Interaction Of Platelets Research Articles

Platelets Preferentially Bind to Myeloma Cells Bearing Sialofucosylated Structures and Protect Them from Natural Killer Cell-Mediated Cytotoxicity

IntroductionPlatelets have been implicated in promoting metastasis in a number of cancers. In many solid tumours including colon and ovarian cancer, thrombocytosis has been demonstrated as an adverse factor for disease metastasis and survival. A number of mechanisms have been proposed including platelet “cloaking” of tumour cells to evade recognition by immune cells, production of cytokines which promote tumour growth and the role of platelets in enabling tethering and migration of metastatic cells. In vitro studies have demonstrated the dependence on platelets for successful solid tumour metastasis and implicated the role of P-Selectin in tumour adhesion to platelets. There have been no published studies demonstrating the interaction of platelets with Myeloma cells and their role in enabling Multiple Myeloma (MM) cell metastasis. We sought to determine in vitro whether platelets interacted significantly with MM cell lines and whether they preferentially bind to specific sub-populations of MM cells. We also sought to determine whether this interaction is mediated by P-Selectin and whether “platelet coating” can protect MM cells from Natural Killer (NK)-mediated cell death.MethodsPlatelet rich plasma (PRP) sample was isolated from freshly drawn peripheral blood samples from healthy participants by FICOLL gradient separation. All samples were collected with informed consent and in accordance with the declaration of Helsinki. The MM1S cell line and its derivative HECA-452 MM1S, which is enriched for the sialofucosylated structure sialyl Lewis X (SLex), were used for this study. Cells were co-cultured with PRP at a ratio of 50 platelets/ 1 MM cell, washed and stained with CD41, CD138, and HECA-452. FACS was used to assess degree of platelet/MM cell binding. Platelet/MM cell interaction was further confirmed using Image Stream. In some experiments, a P-Selectin blocking antibody was used to determine the dependency of platelet/MM cell binding on P-Selectin. For functional studies, MM cells were co-cultured with PRP at different platelet/cell ratios (50:1, 100:1 and 500:1) for 24 h and then incubated with either the NK cell line KHYG-1 or freshly isolated autologous NK cells. Cytotoxicity assays were performed after 24 h by FACS using Propidium Iodide (PI) staining. NK cells were discriminated from MM cells by staining them with cell trace violet dye.ResultsPlatelets displayed preferential binding to the HECA-452 MM1S cells compared to parental cells, suggesting a requirement for sialofucosylated structures for efficient binding. The interaction between platelets and HECA-452 MM1S cells occurred as early as 30 min and remained stable at 24 h post incubation. Moreover, platelets bound to the surface of MM cells, as demonstrated by Image Stream (Figure 1). This binding could be significantly blocked by a P-Selectin blocking antibody, indicating a dependence upon P-Selectin (P=0.0043 at 50:1; P=0.0027 at 100:1; P=0.0163 at 500:1 ratio). Importantly, co-incubation of platelets and MM cells at 500:1 ratio significantly reduced KHYG1-mediated cytotoxicity on HECA-452 MM1S cells at 24 h post incubation (P=0.0078 at 0.25:1 and P=0.0072 at 0.5:1 ratio). Platelets could also protect HECA-452 MM1S cells from primary NK-mediated cytotoxicity, although this didn't quite reach statistical significance.ConclusionsWe demonstrate for the first time a physical interaction between MM cells and platelets. Efficient binding requires sialofucosylated structures, as shown by almost exclusive binding of platelets to the HECA-452 MM1S compared to parental cells. Moreover, platelets were able to protect HECA-452 MM1S cells from NK-mediated cell death, suggesting that in vivo these cells may escape the immune system and promote MM spreading and metastasis. Notably, platelet-MM cell binding was reverted by a P-Selectin blocking antibody, suggesting that this interaction can be therapeutically targeted in order to restrict MM metastasis and re-sensitize MM cells to NK cells. [Display omitted] DisclosuresO'Dwyer:Abbvie: Membership on an entity's Board of Directors or advisory committees; Glycomimetics: Research Funding; BMS: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Onkimmune: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Research Funding.

