Regulation of Mitochondria on Platelet Apoptosis and Activation / 中国实验血液学杂志

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 / 中国实验血液学杂志

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 / 中国实验血液学杂志

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.

The Role of Zyxin in Regulating Platelet Cytoskeleton Distribution / 中国实验血液学杂志

OBJECTIVE@#To investigate the regulatory effect of zyxin on the distribution of platelet cytoskeleton.@*METHODS@#Platelets were isolated from zyxin-knockout (Zyx@*RESULTS@#After zyxin gene was knockout, the expressions of cytoskeleton proteins β-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@*CONCLUSION@#Zyxin significantly regulates the distribution of platelet cytoskeleton, which plays an important role in maintaining platelet cytoskeleton homeostasis.

Effect of Protein Kinase A Activation on Aggregation Function of Platelets / 中国实验血液学杂志

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.

Main Factors Influencing FeCl-Induced Mouse Mesenteric Arteriole Thrombosis Model / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the factors that influence FeCl-induced mouse mesenteric arteriole thrombosis model.</p><p><b>METHODS</b>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 (1×10, 1×10and 2×10platelets) and concentrations of FeCl(6%, 12%, 24% and 48%) on FeCl-induced thrombosis model were compared.</p><p><b>RESULTS</b>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 1×10and 2×10of pletclets was 15-18 min, which was shorter than that of transfusion 1×10platelets (30 mins). The occlusion time resulting from transfusion of 6% and 12% FeClwas from 15 to 20 min, however the transfusion of 24% and 48% FeClall in all leads to vessel occlusion within 10 min.</p><p><b>CONCLUSION</b>The factors influencing the success of FeCl-induced mouse thrombosis model are more. Transfusion of 1×10to 2×10labeled platelets to 3-6 week-old mice, and 6% to 12% of FeClshould 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.</p>

Regulatory Effect of Protein Disulfide Isomerase on Platelet GPIbα Ectodomain Shedding / 中国实验血液学杂志

<p><b>OBJECTIVE</b>This study was aimed to investigate the regulatory effect of protein disulfide isomerase (PDI) on platelet GPIbα ectodomain shedding.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>

Protein kinase C activation induces platelet apoptosis / 中国实验血液学杂志

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.

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 / 中华血液学杂志

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSIONS</b>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.</p>

Inhibition of protein kinase A leads to cleavage of platelet GP I balpha and downregulation of GP I b-dependent platelet aggregation / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the regulatory role of protein kinase A (PKA) in platelet surface glycoprotein (GP) I balpha expression.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>

Establishment of Chinese hamster ovary cell line expressing recombinant GPIb-IX complex / 中国实验血液学杂志

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.