Augmentation of the Anticoagulant Effects of Heparin By a Tri-Block Polymer MST-188

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5080-5080
Author(s):  
Daniel Dansdill ◽  
Jae Cho ◽  
Daneyal Syed ◽  
Jawed Fareed ◽  
Martin Emanuele
Keyword(s):  
Blood Vessel ◽  
Bleeding Time ◽  
Jugular Vein ◽  
Ex Vivo ◽  
Rat Plasma ◽  
Acute Limb Ischemia ◽  
Thrombin Time ◽  
Blood Samples

Abstract Introduction: MST-188 (purified poloxamer 188) is a tri-block co-polymer with high affinity to hydrophobic cellular surfaces that inhibits hydrophobic adhesive interactions in the circulation. It also facilitates blood flow by reducing blood viscosity and reportedly exhibits anti-adhesion and anti-inflammatory properties. Currently, this agent is under study in with patients experiencingsickle cell crisis and in patients with acute limb ischemia. Since MST-188 may be administered with other anti-coagulant agents such as heparin its potential interaction to modulate the anti-coagulant effects of this drug require experimental validation. These studies are designed to investigate the potential interactions between heparin and MST-188 in a rat model of tail transection bleeding and jugular vein clamping induced thrombosis model. Materials and Methods: The in vitro interactions between MST-188 were investigated by supplementing this agent to normal rat plasma (NRP) and heparinized rat plasma at a fixed concentrations of 1.25 and 2.50 mg/mL. The concentration of heparin was kept at 1.25 and 2.50 μg/mL. In the in vivo studies individual groups of rats (n=6-8) were administered with saline as a control, heparin in the dosage range of 125-500 ug/kg intravenously and MST-188 at 25 mg/kg IV followed by heparin at the 125-500 ug/kg dosages. Rat tail resection time was measured 5 minutes after administration of heparin and clot occlusion index was measured as number of jugular vein clamping required to occlude the blood vessel. After the completion of the procedure blood samples were obtained through cardiac puncture and used for ex vivo analysis of PT, aPTT, heptest and thrombin time. Results: In the in vitro studies heparin produced a concentration dependent prolongation of the aPTT, heptest and thrombin time. MST-188 did not produce any effects on the aPTT and heptest time, however it decreased the thrombin time. MST-188 at a higher concentration of 2.5 and 5.0 mg/ml produced a shorting of heparins anticoagulant responses, as measured by aPTT and thrombin time. Heparin produced a dose dependent increase in both the bleeding time (p<0.0001) and number of jugular vein clamps to occlude the blood vessel (p<0.0001). MST-188 at dosages of 25 mg/kg produced a significant increase on both bleeding time (p <0.05) and number of clampings required to occlude the blood vessel (p<0.0001). When MST-188 was administered simultaneously with heparin it augmented the bleeding time (p < 0.05) and increased the number of jugular vein clamps required to occlude the blood vessel (p < 0.05). The ex vivo analysis of blood samples collected from rats treated in different regimens did not exhibit any anti-coagulant effects as measured by PT, aPTT, heptest and thrombin time. Conclusion: These studies suggest a differential response of MST-188. While in vitro it exhibits a prohemostatic response as evident by shortening of thrombin time, in the in vivo setting it enhances the anticoagulant effects of heparin as evident by increased bleeding time and increased number of jugular vein clamps required to occlude the vessel. Thus, both of these mechanisms are involved in the mediation of the beneficial effects observed with this agent in vaso-occlusive and thrombotic processes. Disclosures Emanuele: Mast Therapeutics: Employment.

scholarly journals Image-based modelling of skeletal muscle oxygenation

2017 ◽  
Vol 14 (127) ◽  
pp. 20160992 ◽  
Author(s):  
B. Zeller-Plumhoff ◽  
T. Roose ◽  
G. F. Clough ◽  
P. Schneider
Keyword(s):  
Skeletal Muscle ◽  
Blood Vessel ◽  
Ex Vivo ◽  
Imaging Techniques ◽  
Muscle Oxygenation ◽  
Skeletal Muscle Oxygenation ◽  
Health And Disease ◽  
Vessel Networks