Role of Platelets in Leukocyte Recruitment and Resolution of Inflammation.

Platelets are most often recognized for their crucial role in the control of acute hemorrhage. However, current research has greatly expanded the appreciation of platelets beyond their contribution to primary hemostasis, indicating that platelets also actively participate in leukocyte recruitment and the regulation of the host defense in response to exogenous pathogens and sterile injury. Early recruitment of leukocytes, especially neutrophils, is the evolutionary stronghold of the innate immune response to successfully control exogenous infections. Platelets have been shown to physically interact with different leukocyte subsets during inflammatory processes. This interaction holds far-reaching implications for the leukocyte recruitment into peripheral tissues as well as the regulation of leukocyte cell autonomous functions, including the formation and liberation of neutrophil extracellular traps. These functions critically depend on the interaction of platelets with leukocytes. The host immune response and leukocyte recruitment must be tightly regulated to avoid excessive tissue and organ damage and to avoid chronification of inflammation. Thus, platelet-leukocyte interactions and the resulting leukocyte activation and recruitment also underlies tight regulation by several inherited feedback mechanisms to limit the extend of vascular inflammation and to protect the host from collateral damage caused by overshooting immune system activation. After the acute inflammatory phase has been overcome the host defense response must eventually be terminated to allow for resolution from inflammation and restoration of tissue and organ function. Besides their essential role for leukocyte recruitment and the initiation and propagation of vascular inflammation, platelets have lately also been implicated in the resolution process. Here, their contribution to phagocyte clearance, T cell recruitment and macrophage reprogramming is also of outmost importance. This review will focus on the role of platelets in leukocyte recruitment during the initiation of the host defense and we will also discuss the participation of platelets in the resolution process after acute inflammation.

The Inter-Relationship of Platelets with Interleukin-1β-Mediated Inflammation in Humans.

Inflammation and coagulation are key processes in cardiovascular diseases (CVDs). The Canakinumab Anti-inflammatory Thrombosis Outcome Study trial affirmed the importance of inflammation in CVD by showing that inhibition of the interleukin (IL)-1β pathway prevents recurrent CVD. A bi-directional relationship exists between inflammation and coagulation, but the precise interaction of platelets and IL-1β-mediated inflammation is incompletely understood. We aimed to determine the inter-relationship between platelets and inflammation-and especially IL-1β-in a cohort of healthy volunteers. We used data from the 500-Human Functional Genomics cohort, which consists of approximately 500 Caucasian, healthy individuals. We determined associations of plasma levels of IL-1β and other inflammatory proteins with platelet number and reactivity, the association of platelet reactivity with ex vivo cytokine production as well as the impact of genetic variations through a genome-wide association study (GWAS). Platelets were associated with IL-1β on different levels. First, platelet number was positively associated with plasma IL-1β concentrations (p = 8.9 × 10-9) and inversely with concentrations of α-1-anti-trypsin (p = 1.04 × 10-18), which is a known antagonist of IL-1β. Second, platelet degranulation capacity, as determined by agonist-induced P-selectin expression, was associated with ex vivo IL-1β and IL-6 production. Third, several platelet single-nucleotide polymorphisms (SNPs) were associated with cytokine production and there was a significant platelet SNP enrichment in specific biological important pathways. Finally, platelet SNPs were enriched among SNPs earlier identified in GWAS studies in blood-related diseases and immune-mediated diseases. This comprehensive assessment of factors associated with platelet number and reactivity reinforces the important inter-relationship of platelets and IL-1β-mediated inflammation.

Platelet biology and functions: new concepts and clinical perspectives.

