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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., Competing Interests: The authors declare no conflict of interest.

Cerebral amyloid angiopathy (CAA) is characterized by cerebrovascular amyloid deposition. It contributes to the rate of cognitive decline in older individuals and is present in >90% of patients with Alzheimer's disease (AD), with no cure so far. Molecular modifications during CAA should be elucidated to improve its diagnosis and treatment. In this study, amyloid-β (Aβ) aggregates in platelet membranes from 65 patients with CAA and 66 healthy volunteers (controls) were confirmed through thioflavin T (ThT) assay and Western blot analysis. Further, post-translational modifications (PTMs) of Aβ in platelet membranes were analyzed using ultra-performance liquid chromatography/electrospray ionization quadruple time-of-flight mass spectrometry (UPLC/ESI-Q-TOF/MS). ThT assay results indicated that there were amyloid components in platelets from both patients with CAA and controls. Western blot analysis showed that different molecular weight (MW) Aβ aggregates were found in platelet membranes. LC-MS analysis showed that PTMs including methylation, phosphopantetheine, phosphorylation, deamidation, and acetylation, occurred in Aβ peptide in platelet membranes from both patients with CAA and controls, while Met 35 oxidation (Met OX ) and Gln 15 deamidation were identified only from patients with CAA. Thus, this study identified potential biomarkers of CAA and characterized the mechanism underlying amyloidogenesis in CAA., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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.

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., (Copyright © 2017 by the Research Society on Alcoholism.)

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.

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.

Many immune thrombocytopenia (ITP) patients, particularly patients with anti-glycoprotein (GP) Ib-IX autoantibodies, do not respond to the conventional treatments such as splenectomy. However, the underlying mechanism remains unclear. Here we found that anti-GPIbα N-terminus antibody AN51, but not other anti-GPIbα antibodies (AK2, HIP1, VM16d, or WM23), induced GPIbα clustering that led to integrin αIIbβ3-dependent platelet aggregation. After intravenous injection, AN51 dose-dependently induced thrombocytopenia in guinea pigs, and the platelets were mainly removed by macrophages in the liver. N-acetyl-D-glucosamine, previously shown to inhibit integrin αMβ2-mediated phagocytosis of refrigerated platelets, dose-dependently inhibited AN51-induced platelet clearance. Furthermore, AN51 but not VM16d, induced rapid platelet clearance in the liver of cynomolgus macaques. Five of 22 chronic ITP patients had anti-GPIbα autoantibodies, and the autoantibodies from four of the five patients competed with AN51 for binding to platelets. These data indicate that GPIbα clustering induced by anti-GPIbα N-terminus antibody causes integrin αIIbβ3-dependent platelet aggregation, phagocytosis, and rapid platelet clearance in the liver. Our findings reveal a novel Fc-independent mechanism underlying the pathogenesis of ITP, and suggest new therapeutic strategies for ITP patients with anti-GPIbα autoantibodies.

Objective: To investigate the in vitro effects of immune inhibitor tacrolimus on platelet function., Methods: Fresh venous blood was collected from healthy volunteers at ages of 18-25 years old, who are not taking antiplatelet drugs within two weeks. The platelets were isolated from the blood and incubated with different concentrations of tacrolimus (0.06, 0.6, 6, 60, 120, 240 μmol/L) at 37 °C for 2 hours, and then the changes of mitochondrial membrane potential and P-selection of platelets were detected by flow cytometry, the expression of apoptosis related protein by Western Blot, and the change of the platelet aggregation function by platelet aggregation analyzer., Results: Tacrolimus at concentration of 0.06 μmol/L could promote collagen induced platelet aggregation, inhibit thrombin induced platelet aggregation, have no effect on ristocetin and vWF induced platelet aggregation function. Tacrolimus at concentration of 120 μmol/L and 240 μmol/L could reduce the platelet mitochondrial membrane potential and induce the expression of apoptosis protein caspase-3., Conclusion: In vitro experimental results showed that high concentration of tacrolimus could lead to platelet apoptosis. But the current therapeutic dose of tacrolimus at 0.06 μmol/L (which is equivalent to 50 ng/ml blood concentration) could have different effects on platelet aggregation function according to different stimulating agents.

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.

