The role of PKC in X-ray-induced megakaryocyte apoptosis and thrombocytopenia.

Thrombocytopenia is a critical complication after radiation therapy and exposure. Dysfunction of megakaryocyte development and platelet production are key pathophysiological stages in ionizing radiation (IR)-induced thrombocytopenia. Protein kinase C (PKC) plays an important role in regulating megakaryocyte development and platelet production. However, it remains unclear how PKC regulates IR-induced megakaryocyte apoptosis. In this study, we found that pretreatment of PKC pan-inhibitor Go6983 delayed IR-induced megakaryocyte apoptosis, and inhibited IR-induced mitochondrial membrane potential and ROS production in CMK cells. Moreover, suppressing PKC activation inhibited cleaved caspase3 expression and reduced p38 phosphorylation levels, and IR-induced PKC activation might be regulated by p53. In vivo experiments confirmed that Go6983 promoted platelet count recovery after 21 days of 3 Gy total body irradiation. Furthermore, Go6983 reduced megakaryocyte apoptosis, increased the number of megakaryocyte and polyploid formation in bone marrow, and improved the survival rate of 6 Gy total body irradiation. In conclusion, our results provided a potential therapeutic target for IR-induced thrombocytopenia.

Activation of AMPK in platelets promotes the production of offspring.

Platelets are terminally differentiated anucleated cells, but they still have cell-like functions and can even produce progeny platelets. However, the mechanism of platelet sprouting has not been elucidated so far. Here, we show that when platelet-rich plasma(PRP) was cultured at 37°C, platelets showed a spore phenomenon. The number of platelets increased when given a specific shear force. It is found that AMP-related signaling pathways, such as PKA and AMPK are activated in platelets in the spore state. Meanwhile, the mRNA expression levels of genes, such as CNN3, CAPZB, DBNL, KRT19, and ESPN related to PLS1 skeleton proteins also changed. Moreover, when we use the AMPK activator AICAR(AI) to treat washed platelets, cultured platelets can still appear spore phenomenon. We further demonstrate that washed platelets treated with Forskolin, an activator of PKA, not only platelet sprouting after culture but also the AMPK is activated. Taken together, these data demonstrate that AMPK plays a key role in the process of platelet budding and proliferation, suggesting a novel strategy to solve the problem of clinical platelet shortage.

What is new? In this study, we showed that when platelet-rich plasma(PRP) was cultured at 37°C, platelets showed spore phenomenon and increased.It was found that AMP-related signaling pathways, such as PKA and AMPK were activated in platelets in the spore state.In addition, we found that PKA acts as an upstream kinase of AMPK.In the process of platelet sprouting and proliferation, the mRNA expression levels of skeleton protein PLS1 and its related genes, such as CNN3, CAPZB, DBNL, KRT19, andESPN also changed.What is the impact? Our study proposes a new strategy to solve the problem of clinical platelet shortage.

X-rays Stimulate Granular Secretions and Activate Protein Kinase C Signaling in Human Platelets.

X-rays can induce morphological as well as functional changes in cells. Platelets are anuclear cellular fragments originating from megakaryocytes and are the major regulators in hemostasis and thrombosis. Platelet products are irradiated to avoid medical complications associated with platelet transfusion. So far, gamma, UV, and laser radiation have been used for this purpose. However, scientists are divided about the effects of radiation on platelet quality. The present study was designed to explore the possible effects of X-rays in washed human platelets and understand the molecular mechanism behind them. In the present study, we exposed washed human platelets to 10 or 30 Gy X-rays at 0.25 Gy/min. Flow cytometry, aggregometry, and western blot were performed to investigate the effect of X-rays on platelet degranulation, integrin activation, platelet aggregation, and apoptosis. It was found that X-rays immediately induced granular secretions with no effect on GP IIb/IIIa activation. Not surprisingly, due to granule secretions in irradiated platelets, platelet aggregation was significantly reduced. In contrast to granular secretions and platelet aggregation, X-rays induced mitochondrial transmembrane potential depolarization in a time-dependent manner to induce apoptosis and activated protein kinase C (PKC) signaling. This study revealed and explained the molecular mechanism activated by X-rays in washed human platelets. Here we also introduced Gö 6983, a PKC inhibitor, as an agent that counteracts X-ray-induced changes and maintains the integrity of platelets.

Alantolactone induces platelet apoptosis by activating the Akt pathway.