The supply of oxygen in sufficient quantity is vital for the correct functioning of all organs in the human body, in particular for skeletal muscle during exercise. Disease is often associated with both an inhibition of the microvascular supply capability and is thought to relate to changes in the structure of blood vessel networks. Different methods exist to investigate the influence of the microvascular structure on tissue oxygenation, varying over a range of application areas, i.e. biological in vivo and in vitro experiments, imaging and mathematical modelling. Ideally, all of these methods should be combined within the same framework in order to fully understand the processes involved. This review discusses the mathematical models of skeletal muscle oxygenation currently available that are based upon images taken of the muscle microvasculature in vivo and ex vivo . Imaging systems suitable for capturing the blood vessel networks are discussed and respective contrasting methods presented. The review further informs the association between anatomical characteristics in health and disease. With this review we give the reader a tool to understand and establish the workflow of developing an image-based model of skeletal muscle oxygenation. Finally, we give an outlook for improvements needed for measurements and imaging techniques to adequately investigate the microvascular capability for oxygen exchange.

scholarly journals Effects of Juglans regia Root Bark Extract on Platelet Aggregation, Bleeding Time, and Plasmatic Coagulation: In Vitro and Ex Vivo Experiments

2018 ◽  
Vol 2018 ◽  
pp. 1-7 ◽  
Author(s):  
A. Amirou ◽  
M. Bnouham ◽  
A. Legssyer ◽  
A. Ziyyat ◽  
M. Aziz ◽  
...  
Keyword(s):  
Cardiovascular Diseases ◽  
Bleeding Time ◽  
Ex Vivo ◽  
Juglans Regia ◽  
Anticoagulant Effect ◽  
Bark Extract ◽  
Root Bark ◽  
Thrombin Time ◽  
Induced Aggregation

Platelets have an important role in thrombosis and haemostasis. Hyperactivity of the platelets has been associated with thromboembolic diseases and represents the main cause of complications of cardiovascular diseases. Crude aqueous extract (CAE) of Juglans regia root bark was evaluated for bleeding time, antiaggregant activity by using agonists, thrombin, ADP, collagen, or arachidonic acid (in vitro and ex vivo), and anticoagulant activity by measuring the clotting parameters: activated partial thromboplastin time, prothrombin time, thrombin time, and fibrinogen dosage (in vitro and ex vivo). The result of this study reported that the strongest antiaggregant effect of CAE in vitro was observed on the ADP-induced aggregation with inhibitions up to 90 %, while, in ex vivo experiments, the inhibition (more than 80 %) was observed with all agonists. Anticoagulant effect of CAE significantly prolonged the TT and decreased the fibrinogen level in vitro and ex vivo without interfering with APTT and PT. The bleeding time in mice and rats was significantly increased by CAE. The antiplatelet and anticoagulant effect observed in this study suggest that Juglans regia could have antithrombotic and/or thrombolytic activities and provide an alternative therapy against thrombotic complications related to cardiovascular diseases.

Comparison Of The Effects Of Aspirin In Humans Ex Vivo In Platelet-Rich Plasma And Whole Blood

1981 ◽  
Author(s):  
Barbara Nunn
Keyword(s):  
Whole Blood ◽  
Ex Vivo ◽  
Platelet Rich Plasma ◽  
Trisodium Citrate ◽  
Blood Platelet ◽  
Maximal Response ◽  
Blood Samples ◽  
Platelet Concentration

The effect of aspirin on human platelet function is usually assessed using platelet-rich plasma (PRP). Some preliminary results in vitro suggested that the effect of aspirin appears to be greater in PRP than whole blood. To explore this possibility further, a comparison of the effect of aspirin in humans ex vivo has been made taking measurements simultaneously in whole blood and PRP at 2 platelet concentrations. Blood samples (36ml) were drawn from 7 male volunteers after a light breakfast. Each took 300mg soluble aspirin and blood samples were drawn again 2 hours later. Blood was mixed with 0.1 volumes 129nM trisodium citrate. Some (30ml) was then centrifuged to prepare PRP and platelet -poor plasma (PPP) by standard techniques. Platelet concentration of some PRP was adjusted with PPP to equal that of the corresponding blood sample; the rest was adjusted to 350,000 per μl. Aggregation in response to collagen (Horm, Munich) was measured photometrically at 37°. Aggregation in 0.5ml aliquots of whole blood was measured after 4 min stirring with 154mM NaCl (control) or collagen at 37° as the fall in single platelet count determined using an Ultraflo- 100 whole blood platelet counter (Clay Adams). The concentrations of collagen producing a 50% maximal response (EC50) in PRP and blood were determined. Dose-ratios for each volunteer were calculated by dividing the EC50 obtained after aspirin by the corresponding value obtained before aspirin.The effect of aspirin was significantly (p<0.001) less in blood than PRP. Whether or not the results in whole blood more closely reflect the effect of aspirin in vivo remains to be determined.

