Plasma Clotting Time Research Articles

Hemocompatibility of Silver Nanoparticles Based on Conjugate of Quaternized Chitosan with Gallic Acid in In Vitro Experiments.

We studied hemocompatibility of silver nanoparticles synthesized on the basis of a conjugate of quaternized chitosan with gallic acid (QChit-Gal). For the three variants of silver particles (Nos. 1, 2, and 3), the QChit-Gal:AgNO3 ratio was 5:1, 5:3, and 1:1, respectively. Anticoagulant activity of all samples of silver nanoparticles was lower than that of the conjugate. Samples of nanoparticles Nos. 1 and 2 in a concentration of 0.0233 mg/ml did not affect plasma clotting time and can be used for intravenous administration. However, their concentration in the blood should not exceed 0.01 mg/ml, because in this concentration they do not affect erythrocyte membrane, do not induce platelet aggregation, and do not affect platelet aggregation induced by ADP.

Prothrombin Is Responsible for the Lupus Cofactor Phenomenon in a Patient with Lupus Anticoagulant/Hypoprothrombinemia Syndrome.

Lupus anticoagulant is a misnomer as it is commonly associated with thromboembolic events. In few cases, the name retains its literal meaning when it characterizes patients with a bleeding disorder. We describe a patient with lupus anticoagulant, hypoprothrombinemia, and major bleeding (lupus anticoagulant/hypoprothrombinemia syndrome). Immunological studies revealed a huge amount of circulating monoclonal immunoglobulin M lambda (IgMλ) antiphosphatidylserine/prothrombin antibodies (14,400 U/mL). Affinity purified monoclonal antibodies (440 U/mL) prolonged the coagulation time of normal plasma by 12.2 seconds (diluted Russell viper venom time) and 25.5 seconds (silica clotting time). The original patient's plasma mixed 1:1 with normal plasma showed a marked prolongation of coagulation times (lupus cofactor) from a ratio of 2.94 to 5.23 in diluted Russel viper venom time and from 2.30 to 3.00 using the silica clotting time. Human prothrombin added to original patient's plasma caused a marked prolongation of coagulation times in diluted Russell viper venom test thus unequivocally explaining the lupus cofactor phenomenon. In conclusion, we have shown that lupus anticoagulant/hypoprothrombinemia syndrome is attributable to monoclonal IgMλ antibodies directed to phosphatidylserine/prothrombin and that prothrombin is the protein responsible for the observed lupus cofactor phenomenon.

Protein structure of the venom in nine species of snake: from bio-compounds to possible healing agents.

Due to its various structures in bio-compounds, snake venom is the indisputable result of evolutionary stages of molecules with an increasingly complex structure, high specificity, and of great importance for medicine because of their potential. The present study proposed an underpinning examination of venom composition from nine species of venomous snakes using a useful and replicable methodology. The objective was the extension of the evaluation of protein fractions in the field up to 230 kDa to permit possible identification of some fractions that are insufficiently studied. The gel capillary electrophoresis method on the chip was performed using an Agilent 2100 bioassay with the 80 and 230-LabChip Protein kits. Interpretation of electrophoresis was performed using the Protein 2100 expert (Agilent) test software as follows: a) Protein 80 (peak size scale): 1.60, 3.5, 6.50, 15.00, 28.00, 46.00, 63.00, 95.00 kDa; b) Protein 230 (peak size scale): 4.50, 7.00, 15.00, 28.00, 46.00, 63.00, 95.00, 150.00, 240.00 kDa. The screening revealed the presence of compounds with a molecular weight greater than 80 kDa, in the case of Vipera aspis and Vipera xantina palestinae. For V. aspis, a 125 kDa molecular weight pro-coagulant protein was identified, known as being involved in the reduction of plasma clotting time without any direct activity in the fibrinogen coagulation process. The samples examined on the Protein 230-LabChip electrophoresis chip can be considered as a novelty with possible uses in medicine, requiring further approaches by advanced proteomics techniques to confirm the intimate structural features and biological properties of snake venoms.

Per oral rat treatment with glyconjugate fractions of Genipa americana leaves protects thrombus formation.

: The current study evaluated the effect of the arabinogalactan-glycoconjugate fractions (FI and FII) isolated from Genipa americana leaves given per oral in rat hemostasis protocols. Rats received daily treatment with FI or FII during 7 days and were evaluated for coagulation, platelet aggregation, venous thrombosis and bleeding tendency 1 h after the last treatment. FII prolonged in 5.5-fold the rat plasma coagulation time (activated partial thromboplastin time test). FI inhibited by 46% the platelet aggregation. Both FI and FII prevented thrombus formation by 33 and 28%, respectively. However, the bleeding time was not altered by any fractions, showing an advantage in relation to acetylsalicylic acid or warfarin that increased the bleeding time in 3.6 and 2.9-fold, respectively. Per oral treatment with the arabinogalactan-glyconjugate fractions FI and FII of G. americana leaves in rats prevents thrombus formation, being devoid of hemorrhagic risk. These results bring novel therapeutic possibilities for thromboembolic diseases.