Objective: To study the effect of reactive oxygen species (ROS) on the regulation of platelet apoptosis., Methods: Washed healthy human platelets were pre-incubated with N-caetyl-Lcysteine (NAC), and then stimulated with dibucaine or thrombin. The production of ROS and depolarization of mitochondrial membrane potential (∆ ψm) were detected by flow cytometry. The activation of caspase-3 and expression of Bcl-xL were analyzed by Western blot., Results: (1)The average ROS fluorescence value of NAC+dibucaine group was lower than that of dibucaine group(0.66 ± 0.11 vs 1.06 ± 0.08, P<0.01), while that of NAC+thrombin group was also lower than that of thrombin group(0.45 ± 0.05 vs 0.71 ± 0.11, P=0.001). (2)The percentage of platelets with normal ∆ψm in NAC+Dibucaine group was higher than that of dibucaine group[(86.30 ± 9.37)% vs (13.52 ± 3.01)%, P=0.000], while that of NAC+thrombin group was also higher than that of thrombin group[(93.00 ± 3.03)% vs (76.58 ± 5.28)%, P=0.000]. (3)Fragmentation generated by caspase-3 activation in dibucaine group was much more than that in DMSO control group, while the fragmentation in NAC+dibucaine group was significantly decreased. (4)The expression of anti-apoptosis protein Bcl-xL of NAC+dibucaine group was significantly higher than that of the dibucaine group, while that of NAC+thrombin group was also higher than that of thrombin group., Conclusion: Through the regulation of ROS, NAC could inhibit the platelet apoptosis induced by dibucaine or thrombin.

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.

Glycoprotein (GP) Ibα ectodomain shedding has become a generally accepted negative regulatory mechanism of platelet function. Stimulation of platelet with either physiological or chemical compound results in GPIbα ectodomain shedding in vitro and in vivo, the mechanism, however, is not totally understood. Here we show, collagen, thrombin, and calcium ionophore A23187 induce reactive oxygen species (ROS) generation, and simultaneously incur GPIbα ectodomain shedding. ROS scavengers N-acetylcysteine (NAC) and dithiothreitol (DTT) abolish not only collagen, thrombin, and A23187 induced ROS production, but also GPIbα ectodomain shedding. Interestingly, a recognized calpain activator, dibucaine, induces both ROS production and GPIbα shedding, which are also obviously reduced by NAC and DTT. Furthermore, calpain inhibitors calpain inhibitor I and carbobenzoxy-valinyl-phenylalaninal, obviously reduce dibucaine, thrombin, and A23187-induced ROS generation. These data indicate that ROS plays a key role in collagen, thrombin, and A23187-induced GPIbα ectodomain shedding. Calpain is an up-stream regulator that regulates ROS-mediated GPIbα shedding., (© 2013.)

Cisplatin (cis-diamminedichloroplatinum II) is one of the most widely used anti-tumor agents. However, cisplatin-based chemotherapy is usually accompanied by adverse side effects such as thrombocytopenia, and the mechanism remains unclear. Here we show that cisplatin induced several platelet apoptotic events including up-regulation of Bax and Bak, down-regulation of Bcl-2 and Bcl-X(L), mitochondrial translocation of Bax, mitochondrial inner transmembrane potential depolarization, caspase-3 activation and phosphatidylserine (PS) exposure. Cisplatin dose-dependently induced activation of extracellular signal-regulated protein kinase (ERK) in platelets. Caspase-3 inhibitor z-DEVD-fmk dramatically inhibited cisplatin-induced caspase-3 activation and PS exposure without affecting ERK activation. Blockade of the ERK pathway significantly prevented platelet apoptosis. Furthermore, levels of reactive oxygen species (ROS) and Ca(2+) were significantly elevated by cisplatin, and scavenging of ROS and Ca(2+) obviously inhibited platelet apoptosis induced by cisplatin. In addition, cisplatin did not induce platelet activation, whereas it obviously impaired platelet functions. These data indicate that cisplatin induces platelet apoptosis through the ERK signaling pathway, which might contribute to cisplatin-related haematological toxicity., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Calcium-dependent calpains are a family of cysteine proteases that have been demonstrated to play key roles in both platelet glycoprotein Ibα shedding and platelet activation and altered calpain activity is associated with thrombotic thrombocytopenic purpura. Calpain activators induce apoptosis in several types of nucleated cells. However, it is not clear whether calpain activators induce platelet apoptosis. Here we show that the calpain activator dibucaine induced several platelet apoptotic events including depolarization of the mitochondrial inner transmembrane potential, up-regulation of Bax and Bak, down-regulation of Bcl-2 and Bcl-X(L), caspase-3 activation and phosphatidylserine exposure. Platelet apoptosis elicited by dibucaine was not affected by the broad spectrum metalloproteinase inhibitor GM6001. Furthermore, dibucaine did not induce platelet activation as detected by P-selectin expression and PAC-1 binding. However, platelet aggregation induced by ristocetin or α-thrombin, platelet adhesion and spreading on von Willebrand factor were significantly inhibited in platelets treated with dibucaine. Taken together, these data indicate that dibucaine induces platelet apoptosis and platelet dysfunction.

