Objective To evaluate whether Vibrio vulnificus secreted exotoxin-hemolysin (VVH) can activate platelet, an important blood immune cell, and to explore the possible molecular mechanism of platelet activation by VVH. Methods Transcriptomics and immunohistochemistry were used to analyze whether Vibrio vulnificus infection caused platelet activation in mice. Then, flow cytometry was used to identify whether VVH was the main stimulator of platelet activation. Naturally expressed VVH toxin was purified and prepared. The effects of extracellular and intracellular Ca²⁺ signal inhibitors on VVH activated platelets were evaluated by flow cytometry and Western blotting. The immune activation effect of VVH in the early stage of Vibrio vulnificus infection was analyzed in vivo. Results VVH was the main stimulator of platelet activation in Vibrio vulnificus culture supernatant. Natural VVH can induce the increase of P-selectin (CD62P) on platelet surface, the formation of platelet-neutrophil complex (PNC), and the release of platelet microvesicles. The activation mechanism may be related to the VVH pore-dependent Ca²⁺-calmodulin (CaM) -myosin light chain kinase (MLCK) signaling pathway, which led to the release of platelet alpha particles and cascade activation of platelets. In a mouse model of ALD infected by Vibrio vulnificus gavage, VVH was strongly associated with platelet activation. Conclusion This study shows that VVH is an important platelet activating molecule in the early stage of Vibrio vulnificus infection, and its induction of platelet activation may be related to the pathogenic process.
Animals ; Platelet Activation/drug effects* ; Hemolysin Proteins/pharmacology* ; Vibrio vulnificus/metabolism* ; Mice ; Blood Platelets/drug effects* ; Vibrio Infections/immunology* ; P-Selectin/metabolism* ; Bacterial Proteins ; Female

OBJECTIVE: To investigate the effects of oridonin on platelet function and related mechanisms. METHODS: Washed platelets from healthy adults and mice were incubated with different concentrations of oridonin (2.5, 5 and 10 μmol/L) in vitro . The surface expression level of P-selectin and the activation of integrin αIIbβ3 in platelets were detected by flow cytometry, and the aggregation ability of platelets under the stimulation by various agonists was detected by light transmission aggregometry. The expression of P-AKT (Ser473) was detected by protein immunoblotting. Arterial thrombosis model was established in mice with mesenteric injury induced by ferric chloride, and tail hemorrhage model was established by cutting off the tail of mice. The effect of intraperitoneal injection of oridonin (10 mg/kg) on thrombosis and haemostasis was tested. RESULTS: Oridonin inhibited platelet P-selectin expression and integrin αIIbβ3 activation. In the presence of different stimulants, oridonin inhibited platelet aggregation in a concentration-dependent manner. The phosphorylation level of AKT Ser473 was reduced in the groups treated with different concentrations of oridonin. Oridonin significantly prolonged the time of mesenteric artery thrombosis in mice, but did not affect the tail bleeding time. CONCLUSION Oridonin inhibits platelet activation, aggregation, and thrombosis by inhibiting AKT phosphorylation, and may be used as a potential antiplatelet drug.
Diterpenes, Kaurane/pharmacology* ; Animals ; Mice ; Blood Platelets/drug effects* ; Platelet Aggregation/drug effects* ; P-Selectin/metabolism* ; Thrombosis ; Platelet Glycoprotein GPIIb-IIIa Complex/metabolism* ; Proto-Oncogene Proteins c-akt/metabolism* ; Humans ; Phosphorylation ; Platelet Activation/drug effects*

