Dual antithrombotic therapy dose-dependently alters hemostatic plug structure and function.

BACKGROUND: Antithrombotic medications carry an inherent risk of bleeding, which may be exacerbated when anticoagulant and antiplatelet therapeutics are combined. Prior studies have shown different effects of antiplatelet vs anticoagulant drugs on the structure and function of hemostatic plugs in vivo. OBJECTIVES: We examined whether dual antithrombotic treatment consisting of combined antiplatelet and anticoagulant therapeutics alters hemostatic plug structure and function differently from treatment with either therapeutic alone. METHODS: Mice were treated with the P2Y12 antagonist clopidogrel and the factor Xa inhibitor rivaroxaban across a range of doses, either alone or in combination. The hemostatic response was assessed using a mouse jugular vein puncture injury model. Platelet accumulation and fibrin deposition were evaluated using quantitative multiphoton fluorescence microscopy, and bleeding times were recorded. RESULTS: Mice treated with clopidogrel alone exhibited a decrease in platelet accumulation at the site of injury, with prolonged bleeding times only at the highest doses of clopidogrel used. Mice treated with rivaroxaban alone instead showed a reduction in fibrin deposition with no impact on bleeding. Mice treated with both clopidogrel and rivaroxaban exhibited platelet and fibrin accumulation that was similar to that with either drug given alone; however, dual antithrombotic therapy resulted in impaired hemostasis at doses that had no impact on bleeding when given in isolation. CONCLUSION: Combined administration of antiplatelet and anticoagulant therapeutics exacerbates bleeding as compared to that with either drug alone, potentially via combined loss of both adenosine 5'-diphosphate- and thrombin-mediated platelet activation. These findings enhance our understanding of the bleeding risk associated with dual antithrombotic therapy.

Facile green approach towards the synthesis of some phenyl piperazine based dithiocarbamates as potent hemolytic and thrombolytic agents.

The facile and efficient protocol for the synthesis of N-phenyl piperazine based di-thio-carbamates has been reported under neat conditions. A library of novel piperazine based di-thio-carbamates (3a-h) in excellent yields has been prepared. Solvent free, catalyst free and easy work up conditions make this protocol an attractive synthetic protocol to achieve novel biologically active di-thio-carbamates. The synthesized molecules have been characterized by FT-IR, 1HNMR and 13CNMR spectroscopic techniques. The pharmacological aspects of these derivatives have been evaluated via hemolysis and thrombolysis. All the target molecules (3a-h) exhibit mild to medium potential as hemolytic and thrombolytic agents. Among the synthesized derivatives, compound 3c showed least cytotoxicity and better thrombolytic potential.

A honokiol-mediated robust coating for blood-contacting devices with anti-inflammatory, antibacterial and antithrombotic properties.

Thrombus, bacterial infections, and severe inflammation are still serious problems that have to be faced with blood-contacting materials. However, it is a great challenge to simultaneously meet the above functional requirements in a simple, economical and efficient method. As such, we put forward a robust and versatile coating strategy by covalently modifying the multi-pharmacological drug honokiol (HK) with an amine-rich polydopamine/polyethyleneimine coating, through which anticoagulant, antibacterial and anti-inflammatory properties were obtained (DPHc) simultaneously. The amine content in the DPHc coating was lower than the detection limit, while it contained abundant phenolic hydroxyl groups (49 µmol cm-2). Meanwhile, the 30 day drug release test confirmed that the drug was firmly modified on the surface of the coating without release. A systematic in vitro and ex vivo evaluation confirmed that the coating had significant anti-thrombotic properties. The antibacterial rates of the DPHc coating against Staphylococcus aureus and Escherichia coli reached 99.98% and 99.99%, respectively. In addition, subcutaneous implantation indicated that the DPHc coating also has excellent histocompatibility. To the best of our knowledge, this is the first study using HK as a coating material that can not only combat thrombosis and infection but also significantly inhibit inflammation associated with the use of blood-contacting materials, thus expanding the application of HK in the field of biomaterials.

Chemical, biological and protein-receptor binding profiling of Bauhinia scandens L. stems provide new insights into the management of pain, inflammation, pyrexia and thrombosis.

