Novel Strategy of Gene Delivery System Based on Dendrimer Loaded Recombinant Hirudine Plasmid for Thrombus Targeting Therapy.

This study proposed a new nonviral gene delivery system for thrombus targeting therapy based on PEGlyation polyamides dendrimer (PAMAM) modified with RGDyC to condense the pDNA with recombinant hirudine (rHV) gene (RGDyC-rHV-EGFP). The RGDyC-mPEG-PAMAM was synthesized and characterized by 1H NMR, PAMAM/pDNA was characterized by particle size, zeta potential, cellular uptake, and gel retraction assay. The transfection was carried out between lipofectamine 2000 and PAMAM/pDNA on HUVEC cells at various N/P ratios. The antithrombotic effect in vivo was evaluated by venous thrombosis model on Wistar rats. It showed that the drug delivery system of RGDyC modified PAMMA, which entrapped pDNA could significantly improve the transfection efficiency. It was about 7.56-times higher than that of lipofectamine 2000. In addition, the expression level of hirudine fusion protein was the highest at N/P ratio of 0.5. The results of antithrombotic effect showed that the weight of thrombus was reduced in RGDyC modified group; compared with heparin group, there was no significant difference ( P > 0.05). Overall, we take the advantage of the unique advantages of hirudine, combining the genetic engineering, nanocarriers, and targeting technology, to achieve the targeted enrichment and activation the hirudine fusion protein in the thrombus site, to improve the concentration of drugs in the thrombus site, finally increasing the curative effect and reduce the risk of bleeding. The strategy of gene delivery system holds unique properties as a gene delivery system and has great promises in thrombus targeting therapy.

Prognostic Value of Red Blood Cell Distribution Width on Bleeding Events in Nonvalvular Atrial Fibrillation Patients Taking Dabigatran (110 mg b.i.d.) after Catheter Ablation.

OBJECTIVES: Recent research has indicated that red blood cell distribution width (RDW) is associated with the prognosis of cardiovascular diseases such as chronic heart failure and coronary heart disease. We aimed to study the predictive value of RDW for bleeding events in patients with nonvalvular atrial fibrillation (NVAF) during the administration of 110 mg of dabigatran twice a day after catheter ablation. METHODS: One hundred and seventy-two NVAF patients who were hospitalized and received catheter ablation in Jiangsu Provincial People's Hospital from January 2014 to January 2015 were enrolled (110 mg of dabigatran was administered orally to outpatients preoperatively twice a day for 3 weeks). The enrolled patients were divided into the high RDW (>12.8%) group (n = 85) and the low RDW (≤12.8%) group (n = 87) according to the median RDW. The activated partial thromboplastin time (APTT) at dabigatran trough concentration was also detected. Patients were followed up for 3 months to observe the occurrence of bleeding events, and the predictive value of RDW as well as APTT for bleeding events was assessed from receiver-operating characteristic (ROC) analyses. RESULTS: In all patients, preoperatively, no bleeding events were observed and the APTT did not exceed twice the normal upper limit. Thirteen cases of bleeding events, all minor bleeding, occurred after a 3-month follow-up: 3 of gingival bleeding, 3 of urinary tract bleeding, 3 conjunctival hemorrhages and 4 subcutaneous hemorrhages. The incidence of bleeding events in the low RDW group was lower than in the high RDW group (3.4 vs. 11.8%, p = 0.039). The areas under the ROC curve for RDW and APTT to predict the occurrence of bleeding events were 0.737 (cutoff point 13.25%; p < 0.05) and 0.558 (p > 0.05), respectively. CONCLUSION: RDW was associated with the occurrence of bleeding events in NVAF patients on dabigatran (110 mg twice a day) after ablation, while also being an independent predictor of bleeding events. RDW had superior predictive value for bleeding events over APTT when APTT did not exceed twice the normal upper limit.

Management of Heparin-Resistant Patients with Benefits? Maximizing Biocompatibility in Cardiopulmonary Bypass: Combining ATryn® Recombinant Antithrombin III and Carmeda® Heparin-Bonded Perfusion Circuits: A Case Series.