scholarly journals Synthesis and Thrombin, Factor Xa and U46619 Inhibitory Effects of Non-amidino and Amidino N2-Thiophenecarbonyl- and N2-Tosylanthranilamides

2017 ◽  
Author(s):  
Soo Hyun Lee ◽  
Wonhwa Lee ◽  
Nguyen Thi Ha ◽  
Il Soo Um ◽  
Jong-Sup Bae ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Bleeding Time ◽  
Ex Vivo ◽  
Factor Xa ◽  
Inhibitory Effects ◽  
Human Endothelial Cells ◽  
Antithrombotic Activity ◽  
Pharmacological Targets

Thrombin (factor IIa) and factor Xa (FXa) are key enzymes at the junction of the intrinsic and extrinsic coagulation pathways and are the most attractive pharmacological targets for the development of novel anticoagulants. Twenty non-amidino N2-thiophencarbonyl- and N2-tosyl anthranilamides 1-20 and six amidino N2-thiophencarbonyl- and N2-tosylanthranilamides 21-26 were synthesized and evaluated prothrombin time (PT) and activated partial thromboplastin time (aPTT) using human plasma at concentration 30 &mu;g/mL in vitro. From these results, compounds 5, 9, and 21-23 were selected to study the further antithrombotic activity. The anticoagulant properties of 5, 9, and 21-23 significantly exhibited a concentration-dependent prolongation of in vitro PT and aPTT, in vivo bleeding time, and ex vivo clotting time. These compounds concentration-dependently inhibited the activities of thrombin and FXa and inhibited the generation of thrombin and FXa in human endothelial cells. In addition, data showed that 5, 9, and 21-23 significantly inhibited thrombin catalyzed fibrin polymerization and mouse platelet aggregation and inhibited platelet aggregation induced U46619 in vitro and ex vivo. N-(3'-Amidinophenyl)-2-((thiophen-2''-yl)carbonyl amino)benzamide (21) was most active.

Antithrombotic Effect of a Recombinant von Willebrand Factor, VCL, on Nitrogen Laser-Induced Thrombus Formation in Guinea Pig Mesenteric Arteries

1995 ◽  
Vol 73 (02) ◽  
pp. 318-323 ◽  
Author(s):  
K Azzam ◽  
L I Garfinkel ◽  
C Bal dit Sollier ◽  
M Cisse Thiam ◽  
L Drouet
Keyword(s):  
Platelet Aggregation ◽  
Bleeding Time ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Mesenteric Arteries ◽  
Antithrombotic Effects ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryTo assess the antithrombotic effectiveness of blocking the platelet glycoprotein (GP) Ib/IX receptor for von Willebrand factor (vWF), the antiaggregating and antithrombotic effects were studied in guinea pigs using a recombinant fragment of vWF, Leu 504-Lys 728 with a single intrachain disulfide bond linking residues Cys 509-Cys 695. The inhibitory effect of this peptide, named VCL, was tested in vitro on ristocetin- and botrocetin-induced platelet aggregation and compared to the ADP-induced platelet aggregation. In vivo, the antithrombotic effect of VCL was tested in a model of laser-injured mesentery small arteries and correlated to the ex vivo ristocetin-induced platelet aggregation. In this model of laser-induced thrombus formation, five mesenteric arteries were studied in each animal, and the number of recurrent thrombi during 15 min, the time to visualization and time to formation of first thrombus were recorded.In vitro, VCL totally abolished ristocetin- and botrocetin-induced platelet aggregation, but had no effect on ADP-induced platelet aggregation. Ex vivo, VCL (0.5 to 2 mg/kg) administered as a bolus i. v. injection inhibits ristocetin-induced platelet aggregation with a duration of action exceeding 1 h. The maximum inhibition was observed 5 min after injection of VCL and was dose related. The same doses of VCL had no significant effect on platelet count and bleeding time. In vivo, VCL (0.5 to 2 mg/kg) had no effect on the appearance of the thrombi formed but produced dose-dependent inhibition of the mean number of recurrent thrombi (the maximal effect was obtained at 5 min following i. v. injection of the highest dose: 0.8 ± 0.2 thrombi versus 4 ± 0.4 thrombi in controls). The three doses of VCL increased the time in which the first thrombus in a concentration-dependent manner was formed. However, the time to visualize the first thrombus was only prolonged in the higher dose-treated group.These in-vivo studies confirm that VCL induces immediate, potent, and transient antithrombotic effects. Most importantly, this inhibition was achieved without inducing thrombocytopenia nor prolongation of the bleeding time.

