains inadequately explored. Purpose To evaluate the influence of plasma derived from individuals with severe AS on aortic VECs. Methods Plasma samples obtained from patients with severe AS undergoing TAVR (n=100) were collected immediately before the procedure, alongside plasma samples from healthy individuals. Factor (F)Xa activity within the plasma samples was quantified using fluorometer-based method. Porcine VECs were then exposed to either plasma (at 10% concentration) or isolated FXa (1 nM), with or without different pharmacological modulators. Protein expression levels were assessed by Western blot analyses. The level of reactive oxygen species (ROS) was evaluated by dihydroethidium staining. Results Exposure of VECs to plasma from severe AS patients promoted ROS formation compared to plasma from healthy individuals (1402 ± 438 AU vs 732 ± 137 AU, p=0.001) (Figure 1). The heightened oxidative stress elicited by plasma from severe AS patients was attenuated by empagliflozin, perindoprilat, losartan, neutralizing antibodies directed against IL-1, IL-6 and TNF-α. Plasma from severe AS patients showed elevated FXa activity compared to plasma from healthy individuals (179.4 ± 101.3 ng/mL vs 63.2 ± 87.2 ng/mL, p=0.02). Stimulation of VECs with isolated FXa resulted in a sustained formation of ROS, which was blunted by empagliflozin, perindoprilat, losartan, rivaroxaban, dabigatran and PAR-1, 2 and 4 antagonists. Quantification of protein expression in VECs stimulated by isolated FXa revealed an endothelial dysfunction phenotype characterized by increased expression of SGLT2, VCAM-1 and COX-1, and decreased expression of eNOS. Conclusions Plasma from patients with severe AS exerts a detrimental effect on aortic VECs, characterized by a pronounced oxidative stress response. This adverse impact is mediated by multiple mechanisms, including activation of the AT1R/NADPH oxidase/SGLT2 pathway, upregulation of pro-inflammatory cytokines, and activation of PAR. FXa plays a pivotal role by promoting oxidative stress in VECs and contributing to valvular endothelial dysfunction.Pro-oxidant state in VECs.

Laminin G1 residues of protein S mediate its TFPI cofactor function and are competitively regulated by C4BP

AbstractProtein S is a cofactor in the tissue factor pathway inhibitor (TFPI) anticoagulant pathway. It enhances TFPIα-mediated inhibition of factor (F)Xa activity and generation. The enhancement is dependent on a TFPIα-protein S interaction involving TFPIα Kunitz 3 and protein S laminin G-type (LG)-1. C4b binding protein (C4BP), which binds to protein S LG1, almost completely abolishes its TFPI cofactor function. However, neither the amino acids involved in TFPIα enhancement nor the mechanisms underlying the reduced TFPI cofactor function of C4BP-bound protein S are known. To screen for functionally important regions within protein S LG1, we generated 7 variants with inserted N-linked glycosylation attachment sites. Protein S D253T and Q427N/K429T displayed severely reduced TFPI cofactor function while showing normal activated protein C (APC) cofactor function and C4BP binding. Based on these results, we designed 4 protein S variants in which 4 to 6 surface-exposed charged residues were substituted for alanine. One variant, protein S K255A/E257A/D287A/R410A/K423A/E424A, exhibited either abolished or severely reduced TFPI cofactor function in plasma and FXa inhibition assays, both in the presence or absence of FV-short, but retained normal APC cofactor function and high-affinity C4BP binding. The C4BP β-chain was expressed to determine the mechanisms behind the reduced TFPI cofactor function of C4BP-bound protein S. Like C4BP-bound protein S, C4BP β-chain-bound protein S had severely reduced TFPI cofactor function. These results show that protein S Lys255, Glu257, Asp287, Arg410, Lys423, and Glu424 are critical for protein S-mediated enhancement of TFPIα and that binding of the C4BP β-chain blocks this function.

