Platelet Release Research Articles

Drug-Induced Thrombocytopenia: Mechanisms and Laboratory Diagnostics.

Thrombocytopenia is a condition characterized by a decreased number of platelets in peripheral blood, which can be caused by a myriad of both congenital and acquired disorders. Drug-induced thrombocytopenia (DIT) deserves a special focus since its cumulative incidence can be as high as 10 cases per million population per year, with a prevalence of approximately 25% in critically ill patients. This condition is usually suspected following identification of an acute and severe decrease in platelet count, with values usually < 50 ×109/L, thus potentially exposing patients to an increased risk of developing spontaneous hemorrhages. Conversely, however, some drug-related thrombocytopenias are instead (and perhaps counterintuitively) associated with increased thrombosis risk. Although a vast number of drugs have been implicated in DIT, the underlying pathogenetic mechanisms are essentially bifold, encompassing reduced platelet production due to bone marrow suppression (thus insufficient maturation or inefficient expansion of megakaryocytes, impaired release of platelets, or accelerated platelet apoptosis) or accelerated clearance of platelets from the circulation. This second form of DIT can be sustained by nonimmune, immune-mediated, or autoimmune mechanisms. An early and accurate diagnosis of DIT, which is crucial for reversing an otherwise unfavorable clinical outcome, is essentially based on the complete blood cell count, blood smear analysis, and performance of specific functional or immunochemical tests aimed at demonstrating the presence of antiplatelet antibodies.

Microvesicles promote megakaryopoiesis by regulating DNA methyltransferase and methylation of Notch1 promoter.

Megakaryopoiesis is the process of formation of mature megakaryocytes that takes place in the bone marrow niche resulting in the release of platelets into the peripheral blood. It has been suggested that cell to cell communication in this dense bone marrow niche may influence the fate of the cells. Numerous studies point to the role of exosomes and microvesicles not only as a messenger of the cellular crosstalk but also in growth and developmental process of various cell types. In the current study, we explored the effects of megakaryocyte-derived microvesicles in hematopoietic cell lines in the context of differentiation. Our study demonstrated that microvesicles isolated from the induced megakaryocytic cell lines have the ability to stimulate noninduced cells specifically into that particular lineage. We showed that this lineage commencement comes from the change in the methylation status of Notch1 promoter, which is regulated by DNA methyltransferases.

Single-center clinical analysis of hereditary thrombocytopenia in children with chronic thrombocytopenia

Objective To know the detection rate of hereditary thrombocytopenia (HT) in children with chronic thrombocytopenia and its clinical and laboratory characteristics for an early clinical identification and diagnosis of HT in future. Methods Data of the children with thrombocytopenia, who had been treated in Beijing Children′s Hospital from April 2016 to May 2018 and whose present history lasted for more than 1 year and had poor response to immunotherapy were retrospectively collected.HT was screened in these patients by adopting next generation sequencing (NGS). Finally, clinical and laboratory characteristics of these children with HT were summarized and analyzed. Results A total of 161 children with chronic thrombocytopenia were included.Forty-three cases (26.7%) were found to have gene mutations.The genetic rules of the mutant gene, the family verification and the clinical manifestations of the proband and some related laboratory tests were analyzed and 24 cases (14.9%) can be diagnosed as HT.Among the HT patients, the proportion of males and females was 159, and the median onset of age was 0.58 years, which was significantly lower than that of non-HT cases (the median onset of age was 4.36 years), and the difference was statistically significant (P<0.001); the proportion of mucosal hemorrhage and visceral hemorrhage (31.8% and 13.7%) of HT was significantly higher than non-HT cases (15.3% and 0.6%), and the difference was statistically significant (P<0.001). Fifty percent of (12/24 cases) cases of HT had positive family history; according to the ave-rage platelet volume and platelet morphology in peripheral blood smear, HT could be divided into small platelet HT, po-sitive platelet HT and large platelet HT.Some cases had well response to immunotherapy but seemed easy to relapse du-ring the withdrawal period, while the others responded poorly to therapy.Different clinical manifestations of HT suggest different pathogenesis, which can be divided into the related types of megakaryocyte differentiation defect, megakaryocyte maturation defect, platelet release defect and platelet survival time shortening. Conclusions The pathogenesis and cli-nical phenotype of HT was different.Some of them were effective for immunotherapy, which were easily confused with immune thrombocy-topenla(ITP). It is clinically necessary to perform NGS in children with thrombocytopenia at early onset, abnormal platelet morphology, prolonged disease course or severe mucosal/visceral hemorrhage, in order to recognize HT timely to avoid delay in diagnosis and poor prognosis. Key words: Child; Hereditary thrombocytopenia; Next generation sequencing