scholarly journals Calin from Hirudo medicinalis, an inhibitor of platelet adhesion to collagen, prevents platelet-rich thrombosis in hamsters

Blood ◽  
1995 ◽  
Vol 85 (3) ◽  
pp. 712-719 ◽  
Author(s):  
H Deckmyn ◽  
JM Stassen ◽  
I Vreys ◽  
E Van Houtte ◽  
RT Sawyer ◽  
...  
Keyword(s):  
Platelet Adhesion ◽  
Bleeding Time ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Femoral Vein ◽  
Video Recording ◽  
Hirudo Medicinalis ◽  
Thrombosis Model

Interaction between exposed collagen and platelets and/or von Willebrand factor is believed to be one of the initiating events for thrombus formation at sites of damaged endothelium. Interference with this mechanism may provide an anti-thrombotic potential. Calin, a product from the saliva of the leech Hirudo medicinalis, was tested in vitro and for its in vivo activity in a thrombosis model in hamsters. Calin specifically and dose dependently (IC50:6.5 to 13 micrograms/mL) inhibited human platelet aggregation induced by collagen. In addition, specific platelet adhesion onto microtiter wells coated with collagen and detected with a monoclonal antiglycoprotein IIb/IIIa antibody- conjugated with horseradish peroxidase, could be completely prevented with Calin (IC50:22 micrograms/mL). A dose-response curve was constructed in groups of six hamsters in whom a standardized trauma was induced on the femoral vein. Thrombus formation was followed continuously using video recording and processing of the image obtained upon transillumination of the vessel. Intravenous Calin dose- dependently inhibited platelet-rich thrombus formation in this model with an ED50 of 0.07 mg/kg and complete inhibition with 0.2 mg/kg. No effects were seen on coagulation tests or bleeding times, whereas ex vivo aggregation induced by collagen was inhibited dose dependently. Local application of leech saliva, Calin, hirudin, or the combination of the latter two into the bleeding time wound of hamsters resulted in a mild prolongation of the bleeding time (twofold to threefold). A similar experiment in baboons did not cause any prolongation of the bleeding time. This is in sharp contrast with the long-lasting bleeding after a leech bite itself in both species. Calin from the leech Hirudo medicinalis is able, by binding to collagen, to effectively interfere with platelet-collagen interaction, which results in an antithrombotic effect observed in a platelet-rich thrombosis model in hamsters.

The Impact of NFAT Inhibition on Neutrophil Effector Functions in Patients after Allogeneic Hematopoietic Stem Cell Transplantation and on Neutrophil Antifungal Defense and Myelopoiesis in Cyclosporin Α Treated and NFATc1LysM Mice

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3679-3679
Author(s):  
Daniel Teschner ◽  
Katharina Plein ◽  
Christian Michel ◽  
Steve Pruefer ◽  
Matthias Bros ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Ex Vivo ◽  
Pulmonary Inflammation ◽  
Blood Samples ◽  
Effector Functions ◽  
Myeloid Progenitor ◽  
Healthy Donors ◽  
Myeloid Progenitor Cells

