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Platelet GPIbα–von Willebrand factor (VWF) interaction initiates platelet adhesion, activation, and thrombus growth, especially under high shear conditions. Therefore, the GPIb–VWF axis has been suggested as a promising target against arterial thrombosis. The polysaccharide fucoidan has been reported to have opposing prothrombotic and antithrombotic effects; however, its binding mechanism with platelets has not been adequately studied.

The role of gut microbiota in terms of host health is becoming increasingly important. In this study, the comparative effects of tea polyphenols (TPs) on weight loss and lipid metabolism on conventionalized mice (CVZ) and pseudo germ-free (PGF) mice (treated with antibiotics) were investigated. Our findings revealed that high fat (HF) diet considerably increased the body weight, total fat and upsurge lipid indices in CVZ mice but PGF mice were not sensitive to the effect of HF diet as CVZ mice. After the dietary administration of TP, body weight, perirenal fat and epididymal fat, liver weight, glucose (GLU) level, total chloestrol (TC level), high density lipoprotein-cholesterol (HDL-C) level significantly lowered in PGF mice as compared to CVZ mice group. However, the area of fat cells and triglyceride (TG) level were significantly increased in PGF mice. In CVZ mice, TP intervention resulted in a considerable drop in the Firmicutes/Bacteroides ratio as compared to PGF mice. The intestinal flora of PGF mice was severely reduced after antibiotic treatment, while TP administration restored intestinal diversity; the abundance of Akkermansiaand Lactobacillusincreased, whereas the abundance of Enterobacteriaceae and Prevotellareduced. Overall, we can assume that PGF obese mice administered with TP have less anti-obesity effects compared to obese CVZ mice.

PI3K has been indicated in regulating microvascular permeability changes during inflammation. However, its role in neutrophil‐driven microvascular leakage in acute inflammation remains unclear. Using intravital microscopy in mice, we examined the role of PI3Kγ and PI3Kδ in formyl peptide WKYMVm‐ and chemokine CXCL2‐induced permeability changes and assessed simultaneously neutrophil adhesion and emigration in post‐capillary venules of murine cremaster muscle. We found a PI3Kγ‐specific mechanism in WKYMVm‐induced but not CXCL2‐induced microvascular hyperpermeability. The increased microvascular permeability triggered by WKYMVm was not entirely due to neutrophil adhesion and emigration in cremasteric microvasculature in different PI3K transgenic mouse strains. The PI3Kγ‐specific hyperpermeability was neutrophil‐mediated as this was reduced after depletion of neutrophils in mouse circulation. Chimeric mice with PI3Kγ‐deficient neutrophils but wild‐type endothelium also showed reduced hyperpermeability. Furthermore, we found that the catalytic function of PI3Kγ was required for reactive oxygen species (ROS) generation in neutrophils stimulated with WKYMVm. Pharmacological scavenging PI3Kγ‐dependent ROS in the tissue eliminated the discrepancy in hyperpermeability between different PI3K transgenic mice and alleviated WKYMVm‐induced microvascular leakage in all mouse strains tested. In conclusion, our study uncovers the critical role for PI3Kγ‐dependent ROS generation by neutrophils in formyl peptide‐induced microvascular hyperpermeability during neutrophil recruitment. PI3Kγ‐dependent ROS generation by neutrophils has a critical role in formyl peptide‐induced microvascular hyperpermeability during neutrophil recruitment.

Integrins are a large family of heterodimeric transmembrane receptors differentially expressed on almost all metazoan cells. Integrin β subunits contain a highly conserved plexin-semaphorin-integrin (PSI) domain. The CXXC motif, the active site of the protein-disulfide-isomerase (PDI) family, is expressed twice in this domain of all integrins across species. However, the role of the PSI domain in integrins and whether it contains thiol-isomerase activity have not been explored. Here, recombinant PSI domains of murine β3, and human β1 and β2 integrins were generated and their PDI-like activity was demonstrated by refolding of reduced/denatured RNase. We identified that both CXXC motifs of β3 integrin PSI domain are required to maintain its optimal PDI-like activity. Cysteine substitutions (C13A and C26A) of the CXXC motifs also significantly decreased the PDI-like activity of full-length human recombinant β3 subunit. We further developed mouse anti-mouse β3 PSI domain monoclonal antibodies (mAbs) that cross-react with human and other species. These mAbs inhibited αIIbβ3 PDI-like activity and its fibrinogen binding. Using single-molecular Biomembrane-Force-Probe assays, we demonstrated that inhibition of αIIbβ3 endogenous PDI-like activity reduced αIIbβ3-fibrinogen interaction, and these anti-PSI mAbs inhibited fibrinogen binding via different levels of both PDI-like activity-dependent and -independent mechanisms. Importantly, these mAbs inhibited murine/human platelet aggregation in vitro and ex vivo, and murine thrombus formation in vivo, without significantly affecting bleeding time or platelet count. Thus, the PSI domain is a potential regulator of integrin activation and a novel target for antithrombotic therapies. These findings may have broad implications for all integrin functions, and cell-cell and cell-matrix interactions.

