Essential role of glycoprotein Ibα in platelet activation.

Glycoprotein (GP) Ib, 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 GPIb interacts with 14-3-3, regulate ng the VWF-GPIb-elicited signal transduction and VWF binding function of GPIb. However, we unexpectedly found that the GPIb-14-3-3 association, beyond VWF-dependent function, is essential for general platelet activation. We found that the GPIb cytoplasmic tail peptide MPC, a potential GPIb inhibitor, by itself induced platelet aggregation, integrin αIIbß3 activation, granule secretion, and phosphatidylserine (PS) exposure. Conversely, the deletion of the cytoplasmic tail of GPIb in mouse platelets (10aa-/-) decreased platelet aggregation, integrin IIb3 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 MPC-treated platelets. MPC-induced platelet activation was abolished by the pan-PKC inhibitor and PKC deletion. Decreased PKC activity was observed in both resting and agonist-stimulated 10aa-/- platelets. GPIb regulates PKC activity by sequestering 14-3-3 from PKC. In vivo, the deletion of the GPIb cytoplasmic tail impaired mouse hemostasis and thrombus formation and protected against platelet-dependent pulmonary thromboembolism. Therefore, our findings demonstrate an essential role for the GPIb cytoplasmic tail in regulating platelet general activation and thrombus formation beyond the VWF-GPIb axis.

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.

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.

Monascuspiloin from Monascus-Fermented Red Mold Rice Alleviates Alcoholic Liver Injury and Modulates Intestinal Microbiota.

Monascus-fermented red mold rice (RMR) has excellent physiological efficacy on lipid metabolism and liver function. This study investigated the ameliorative effects of monascuspiloin (MP) from RMR on alcoholic liver injury in mice, and further clarified its mechanism of action. Results showed that MP intervention obviously ameliorated lipid metabolism and liver function in mice with over-drinking. In addition, dietary MP intervention reduced liver MDA levels and increased liver CAT, SOD, and GSH levels, thus alleviating liver oxidative stress induced by excessive drinking. 16S rRNA amplicon sequencing showed that MP intervention was beneficial to ameliorate intestinal microbiota dysbiosis by elevating the proportion of norank_f_Lachnospiraceae, Lachnoclostridium, Alistipes, Roseburia, Vagococcus, etc., but decreasing the proportion of Staphylococcus, norank_f_Desulfovibrionaceae, Lachnospiraceae_UCG-001, Helicobacter, norank_f_Muribaculaceae, unclassified_f_Ruminococcaceae, etc. Additionally, correlation network analysis indicated that the key intestinal bacterial taxa intervened by MP were closely related to some biochemical parameters of lipid metabolism, liver function, and oxidative stress. Moreover, liver metabolomics analysis revealed that dietary MP supplementation significantly regulated the levels of 75 metabolites in the liver, which were involved in the synthesis and degradation of ketone bodies, taurine, and hypotaurine metabolism, and other metabolic pathways. Furthermore, dietary MP intervention regulated gene transcription and protein expression associated with hepatic lipid metabolism and oxidative stress. In short, these findings suggest that MP mitigates alcohol-induced liver injury by regulating the intestinal microbiome and liver metabolic pathway, and thus can serve as a functional component to prevent liver disease.

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.

Essential role of zyxin in platelet biogenesis and glycoprotein Ib-IX surface expression.

Platelets are generated from the cytoplasm of megakaryocytes (MKs) via actin cytoskeleton reorganization. Zyxin is a focal adhesion protein and wildly expressed in eukaryotes to regulate actin remodeling. Zyxin is upregulated during megakaryocytic differentiation; however, the role of zyxin in thrombopoiesis is unknown. Here we show that zyxin ablation results in profound macrothrombocytopenia. Platelet lifespan and thrombopoietin level were comparable between wild-type and zyxin-deficient mice, but MK maturation, demarcation membrane system formation, and proplatelet generation were obviously impaired in the absence of zyxin. Differential proteomic analysis of proteins associated with macrothrombocytopenia revealed that glycoprotein (GP) Ib-IX was significantly reduced in zyxin-deficient platelets. Moreover, GPIb-IX surface level was decreased in zyxin-deficient MKs. Knockdown of zyxin in a human megakaryocytic cell line resulted in GPIbα degradation by lysosomes leading to the reduction of GPIb-IX surface level. We further found that zyxin was colocalized with vasodilator-stimulated phosphoprotein (VASP), and loss of zyxin caused diffuse distribution of VASP and actin cytoskeleton disorganization in both platelets and MKs. Reconstitution of zyxin with VASP binding site in zyxin-deficient hematopoietic progenitor cell-derived MKs restored GPIb-IX surface expression and proplatelet generation. Taken together, our findings identify zyxin as a regulator of platelet biogenesis and GPIb-IX surface expression through VASP-mediated cytoskeleton reorganization, suggesting possible pathogenesis of macrothrombocytopenia.

