[Regulation of Mitochondria on Platelet Apoptosis and Activation].

OBJECTIVE: To explore the regulation of mitochondria on platelet apoptosis and activation, and the relationship between platelet apoptosis and activation. METHODS: Platelets were isolated from peripheral venous blood of healthy volunteers. Cyclosporin A (CsA), which has a protective effect on the function of platelet mitochondria, BAPTA, which can chelate calcium ions across membranes in platelets, and NAC, an antioxidant that reduces the level of intracellular reactive oxygen species, were selected for coincubation with washed platelets, respectively. By flow cytometry, platelet aggregator was used to detect the changes of platelet mitochondrial function and platelet activation indexes after different interventions. RESULTS: H89, staurosporine, and A23187 led to platelet mitochondrial abnormalities, while CsA could effectively reverse the decline of platelet mitochondrial membrane potential caused by them. Antioxidant NAC could reverse platelet mitochondrial damage correspondingly, and completely reverse platelet shrinkage and phosphatidylserine eversion induced by H89. BAPTA, prostaglandin E1, acetylsalicylic acid and other inhibitors could not reverse the decline of platelet mitochondrial membrane potential. CONCLUSION: Mitochondrial function plays an important role in platelet apoptosis and activation. Abnormal mitochondrial function causes the imbalance of reduction/oxidation state in platelets, which leads to platelet apoptosis. Platelet apoptosis and activation are independent signal processes.

[The Effect of Ena/VASP Family on the Expression of GPIb-IX Complex in Human Megakaryoblastic Leukemia Dami Cells].

OBJECTIVE: To explore the effects of Ena/VASP gene family on the expression of glycoprotein (GP) Ib-IX complex in human megakaryoblastic leukemia Dami cells. METHODS: SiRNAs targeting Ena/VASP gene family were designed and synthesized to interfere Enah, EVL and VASP gene expression. When the siRNAs were transfected into Dami cells by using LipofectamineTM 2000 for 48 h, the expression of GPIb-IX complex was detected by quantitative real-time PCR, Western blot and flow cytometry. RESULTS: We successfully established siVASP , siEVL and si Enah Dami cell lines. And it was found that the expression of GPIb-IX complex had no evident reduction in siEVL or siVASP Dami cells at both mRNA and protein level, while the total protein and membrane protein of GPIb-IX complex were obviously reduced when Enah was knocked down. CONCLUSION: Enah could affect the expression of GPIb-IX complex in human megakaryoblastic leukemia Dami cells, but the underlying mechanism still needs to be further explored.

[Main Factors Influencing the Platelet Spreading].

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 ï¼»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.

[The Role of Zyxin in Regulating Platelet Cytoskeleton Distribution].

OBJECTIVE: To investigate the regulatory effect of zyxin on the distribution of platelet cytoskeleton. METHODS: Platelets were isolated from zyxin-knockout (Zyx-/-) and wild type (WT) mice respectively and corresponding platelet cytoskeleton components were separated. The expressions of ß-actin, α-actinin, filamin A and myosin ⅡA in cytoskeleton components were detected by Western blot. Actin polymerization was induced by the actin polymerization inducer Jasplakinolide (Jas) in WT and Zyx-/- platelets. Also, the expressions of the cytoskeleton proteins in cytoskeleton components were detected by Western blot. WT and Zyx-/- platelets were allowed to spread on fibrinogen-coated surface. Platelet F-actin was labeled with Alexa Fluor 488-conjugated phalloidin and the fluorescent intensity was compared between WT and Zyx-/- platelets. RESULTS: After zyxin gene was knockout, the expressions of cytoskeleton proteins ß-actin, α-actinin, filamin A, and myosin Ⅱ A in resting and Jas-induced platelets were significantly increased. In the platelet spreading on fibrinogen surface, F-actin was increased in Zyx-/- platelets as compared with that in WT platelets. CONCLUSION: Zyxin significantly regulates the distribution of platelet cytoskeleton, which plays an important role in maintaining platelet cytoskeleton homeostasis.

[Inducing Effect of PKA Inhibitor at Different Concentrations on Platelet Apoptosis and Its Mechanism].