Polyketides from Alternaria alternata MT-47, an endophytic fungus isolated from Huperzia serrata

Four new polyketides, alternatains A-D (1–4), along with 17 known compounds (5–21) were obtained from the solid substrate fermentation cultures of Alternaria alternata MT-47, an endophytic fungus isolated from the medicinal plant of Huperzia serrata. Their structures were elucidated by extensive spectroscopic and spectrometric techniques (1D and 2D NMR, IR, and HRESIMS) and calculated electronic circular dichroism (ECD) method. Compounds 4, 6, 15, and 21 exhibited inhibitory activities on ATP release of thrombin-activated platelets with IC50 values in the range of 18.2–68.8 μM.

Development of a Nonradioactive Platelet Serotonin Uptake and Release Assay by Micro-Liquid Chromatography Tandem Mass Spectrometry Using Minimal Blood Volume.

Analysis of platelet functional responses to stimuli is presently quite limited with respect to measurement of dense granule secretion. We sought to develop a nonradioactive assay of stimulated serotonin release using liquid chromatography tandem mass spectrometry (LC-MS/MS). Citrated whole blood (200 μL) was incubated with deuterated serotonin (d45-HT). Following uptake by platelets, blood was diluted 10-fold and aliquots were incubated with platelet stimuli. Following stimulation, blood was further diluted, centrifuged, and supernatant was assayed for released d45-HT by micro-LC-MS/MS. This study demonstrated a broad linear range of 50 to 2,000 pg/mL d45-HT, with a total precision of less than 15.0% coefficient of variation at all quality control levels and a limit of quantitation of 50 pg/mL. Quantification of d45-HT by micro-LC-MS/MS assay offers a highly sensitive, nonradioactive methodology for quantitating platelet serotonin uptake and dense granule secretion, requiring only small volumes of patient blood.

Haemostatic responsiveness and release of biological response modifiers following cryopreservation of platelets treated with amotosalen and ultraviolet A light.

Due to the risk of replication of contaminating pathogens, platelets have a limited storage time of 5 days, which can be prolonged to 7 days by the use of pathogen inactivation technologies. Cryopreservation (CP) may be an alternative to permit longer storage periods and increased availability. However, the preparation of platelets can result in secretion of biological response modifiers (BRM), which can cause adverse transfusion reactions in the recipient. We investigated the impact of CP on platelet function and release of BRM in untreated (conventional) and pathogen-inactivated (PI) platelet concentrates. Twelve buffy coat-derived platelet units were treated with amotosalen and ultraviolet A light to inactivate pathogens. Twelve untreated units were used as controls. The 24 units were cryopreserved and in vitro variables were analysed before and after CP. The in vitro variables investigated included platelet surface receptors and activation markers by flow cytometry, and coagulation time by viscoelastography. A panel of BRM, including cytokines, was investigated. CP of both conventional and PI platelets resulted in a significant increase of BRM with similar increases of most of the BRM after CP of conventional and PI platelet concentrates. The increase in some of the BRM correlated significantly with shortened coagulation time, increased P-selectin expression, reduced mitochondrial transmembrane potential, and reduced capacity to respond to stimulation with ADP and collagen. Cryopreservation of both conventional and PI platelets results in secretion of BRM. The increase in some of the BRM correlated with changes in platelet function variables and suggests that BRM release is affected, in part, in a similar way by CP as are changes in platelet function variables. PI with amotosalen and ultraviolet A light in combination with CP did not affect the release of immunomodulatory factors more than CP alone did.

Platelet Contributions to Myocardial Ischemia/Reperfusion Injury.