Carbamazepine Induces Platelet Apoptosis and Thrombocytopenia Through Protein Kinase A.

Carbamazepine is extensively used worldwide to treat a wide range of disorders such as epilepsy, peripheral neuralgia and bipolar disorder. Thrombocytopenia and hemorrhage have been identified in multiple carbamazepine-treated patients. However, the underlying mechanism remains poorly understood. Here, we show that platelets undergo apoptosis after carbamazepine treatment. The apoptotic platelets induced by carbamazepine are rapidly removed in vivo, which accounts for thrombocytopenia. We found that carbamazepine treatment attenuates the phosphorylation level of bcl-xl/bcl-2-associated death promoter (BAD), vasodilator-associated stimulated phosphoprotein (VASP) and GPIbß in platelets, indicating an inhibition effect on protein kinase A (PKA). We further demonstrated that carbamazepine reduced PKA activity through PI3K/Akt/PDE3A signaling pathway. Pharmacological activation of PKA or inhibition of PI3K/Akt/PDE3A protects platelets from apoptosis induced by carbamazepine. Importantly, PDE3A inhibitors or PKA activator ameliorates carbamazepine-mediated thrombocytopenia in vivo. These findings shed light on a possible mechanism of carbamazepine-induced thrombocytopenia, designating PDE3A/PKA as a potential therapeutic target in the treatment of carbamazepine-induced thrombocytopenia.

Comparative Study on the Antioxidant Activity of Monascus Yellow Pigments From Two Different Types of Hongqu-Functional Qu and Coloring Qu.

This study is the first to investigate the difference in the composition of Monascus azaphilone pigments (MonAzPs) between functional Qu (FQ) and coloring Qu (CQ) and analyze their relationships with antioxidant activity. The composition of key active components and antioxidant activity of the ethanol extracts of FQ and CQ were analyzed by Uv-vis, HPLC, and chemical antioxidant tests. The composition of MonAzPs of the ethanol extracts was further analyzed by HPLC-MS. Seven Monascus yellow pigments (MYPs) with high abundance were successfully purified for the antioxidation evaluation in vitro and in the cell. Correlation analysis between the metabolites and the antioxidant activity of Hongqu indicated that MonAzPs might play an essential role in the antioxidant activity (r > 0.80). By contrast, the monacolin K (MK), polysaccharide, ergosterol, and γ-aminobutyric acid (GABA) were not significantly correlated with the antioxidant activity. Orthogonal partial least squares discriminant analysis (OPLS-DA) based on the composition of MonAzPs revealed that the abundance of MYPs is significantly different between FQ and CQ (P < 0.05 and VIP > 1.0). Seven MYPs (monasfluore A, monaphilone B, monascuspilion, monascin, monaphilone A, ankaflavin, and new yellow pigment) with high abundance were successfully purified for the antioxidation evaluation. Chemical antioxidant tests revealed that the antioxidant activities of monaphilone A, ankaflavin, and new yellow pigment only from CQ were significantly more potent than monasfluore A and monascuspilion only separated from FQ. The cellular antioxidant assay (CAA) showed that the new yellow pigment had the best antioxidant activity (quercetin equivalent 7.23 µM), followed by monasfluore A and monaphilone B, all of which were significantly better than monascin and ankaflavin, the two most frequently reported MYPs. Research on the structure-activity relationship demonstrated that alterations of the hydroxyl that occurred on C-3' or C-11 obviously affected the antioxidant activities of MYPs. Our findings provide evidence that MYPs may be the key active components for CQ to have a more potent antioxidant capacity than FQ. The alterations of the hydroxyl that occurred on C-3' or C-11 obviously affected the antioxidant activities of MYPs.

Development of a Novel Restrictive Medium for Monascus Enrichment From Hongqu Based on the Synergistic Stress of Lactic Acid and Ethanol.