Bauhinia scandens L. (Family, Fabaceae) is a medicinal plant used for conventional and societal medication in Ayurveda. The present study has been conducted to screen the chemical, pharmacological and biochemical potentiality of the methanol extracts of B. scandens stems (MEBS) along with its related fractions including carbon tetrachloride (CTBS), di-chloromethane (DMBS) and n-butanol (BTBS). UPLC-QTOF-MS has been implemented to analyze the chemical compounds of the methanol extracts of Bauhinia scandens stems. Additionally, antinociceptive and anti-inflammatory effects were performed by following the acetic acid-induced writhing test and formalin-mediated paw licking test in the mice model. The antipyretic investigation was performed by Brewer Yeast induced pyrexia method. The clot lysis method was implemented to screen the thrombolytic activity in human serum. Besides, the in silico study was performed for the five selected chemical compounds of Bauhinia scandens, found by UPLC-QTOF-MS By using Discover Studio 2020, UCSF Chimera, PyRx autodock vina and online tools. The MEBS and its fractions exhibited remarkable inhibition in dose dependant manner in the antinociceptive and antiinflammatory investigations. The antipyretic results of MEBS and DMBS were close to the standard drug indomethacin. Investigation of the thrombolytic effect of MEBS, CTBS, DMBS, and BTBS revealed notable clot-lytic potentials. Besides, the phenolic compounds of the plant extracts revealed strong binding affinity to the COX-1, COX-2, mPGES-1 and plasminogen activator enzymes. To recapitulate, based on the research work, Bauhinia scandens L. stem and its phytochemicals can be considered as prospective wellsprings for novel drug development and discovery by future researchers.

Safety assessment of subtilisin QK in rats.

BACKGROUND: Subtilisin QK is a serine protease in the subtilisin family, and is fermented by Bacillus subtilis QK02. The fibrinolytic activity of subtilisin QK was measured by detecting low molecular weight degradation products using a spectrophotometric method developed by Japan Bio Science Laboratory Co., Ltd. Subtilisin QK powder can maintain its fibrinolytic activity for more than 24 months when it is stored at room temperature and protected from light. Our previous results showed that subtlisin QK directly degraded cross-linked fibrins in the fibrin plate assay and effectively inhibited thrombosis in the mouse thrombus model. The aim of this study was to determine the acute toxicity, potential subchronic toxicity, and safety pharmacology of subtilisin QK in Sprague-Dawley (SD) rats. METHODS: In the acute toxicity study, a single oral dose of 100,000 FU/kg was administered to 10 female and 10 male SD rats. In the 28-day subchronic toxicity, 60 female and 60 male SD rats were randomly assigned to four experimental groups (daily oral dose of 0, 2500, 7500 and 25,000 FU/kg). In the safety pharmacology study, 20 female and 20 male SD rats were randomly assigned to four experimental groups (single oral dose of 0, 500, 1500 and 5000 FU/kg). RESULTS: No death occurred and no adverse effects were observed in the acute toxicity study at a dose of 100,000 FU/kg. In the 28-day subchronic toxicity study, several hematological and blood biochemical parameters showed increases or decreases; however, due to the lack of a dose-response relationship, these differences were considered unrelated to treatment. In the safety pharmacology study, no adverse effects were observed on the central nervous of SD rats post-administration up to a dose of 5000 FU/kg subtilisin QK. CONCLUSION: The results showed that oral consumption of subtilisin QK is of low toxicological concern. No adverse effects were observed at doses of 2500, 7500, and 25,000 FU/kg in the 28-day subchronic toxicity, and the no-observed-adverse-effect level (NOAEL) of subtilisin QK was 25,000 FU/kg.

Synthesis and Bioactivities of Marine Pyran-Isoindolone Derivatives as Potential Antithrombotic Agents.