Obstruction of a coronary artery causes ischemia of heart tissue leading to myocardial infarction. Prolonged oxygen deficiency provokes tissue necrosis, which can result in heart failure and death of the patient. Therefore, restoration of coronary blood flow (reperfusion of the ischemic area) by re-canalizing the affected vessel is essential for a better patient outcome. Paradoxically, sudden reperfusion also causes tissue injury, thereby increasing the initial ischemic damage despite restoration of blood flow (=ischemia/reperfusion injury, IRI). Myocardial IRI is a complex event that involves various harmful mechanisms (e.g., production of reactive oxygen species and local increase in calcium ions) as well as inflammatory cells and signals like chemokines and cytokines. An involvement of platelets in the inflammatory reaction associated with IRI was discovered several years ago, but the underlying mechanisms are not yet fully understood. This mini review focusses on platelet contributions to the intricate picture of myocardial IRI. We summarize how upregulation of platelet surface receptors and release of immunomodulatory mediators lead to aggravation of myocardial IRI and subsequent cardiac damage by different mechanisms such as recruitment and activation of immune cells or modification of the cardiac vascular endothelium. In addition, evidence for cardioprotective roles of distinct platelet factors during IRI will be discussed.

PF684 ANAGRELIDE MODULATES PROPLATELET FORMATION RESULTING IN DECREASED NUMBER AND INCREASED SIZE OF PLATELETS

Background:In patients with essential thrombocythemia (ET) who have high risk factors for thrombotic events, cytoreductive therapy with anagrelide (ANA) or hydroxycarbamide (HC) is one of the treatment options. Despite the clinically evident platelet‐lowering effect of ANA, the detailed mechanism of its effect remains to be elucidated. In addition to the previously reported inhibition of megakaryocytic differentiation, we hypothesized that ANA affects the formation of proplatelets, the final process of platelet release.Aims:To determine platelet size change and proplatelet formation with ANA treatment, we retrospectively analyzed patients’ data and analyzed data obtained from an experimental model using a human megakaryocytic cell line (MEG‐01 cells), an accepted model of thrombopoiesis.Methods:We retrospectively evaluated 48 patients diagnosed with ET in Hokkaido University Hospital (male/female: 14/34, age, 18 to 87 years; median age, 61.5 years; ANA only, 3 patients; ANA after HC due to insufficient decrease in platelets, 11; HC only, 18; observed without ANA or HC, 16). We analyzed changes in platelet counts and mean platelet volume (MPV) after administering each medicine. This study was approved by the Institutional Review Board of Hokkaido University Hospital. Megakaryocytic differentiation of MEG‐01 cells was induced by PMA treatment with or without ANA. Morphological change of proplatelets and size of platelet‐like particles (PLPs) were analyzed.Results:The figure shows the relationship between decrease in platelet count and MPV change in each patient at the time of maximum response to ANA or HC. In patients treated with ANA (n = 14), the degree of platelet count reduction (‐83.4% to −27.1%; median, −56.6%) was strongly correlated with increase of MPV (‐6.6% to +28.7%; median, +16.7%) (left panel; R = 0.777, P = 0.001). On the other hand, in patients treated with HC (n = 29), the degree of platelet count reduction (‐78.7% to −20.8%; median, −55.5%) was not correlated with change of MPV (‐10.3% to +12.9%; median, +1.0%) (right panel; R = 0.245, P = 0.21). PMA‐induced megakaryocytic differentiation of MEG‐01 cells promoted production of PLPs. The number of PLPs was decreased in PMA + ANA‐treated MEG‐01 cells (P &lt; 0.01 vs. PMA alone). PLP size measured by flow cytometry was significantly increased in PMA + ANA‐treated MEG‐01 cells (P &lt; 0.05 vs. PMA alone). These results are consistent with the clinical findings. MGG staining of MEG‐01 cells revealed that ANA modified the formation of cytoplasmic protrusions (proplatelets). We compared morphological parameters including the length, width and number of protrusions. The protrusions were shorter and thicker and the number of protrusions was decreased in PMA + ANA‐treated MEG‐01 cells (P &lt; 0.01 vs. PMA alone).The FAK‐RhoA‐ROCK‐MLC2‐myosin IIA pathway is an important pathway for the formation of proplatelets and production of platelets from megakaryocytes. We evaluated the effect of ANA on phosphorylation of FAK and MLC2. Western blot analysis showed that ANA treatment caused reduced phosphorylation of FAK and increased phosphorylation of MLC2 (PMA vs. PMA+ANA 5 μM, P &lt; 0.01), which presumably resulted in suppression of actin remodeling.Summary/Conclusion:ANA reduces platelet counts and increases platelet size in ET patients. Analysis of ANA‐treated MEG‐01 cells suggested that suppression of proplatelet formation through modulation of the FAK‐RhoA‐ROCK‐MLC2‐myosin IIA pathway is a key mechanism. Morphological observation suggested that ANA induces premature release of platelets, resulting in increased platelet size.image