Platelets - blood cells continuously produced from megakaryocytes mainly in the bone marrow - are implicated not only in haemostasis and arterial thrombosis, but also in other physiological and pathophysiological processes. This Review describes current evidence for the heterogeneity in platelet structure, age, and activation properties, with consequences for a diversity of platelet functions. Signalling processes of platelet populations involved in thrombus formation with ongoing coagulation are well understood. Genetic approaches have provided information on multiple genes related to normal haemostasis, such as those encoding receptors and signalling or secretory proteins, that determine platelet count and/or responsiveness. As highly responsive and secretory cells, platelets can alter the environment through the release of growth factors, chemokines, coagulant factors, RNA species, and extracellular vesicles. Conversely, platelets will also adapt to their environment. In disease states, platelets can be positively primed to reach a pre-activated condition. At the inflamed vessel wall, platelets interact with leukocytes and the coagulation system, interactions mediating thromboinflammation. With current antiplatelet therapies invariably causing bleeding as an undesired adverse effect, novel therapies can be more beneficial if directed against specific platelet responses, populations, interactions, or priming conditions. On the basis of these novel concepts and processes, we discuss several initiatives to target platelets therapeutically.

Platelet Function Changes in a Time-Dependent Manner Following Traumatic Brain Injury in a Murine Model.

Traumatic brain injury (TBI) results in systemic changes in coagulation and inflammation that contribute to post-traumatic morbidity and mortality. The potential interaction of platelets and pro-inflammatory cytokines in the modulation of coagulation, microthrombosis, and venous thromboembolic events after moderate TBI has not been determined. Using a murine model, we hypothesized that the degree of platelet-induced coagulation varies depending on the platelet aggregation agonist platelet-induced coagulation changes in a time-dependent manner following TBI, and changes in platelet-induced coagulation are mirrored by changes in the levels of circulating pro-inflammatory cytokines. An established weight-drop model was used to induce TBI in anesthetized mice. Blood samples were collected at intervals after injury for measurements of platelet count, serum fibrinogen, pro-inflammatory cytokines, and determination of soluble P-selectin levels. Thromboelastometry was used to evaluate changes in hemostasis. Platelet function was determined using whole blood impedance aggregometry. Ten minutes following TBI, adenosine diphosphate-induced platelet aggregation decreased as measured by platelet aggregometry. Despite no changes in platelet counts and serum fibrinogen, platelet aggregation, pro-inflammatory cytokines, and soluble P-selectin were increased at 6 h after TBI. Rotation thromboelastometry demonstrated increased maximal clot firmness at 6 h. Platelet function and coagulability returned to baseline levels 24 h following head injury. Our data demonstrate that after TBI, acute platelet dysfunction occurs followed by rebound platelet hyperaggregation. Alterations in post-TBI platelet aggregation are reflected in whole blood thromboelastometry and are temporally associated with the systemic pro-inflammatory response.

Unfractionated and Low Molecular Weight Heparin Reduce Platelet Induced Epithelial-Mesenchymal Transition in Pancreatic and Prostate Cancer Cells.

The interaction with platelets is of crucial importance for tumor cells passing through hematogenous metastasis. Platelets protect cancer cells from immune surveillance and exhibit many other prometastatic effects. Notably, platelets can change the epithelial tumor phenotype, a process termed epithelial-mesenchymal transition (EMT), which confers stem cell-like properties onto tumor cells associated with an increased motility and drug resistance. The aim of the study is to investigate the impact of heparin on the platelet induced EMT program in pancreatic and prostate tumor cells. Platelet activation and interaction with cancer cells were determined by static adhesion assays. Applying ELISAs, the platelet release of EMT inducing mediators was quantified. EMT marker protein expression by tumor cells was explored by western blot and qPCR. Our data show that different tumor cell entities have different platelet binding capacities and also that a weak interaction is sufficient to change tumor cell phenotype. Additionally, unfractionated heparin (UFH) as well as low molecular weight heparin (LMWH) reduced tumor cell platelet interaction. Subsequently, attenuated platelet-derived mediator release resulted in reduced EMT marker protein and transcription factor expression by the cancer cells and decreased cell migration. These data suggest that heparin reduces platelet induced EMT program and prevents the formation of cancer cells with stem cell-like properties. This additional mechanism argues for the use of heparin in oncological applications.