Effect of enoxaparin on plasma fibrin clot properties and fibrin structure in patients with acute pulmonary embolism

BackgroundLow-molecular-weight heparins (LMWHs) influence the fibrin network structure in in vitro models. There have been no reports on LMWH-induced modifications of fibrin clot characteristics and their determinants in acute pulmonary embolism (PE). AimWe investigated how enoxaparin alters fibrin clot properties in acute PE patients. MethodsClots were generated from plasma of 46 acute PE patients, aged 47-77 years treated with enoxaparin 1 mg/kg bid. Fibrin clot permeability (Ks) and clot lysis time (CLT), along with coagulation and fibrinolysis proteins were determined. Plasma fibrin clot nanostructure was assessed using scanning electron microscopy (SEM). ResultsBoth Ks and CLT were associated with anti-factor (F)Xa activity (r = 0.75, p < 0.0001 and r = −0.37, p = 0.011). Anti-FXa was positively associated with fibrin fiber diameter and the pore area, and inversely with fibrin fiber density on SEM images. Multiple regression analysis adjusted for age, body-mass index, and fibrinogen levels showed that anti-FXa activity, antithrombin activity, and FVIII activity determined Ks, while anti-FXa activity, plasminogen activator inhibitor-1 level, and presence of right ventricular dysfunction determined CLT. ConclusionsWe identified new laboratory and clinical factors contributing to prothrombotic plasma fibrin clot characteristics during enoxaparin treatment, which might help elucidate mechanisms underlying therapy failure in patients with acute PE.

Anti-Factor Xa Activity of Fixed-Dose Fondaparinux in Low-Body-Weight Patients With Acute Coronary Syndrome.

Fixed-dose 2.5 mg of fondaparinux subcutaneous injection once daily has been recommended in treatment of non-ST-segment elevation acute coronary syndrome (NSTE-ACS) irrespective of body weight (BW). However, data on anti-factor Xa (anti-FXa) activity of fondaparinux are scarce in low-BW patients. We aimed to assess anti-FXa activity of fondaparinux in low-BW patients (BW < 50 kg) compared with normal-BW patients (BW ≥ 50 kg) who presented with NSTE-ACS. This is a prospective cohort study of patients with NSTE-ACS receiving fondaparinux. Anti-FXa activity was measured 4 hours after 2.5 mg subcutaneous injection of fondaparinux after the first 2 doses. Among 87 enrolled patients, 18 (21%) had BW <50 kg. Patients in the low-BW group were older and had lower creatinine clearance. Median duration of fondaparinux therapy was 3 (IQR 2-4) days. Anti-FXa activity after the first dose of fondaparinux was similar between the low-BW and normal-BW groups (0.40 ± 0.15 vs 0.40 ± 0.17 mg/L, P = 0.914). However, anti-FXa activity after the second dose of fondaparinux was significantly higher in the low-BW group as compared with the normal-BW group (0.53 ± 0.10 vs 0.44 ± 0.16 mg/L, P = 0.011). Multivariate analysis showed that BW was the only independent factor that inversely correlated with anti-FXa activity. There was only 1 bleeding event during hospitalization in the normal-BW group and none in the low-BW group. Anti-FXa activity of the second dose of fondaparinux was higher in low-BW patients but still within the expected range.

SULFATED DERIVATIVES OF ARABINOGALACTAN AND THEIR ANTICOAGULANT ACTIVITY

The IR spectra and molecular mass distribution of arabinogalactan sulfates in the form of sodium and ammonium salts, obtained using various sulfating reagents, were compared.&#x0D; According to the obtained data, the sulfated derivatives of arabinogalactan differ from each other by the nature of the hydrogen bonds and the molecular weight distribution.&#x0D; Using coagulological tests at the activation of coagulation of platelet-poor human plasma, in vitro study of the anticoagulant properties of arabinogalactan sulfated derivatives in various salt forms differing in methods of preparation, degree of sulfation and molecular weight was conducted.&#x0D; It was established that the sample in the form of arabinogalactan sodium salt (SAG 1) with a sulfur content of 13.2 wt.% аnd a polydispersity degree of 1.52 showed 2 times more anticoagulant activity than the sample in the form of ammonium salt of arabinogalactan (SAG 2) with the sulfur content of 6.6 wt.% and the degree of polydispersity of 1.30.&#x0D; Antithrombin (aIIa) activity of samples obtained by sulfation with pyridine and sulfuric anhydride complex (SAG 1) and sulfamic acid complex (SAG 2) was, respectively, 23.42±1.86 and 10.20±1.50 U/mg; the anti-factor Xa activity of SAG 1 and SAG 2 was 2.13±0.42 and 0.37±0.08 U/mg; and the ratio aIIa/aXa for SAG1 and SAG 2 was 11 and 28, respectively.&#x0D; The less activity of antifactor Xa(aXA) of SAG as compared to unfractionated heparin (UFG) and higher rations of activities aIIa/aXa may contribute the less provocation of bleeding by SAG samples in the comparition with UFG.

