OBJECTIVE: To explore the main factors of platelet spreading and provide the foundation for related research. METHODS: Platelets (2×10⁷/ml) were draw from C57BL/6J mouse and kept at 22 ℃ for 1-2 hours. Platelets (2×10⁷/ml) were were allowed to adhere and spread on the fibrinogen-coated slides, after staining F-actin in platelets, the platelets were observed with the confocal microscopy. The effects of different concentrations of fibrinogen (10 μg/ml, 30 μg/ml, 100 μg/ml) and kinds of agonists ［thrombin(0.01,0.05,0.1 U/ml), ADP（5,10,20 μmol/L）, U46619（0.125,0.25,0.5 μmol/L）］ on platelets were analyzed. The platelet spreading was successful if the spreading rate was higher after treated with agonists. RESULTS: Compared to the group which coated with 10 μg/ml and 100 μg/ml fibrinogen, the platelet density is optimal when coated with 30 μg/ml fibrinogen. In addition, under the stimulation of thrombin, ADP and U46619, the spreading rate of platelets showed a certain concentration-dependent increasing. CONCLUSION The platelet spreading is easily influenced by various factors, the platelet spreading can be induced successfully at 0.1 U/ml thrombin, 20 μmol/L ADP and 0.5 μmol/L U46619 on the slide coated with 30 μg/ml fibrinogen.
15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid/pharmacology* ; Adenosine Diphosphate ; Animals ; Blood Platelets/physiology* ; Fibrinogen ; Humans ; Mice ; Mice, Inbred C57BL ; Platelet Adhesiveness/physiology* ; Thrombin/pharmacology*

Deep venous thrombosis (DVT) poses a major challenge to public health worldwide. Endothelial cell injury evokes inflammatory and oxidative responses that contribute to thrombus formation. Tea polyphenol (TP) in the form of epigallocatechin-3-gallate (EGCG) has anti-inflammatory and oxidative effect that may ameliorate DVT. However, the precise mechanism remains incompletely understood. The current study was designed to investigate the anti-DVT mechanism of EGCG in combination with warfarin (an oral anticoagulant). Rabbits were randomly divided into five groups. A DVT model of rats was established through ligation of the inferior vena cava (IVC) and left common iliac vein, and the animals were orally administered with EGCG, warfarin, or vehicle for seven days. In vitro studies included pretreatment of human umbilical vein endothelial cells (HUVECs) with different concentrations of EGCG for 2 h before exposure to hydrogen peroxide. Thrombus weight and length were examined. Histopathological changes were observed by hematoxylin-eosin staining. Blood samples were collected for detecting coagulation function, including thrombin and prothrombin times, activated partial thromboplastin time, and fibrinogen levels. Protein expression in thrombosed IVCs and HUVECs was evaluated by Western blot, immunohistochemical analysis, and/or immunofluorescence staining. RT-qPCR was used to determine the levels of AGTR-1 and VEGF mRNA in IVCs and HUVECs. The viability of HUVECs was examined by CCK-8 assay. Flow cytometry was performed to detect cell apoptosis and ROS generation was assessed by 2',7'-dichlorofluorescein diacetate reagent. In vitro and invivo studies showed that EGCG combined with warfarin significantly reduced thrombus weight and length, and apoptosis in HUVECs. Our findings indicated that the combination of EGCG and warfarin protects HUVECs from oxidative stress and prevents apoptosis. However, HIF-1α silencing weakened these effects, which indicated that HIF-1α may participate in DVT. Furthermore, HIF-1α silencing significantly up-regulated cell apoptosis and ROS generation, and enhanced VEGF expression and the activation of the PI3K/AKT and ERK1/2 signaling pathways. In conclusion, our results indicate that EGCG combined with warfarin modifies HIF-1α and VEGF to prevent DVT in rabbits through anti-inflammation via the PI3K/AKT and ERK1/2 signaling pathways.
Animals ; Anticoagulants/pharmacology* ; Catechin/analogs & derivatives* ; Eosine Yellowish-(YS)/pharmacology* ; Fibrinogen/pharmacology* ; Hematoxylin/pharmacology* ; Human Umbilical Vein Endothelial Cells ; Humans ; Hydrogen Peroxide/pharmacology* ; Hypoxia-Inducible Factor 1, alpha Subunit/metabolism* ; MAP Kinase Signaling System ; Phosphatidylinositol 3-Kinases/metabolism* ; Polyphenols/pharmacology* ; Proto-Oncogene Proteins c-akt/metabolism* ; RNA, Messenger ; Rabbits ; Rats ; Reactive Oxygen Species/metabolism* ; Signal Transduction ; Sincalide/pharmacology* ; Tea ; Thrombin/pharmacology* ; Vascular Endothelial Growth Factor A/metabolism* ; Venous Thrombosis/pathology* ; Warfarin/pharmacology*