Sushi Domain Containing 2 (SUSD2) inhibits platelet activation and binding to high-grade serous ovarian carcinoma cells

Platelets play a central role in primary hemostasis affecting tumor survival and metastases. Tumors induce platelets to aggregate and bind to the cancer cells, resulting in protection from immune surveillance and often leading to thrombocytosis. In ovarian cancer (OvCa), one-third of patients present with thrombocytosis, a diagnosis that correlates with shorter survival. SUSD2 (SUShi Domain containing 2), a type I transmembrane protein, shown to inhibit metastatic processes in high-grade serous ovarian carcinoma (HGSOC), is expressed on endothelial cells and thus may influence platelet reactivity. As such, we hypothesized that SUSD2 levels in ovarian cancer-derived cell lines influence platelet activation. We incubated OvCa non-targeting (NT) and SUSD2 knockdown (KD) cell lines with labeled platelets and quantified platelet binding, as well as GPIIb/IIIa integrin activation. The role of GPIIb/IIIa in tumor cell/platelet interaction was also examined by measuring cell–cell adhesion in the presence of eptifibatide. We found that platelets exposed to OvCa cells with low SUSD2 expression display increased tumor cell-platelet binding along with an increase in GPIIb/IIIa receptor activation. As such, platelet activation and binding to HGSOC cells was inversely correlated with the presence of SUSD2. This represents one of the first tumor proteins known to provide differential platelet interaction based on protein status.

Interaction between platelets and endothelium: from pathophysiology to new therapeutic options.

Platelets were traditionally considered to purely have a role in the maintenance of haemostasis. Recently their role in vasomotor function, inflammation and atherosclerosis has been very well-recognized. Endothelium which was originally considered as a simple passive barrier, it is now viewed as an organ whose normal functioning is crucial for maintaining vascular health. When endothelial balance is disturbed, vascular disease initiates. Platelet interactions with endothelium have an important contribution in this process. Low-grade inflammation, endothelial dysfunction, and platelet hyper-reactivity are all independently associated with an increased risk of cardiovascular events. Older antiplatelet agents like aspirin and clopidogrel and newer more potent agents like prasugrel and ticagrelor have been proven effective in all the clinical spectrum of coronary artery disease patients. Current antiplatelet medications and especially newer generation P2Y12 inhibitor ticagrelor, offer clinical benefits not only due to their well-recognized antithrombotic effect, but also via the attenuation of platelet inflammatory action, impediment of P2Y12 activation effects in other cells and through other complex and sometimes undefined pathways. Future research is expected to better define platelet-endothelium interactions and the multiple impact of current antiplatelet therapy on them.

The Mutual Relation of Platelet Activation and Innate Immunity.

Platelets are known to be central regulators of haemostasis, inflammation and immune response. Formed by megakaryocytes in the bone marrow and the lungs, platelets express a broad range of adhesion receptors and release cytokines and platelet microparticles which enable them to interact with both immune cells and pathogens. In bacterial and viral infections, thrombophilia and thrombocytopenia are commonly seen symptoms, indicating the close relationship between haemostasis and immune defence. Indeed, platelets contribute both directly and via immune mediation to pathogen clearance. In sterile inflammation, a pathogen-free process which is often triggered by cell necrosis and autoimmune reactions, platelets are also of central importance. Recently, platelet inflammasome has been extensively studied in this context. Both sterile inflammation and infection are affected by the interactions of platelets and innate immunity, notably the complement system. Although the general elements of this interplay have been known for long, more and more insights into disease-specific mechanisms could be gained recently. This review gives an outline of the current findings in the field of platelet-immune cell interactions and points out possible implications for clinical therapy.

The dual effect of C-peptide on cellular activation and atherosclerosis: Protective or not?