P1624Blockade of protease activated receptor-1 signaling attenuates cardiac hypertrophy and fibrosis in renin-overexpressing hypertensive mice

Abstract Introduction Recent evidences have demonstrated that coagulation pathway is involved in cardiovascular remodeling induced by renin-angiotensin system (RAS), which finally leads to heart failure. Protease activated receptor-1 (PAR-1) is widely expressed in the vasculature and the heart, and plays important roles in pro-inflammatory process in the cardiovascular system. Recently, we demonstrated that the activity of factor Xa (FXa), which functions not only as a coagulation factor but as an agonist for PAR-1, was enhanced in renin-overexpressing hypertensive mice (Ren-Tg). Purpose The purpose of this study was to investigate whether inhibition of PAR-1 signaling has protective effects on the progression of heart failure induced by chronic RAS activation in Ren-Tg. Methods and results We treated 12–16 weeks-old male wild type mice (WT) and Ren-Tg with continuous subcutaneous infusion of PAR-1 antagonist SCH79797 (25mg/kg/day) or vehicle for 4 weeks. After treatment period, left ventricular (LV) wall thickness calculated as interventricular septum plus posterior wall thickness measured by echocardiography was greater in Ren-Tg than in WT (0.25±0.003 versus 0.18±0.002 mm), and SCH79797 attenuated the increase to 0.22±0.01 mm in Ren-Tg (both p&lt;0.05, respectively). The ratio of heart weight to body weight was greater in Ren-Tg than in WT (6.1±0.4 versus 4.6±0.7 mg/g), and SCH79797 attenuated the increase to 5.2±0.1 mg/g (both p&lt;0.05). The area of cardiac fibrosis evaluated by Masson-trichrome staining was greater in Ren-Tg than in WT (2.6±0.2 versus 1.4±0.3%), and SCH79797 attenuated it to 1.6±0.3% in Ren-Tg (both p&lt;0.05). Cardiac mRNA expressions of tumor necrosis factor-α, transforming growth factor-β1, and β-myosin heavy chain were all greater in Ren-Tg than in WT, and SCH79797 attenuated the increases in Ren-Tg (all p&lt;0.05). Conclusions Inhibition of PAR-1 signaling attenuates cardiac hypertrophy and fibrosis in Ren-Tg via inhibition of inflammatory cytokines production. These results support the involvement of PAR signaling in the development of heart failure induced by RAS, and may provide novel therapeutic insights for the treatment of hypertensive heart failure.

To clot, or not to clot – Antithrombotic therapy is the question

Haemostasis and thrombosis rely on three components namely the vascular endothelial wall, blood platelets and the coagulation cascade. Non-physiologic excessive thrombosis occurs when haemostatic processes are dysfunctional, causing undue clot formation or reduced clot lysis. Antithrombotic agents including antiplatelet, anticoagulation and fibrinolytic agents are essential for the prophylaxis and pharmacological management of venous thromboembolism and arterial thrombosis. Anticoagulation treatment options have expanded steadily over the past few decades, providing a greater number of agents. Anticoagulants that directly target the enzymatic activity of thrombin and factor Xa have recently been developed to address the inadequacies of traditional vitamin K antagonists. Appropriate use of these agents requires knowledge of their individual characteristics, risks, and benefits.

Effect of IgG from multiple sclerosis patients on amidolytic activity of coagulation and anticoagulation factors of hemostasis

Background: Immunoglobulin G (IgG) is a major immunoglobulin (Ig) in blood that accumulates to a greater extent in the bloodstream of patients impacted by neuroimmunological disorders such as multiple sclerosis (MS). The aim of this study was to determine the effect of IgG obtained from MS patients on the amidolytic activity of coagulation and on anticoagulation factors, and to compare those effects to the effects of IgG from healthy donors.&#x0D; Methods: Spectrophotometric hydrolysis of specific chromogenic substrate by key haemostasis factors was examined.&#x0D; Results: Our study shows that unlike healthy individuals, patients suffering from MS express IgG which enhances the amidolytic activity of thrombin and protein C, but inhibits the activity of factor Xa.&#x0D; Conclusion: Our study shows that IgG and coagulation factors, indeed, interact with each other. IgG may be key mediators of neuroinflammation and, therefore, may serve as a potential target for therapeutic strategies for MS and other neuroimmunological diseases.