Modulation of megakaryopoiesis and platelet production during inflammation

Megakaryocytes (MKs) are widely known as the progenitor cells of platelets. These large, polyploid cells are a derivative of the hematopoietic stem cell (HSC), and reside in the bone marrow, lining blood vessel walls where they release their platelet progeny into circulation. Although little is known about how MKs differ under various environmental stressors, both chronic and acute inflammation alter the differentiation and molecular content of MKs. Furthermore, evidence suggests that the release of inflammatory cytokines may induce MK rupture and rapid release of platelets as a mechanism to quickly replenish diminished platelet counts in response to inflammation. Similarities between MKs and their close relatives, white blood cells, have introduced the notion that MKs may play a role in combating infection by engulfing and presenting antigens, and passing this information to circulating platelets. In addition, MKs exposed to varying bone marrow environments produce different platelets which enter circulation primed to respond to and combat inflammation, infection, or injury. This review focuses on how inflammation alters MK production, maturation, and platelet production. In addition, it introduces the idea that inflammation reprograms MKs to create different, more pathogenic platelets and leads them to take on different roles as responders to deleterious conditions. In the future, studies determining how platelets are altered in disease states may lead to novel MK- and platelet-based therapeutic targets to mitigate inflammation-related morbidity and mortality.

Current Insights into the Potential Misuse of Platelet-based Applications for Doping in Sports.

Platelet-based applications are currently used for the delivery of growth factors and other biomolecules as autologous biomaterials in regenerative medicine and cosmetic therapies. Many studies have revealed that platelet-based applications such as platelet-rich plasma and platelet releasate exhibit beneficial biological effects after a sports injury or trauma when administered locally by intramuscular injections. At present, treatment of the public, patients and athletes with platelet-based applications is permitted and regulated by the Food and Drug Administration and the World Anti-Doping Agency. Since 2011 the use of autologous platelet-rich plasma is permitted in competitive sports by the World Anti-Doping Agency, due to the lack of evidence in performance enhancement and anabolic effects. However, accumulating research has recently shed light on the role of platelet-derived growth factors in wound healing, skeletal myogenesis, muscle stem cell function and tissue regeneration. Although any ergogenic potential of platelet-rich plasma and platelet releasate on intact skeletal muscle and human sports performance remain to be established, novel evidence suggests that platelet-derived growth factors can modulate muscle, tendon, ligament, protein synthesis/degradation, vascularization, energy utilization and regenerative capacity in various experimental settings. Since platelet-based applications are currently not prohibited, they constitute a tool for potential abuse and doping in sports. The aim of this review is to critically discuss and assimilate current insights and biological evidence that set the ground for exploitation and misuse in competitive sports, and develop strategies to combat these activities.

432 Patient and device generated variability in platelet-derived growth factors during platelet-rich plasma therapy for hair loss

Platelets contain α-granules that are reservoirs of critical growth factors (GFs) regulating cellular proliferation, migration, differentiation and angiogenesis. Platelet-rich plasma (PRP) can be effective in treating hair loss disorders as reported from several clinical trials, however the response within a study and between studies has been inconsistent. It is possible that this is related to quantitative and qualitative differences in platelet-derived GFs in PRP. It is currently unclear whether these differences result from inherent patient-to-patient variability or from the devices used to prepare PRP or both. To test these variables, we first compared platelet expression and release of prominent GFs (TGFβ1, PDGF-A, PDGF-BB, VEGF, FGF2 and EGF) between patient volunteers undergoing PRP therapy for hair loss. PRP was prepared using an FDA-cleared device which utilizes a density-based separation technique on a patented separator gel. Platelet GF expression and their levels in PRP were highly variable between patients (n=34) and failed the Shapiro-Wilk test for normality, suggesting an inherent patient-to-patient variability. The variability was more pronounced for VEGF and EGF while it was least for TGFβ1. We then compared GF levels in PRP to those prepared using another FDA-cleared device (n=7) which utilizes a dual spin protocol wherein the first soft spin removes the red blood cells while the second spin concentrates the platelets. A final PRP volume of 7-10 ml was produced in both devices although the original volume of blood used and platelet enrichment in PRP was different between them. Significant individual differences in GF levels were also detected when PRP prepared from both devices was compared. Thus, our study suggests that quantitative and qualitative differences in GFs are not only inherent between patients but may also be driven by the devices used to prepare PRP. This may affect the clinical data reported in the literature contributing to inconsistent clinical responses.