2,5-Bis-[8-(4,8-dimethyl-nona-3,7-dienyl)-5,7-dihydroxy-8-methyl-3-keto-1,2,7,8-teraahydro-6H-pyran[a]isoindol-2-yl]-pentanoic acid (FGFC1) is a marine pyran-isoindolone derivative isolated from a rare marine microorganism Stachybotrys longispora FG216, which showed moderate antithrombotic(fibrinolytic) activity. To further enhance its antithrombotic effect, a series of new FGFC1 derivatives (F1-F7) were synthesized via chemical modification at C-2 and C-2' phenol groups moieties and C-1″ carboxyl group. Their fibrinolytic activities in vitro were evaluated. Among the derivatives, F1-F4 and F6 showed significant fibrinolytic activities with EC50 of 59.7, 87.1, 66.6, 82.8, and 42.3 µM, respectively, via enhancement of urokinase activity. Notably, derivative F6 presented the most remarkable fibrinolytic activity (2.72-fold than that of FGFC1). Furthermore, the cytotoxicity of derivative F6 was tested as well as expression of Fas/Apo-1 and IL-1 on HeLa cells. The results showed that, compared to FGFC1, derivative F6 possessed moderate cytotoxicity and apoptotic effect on HeLa cells (statistical significance p > 0.1), making F6 a potential antithrombotic agent towards clinical application.

Acute and Subchronic Toxicities and Safety Pharmacology Studies of a Bacillus Subtilisin in Dogs.

Subtilisin NAT, a Bacillus subtilisin, is widely applied as a functional food and considered to be one of the most exploitable potential oral thrombolytic agents. Subtilisin QK, another Bacillus subtilisin, is a serine protease fermented by Bacillus subtilis 02 and has a better thrombolytic effect. Therefore, subtilisin QK is typically used for evaluating the safety of Bacillus subtilisins. Here, we conduct several good laboratory practice (GLP)-compliant studies in non-rodent animal, i.e., in Beagle dogs, including acute toxicity, subchronic toxicity, and safety pharmacology studies. No adverse effects were evident in the acute and 28-d subchronic toxicity studies at doses up to 40000 FU/kg and 16000 FU/kg/d, respectively. In evaluating the pharmacological safety of up to 2000FU/kg subtilisin QK, we found no significant differences between the electrocardiograms, blood pressures, and respiration of beagle dogs. These findings suggest the safety of Bacillus subtilisin, providing reliable pharmacological and toxicological data for its development and popularization as a functional food and drug.

Sparganii Rhizoma: A review of traditional clinical application, processing, phytochemistry, pharmacology, and toxicity.

ETHNOPHARMACOLOGICAL RELEVANCE: Sparganii Rhizoma (SR), a traditional Chinese medicine (TCM), is the rhizome of Sparganium stoloniferum Buch.-Ham. mainly distributed in East Asia. It has been used for eliminating blood stasis, promoting the flow of Qi, removing the retention of undigested food and relieving pain in China for hundreds of years. AIM OF THE REVIEW: This review summarizes comprehensive information in traditional clinical application, processing, phytochemistry, pharmacology, quality control and toxicity of SR, in exploring future scientific and therapeutic potentials. MATERIALS AND METHODS: Pertinent information was systematically collected from several electronic scientific databases (e.g., Web of Science, PubMed, China Knowledge Resource Integrated, Springer, Elsevier, ScienceDirect, and Google Scholar), PhD and MS dissertations, and classic Chinese medical books. RESULTS: SR is a gynecological drug which is often used to treat dysmenorrhea, mass in the abdomen, amenorrhea due to blood stasis, and abdominal distension in TCM. Two kinds of processed products of SR are included in Chinese Pharmacopoeia, which have better pharmacological effects than the crude herb. Approximately 180 compounds have been identified from SR, including phenylpropanoids, flavonoids, anthraquinones, organic acids, alkaloids, steroids, volatile oils, diarylheptanes, etc. The crude extracts and isolated components of SR have been reported to have anti-tumor, antithrombotic, estrogen antagonistic , anti-inflammatory, analgesic, antioxidant, anti organ fibrosis and other pharmacological activities. SR also has reproductive toxicity. CONCLUSIONS: As an important TCM, SR has been demonstrated by modern pharmacological researches to have significant bioactivities, especially on anti-tumor, antithrombotic, and estrogen antagonistic activities. These activities provide prospects for the development of new drugs and therapeutics for future applications. Nevertheless, quality control and evaluation, in-depth pharmacological mechanism, and toxicological effect of SR require further detailed research.

Targeting Thymidine Phosphorylase With Tipiracil Hydrochloride Attenuates Thrombosis Without Increasing Risk of Bleeding in Mice.

