scholarly journals A Bioassay-Based Approach for the Batch-To-Batch Consistency Evaluation of Xuesaitong Injection on a Zebrafish Thrombosis Model

2021 ◽  
Vol 12 ◽  
Author(s):  
Xiangwei Ma ◽  
Yanyu Chen ◽  
Shumin Jiang ◽  
Xiaoping Zhao
Keyword(s):  
Quality Control ◽  
High Performance ◽  
Green Fluorescence Protein ◽  
Coagulation Cascade ◽  
Dependent Manner ◽  
Zebrafish Model ◽  
Ginsenoside Rd ◽  
Thrombosis Model ◽  
Fluorescence Protein

Quality control of Chinese medicine (CM) is mainly based on chemical testing, which sometimes shows weak correlation to pharmacological effects. Thus, there is a great demand to establish bioactivity-based assays to ensure the quality of CM. The aim of the present study was to establish a bioassay-based approach to evaluate the biological activity of Xuesaitong injection (XST) based on an in vivo zebrafish model. Zebrafish larvae with arachidonic acid (AA)-induced thrombus were applied to evaluate anti-thrombosis effects of XST and explore the potential mechanism of XST. Analysis of major components in normal and abnormal XST samples was performed by high performance liquid chromatography (HPLC). The results indicate that XST could significantly restore heart red blood cells (RBCs) intensity of thrombotic zebrafish in a dose-dependent manner, whilst decreasing RBCs accumulation in the caudal vein. The results were confirmed using a green fluorescence protein (GFP)-labeled zebrafish thrombosis model. Moreover, we could show that XST downregulates the expression of the fibrinogen alpha chain (fga) gene to inhibit the coagulation cascade during the process of thrombosis in zebrafish. Notoginsenoside R1, ginsenoside Rg1, ginsenoside Rb1 and ginsenoside Rd, which were considered to be the major components of XST, also showed moderate anti-thrombosis efficacy. Further results showed that the zebrafish thrombosis model could efficiently distinguish five abnormal batches of XST from 24 normal batches. Furthermore, the inhibition rates of different batches were correlated with the content level of major components. Our results suggested that the proposed zebrafish thrombosis model could be successfully used to evaluate the batch-to-batch consistency of XST, which provided an alternative way for the quality control of CM.

scholarly journals Protective Effect of Phillyrin on Lethal LPS-Induced Neutrophil Inflammation in Zebrafish

2017 ◽  
Vol 43 (5) ◽  
pp. 2074-2087 ◽  
Author(s):  
Liling Yang ◽  
Xiangjun Zhou ◽  
Weijuan Huang ◽  
Qin Fang ◽  
Jianlan Hu ◽  
...  
Keyword(s):  
Signalling Pathway ◽  
Dependent Manner ◽  
Zebrafish Model ◽  
Lps Challenge ◽  
Cell Counts ◽  
Tnf Α ◽  
Forsythia Suspensa ◽  
Anti Inflammatory

Background/Aims: Forsythia suspensa Vahl. (Oleaceae) fruits are widely used in traditional Chinese medicine to treat pneumonia, typhoid, dysentery, ulcers and oedema. Antibacterial and anti-inflammatory activities have been reported for phillyrin (PHN), the main ingredient in Forsythia suspensa Vahl fruits, in vitro. However, the underlying mechanisms in vivo remain poorly defined. In this study, we discovered that PHN exerted potent anti-inflammatory effects in lethal LPS-induced neutrophil inflammation by suppressing the MyD88-dependent signalling pathway in zebrafish. Methods: LPS-yolk microinjection was used to induce a lethal LPS-infected zebrafish model. The effect of PHN on the survival of zebrafish challenged with lethal LPS was evaluated using survival analysis. The effect of PHN on neutrophil inflammation grading in vivo was assessed by tracking neutrophils with a transgenic line. The effects of PHN on neutrophil production and migration were analysed by SB+ cell counts during consecutive hours after modelling. Additionally, key cytokines and members of the MyD88 signalling pathway that are involved in inflammatory response were detected using quantitative RT-PCR. To assess gene expression changes during consecutive hours after modelling, the IL-1β, IL-6, TNF-α, MyD88, TRIF, ERK1/2, JNK, IκBa and NF-κB expression levels were measured. Results: PHN could protect zebrafish against a lethal LPS challenge in a dose-dependent manner, as indicated by decreased neutrophil infltration, reduced tissue necrosis and increased survival rates. Up-regulated IL-1β, IL-6 and TNF-α expression also showed the same tendencies of depression by PHN. Critically, PHN significantly inhibited the LPS-induced activation of MyD88, IκBa, and NF-κB but did not affect the expression of ERK1/2 MAPKs or JNK MAPKs in LPS-stimulated zebrafish. Additionally, PHN regulated the MyD88/IκBα/NF-κB signalling pathway by controlling IκBα, IL-1β, IL-6, and TNF-α expression. Conclusion: This study provides a rationale for the clinical application of PHN as an anti-inflammatory agent.