Integrins are a large family of heterodimeric transmembrane receptors differentially expressed on almost all metazoan cells. Integrin β subunits contain a highly conserved plexin-semaphorin-integrin (PSI) domain. The CXXC motif, the active site of the protein-disulfide-isomerase (PDI) family, is expressed twice in this domain of all integrins across species. However, the role of the PSI domain in integrins and whether it contains thiol-isomerase activity have not been explored. Here, recombinant PSI domains of murine β3, and human β1 and β2 integrins were generated and their PDI-like activity was demonstrated by refolding of reduced/denatured RNase. We identified that both CXXC motifs of β3 integrin PSI domain are required to maintain its optimal PDI-like activity. Cysteine substitutions (C13A and C26A) of the CXXC motifs also significantly decreased the PDI-like activity of full-length human recombinant β3 subunit. We further developed mouse anti-mouse β3 PSI domain monoclonal antibodies (mAbs) that cross-react with human and other species. These mAbs inhibited αIIbβ3 PDI-like activity and its fibrinogen binding. Using single-molecular Biomembrane-Force-Probe assays, we demonstrated that inhibition of αIIbβ3 endogenous PDI-like activity reduced αIIbβ3-fibrinogen interaction, and these anti-PSI mAbs inhibited fibrinogen binding via different levels of both PDI-like activity-dependent and -independent mechanisms. Importantly, these mAbs inhibited murine/human platelet aggregation in vitro and ex vivo, and murine thrombus formation in vivo, without significantly affecting bleeding time or platelet count. Thus, the PSI domain is a potential regulator of integrin activation and a novel target for antithrombotic therapies. These findings may have broad implications for all integrin functions, and cell-cell and cell-matrix interactions.

As a flexible AC transmission system equipment, controlled shunt reactor is becoming increasingly important in the grid with the development of EHV / UHV transmission systems. Since currently rated voltage of controlled shunt reactor has developed to 1100kV, as its capacity control system, or valve-control system, stability and reliability test is very important. In this paper, the test circuit and method for the valve-control system in the laboratory or the test station for combined adjustment test is put forward. By adjustment of power supply and the device parameter, the valve-control system can put the voltage and current of the device to be the same as the on-set operation. Actual test of the world's first 1100kV controlled shunt reactor valve-control system shows that, the test method in this paper is correct and feasible, and the objective of assessment can be achieved.

This paper focuses on spatial data characteristics of coalfield geological information and uses GIS technology to analyze and manage geological information resources of the coalfield efficiently. It also designs the geographic information system of coalfield and receives the secondary development of GIS database system using ArcGIS. In the development and design process of database system, this paper uses the ArcEngine component and develops data system in the Net platform of MircoSoft Company and uses programming control method of C # programming language to manage coalfield geology data. Finally, this paper establishes the contact between the data content, data attribute and geographic information system. It designs visual information view mode of coalfield geology. It improves management efficiency of coalfield’s geological information and provides technical reference for the study of coalfield’s geology information.

Background:Integrins are α and β subunit heterodimeric receptors expressed ubiquitously on metazoan cells that play key roles in cell adhesion, movement, and signaling. The αIIbβ3 integrin on platelets is essential for thrombosis and hemostasis through its binding of fibrinogen and other ligands to mediate platelet adhesion and aggregation. The plexin-semaphorin-integrin (PSI) domain is an approximately 54 amino acid sequence at the N-terminus of the β3 subunit located in close proximity to the “knee” region. Our lab recently discovered that two “CXXC” motifs within PSI domains in the integrin family exert endogenous thiol isomerase activity, and blockade of this activity in β3 integrin with our novel anti-PSI domain monoclonal antibodies (mAbs) significantly attenuated platelet adhesion and aggregation without impairing hemostasis (Blood,2017). Interestingly, intravital microscopy studies demonstrate that our anti-PSI antibodies inhibited thrombosis in vivo10-30 fold more potent than their effects in vitrounder anti-coagulant conditions, suggesting a possible inhibitory effect on blood coagulation. Notably, A L33P polymorphism (HPA-1b) within the β3 integrin PSI domain is linked to an approximately two-fold increased risk for cardiovascular disease (CVD), however, the underlying mechanism for this remains unclear.