OBJECTIVE: Current antiplatelet medications increase the risk of bleeding, which leads to a clear clinical need in developing novel mechanism-based antiplatelet drugs. TYMP (Thymidine phosphorylase), a cytoplasm protein that is highly expressed in platelets, facilitates multiple agonist-induced platelet activation, and enhances thrombosis. Tipiracil hydrochloride (TPI), a selective TYMP inhibitor, has been approved by the Food and Drug Administration for clinical use. We tested the hypothesis that TPI is a safe antithrombotic medication. Approach and Results: By coexpression of TYMP and Lyn, GST (glutathione S-transferase) tagged Lyn-SH3 domain or Lyn-SH2 domain, we showed the direct evidence that TYMP binds to Lyn through both SH3 and SH2 domains, and TPI diminished the binding. TYMP deficiency significantly inhibits thrombosis in vivo in both sexes. Pretreatment of platelets with TPI rapidly inhibited collagen- and ADP-induced platelet aggregation. Under either normal or hyperlipidemic conditions, treating wild-type mice with TPI via intraperitoneal injection, intravenous injection, or gavage feeding dramatically inhibited thrombosis without inducing significant bleeding. Even at high doses, TPI has a lower bleeding side effect compared with aspirin and clopidogrel. Intravenous delivery of TPI alone or combined with tissue plasminogen activator dramatically inhibited thrombosis. Dual administration of a very low dose of aspirin and TPI, which had no antithrombotic effects when used alone, significantly inhibited thrombosis without disturbing hemostasis. CONCLUSIONS: This study demonstrated that inhibition of TYMP, a cytoplasmic protein, attenuated multiple signaling pathways that mediate platelet activation, aggregation, and thrombosis. TPI can be used as a novel antithrombotic medication without the increase in risk of bleeding.

Identification and Characterization of Plasmin-Independent Thrombolytic Enzymes.

RATIONALE: Current thrombolytic agents activate plasminogen to plasmin which triggers fibrinolysis to dissolve thrombi. Since plasmin is a nonspecific proteolytic enzyme, all of the current plasmin-dependent thrombolytics lead to serious hemorrhagic complications, demanding a new class of fibrinolytic enzymes independent from plasmin activation and undesirable side effects. We speculated that the mammalian version of bacterial heat-shock proteins could selectively degrade intravascular thrombi, a typical example of a highly aggregated protein mixture. OBJECTIVE: The objective of this study is to identify enzymes that can dissolve intravascular thrombi specifically without affecting fibrinogen and fibronectin so that the wound healing processes remain uninterrupted and tissues are not damaged. In this study, HtrA (high-temperature requirement A) proteins were tested for its specific proteolytic activity on intravascular thrombi independently from plasmin activation. METHODS AND RESULTS: HtrA1 and HtrA2/Omi proteins, collectively called as HtrAs, lysed ex vivo blood thrombi by degrading fibrin polymers. The thrombolysis by HtrAs was plasmin-independent and specific to vascular thrombi without causing the systemic activation of plasminogen and preventing nonspecific proteolysis of other proteins including fibrinogen and fibronectin. As expected, HtrAs did not disturb clotting and wound healing of excised wounds from mouse skin. It was further confirmed in a tail bleeding and a rebleeding assay that HtrAs allowed normal clotting and maintenance of clot stability in wounds, unlike other thrombolytics. Most importantly, HtrAs completely dissolved blood thrombi in tail thrombosis mice, and the intravenous injection of HtrAs to mice with pulmonary embolism completely dissolved intravascular thrombi and thus rescued thromboembolism. CONCLUSIONS: Here, we identified HtrA1 and HtrA2/Omi as plasmin-independent and highly specific thrombolytics that can dissolve intravascular thrombi specifically without bleeding risk. This work is the first report of a plasmin-independent thrombolytic pathway, providing HtrA1 and HtrA2/Omi as ideal therapeutic candidates for various thrombotic diseases without hemorrhagic complications.

Towards the Therapeutic Use of Thrombospondin 1/CD47 Targeting TAX2 Peptide as an Antithrombotic Agent.