OBJECTIVE: To investigate the inducing effect of PKA inhibitor H89 of different concentrations on platelet apoptosis and its mechanism. METHODS: Platelets were isolated from peripheral venous blood of healthy volunteers. Different concentrations gradient PKA inhibitor H89 were co-incubated with washing platelets, and the effects of PKA inhibitor H89 at different concentrations on platelet mitochondrial membrane potential, phosphatidylserine and reactive oxygen species(ROS) were determined by ELISA and flow cytometry. RESULTS: Different concentration of PKA inhibitor H89 could induce the depolarization of mitochondrial membrane potential and PS exposure of platelet. However, high concentration(100 µmol/L) PKA inhibitor H89 could induce the production of ROS in platelets, but medium and low concentrations did not induce the production of ROS in platelets. And several ROS inhibitors could inhibit the apoptosis induced by high concentration PKA inhibitor H89. CONCLUSION: High concentration H89 can induce platelet apoptosis, however the mechanism of platelet apoptosis caused by H89 of high concentration is different from H89 at medium and low concentrations.

[Regulatory Role of Mitochondria in the Shedding of Platelet Membrane Protein GPIbα].

OBJECTIVE: To investigate the role of mitochonaria in the regulation of platelet membrane protein GPIbα shedding and its mechanisms. METHODS: The washed platelets were obtained from peripheral blood in healthy volunteers and co-incubated with mitochondrial inhibitor carbonyl cyanide m-chlorophenyl hydrazone (CCCP), mitochondrial protector cyclosporin A (CsA) or matrix metalloproteinases inhibitor GM6001. After the platelets was stimulated, the effect of mitochondria to the shedding in platelet membrane protein GPIbα was detected by flow cytometry. RESULTS: Depolarization of mitochondrial membrane potential and the respiratory function of mitochondrial could be induced and destroyed by the uncoupling agent CCCP. At the same time, the shedding of GPIbα was detected out, and the result showed a statistical significance, which showed that the shedding of GPIbα could be activated by the damaged of mitochondrial in platelets. After the mitochondrial was protected by CsA, the shedding of GPIbα was inhibited significantly. GM6001 could only inhibited the shedding of GPIbα, but showed no inhibitation to the function of mitochondrial, which showed that the shedding of GPIbα was regulated at the mitochondrial, and the regulatory enzyme of receptor shedding (ADAM17) was located in the pathway of downstream of mitochondria. After the oxidative damage in cells was inhibited by NAC, and the changes of GPIbα shedding was detected, the result showed that the GPIbα shedding could be inhibited by NAC, which showed a dose-dependent manner. CONCLUSION: The GPIbα shedding could be caused by abnormality function of metabolic, and the metabolic imbalance of ROS is caused by the abnormallity function of mitochondria.

[Effect of Protein Kinase A Activation on Aggregation Function of Platelets].

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.

Effects of remote ischemic post-conditioning on platelet activation of AMI patients.

The recovery of ischemic myocardium blood perfusion is the main treatment option for acute myocardial infarction (AMI). However, this treatment option has multiple side effects that directly affect the quality of life of the patients. The activation of platelet function plays an important role in the occurrence, development and treatment of AMI. The aim of the present study was to analyze the effects of remote ischemic post-conditioning on platelet activation of AMI patients with primary PCI treatment and clinical prognosis. A total of 71 patients with AMI were treated with primary percutaneous coronary intervention (PCI). They were randomly divided into control group (n=34) and observation group (n=37). The patients in the observation group were treated with remote ischemic post-conditioning. Further, flow cytometer was used to detect the platelet alpha granule membrane glycoprotein (CD62P) and the percentages of activated IIb/IIIa (PAC-1). The maximum platelet aggregation rate induced by adenosine diphosphate (ADP) and arachidonic acid (AA) was measured by light transmittance aggrometer. The incidence of major adverse cardiac events (MACE) was compared between the two groups during the follow-up period of 6 months. The percentage of CD62P (24 h after PCI) in the observation group was significantly lower than control group (P<0.05). Further, the incidence of MACE in the observation group was also lower than that of the control group (P<0.05). Remote ischemic post-conditioning could reduce the incidence of MACE in patients with AMI after primary PCI treatment. Moreover, the above observation may be related to the improvement of platelet CD62P activation.

Chinese guidelines for treatment of adult primary immune thrombocytopenia.

Primary immune thrombocytopenia (ITP) is a bleeding disorder commonly encountered in clinical practice. The International Working Group (IWG) on ITP has published several landmark papers on terminology, definitions, outcome criteria, bleeding assessment, diagnosis, and management of ITP. The Chinese consensus reports for diagnosis and management of adult ITP have been updated to the 4th edition. Based on current consensus positions and new emerging clinical evidence, the thrombosis and hemostasis group of the Chinese Society of Hematology issued Chinese guidelines for management of adult ITP, which aim to provide evidence-based recommendations for clinical decision making.