As many as 25% of our cardiopulmonary bypass (CPB) patients have a diminished heparin response and fail to reach a therapeutic activated clotting time (ACT). We treat a majority of these patients with antithrombin III (ATryn®, recombinant antithrombin III [rhAT], rEVO Biologics). Our current CPB circuit uses Medtronic Carmeda® coating. We observed less post-operative bleeding in a number of patients treated with rhAT. We theorized that adding rhAT would allow patients with diminished heparin response to safely achieve a therapeutic ACT. On the basis of our postoperative bleeding observations, we wondered if using rhAT with a heparin-bonded CPB circuit enhanced its biocompatibility and perhaps improved patient outcomes. Data were collected on 15 patients undergoing CPB who received antithrombin III (AT) replacement therapy for diminished heparin response. We used patient data from 2012, prior to rhAT usage for comparison. All patients achieved therapeutic ACT after rhAT administration. We also observed decreased postoperative atrial fibrillation rates, improved platelet preservation, decreased intensive care unit and ventilator times in patients receiving rhAT compared to rates commonly observed at our center. Heparin-resistant patients can be treated with rhAT to achieve therapeutic ACTs. Our observations suggest that the use of rhAT in conjunction with Carmeda® heparin-bonded circuits may also have a positive benefit on some of the well-established negative clinical consequences of CPB and improve patient outcomes.

Managing dentoalveolar surgical procedures in patients taking new oral anticoagulants.

The development of new orally administered anticoagulants, such as dabigatran, rivaroxaban, and apixaban, in the past few years has focused on avoiding some of the drawbacks associated with warfarin. This work aims to illustrate the main features of the most commonly used new oral anticoagulants, reviewing the current literature on the management of patients taking these drugs and needing oral and implant surgery, and discussing the currently proposed related guidelines.

Clot-Targeted Nanogels for Dual-Delivery of AntithrombinIII and Tissue Plasminogen Activator to Mitigate Disseminated Intravascular Coagulation Complications.

Disseminated intravascular coagulation (DIC) is a pathologic state that follows systemic injury and other diseases. Often a complication of sepsis or trauma, DIC causes coagulopathy associated with paradoxical thrombosis and hemorrhage. DIC upregulates the thrombotic pathways while simultaneously downregulating the fibrinolytic pathways that cause excessive fibrin deposition, microcirculatory thrombosis, multiorgan dysfunction, and consumptive coagulopathy with excessive bleeding. Given these opposing disease phenotypes, DIC management is challenging and includes treating the underlying disease and managing the coagulopathy. Currently, no therapies are approved for DIC. We have developed clot-targeted therapeutics that inhibit clot polymerization and activate clot fibrinolysis to manage DIC. We hypothesize that delivering both an anticoagulant and a fibrinolytic agent directly to clots will inhibit active clot polymerization while also breaking up pre-existing clots; therefore, reversing consumptive coagulopathy and restoring hemostatic balance. To test this hypothesis, we single- and dual-loaded fibrin-specific nanogels (FSNs) with antithrombinIII (ATIII) and/or tissue plasminogen activator (tPA) and evaluated their clot preventing and clot lysing abilities in vitro and in a rodent model of DIC. In vivo, single-loaded ATIII-FSNs decreased fibrin deposits in DIC organs and reduced blood loss when DIC rodents were injured. We also observed that the addition of tPA in dual-loaded ATIII-tPA-FSNs intensified the antithrombotic and fibrinolytic mechanisms, which proved advantageous for clot lysis and restoring platelet counts. However, the addition of tPA may have hindered wound healing capabilities when an injury was introduced. Our data supports the benefits of delivering both anticoagulants and fibrinolytic agents directly to clots to reduce the fibrin load and restore hemostatic balance in DIC.

Implications of Dabigatran, a direct thrombin inhibitor, for oral surgery practice.