OBJECTIVE: TSP-1 (thrombospondin 1) is one of the most expressed proteins in platelet α-granules and plays an important role in the regulation of hemostasis and thrombosis. Interaction of released TSP-1 with CD47 membrane receptor has been shown to regulate major events leading to thrombus formation, such as, platelet adhesion to vascular endothelium, nitric oxide/cGMP (cyclic guanosine monophosphate) signaling, platelet activation as well as aggregation. Therefore, targeting TSP-1:CD47 axis may represent a promising antithrombotic strategy. Approach and Results: A CD47-derived cyclic peptide was engineered, namely TAX2, that targets TSP-1 and selectively prevents TSP-1:CD47 interaction. Here, we demonstrate for the first time that TAX2 peptide strongly decreases platelet aggregation and interaction with collagen under arterial shear conditions. TAX2 also delays time for complete thrombotic occlusion in 2 mouse models of arterial thrombosis following chemical injury, while Thbs1-/- mice recapitulate TAX2 effects. Importantly, TAX2 administration is not associated with increased bleeding risk or modification of hematologic parameters. CONCLUSIONS: Overall, this study sheds light on the major contribution of TSP-1:CD47 interaction in platelet activation and thrombus formation while putting forward TAX2 as an innovative antithrombotic agent with high added-value.

tPA Mobilizes Immune Cells That Exacerbate Hemorrhagic Transformation in Stroke.

RATIONALE: Hemorrhagic complications represent a major limitation of intravenous thrombolysis using tPA (tissue-type plasminogen activator) in patients with ischemic stroke. The expression of tPA receptors on immune cells raises the question of what effects tPA exerts on these cells and whether these effects contribute to thrombolysis-related hemorrhagic transformation. OBJECTIVE: We aim to determine the impact of tPA on immune cells and investigate the association between observed immune alteration with hemorrhagic transformation in ischemic stroke patients and in a rat model of embolic stroke. METHODS AND RESULTS: Paired blood samples were collected before and 1 hour after tPA infusion from 71 patients with ischemic stroke. Control blood samples were collected from 27 ischemic stroke patients without tPA treatment. A rat embolic middle cerebral artery occlusion model was adopted to investigate the underlying mechanisms of hemorrhagic transformation. We report that tPA induces a swift surge of circulating neutrophils and T cells with profoundly altered molecular features in ischemic stroke patients and a rat model of focal embolic stroke. tPA exacerbates endothelial injury, increases adhesion and migration of neutrophils and T cells, which are associated with brain hemorrhage in rats subjected to embolic stroke. Genetic ablation of annexin A2 in neutrophils and T cells diminishes the effect of tPA on these cells. Decoupling the interaction between mobilized neutrophils/T cells and the neurovascular unit, achieved via a S1PR (sphingosine-1-phosphate receptor) 1 modulator RP101075 and a CCL2 (C-C motif chemokine ligand 2) synthesis inhibitor bindarit, which block lymphocyte egress and myeloid cell recruitment, respectively, attenuates hemorrhagic transformation and improves neurological function after tPA thrombolysis. CONCLUSIONS: Our findings suggest that immune invasion of the neurovascular unit represents a previously unrecognized mechanism underlying tPA-mediated brain hemorrhage, which can be overcome by precise immune modulation during thrombolytic therapy.

Phosphorylcholine- and cation-bearing copolymer coating with superior antibiofilm and antithrombotic properties for blood-contacting devices.

Nosocomial infections resulting from bacterial attachment on blood-contacting medical devices, as well as biofilm and thrombus formation caused by fibrin crosslinking and platelet accumulation/activation are a major health concern and may lead to severe morbidity and mortality. Therefore, there is an urgent need to develop facile and efficient surface coatings with both antibiofilm and antithrombotic properties to prevent medical-device associated infections as well as thrombus formation. In this study, the copolymers containing quaternary ammonium (QA) and phosphorylcholine (PC) groups were synthesized through traditional free-radical copolymerization. The cationic group of QA provides bactericidal properties, and the cell membrane-mimicking group of PC provides antithrombotic and antifouling properties. Long-term stability of the copolymer coating was achieved via simple dip coating. X-ray photoelectron spectroscopy and water contact angle measurement demonstrated that the QA and PC groups possessed inversion properties once in contact with water allowing for long-term stability. Scanning electron microscopy and confocal laser scanning microscopy demonstrated that the copolymer coating could maintain antibiofilm properties for one week in a nutrient-rich environment. Furthermore, the copolymer coating significantly decreased platelet adhesion/activation and did not cause hemolysis. The ex vivo blood circulation showed no thrombus formation which confirmed the excellent antithrombotic property of the copolymer coating. Such coatings that maintain high cell viability and exhibit both antibiofilm and antithrombotic properties present potential applications for blood-contacting devices.