[Main Factors Influencing FeCl3-Induced Mouse Mesenteric Arteriole Thrombosis Model].

OBJECTIVE: To investigate the factors that influence FeCl3-induced mouse mesenteric arteriole thrombosis model. METHODS: Platelets were isolated from donor mice and labeled with Calcein-AM. Mice were transfused intravenously with Calcein-AM labeled platelets. The influence of mouse ages (3-6 weeks, 6-10 weeks and >10 weeks), transfused platelets counts (1×107, 1×108 and 2×108 platelets) and concentrations of FeCl3 (6%, 12%, 24% and 48%) on FeCl3-induced thrombosis model were compared. RESULTS: The occlusion time was 16 min for mice aged 3-6 weeks, which was shorter than that for 6 mice aged 6-10 weeks(25 min)(P<0.05) and that for mice aged >10 weeks(38 min)(P<0.01). The occlusion time resulting from transfusion of 1×108 and 2×108 of pletclets was 15-18 min, which was shorter than that of transfusion 1×107 platelets (30 mins). The occlusion time resulting from transfusion of 6% and 12% FeCl3 was from 15 to 20 min, however the transfusion of 24% and 48% FeCl3 all in all leads to vessel occlusion within 10 min. CONCLUSION: The factors influencing the success of FeCl3-induced mouse thrombosis model are more. Transfusion of 1×108 to 2×108 labeled platelets to 3-6 week-old mice, and 6% to 12% of FeCl3 should be used to induce thrombosis, and the experimental conditions should be optimized for this animal model, therefore, it is easier for us to set up a mouse mesenteric arteriole thrombosis model.

A novel STAT3 inhibitor negatively modulates platelet activation and aggregation.

The signal transducer and activator of transcription 3 (STAT3) plays a critical role in platelet functions. This study sought to understand the effects of the STAT3 inhibitor SC99 on platelet activation and aggregation. Immunoblotting assays were applied to measure the effects of SC99 on the STAT3 signaling pathway. A ChronoLog aggregometer was used to evaluate platelet aggregation. A flow cytometer was used to evaluate P-selectin expression in the presence of SC99. AlamarBlue and Annexin-V staining were used to evaluate platelet viability and apoptosis, respectively. A fluorescence microscope was applied to analyze platelet spreading. SC99 inhibited the phosphorylation of JAK2 and STAT3 in human platelets but had no effects on the phosphorylation of AKT, p65 or Src, all of which are involved in platelet activation. Further studies revealed that SC99 inhibited human platelet aggregation induced by collagen and thrombin in a dose-dependent manner. SC99 inhibited thrombin-induced P-selectin expression and fibrinogen binding to single platelets. Moreover, SC99 inhibited platelet spreading on fibrinogen and clot retraction mediated by outside-in signaling. SC99 inhibited platelet aggregation in mice but it did not significantly prolong the bleeding time. Taken together, the present study revealed that SC99 inhibited platelet activation and aggregation as a STAT3 inhibitor. This agent can be developed as a promising treatment for thrombotic disorders.

[Regulatory Effect of Protein Disulfide Isomerase on Platelet GPIbα Ectodomain Shedding].

OBJECTIVE: This study was aimed to investigate the regulatory effect of protein disulfide isomerase (PDI) on platelet GPIbα ectodomain shedding. METHODS: The washed platelets were obtained from healthy volunteers. Platelets were incubated with PDI inhibitor bacitracin before stimulation with PMA (Phorbol-12-myristate-13-acetate), dibucaine and collagen. The N-terminal domain of GPIbα in supernatant was detected by Western blot, the GPIbα expression and the intraplatelet ROS levels were measured by flow cytometry. RESULTS: neither GC content nor GPIbα expression was changed after the washed platelets from the healthy donors were incubated only with PDI inhibitor. The washed platelets were incubated with PDI inhibitor before stimulation with different stimulin, PMA, dibucaine or collagen, and then GPIbα was cleaved and ROS levels were elevated more than that in the controls. CONCLUSION: PDI participates in the induced GPIbα ectodomein shedding, and the effect of PDI in this process maybe depend on the change of ROS level inside platelets. These results might provide a new point of view for the platelet drug development.