scholarly journals A Mechano-Activated Cell Reporter System as a Proxy for Flow-Dependent Endothelial Atheroprotection

2018 ◽  
Vol 23 (8) ◽  
pp. 869-876
Author(s):  
Bendix R. Slegtenhorst ◽  
Oscar R. Fajardo Ramirez ◽  
Yuzhi Zhang ◽  
Zahra Dhanerawala ◽  
Stefan G. Tullius ◽  
...  
Keyword(s):  
Vascular Endothelium ◽  
Critical Role ◽  
Green Fluorescence Protein ◽  
Dependent Manner ◽  
Reporter System ◽  
Health And Disease ◽  
Fluorescence Protein ◽  
Biomechanical Stimuli ◽  
Dose Dependent

The vascular endothelium plays a critical role in the health and disease of the cardiovascular system. Importantly, biomechanical stimuli generated by blood flow and sensed by the endothelium constitute important local inputs that are translated into transcriptional programs and functional endothelial phenotypes. Pulsatile, laminar flow, characteristic of regions in the vasculature that are resistant to atherosclerosis, evokes an atheroprotective endothelial phenotype. This atheroprotective phenotype is integrated by the transcription factor Kruppel-like factor-2 (KLF2), and therefore the expression of KLF2 can be used as a proxy for endothelial atheroprotection. Here, we report the generation and characterization of a cellular KLF2 reporter system, based on green fluorescence protein (GFP) expression driven by the human KLF2 promoter. This reporter is induced selectively by an atheroprotective shear stress waveform in human endothelial cells, is regulated by endogenous signaling events, and is activated by the pharmacological inducer of KLF2, simvastatin, in a dose-dependent manner. This reporter system can now be used to probe KLF2 signaling and for the discovery of a novel chemical-biological space capable of acting as the “pharmacomimetics of atheroprotective flow” on the vascular endothelium.

“M-AAA-Thrombin”, a New Thrombin Mutant with Potent Anticoagulant and Antiplatelet Properties.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4154-4154
Author(s):  
Kazuya Hosokawa ◽  
Tomoko Ohnishi ◽  
Hiroyuki Matsuda ◽  
Kousuke Kashima ◽  
Takehiko Koide
Keyword(s):  
Platelet Aggregation ◽  
Thrombus Formation ◽  
Antiplatelet Agent ◽  
Dependent Manner ◽  
In Vivo Experiments ◽  
Thrombosis Model ◽  
Venous Shunt ◽  
Prolonged Aptt ◽  
Antithrombotic Efficacy

Abstract Thrombosis is a major cause of morbidity and mortality, and thrombin is a major inducer of thrombus formation. Thus several antithrombotic agents targeting thrombin have been developed. We previously reported an anticoagulant and antiplatelet thrombin derivative, ‘M-anhydrothrombin’ prepared by chemical modifications. In this study, we prepared a new thrombin mutant, specificity of which was highly modulated with substantially improved antithrombotic efficacy. The thrombin mutant designated “AAA-Thrombin” in which Lys65, His43 and Ser205 in B-chain have been replaced by Ala revealed higher affinity and specificity for factor VIII with no enzymatic activity. AAA-Thrombin prolonged APTT much more than anhydrothrombin in a dose dependent manner without affecting PT and TT. Platelet aggregation induced by activation of PAR-1 was also effectively suppressed by AAA-Thrombin. “M-AAA-Thrombin” prepared by further chemical modification of carboxyl groups in AAA-Thrombin enhanced its antithrombotic efficacy. M-AAA-Thrombin (250nM) prolonged APTT approx. two times, and suppressed platelet aggregation by PAR-1 activation, while AAA-Thrombin did not at the same concentration. M-AAA-Thrombin also suppressed ristocetin-induced platelet aggregation. In vivo experiments, M-AAA-Thrombin demonstrated significant antithrombotic property in the arterio-venous shunt thrombosis model and the FeCl3-induced carotid artery thrombosis model in guinea pigs. These results indicate that M-AAA-Thrombin would be a candidate for quite an innovative anticoagulant and antiplatelet agent for both arterial and venous thromboses. Further optimization of mutagenesis and modification, in terms of efficacy and safety is in progress in our laboratory.