Oscillatory shear potentiates latent TGF-\u03b21 activation more than steady shear as demonstrated by a novel force generator

Cardiovascular mechanical stresses trigger physiological and pathological cellular reactions including secretion of Transforming Growth Factor β1 ubiquitously in a latent form (LTGF-β1). While complex shear stresses can activate LTGF-β1, the mechanisms underlying LTGF-β1 activation remain unclear. We hypothesized that different types of shear stress differentially activate LTGF-β1. We designed a custom-built cone-and-plate device to generate steady shear (SS) forces, which are physiologic, or oscillatory shear (OSS) forces characteristic of pathologic states, by abruptly changing rotation directions. We then measured LTGF-β1 activation in platelet releasates. We modeled and measured flow profile changes between SS and OSS by computational fluid dynamics (CFD) simulations. We found a spike in shear rate during abrupt changes in rotation direction. OSS activated TGF-β1 levels significantly more than SS at all shear rates. OSS altered oxidation of free thiols to form more high molecular weight protein complex(es) than SS, a potential mechanism of shear-dependent LTGF-β1 activation. Increasing viscosity in platelet releasates produced higher shear stress and higher LTGF-β1 activation. OSS-generated active TGF-β1 stimulated higher pSmad2 signaling and endothelial to mesenchymal transition (EndoMT)-related genes PAI-1, collagen, and periostin expression in endothelial cells. Overall, our data suggest variable TGF-β1 activation and signaling occurs with competing blood flow patterns in the vasculature to generate complex shear stress, which activates higher levels of TGF-β1 to drive vascular remodeling.

Novel therapeutic approaches in utilizing platelet lysate in regenerative medicine: Are we ready for clinical use?

Hemoderivative materials are used to treat different diseases. These derivatives include platelet-rich plasma, serum, platelet gel, and platelet lysate (PL). Among them, PL contains more growth factors than the others and its production is inexpensive and easy. PL is one of the proper sources of platelet release factors. It is used in cells growth and proliferation and is a good alternative to fetal bovine serum. In recent years, the clinical use of PL has gained more appeal by scientists. PL is a solution saturated by growth factors, proteins, cytokines, and chemokines and is administered to treat different diseases such as wound healing, bone regeneration, alopecia, oral mucositis, radicular pain, osteoarthritis, and ocular diseases. In addition, it can be used in cell culture for cell therapy and tissue transplantation purposes. Platelet-derived growth factor, fibroblast growth factor, insulin-like growth factor, transforming growth factor β, and vascular endothelial growth factor are key PL growth factors playing a major role in cell proliferation, wound healing, and angiogenesis. In this paper, we scrutinized recent advances in using PL and PL-derived growth factors to treat diseases and in regenerative medicine, and the ability to replace PL with other hemoderivative materials.

Dual actions of group B Streptococcus capsular sialic acid provide resistance to platelet-mediated antimicrobial killing

Circulating platelets have important functions in thrombosis and in modulating immune and inflammatory responses. However, the role of platelets in innate immunity to bacterial infection is largely unexplored. While human platelets rapidly kill Staphylococcus aureus, we found the neonatal pathogen group B Streptococcus (GBS) to be remarkably resistant to platelet killing. GBS possesses a capsule polysaccharide (CPS) with terminal α2,3-linked sialic acid (Sia) residues that mimic a common epitope present on the human cell surface glycocalyx. A GBS mutant deficient in CPS Sia was more efficiently killed by human platelets, thrombin-activated platelet releasate, and synthetic platelet-associated antimicrobial peptides. GBS Sia is known to bind inhibitory Sia-recognizing Ig superfamily lectins (Siglecs) to block neutrophil and macrophage activation. We show that human platelets also express high levels of inhibitory Siglec-9 on their surface, and that GBS can engage this receptor in a Sia-dependent manner to suppress platelet activation. In a mouse i.v. infection model, antibody-mediated platelet depletion increased susceptibility to platelet-sensitive S. aureus but did not alter susceptibility to platelet-resistant GBS. Elimination of murine inhibitory Siglec-E partially reversed platelet suppression in response to GBS infection. We conclude that GBS Sia has dual roles in counteracting platelet antimicrobial immunity: conferring intrinsic resistance to platelet-derived antimicrobial components and inhibiting platelet activation through engagement of inhibitory Siglecs. We report a bacterial virulence factor for evasion of platelet-mediated innate immunity.