Antiplatelet Agents and Anticoagulants in Patients with Chronic Kidney Disease - from Pathophysiology to Clinical Practice.

Progressive impairment of renal function can lead to uremia, which is associated with an increased risk of bleeding as well as thrombosis. Furthermore, many patients with chronic kidney disease (CKD) have an indication for an anticoagulation or antiplatelet therapy due to atrial fibrillation, coronary artery disease, thromboembolic disease, or peripheral artery disease. The treatment usually includes vitamin-K antagonists (VKAs) and/or platelet aggregation inhibitors. The direct oral anticoagulants (DOACs) inhibiting factor Xa or thrombin activity represent an alternative for heparins and VKAs. However, DOACs can further aggravate the bleeding risk in CKD patients. This is related to a combination of an accumulation of the substance due to the reduced renal clearance, an inhibition of thrombin-mediated platelet activation, and uremia associated factors such as impaired coagulation, platelet function, and platelet-vessel wall interactions. Furthermore, platelet aggregation inhibitors can also influence the bleeding risk, particularly if they are administered in combination with anticoagulants in patients with advanced CKD. In this review we discuss the different mechanisms leading to the increased risk of bleeding and thrombosis as well as the different options and problems related to an antiplatelet or anticoagulation therapy in CKD patients.

Purification and antithrombotic activity of wulfase, a fibrinolytic enzyme from the fruit bodies of the edible and medicinal mushroom Sparassis crispa Wulf. ex. Fr.

A fibrinolytic protease, wulfase, exhibiting antithrombotic potency was purified from the fruit bodies of Sparassis crispa Wulf. ex. Fr. Wulfase showed a single band of approximately 90 kDa by SDS-PAGE and fibrin zymography. The fibrinolytic activity of wulfase determined by fibrin plate assay revealed that it could directly degrade fibrin clot. The enzyme could inhibit activities of factor Xa and thrombin. Turbidity and electron-microscopy analysis using fluorescent conjugate demonstrated that wulfase also inhibited fibrin polymer formation. The anticoagulant effect of the enzyme was further confirmed in human plasma. These results suggest that wulfase may be useful for reducing or preventing thrombotic challenge.

Abstract 594: Characterization of Anti-thrombotic and Anti-inflammatory Properties of New Synthetic, Protamine Reversible Low Molecular Weight Heparin

Low-molecular-weight heparins (LMWHs) are carbohydrate-based anticoagulants clinically used to treat thrombotic disorders; however, lack of full reversibility with protamine and clearance by kidney limit treatment options. Recently we have developed a chemoenzymatic method to synthesize a new synthetic dodecasaccharide (12mer) with high anti-factor Xa (FXa) activity which was protamine reversible in vitro and in vivo. In the current study, we further characterized the pharmacokinetic, anti-thrombotic and anti-inflammatory properties of this compound using various mouse models. First, time course (0.5, 1, 2, 4, 8 hours, n=4) and dose response (0.5, 1, 1.5, 2.0 mg/kg, n=4) studies in C57Bl/6J mice showed that FXa activity was inhibited by 12mer in vivo and the half-life of the compound injected s.c. at the dose of 1.5 mg/kg was 3 hours. 12mer was cleared from the circulation by 8 hours. Furthermore, fibrin accumulation observed in the femoral vein 60 minutes after electrolytic injury (2.99 ± 1.16; p&lt;0.05; normalized relative fluorescent intensity to control mouse, mean ± SD), was similarly reduced by 12mer (0.6 mg/kg, 1.63± 0.48), UFH (150 U/kg, 1.26 ± 0.35) and enoxaparin (1 mg/kg, 1.39 ± 0.85, p&lt;0.05 for all). Importantly, protamine reversed the anti-thrombotic effect of 12mer and returned fibrin accumulation to normal levels (2.69 ± 0.87; p &lt; 0.05 vs.12mer alone). Finally, we investigated if 12mer can inhibit thrombotic and inflammatory effect of FXa, previously demonstrated by us in the Berkeley mouse model of sickle cell disease. Sickle mice received subcutaneous injection of saline (n=6) or 12-mer (n=6; 2.0 mg/kg) every 8 hours for 7 days. Compared to saline, 12mer reduced plasma levels of thrombin anti-thrombin complexes (8.2±1.4 vs 4.4±1.3 ng/mL; p=0.07, mean ± S.E.M), IL-6 (5.5±1.5 vs 1.1±0.4 pg/mL, p&lt;0.05) and sVCAM1 (1.1±0.2 vs 0.6±0.07 μg/mL, p&lt; 0.05) in sickle mice. Together, our data demonstrates that chemoenzymatic synthesis offers a promising approach toward obtaining new, protamine-reversible LMWHs with excellent anti-coagulant and anti-inflammatory properties.