C-peptide is a cleavage product of proinsulin that acts on different type of cells, such as blood and endothelial cells. C-peptide biological effects may be different in type 1 and type 2 diabetes. Besides, there are further evidence for a functional interaction between C-peptide and insulin. In this way, C-peptide has ambiguous effects, acting as an antithrombotic or thrombotic molecule, depending on the physiological environment and disease conditions. Moreover, C-peptide regulates interaction of leucocytes, erythrocytes, and platelets with the endothelium. The beneficial effects include stimulation of nitric oxide production with its subsequent release by platelets and endothelium, the interaction with erythrocytes leading to the generation of adenosine triphosphate, and inhibition of atherogenic cytokine release. The undesirable action of C-peptide includes the chemotaxis of monocytes, lymphocytes, and smooth muscle cells. Also, C-peptide was related with increased lipid deposits and elevated smooth muscle cells proliferation in the vessel wall, contributing to atherosclerosis. Purpose of this review is to explore these dual roles of C-peptide on the blood, contributing at one side to haemostasis and the other to atherosclerotic process.

Dynamic platelet function is markedly different in patients with cancer compared to healthy donors

Despite a fivefold increased risk of thromboembolism in patients with cancer, the mechanism of arterial thromboembolism is poorly understood. To address this, we investigated platelet function in cancer patients and healthy controls using an assay that mimics the arterial vasculature. Blood samples from cancer patients (n = 36) and healthy controls (n = 22) were perfused through custom-made parallel-plate flow chambers coated with von Willebrand factor (VWF) under arterial shear (1,500 s−1). Multiparameter measurements of platelet interactions with the immobilized VWF surface were recorded by digital-image microscopy and analyzed using custom-designed platelet-tracking software. Six measured parameters that characterize in detail the surface motion and surface binding of several hundred platelets per blood sample differed significantly in those with cancer from the healthy donors. In particular, it was found that patients with cancer had decreased numbers of platelets interacting, translocating and adhering to VWF. There were also reductions in the speed and distances that platelets traveled on VWF in comparison to healthy controls. Platelet function differed between those with early-stage cancer compared to those with later stage cancer. Patients with advanced cancer had an increased number of platelets stably adhering to VWF and greater platelet surface coverage after a given time of interaction. To the best of our knowledge, our results demonstrate for the first time that dynamic platelet function is markedly different in patients with cancer compared to healthy donors.

Platelets mediate protective neuroinflammation and promote neuronal plasticity at the site of neuronal injury

It is generally accepted that inflammation within the CNS contributes to neurodegeneration after traumatic brain injury (TBI), but it is not clear how inflammation is initiated in the absence of infection and whether this neuroinflammation is predominantly beneficial or detrimental. We have previously found that brain-enriched glycosphingolipids within neuronal lipid rafts (NLR) induced platelet degranulation and secretion of neurotransmitters and pro-inflammatory factors. In the present study, we compared TBI-induced inflammation and neurodegeneration in wild-type vs. St3gal5 deficient (ST3−/−) mice that lack major CNS-specific glycosphingolipids. After TBI, microglial activation and CNS macrophage infiltration were substantially reduced in ST3−/− animals. However, ST3−/− mice had a larger area of CNS damage with marked neuronal/axonal loss. The interaction of platelets with NLR stimulated neurite growth, increased the number of PSD95-positive dendritic spines, and intensified neuronal activity. Adoptive transfer and blocking experiments provide further that platelet-derived serotonin and platelet activating factor plays a key role in the regulation of sterile neuroinflammation, hemorrhage and neuronal plasticity after TBI.

Parameter Estimation of Platelets Deposition: Approximate Bayesian Computation With High Performance Computing.