OBJECTIVE: To construct a mouse model of Glanzmann's thrombasthenia (GT) with ITGA2B c.2659 C>T (p.Q887X) nonsense mutation by CRISPR/Cas9 technology, and then further explore the expression and function of glycoprotein αIIbβ3 on the surface of platelet membrane. METHODS: The donor oligonucleotide and gRNA vector were designed and synthesized according to the ITGA2B gene sequence. The gRNA and Cas9 mRNA were injected into fertilized eggs with donor oligonucleotide and then sent back to the oviduct of surrogate mouse. Positive F0 mice were confirmed by PCR genotyping and sequence analysis after birth. The F1 generation of heterozygous GT mice were obtained by PCR and sequencing from F0 bred with WT mice, and then homozygous GT mice and WT mice were obtained by mating with each other. The phenotype of the model was then further verified by detecting tail hemorrhage time, saphenous vein bleeding time, platelet aggregation, expression and function of αIIbβ3 on the surface of platelet. RESULTS: The bleeding time of GT mice was significantly longer than that of WT mice (P<0.01). Induced by collagen, thrombin, and adenosine diphosphate (ADP), platelet aggregation in GT mice was significantly inhibited (P<0.01, P<0.01, P<0.05). Flow cytometry analysis showed that the expression of αIIbβ3 on the platelet surface of GT mice decreased significantly compared with WT mice (P<0.01), and binding amounts of activated platelets to fibrinogen were significantly reduced after thrombin stimulation (P<0.01). The spreading area of platelet on fibrinogen in GT mice was significantly smaller than that in WT mice (P<0.05). CONCLUSION A GT mouse model with ITGA2B c.2659 C>T (p.Q887X) nonsense mutation has been established successfully by CRISPR/Cas9 technology. The aggregation function of platelet in this model is defective, which is consistent with GT performance.
Animals ; CRISPR-Cas Systems ; Codon, Nonsense ; Disease Models, Animal ; Fibrinogen/genetics* ; Humans ; Integrin alpha2/genetics* ; Mice ; Oligonucleotides ; Platelet Glycoprotein GPIIb-IIIa Complex/genetics* ; RNA, Guide ; Thrombasthenia/genetics* ; Thrombin/genetics*

OBJECTIVE: To investigate the effect of protein kinase A (PKA) activation on aggregation funetion of platelets in vitro. METHODS: The peripheral blood of healthy adults were collected, and the washed platelets were gained from collected peripheral blood. The washed platelets were treated with PKA activator Forskolin, then the platelet aggregation was induced by using Ristocetin, Thrombin, Collagen and ADP respectively, the platelet aggregation level was detected by the platelet aggregator. RESULTS: Compared with the controls, 5 μmol/L forskolin significantly inhibited ADP and collagen-induced platelet aggregation (P＜0.001), and showed mild inhibiting effect on Thrombin-induced platelet aggregation (P＜0.05). 2.5-10 μmol/L forskolin significantly inhibited ADP and Collagen -induced platelet aggregation (P＜0.001); but not showed significantly inhibitory effects on Ristocetin-induced platelet aggregation (P＞0.05). CONCLUSION PKA activation inhibits agonists-induced platelet aggregation.
Blood Platelets ; Cyclic AMP-Dependent Protein Kinases ; Humans ; Platelet Aggregation ; Platelet Aggregation Inhibitors ; Ristocetin ; Thrombin

Platelet activation has a major role in hemostasis and thrombosis. Various agonists including adenosine diphosphate (ADP) and thrombin interact with G protein-coupled receptors (GPCRs) which transduce signals through various G proteins. Recent studies have elucidated the role of GPCRs and their corresponding G proteins in the regulation of events involved in platelet activation. However, agonist-induced platelet activation in companion animals has not been elucidated. This study was designed to characterize the platelet response to various agonists in dog platelets. We found that 2-methylthio-ADP-induced dog platelet aggregation was blocked in the presence of either P2Y₁ receptor antagonist MRS2179 or P2Y₁₂ receptor antagonist AR-C69931MX, suggesting that co-activation of both the P2Y₁ and P2Y₁₂ receptors is required for ADP-induced platelet aggregation. Thrombin-induced dog platelet aggregation was inhibited in the presence of either AR-C69931MX or the PKC inhibitor GF109203X, suggesting that thrombin requires secreted ADP to induce platelet aggregation in dog platelets. In addition, thrombin-mediated Akt phosphorylation was inhibited in the presence of GF109203X or AR-C69931MX, indicating that thrombin causes Gi stimulation through the P2Y₁₂ receptor by secreted ADP in dog platelets. Unlike human and murine platelets, protease-activated receptor 4 (PAR4)-activating peptide AYPGKF failed to cause dog platelet aggregation. Moreover, PAR1-activating peptide SFLLRN or co-stimulation of SFLLRN and AYPGKF failed to induce dog platelet aggregation. We conclude that ADP induces platelet aggregation through the P2Y₁ and P2Y₁₂ receptors in dogs. Unlike human and murine platelets, selective activation of the PAR4 receptor may be insufficient to cause platelet aggregation in dog platelets.
Adenosine Diphosphate ; Animals ; Blood Platelets ; Dogs ; GTP-Binding Proteins ; Hemostasis ; Humans ; Pets ; Phosphorylation ; Platelet Activation ; Platelet Aggregation ; Receptors, Proteinase-Activated ; Thrombin ; Thrombosis