[Protein kinase C activation induces platelet apoptosis].

Platelet apoptosis elucidated by either physical or chemical compound or platelet storage occurs wildly, which might play important roles in controlling the numbers and functions of circulated platelets, or in the development of some platelet-related diseases. However, up to now, a little is known about the regulatory mechanisms of platelet apoptosis. Protein kinase C (PKC) is highly expressed in platelets and plays central roles in regulating platelet functions. Although there is evidence indicating that PKC is involved in the regulation of apoptosis of nucleated cells, it is still unclear whether PKC plays a role in platelet apoptosis. The aim of this study was to investigate the role of PKC in platelet apoptosis. The effects of PKC on mitochondrial membrane potential (ΔΨm), phosphatidylserine (PS) exposure, and caspase-3 activation of platelets were analyzed by flow cytometry and Western blot. The results showed that the ΔΨm depolarization in platelets was induced by PKC activator in time-dependent manner, and the caspase-3 activation in platelets was induced by PKC in concentration-dependent manner. However, the platelets incubated with PKC inhibitor did not results in ΔΨm depolarization and PS exposure. It is concluded that the PKC activation induces platelet apoptosis through influencing the mitochondrial functions and activating caspase 3. The finds suggest a novel mechanism for PKC in regulating platelet numbers and functions, which has important pathophysiological implications for thrombosis and hemostasis.

Cantharidin and norcantharidin inhibit the ability of MCF-7 cells to adhere to platelets via protein kinase C pathway-dependent downregulation of α2 integrin.

Cancer metastasis is a highly coordinated and dynamic multistep process in which cancer cells interact with a variety of host cells. Morphological studies have documented the association of circulating tumor cells with host platelets, where a surface coating of platelets protects tumor cells from mechanical trauma and the immune system. Cantharidin is an active constituent of mylabris, a traditional Chinese medicine. Cantharidin and norcantharidin are potent protein phosphatase 2A (PP2A) inhibitors that exhibit in vitro and in vivo antitumor activity against several types of cancer, including breast cancer. We investigated whether cantharidin and norcantharidin could repress the ability of MCF-7 breast cancer cells to adhere to platelets. Using MTT, clone formation, apoptosis, adhesion and wound-healing assays, we found that cantharidin and norcantharidin induced apoptosis and repressed MCF-7 cell growth, adhesion and migration. Moreover, we developed a flow cytometry-based analysis of tumor cell adhesion to platelets. We proved that cantharidin and norcantharidin repressed MCF-7 cell adhesion to platelets through downregulation of α2 integrin, an adhesion molecule present on the surface of cancer cells. The repression of α2 integrin expression was found to be executed through the protein kinase C pathway, the activation of which could have been due to PP2A inhibition.

[The role of amino acid sequence between 551 and 565 in the cytoplasmic domain of glycoprotein (GP) I b alpha in the regulation of the VWF binding to GP I b alpha].

OBJECTIVE: To explore the role of the amino acids between 551 and 565 in the cytoplasmic domain of glycoprotein (GP) I b alpha in the VWF binding to GP I b alpha. METHODS: The VWF binding to GP I b alpha induced by ristocetin was analyzed by flow cytometry, in three GP I b-IX-expressing Chinese hamster ovary (CHO) cell lines 1b9, delta 565 and delta 551, adhesion of above cells on VWF by flow chamber analysis at shear rate of 200 s(-1). The spread of GP I b-IX-expressing cells were stimulated with botrocetin on VWF-coated coverslips by confocal microscope. RESULTS: The VWF binding to GP I b alpha was higher in delta 565 cells stimulated by ristocetin than in delta 551 or 1b9 cells. The number of delta 565 cells adhered on the VWF-coated-chamber was more than that of controls at shear rate of 200 s(-1). Moreover, the surface spreading areas of delta 565 cells were greater than that of the controls on VWF-coated coverslips. CONCLUSIONS: The amino acids between 551 and 565 in the cytoplasmic domain of GP I b alpha regulates the VWF binding to GP I b alpha.

[Establishment of Chinese hamster ovary cell line expressing recombinant GPIb-IX complex].