Direct thrombin inhibitors, specifically orally administered dabigatran etexilate, are emerging as alternatives to warfarin for anticoagulation in the management of atrial fibrillation and venous thromboembolism. The risk associated with bleeding events while taking dabigatran has been documented in multiple randomized controlled trials, but to date, no studies have focused on the risk of bleeding after dental extraction. Extraction of teeth is one of the most common surgical procedures and may cause significant bleeding, so a thorough understanding of the pharmacology of anticoagulant medications is required to prevent complications. With the increasing use of direct thrombin inhibitors, the safe management of patients taking these anticoagulants must be delineated. This review compares dabigatran and warfarin, especially in terms of their effects on dental and oral surgery practice, and examines best management of these patients in light of the existing literature.

A semi-mechanistic modeling strategy to link in vitro and in vivo drug release for modified release formulations.

PURPOSE: To develop a semi-mechanistic model linking in vitro to in vivo drug release. METHODS: A nonlinear mixed-effects model describing the in vitro drug release for 6 hydrophilic matrix based modified release formulations across different experimental conditions (pH, rotation speed and ionic strength) was developed. It was applied to in vivo observations of drug release and tablet gastro intestinal (GI) position assessed with magnetic marker monitoring (MMM). By combining the MMM observations with literature information on pH and ionic strength along the GI tract, the mechanical stress in different parts of the GI tract could be estimated in units equivalent to rotation speed in the in vitro USP 2 apparatus. RESULTS: The mechanical stress in the upper and lower stomach was estimated to 94 and 134 rpm, respectively. For the small intestine and colon the estimates of mechanical stress was 93 and 38 rpm. Predictions of in vivo drug release including between subject/tablet variability was made for other newly developed formulations based on the drug release model and a model describing tablet GI transit. CONCLUSION: The paper outlines a modeling approach for predicting in vivo behavior from standard in vitro experiments and support formulation development and quality control.

Antithrombin supplementation did not impact the incidence of pegylated asparaginase-induced venous thromboembolism in adults with acute lymphoblastic leukemia.

Pegylated asparaginase (PEG-Asp), a key component in the treatment of acute lymphoblastic leukemia (ALL), is associated with coagulopathy and an increased risk of venous thromboembolism (VTE). PEG-Asp also lowers antithrombin (AT) levels. Between April 2014 and October 2017, 75 adult ALL patients were identified to have received at least one dose of PEG-Asp. Patients were assigned to the AT group if a physician monitored AT levels with an intention to correct low AT levels (<60%). Incidence of VTE was not significantly different, with 17% (8/47) of the AT patients and 11% (3/28) of the control patients experiencing a VTE event (p = .52). The occurrence of coagulopathies was not significantly different. Within the AT group, 37 patients (78%) received AT, and median AT% prior to supplementation was 49%. The median number of AT doses received was 2 (range 0-12) and the mean cost of AT per patient was $11,847.

A pharmacotherapy review of the novel, oral antithrombotics.

Coagulation disorders account for a high incidence of death in the U.S. due to stroke, myocardial infarction, and venous thromboembolism. In the past few years, numerous agents have been brought to market for the treatment of thromboembolism or prevention of thromboembolism. Similar to warfarin, these agents can cause bleeding disorders, which may exacerbate dental care treatment plans. This literature review examines the newer agents for the treatment of thromboembolism disorders, common side effects and drug interactions, the specific medical conditions each agent treats, and the dental perspective on how to medically management patients prescribed these medications.

An evidence summary of the management of patients taking direct oral anticoagulants (DOACs) undergoing dental surgery.

Direct oral anticoagulant (DOAC) drugs (dabigatran, rivaroxaban, and apixaban) have emerged in the last decade to overcome some of the drawbacks of existing medications. To date, little is known about the dental management of patients taking these drugs. This study was undertaken to establish the evidence for the management of patients undergoing dental procedures while taking these medications. A rapid review approach was used to identify clinical and scientific research related to dental surgery performed in patients taking DOACs in order to produce an evidence summary. The rapid review did not identify any systematic reviews or original clinical trials and the overall quality of evidence found was poor. Most of the literature consisted of non-structured review articles and guidance documents based on assumptions from non-dental data and expert opinion, and recommendations on best practice varied throughout. The findings from the review of the literature varied considerably. Currently, recommendations are based on poor quality scientific data and clinical trials are required to establish best evidence-based practice guidance.