Procoagulant and prothrombotic activation of human erythrocytes by phosphatidic acid

2010 ◽  
Vol 299 (2) ◽  
pp. H347-H355 ◽  
Author(s):  
Ji-Yoon Noh ◽  
Kyung-Min Lim ◽  
Ok-Nam Bae ◽  
Seung-Min Chung ◽  
Sang-Wook Lee ◽  
...  
Keyword(s):  
Phosphatidic Acid ◽  
Thrombus Formation ◽  
Biological Activities ◽  
Human Erythrocytes ◽  
Erythrocyte Aggregation ◽  
Dependent Manner ◽  
Disease States ◽  
Thrombosis Model ◽  
Intracellular Ca

Increased phosphatidic acid (PA) and phospholipase D (PLD) activity are frequently observed in various disease states including cancers, diabetes, sepsis, and thrombosis. Previously, PA has been regarded as just a precursor for lysophosphatidic acid (LPA) and diacylglycerol (DAG). However, increasing evidence has suggested independent biological activities of PA itself. In the present study, we demonstrated that PA can enhance thrombogenic activities in human erythrocytes through phosphatidylserine (PS) exposure in a Ca2+-dependent manner. In freshly isolated human erythrocytes, treatment of PA or PLD induced PS exposure. PA-induced PS exposure was not attenuated by inhibitors of phospholipase A2or phosphatidate phosphatase, which converts PA to LPA or DAG. An intracellular Ca2+increase and the resultant activation of Ca2+-dependent PKC-α appeared to underlie the PA-induced PS exposure through the activation of scramblase. A marginal decrease in flippase activity was also noted, contributing further to the maintenance of exposed PS on the outer membrane. PA-treated erythrocytes showed strong thrombogenic activities, as demonstrated by increased thrombin generation, endothelial cell adhesion, and erythrocyte aggregation. Importantly, these procoagulant activations by PA were confirmed in a rat in vivo venous thrombosis model, where PA significantly enhanced thrombus formation. In conclusion, these results suggest that PA can induce thrombogenic activities in erythrocytes through PS exposure, which can increase thrombus formation and ultimately contribute to the development of cardiovascular diseases.

scholarly journals Actin-dependent Lamellipodia Formation and Microtubule-dependent Tail Retraction Control-directed Cell Migration

2000 ◽  
Vol 11 (9) ◽  
pp. 2999-3012 ◽  
Author(s):  
Christoph Ballestrem ◽  
Bernhard Wehrle-Haller ◽  
Boris Hinz ◽  
Beat A. Imhof
Keyword(s):  
Cell Migration ◽  
Cell Body ◽  
Actin Polymerization ◽  
Green Fluorescence Protein ◽  
Time Lapse ◽  
Dependent Manner ◽  
Directed Cell Migration ◽  
Cell Front ◽  
Fluorescence Protein

Migrating cells are polarized with a protrusive lamella at the cell front followed by the main cell body and a retractable tail at the rear of the cell. The lamella terminates in ruffling lamellipodia that face the direction of migration. Although the role of actin in the formation of lamellipodia is well established, it remains unclear to what degree microtubules contribute to this process. Herein, we have studied the contribution of microtubules to cell motility by time-lapse video microscopy on green flourescence protein-actin- and tubulin-green fluorescence protein–transfected melanoma cells. Treatment of cells with either the microtubule-disrupting agent nocodazole or with the stabilizing agent taxol showed decreased ruffling and lamellipodium formation. However, this was not due to an intrinsic inability to form ruffles and lamellipodia because both were restored by stimulation of cells with phorbol 12-myristate 13-acetate in a Rac-dependent manner, and by stem cell factor in melanoblasts expressing the receptor tyrosine kinase c-kit. Although ruffling and lamellipodia were formed without microtubules, the microtubular network was needed for advancement of the cell body and the subsequent retraction of the tail. In conclusion, we demonstrate that the formation of lamellipodia can occur via actin polymerization independently of microtubules, but that microtubules are required for cell migration, tail retraction, and modulation of cell adhesion.