The aim of this study was to construct Chinese Hamster Ovary (CHO) cell models expressing recombinant wild-type GPIb-IX and mutant GPIb-IX complex, so as to provide the platform to study the related physiologic functions of GPIb-IX. The plasmids were extracted from E.coli expressing wild-type or deletion mutant GPIbalpha and were identified by digestion with EcoR I. Three plasmids containing GPIbalpha, GPIbbeta, and GPIX genes were co-transfected into CHO cells, and then the expression of GPIb-IX complex was detected by immune coprecipitation, Western blot and flow cytometry. The results showed that the expression of GPIb-IX complex could be detected in the lysate and on the surface of CHO cells at 48 hours after transfection. In conclusion, CHO cell models expressing recombinant wild-type or mutation GPIb-IX complex has been successfully constructed.

[Inhibition of protein kinase A leads to cleavage of platelet GP I balpha and downregulation of GP I b-dependent platelet aggregation].

OBJECTIVE: To explore the regulatory role of protein kinase A (PKA) in platelet surface glycoprotein (GP) I balpha expression. METHODS: Washed platelets from healthy volunteers were incubated with PKA inhibitor. The N-terminal fragment of GP I balpha (glycocalicin, GC) in the supernatant of platelet suspensions was detected by Western blot and GP I balpha surface expression by flow cytometry. Calpain activity was determined by cytoskeletal proteins proteolysis and calpain surface expression by flow cytometry. The effect of PKA inhibitor on ristocetin-induced platelet aggregation was measured by platelet aggregometer. RESULTS: After PKA was inhibited in washed platelets, GP I balpha was cleaved and released to the supernatant, which significantly decreased the surface expression of GP I balpha (P < 0.05). The event was suppressed by pre-treatment with various calpain inhibitors, indicating that PKA inhibitor-mediated shedding was calpain dependent. The actin-binding protein (ABP) and talin proteolysis demonstrated that calpain was activated by PKA inhibitor and expressed on the platelet membrane. Ristocetin-induced aggregation was inhibited by PKA inhibitor. CONCLUSION: PKA inhibition results in calpain-dependent GP I balpha shedding, which thus reduces GP I balpha surface expression and GP I balpha-dependent platelet aggregation. These results might provide a view to develop new drugs for thrombotic diseases.

[Preparation and preliminary application of rabbit polyclonal antibodies against intracellular peptides of human platelet glycoprotein GPIbalpha].

OBJECTIVE: To prepare rabbit polyclonal antibodies against intracellular peptides of human platelet glycoprotein GPIbalpha. METHODS: Two peptides corresponding to human platelet GPIbalpha C-terminus were synthesized and purified by high-performance liquid chromatography (HPLC). The peptides were cross-linked with keyhole limpet hemocyanin (KLH). Two New Zealand white rabbits were immunized with conjugated peptides for 3 times. The polyclonal antibodies were purified by Ammonium Sulfate Precipitation and identified by dot blotting and ELISA. GPIbalpha intracellular peptides phosphorylation was tested with these polyclonal antibodies by ELISA. RESULTS: The titers of the two polyclonal antibodies against the GPIbalpha C-terminus peptides were 1:32 000 and 1:64 000 respectively and both of these antibodies reacted with purified GPIbalpha. CONCLUSIONS: Two rabbit polyclonal antibodies against C-terminal peptides of human platelet GPIbalpha have been prepared successfully, providing a way for the preparation of these kinds of antibody. Both phosphorylation and dephosphorylation states exist in the intracellular peptide of human platelets.

[Construction and expression of mouse-human chimeric Fab fragment gene of monoclonal antibody SZ-2 against platelet].

AIM: To reduce immunogenicity of a monoclonal antibody SZ-2 specific for human platelet. METHODS: Reverse transcription and polymerize chain reaction were used to amplify the variable region genes of monoclonal antibody SZ-2. The cloned V(H) and V(L) genes were sequenced and fused to human IgG1 constant region gene CH1 and Ckappa in plasmid pSW1. The recombinant plasmid were transformed into E. coli. The expressed recombinant proteins were analysed. RESULTS: The V(H) and V(L) genes were homologous with the published gene sequences of mouse antibody variable region. The concentration of chimeric Fab fragment in expression supernatant was about 180 microg/L detected by ELISA. Western blot analysis showed that SZ-2 Fab/Hu maintained the binding activity to human platelet GPIb. The recombinant proteins could suppress platelet aggregation induced by Ristocatin. CONCLUSION: The variable region genes of SZ-2 are cloned and the mouse-human chimeric Fab fragment is expressed successfully in E. coli.