Warfarin and Newer Agents: What the Oral Surgeon Needs to Know.

The new direct oral anticoagulants-dabigatran etexilate, rivaroxaban, and apixaban- have predictable pharmacokinetic and pharmacodynamic profiles and are alternatives to warfarin. However, many surgeons are wary of these drugs, as there is limited evidence on how to manage bleeding in patients taking them, and only recently has a specific antidote been developed to reverse their anticoagulant effect. Management of the newer agents requires careful adherence to primary measures of bleeding care, knowledge of their mechanism of action, and familiarity with the unapproved and untested reversal strategies that may be required in patients with life-threatening bleeding.

Inhibition of neointima formation after experimental coronary artery stenting: a new biodegradable stent coating releasing hirudin and the prostacyclin analogue iloprost.

BACKGROUND: To minimize acute stent thrombosis and development of restenosis, stents coated with biodegradable and nonbiodegradable polymers have been proposed to serve as sustained-release drug carriers. METHODS AND RESULTS: In both a sheep and a pig model, we examined the vascular response to standard and high-pressure implantation of coronary Palmaz-Schatz stents coated with a 10-microm layer of polylactic acid (MW 30 kDa) releasing recombinant polyethylene glycol (r-PEG)-hirudin and the prostacyclin analogue iloprost, both drugs with antithrombotic and potentially antiproliferative effects. Study observation time was 28 days. Between the corresponding stent groups, no differences were observed with regard to preplacement and postplacement implantation parameters. The morphometric analysis demonstrated that the coating was associated with a greater lumen diameter through a reduction in the mean restenosis area by 22.9% (P<0.02) in the standard-pressure model (sheep) and by 24.8% (P<0.02) in the overstretch pig model compared with uncoated control stents without inducing a local inflammatory response. CONCLUSIONS: The results from this study demonstrate beneficial effects of a polymeric stent coating with polylactic acid releasing r-PEG-hirudin and iloprost on the development of restenosis after coronary stent placement at 4 weeks, independent of the extent of vascular injury. Future studies are proposed to investigate the integration of other substances to further enhance the potential of the stent coating on reducing neointimal formation.

Local delivery of a synthetic antithrombin with a hydrogel-coated angioplasty balloon catheter inhibits platelet-dependent thrombosis.

OBJECTIVES: This study evaluated the efficacy of local administration of an antithrombin agent with a hydrogel-coated percutaneous transluminal coronary angioplasty balloon catheter. BACKGROUND: Intravenous infusion of antithrombin compounds has been shown to inhibit platelet-dependent thrombosis. However, hemorrhage is a common side effect associated with the systemic administration of antithrombin compounds. METHODS: The potent, irreversible thrombin inhibitor D-Phe-L-Pro-L-Arginyl chloromethyl ketone (PPACK) was used to inhibit thrombus formation in chronic porcine arteriovenous shunts. Platelet deposition was quantitated with gamma camera imaging of 111In-labeled platelets. RESULTS: Intravenous administration of PPACK in swine, in doses sufficient to maximally inhibit thrombus formation, was associated with prolongation of bleeding parameters. The inhibition of thrombosis associated with intravenous PPACK was dose related. The amount of intravenous PPACK necessary for maximal inhibition of thrombus formation for a period of 45 min was 16.9 mg. In contrast, local delivery of PPACK with a hydrogel-coated angioplasty balloon deployed at the site of the thrombus inhibited platelet deposition for at least 45 min after the balloon was removed. Using 3H-labeled PPACK, the calculated amount of PPACK delivered was 33.5 micrograms. There was no change in bleeding time or activated partial thromboplastin time when swine received an intravenous bolus greater than the total amount of PPACK adsorbed onto the balloon (70 micrograms). CONCLUSIONS: These results suggest that in this model, a hydrogel-coated coronary angioplasty balloon catheter can be used to deliver enough antithrombin agent to inhibit platelet-dependent thrombosis for at least 45 min at doses that are several orders of magnitude less than those required for systemic administration. In addition, local delivery can provide effective inhibition of thrombus formation without alteration of bleeding parameters.