OBJECTIVES: To develop an E-selectin-targeting nanomedicine delivery system that competitively inhibits E-selectin-neutrophil ligand binding to block neutrophil adhesion to vessels and suppress their recruitment to the lesion sites. METHODS: Doxorubicin hydrochloride (DOX)-loaded liposomes (IEL-Lip/DOX) conjugated with E-selectin-affinity peptide IELLQARC were developed using a post-insertion method. Two formulations [2-1P: Mol(PC): Mol(DPI)=100:1; 2-3P: 100:3] were prepared and their modification density and in vitro release characteristics were determined. Their targeting efficacy was assessed in a cell model of LPS-induced inflammation, a mouse model of acute lung injury (ALI), a rat femoral artery model of physical injury-induced inflammation, and a zebrafish model of local inflammation. RESULTS: The prepared IEL-Lip/DOX 2-1P and 2-3P had peptide modification densities of 4.76 and 7.57 pmoL/cm², respectively. Compared with unmodified liposomes, IEL-Lip/DOX exhibited significantly reduced 48-h cumulative release rates at pH 5.5. In the inflammation cell model, IEL-Lip/DOX showed increased uptake by activated inflammatory endothelial cells, and 2-1P exhibited a higher trans-endothelial ability. In ALI mice, the fluorescence intensity of IEL-Lip/Cy5.5 increased significantly in lung tissues by 53.71% [Z-(2-1P)] and 93.41% [Z-(2-3P)], and 2-1P had an increased distribution by 24.19% in the inflammatory lung tissue compared to normal mouse lung tissue. In rat femoral artery models, 2-1P had greater injured/normal vessel fluorescence intensity contrast. In the zebrafish models, both 2-1P and 2-3P showed increased aggregation at the site of inflammation. CONCLUSIONS This E-selectin-targeting nanomedicine delivery system efficiently targets activated inflammatory endothelial cells to increase drug concentration at the inflammatory site, which sheds light on new strategies for treating neutrophil-mediated inflammatory diseases and practicing the concept of "one drug for multiple diseases".
Animals ; Liposomes ; Rats ; Nanomedicine ; E-Selectin ; Drug Delivery Systems ; Inflammation/drug therapy* ; Mice ; Doxorubicin/analogs & derivatives* ; Zebrafish ; Acute Lung Injury/drug therapy*

OBJECTIVE: To investigate the effect of vitamin D3 to platelet activation by tumor cell culture medium. METHODS: The peripheral blood platelets of BALB/c mice were isolated. The platelets were activated in 4T1 culture fluid for 24 h. The platelets were divided into 7 groups: control group, activation group, 1 nmol/L vitamin D3 group, 10 nmol/L vitamin D3 group, 50 nmol/L vitamin D3 group, 100 nmol/L vitamin D3 group, and positive drug (0.1 μmol/L eptifibatide) group. CCK-8 assay was used to detect the platelet proliferation at 24, 48 and 72 h. Flow cytometry was used to detect the expression of CD61 and CD62p and receptor for advanced glycation end products (RAGE) at 24, 48 and 72 h. ELISA was used to detect the level of platelet-endothelial cell adhesion molecule-1 (PECAM-1) at 24, 48 and 72 h. RESULTS: The CD41 CONCLUSION Vitamin D3 shows antiplatelet effect and can inhibit platelet proliferation and activation.
Animals ; Blood Platelets ; Cell Culture Techniques ; Cholecalciferol/pharmacology* ; Flow Cytometry ; Mice ; Mice, Inbred BALB C ; P-Selectin ; Platelet Activation