Alantolactone (ALT), a sesquiterpene lactone compound isolated from Inula helenium L., has recently attracted much attention for its anti-tumor properties. ALT reportedly functions by regulating the Akt pathway, which has been shown to be involved in programmed platelet death (apoptosis) and platelet activation. However, the precise effect of ALT on platelets remains unclear. In this study, washed platelets were treated with ALT in vitro, and apoptotic events and platelet activation were detected. In vivo, platelet transfusion experiments were employed to detect the effect of ALT on platelet clearance. Platelet counts were examined after intravenous injection of ALT. We found that ALT treatment induced Akt activation and Akt-mediated apoptosis in platelets. ALT-activated Akt elicited platelet apoptosis by activating phosphodiesterase (PDE3A) and PDE3A-mediated protein kinase A (PKA) inhibition. Pharmacological inhibition of the PI3K/Akt/PDE3A signaling pathway or PKA activation was found to protect platelets from apoptosis induced by ALT. Moreover, ALT-induced apoptotic platelets were removed faster in vivo, and ALT injection resulted in the platelet count decline. Either PI3K/Akt/PDE3A inhibitors or a PKA activator could protect platelets from clearance, ultimately ameliorating the ALT-induced decline in platelet count in the animal model. These results reveal the effects of ALT on platelets and their related mechanisms, suggesting potential therapeutic targets for the prevention and alleviation of possible side effects resulting from ALT treatments.

What is the context? In the past several decades, natural products, including traditional Chinese medicine (TCM), have been developed for the treatment of a variety of diseases.Alantolactone (ALT), a natural herb compound mainly extracted from the root of Inula helenium L., is the essential active component in many TCM formulas. ALT has attracted extensive attention because of its anti-cancer capacity recently.However, adverse events (AEs) induced by drugs are common in chemotherapy, and the side effects of ALT treatment remain unclear.What is new? In this study, experiments were conducted to clarify the precise effect of ALT on platelets. We demonstrated for the first time that ALT induces platelet apoptosis and platelet count decline, suggesting possible side effects of ALT treatment.ALT-activated Akt elicited platelet apoptosis by activating phosphodiesterase (PDE3A) and PDE3A-mediated protein kinase A (PKA) inhibition.Our work provides experimental evidence supporting the hypothesis that the effects of ALT on Akt may vary depending on cell types. Therefore. More research is needed to explore the side effects of ALT on other cells before clinical application.What is the impact? This study reveals possible side effects of ALT treatment, providing the reference for clinic drug administrate and estimation of medicine safety. Significantly, our findings demonstrated relevant molecular mechanisms, providing strategies for controlling or alleviating these side effects in the future.

Actin polymerization regulates glycoprotein Ibα shedding.

Glycoprotein (GP) Ibα shedding mediated by ADAM17 (a disintegrin and metalloproteinase 17) plays an important role in negatively regulating platelet function and thrombus formation. However, the mechanism of GPIbα shedding remains elusive. Here, we show that jasplakinolide (an actin-polymerizing peptide)-induced actin polymerization results in GPIbα shedding and impairs platelet function. Thrombin and A23187-induced GPIbα shedding is increased by jasplakinolide; in contrast, GPIbα shedding is reduced by a depolymerization regent (cytochalasin B). We find that actin polymerization activates calpain leading to filamin A hydrolyzation. We further demonstrate that the interaction of filamin A with the cytoplasmic domain of GPIbα plays a critical role in regulating actin polymerization-induced GPIbα shedding. Taken together, these data demonstrate that actin polymerization regulates ADAM17-mediated GPIbα shedding, suggesting a novel strategy to negatively regulate platelet function.

Identification of seven tumor-educated platelets RNAs for cancer diagnosis.