Cardio/cerebrovascular diseases (CVD) have become one of the major health issue in our societies. Recent studies show the existing clinical tests to detect CVD are ineffectual as they do not consider different stages of platelet activation or the molecular dynamics involved in platelet interactions. Further they are also incapable to consider inter-individual variability. A physical description of platelets deposition was introduced recently in Chopard et al. (2017), by integrating fundamental understandings of how platelets interact in a numerical model, parameterized by five parameters. These parameters specify the deposition process and are relevant for a biomedical understanding of the phenomena. One of the main intuition is that these parameters are precisely the information needed for a pathological test identifying CVD captured and that they capture the inter-individual variability. Following this intuition, here we devise a Bayesian inferential scheme for estimation of these parameters, using experimental observations, at different time intervals, on the average size of the aggregation clusters, their number per mm2, the number of platelets, and the ones activated per μℓ still in suspension. As the likelihood function of the numerical model is intractable due to the complex stochastic nature of the model, we use a likelihood-free inference scheme approximate Bayesian computation (ABC) to calibrate the parameters in a data-driven manner. As ABC requires the generation of many pseudo-data by expensive simulation runs, we use a high performance computing (HPC) framework for ABC to make the inference possible for this model. We consider a collective dataset of seven volunteers and use this inference scheme to get an approximate posterior distribution and the Bayes estimate of these five parameters. The mean posterior prediction of platelet deposition pattern matches the experimental dataset closely with a tight posterior prediction error margin, justifying our main intuition and providing a methodology to infer these parameters given patient data. The present approach can be used to build a new generation of personalized platelet functionality tests for CVD detection, using numerical modeling of platelet deposition, Bayesian uncertainty quantification, and High performance computing.

Surface fluorination of polylactide as a path to improve platelet associated hemocompatibility

Surface-induced thrombosis is still a significant clinical concern for many types of blood-contacting medical devices. In particular, protein adsorption and platelet adhesion are important events due to their ability to trigger the coagulation cascade and initiate thrombosis. Poly(lactic acid) (PLA) has been the predominant polymer used for making bioresorbable stents. Despite long-term advantages, these stents are associated with higher rates of early thrombosis compared with permanent metallic stents. To address this issue, we modified the surface of PLA with a perfluoro compound facilitated by surface activation using radio frequency (RF) plasma. Fluoropolymers have been extensively used in blood contacting materials, such as blood vessel replacements due to their reduced thrombogenicity and reduced platelet reactivity. The compositions of plasma-treated surfaces were determined by electron spectroscopy for chemical analysis (ESCA). Also, contact angle measurements, cell cytotoxicity and the degradation profile of the treated polymers are presented. Finally, relevant blood compatibility parameters, including plasma protein adsorption, platelet adhesion and morphology, were evaluated. We hypothesized that tight binding of adsorbed albumin by fluoropolymers enhances its potential for blood-contacting applications. Statement of SignificanceAlthough bioresorbable stents made from poly(lactic acid) (PLA) may have long-term clinical advantages, they have shown higher rates of early thrombosis as compared with permanent metallic stents. To improve the thromboresistance of PLA, we developed a novel method for surface fluorination of this polymer with a perfluoro compound. Fluoropolymers (e.g., expanded polytetrafluoroethylene) have long been used in blood-contacting applications due to their satisfactory clinical performance. This is the first report of PLA surface fluorination which might be applied to the fabrication of a new generation of fluorinated PLA stents with improved platelet interaction, tunable degradability and drug release capabilities. Also, we describe a general strategy for improving the platelet interactions with biomaterials based on albumin retention.

Abstract 4081: Podoplanin expression in Kupffer cells and platelet deposition on the hepatic sinusoidal cells in the liver of transgenic mice with a hepatocyte-specific human BRAFV600E mutation