OBJECTIVE: To investigate the predictive effect of platelet activation index expression before and after adenosine bisphosphate activation on bleeding risk in patients with primary immune thrombocytopenia (ITP). METHODS: Eighty-nine patients with ITP admitted in our hospital from January 2017 to October 2018 were selected and inrolled in ITP group, the bleeding scoreing and grading were performed by using the ITP-BAT for ITP patients, then 89 ITP patients were divided into 4 subgroups: nothing bleeding symptom group, mild bleeding symprom group, mode rate bleeding symptom group and severe bleeding symptom group according to bleeding scores and grades obtained from ITP-BAT detection. At the same time, 22 persons underwent the health physical examination were selected and enrolled in control group. The adenosine diphosphate (ADP) was used as activator for all patients and controls. The flow cytonetry was used to analyze the expression of platelet membranc glyco protein (GPⅠb, GPⅡb /Ⅲ a) and P-selectin before and after ADP activation, the multiple linear person's correlation analysis was used to analyze the correlation of bleeding degree of ITP patients before and after ADP acbivation with the expression levels of GPⅠb, GPⅡb/Ⅲa and P-selectin. RESULTS: After the ADP activation, the expression level of GPⅠb significantly decreased, while the expression levels of GPⅠb, GPⅡb/Ⅲ a and P-selectin significantly increased in control group, nothing bleeding symptom group and mild bleeding symptom group; but the expression level of GPⅠb significantly increased, while the expression level of GPⅡb/Ⅲ a significantly decreased in moderate and severe bleeding symptom group, the both differences were statistically significant (P＜0.05). however, the expression level of P-selectin in moderate and severe bleeding symptom groups before and after ADP activation was not statistivally significant (P＞0.05). Before ADP activation, the expression level of GPⅠb in ITP subgroups was lower than that in control group, the expression level of GPⅡb/Ⅲ a in ITP subgroups was higher than that in control group, the expression level of P-selectin in moderate and severe bleeding symptom groups was higher than that in control group (P＜0.05). After ADP activation, the expression levels of GPⅠb and P-selectin in ITP subgroups both were lower than those in control group, the expression level of GPⅡb/Ⅲa in ITP subgroups was higher than that in control group (P＜0.05). The comparison among ITP subgroups showed that before ADP activation, the expression level of GPⅠb in moderate and severe bleeding symptom groups was lower than that in nothing bleeding symotom and mild bleeding symptom groups, while the expression levels of GPⅡb/Ⅲa and P-selectin were higher than those in nothing bleeding symptom and mild bleeding symptom groups (P＜0.05), however, after ADP activation, the expression level of GPⅠb in moderate and severe bleeding symptom groups was higher than that in nothing bleeding symptom and mild bleeding symptom groups, while the expression levels of GPⅡb/Ⅲ a and P-selection in moderate and severe bleeding symptom groups were lower than those in nothing and mild bleeding symptom groups (P＜0.05). The correlation analysis showed that before ADP activation, the expression levels of GPⅠb and GPⅡb/Ⅲa positivdy correlated with the bleeding risk (r=0.483, 0.504), and the P-selectin not correlated with the bleeding risk (r=0.000); however, after ADP activation, the expression level of GPⅠb and GPⅡb/Ⅲ a negatively correlated with the bleeding risk (r=-0.627, -0.406, -0.108). CONCLUSION The expression level of platelet activation indicators before and after ADP activation is of certain value for prevention of bleeding risk in ITP patients and can be used as a reference indicator for the treatment and efficacy evaluation.
Adenosine ; Blood Platelets ; Humans ; P-Selectin ; Platelet Activation ; Platelet Count ; Purpura, Thrombocytopenic, Idiopathic