BACKGROUND: Tumor-educated platelets (TEPs) may enable blood-based cancer diagnosis. This study aimed to identify diagnostic TEPs genes involved in carcinogenesis. MATERIALS AND METHODS: The TEPs differentially expressed genes (DEGs) between healthy samples and early/advanced cancer samples were obtained using bioinformatics. Gene ontology (GO) analysis and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis were used to identify the pathways and functional annotation of TEPs DEGs. Protein-protein interaction of these TEPs DEGs was analyzed based on the STRING database and visualized by Cytoscape software. The correlation analysis and diagnostic analysis were performed to evaluate the diagnostic value of TEPs mRNAs expression for early/advanced cancers. Quantitative real-time PCR (qRT-PCR) was applied to validate the role of DEGs in cancers. RESULTS: TEPs mRNAs were mostly involved in protein binding, extracellular matrix, and cellular protein metabolic process. RSL24D1 was negatively correlated to early-stage cancers compared to healthy controls and may be potentially used for early cancer diagnosis. In addition, HPSE, IFI27, LGALS3BP, CRYM, HBD, COL6A3, LAMB2, and IFITM3 showed an upward trend in the expression from early to advanced cancer stages. Moreover, ARL2, FCGR2A, and KLHDC8B were positively associated with advanced, metastatic cancers compared to healthy controls. Among the 12 selected DEGs, the expression of 7 DEGs, including RSL24D1, IFI27, CRYM, HBD, IFITM3, FCGR2A, and KLHDC8B, were verified by the qRT-PCR method. CONCLUSION: This study suggests that the 7-gene TEPs liquid-biopsy biomarkers may be used for cancer diagnosis and monitoring.

Akt-mediated platelet apoptosis and its therapeutic implications in immune thrombocytopenia.

Immune thrombocytopenia (ITP) is an autoimmune disorder characterized by low platelet count which can cause fatal hemorrhage. ITP patients with antiplatelet glycoprotein (GP) Ib-IX autoantibodies appear refractory to conventional treatments, and the mechanism remains elusive. Here we show that the platelets undergo apoptosis in ITP patients with anti-GPIbα autoantibodies. Consistent with these findings, the anti-GPIbα monoclonal antibodies AN51 and SZ2 induce platelet apoptosis in vitro. We demonstrate that anti-GPIbα antibody binding activates Akt, which elicits platelet apoptosis through activation of phosphodiesterase (PDE3A) and PDE3A-mediated PKA inhibition. Genetic ablation or chemical inhibition of Akt or blocking of Akt signaling abolishes anti-GPIbα antibody-induced platelet apoptosis. We further demonstrate that the antibody-bound platelets are removed in vivo through an apoptosis-dependent manner. Phosphatidylserine (PS) exposure on apoptotic platelets results in phagocytosis of platelets by macrophages in the liver. Notably, inhibition or genetic ablation of Akt or Akt-regulated apoptotic signaling or blockage of PS exposure protects the platelets from clearance. Therefore, our findings reveal pathogenic mechanisms of ITP with anti-GPIbα autoantibodies and, more importantly, suggest therapeutic strategies for thrombocytopenia caused by autoantibodies or other pathogenic factors.

Receptor-interacting protein kinase 3 promotes platelet activation and thrombosis.

Previous studies have shown that receptor-interacting protein kinase 3 (RIP3) is involved in many important biological processes, including necroptosis, apoptosis, and inflammation. Here we show that RIP3 plays a critical role in regulating platelet functions and in vivo thrombosis and hemostasis. Tail bleeding times were significantly longer in RIP3-knockout (RIP3-/-) mice compared with their wild-type (WT) littermates. In an in vivo model of arteriole thrombosis, mice lacking RIP3 exhibited prolonged occlusion times. WT mice repopulated with RIP3-/- bone marrow-derived cells had longer occlusion times than RIP3-/- mice repopulated with WT bone marrow-derived cells, suggesting a role for RIP3-deficient platelets in arterial thrombosis. Consistent with these findings, we observed that RIP3 was expressed in both human and mice platelets. Deletion of RIP3 in mouse platelets caused a marked defect in aggregation and attenuated dense granule secretion in response to low doses of thrombin or a thromboxane A2 analog, U46619. Phosphorylation of Akt induced by U46619 or thrombin was diminished in RIP3-/- platelets. Moreover, RIP3 interacted with Gα13 Platelet spreading on fibrinogen and clot retraction were impaired in the absence of RIP3. RIP3 inhibitor dose-dependently inhibited platelet aggregation in vitro and prevented arterial thrombus formation in vivo. These data demonstrate a role for RIP3 in promoting in vivo thrombosis and hemostasis by amplifying platelet activation. RIP3 may represent a novel promising therapeutic target for thrombotic diseases.

Ethanol Induces Platelet Apoptosis.