Antithrombogenic coating of stents using a biodegradable drug delivery technology.

To reduce the thrombogenic properties of coronary artery stents, a biodegradable polylactic acid (PLA) stent coating with an incorporated thrombin inhibitor and a platelet aggregation inhibitor has been developed. In an ex vivo human stasis model, its effect on platelets, plasmatic coagulation and its release characteristics were studied using whole blood. Bare steel and bare gold-surface stents were compared to steel and gold-surface stents coated with PLA (30 kDa) containing 5% polyethyleneglycol (PEG)-hirudin and 1% iloprost, with an empty tube as control. Markers of activated coagulation (prothrombin fragment F1-2 and thrombin-antithrombin III complex, TAT), were assayed and the release of drugs from the coating was assessed by aPTT and collagen-induced platelet aggregation. Bare steel and gold stents were completely covered by a blood clot, and high levels of coagulation markers (F1-2 fragment and TAT) were detected. No differences in the thrombogenic properties were found between bare gold or steel stents. Coated stents were free of blood clots and only minor elevations of markers were detected. Release data from in-vitro studies over 90 days showed a gradual release of the drugs with an initial exponential release characteristic for PEG-hirudin, slow release of iloprost and a 10% degradation of the PLA carrier. This drug releasing biodegradable coating effectively reduced thrombus formation independent of the metallic surface.

Surface modification of polyethylene balloon catheters for local drug delivery.

Local drug delivery is an attractive approach to the associated problems of percutaneous transluminal coronary angioplasty (PTCA), including arterial injury. The objective of the present research was to deliver a high concentration of a potent anti-thrombin agent, argatroban (ARG), to the vessel wall in order to reduce arterial injury. Local delivery was accomplished by the ionic attachment of drug particles to a modified balloon surface. Surface graft polymerization of ionic monomers to a high-density poly(ethylene) (PE) substrate was performed utilizing ultra-violet (UV) methods. Acrylic acid (AAc) and 2(dimethylamino) ethyl methacrylate (DMAEMA) were successfully grafted onto PE surfaces. Surface grafting was verified by contact angle, X-ray photoelectron spectroscopy, and zeta potential measurements. The amount of ARG adsorbed onto the modified PE surface was highly dependent on the pH of the drug media for both anionic and cationic grafted monomers. The efficacy of local drug delivery to the arterial wall was analyzed using drug-immobilized PE balloon catheters in the rabbit common carotid artery model. High concentrations of ARG (280 nmol/g tissue) were found within the ballooned arterial segment immediately after angioplasty, followed by a decrease after blood flow was restored.

Argatroban in adult extracorporeal membrane oxygenation.

This case report addresses the use of Argatroban, an anticoagulant and thrombin inhibitor for treatment of thrombocytopenia in an adult patient on extracorporeal membrane oxygenation (ECMO). After 5 days on ECMO, the patient showed signs of heparin-induced thrombocytopenia (HIT) with a platelet count of 20K. Argatroban was initiated to decrease progression of HIT and continue treatment with ECMO proved to be successful. Given the occurrence of HIT with heparin therapy, a need for alternate drug therapy is required for patients requiring treatment with ECMO. The use of Argatroban in adult ECMO is outlined and includes dosage, monitoring, and patient treatment.

[Prevention of postoperative thromboembolism in Denmark. A questionnaire study on principles of prevention of thrombosis at hospital surgery departments]. / Forebyggelse af postoperativ tromboemboli i Danmark. Spørgeskemaundersøgelse vedrørende principper for anvendelsen af tromboseprofylakse ved kirurgisk arbejdende sygehusafdelinger.