scholarly journals A Naphthalenic Derivative ND-1 Inhibits Thrombus Formation by Interfering the Binding of Fibrinogen to Integrin αIIbβ3

2016 ◽  
Vol 2016 ◽  
pp. 1-8 ◽  
Author(s):  
Xue Ding ◽  
Tong-dan Liu ◽  
Zhou-ling Xie ◽  
Qi Zhao ◽  
Yuan Cao ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Thrombus Formation ◽  
Antiplatelet Agents ◽  
Bleeding Risk ◽  
Dependent Manner ◽  
Antithrombotic Effect ◽  
Thrombosis Model ◽  
Prolonged Bleeding Time ◽  
Cutting Model

Integrin αIIbβ3 plays a crucial role in the process of platelet aggregation. Three integrin αIIbβ3 antagonists (abciximab, eptifibatide, and tirofiban) have been approved by FDA for clinical use. Unfortunately, they all showed severe side effects such as thrombocytopenia and bleeding risk. Thus, researches on the development of more effective and safer antiplatelet agents are needed. In this manuscript we reported a novel naphthalenic derivative compound ND-1 with potent antithrombotic effect and lower bleeding risk. ND-1 inhibited ADP-, collagen-, thrombin-, and U46619-induced platelet aggregation with IC50 values of 1.29, 14.46, 12.84, and 40.24 μM, respectively. Mechanism studies indicated that ND-1 inhibited the binding of fibrinogen to integrin αIIbβ3 in a dose-dependent manner with an IC50 value of 3.12 μM. ND-1 inhibited P-selectin expression induced by ADP, collagen, thrombin, and U46619 on the surface of platelets. Additionally, this compound reduced platelets spreading to the immobilized fibrinogen. In vivo, ND-1 potently decreased thrombus formation in an arteriovenous shunt thrombosis model in rats and slightly prolonged bleeding time in a tail cutting model in mice. Taken together, our results reveal that ND-1 is a novel antagonist of αIIbβ3 with strong antithrombotic effect and lower bleeding risk.

scholarly journals Hypoglycemic Activity of Aqueous Extract of Latex from Hancornia speciosa Gomes: A Study in Zebrafish and In Silico

2021 ◽  
Vol 14 (9) ◽  
pp. 856
Author(s):  
Rosana Tomazi ◽  
Ângela Costa Figueira ◽  
Adriana Maciel Ferreira ◽  
Diego Quaresma Ferreira ◽  
Gisele Custódio de Souza ◽  
...  
Keyword(s):  
Aqueous Extract ◽  
In Silico ◽  
High Performance ◽  
Therapeutic Potential ◽  
In Silico Analysis ◽  
Hypoglycemic Activity ◽  
Zebrafish Model ◽  
Phytochemical Study ◽  
Hancornia Speciosa

Hancornia speciosa Gomes is a tree native to Brazil and has therapeutic potential for several diseases. Ethnopharmacological surveys have reported that the plant is used as a hypoglycemic agent and to lose weight. This study aimed to evaluate the effects of the aqueous extract from H. speciosa latex (LxHs) in a zebrafish model of diabetes. The extract was evaluated through high-performance thin-layer chromatography (HTPLC), nuclear magnetic resonance (NMR), and Fourier-transform infrared spectroscopy (FT-IR). We then tested treatments with LxHs (500, 1000, and 1500 mg/kg) by assessing blood glucose levels in alloxan-induced diabetic animals, and metformin was used as a control. The toxicity was evaluated through histopathology of the pancreas and biochemical assessment of serum levels of AST, ALT, creatinine, and urea. The extract was also assessed for acute toxicity through several parameters in embryos and adult animals. Finally, we performed in silico analysis through the SEA server and docking using the software GOLD. The phytochemical study showed the compounds cornoside, dihydrocornoide, and 1-O-methyl-myoinositol (bornesitol). The treatment with all doses of LxHs significantly decreased alloxan-induced hyperglycemia without any significant histological or biochemical abnormalities. No significant frequency of teratogenesis was observed in the embryos exposed to the extract, and no significant behavioral changes or deaths were observed in adult animals. In silico, the results showed a potential interaction between inositol and enzymes involved in carbohydrates’ metabolism. Overall, the results show a hypoglycemic activity of the extract in vivo, with no apparent toxicity. The computational studies suggest this could be at least partially due to the presence of bornesitol, since inositols can interact with carbohydrates’ enzymes.