YiQiFuMai Lyophilized Injection ameliorates tPA-induced hemorrhagic transformation by inhibiting cytoskeletal rearrangement associated with ROCK1 and NF-κB signaling pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Thrombolytic therapy with tissue plasminogen activator (tPA) after ischemic stroke exacerbates blood-brain barrier (BBB) breakdown and leads to hemorrhagic transformation (HT). YiQiFuMai Lyophilized Injection (YQFM) is a modern preparation derived from Sheng-mai San (a traditional Chinese medicine). YQFM attenuates the BBB dysfunction induced by cerebral ischemia-reperfusion injury. However, whether YQFM can suppress tPA-induced HT remains unknown. AIM OF THE STUDY: We investigated the therapeutic effect of YQFM on tPA-induced HT and explored the underlying mechanisms in vivo and in vitro to improve the safety of tPA use against stroke. METHODS: Male C57BL/6J mice were subjected to 45 min of ischemia and 24 h of reperfusion. tPA (10 mg/kg) were infused 2 h after occlusion and YQFM (0.671 g/kg) was injected 2.5 h after occlusion. The in vitro effect of YQFM (100, 200, 400 µg/mL) on tPA (60 µg/mL)-induced dysfunction of the microvascular endothelial barrier in the brain following oxygen-glucose deprivation/reoxygenation (OGD/R) was observed in bEnd.3 cells. RESULTS: YQFM suppressed tPA-induced high hemoglobin level in the brain, mortality, neurologic severity score, BBB permeability, expression and activation of matrix metalloproteinase (MMP)-9 and MMP-2, and degradation of tight-junction proteins. Furthermore, YQFM significantly blocked tPA-induced brain microvascular endothelial permeability and phosphorylation of Rho-associated kinase (ROCK)1, myosin light chain (MLC), cofilin and p65 in vivo and in vitro. CONCLUSION: YQFM suppressed tPA-induced HT by inhibiting cytoskeletal rearrangement linked with ROCK-cofilin/MLC pathways and inhibiting the nuclear factor-kappa B pathway to ameliorate BBB damage caused by tPA.

Improved Antithrombotic Activity and Diminished Bleeding Side Effect of a PEGylated αIIbß3 Antagonist, Disintegrin.

Polymer polyethylene glycol (PEG), or PEGylation of polypeptides improves protein drug stability by decrease degradation and reduces renal clearance. To produce a pharmaceutical disintegrin derivative, the N-terminal PEGylation technique was used to modify the disintegrin derivative [KGDRR]trimucrin for favorable safety and pharmacokinetic profiles and antithrombotic efficacy. We compared intact [KGDRR]trimucrin (RR) and PEGylated KGDRR (PEG-RR) by in vitro and in vivo systems for their antithrombotic activities. The activity of platelet aggregation inhibition and the bleeding tendency side effect were also investigated. PEG-RR exhibited optimal potency in inhibiting platelet aggregation of human/mouse platelet-rich plasma activated by collagen or ADP with a lower IC50 than the intact derivative RR. In the illumination-induced mesenteric venous thrombosis model, RR and PEG-RR efficaciously prevented occlusive thrombosis in a dose-dependent manner. In rotational thromboelastometry assay, there was no effect of PEG-RR in human whole blood coagulation even given at a higher concentration (30 µg/mL), while RR slightly prolonged clotting time. However, RR and PEG-RR were not associated with severe thrombocytopenia or bleeding in FcγRIIa-transgenic mice at equally efficacious antithrombotic dosages. We also found the in vivo half-life of PEGylation was longer than RR (RR: 15.65 h vs. PEG-RR: 20.45 h). In conclusion, injectable PEG-RR with prolonged half-life and decreased bleeding risk is a safer anti-thrombotic agent for long-acting treatment of thrombus diseases.

Evaluation of carbon tetrachloride fraction of Actinodaphne angustifolia Nees (Lauraceae) leaf extract for antioxidant, cytotoxic, thrombolytic and antidiarrheal properties.