Dose Adjustment Of Low Molecular Weight Heparins (LMWH) Improves Pregnancy Outcome In Thrombophilic Women

Introduction Acquired and inherited thrombophilias are associated with unfavourable pregnancy outcome. Low-molecular-weight heparin (LMWH) has demonstrated utility in the prevention and treatment of thrombosis during the pregnancy. There is no consensus on the dose of LMWH for prophylaxis, between fixed dose and “adjusted” dose according to anti-Xa levels. We report our experience the management of heparin-therapy during pregnancy in thrombophilic women. Methods We performed a retrospective analysis of patients with high risk of venous thromboembolism (VTE) and obstetric complications during pregnancy. Table 1 shows the risk factors of VTE in pregnancy. The obstetric complications considered were: preeclampsia, fetal growth restriction, fetal death and recurrent miscarriages. All patients were prescribed enoxaparin 70-100 IU /Kg SC once daily. To determine the dose of LMWH we evaluated levels of anti-factor Xa (chromogenic test) and the utero-placental circulation with color Doppler ultrasound. Dose adjustment was performed if the activity of factor Xa was suboptimal (prophylaxis range &gt; 0.4IU/ml and &lt; 0.8 IU/ml anti-Xa value) or were detected alteration in the utero-placental circulation. Outcome Measures Time of delivery, birth weights and Apgar-index. Results 70 pregnancies for 68 women were recruited between January 2008 and December 2012. The median age of patients was 35 years (range 25- 43years) and 7 (10%) were over 40. Twenty-four patients had a previous VTE and thirty-six had an obstetric complications. No one patients reported thrombotic and/or hemorrhagic complications during heparin treatment. In 97 % of cases the time of delivery occurred after 36th weeks of pregnancy, with a birth weight greater than 2600 grams and Apgar score index higher than 7. The dose of LMWH had to be adjusted at least once during the course of the pregnancy and the mean daily dose was increased from 4.000UI to 8.000UI between the 12th and 32th week of gestation. Conclusion In pregnancy LMWH thromboprophylaxis enables the modulation of systemic haemostatic parameters by the inhibition of factor Xa and by increasing TFPI levels. In the literature-in some high risk pregnancy- the weight-based dosing of LMWH could seem to fail achieving a prophylactic anticoagulation. In our experience adjusted doses prevent unfavourable events and improve fetal growth. °Carriage of defects of antithrombin, protein C or S, factor V Leiden, G20210A prothrombin mutation, antiphospholipid syndrom Disclosures: No relevant conflicts of interest to declare.

Novel Point-Of-Use FXa Assay For Monitoring Anticoagulant Therapy By Oral Factor Xa Inhibitors and Heparin