OBJECTIVE: This study aimed to determine whether simultaneous decreases in the serum levels of cell adhesion molecules (intracellular cell adhesion molecule-1 [ICAM-1], vascular cell adhesion molecule-1 [VCAM-1], and E-selectin) and S100 proteins within the first 24 hours after the return of spontaneous circulation were associated with good neurological outcomes in cardiac arrest survivors. METHODS: This retrospective observational study was based on prospectively collected data from a single emergency intensive care unit (ICU). Twenty-nine out-of-hospital cardiac arrest survivors who were admitted to the ICU for post-resuscitation care were enrolled. Blood samples were collected at 0 and 24 hours after ICU admission. According to the 6-month cerebral performance category (CPC) scale, the patients were divided into good (CPC 1 and 2, n=12) and poor (CPC 3 to 5, n=17) outcome groups. RESULTS: No difference was observed between the two groups in terms of the serum levels of ICAM-1, VCAM-1, E-selectin, and S100 at 0 and 24 hours. A simultaneous decrease in the serum levels of VCAM-1 and S100 as well as E-selectin and S100 was associated with good neurological outcomes. When other variables were adjusted, a simultaneous decrease in the serum levels of VCAM-1 and S100 was independently associated with good neurological outcomes (odds ratio, 9.285; 95% confidence interval, 1.073 to 80.318; P=0.043). CONCLUSION: A simultaneous decrease in the serum levels of soluble VCAM-1 and S100 within the first 24 hours after the return of spontaneous circulation was associated with a good neurological outcome in out-of-hospital cardiac arrest survivors.
Blood-Brain Barrier ; Cardiopulmonary Resuscitation ; Cell Adhesion ; Cell Adhesion Molecules ; E-Selectin ; Emergencies ; Endothelium ; Heart Arrest* ; Humans ; Intensive Care Units ; Intercellular Adhesion Molecule-1 ; Observational Study ; Out-of-Hospital Cardiac Arrest ; Prospective Studies ; Retrospective Studies ; S100 Proteins ; Survivors* ; Vascular Cell Adhesion Molecule-1*

Gene therapies have been applied to the treatment of cardiovascular disease, but their use is limited by the need to deliver them to the right target. We have employed targeted contrast ultrasound-mediated gene transfection (TCUMGT) via ultrasound-targeted microbubble destruction (UTMD) to transfer therapeutic genes to specific anatomic and pathological targets. Phospholipid microbubbles (MBs) with pcDNA_3.1-human vascular endothelial growth factor 165 (pcDNA_3.1-hVEGF₁₆₅) plasmids targeted to P-selectin (MB+P+VEGFp) were created by conjugating monoclonal antibodies against P-selectin to the lipid shell. These microbubbles were divided into four groups: microbubble only (MB), microbubble+P-selectin (MB+P), microbubble+pcDNA_3.1-hVEGF₁₆₅ plasmid (MB+VEGFp), and microbubble+ P-selectin+pcDNA_3.1-hVEGF₁₆₅ plasmid (MB+P+VEGFp). The reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) results showed that the VEGF gene was successfully transfected by TCUMGT and the efficiency is increased with P-selectin targeting moiety. UTMD-mediated delivery of VEGF increased myocardial vascular density and improved cardiac function, and MB+P+VEGFp delivery showed greater improvement than MB+VEGFp. This study drew support from TCUGMT technology and took advantage of targeted ultrasound contrast agent to identify ischemic myocardium, release pcDNA_3.1-hVEGF₁₆₅ recombinant plasmid, and improve the myocardial microenvironment, so promoting the restoration of myocardial function.
Animals ; Genetic Therapy/methods* ; Male ; Microbubbles ; Myocardial Ischemia/therapy* ; P-Selectin/genetics* ; Rats ; Rats, Sprague-Dawley ; Transfection/methods* ; Ultrasonics ; Vascular Endothelial Growth Factor A/genetics*

BACKGROUND/AIMS: Although epigallocatechin-3-gallate (EGCG), which is found in high contents in the dried leaves of green tea, has been reported to have an anti-platelet effect, synergistic effects of EGCG in addition to current anti-platelet medications remains to be elucidated. METHODS: Blood samples were obtained from 40 participants who took aspirin (ASA, n = 10), clopidogrel (CPD, n = 10), ticagrelor (TCG, n = 10) and no anti-platelet medication (Control, n = 10). Ex vivo platelet aggregation and adhesion under various stimulators were analyzed by multiple electrode aggregometry (MEA) and Impact-R systems. PAC-1 and P-selectin expressions in human platelets were analyzed by flow cytometry. RESULTS: In MEA analysis, adenosine diphosphate (ADP) and thrombin receptor activating peptide (TRAP)-induced platelet aggregations were lower in the CPD and the TCG groups; arachidonic acid (AA)-induced platelet aggregation was lower in the ASA group, whereas collagen (COL)-induced platelet aggregations were comparable among four groups. EGCG significantly reduced ADP- and COL-induced platelet aggregation in dose-dependent manner (ADP, p = 0.04; COL, p < 0.01). There were no additional suppressions of platelet aggregation stimulated by AA in the ASA group, and by ADP in the CPD and TCG groups. Moreover, EGCG suppressed shear stress-induced platelet adhesion on Impact-R, and had no effect on P-selectin and PAC-1 expressions. CONCLUSIONS: Ex vivo treatment of EGCG inhibited platelet adhesion and aggregation without changes in P-selectin and PAC-1 expression. There was no additional suppressions in platelet aggregation stimulated by AA in the ASA group and ADP in the CPD and TCG groups.
Adenosine Diphosphate ; Arachidonic Acid ; Aspirin* ; Blood Platelets ; Catechin ; Collagen ; Electrodes ; Flow Cytometry ; Humans ; P-Selectin ; Platelet Aggregation ; Platelet Aggregation Inhibitors ; Receptors, Thrombin ; Tea