Platelet-derived β2M regulates monocyte inflammatory responses.

β-2 Microglobulin (β2M) is a molecular chaperone for the major histocompatibility class I (MHC I) complex, hemochromatosis factor protein (HFE), and the neonatal Fc receptor (FcRn), but β2M may also have less understood chaperone-independent functions. Elevated plasma β2M has a direct role in neurocognitive decline and is a risk factor for adverse cardiovascular events. β2M mRNA is present in platelets at very high levels, and β2M is part of the activated platelet releasate. In addition to their more well-studied thrombotic functions, platelets are important immune regulatory cells that release inflammatory molecules and contribute to leukocyte trafficking, activation, and differentiation. We have now found that platelet-derived β2M is a mediator of monocyte proinflammatory differentiation through noncanonical TGFβ receptor signaling. Circulating monocytes from mice lacking β2M only in platelets (Plt-β2M-/-) had a more proreparative monocyte phenotype, in part dependent on increased platelet-derived TGFβ signaling in the absence of β2M. Using a mouse myocardial infarction (MI) model, Plt-β2M-/- mice had limited post-MI proinflammatory monocyte responses and, instead, demonstrated early proreparative monocyte differentiation, profibrotic myofibroblast responses, and a rapid decline in heart function compared with WT mice. These data demonstrate a potentially novel chaperone-independent, monocyte phenotype-regulatory function for platelet β2M and that platelet-derived 2M and TGFβ have opposing roles in monocyte differentiation that may be important in tissue injury responses.

Bone marrow sinusoidal endothelium as a facilitator/regulator of cell egress from the bone marrow.

Despite more attention to cell migration from circulation into the bone marrow (BM), particularly homing of haematopoietic stem/progenitor cells, the process and mechanisms of cell mobilisation from the BM into the circulation remain largely underexplored. The process of cell mobilisation or transcellular cell migration from BM into the circulation (cell egress) is a crucial biological process in mammals as it is important to maintain homeostasis of various physiological functions including, but not limited to, the immune system, erythropoiesis, platelet release, and stem cell migration. The BM microvascular system composes of a monolayer of specialized endothelial cells, called sinusoidal endothelial cells (SECs). While it is very well evident that the process of cell egress occurs exclusively through BM SECs, there is a lack of systematic analyses addressing the extent of contribution of BM SECs to the process of cell egress from the BM. Therefore, this review aims to address the potential ability of BM SECs in regulating and/or facilitating the process of cell egress from BM. In this review, we address, firstly, the unique ultra-/structural and molecular features of BM SECs and discuss the possible biological interactions between BM SECs and various egressing cells in physiological conditions. Secondly, we propose the potential role of BM SECs in egress of leukemic cells from BM into the circulation. Finally, we discuss the potential role of BM SECs in homing of haematopoietic stem cells. Collectively, the current review suggests that the BM SECs may not be merely a neutral gatekeeper for cell intravasation and extravasation, but rather is a dynamic trafficking surveillance system, thereby the process of BM cell egress/mobilisation can be regulated.

Application of pulsed electric fields for the valorization of platelets with no therapeutic value for transfusion medicine

Platelets are blood components with high biomedical potential due to their physiological role in wound healing and their rich growth factor content. This paper describes proof-of-concept experiments aimed to produce a new blood-derived product by applying pulsed electric fields (PEF) to platelet concentrates (PC) with no therapeutic value for transfusion medicine. A human platelet concentrate suspension was subjected to a PEF treatment of 1 pulse, 5[Formula: see text]kV/cm, for 2[Formula: see text][Formula: see text]s. Release of platelet-derived growth factor (PDGF) from the electroporated platelets was measured by ELISA. Furthermore, the biological activity of the obtained blood-derived product was characterized. Human mesenchymal stem cells (hMSC) were cultured in the presence of the proteins released from the platelets after PEF application and evaluated for their expansion potential. Results show that platelet concentrates subjected to a single PEF treatment can release PDGF to the supernatant. Protein release from the single and transient PEF cycle was confirmed by the expansion of hMSC cultured with a medium supplemented with the platelet releasate obtained from electroporated platelets. These results demonstrate the potential of a new application of PEF for the valorization of PC into a biomedical product with therapeutic value.