scholarly journals Monitoring Compound-Related Effects on Coagulability in Rats and Cynomolgus and Rhesus Monkeys by Thrombin Generation Kinetic Measurement

2020 ◽  
Vol 39 (3) ◽  
pp. 207-217
Author(s):  
F. Poitout-Belissent ◽  
D. Culang ◽  
D. Poulin ◽  
R. Samadfan ◽  
S. Cotton ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Thrombin Generation ◽  
In Vivo Studies ◽  
Coagulation Cascade ◽  
Dependent Manner ◽  
Kinetic Assay ◽  
Thrombin Generation Assay ◽  
Coefficients Of Variation

Thrombin generation assay (TGA) is a sensitive method for the assessment of the global clotting potential of plasma. This kinetic assay can detect both hypocoagulable and hypercoagulable conditions: delayed or reduced thrombin generation leading to a prolonged clotting time, or induced thrombin activity, shifting the coagulation cascade toward thrombosis. The purpose of this study is to qualify the TGA in nonhuman primates (NHP) and rats for its use during nonclinical in vivo and in vitro studies. Blood was drawn from nonanesthetized animals, and platelet-poor plasma was obtained after double centrifugation; coefficients of variation were <10% for all derived parameters of thrombin generation assessed with 5 pM of tissue factor. Thrombin generation was evaluated in vitro in rat and NHP plasmas with ascending doses of unfractionated heparin (UFH), recombinant tissue factor, and anticoagulant compounds. Thrombin generation was decreased with UFH and anticoagulant compounds, but was increased in the presence of tissue factor, in a dose-dependent manner. In a rat model of inflammation, animals were administered a low dose of lipopolysaccharides. Thrombin generation measurements were decreased 3 hours post-LPS administration with a nadir at 24 hours, while thrombin–antithrombin complexes reached a peak at 8 hours, supporting an earlier production of thrombin. In conclusion, these data demonstrated that TGA can be performed in vitro for screening of compounds expected to have effects on coagulation cascade, and thrombin generation can be measured at interim time points during nonclinical in vivo studies in rats and NHP.

Simultaneous Determination of Saponins and Lignans in Rat Plasma by UPLC-MS/MS and its application to a pharmacokinetic study of Shenqi Jiangtang Granule

2021 ◽  
Vol 22 ◽  
Author(s):  
Hui Zhang ◽  
Ruoyu Chen ◽  
Cong Xu ◽  
Ya Zhang ◽  
Qinghua Tian ◽  
...  
Keyword(s):  
Oral Administration ◽  
Simultaneous Determination ◽  
High Performance ◽  
Negative Ion ◽  
Schisandrin B ◽  
Limit Of Quantification ◽  
Ginsenoside Re ◽  
Ginsenoside Rd

Background: Shenqi Jiangtang Granule (SJG), a classical prescription of traditional Chinese medicine, is widely used to treat diabetes and its complications. Despite the clinical efficacy of SJG is effective, pharmacokinetic behavior of various substance in plasma of SJG are unknown. Objective: The aim of this study was to investigate the plasma pharmacokinetics during absorption of SJG after oral administration in rats. Methods: A rapid and accurate ultra-high performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) method was developed for the simultaneous determination of eight analytes in SJG, including gomisin D, schisandrin A, schisandrin B, schizandrol A, schizandrol B, ginsenoside Rd, ginsenoside Re and notoginsenoside Ft1. The analysis was carried out on a BEH C18 column (2.1 mm × 50 mm, 1.7 μm) with gradient elution at a flow rate of 0.2 mL/min in a mobile phase consisting of 0.1% formic acid water and acetonitrile. In addition, lignans and saponins were detected in positive ion mode and negative ion mode, respectively. Results: Eight analytes in SJG, including gomisin D, schisandrin A, schisandrin B, schizandrol A, schizandrol B, ginsenoside Rd, ginsenoside Re and notoginsenoside Ft1, showed good linearity (R2 in the range of 0.9955~0.9999). The lower limit of quantification (LLOQ) was 5, 0.8, 0.8, 8, 0.8, 5, 0.6 and 10 ng/mL. Accuracy and precision of all analytes were at ±15%. Matrix effect and average extraction recovery were > 85%. All analytes performed well under four storage conditions. Conclusions: The results showed that in vivo absorption and exposure of gomisin D and ginsenoside Rd were better than other analytes, while schizandrol B and notoginsenoside Ft1 were poorly absorbed. This approach could be applied to study the pharmacokinetic characteristics of various analytes in plasma after oral administration of SJG in rats.