Влияние аргининсодержащих пептидов на антикоагулянтную и фибринолитическую активность плазмы крови крыс в условиях <i>in vitro</i>

Исследовано действие олигопептидов глипролинового ряда — His-Phe-Arg-Trp-Pro-Gly-Pro (АКТГ(6-9)PGP), Thr-Lys-Pro-Arg-Pro-Gly-Pro (селанк) и Pro-Gly-Pro (PGP) – в различных концентрациях от 1 до 10 – 5 мг/мл в условиях in vitro на плазму крови крыс посредством тестов АЧТВ (активированное частичное тромбопластиновое время) и определения пластин нестабилизированного фибрина. Удлинение времени свертывания плазмы (по тесту АЧТВ) от 158 до 210 % в зависимости от концентрации пептидов при p < 0,01 указывает на гипокоагуляцию под действием АКТГ(6-9)PGP и селанка. Выявлены суммарные фибринолитические эффекты пептидов в концентрациях от 1 до 10–5 мг/мл методом измерения зоны лизиса на пластинах нестабилизированного фибрина в норме и под влиянием пептидов. Наибольшей фибриндеполимеризационной активностью по данным суммарной фибринолитической активности обладал пептид АКТГ(6-9)PGP от 117 до 148 % в концентрациях от 10–3 до 1 мг/мл при p < 0,05.

Preliminary study of thrombogenicity induced by the nanoparticle surface coating of intracranial stents

Endovascular treatment of intracranial aneurysms with intracranial stents was proven to be clinically safe and effective, but is still associated with a risk of thromboembolic complications. Stent thrombosis could be a sever complication associated with specific stent surface coatings and designs. Standardized in vitro tests for investigation of thrombogenicity induced by different nanomaterials were used as the basic method in carrying out the present study. Therefore, the aim of this study was to evaluate the thrombogenicity of three different nanomaterials (ZnO, TiO2 si Fe3O4) possible used as surface coating for intracranial stents. This study is based on a procedure for in vitro analyses of plasma coagulation time. To measure the plasma coagulation time, platelet-poor plasma from human whole blood was in vitro exposed to nanoparticles and analysed in prothrombin (PT) and activated partial thromboplastin (APTT).

Design of a thrombin inhibitory staphylokinase based plasminogen activator with anti-reocclusion potential

In the quest of a therapeutically improved thrombolytic drug, we designed and expressed a Staphylokinase based, recombinant fusion protein with fibrin specific clot lysis and anti-thrombin activities derived from human thrombomodulin, a transmembrane glycoprotein with anticoagulant properties, known to form a complex with thrombin and then activating Protein C. The fusion construct was expressed in the yeast Pichia pastoris for cost effective and facile production. The purified construct had comparable plasminogen activation and clot lysis capability as compared to native SAK in that it showed plasmin dependent Plasminogen activation, but exhibited significantly lower fibrinogenolysis (an indicator of fibrin-specific action) even at much higher doses than SAK. In addition, its successful activation of the thrombin-mediated Protein C anticoagulant pathway was reflected in increased in vitro plasma clotting time, as well as inhibition of clot bound thrombin, which led to nearly 15–25% lesser re-formation of fibrin clots after initial successful clot lysis in a specially designed in vitro assay for re-occlusion. Thus, this novel chimeric protein could be of high therapeutic potential as a thrombolytic drug with enhanced fibrin specific clot dissolving properties along with lower bleeding and re-thrombosis complications- a dire need today, especially in the treatment of thrombotic strokes.