BACKGROUND: Alcohol abuse incurs severe medical conditions, such as thrombocytopenia and hemorrhage, but the pathogenesis is not totally understood. Alcohol has been reported to induce apoptosis in eukaryotic cells, such as hepatocyte, nerve cell, corneal fibroblasts. However, it is still unclear whether alcohol induces platelet apoptosis. METHODS: Washed human platelets were pretreated with ethanol (EtOH), and apoptotic events and platelet function were detected. In in vivo experiments, C57BL/6J mice were given EtOH by gavage. Platelet counts, tail bleeding time, and the stomach were examined. RESULTS: EtOH dose dependently induces depolarization of mitochondrial inner transmembrane potential, up-regulation of Bax, down-regulation of Bcl-2, and caspase-3 activation. EtOH does not induce surface expression of P-selectin or PAC-1 binding, whereas significantly reduces collagen-, thrombin-, and ADP-induced platelet aggregation. Moreover, EtOH induces c-Jun NH2-terminal kinase activation. In an in vivo mouse model of the acute alcoholism, EtOH significantly reduces the number of circulating platelets, prolongs the tail bleeding time, and causes gastric mucosa hemorrhage. CONCLUSIONS: These data demonstrate that EtOH induces mitochondria-mediated intrinsic platelet apoptosis, results in the reduction of the number of circulating platelets, and impairs in vivo hemostasis. These findings reveal the possible pathogenesis of hemorrhagic symptoms in patients experiencing acute alcohol intoxication.

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.

Podoplanin requires sialylated O-glycans for stable expression on lymphatic endothelial cells and for interaction with platelets.

O-glycosylation of podoplanin (PDPN) on lymphatic endothelial cells is critical for the separation of blood and lymphatic systems by interacting with platelet C-type lectin-like receptor 2 during development. However, how O-glycosylation controls endothelial PDPN function and expression remains unclear. In this study, we report that core 1 O-glycan-deficient or desialylated PDPN was highly susceptible to proteolytic degradation by various proteases, including metalloproteinases (MMP)-2/9. We found that the lymph contained activated MMP-2/9 and incubation of the lymph reduced surface levels of PDPN on core 1 O-glycan-deficient endothelial cells, but not on wild-type ECs. The lymph from mice with sepsis induced by cecal ligation and puncture, which contained bacteria-derived sialidase, reduced PDPN levels on wild-type ECs. The MMP inhibitor, GM6001, rescued these reductions. Additionally, GM6001 treatment rescued the reduction of PDPN level on lymphatic endothelial cells in mice lacking endothelial core 1 O-glycan or cecal ligation and puncture-treated mice. Furthermore, core 1 O-glycan-deficient or desialylated PDPN impaired platelet interaction under physiological flow. These data indicate that sialylated O-glycans of PDPN are essential for platelet adhesion and prevent PDPN from proteolytic degradation primarily mediated by MMPs in the lymph.

Platelet Integrin αIIbß3 Inhibitor Rescues Progression of Apoptosis in Human Platelets.

BACKGROUND Apoptosis plays an important role in the physiology of platelet function. We aimed to detect the effect of the platelet integrin αIIbß3 inhibitor, tirofiban, on apoptotic events, including mitochondrial inner-membrane potential (ΔΨm), phosphatidylserine (PS) exposure on platelet surface, and the generation of reactive oxygen species (ROS), when washed platelets were stimulated with thrombin. MATERIAL AND METHODS The study included washed platelets from healthy humans, divided into 4 groups: vehicle, and tirofiban (0.05 µg/ml, 0.25 µg/ml, and 0.5 µg/ml). Platelets were pretreated with vehicle or tirofiban and incubated at 37°C with agitation for 6 h and 24 h. Before thrombin addition, the vehicle group divided into 2 equal groups. Except one vehicle group, the other 4 groups were all stimulated with thrombin (1 U/ml) for 30 min at 37°C. Using flow cytometry, we studied the DYm and PS exposure on platelet surfaces, and the generation of ROS in platelets. RESULTS We observed that at the time of 6 h and 24 h, thrombin-stimulated vehicle platelets induced significant depo-larization of ΔΨm, higher PS exposure, and increased ROS production compared with the vehicle group (P<0.01). However, the tirofiban group had significantly more recovery of DYm, PS exposure, and ROS production compared with the thrombin group (P<0.01). CONCLUSIONS The platelet integrin αIIbß3 inhibitor, tirofiban, inhibits the depolarization of DYm, PS exposure on platelet surface, and ROS production when stimulated with thrombin. These results suggest that αIIbß3 inhibitor inhibits the initiation of apoptosis in platelets, showing a potential clinical application of tirofiban as an apoptosis inhibitor.

Carmustine induces platelet apoptosis.