Abstract In hepatocarcinogenesis induced by diethylnitrosamine (DEN) treatment in neonatal B6C3F1 mice, the BrafV637E mutation, corresponding to the human BRAFV600E mutation, plays a pivotal role. We produced the transgenic mice expressing human BRAFV600E mutation specifically in hepatocytes by using the albumin-cre method. The Alb-Cre/BRAFV600E transgenic mice showed the enlarged liver whose weight was five times as large as that of normal mice, and the liver entirely consisted of small basophilic hepatocytes resembling the DEN-induced preneoplastic hepatocytes. Although these transgenic mice were healthy after birth, they spontaneously died showing renal and pulmonary diseases. In the transgenic mice, the liver showed thrombopoietin (TPO) overexpression, which is associated with eventual megakaryocytosis/thrombocytosis, and platelets were found activated in the peripheral blood and deposited in hepatic sinusoids. Simultaneous staining for CD61/CD31 or CD61/F4-80 revealed that platelets were sparsely adhered to liver sinusoidal endothelial cells (LSEC) and densely adhered to and incorporated into Kupffer cells. Because the interaction of podoplanin with C-type lectin receptor 2 (CLEC-2) on platelet membranes is an important mechanism to reciprocally activate platelets and the podoplanin-expressing cells, we investigated whether podoplanin is expressed in any of the hepatic cell components in the transgenic mice. Simultaneous staining for podoplanin and F4-80 revealed that podoplanin was expressed in some of the Kupffer cells, indicating that the platelet activation occurred via the interaction of podoplanin on Kupffer cell membrans with C-type lectin receptor 2 (CLEC-2) on the platelet membrane. Additionally, glomerulonephropathy and interstitial pneumonia associated with platelet deposition were observed. Because platelets contribute growth/survival of hepatocytes by activating hepatic sinusoidal cells and directly hepatocytes via releasing various factors, interaction of platelets with hepatic sinusoidal cells was considered to promote the growth/survival of BRAF mutated hepatocytes. Furthermore, podoplanin expression in part of Kupffer cells in the transgenic mice was an important mechanism for platelet activation in the transgenic mice. On the other hand, the aberrant platelet activation was thought to cause glomerulonephropathy and interstitial pneumonia and led to spontaneous death in some of the transgenic mice. Citation Format: Hiroki Tanaka, Kie Horioka, Masahiro Yamamoto, Asari Masaru, Katsuhiro Okuda, Seiji Ohtani, Kosuke Yamazaki, Keiko Shimizu, Katsuhiro Ogawa. Podoplanin expression in Kupffer cells and platelet deposition on the hepatic sinusoidal cells in the liver of transgenic mice with a hepatocyte-specific human BRAFV600E mutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4081.

Platelets Stimulate Liver Sinusoidal Endothelial Cells to Secrete Interleukin-6 During the Early Phase of Liver Regeneration in Mice

Background and Aims Platelets and liver sinusoidal endothelial cells (LSEC) are both known to independently regulate liver regeneration. Our aim was to investigate the role of interactions between platelets and LSEC in a model of liver regeneration. Methods Platelet and LSEC interactions were analyzed in vivo by intravital confocal microscopy in partially hepatectomized mice. Then, highly purified primary mouse LSEC were co-incubated with increasing concentrations of resting platelets, activated platelets, or platelet releasates, and secretion of growth factors was measured. The active fraction of platelet releasates was characterized and its effect on hepatocyte proliferation assessed. Finally, IL-6 serum concentrations were measured in mice with varying platelet counts after partial hepatectomy. Results Following partial hepatectomy, in vivo adhesion of platelets to LSEC was significantly increased, when compared to sham-operated mice. Co-incubation of increasing numbers of resting or activated platelets with LSEC resulted in significantly enhanced IL-6 secretion by LSEC. IL-6 release by LSEC was the highest after incubation with ADP-activated platelets. The effect of platelet releasates on LSEC was similar to that seen with intact, whole platelets. Further, the effect of platelets on LSEC was mediated by a platelet factor, whose action was synergistically enhanced by ADP. ADP-activated platelet releasates increased proliferation of hepatocytes co-cultured with LSEC. Decreased platelet counts resulted in decreased serum levels of IL-6 after partial hepatectomy in vivo. Conclusion We show that platelets modulate LSEC to constitutively secrete IL-6, a key player in liver regeneration. After partial hepatectomy, platelets adhere to LSEC and serum IL-6 levels depend on platelet concentration. Interactions between platelets and LSEC modulate IL-6 release by LSEC and increase hepatocyte proliferation. This effect is dependent on ADP.

Amicus or Adversary Revisited: Platelets in Acute Lung Injury and Acute Respiratory Distress Syndrome.