Hongqu is a famous fermented food produced by Monascus and has been used as food coloring, wine starters and food additives for thousands of years in China. Excellent Monascus strain is an important prerequisite for producing high-quality Hongqu. However, the isolation of Monascus pure culture from Hongqu samples is time-consuming and laborious because it is easily interfered by other microorganisms (especially filamentous fungi). Therefore, the development of restrictive medium for Monascus enrichment from Hongqu is of great significance for the preparation and screening of excellent Monascus strains. Results of this study showed that Monascus has good tolerance to lactic acid and ethanol. Under the conditions of tolerance limits [7.5% lactic acid (v/v) and 12.0% ethanol (v/v)], Monascus could not grow but it still retained the vitality of spore germination, and the spore activity gradually decreased with the increasing concentrations of lactic acid and ethanol. More interestingly, the addition of lactic acid and ethanol significantly changed the microbial community structure in rice milk inoculated with Hongqu. After response surface optimization, Monascus could be successfully enriched without the interference of other microorganisms when 3.98% (v/v) lactic acid and 6.24% (v/v) ethanol were added to rice milk simultaneously. The optimal enrichment duration of Monascus by the restrictive medium based on the synergistic stress of lactic acid and ethanol is 8∼24 h. The synergistic stress of lactic acid and ethanol had no obvious effects on the accumulation of major metabolites in the progeny of Monascus, and was suitable for the enrichment of Monascus from different types of Hongqu. Finally, the possible mechanisms on the tolerance of Monascus to the synergistic stress of lactic acid and ethanol were preliminarily studied. Under the synergistic stress of lactic acid and ethanol, the cell membrane of Monascus defends against lactic acid and ethanol into cells to some extent, and the superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) activities of Monascus were higher than those of other fungi, which significantly reduced the degree of lipid peroxidation of cell membrane, while secreting more amylase to make reducing sugars to provide the cells with enough energy to resist environmental stress. This work has great application value for the construction of Monascus strain library and the better development of its germplasm resources.

A novel method to detect autoantibodies against platelets in patients with immune thrombocytopenia.

BACKGROUND AND OBJECTIVES: To examine anti-platelet autoantibodies in patients with immune thrombocytopenia (ITP) not only provides solid evidence for diagnosis, and also helps to select an individualized strategy for the treatment. The aim of this study is to develop a novel cell-based assay to detect autoantibodies in ITP patients. METHODS/PATIENTS: The DNA sequences of human platelet membrane protein GPIbα, GPIbß, GP IX, GPIIb and GPIIIa subunits were obtained from NCBI database and synthesized. The synthetic fragments were ligated into pcDNA 3.3 and constructed the recombinant plasmids and transfected into Chinese hamster ovary (CHO) cells to establish cell lines stable expressing GPIb-IX and/or GPIIb/IIIa complexes. One hundred and two ITP patients with different anti-platelet autoantibodies, 57 patients with other kinds of autoimmune diseases and 104 healthy control were selected to examine sensitivity, specificity and accuracy of this method. RESULTS: CHO cells stable expressing GPIb-IX and/or GPIIb/IIIa proteins were established. The cells were fixed with 4% paraformaldehyde and stored at -80 ℃, more than 80% of the cells were still expressed target proteins after 180 days of storage. The concentrations of target antibody from 0.1 to 100 µg/ml were detectable by this method, and 10-50 µg/ml antibody binding to the CHO cells yielded higher distinguishable fluorescent intensities. Inter-assay and intra-assay coefficients of variation and receiver operating characteristic curve analysis showed that this method had relatively higher reproducibility and specificity. Compared with Flow Cytometric Immunobead Array, this method has relatively higher specificity (95.2%) and accuracy (90.8%) in detection of 102 ITP patients. CONCLUSION: A novel cell-based assay to detect autoantibodies in ITP patients is established, which appears to be a promising method to diagnose ITP.

[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.

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.

Protein kinase A determines platelet life span and survival by regulating apoptosis.

Apoptosis delimits platelet life span in the circulation and leads to storage lesion, which severely limits the shelf life of stored platelets. Moreover, accumulating evidence indicates that platelet apoptosis provoked by various pathological stimuli results in thrombocytopenia in many common diseases. However, little is known about how platelet apoptosis is initiated or regulated. Here, we show that PKA activity is markedly reduced in platelets aged in vitro, stored platelets, and platelets from patients with immune thrombocytopenia (ITP), diabetes, and bacterial infections. Inhibition or genetic ablation of PKA provoked intrinsic programmed platelet apoptosis in vitro and rapid platelet clearance in vivo. PKA inhibition resulted in dephosphorylation of the proapoptotic protein BAD at Ser155, resulting in sequestration of prosurvival protein BCL-XL in mitochondria and subsequent apoptosis. Notably, PKA activation protected platelets from apoptosis induced by storage or pathological stimuli and elevated peripheral platelet levels in normal mice and in a murine model of ITP. Therefore, these findings identify PKA as a homeostatic regulator of platelet apoptosis that determines platelet life span and survival. Furthermore, these results suggest that regulation of PKA activity represents a promising strategy for extending platelet shelf life and has profound implications for the treatment of platelet number-related diseases and disorders.