Carmustine is one of the alkylating chemotherapeutic agents, which are used to treat various types of cancers, such as brain tumors, Hodgkins and non-Hodgkins lymphoma and multiple myeloma. However, carmustine has the side effect of thrombocytopenia, and the mechanism is not completely understood. In this study, we show that carmustine dose-dependently induced depolarization of mitochondrial inner transmembrane potential (ΔΨm), up-regulation of Bax, down-regulation of Bcl-2 and caspase-3 activation. Carmustine did not induce surface expression of P-selectin or PAC-1 binding, whereas, obviously reduced collagen and thrombin-induced platelet aggregation. Dicumarol, c-Jun NH2-terminal kinase-specific inhibitor, reduced carmustine-induced ΔΨm depolarization in platelets. The numbers of circulating platelets were reduced, and the tail bleeding time was significantly increased in mice that were injected with carmustine. Taken together, these data indicate that carmustine induced platelet apoptosis, suggesting the possible pathogenesis of thrombocytopenia in patients treated with carmustine.

Staurosporine Induces Platelet Apoptosis Through p38 Mitogen-Activated Protein Kinase Signaling Pathway.

BACKGROUND: Staurosporine (STS), a microbial alkaloid and potent PKC inhibitor, has become one of the most promising anti-cancer drugs. STS effectively induces apoptosis in many nucleated cells; however, it is still unclear whether STS induces apoptosis in enucleated platelets. METHODS: Apoptotic events in platelets treated with STS were assessed by flow cytometry or western blotting. RESULTS: STS induced depolarization of mitochondrial inner transmembrane potential (ΔΨm), up-regulation of Bax and Bak, phosphatidylserine (PS) exposure, release of mitochondrial cytochrome c, and activation of caspase-8 and caspase-9 in human platelets. Furthermore, STS stimulation induced phosphorylation of p38 mitogen-activated protein kinase (MAPK). Inhibition of p38 MAPK activation significantly reduced ΔΨm depolarization and PS exposure in platelets stimulated with STS. CONCLUSIONS: These data indicate that STS induces platelet apoptosis via the p38 MAPK signaling pathway. These findings suggest that platelet apoptosis-related hemorrhage should be noticed in STS and its derivatives in clinical tests.

Enhanced platelet apoptosis in chronic uremic patients.

BACKGROUND: There is a paucity of research on platelet apoptosis and its contribution to platelet dysfunction in uremic patients. The present study sought to analyze platelets apoptosis in uremic patients who underwent different dialysis modalities. METHODS: Sixteen chronic uremic patients (5 on hemodialysis, 6 on peritoneal dialysis and 5 on non-dialysis) and 16 controls were studied. Platelet-rich plasma was detected for apoptotic events including depolarization of mitochondrial inner membrane potential (ΔΨm), phosphatidylserine (PS) exposure, activation of caspases-3 and Bcl-2 family proteins variations by Flow Cytometry or by Western-Blot. Washed normal platelets were incubated with normal or uremic platelet poor plasma and then were detected apoptotic events. Platelets function was assessed by ristocetin induced aggregative function test. RESULTS: Compared to controls, uremic platelets demonstrated greater apoptosis for the ΔΨm depolarization (43.48 ± 9.58 vs. 52.76 ± 15.36, p = 0.005) as well as PS exposure (1.36 ± 0.51 vs. 0.99 ± 0.27, p < 0.001). There was no significant difference among different treatment groups (for the ΔΨm depolarization f = 0.16, p = 0.85; for the PS exposure f = 1.06, p = 0.36). Western Blot analyses showed caspase-3 activation and pro-apoptotic Bcl-2 family proteins expression. Platelets exposed to uremic plasma exhibited distinct apoptosis phenomena. Ristocetin induced platelet aggregation was markedly diminished in uremic patients and treated platelets. CONCLUSIONS: These findings indicate that platelets are incurred apoptosis in uremia patients. Uremic plasma accelerates apoptosis of normal platelets, resulting in a dysfunctional pattern of platelets in uremia. Uremic platelets apoptosis has no relationship with dialysis modality.

Essential role of glycoprotein Ibα in platelet activation.