Potency Equated Porcine and Bovine Mucosal Heparin Are Bioequivalent in Terms of Biochemical and Pharmacological Effects

Introduction: Currently, there is a shortage of porcine heparin due to limited availability of porcine mucosa and supply chain issues. Bovine heparin has been used for clinical purposes globally and is being considered for reintroduction in the U.S. On a mass basis, commercially available porcine heparins exhibit a higher potency (180-220 units/mg) than their bovine counterpart (130-150 units/mg). Therefore, at gravimetric levels, the porcine heparins exhibit stronger biochemical and pharmacological effects in comparison to bovine heparin. Since heparin is standardized in biologic units relative to the USP or EP Standard, it is hypothesized that potency equated porcine and bovine heparin will exhibit similar biologic activities in laboratory assays. The purpose of this study was to compare the biologic properties of the porcine and bovine heparin at USP potency equated levels in standardized laboratory assays used for measuring and monitoring heparins. Materials and Methods: Active pharmaceutical ingredient (API) porcine mucosal heparin (200 units/mg) of U.S. origin was obtained from Medefil Inc. (Glendale Heights, IL). Bovine heparin (140 units/mg) was obtained from KinMaster Produtos Químicos (Passo Fundo, Brazil). Whole blood Activated Clotting Time (ACT) and Thrombelastographic analyses were performed. Clot based assays such as aPTT, TT, and prothrombinase induced clotting time (PiCT) were performed using citrated human plasma supplemented with heparins up to 1 anti-Xa unit/mL. Anti-Xa and anti-IIa activities were determined using chromogenic antiprotease assays (Biophen Kits). In a purified antithrombin-supplemented system, the inhibitory effects of these agents were measured in terms of anti-Xa and anti-IIa activities and expressed as IC50 values. Thrombin generation inhibition was measured using a kinetic assay (CAT system, Diagnostica Stago, Paris, France). Protamine and platelet factor 4 neutralization profiles were investigated in plasma-based systems. The heparin induced thrombocytopenia antibody (HIT) interaction profile was assessed using platelet aggregometry. Results: Both heparins produced comparable prolongation of the ACT to a range of 200-250 seconds. The thrombelastographic profile was comparable at concentrations of 0.125 unit/mL and 2.5 units/mL. Whole blood and plasma clotting times (aPTT and TT assays) were comparable at concentrations up to 1 unit/mL. In the chromogenic anti-Xa and anti-IIa assays, the behavior of both agents was also comparable. In the purified system, the IC50 values for both agents for the anti-Xa activity ranged from 0.33-0.40 unit/mL, whereas the anti-IIa values ranged from 0.38-0.42 unit/mL; no significant differences were noted between the porcine and bovine heparins. In the thrombin generation assays, in terms of peak thrombin generation, area under the curve, and lag time, both the porcine and bovine heparins showed comparable effects. The protamine neutralization profiles of the porcine and bovine heparin exhibited variable assay dependent results. Potency adjusted bovine heparin required higher amount of protamine for the complete neutralization of the biologic effects in comparison to the porcine heparin. Similar results were obtained in the platelet factor 4 neutralization studies. In the functional HIT screening assay, both heparins exhibited a similar concentration dependent aggregation of human platelets. These results are summarized in the Table. Summary and Conclusion: These results show that at potency adjusted concentrations, porcine and bovine heparin exhibit comparable biochemical and anticoagulant responses in whole blood, plasma based, and purified biochemical assays. Most notably, both the porcine and bovine heparin produced comparable HIT antibody mediated aggregation responses. Thus, the proposed approach to standardize heparins against a common standard using a biologic assay such as the USP method is valid. These results warrant additional validation studies to reintroduce bovine heparin for clinical use. Table Disclosures Jeske: KinMaster Produtos Quimicas: Research Funding.

A Lipid Peroxidation Product, 4-Hydroxy-2-Nonenal, Triggers Intravascular Coagulation and Inflammation through Generation of Tissue Factor-Positive Microvesicles