Introduction A highly specific, rapid, robust and sensitive assay for Factor Xa (FXa) activity will greatly improve monitoring and optimization of anticoagulant therapy by direct FXa inhibitors and by low molecular weight (LMW) heparins. We have developed a new point-of-use diagnostic system that will provide results in less than 30 minutes, enabling rapid medical decisions based on quantitation of FXa activity and anticoagulant drugs in patients’ blood. Anticoagulant therapy with unfractionated or LMW heparins needs aggressive monitoring in pregnant women, patients with renal insufficiency, and neonates, in particular in cases of treatment-related side effects, including heparin-induced thrombocytopenia and bleeding tendency (Chest, 2008). New oral anticoagulants rivaroxaban (Xarelto®) and apixaban (Eliquis®) have been effective in reducing risk of stroke and systemic embolism in patients with nonvalvular atrial fibrillation, and in preventing and treating deep vein thrombosis. Both drugs directly inhibit FXa and have significant therapeutic potential as an alternative to warfarin. Unlike warfarin, these drugs do not require constant monitoring and dose adjustment to maintain anticoagulation within the therapeutic interval. However, excessive anticoagulation and consequent hemorrhage risks have been sufficiently common problems to warrant clinical development of specific antidotes for FXa inhibitors. Currently available anti-Xa heparin assays may not be appropriate for rivaroxaban and apixaban measurements. Additionally, recently published data indicate that new oral anticoagulants interfere with the measurement of common test parameters, expanding the need for more specific diagnostic tests (Clinical Chemistry, February 2013). This novel rapid and specific FXa assay will allow monitoring LMW heparins and the new FXa inhibitors, identify heparin-refractory patients as well as those requiring antidotes to oral Factor Xa inhibitors, in addition to directly measuring FXa activity in patients with inherited deficiency of Factor X, congenital antithrombin deficiency, and acquired FXa deficiencies. Method A diagnostic assay to measure FXa activity in human plasma using electrochemiluminescence (ECL) technology was developed at Wellstat Diagnostics, LLC. The assay utilizes a synthetic peptide containing an FXa substrate sequence that is recognized by specific antibodies (labeled with an ECL-active ruthenium chelate) only after specific cleavage by FXa protease. The resulting ECL signal is directly proportional to FXa activity within the physiological range. The assay has been adapted to also provide quantitative measurement of activity of anticoagulant drugs that inhibit FXa. In the modified format the resulting ECL signal is inversely proportional to the concentration of rivaroxaban, apixaban or heparin in plasma. Results The ECL-based assay measures activity of anticoagulant drugs on FXa in human plasma with high intra- and inter-assay reproducibility, accuracy and specificity. Using FX-deficient plasma spiked with Factor X in the range 1-100 ng/mL, we have demonstrated linear increases of ECL signal directly proportional to the concentration of spiked Factor X activated by a specific Russell Viper Venom activator. Serial dilutions of anticoagulant drugs rivaroxaban, apixaban and enoxaparin (LMW heparin) in normal donor plasma showed exponential decreases of ECL signal in the range of concentrations relevant to the therapeutic doses of all three drugs. The dynamic range of the drug detection was 15 - 500 ng/mL for rivaroxaban and apixaban, and 0.2 - 2 IU/mL of enoxaparin. The assay was specific to activity of FXa, while insensitive to variable concentrations of other clotting factors in human plasma, as well as to the presence of other drugs in plasma that don’t directly inhibit FXa. Conclusions The point-of-use FXa assay is shown to be highly specific, robust, rapid and reproducible for measurements of anticoagulant activity of apixaban, rivaroxaban and enoxaparin in human plasma. Use of this point-of-use system for monitoring anticoagulant therapy may be further extended to measure other blood clotting factors (for example, Factor IXa, VIIa, FVIII), for monitoring activity of drugs acting on these factors and for rapid diagnosis of coagulation disorders. Disclosures: No relevant conflicts of interest to declare.

The Impact Of Dietary Vitamin K On The Pharmacological Activity Of FXa Inhibitor, Rivaroxaban, In Man