Abstract Background and Aims: Immunosuppressive medication e.g. by calcineurin inhibitors substantially contributes to the risk for opportunistic fungal infections in patients after allogeneic transplantation (HSCT). It is well known that the nuclear factor of activated T cells (NFAT) is an important transcription factor downstream of calcineurin especially in T cells. Additionally, recent data in rodent models indicate that NFAT also seems to play a relevant role in innate antifungal immune responses by polymorphonuclear neutrophils (PMN), as well as in regulation of myelopoiesis and myeloid differentiation. Methods: Firstly, isolated PMN from healthy donors were analyzed in vitro in absence or presence of CsA regarding their effector functions and activation-induced release of inflammatory mediators. Consecutively, blood samples of CsA-treated patients after allogeneic HSCT (n=17) and healthy donors (n=8) were analyzed ex vivo at two different time points as described above. Secondly, we used a murine IPA model (C57BL/6) and treated mice with CsA (18 mg/kg/d) or vehicle and challenged them with Aspergillus fumigatus (A. f.) conidia intratracheally. PMN recruitment to the lungs and pulmonary fungal clearance were examined by analyzing bronchoalveolar lavages (BAL) and peripheral blood (PB) using flow cytometry and cytometric bead array and murine lungs by fungal culture assays and histopathologic examination. Furthermore, survival was studied with neutropenic animals serving as positive controls. Moreover, LysM-specific NFATc1 knockout (NFATc1LysM) mice were bred lacking NFATc1 expression solely in myelomonocytic cells. These animals were also infected with A. f. and analyzed as further mentioned. In addition, we investigated myelopoiesis and myeloid differentiation by quantifying bone marrow derived myeloid progenitor cells from CsA treated or NFATc1LysMmice using flow cytometry and simultaneously counting PMN in PB under steady state conditions. Results: CsA enhanced phagocytosis of PMN in vitro and ex vivo in patients' blood samples (54.2 % +/- 4.1 (patients) vs. 43.8 +/- 1.5, LPS, p=0.006). Moreover, PMNs migratory capabilities were reduced in vitro, whereas other effector functions or release of IL-8 were rather unaffected. PMNs of CsA-treated patients showed increased activation, degranulation and production of inflammatory mediators, but production of ROS was slightly decreased. In our in vivo model, IPA was lethal in neutropenic mice whereas solely CsA or vehicle treated mice survived the infection. CsA treatment resulted in enhanced PMN recruitment in BAL by trend, while pulmonary inflammation and PMN counts in PB remained stable. Indeed, fungal clearance was clearly constrained in CsA　treated animals (2.1 x 105 CFU/lung +/- 0.5 (CsA) vs. 1.7 x 105 +/- 0.2, p<0.005). In our murine knockout model, NFATc1LysM mice infected with A. f. showed unimpaired survival without displaying detectable differences in PMN recruitment or fungal clearance. However, pulmonary inflammation and PMN counts in PB seemed to be more pronounced in knockout mice. Interestingly, BALs of CsA treated mice showed increased levels of IL-6 by trend (4634 pg/mL +/- 1073 (CsA) vs. 3108 +/- 729, p=0.48) but decreased levels of MCP-1 and TNF-α. In contrast, MCP-1, RANTES and TNF-α were enhanced by trend in BALs of NFATc1LysM mice, while IL-6 was reduced compared to wild type controls (3762 pg/mL +/- 729 vs. 4770 +/- 1613, p=0.81). PMN counts in PB were unaffected in NFATc1LysM mice but distribution of bone marrow derived murine myeloid progenitor cells was clearly impaired especially in megakaryocyte-erythroid progenitor cells (1.2 x 105 cells +/- 0.2 (NFATc1LysM) vs. 2.7 +/- 0.6, p=0.015), whereas solely CsA treatment had no influence. Conclusion: Results from our in vitro and ex vivo studies on patients' blood samples as well as from our murine in vivo IPA model indicate that NFAT regulates not only myelopoiesis, but also PMN functionalities in mice and humans. Nevertheless, these interactions are obviously multidimensional and potentially derive from involvement of different pathways. The underlying molecular mechanisms and clinical relevance of our findings in HSCT remain to be determined. Disclosures No relevant conflicts of interest to declare.