Tissue factor (TF) is a cellular receptor for the clotting protease factor VIIa (FVIIa), and TF-FVIIa complex initiates activation of the coagulation cascade. TF-FVIIa also influences non-hemostatic functions via direct or indirect cell signaling. Constitutive expression of TF is primarily restricted to the surface of perivascular cells, sequestered from circulating blood clotting factors, to prevent aberrant intravascular clotting. It has been suggested that TF is also present in the blood associated with microvesicles (MVs). These circulating TF, however, do not induce any thrombotic complications as they are either cleared from the system before they could accumulate to a pathological level or lack procoagulant activity. However, various pathological conditions such as sepsis, diabetes, bacterial infection, atherosclerosis, and cancer can increase the levels of circulating TF+ MVs, which can then trigger intravascular coagulation and inflammation. We hypothesized that oxidative stress, a pathological condition, central to most of the diseases mentioned above, plays a role in the generation of TF+ MVs in vivo, which could contribute to intravascular coagulation and inflammation. We recently showed that 4-hydroxy-2-nonenal (HNE), the most abundant and stable unsaturated aldehyde produced by the oxidation of ω-6 polyunsaturated fatty acids, enhances TF activity in monocytic cells and macrophages. However, the role of HNE in activation of intravascular coagulation is unknown. To investigate this, C57Bl/6J wild-type mice were administered with HNE intraperitoneally (10 mg/kg body weight) and generation of TF+ MVs, activation of intravascular coagulation, and inflammation was assessed. HNE administration markedly increased TF activity associated with MVs. Nanoparticle tracking analysis (NTA) showed a 3-fold increase in the number of MVs in the plasma of HNE-treated mice. Measurement of thrombin-antithrombin (TAT) complexes revealed a significant increase in TAT levels in HNE treated mice. Pre-treatment of the mice with anti-murine TF antibody attenuated the HNE-induced increase in TAT levels. Measurement of plasma clotting times showed a significant reduction in HNE treated mice (118.3 ± 10.42 sec) compared to saline treated control mice (> 300 sec). Incubation of plasma with anti-TF antibody reversed the reduction observed in the plasma clotting time of HNE treated mice. Assessment of clotting in the saphenous vein injury bleeding model showed an increased number of clots, and reduced blood loss in HNE-administered mice compared to saline treated mice. Blockade of TF with 1H1 anti-TF antibody negated the HNE-mediated reduction in the clotting time. Next, we investigated whether HNE administration also induces a proinflammatory response in mice. Blood cells analysis showed approximately a 4-fold increase in circulating neutrophils in HNE-treated mice at 6 h following HNE treatment. Immunohistochemistry studies showed a robust infiltration of neutrophils into the lungs. Cytokine analysis of plasma showed a significant increase in IL-6 and CXCL1 levels in HNE-treated mice. Blockade of TF using 1H1 anti-TF antibody significantly reduced the HNE-mediated neutrophil infiltration into the lungs and completely abrogated the increase in plasma IL-6 levels. In additional studies, we attempted to identify the source of TF+ MVs. It is unlikely that they are originated from monocytes or endothelial cells as HNE did not induce TF expression in these cell types. LPS, used as a positive control, showed significant induction of TF on both PBMCs and the endothelium Immunohistochemistry revealed that HNE disrupted endothelial barrier integrity of vessel wall, raising a possibility that TF+ MVs could have come from perivascular cells. In summary, our data indicate that oxidative stress could induce the release of TF+ MVs, and this would contribute to intravascular coagulation and inflammation. Our studies also suggest that TF-positive microvesicles can be a useful therapeutic target in the treatment of thromboinflammatory diseases. Disclosures No relevant conflicts of interest to declare.

Plasma Kallikrein Contributes to Ellagic Acid-Induced Coagulation in the Absence of FXI By Activating Factor IX

Background - Factor XI (FXI) plays a critical role in thrombosis, as its inhibition reduces clot formation in animal models, and human congenital FXI deficiency appears to protect against venous thromboembolism and stroke. In contrast, in both settings, the impact on hemostasis is rather low, especially compared to FIX deficiency. Little is known about the cause of this difference. As thrombin generation can be observed in FXI-deficient plasma, we speculated that intrinsic activation of FIX is not solely dependent on FXI(a). Aim - Evaluate whether plasma kallikrein contributes to FIX activation and subsequent thrombin generation in FXI deficiency. Methods - Kaolin-initiated coagulation and fibrin formation was assessed in different plasma types in the presence or absence of an active site inhibitor of human plasma kallikrein (PKa) using the activated partial thromboplastin time (aPTT). Thrombin generation was measured in ellagic acid-induced contact activation in human FXI-deficient plasma in the presence or absence of the PKa inhibitor. Levels of FIXa-antithrombin (FIXa-AT) or FXa-AT were determined at defined time points using in-house developed ELISAs. The role of thrombin and its substrate FVIII in ellagic acid-induced contact activation in the absence of FXI was investigated using dabigatran or a human monoclonal FVIII antibody and assessing the FIXa-AT and FXa-AT plasma levels. In in vivo experiments, F11-/- mice were treated with ellagic acid in the presence or absence of the PKa inhibitor and FXIa-AT levels were assessed 30 min after administration. Results - Plasma kallikrein inhibition dose-dependently prolonged kaolin-triggered clotting time in human FXI-deficient plasma but not in normal pooled or plasma prekallikrein depleted plasma, indicating a role for PKa in coagulation in the absence of FXI. In FXI-deficient plasma, ellagic acid activated the coagulation system, as could be demonstrated by measurement of thrombin generation, and FIXa-AT and FXa-AT levels, which immediately reached the upper detection limit of 1.6 nM suggesting a FXI bypass route towards FIX activation. Addition of a PKa inhibitor caused a delayed and diminished FXa-AT and FIXa-AT complex formation and led to a reduction in thrombin peak height (EC50: 3 µM). This indicates that PKa might contribute to FXI-independent coagulation by activating FIX. In the presence of dabigatran FIXa-AT complex formation in FXI-deficient plasma was completely inhibited, which could be reversed by addition of purified thrombin. In the presence of purified thrombin, a FVIII antibody inhibited thrombin-dependent FIXa-AT complex formation suggesting that the activation of FVIII by thrombin is essential for FIX activation by PKa or to support the activity of FIXa. In line with this, thrombin generation in FXI-defcient plasma was inhibited by the FVIII antibody. In ellagic acid-treated F11-/- mice, FIXa-AT complex formation was significantly increased compared to controls (612 pM vs. 121 pM; P < 0.001), and was attenuated upon inhibition of PKa. Conclusions - We demonstrate that, activation of FXII leads to thrombin generation via FIX activation by plasma kallikrein in the absence of FXI. In addition, the activation of FVIII by thrombin is essential for the FXI bypass route towards FIX activation. These findings support the idea that FXI and FIX have distinctly different roles within the intrinsic coagulation cascade upon FXII activation and may contribute to variations in bleeding in FXI-deficient subjects due to different prekallikrein plasma levels. Disclosures Visser: Bayer AG: Employment. Laux:Bayer AG: Employment. Heitmeier:Bayer AG: Employment.