<p>OBJECTIVETo investigate the coagulation abnormality and tumor-associated hypercoagulable state in lymphoma-bearing mice by measuring the changes in coagulation indices (D-D, vWF, TF) and platelet activation indices (P-selectin, GPIIbIIIa).p><p>METHODSThe mouse model with lymphoma was established by the subcutaneous injection of 38B9 lymphoma cells into BALB/c mice, and the tumor formation was evaluated by using MRI and B ultrasonography. The D-D, vWF and TP levels of blood samples from inner canthal vein of tumor-bearing mice on 1 d, 14 d and 21 d were detected by using ELISA, the platelet activation indices (P-selectin, GPIIbIIIa) were detected by using flow cytometry.p><p>RESULTSThe lymphoma-bearing mouse model was successfully established. The levels of D-D, vWF and TF as well as platelet activation indices P-selectin and GPIIbIIIa in the peripheraI blood were significantly higher than those of control group (P<0.05).p><p>CONCLUSIONLymphoma-bearing mice showed abnormalities of coagulation and platelet activation, which relates with the tumor hypercoagulable state in lymphoma-bearing mice.p>
Animals ; Blood Coagulation ; Lymphoma ; Mice ; Mice, Inbred BALB C ; P-Selectin ; Platelet Activation ; Thrombophilia

PURPOSE: Intra-abdominal adhesions (IAA) are among the most frequently seen pathologies in general surgery practice with an increased morbidity and mortality. In the present study, we investigated the effect of locally applied mesenchymal stem cells (MSCs) on IAA. METHODS: Twenty-four Wistar Albino rats were used in the study. The rats were divided into three groups including: Sham, control, and MSCs group. On day 0, cecum was reached under anesthesia in all groups, except the Sham group. Scraping with a sponge was performed until petechial bleeding occurred. The control group received no treatment. In the stem cell group, MSCs were applied topically immediately after surgery on adhesions. The rats were sacrificed on day 10 and colon tissues and blood samples were collected for macroscopic, histopathological, and biochemical analysis. RESULTS: In our study, E-selectin, P-selectin, TNF-α and IL-1 levels were statistically significantly lower in the MSC group than the control group, while the sham group has the lowest levels. In both the macroscopic and histopathological analyses (Zühlke's scale), the least amount of adhesion was observed in the Sham group. In addition, although there was less adhesion in the MSC group than the control group, the difference did not reach statistical significance. CONCLUSION: Topical MSC application immediately after surgery suppresses the inflammatory process. However it was found to be ineffective in histopathological and macroscopic examinations performed on the 10th day.
Anesthesia ; Animals ; Cecum ; Colon ; E-Selectin ; Hemorrhage ; Interleukin-1 ; Mesenchymal Stromal Cells ; Models, Animal ; Morphological and Microscopic Findings ; Mortality ; P-Selectin ; Pathology ; Porifera ; Rats ; Selectins ; Stem Cells