Rat model of blood stasis syndrome was prepared by subcutaneous injecting of epinephrine hydrochlorid,then the model rats were administrated by Yunnan Baiyao for 15 days. Blood rheology,coagulation function and histopathology were chosen as indicators to evaluate the successful replication of blood stasis syndrome model and the treatment effect of Yunnan Baiyao. UPLC-Q-TOF-MS was used to rapidly analyze the serum samples of blood stasis syndrome rat after 15 days Yunnan Baiyao treatment,Progenesis QI software was employed to identify the alkaloids components. The results showed that Yunnan Baiyao reduced the plasma viscosity and whole blood viscosity of rats with blood stasis syndrome,prolonged thrombin and prothrombin time,reduced fibrinogen content,and effectively improved pathological state such as inflammatory cell infiltration,blood stasis,congestion and edema of various organs in rats with blood stasis syndrome. Seven alkaloids components from Aconitum kusnezoffii,including karacolidine,senbusine B,isotalatizidine,karakoline,denudatine,talatisamine and chasmanine were found in the rat serum after Yunnan Baiyao treatment. Based on the effectiveness of Yunnan Baiyao in the treatment of blood stasis syndrome induced by epinephrine hydrochloride in rats,alkaloids components from the root of A. kusnezoffii absorbed into blood after Yunnan Baiyao treatment were clarified rapidly and accurately with the help of UPLC-Q-TOF-MS. Karacolidine,senbusine B,isotalatizidine,karakoline,denudatine,talatisamine and chasmanine are the pharmacodynamic material basis of the root of A. kusnezoffii for activating blood circulation and removing blood stasis.
Aconitum ; chemistry ; Animals ; Blood Circulation ; drug effects ; Blood Viscosity ; Drugs, Chinese Herbal ; pharmacology ; Prothrombin Time ; Rats ; Thrombin Time

OBJECTIVE: To develop methods of extraction and purification of Cterminal NUDT9 homology domain of human transient receptor potential melastatin 2 (TRPM2) channel. METHODS: After sonication and centrifuge of strain Rosetta (DE3) which was induced by isopropylthio-β-D-galactoside, GST-NUDT9-H was collected after the binding of supernatant with GST beads and eluted with reduced glutathione. Then the elution buffer containing fusion protein was purified by size exclusion chromatography after concentration and centrifuge. Finally, with the cleavage of thrombin and binding with the GST beads, NUDT9-H with high purity in supernatant was collected. RESULTS: The GST-NUDT9-H fusion protein was stabilized with lysis buffer containing 0.5% n-dodecyl -β-d-maltoside (DDM), and wash buffer containing 0.025% DDM in size-exclusion chromatography system, and finally the NUDT9-H with high purity was obtained after cleaved by thrombin (1 U/2 mg fusion protein) for 24 h. CONCLUSIONS Due to the poor stability of NUDT9-H, it is necessary to add DDM in extraction and purification buffer to stabilize the conformation of NUDT9-H, so as to increase its yields and purity.
Escherichia coli ; genetics ; Glucosides ; chemistry ; Humans ; Protein Domains ; Protein Stability ; Pyrophosphatases ; chemistry ; genetics ; isolation & purification ; Recombinant Fusion Proteins ; chemistry ; isolation & purification ; TRPM Cation Channels ; chemistry ; isolation & purification ; Thrombin ; metabolism