Antithrombotic Effect of Artemisia princeps Pampanini Extracts in Vitro and in FeCl3 -Induced Thrombosis Rats.

Extracts of several plants possess antithrombotic effects. Herein, we examined the antithrombotic effects of different extracts of Artemisia princeps Pampanini prepared using distilled water, hot distilled water, 70% ethanol, or subcritical water. The antithrombotic effects were determined using a co-culture system consisting of tumor necrosis factor-alpha (TNF-α)-treated EA.hy926 cells and THP-1 cells. In addition, the coagulation time of plasma collected from healthy volunteers was evaluated in terms of the prothrombin time and activated partial thromboplastin time. A carotid arterial thrombosis model was induced by ferric chloride in Sprague Dawley rats. The rats were treated with either sterile water or three different doses of the subcritical water extract for 2 weeks. The thrombus weight, gene expression of cell adhesion molecules, and histological characteristics were assessed. The results of in vitro studies revealed a significant inhibition in the adhesion of monocytes to EA.hy926 cells stimulated by TNF-α in the subcritical water extract-treated group. We also observed considerable suppression of the occlusion and mRNA expression of cell adhesion molecules in the in vivo experiments. This study suggests that Artemisia princeps Pampanini may have the potential to improve blood coagulation.

Bioinspired, Injectable, Quaternized Hydroxyethyl Cellulose Composite Hydrogel Coordinated by Mesocellular Silica Foam for Rapid, Noncompressible Hemostasis and Wound Healing

Massive bleeding control and anti-infection are the major challenges for urgent trauma with deep and noncompressible hemorrhage in both clinic and battlefield. Inspired by the coordinated primarily blood clot formation and secondly coagulation cascade activation in natural hemostasis process, an injectable, quaternized hydroxyethyl cellulose/mesocellular silica foam (MCF) hydrogel sponge (QHM) for both hemorrhage control and antibacterial activities were prepared via one-pot radical graft copolymerization. The as-prepared QHMs exhibited instant water-triggered expansion and superabsorbent capacity and thereby effectively facilitated blood components concentration. Moreover, the QHM1 with appropriate amount of MCF (9.82 w/w %) could further activate the coagulation factors. Synergistically, the QHM1 could reduce the plasma clotting time to 59 ± 4% in vitro and showed less blood loss than commercially available hemostatics in vivo noncompressible hemorrhage models of lethal rabbit-liver defect. Furthermore, the QHM with a quaternary ammonium groups density of 2.732 mmol/g exhibited remarkable antibacterial activities and excellent cytocompatibility. With the efficient hemostasis efficacy and excellent antibacterial behavior, QHM dramatically facilitated the wound healing in a full-thickness skin defect model in vivo. Thus, this QHM represents a promising hemostatic in more widespread clinical application.

Interaction of blood plasma proteins with superhemophobic titania nanotube surfaces

The need to improve blood biocompatibility of medical devices is urgent. As soon as blood encounters a biomaterial implant, proteins adsorb on its surfaces, often leading to several complications such as thrombosis and failure of the device. Therefore, controlling protein adsorption plays a major role in developing hemocompatible materials. In this study, the interaction of key blood plasma proteins with superhemophobic titania nanotube substrates and the blood clotting responses was investigated. The substrate stability was evaluated and fibrinogen adsorption and thrombin formation from plasma were assessed using ELISA. Whole blood clotting kinetics was also investigated, and Factor XII activation on the substrates was characterized by an in vitro plasma coagulation time assay. The results show that superhemophobic titania nanotubes are stable and considerably decrease surface protein adsorption/Factor XII activation as well as delay the whole blood clotting, and thus can be a promising approach for designing blood contacting medical devices.