Rationale: The interaction between platelet glycoprotein (GP) Ib-IX and von Willebrand factor (VWF) is initiated by conformational changes in immobilized VWF and is also regulated by the intraplatelet proteins 14-3-3zeta and filamin A. Both 14-3-3zeta and filamin A associate with the cytoplasmic domain of GPIbalpha, whereas little is known about their relationship in regulating the VWF binding function of GPIb-IX., Objective: To explore the mechanism underlying the roles of 14-3-3zeta and filamin A in regulating the VWF binding function of GPIb-IX., Methods and Results: A truncation mutant of GPIbalpha (Delta565) deleting the C-terminal 14-3-3zeta binding sites retained 14-3-3zeta binding function, in contrast, deletion of the C-terminal residues 551 to 610 of GPIbalpha totally abolished 14-3-3zeta binding, indicating that the residues 551 to 564 of GPIbalpha are important in the interaction between 14-3-3zeta and GPIb-IX. An antibody recognizing phosphorylated R557GpSLP561 sequence reacted with GPIbalpha suggesting phosphorylation of a population of GPIbalpha molecules at Ser559, and a membrane permeable phosphopeptide (MP-P), R557GpSLP561 corresponding to residues 557 to 561 of GPIbalpha eliminated the association of 14-3-3zeta with Delta565. MP-P also promoted GPIb-IX association with the membrane skeleton, and inhibited ristocetin-induced platelet agglutination, VWF binding to platelets and platelet adhesion to immobilized VWF. Furthermore, a GPIb-IX mutant replacing Ser559 of GPIbalpha with alanine showed an enhanced association with the membrane skeleton, reduced ristocetin-induced VWF binding, and diminished ability to mediate cell adhesion to VWF under flow conditions., Conclusions: These data suggest a phosphorylation-dependent binding of 14-3-3zeta to central filamin A binding site of GPIbalpha, and the dimeric 14-3-3zeta binding to both the C-terminal site and central RGpSLP site inhibits GPIb-IX association with the membrane skeleton and promotes the VWF binding function of GPIb-IX.

Currently, most primary hospitals cannot routinely perform liver stiffness measurements (LSMs) and spleen stiffness measurements (SSMs), which are recommended by guidelines to exclude high-risk varices (HRVs). We tried to find more convenient indicators for HRV screening. We enrolled 213 cirrhosis patients as the training cohort (TC) and 65 primary biliary cirrhosis patients as the validation cohort (VC). We included indicators such as SSM by two-dimensional shear wave elastography, LSM by transient elastography, and other imaging and laboratory tests. Variable analysis revealed SSM, platelets (PLT), and spleen thickness (ST) as independent risk indicators for HRV. In TC, ST+PLT (ST < 42.2 mm and PLT > 113.5 × 109/L) could avoid 35.7% of the esophagogastroduodenoscopies (EGDs), with a 2.4% missed HRV rate. Although the proportion of EGDs spared by ST+PLT was less than SSM+PLT (SSM < 29.89 kPa + PLT > 113.5 × 109/L) (35.7% vs. 44.1%), it was higher than that of the Baveno VI criteria (B6) (35.7% vs. 28.2%). We did not validate SSM+PLT in VC considering our aims. ST+PLT safely spared 24.6% of EGDs in VC, identical to B6. Conclusions: The ability of ST+PLT to exclude HRVs was superior to B6 but slightly inferior to SSM+PLT. When SSM cannot be routinely performed, ST+PLT provides an extra option for patients to exclude HRVs as a more convenient model. [ABSTRACT FROM AUTHOR]

Few studies have reported on acoustic radiation force impulse (ARFI) for varices screening. Our study aimed to identify a strategy based on liver stiffness measurement (LSM) and spleen stiffness measurement (SSM) by ARFI combined with platelet count (PLT), named the ARP strategy, for ruling out high‐risk varices (HRV) and avoiding unnecessary esophagogastroduodenoscopy (EGD) in patients with compensated cirrhosis. We retrospectively reviewed patients who underwent ARFI from a previous cohort (NCT04307264). Of them, patients between 2017 and 2019 composed the training cohort to develop the ARP strategy. The validation cohort consisted of others between 2015 and 2016 to validate and compare it with Baveno VI criteria about the performance for varices screening. Primary outcomes were the rates of spared EGDs and HRV missed. A total of 741 consecutive patients were included in the final analysis. Of them, 576 patients were included in the training cohort and 165 patients in the validation cohort. In the training cohort, ARP strategy was defined as LSM < 1.805 m/s or SSM < 2.445 m/s and PLT > 110 × 109/L. ARP strategy could spare 234 (40.6%) EGDs with a missed HRV rate of 3.4% (8 of 234). In the validation cohort, compared with Baveno VI criteria, the ARP strategy improved the proportion of avoided EGDs (49.7% vs. 34.5%; p < 0.001) and lowered the rate of misclassified HRV (1.2% vs. 3.5%; p < 0.001). Conclusion: The ARP strategy was an efficient and safe tool for varices screening in compensated cirrhosis, and it might be an auxiliary or even alternative to Baveno VI criteria. [ABSTRACT FROM AUTHOR]