To compare the blood-cooling and hemostasis effects of Rehmanniae Radix before and after carbonizing on rats with blood heat and hemorrhage syndrome. The blood heat and hemorrhage syndrome rat model was established. Indexes including rectal temperature,whole blood viscosity,plasma viscosity,thrombin time(TT),activated partial thromboplastin time(APTT),prothrombin time(PT),fibrinogen content(FIB),red blood cell(RBC),hemoglobin(Hb),hematocrit(HCT),blood platelet count(PLT),mean platelet volume(MPV),serum IL-1,serum IL-6 and lung histopathology were detected to investigate the blood-cooling and hemostasis effects of Rehmanniae Radix and its carbonized products. Compared with the blank control group,the rectal temperature was significantly increased with rise of the high,middle and low whole blood viscosities and plasma viscosity(P<0.05); both the high and low whole blood restore viscosity and the high and low whole blood relative viscosity were increased significantly(P< 0.05); TT,APTT and PT were notably prolonged with the increase in FIB content(P<0.05); RBC,Hb and HCT increased significantly(P< 0.05); concentrations of serum IL-1 and IL-6 were also increased(P< 0.05) in model group. Additionally,obvious hemorrhages in lung and stomach were observed in rats of the model group. Rehmanniae Radix and its carbonized products can significantly reduce rectal temperature,high middle and low whole blood viscosities and plasma viscosity(P<0.05). TT and APTT were shortened,with lower expression of FIB in group of Rehmannia Radix and its carbonized products. Hemorrhages of lung and stomach were improved by Rehmannia Radix and its carbonized products. The results indicated that Rehmannia Radix before and after carbonizing had the hemostasis and blood-cooling effects by promoting coagulation,improving blood rheology and inhibiting expressions of IL-1 and IL-6.
Animals ; Blood Coagulation ; Blood Viscosity ; Body Temperature ; Drugs, Chinese Herbal ; pharmacology ; Hemorrhage ; drug therapy ; Hemostasis ; Interleukin-1 ; metabolism ; Interleukin-6 ; metabolism ; Partial Thromboplastin Time ; Plant Roots ; Rats ; Rehmannia ; chemistry ; Thrombin Time

BACKGROUND: Topical hemostatic agents are used when ligation, electrocauterization, or other conventional hemostatic methods are impractical. Because a hemostatic agent is a foreign body, it can cause foreign body reactions, inflammation, and infections that can interfere with the wound healing process. Therefore, we should select hemostatic agents after considering their effects on wound healing. Here, we compared the effects of hemostatic agents on wound healing in a rectus abdominis muscle defect in rats. METHODS: Twelve Sprague Dawley rats were subjected to creation of a 6 × 6 mm defect in the rectus abdominis muscle and divided into four groups: control group; group A, Tachosil fibrin sealant patch; group B, Surgicel Fibrillar oxidized regenerated cellulose; and group C, Surgicel Snow oxidized regenerated cellulose. For the histologic analysis, biopsies were performed on the 3rd, 7th, and 27th days. RESULTS: The foreign body reaction was the weakest in group A and most significant in group C. The inflammatory cell infiltration was the weakest in group A and similar in groups B and C. Muscle regeneration differed among periods. The rats in group A were the most active initially, while those in group C showed prolonged activity. CONCLUSION: Tachosil and Surgicel administration increased inflammation via foreign body reactions, but the overall wound healing process was not significantly affected. The increased inflammation in the Surgicel groups was due to a low pH. We recommend using Tachosil, because it results in less intense foreign body reactions than Surgicel and faster wound healing due to the fibrin action.
Animals ; Biopsy ; Cellulose ; Cellulose, Oxidized ; Fibrin Tissue Adhesive ; Fibrin ; Fibrinogen ; Foreign Bodies ; Foreign-Body Reaction ; Hemostatics ; Hydrogen-Ion Concentration ; Inflammation ; Ligation ; Rats ; Rats, Sprague-Dawley ; Rectus Abdominis ; Regeneration ; Snow ; Thrombin ; Wound Healing ; Wounds and Injuries

Leech has a good anticoagulant activity and is one of the raw materials for treatment of many cardiovascular and cerebrovascular diseases. This study was based on in vitro anticoagulant experiments( APTT and PT) to investigate the effects of lead contamination on the anticoagulant effect of leech. At present,the Hirudo circulating in the market are dominated by Whitmania pigra,therefore Wh. pigra were cultivated under a different lead pollution for 50 days. Then,the effects of Wh. pigra extract,extracting from different cultivating environment,on activated partial thrombin time( APTT) and prothrombin time( PT) were determined by automatic coagulation instrument. The results showed that the Wh. pigra extract significantly prolonged the APTT compared with the saline group.The APTT of the lead-high residual Wh. pigra was shorter than that of the blank Wh. pigra. The Wh. pigra extracts from different treatment groups had little effect on PT. The results showed that the lead residue in the Wh. pigra increased with the increase of lead in the cultured soil,the lead residual of the Pb-H group was( 10. 66±2. 79) mg·kg~(-1),which exceeded the lead limit specified in the 2015 edition of the Chinese Pharmacopoeia. The results indicated that growth environment pollution is one of the important factors causing excessive lead in Wh. pigra. Lead pollution will reduce the anticoagulant effect of Wh. pigra and affect its clinical efficacy.
Animals ; Anticoagulants ; Biological Products/pharmacology* ; Blood Coagulation ; Environmental Pollution ; Lead/toxicity* ; Leeches/drug effects* ; Prothrombin Time ; Thrombin Time