Low-dose chidamide restores immune tolerance in ITP in mice and humans

AbstractIncreased macrophage phagocytosis of antibody-coated platelets, as well as decreased numbers and/or impaired function of CD4+CD25+Foxp3+ regulatory T (Treg) cells, has been shown to participate in the pathogenesis of immune thrombocytopenia (ITP). Low-dose histone deacetylase inhibitors (HDACi's) are anti-inflammatory and immunomodulatory agents that can enhance immunosuppression in graft-versus-host disease by increasing the number and function of Foxp3+ Treg cells, but it is unclear whether they have the potential to promote immune tolerance and platelet release in ITP. In this study, we performed in vitro and in vivo experiments and found that a low-dose HDACi (chidamide) alleviated thrombocytopenia in passive and active murine models of ITP. Further, low-dose HDACi's attenuated macrophage phagocytosis of antibody-coated platelets, stimulated the production of natural Foxp3+ Treg cells, promoted the peripheral conversion of T cells into Treg cells, and restored Treg cell suppression in vivo and in vitro. Finally, we confirmed that low-dose HDACi's could regulate CTLA4 expression in peripheral blood mononuclear cells through modulation of histone H3K27 acetylation. Low-dose HDACi treatment in ITP could be offset by blocking the effect of CTLA4. Therefore, we propose that low-dose chidamide administration has potential as a novel treatment for ITP in the clinic.

Aspirin inhibits platelets from reprogramming breast tumor cells and promoting metastasis

AbstractIt is now recognized that compounds released from tumor cells can activate platelets, causing the release of platelet-derived factors into the tumor microenvironment. Several of these factors have been shown to directly promote neovascularization and metastasis, yet how the feedback between platelet releasate and the tumor cell affects metastatic phenotype remains largely unstudied. Here, we identify that breast tumor cells secrete high levels of interleukin 8 (IL-8, CXCL8) in response to platelet releasate, which promotes their invasive capacity. Furthermore, we found that platelets activate the Akt pathway in breast tumor cells, and inhibition of this pathway eliminated IL-8 production. We therefore hypothesized inhibiting platelets with aspirin could reverse the prometastatic effects of platelets on tumor cell signaling. Platelets treated with aspirin did not activate the Akt pathway, resulting in reduced IL-8 secretion and impaired tumor cell invasion. Of note, patients with breast cancer receiving aspirin had lower circulating IL-8, and their platelets did not increase tumor cell invasion compared with patients not receiving aspirin. Our data suggest platelets support breast tumor metastasis by inducing tumor cells to secrete IL-8. Our data further support that aspirin acts as an anticancer agent by disrupting the communication between platelets and breast tumor cells.

Mechanisms of thrombocytopenia in platelet-type von Willebrand disease

Platelet-type von Willebrand disease is an inherited platelet disorder characterized by thrombocytopenia with large platelets caused by gain-of-function variants in GP1BA leading to enhanced GPIbα-von Willebrand factor (vWF) interaction. GPIbα and vWF play a role in megakaryocytopoiesis, thus we aimed to investigate megakaryocyte differentiation and proplatelet-formation in platelet-type von Willebrand disease using megakaryocytes from a patient carrying the Met239Val variant and from mice carrying the Gly233Val variant. Platelet-type von Willebrand disease megakaryocytes bound vWF at an early differentiation stage and generated proplatelets with a decreased number of enlarged tips compared to control megakaryocytes. Moreover, they formed proplatelets upon contact with collagen, differently from normal megakaryocytes. Similarly, collagen triggered megakaryocytes showed defective activation of the RhoA-MLC2 axis, which prevents proplatelet formation, and increased phosphorylation of Lyn, which acts as a negative regulator of GPVI signaling, thus preventing ectopic proplatelet-formation on collagen. Consistently, human and murine bone marrow contained an increased number of extravascular platelets compared to controls. In addition, platelet survival of mutant mice was shortened compared to control mice, and the administration of desmopressin, raising circulating vWF, caused a marked drop in platelet count. Taken together, these results show for the first time that thrombocytopenia in platelet-type von Willebrand disease is due to the combination of different pathogenic mechanisms, i.e. the formation of a reduced number of platelets by megakaryocytes, the ectopic release of platelets in the bone marrow, and the increased clearance of platelet/vWF complexes.