A COMPARISON BETWEEN FIBRINOLYSIS OF FRESH AND AGED CLOTS OR THROMBI BY rt-PA IN VIVO, EX VIVO AND IN VITRO

1987 ◽  
Author(s):  
U Nauland ◽  
W Haarmann ◽  
T H Müller ◽  
W G Eisert
Keyword(s):  
Carotid Artery ◽  
Whole Blood ◽  
Jugular Vein ◽  
Ex Vivo ◽  
Clot Retraction ◽  
Therapeutic Applications ◽  
Blood Clots ◽  
Better Than

In view of the therapeutic applications of rt-PA it is of interest to investigate whether there is any difference in the lysability between fresh and aged thrombi. The efficiency of fibrinolysis by rt-PA was studied in 3 different ways: In vivo, by measuring the thrombus weight of fresh (1 h) or aged (24 h) thrombi in the carotid artery of rabbits which had been treated with rt-PA (0.4 mg/kg) or saline for 1 h. Ex vivo, by measuring I125-release of in vivo fresh (1 h) and aged (24 h) thrombi (labelled with I125-fibrinogen) suspended in vitro in plasma containing rt-PA (1 μg/ml) ; the thrombi were formed in the jugular vein and the carotid artery of each rabbit. In vitro, by measuring I125-release of fresh (1 h) and aged (6 or 24 h) human native whole blood clots, PPP-clots, PRP-clots and squeezed PPP-clots which were formed and lysed in vitro with rt-PA (1 μg/ml) . In vivo as well as ex vivo rt-PA lysed fresh (1 h) thrombi much better than aged (24 h) thrombi. This difference was more pronounced immediately after the onset of fibrinolysis, but decreased with time. However, in vitro relatively little difference was observed in fibrinolysis efficiency between fresh (1 h) and aged (6 or 24 h) clots; fibrinolysis of these clots was decreased (PPP > whole blood > PRP) with increasing clot retraction, which was almost complete within 1 h. This result was also confirmed when PPP-clots were “retracted” by simply squeezing them just before lysis. Therefore we conclude that a considerable difference in lysis efficiency between fresh and aged thrombi was only observed when thrombi were formed and aged in vivo. This difference was less pronounced with increasing fibrinolysis time.

scholarly journals Blood vessels guide Schwann cell migration in the adult demyelinated CNS through Eph/ephrin signaling

2018 ◽  
Author(s):  
Beatriz Garcia-Diaz ◽  
Corinne Bachelin ◽  
Fanny Coulpier ◽  
Gaspard Gerschenfeld ◽  
Cyrille Deboux ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Blood Vessel ◽  
Ex Vivo ◽  
Migration Route ◽  
Eph Receptors ◽  
The Central Nervous System ◽  
Vessel Network ◽  
First Time

ABSTRACTSchwann cells (SC) enter the central nervous system (CNS) in pathophysiological conditions. However, how SC invade the CNS to remyelinate central axons remains undetermined. We studied SC migratory behaviorex vivoandin vivoafter exogenous transplantation in the demyelinated spinal cord. Data highlight for the first time that SC migrate preferentially along blood vessel in perivascular ECM, avoiding CNS myelin. We demonstratein vitroandin vivothat this migration route occurs by virtue of a dual mode of action of Eph/ephrin receptor. Indeed, EphrinB3, enriched in myelin, interacts with SC Eph receptors, to drive SC away from CNS myelin, and triggers their preferential adhesion to ECM components, such as fibronectin via integrinβ1 interactions. This complex interplay enhances SC migration along the blood vessel network and together with lesion-induced vascular remodeling facilitates their timely invasion of the lesion site. These novel findings elucidate the mechanism by which SC invade and contribute to spinal cord repair.

scholarly journals Drug Interactions of Newer Oral Anticoagulants Dabigatran, Rivaroxaban, and Apixaban with Routinely Used Nonanticoagulant/Antiplatelet Drugs

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4267-4267 ◽  
Author(s):  
Shermin Sayani ◽  
Omer Iqbal ◽  
Debra Hoppensteadt ◽  
Jawed Fareed
Keyword(s):  
Platelet Aggregation ◽  
Tranexamic Acid ◽  
Oral Anticoagulant ◽  
Bleeding Time ◽  
Ex Vivo ◽  
Fixed Concentration ◽  
Anticoagulant Drugs ◽  
Rat Tail