Evaluating the Impact of Variable Phosphorothioate Content in Tricyclo-DNA Antisense Oligonucleotides in a Duchenne Muscular Dystrophy Mouse Model.

Antisense oligonucleotides (ASOs) hold promise for therapeutic splice switching correction for genetic diseases, in particular for Duchenne muscular dystrophy (DMD), for which ASO-exon skipping represents one of the most advanced therapeutic strategies. We have previously reported the therapeutic potential of tricyclo-DNA (tcDNA) in mouse models of DMD, highlighting the unique pharmaceutical properties and unprecedented uptake in many tissues after systemic delivery, including the heart and central nervous system. TcDNA-ASOs demonstrate an encouraging safety profile and no particular class-related toxicity, however, when administered in high doses for several months, mild renal toxicity is observed secondary to predictable phosphorothioate (PS)-ASO accumulation in kidneys. In this study, we investigate the influence of the relative content of PS linkages in tcDNA-ASOs on exon skipping efficacy. Mdx mice were injected intravenously once weekly for 4 weeks with tcDNA carrying various amounts of PS linkages (0%, 25%, 33%, 50%, 67%, 83%, and 100%). The results indicate that levels of exon-23 skipping and dystrophin rescue increase with the number of PS linkages in most skeletal muscles except in the heart. As expected, plasma coagulation times are shortened with decreasing PS content, and tcDNA-protein binding in serum directly correlates with the number of PS linkages on the tcDNA backbone. Altogether, these data contribute in establishing the appropriate sulfur content within the tcDNA backbone for maximal efficacy and minimal toxicity of the oligonucleotide.

Nano- and Micropatterned Polycaprolactone Cellulose Composite Surfaces with Tunable Protein Adsorption, Fibrin Clot Formation, and Endothelial Cellular Response.

This work describes the interaction of the human blood plasma proteins albumin, fibrinogen, and γ-globulins with micro- and nanopatterned polymer interfaces. Protein adsorption studies were correlated with the fibrin clotting time of human blood plasma and with the growth of primary human pulmonary artery endothelial cells (hECs) on these patterns. It was observed that blends of polycaprolactone (PCL) and trimethylsilyl-protected cellulose form various thin-film patterns during spin coating, depending on the mass ratio of the polymers in the spinning solutions. Vapor-phase acid-catalyzed deprotection preserves these patterns but yields interfaces that are composed of hydrophilic cellulose domains enclosed by hydrophobic PCL. The blood plasma proteins are repelled by the cellulose domains, allowing for a suggested selective protein deposition on the PCL domains. An inverse proportional correlation is observed between the amount of cellulose present in the films and the mass of irreversibly adsorbed proteins. This results in significantly increased fibrin clotting times and lower masses of deposited clots on cellulose-containing films as revealed by quartz crystal microbalance with dissipation measurements. Cell viability of hECs grown on these surfaces was directly correlated with higher protein adsorption and faster clot formation. The results show that presented patterned polymer composite surfaces allow for a controllable blood plasma protein coagulation and a significant biological response from hECs. It is proposed that this knowledge can be utilized in regenerative medicine, cell cultures, and artificial vascular grafts by a careful choice of polymers and patterns.

Novel Cellulose-Halloysite Hemostatic Nanocomposite Fibers with a Dramatic Reduction in Human Plasma Coagulation Time.

High-performance cellulose-halloysite hemostatic nanocomposite fibers (CHNFs) are fabricated using a one-step wet-wet electrospinning process and evaluated for human plasma coagulation by activated partial thromboplastin time. These novel biocompatible CHNFs exhibit 2.4 times faster plasma coagulation time compared with the industry gold standard QuikClot Combat Gauze (QCG). The CHNFs have superior antileaching property of clay with 3 times higher post-wetting clotting activity compared to QCG. The CHNFs also coagulate whole blood 1.3 times faster than the QCG and retain twice the clotting performance after washing. Halloysite clay is also more effective in plasma coagulation than commercial kaolin clay. The physical and thermal properties of the CHNFs were evaluated using scanning electron microscopy, energy-dispersive X-ray spectroscopy, X-ray diffraction, Brunauer-Emmett-Teller surface area analysis, and thermogravimetric analysis. CHNFs show a 7-fold greater clay loading than QCG and their small average diameter of 450 ± 260 nm affords a greater specific surface area (33.6 m2 g-1) compared with the larger average diameter of 12.6 ± 0.9 μm for QCG with a specific surface area of 1.6 m2 g-1. The CHNFs were shown to be noncytotoxic and human primary fibroblasts proliferated on the composite material. The drastic reduction in coagulation time makes this novel nanocomposite a potential lifesaving material for victims of rapid blood loss such as military personnel and patients undergoing major surgical procedures or to aid in the treatment of unexpected bleeding episodes of patients suffering from hereditary blood clotting disorders. Since a person can die within minutes of heavy bleeding, every second counts for stopping traumatic hemorrhaging.