In Vivo Anti-Thrombotic Potency of Engineered Activated Protein C Variants.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2704-2704
Author(s):  
Laurent O. Mosnier ◽  
Jose A. Fernandez ◽  
Antonella Zampolli ◽  
Xia V. Yang ◽  
Zaverio M. Ruggeri ◽  
...  
Keyword(s):  
Protein C ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Protein S ◽  
Dependent Manner ◽  
Thrombosis Model ◽  
Factor Va ◽  
Cofactor Activity

Abstract Activated protein C (APC) has both anticoagulant activity via inactivation of factors Va and VIIIa and cytoprotective activities on cells that include anti-apoptotic and anti-inflammatory activities, alterations of gene expression profiles and protection of endothelial barrier function. The relative importance of APC’s anticoagulant activity vs. APC’s direct cytoprotective effects on cells for reduction of mortality in severe sepsis patients and protective effects in animal injury models is not entirely clear. In this current study, genetically engineered APC variants with different activity spectra were tested for in vivo anti-thrombotic potency. Recently we made a non-anticoagulant APC variant, 5A-APC (RR229/230AA and KKK191-193AAA), that retains normal in vitro cytoprotective effects and an ability to reduce mortality in murine sepsis models (Kerschen et al, ASH2006, J Exper Med, 2007). In contrast to 5A-APC, mutation of E149 to A in APC increased anticoagulant activity in clotting assays while diminishing cytoprotective effects on cells. Murine APC variants, E149A-APC and 5A-APC (KKK192-194AAA + RR230/231AA) were used to determine in vivo anti-thrombotic potency in an acute carotid artery thrombosis model in mice, using FeCl3-induced injury. Under the conditions employed, first occlusion occurred within 3.5 min (mean: 171 sec; range 150-200 sec) in the absence of APC. Murine wild type (wt)-APC effectively delayed time to first occlusion in a dose-dependent manner (0 to 1.8 mg/kg wt-APC; mean: 561 sec; range 400-960 sec). The E149A-APC variant exhibited potent in vivo anti-thrombotic activity (1.8 mg/kg; mean: 1020 sec; range 540- >1600 sec) and was superior to wt-APC as evident by the absence of appreciable occlusion in 2/6 E149A-APC vs. 0/6 wt-APC treated animals. Thus E149A-APC was hyperactive in plasma clotting assays as well as hyperactive in an acute FeCl3-induced arterial thrombosis model. To test the hypothesis that an increased protein S cofactor activity contributed to its enhanced anticoagulant activity, E149A-APC anticoagulant activity was tested in normal and protein S deficient plasma. Compared to wt-APC, E149A-APC showed 3-fold increased anticoagulant activity in normal plasma but not in protein S deficient plasma. In studies with purified proteins, protein S concentrations required for half-maximal stimulation of factor Va inactivation by E149A-APC were 3-fold lower compared to wt-APC, whereas factor Va inactivation rates were indistinguishable in the absence of protein S. These data support our hypothesis that increased protein S cofactor activity is, at least partially, responsible for the observed hyper anticoagulant and anti-thrombotic potency in vitro and in vivo. In contrast to E149A-APC, 5A-APC was severely deficient in anti-thrombotic activity in vivo. Even at concentrations up to 8 mg/kg, 5A-APC (mean: 245 sec; range 172-300 sec) failed to delay significantly time to first occlusion compared to no APC. These data highlight important distinctions between structural requirements for APC’s anticoagulant, anti-thrombotic and cytoprotective functions. Engineered APC variants with differentially altered activities (e.g. cytoprotective vs. anticoagulant) may lead to safer or better therapeutic APC variants for a variety of indications including sepsis, ischemic stroke or other pathologies.

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