Actinodaphne angustifolia Nees (Family: Lauraceae) is commonly used in folk medicine against urinary disorder and diabetes. The objective of the present study was to evaluate the antioxidant, cytotoxic, thrombolytic, and antidiarrheal activities of carbon tetrachloride (CCl4) fraction of leaves of A. angustifolia (CTFAA) in different experimental models. Antioxidant activity was evaluated by using qualitative and quantitative assays, while antidiarrheal effects assessed with castor oil-induced diarrheal models in mice. The clot lysis and brine shrimp lethality bioassay were used to investigate the thrombolytic and cytotoxic activities, respectively. CTFAA showed antioxidant effects in all qualitative and quantitative procedures. The fraction produced dose-dependent and significant (P<0.05 and P<0.01) activities in castor oil-induced diarrheal models. Moreover, CTFAA significantly (P<0.05) demonstrated a 15.29% clot lysis effect in the thrombolytic test, and the brine shrimp lethality assay LC50 value was 424.16 µg/ml bioassay. In conclusion, the current study showed CTFAA has significant antidiarrheal effects along with modest antioxidant and thrombolytic effects, and these data warrant further experiment to justify and include CTFAA as a supplement to mitigate the onset of diarrheal and cardiovascular disease.

A Pharmacodynamic Study of CN-218, a Novel Antiplatelet and Antithrombotic Agent Primarily Targeting the P2Y12 Receptor.

PURPOSE: Drugs inhibiting the platelet P2Y12 receptor, such as clopidogrel and prasugrel, are potent antithrombotic agents and are widely used in cardiovascular disease. However, the adverse effects of these drugs have limited their clinical use. For example, clopidogrel resistance occurs in approximately one third of patients, while prasugrel increases the risk of major bleeding. Therefore, new generations of such drugs are of clinical interest. METHODS: In this study, the pharmacodynamics of a new P2Y12 antagonist, CN-218, was compared with that of clopidogrel and prasugrel in rats and mice. The differences between CN-218 and clopidogrel include deuteration of the 7-position methyl carboxylate and the introduction of cinnamate in the 2-position of thiophene. RESULTS: CN-218 had an antiaggregatory efficacy that was at least five times more potent than that of clopidogrel but not as potent as that of prasugrel. It had a significant impact on activated partial thromboplastin time (APTT), whereby the APTT of CN-218-treated rats was approximately 9 s longer than that of the vehicle- or clopidogrel-treated group, while it had no impact on prothrombin time (PT) in rats. CN-218 had a similar potent antithrombotic effect to that of prasugrel and clopidogrel and also reduced the risk of bleeding compared to prasugrel. CONCLUSION: CN-218 may be a promising antithrombotic agent, with potent antiplatelet and significant anticoagulant activity, as well as a lower risk of bleeding compared to clopidogrel and prasugrel.

Whole body hypothermia extends tissue plasminogen activator treatment window in the rat model of embolic stroke.

Late treatment with tissue plasminogen activator (tPA) leads to reperfusion injury and poor outcome in ischemic stroke. We have recently shown the beneficial effects of local brain hypothermia after late thrombolysis. Herein, we investigated whether transient whole-body hypothermia was neuroprotective and could prevent the side effects of late tPA therapy at 5.5 h after embolic stroke. After induction of stroke, male rats were randomly assigned into four groups: Control, Hypothermia, tPA and Hypothermia+tPA. Hypothermia started at 5 h after embolic stroke and continued for 1 h. Thirty min after hypothermia, tPA was administrated. Infarct volume, brain edema, blood-brain barrier (BBB) and matrix metalloproteinase-9 (MMP-9) were assessed 48 h and neurological functions were assessed 24 and 48 hour post-stroke. Compared with the control or tPA groups, whole-body hypothermia decreased infarct volume (P < 0.01), BBB disruption (P < 0.05) and MMP-9 level (P < 0.05). However, compared with hypothermia alone a combination of hypothermia and tPA was more effective in reducing infarct volume. While hypothermia alone did not show any effect, its combination with tPA reduced brain edema (P < 0.05). Hypothermia alone or when combined with tPA decreased MMP-9 compared with control or tPA groups (P < 0.01). Although delayed tPA therapy exacerbated BBB integrity, general cooling hampered its leakage after late thrombolysis (P < 0.05). Moreover, only combination therapy significantly improved sensorimotor function as well as forelimb muscle strength at 24 or 48 h after stroke (P < 0.01). Transient whole-body hypothermia in combination with delayed thrombolysis therapy shows more neuroprotection and extends therapeutic time window of tPA up to 5.5 h.