An αIIbβ3- and phosphatidylserine (PS)-binding recombinant fusion protein promotes PS-dependent anticoagulation and integrin-dependent antithrombosis

Blood platelets are required for normal wound healing, but they are also involved in thrombotic diseases, which are usually managed with anticoagulant drugs. Here, using genetic engineering, we coupled the disintegrin protein echistatin, which specifically binds to the platelet integrin αIIbβ3 receptor, to annexin V, which binds platelet membrane-associated phosphatidylserine (PS), to create the bifunctional antithrombotic molecule recombinant echistatin-annexin V fusion protein (r-EchAV). Lipid binding and plasma coagulation studies revealed that r-EchAV dose-dependently binds PS and delays plasma clotting time. Moreover, r-EchAV inhibited ADP-induced platelet aggregation in a dose-dependent manner and exhibited potent antiplatelet aggregation effects. r-EchAV significantly prolonged activated partial thromboplastin time, suggesting that it primarily affects the in vivo coagulation pathway. Flow cytometry results indicated that r-EchAV could effectively bind to the platelet αIIbβ3 receptor, indicating that r-EchAV retains echistatin's receptor-recognition region. In vivo experiments in mice disclosed that r-EchAV significantly prolongs bleeding time, indicating a significant anticoagulant effect in vivo resulting from the joint binding of r-EchAV to both PS and the αIIbβ3 receptor. We also report optimization of the r-EchAV production steps and its purification for high purity and yield. Our findings indicate that r-EchAV retains the active structural regions of echistatin and annexin V and that the whole molecule exhibits multitarget-binding ability arising from the dual functions of echistatin and annexin V. Therefore, r-EchAV represents a new class of anticoagulant that specifically targets the anionic membrane-associated coagulation enzyme complexes at thrombogenesis sites and may be a potentially useful antithrombotic agent.

Evaluation of Coagulation Parameters in Dogs with Overweight or Obesity

Background: Hemostatic alterations have already been well characterized in humans with body fat excess, being included in the list of obesity related comorbidities. Overweight and obesity are common in dogs; however, there is little information about the blood coagulation parameters in dogs with these conditions. The aim of this study was to compare hematological and coagulation parameters between lean dogs and overweight or obese dogs, including platelets count; prothrombin time (PT); activated partial thromboplastin time (aPTT), coagulation time (CT) and plasma fibrinogen concentration.Materials, Methods & Results: A total of 22 dogs (aged 1 to 10 years, neutered) were evaluated, 10 of them presented ideal body condition score (BCS 4-5) and formed the group 1 (control) and 12 were overweight or obese (BCS 7-9) and formed the group 2. The dogs were submitted to clinical evaluation and then to blood collection for the following laboratory tests: blood count (performed on automatic analyzer), quantification of plasma proteins by refractometry, determination PT, aPTT and plasma fibrinogen concentration using specific commercial kits, and CT by Lee-White method. Compared to group 1, group 2 presented a lower leukocytes and lymphocytes counts (P < 0.05) and a higher concentration of plasma fibrinogen (P = 0,026), but compatible with reference values. No difference was observed in the erythrogram, platelets count, total plasma protein concentration, PT, aPTT and CT between the groups. BCS was negatively correlated with leukocytes (r = -0.45) and lymphocytes (r = -0.60) counts and positively with plasma fibrinogen concentration (r = +0.56).Discussion: The reduction in lymphocytes led to a lower leukocytes count in the dogs of group 2. The migration of peripheral blood lymphocytes to adipose tissue has been reported in the early phase of the inflammatory process induced by obesity and could justify the reduction of circulating lymphocytes in overweight or obese dogs evaluated in this study; however, cytopathological assessment of adipose tissue was not performed. Fibrinogen has to be converted to fibrin for clot formation, so the concentration of this plasma protein is an important parameter for the evaluation of hemostasis. However, there are reports of increase in plasma fibrinogen as a result of infammatory processes. In group 2, formed by dogs with overweight or obese, the higher value of plasm fibrinogen concentration, associated with lower lymphocyte count, may suggest an early-stage inflammatory process. Similar results were described in obese humans, but also evidenced in obese dogs. Supporting this suggestion, the correlation analysis indicates that the higher the body fat excess (estimated by the BCS), the higher the fibrinogen concentration and the lower the lymphocyte count in evaluated dogs. Despite the higher concentration of plasma fibrinogen in group 2, no change was observed in the hemostasis of overweight or obese dogs, due to the normal values for platelets count, PT, aPTT and CT, excluding a hypercoagulability condition as already hypothesized by other authors for dogs and obese humans. The difference between our findings and the literature may be in the time of evolution of the disease, since we evaluated younger dogs. In conclusion, the body fat excess did not alter the erythrogram and the activity of the clotting factors, estimated by PT, aPTT and CT, but it interfered in the leukogram and increased the plasma concentration of fibrinogen in the evaluated dogs.