No relevant conflicts of interest to declare.

Introduction:Danshen (Salvia miltiorrhizaroot extracts) have been reported for centuries to control cardiovascular diseases in Chinese medicine. Danshen depside salts are currently approved in China to treat coronary heart disease and angina and are under evaluation in phase II clinical trials outside of China. Salvianolic acids have been identified as the active compounds of danshen and salvianolic acid B (SAB), the most abundant salvianolic acid, has been previously shown to exhibit anti-platelet and anti-thrombotic properties in animal models. However, the mechanism of action has not been adequately explored.Results:We demonstrate that SAB attenuates ADP-, collagen-, and thrombin receptor activating peptide (TRAP)-induced human platelet aggregation. Interestingly, SAB inhibits thrombin-induced platelet aggregation far more potently than other agonists. Furthermore, using our intravital microscopy thrombosis models, we demonstrate that SAB decreases thrombus growth in vivo. Using a series of in vitrocoagulation assays, we found that SAB significantly reduced clot weight in human whole blood, and delayed coagulation in human cell-free plasma using thromboelastography. In addition, SAB reduces fibrin network density in cell-free plasma. Through structural analysis, we found that SAB contains structural similarities to trisubstituted benzimidazole thrombin inhibitors, such as dabigatran. In silicomolecular modeling predicts that SAB binds within the thrombin active site - interacting with similar residues as dabigatran. Using isothermal titration calorimetry and kinetic thrombin inhibition assays, we corroborate these findings and report SAB as a direct competitive thrombin inhibitor.Conclusions:These data establish a novel mechanism of SAB in the inhibition of both platelet aggregation and blood coagulation. These unique characteristics position salvianolic acids as safe and potent herb-derived anti-thrombotic agents.

No relevant conflicts of interest to declare.

Introduction:

No relevant conflicts of interest to declare.

Platelet adhesion and aggregation at sites of vascular injury are key events in thrombosis and hemostasis. Platelet β3 integrins and their ligands are essential in mediating these processes. Therefore the understanding of β3 integrin-ligand interactions is crucial in elucidating mechanisms of thrombosis and hemostasis. In an effort to identify unknown ligands for β3 integrin, we used immobilized human platelet β3 integrin to capture proteins from human plasma. The isolated proteins were further analysed by 2D electrophoresis and mass spectrometry, and apolipoprotein A-IV (apoA-IV) was identified. ApoA-IV is an abundant plasma lipid binding protein secreted by the small intestine during dietary lipid absorption. Several studies in different ethnic populations have suggested that the level of apoA-IV is inversely correlated with cardiovascular diseases. However, the roles of apoA-IV in platelets and thrombosis are completely unknown.