Introduction/Background Over several decades, warfarin has been used as the drug of choice for prevention and treatment of thromboembolic disorders. The impact of dietary vitamin K intake on anticoagulation response to warfarin is well established. However, the effect of dietary vitamin K on the activity of the novel oral anticoagulants is less well known. Since the activation of both factor X and thrombin is inextricably linked to the vitamin K cycle in the coagulation cascade, there is the possibility that the pharmacological activity of agents that inhibit these proteins could be influenced by alterations in vitamin K availability. Aim To examine the influence of dietary vitamin K intake on the pharmacological activity of rivaroxaban in man. Methods The pharmacological activity of rivaroxaban was evaluated ex-vivo. Thirty one medically stable elderly inpatients (12 males) with a poor dietary intake in terms of calories and nutrients including vitamin K (12 males) and 28 healthy subjects (7 males) with adequate diets were enrolled into the study. An overnight fasted venous blood sample (20 ml) was taken from each subject. The plasma was stored at -80 degree C for the later measurement of the effects of rivaroxaban on clotting times and clotting factor activity. Each subject completed a validated dietary questionnaire for quantification of vitamin K content of foods eaten in the past week. The plasma samples from each subject were incubated with a range of rivaroxaban concentrations (100 - 500 ng/ml) in order to simulate the therapeutic drug plasma concentrations. Normal prothrombin time (PT), modified prothrombin time (mPT), and vitamin K dependent clotting factors activity were measured according to established in-house methods. Results The mean±SD age of elderly patients and healthy subjects was 87±6 and 36±10 years, respectively. Rivaroxaban produced a greater prolongation of both PT (P=0.01) and m-PT (P=0.02) in the plasma of elderly subjects than the younger healthy subjects over the drug concentration range studied. The mean difference in PT between the two groups ranged from 1.8s to 5.0s at 100 ng/ml and 500 ng/ml plasma rivaroxaban concentrations respectively. The mean difference in m-PT between the two groups ranged from 10.0s to 34.0s at 100 ng/ml and 500 ng/ml plasma rivaroxaban concentrations respectively. There was also a greater inhibition of factor Xa (P=0.005) activity by rivaroxaban in the elderly subjects compared to the healthy subjects, whereas factor IXa (P=0.03) activity was significantly inhibited in the healthy subjects compared to the elderly subjects. However, there were no significant differences between the two groups in factors II and VII activity and fibrinogen concentration. The analysis of dietary questionnaires for vitamin K intake and plasma for fasting vitamin K levels is in progress and the results will be presented. Conclusion We have previously demonstrated in a proof-of-concept study in rats that vitamin K deficiency significantly enhanced the pharmacological activity of the prototype DTI, ximelagatran [1]. The present study findings suggest that the anticoagulation response to rivaroxaban may be influenced by poor dietary vitamin K intake. Assessment of whether dietary vitamin K influences anticoagulation response as well as any influence of age, co morbidities and drug intake in patients taking rivaroxaban is warranted. [1] Kamali F, Wood P, Ward A. Vitamin K deficiency amplifies anticoagulation response to ximelagatran: possible implications for direct thrombin inhibitors and their clinical safety. Annals of Hematology 2009; 88:141-149. Disclosures: No relevant conflicts of interest to declare.

Acquired factor V deficiency in a patient without evidence of a classical inhibitor

Acquired factor V deficiency in a patient without evidence of a classical inhibitor

New oral anticoagulants: prevention of VTE in phase III studies in total joint replacement surgery and the hospitalized medically-ill patients

Effective venous thromboembolism prophylaxis in hospitalized medically-ill patients and those undergoing orthopaedic surgery remains a challenge for clinicians in the United States. Several new oral anticoagulants, which either directly inhibit the activity of thrombin or factor Xa have been developed and studied for venous thromboembolism (VTE) prevention in phase III trials in these patient populations. These new medications demonstrate several advantages over traditional anticoagulants, including their administration at fixed doses with no requirement for routine coagulation monitoring. Such advantages may potentially be offset by the lack of well-studied methods to reverse their anticoagulant effects and the potential need for standardized testing to monitor their activity in certain situations. This review will provide an overview of the clinical trial results of dabigatran, apixaban and rivaroxaban for VTE prevention in the orthopaedic and medically-ill hospitalized patients.

Efficacy and Safety of Off‐Label Use of Fondaparinux in the Management of Heparin‐Induced Thrombocytopenia with Thrombosis in an Elderly Woman