Mast cell chymase degrades fibrinogen and fibrin.

The accumulation of immunoreactants and fibrinoid necrosis of postcapillary vessel walls are common pathological features of cutaneous immune complex vasculitis. In more advanced lesions, these immunoreactants are subject to proteolysis. Mast cell chymase is a powerful enzyme that can degrade several substrates including the extracellular matrix. Heparin can influence the catalytic properties of chymase. To study the effects of recombinant human (rh) chymase on fibrinogen, coagulation and fibrinolysis, and to relate these effects to the pathogenesis of vasculitis. The colocalization of chymase and fibrin in vasculitis specimens was analysed by immunohistochemical double staining. Fibrinogen and fibrin were treated with rh-chymase and the effects were studied invitro by sodium dodecylsulfate polyacrylamide gel electrophoresis and a variety of clotting and fibrin gel experiments. The effects of rh-chymase on vasculitis cryosections were analysed by direct immunofluorescence. Chymase-positive mast cells were associated with fibrin-positive vessels in vasculitis cryosections. Rh-chymase degraded the alpha-, beta- and gamma-chains of fibrinogen, while heparin enhanced the degradation of the beta-chain. Rh-chymase pretreatment of fibrinogen prolonged thrombin-induced clotting time. Fibrinogen degradation products induced by rh-chymase increased the clotting time of human plasma. Rh-chymase degraded fibrin gel prepared from fibrinogen or human plasma. Immunofluorescence staining positivity of fibrin in vasculitis cryosections decreased after pretreatment with rh-chymase for 24 h, and heparin enhanced this effect. Mast cell chymase may constitute a previously unrecognized endogenous anticoagulant and fibrinolytic enzyme, and may be involved in the clearance of fibrin from vessel walls in aged vasculitis lesions.

The effect of DOAC-Stop on lupus anticoagulant testing in plasma samples of venous thromboembolism patients receiving direct oral anticoagulants.

Background Direct oral anticoagulants (DOACs) cause false positive lupus anticoagulant (LA) results. We assessed the impact of DOAC-Stop, reversing in vitro effects of DOACs, on LA testing in anticoagulated patients. Methods We assessed 75 venous thromboembolism patients aged 44.5±14.6 years. Blood samples were collected 2-28 h since intake of DOACs, including 50 patients on rivaroxaban, 20 on dabigatran and five on apixaban. LA testing was performed at baseline and after DOAC-Stop treatment. Positive LA was defined as the normalized (patient/standard plasma clotting time) LA screening and screening (LA1)/confirmation (LA2) ratios exceeding 1.2. Results LA diluted Russell's viper venom time (dRVVT) normalized screening test revealed abnormal results in 73 (97.3%) and activated partial thromboplastin time (APTT)-LA in 49 (65.3%) patients. In six (8%) patients, antiphospholipid syndrome (APS) was diagnosed. dRVVT LA1/LA2 was abnormal in 35 (50.7%) patients taking DOACs. The APTT ratio was normal in all studied subjects. DOAC-Stop completely removed dabigatran and reduced by 98% rivaroxaban and by 92.3% apixaban concentrations (all p<0.05). After DOAC-Stop screening dRVVT remained prolonged in 34 (49.3%) patients (p<0.001), while dRVVT LA1/LA2 was abnormal in six (8.7%) subjects, with no association with DOAC concentrations at baseline and after DOAC-Stop. The APTT-LA screening test remained prolonged in five (7.2%) patients, while the APTT LA1/LA2 ratio was normal in those subjects. DOAC-Stop did not influence LA testing in APS patients. Conclusions Application of DOAC-Stop effectively reduced plasma DOAC concentrations leading to appropriate dRVVT results in up to 97% of VTE patients.