Characterization of active anticoagulant fraction and a fibrin(ogen)olytic serine protease from leaves of Clerodendrum colebrookianum, a traditional ethno-medicinal plant used to reduce hypertension.

ETHNOPHARMACOLOGICAL RELEVANCE: Cardiovascular diseases are the major cause of mortality and morbidity, causing over 17.9 million deaths a year worldwide. Currently used therapy is often having side effects and expensive, dietary interventions and alternative medicines are required. Clerodendrum colebrookianum has been used to treat cardiac hypertension but anticoagulant potency was not evaluated. AIM OF THE STUDY: To characterize an active anticoagulant fraction (AAFCC) and a 30 kDa fibrin(ogen)olytic serine protease (clerofibrase) isolated from aqueous leave extract of C. colebrookianum. MATERIALS AND METHODS: AAFCC/clerofibrase was subjected to extensive biochemical and pharmacological characterization including LC-MS/MS, amino acid compositional and GC-MS analyses. Interaction between clerofibrase with fibrinogen was studied by spectrofluorometric analysis. In vitro thrombolytic, antiplatelet and cytotoxicity assay were performed. In vivo toxicity, anticoagulant, defibrinogen and antithrombotic activities were determined on Swiss albino mice. RESULTS: The in vitro anticoagulant activity of AAFCC was found to be superior to heparin and clerofibrase and comparable to Nattokinase and warfarin. The proteomics and amino acid composition analyses suggest that clerofibrase is a previously uncharacterized novel plant protease capable of degrading the -αß chains of fibrinogen/fibrin. AAFCC/clerofibrase exerts their anticoagulant action via fibrinogenolytic activity and partially by antiplatelet activity albeit they have no effect on thrombin and FXa inhibition. The spectrofluorometric analysis revealed the binding of clerofibrase to fibrinogen but not to thrombin and FXa. The phytochemical constituents and bioactive components of AAFCC were characterized by biochemical, and GC-MS analyses. The AAFCC and clerofibrase inhibited collagen/ADP-induced mammalian platelet aggregation, showed in vitro thrombolytic activity, and non-cytotoxic to mammalian cells. The AAFCC showed and dose-dependent in vivo plasma defibrinogenating and anticoagulant activities and inhibited k-carrageen-induced thrombus formation in the tails of mice. CONCLUSION: The potent in vivo anticoagulant and antithrombotic effects of AAFCC suggests its pharmacological significance as herbal anticoagulant drug for the prevention and/or treatment of hyperfibrinogenemia- and thrombosis associated cardiovascular disorders.

Differential destructive (non-clotting) fibrinogenolytic activity in Afro-Asian elapid snake venoms and the links to defensive hooding behavior.

Envenomations by venomous snakes have major public health implications on a global scale. Despite its medical importance, snakebite has long been a neglected tropical disease by both governments and medical science. Many aspects of the resulting pathophysiology have been largely under-investigated. Most research on snake venom has focused on the neurological effects, with coagulotoxicity being relatively neglected, especially for venoms in the Elapidae snake family. In order to fill the knowledge gap regarding the coagulotoxic effects of elapid snake venoms, we performed functional activity tests to determine the fibrinogenolytic activity of 29 African and Asian elapid venoms across eight genera. The results of this study revealed that destructive (non-clotting) fibrinogenolytic activity is widespread across the African and Asian elapids. This trait evolved independently twice: once in the Hemachatus/Naja last common ancestor and again in Ophiophagus. Further, within Naja this trait was amplified on several independent occasions and possibly explains some of the clinical symptoms produced by these species. Species within the Hemachatus/Naja with fibrinogenolytic activity only cleaved the Aα-chain of fibrinogen, whereas Ophiophagus venoms degraded both the Aα- and the Bß-chain of fibrinogen. All other lineages tested in this study lacked significant fibrinogenolytic effects. Our systematic research across Afro-Asian elapid snake venoms helps shed light on the various molecular mechanisms that are involved in coagulotoxicity within Elapidae.