Abstract Introduction: The newer non-vitamin K antagonist oral anticoagulant drugs (NOACs) such as dabigatran, apixaban and rivaroxaban are now commonly used for various indications in a large group of patients who are simultaneously managed with several other routinely used drugs. Given the lack of available information on the interaction of newer oral anticoagulant drugs (NOACs) with commonly used non-anticoagulants / anti-platelet drugs, it is important to recognize the impact of these interactions on the safety and efficacy of these agents. We hypothesized that some of the commonly used drugs may modulate the anticoagulant effects of NOACs. This study aims to determine the antiplatelet, anticoagulant, and bleeding effects of the NOACs at varying concentrations with and without routinely used drugs both in the in vivo and in vitro systems. Materials and Methods:Dabigatran (Boehringer Ingelheim, Ridgefield, CT), rivaroxaban (Janssen Pharmaceuticals, Inc., Titusville, NJ), and apixaban (Bristol-Myers Squibb Company, Princeton, NJ and Pfizer Inc., New York, NY); and such routinely used drugs as alendronate sodium, chondroitin sulfate, hydrocodone-acetaminophen, klonopin, penicillin, tacrolimus, tramadol chlorhydrate, and tranexamic acid were commercially obtained and supplemented in citrated plasma at projected therapeutic ranges. Such tests as PT, APTT, dRVVT, TT, Heptest, and Anti- Xa and anti-IIa tests were performed. Agonist induced platelet aggregation studies using ADP, AA, Collagen, Epinephrine, and Thrombin agonists were performed on the Platelet Aggregation Profiler- 8 (PAP-8) (Biodata corporation, Horsham, PA) with dabigatran, apixaban and rivaroxaban alone and with the routinely used drugs. For the in-vivo bleeding studies a model of rat tail transection was used, following ketamine and xylazine anesthesia, 6-8 weeks old male Sprague-Dawley rats weighing 250-300g (n=15) were used to perform the rat tail transection bleeding time using dabigatran alone and dabigatran followed by tranexamic acid. Blood was drawn by cardiac puncture for ex vivo analysis. The collected data from the bleeding and ex vivo studies were tabulated and statistically analyzed using ANOVA. Results: In the in vitro studies, all of the NOACs produced assay dependant anticoagulant and antiprotease effects. Rivaroxaban and apixaban did not exhibit any interactions at the projected therapeutic dosage range when combined with any of the routinely used drugs. However dabigatran at a fixed concentration of 1 µg/ml combined with the commonly used drugs at a fixed concentration of 0.1 µg /ml or 1 µg/ml produced augmented assay-dependent anticoagulant and antiprotease activity. The most pronounced interaction was noticed with tacrolimus (111% difference in PT, 231% difference in APTT, and 46% difference in anti-IIa assay), followed by tramadol (57% difference in PT and 54% difference in Anti-IIa assay). Platelet Aggregation studies revealed no modulation of antiplatelet effects (<10%) with the addition of the commonly used drugs and the NOACs. In the rat tail transection bleeding model, there was a significant difference (p=0.03, α=0.05) between the bleeding time with dabigatran (100 µg/kg) alone (13.1 ±1.5 minutes) intravenously compared to dabigatran with tranexamic acid (10 mg/kg) (10.3 ±1.8 minutes) in each study. Ex-vivo analysis showed a reduction in PT and Heptest assay responses with dabigatran and tranexamic acid by 38% and 80%, respectively, and minimal change (5%) in APTT. Conclusion: In contrast to rivaroxban and apixaban in vitro, dabigatran exhibited stronger interactions with the commonly used drugs and variable assay dependent augmentation of anticoagulant and antiprotease responses. Tacrolimus and tramadol showed the strongest interactions. Agonist induced platelet aggregation studies did not show any interactions. Interestingly, tranexamic acid reduced the anticoagulant effect of dabigatran in the in vivo and ex vivo studies. These results warrant a review of post-marketing surveillance on the reported bleeding in patients concomitantly treated with NOACs and the reported routinely used drugs. Furthermore, these observations underscore the need to screen other commonly used drugs and supplements for their potential interactions with NOACs. Disclosures No relevant conflicts of interest to declare.

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