Platelet adhesion and subsequent aggregation at the site of vascular injury are critical for hemostasis and thrombosis. It has been well accepted that interaction between the GPIb complex and von Willebrand factor (VWF) plays a key role in initiation of platelet adhesion, particularly at high shear. Platelet surface integrin αIIbβ3, through interaction with fibrinogen or other ligands, then mediates platelet aggregation to form a stable hemostatic plug or thrombus. Recently, the indispensable role of the GPIb-VWF interaction in platelet aggregation at extremely high shear (e.g. > 10,000s-1; areas of stenosis following arteriosclerosis and/or thrombus growth) has been highlighted. Therefore, both the GPIb complex and αIIbβ3 are considered major targets for antithrombotic therapies. Interestingly, although several inhibitors of αIIbβ3 have been developed for antithrombotic therapies, no drug has been developed to target the GPIb complex even though there are limitations for anti-αIIbβ3 therapies. The GPIb complex is, therefore, an attractive target for anti-thrombotic therapy. Here, we evaluated the efficacy and safety in vitro and in vivo of Anfibatide, a novel GPIb antagonist, in mice and in a phase I clinical trial.Anfibatide was purified from venom of the Agkistrodon acutus snake and its purity was analyzed by mass spectrometry. The effect of Anfibatide on murine platelet function was assessed by in vitro platelet aggregometry, ex vivo perfusion chamber, and two complementary in vivo intravital microscopy models. The effects of Anfibatide on human platelet aggregation and thrombus formation were studied in vitro, and thrombealastography (TEG) was also performed. Most importantly, we evaluated the safety and efficacy of Anfibatide on platelet function and coagulation in a total of 94 healthy human volunteers in a phase I clinical trial.MALDI-TOF mass spectrometry of Anfibatide showed only one peak and the mass to charge ratio is 29799.7. Anfibatide specifically inhibited ristocetin-induced human platelet aggregation. Interestingly, Anfibatide was not able to inhibit botrocetin-induced murine platelet aggregation in plate-rich plasma (PRP), suggesting that its binding site may differ from other snake venom-derived GPIb antagonists. We found Anfibatide did not affect ADP-, TRAP- or collagen-induced aggregation in PRP, suggesting its specificity to GPIb. In ex vivo perfusion, Anfibatide strongly inhibited murine and human platelet adhesion, aggregation, and thrombus formation on a collagen-coated surface at both high and low shear flow conditions although it is far more sensitive at high shear. Importantly, Anfibatide effectively dissolved the preformed thrombi when we continuously perfused Anfibatide-treated whole blood through perfusion chambers, demonstrating its potential as an anti-thrombotic therapy. In the mesenteric arteriole thrombosis model, Anfibatide strongly inhibited platelet adhesion, thrombus formation, and prevented vessel occlusion in response to FeCl3 injury (P<0.05). At sites of laser-injured cremaster arterioles, Anfibatide also dramatically inhibited platelet accumulation and thrombus growth. Anfibatide did not cause significant murine platelet activation in vitro and had no significant change in coagulation parameters in TEG when we treated human whole blood with Anfibatide, suggesting it had minimal side effects. In the phase I clinical trial, results showed that Anfibatide can occupy approximately 95% of GPIb and inhibit up to 90% of ristocetin specific platelet aggregation. The inhibitory effect was undetectable four hours after Anfibatide was withdrawn. There were no serious adverse events, or deaths that occurred during the study. Anfibatide did not significantly prolong bleeding time, activated partial thromboplastin time (APTT), prothrombin time (PT), or thrombin time (TT). There was also no spontaneous bleeding or bleeding from blood collection sites. Anfibatide did not significantly affect platelet count and no anti-Anfibatide antibodies were detected in the subjects, suggesting that Anfibatide is well-tolerated in healthy individuals.These comprehensive studies in mice and human subjects and in the first clinical trial clearly demonstrated that Anfibatide is a safe and potent anti-platelet reagent with great potential for future anti-thrombotic therapy.Hou: Lee’s pharmaceutical holdings limited: Research Funding. Lei:Lee’s pharmaceutical holdings limited: Research Funding. Zhao:Lee’s pharmaceutical holdings limited: Research Funding. Shen:Lee’s pharmaceutical holdings limited: Research Funding. Zhou:Lee’s pharmaceutical holdings limited: Research Funding. Wang:Lee’s pharmaceutical holdings limited: Research Funding. Marshall:Lee’s pharmaceutical holdings limited: Research Funding. Ni:Lee’s pharmaceutical holdings limited: Research Funding.

Platelet adhesion and subsequent aggregation at the site of vascular injury are critical for hemostasis and thrombosis. It has been well accepted that interaction between the GPIb complex and von Willebrand factor (VWF) plays a key role in initiation of platelet adhesion, particularly at high shear. Platelet surface integrin αIIbβ3, through interaction with fibrinogen or other ligands, then mediates platelet aggregation to form a stable hemostatic plug or thrombus. Recently, the indispensable role of the GPIb-VWF interaction in platelet aggregation at extremely high shear (e.g. > 10,000s-1; areas of stenosis following arteriosclerosis and/or thrombus growth) has been highlighted. Therefore, both the GPIb complex and αIIbβ3 are considered major targets for antithrombotic therapies. Interestingly, although several inhibitors of αIIbβ3 have been developed for antithrombotic therapies, no drug has been developed to target the GPIb complex even though there are limitations for anti-αIIbβ3 therapies. The GPIb complex is, therefore, an attractive target for anti-thrombotic therapy. Here, we evaluated the efficacy and safety in vitro and in vivo of Anfibatide, a novel GPIb antagonist, in mice and in a phase I clinical trial.