Type II heparin‐induced thrombocytopenia (HIT) represents the most common immune‐mediated adverse drug reaction (ADR), during treatment with unfractionated heparin (UFH) or low molecular weight heparins (LMWHs). The frequency of HIT is about 2–3% in surgical divisions (e.g., Cardiac Surgery and Orthopedics) and about 0.5–1% in medical divisions (e.g., Internal Medicine). HIT can develop in 5–10 days after the beginning of heparin and it is induced by the production of antibodies targeting a molecular complex of both “self” protein, that is platelet factor 4 (PF4), and heparin. This induces platelet activation as well as increased thrombin production, that result in a higher risk of venous or arterial thrombosis. When thrombosis is identified, HIT is called HIT‐ associated thrombosis (HITT). Identification of a decrease in platelet count represents the first step in the diagnosis of HIT. However, other clinical and laboratory tests, such as 4Ts score serotonin‐release assay (SRA) and measurement of heparin/PF4 antibodies, must be also used. 4Ts score is a clinical scoring system used to estimate the pretest probability of HIT1, based on its characteristic features (Thrombocytopenia, Timing, Thrombosis) and absence of oTher explanation(s). 4Ts score (Table 1) is also used in the pharmacological management of HIT: score 3 continue heparin, score >4 change heparin to non‐heparin anticoagulants. Among non‐heparin anticoagulants, only lepirudin and argatroban, direct thrombin inhibitors (DTIs), are approved in the treatment of HIT. Another DTI, bivalirudin, is indicated in patients undergoing percutaneous coronary interventions. Danaparoid, a factor Xa inhibitor, is not currently available in Italy, but it is approved for treatment and prevention of HITT in Canada, continental Europe, Australia, New Zealand, and Japan. Finally, fondaparinux sodium, a synthetic antithrombin‐binding pentasaccharide with exclusive anti‐factor Xa activity, has shown favorable results in HIT, although in Italy it is off‐label for this indication. In this paper, we report a case of HITT during treatment with a LMWH, that was successfully treated with an off‐ label administration of fondaparinux sodium.

An assay for anti-factor Xa activity of low molecular weight heparins by high performance liquid size exclusion chromatography

The "gold standard" assay for monitoring low molecular weight heparins (LMWHs) activity is the chromogenic-based anti-factor Xa assay. The methodology of an anti-factor Xa assay is that LMWH is added to a known amount of excess factor Xa and excess antithrombin. It will bind to antithrombin and form a triplet complex with factor Xa, inhibiting the activity of factor Xa. However, the residual factor Xa can still hydrolyze chromogenic peptide substrate, releasing the chromophore for photometric detection. The absorbance is inversely proportional to the amount of heparin/LMWH. The results are given in anticoagulant concentration in units/ mL of anti-factor Xa, such that high values indicate high levels of anticoagulation and low values indicate low levels of anticoagulation. Herein, a novel assay method for anti-FXa activity of LMWHs using high performance liquid size exclusion chromatography (SEC) is reported, in which antithrombin III (AT Ill ) was diluted by the buffer solution contained LMWHs. Subsequently, exogenous FXa and p-nitroaniline coupled peptide substrate were added and incubated for a period, separately. The resulting mixture was separated based on size by SEC, and the free chromophore p-nitroaniline can be detected at an absorption maximum of 385 nm without interference from the absorbance of p-nitroanilide substrates. Moreover, the measurements are not influenced by sample opacity or turbidity, so it is possible to test various complex samples, such as plasma. The assay is robust, sensitive, and cost effective.

Stability of low molecular weight heparin anti‐factor Xa activity in citrated whole blood and plasma

Stability of low molecular weight heparin anti‐factor Xa activity in citrated whole blood and plasma

Reduced Thrombin Generation and Soluble P-selectin After Intravenous Enoxaparin During PCI

The objective of our study was to identify changes in the coagulation and serum concentration of soluble P-selectin (sP-sel) after i.v. bolus of 0.75 mg/kg enoxaparin in a group of 33 patients during PCI. As compared to baseline, i.v. enoxaparin increased anti -Xa activity and FIIa inhibition together with APTT and thrombin time tests within 20 min, that persisted for 60 min. At 6 h, the results of all tests had returned to baseline. In contrast, the level of prothrombin fragments (F1 + 2) decreased persistingly for a period of 6 h (baseline 1.19 ± 0.42 nmol/l, after 20 min 1.03 ± 0.46 nmol/l, after 60 min 1.06 ± 0.43 nmol/l, after 6 h 0.95 ± 0.40 nmol/l, p < 0.001 vs. baseline for all values). In addition, i.v. enoxaparin decreased serum sP-sel level (baseline 111.80 ± 37.05 ng/ml, after 20 min 87.80 ± 33.17 ng/ml, after 60 min 86.45 ± 29.15 ng/ml, after 6 h 92.24 ± 31.34 ng/ml, p < 0.001 vs. baseline value for all). sP-sel level mildly correlated with both F Xa inhibition (r = -0.275, p < 0.05) and F1 + 2 level (r = 0.274, p < 0.05). Intravenous enoxaparin induced target F Xa inhibition (>0.6 IU/ml) for 60 min in 82% of study patients. During the 6 h of monitoring, a decrease of thrombin generation (F1 + 2) and sP-selectin levels were observed.