ABSTRACT: Glycoprotein Ibα (GPIbα), the ligand-binding subunit of platelet GPIb-IX complex, interacts with von Willebrand factor (VWF) exposed at the injured vessel wall, initiating platelet adhesion, activation, hemostasis, and thrombus formation. The cytoplasmic tail of GPIbα interacts with 14-3-3ζ, regulating the VWF-GPIbα-elicited signal transduction and VWF binding function of GPIbα. However, we unexpectedly found that the GPIbα-14-3-3ζ association, beyond VWF-dependent function, is essential for general platelet activation. We found that the myristoylated peptide of GPIbα C-terminus MPαC, a potential GPIbα inhibitor, by itself induced platelet aggregation, integrin αIIbß3 activation, granule secretion, and phosphatidylserine (PS) exposure. Conversely, the deletion of the cytoplasmic tail of GPIbα in mouse platelets (10aa-/-) decreased platelet aggregation, integrin αIIbß3 activation, granule secretion, and PS exposure induced by various physiological agonists. Phosphoproteome-based kinase activity profiling revealed significantly upregulated protein kinase C (PKC) activity in MPαC-treated platelets. MPαC-induced platelet activation was abolished by the pan-PKC inhibitor and PKCα deletion. Decreased PKC activity was observed in both resting and agonist-stimulated 10aa-/- platelets. GPIbα regulates PKCα activity by sequestering 14-3-3ζ from PKCα. In vivo, the deletion of the GPIbα cytoplasmic tail impaired mouse hemostasis and thrombus formation and protected against platelet-dependent pulmonary thromboembolism. Therefore, our findings demonstrate an essential role for the GPIbα cytoplasmic tail in regulating platelet general activation and thrombus formation beyond the VWF-GPIbα axis.

Aspirin Induces platelet apoptosis.

Aspirin is widely used in the treatment of a number of clinical conditions. Although aspirin is being thought to be a relatively "safe" medicine, it also has some side effects, particularly the risk of bleeding which may be severe and lead to death. The mechanisms, however, are not totally understood. It has been reported recently that aspirin induces apoptosis in many cell types. Thus, the aim of the current study is to explore whether aspirin induces platelet apoptosis. The data show that mitochondrial transmembrane potential (ΔΨm) depolarizations and phosphatidylserine (PS) exposures were dose-dependently induced by aspirin in platelets. To further confirm that aspirin incurs platelet apoptosis, caspase-3 activity was measured in platelets, and the result indicated that aspirin induced caspase-3 activation. Furthermore, the mean volume of platelets incubated with aspirin was obviously reduced. Caspase inhibitor z-VAD-fmk inhibited aspirin induced apoptotic platelet shrinkage and ΔΨm depolarization, but had no effect on PS exposure. In addition, platelets incubated with cyclooxygenase inhibitor indomethacin did not incur ΔΨm depolarazation and PS exposure. Taken together, the data indicate that aspirin induces platelet apoptosis via caspase-3 activation.

[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.

Cyclic thrombocytopenia associated with estradiol: a case report.

OBJECTIVES: Cyclic thrombocytopenia (CTP) is a rare blood disorder characterized by periodic fluctuations in platelet counts. CTP usually appears in pre-menopausal women, and these fluctuations of platelets are in phase with the menstrual cycle. CTP is a heterogeneous disease, and the pathogenic mechanism is still unclear. Therefore, it harbors great significance for exploring the association of fluctuations in platelet counts with hormonal-cycle. MATERIALS: Firstly, we washed human platelets from healthy volunteers following the Declaration of Helsinki. Flow cytometer was employed to measure the mitochondrial inner transmembrane potential (ΔΨm) depolarization, PS exposure, P-selectin expression, and GPIIb/IIIa activation in platelets. In addition, western blot detected the related protein expression. The corresponding assay kit measured the caspase-3 and PDE3A activity. Finally, flow cytometry determined mouse platelets labeled with calcein. RESULTS: We find a reverse relationship between the platelet count and serum estradiol (E2) level in a CTP patient. We demonstrated that E2 induces platelet apoptosis in vitro and platelet clearance in vivo. We further discovered that E2 activates phosphodiesterase 3A, which inhibits protein kinase A (PKA), leading to PKA-mediated platelet apoptosis. Activation of PKA protected platelets from E2-induced thrombocytopenia and elevated the number of mice circulatory platelets. CONCLUSIONS: We find that E2 induces platelet apoptosis and clearance through PDE3A-mediated PKA inhibition. Activation of PKA rescues E2-induced thrombocytopenia in mice. Thus, our study reveals a pathogenesis of E2-related CTP and suggests promising therapeutic strategies for the disease.