In a questionnaire survey among surgical hospital departments in Denmark (1990), we assessed the attitudes and practices of use of postoperative thrombosis prophylaxis (TP). Replies were obtained from 92% of departments. Otological and odontological departments seldom used TP. Among departments performing major surgery a total of 88% used TP routinely (general surgery: 91%, orthopedic surgery: 94%, gynecology: 84%). 68% of these departments used TP according to written instructions. Indications for TP, and methods used, are in accordance with the literature, except for the use of TP in emergency surgery (routine in 36% of departments only), or for the continued use of aspirin for TP in 13% of departments.

The effect of a polyurethane coating incorporating both a thrombin inhibitor and nitric oxide on hemocompatibility in extracorporeal circulation.

Nitric oxide (NO) releasing (NORel) materials have been extensively investigated to create localized increases in NO concentration by the proton driven diazeniumdiolate-containing polymer coatings and demonstrated to improve extracorporeal circulation (ECC) hemocompatibility. In this work, the NORel polymeric coating composed of a diazeniumdiolated dibutylhexanediamine (DBHD-N2O2)-containing hydrophobic Elast-eon™ (E2As) polyurethane was combined with a direct thrombin inhibitor, argatroban (AG), and evaluated in a 4 h rabbit thrombogenicity model without systemic anticoagulation. In addition, the immobilizing of argatroban to E2As polymer was achieved by either a polyethylene glycol-containing (PEGDI) or hexane methylene (HMDI) diisocyanate linker. The combined polymer film was coated on the inner walls of ECC circuits to yield significantly reduced ECC thrombus formation compared to argatroban alone ECC control after 4 h blood exposure (0.6 ± 0.1 AG/HMDI/NORel vs 1.7 ± 0.2 cm(2) AG/HMDI control). Platelet count (2.8 ± 0.3 AG/HMDI/NORel vs 1.9 ± 0.1 × 10(8)/ml AG/HMDI control) and plasma fibrinogen levels were preserved after 4 h blood exposure with both the NORel/argatroban combination and the AG/HMDI control group compared to baseline. Platelet function as measured by aggregometry remained near normal in both the AG/HMDI/NORel (63 ± 5%) and AG/HMDI control (58 ± 7%) groups after 3 h compared to baseline (77 ± 1%). Platelet P-selectin mean fluorescence intensity (MFI) as measured by flow cytometry also remained near baseline levels after 4 h on ECC to ex vivo collagen stimulation (16 ± 3 AG/HMDI/NORel vs 11 ± 2 MFI baseline). These results suggest that the combined AG/HMDI/NORel polymer coating preserves platelets in blood exposure to ECCs to a better degree than AG/PEGDI/NORel, NORel alone or AG alone. These combined antithrombin, NO-mediated antiplatelet effects were shown to improve thromboresistance of the AG/HMDI/NORel polymer-coated ECCs and move potential nonthrombogenic polymers closer to mimicking vascular endothelium.

Poly(lactic-co-glycolic) acid microspheres encapsulated in Pluronic F-127 prolong hirudin delivery and improve functional recovery from a demyelination lesion.

Components of the blood have been proposed as potential therapeutic targets for improving cellular regeneration after injury and neurodegenerative disease. In this work, thrombin is shown to increase endogenous neural progenitor proliferation in the intact murine spinal cord. A local injection of heparin before a spinal cord injury reduces cell proliferation and astrogliogenesis associated with scarring. We sought to create depot-formulations of PLGA microsphere and Pluronic F-127 for sustained local delivery of two thrombin inhibitors, heparin and hirudin. Each hydrogel depot-formulation showed delayed drug release compared to microspheres or hydrogel alone. Animals with a lateral demyelination lesion showed a reduction in CD68+ macrophages when treated with hirudin-loaded PLGA/F-127 gels compared to control and heparin-treated animals. Moreover, hirudin-loaded materials showed an accelerated recovery in coordinated stepping and increased oligodendrocyte densities. Together, these data demonstrate that controlled delivery of hirudin accelerates functional recovery from a demyelination lesion in the spinal cord.