Platelets are essential cellular effectors of hemostasis and contribute to disease as circulating effectors of pathologic thrombosis. These are their most widely known biologic activities. Nevertheless, recent observations demonstrate that platelets have a much more intricate repertoire beyond these traditional functions and that they are specialized for contributions to vascular barrier integrity, organ repair, antimicrobial host defense, inflammation, and activities across the immune continuum. Paradoxically, on the basis of clinical investigations and animal models of disease, some of these newly discovered activities of platelets appear to contribute to tissue injury. Studies in the last decade indicate unique interactions of platelets and their precursor, the megakaryocyte, in the lung and implicate platelets as essential effectors in experimental acute lung injury and clinical acute respiratory distress syndrome. Additional discoveries derived from evolving work will be required to precisely define the contributions of platelets to complex subphenotypes of acute lung injury and to determine if these remarkable and versatile blood cells are therapeutic targets in acute respiratory distress syndrome.

Oral Bruton tyrosine kinase inhibitors selectively block atherosclerotic plaque–triggered thrombus formation in humans

AbstractInteraction of von Willebrand factor (VWF) with platelet glycoprotein Ib (GPIb) and interaction of collagen with GPVI are essential for thrombus formation on ruptured or eroded atherosclerotic plaques (atherothrombosis). GPIb and GPVI signal through Bruton tyrosine kinase (Btk), which can be blocked irreversibly by oral application of ibrutinib, an established therapy for chronic lymphocytic leukemia (CLL) with long-term safety. We found that ibrutinib and the novel Btk inhibitors acalabrutinib and ONO/GS-4059 block GPVI-dependent static platelet aggregation in blood exposed to human plaque homogenate and collagen but not to ADP or arachidonic acid. Moreover, Btk inhibitors prevented platelet thrombus formation on human atherosclerotic plaque homogenate and plaque tissue sections from arterially flowing blood, whereas integrin α2β1 and VWF-dependent platelet adhesion to collagen, which is important for physiologic hemostasis, was not affected. This plaque-selective platelet inhibition was also observed in CLL patients taking 450 mg of ibrutinib and in volunteers after much lower and intermittent dosing of the drug. We conclude that Btk inhibitors, by targeting GPIb and GPVI signal transduction, suppress platelet thrombus accretion from flowing blood on atherosclerotic plaque but spare hemostatic platelet function. Btk inhibitors hold promise as the first culprit lesion–focused oral antiplatelet drugs and are effective at low doses.

Acquired Antibodies to αIIbβ3 in Glanzmann Thrombasthenia: From Transfusion and Pregnancy to Bone Marrow Transplants and Beyond.

Patients with the inherited bleeding disorder Glanzmann thrombasthenia (GT) possess platelets that lack αIIbβ3 integrin and fail to aggregate, and have moderate to severe mucocutaneous bleeding. Many become refractory to platelet transfusions due to the formation of isoantibodies to αIIbβ3 with the rapid elimination of donor platelets and/or a block of function. Epitope characterization has shown isoantibodies to be polyclonal and to recognize different epitopes on the integrin with β3 a major site and αvβ3 on endothelial and vascular cells a newly recognized target. Pregnancy in GT can also lead to isoantibody formation when fetal cells with β3 integrins pass into the circulation of a mother lacking them; a consequence is neonatal thrombocytopenia and a high risk of mortality. Antibody removal prior to donor transfusions can provide transient relief, but all evidence points to recombinant FVIIa as the first choice for GT patients either to stop bleeding or as prophylaxis. Promoting thrombin generation by rFVIIa favors GT platelet interaction with fibrin, and the risk of deep vein thrombosis also associated with prolonged immobilization and catheter use requires surveillance. Although having a high risk, allogeneic bone marrow transplantation associated with different stem cell sources and conditioning regimens has proved successful in many cases of severe GT with antibodies, and often, the associated conditioning and immunosuppressive therapy leads to loss of isoantibody production. Animal models of gene therapy for GT show promising results, but isoantibody production can be stimulated and CRISPR/Cas9 technology has yet to be applied. Up-to-date consensus protocols for dealing with isoantibodies in GT are urgently required, and networks providing patient care should be expanded.

Protectin DX Exhibits Protective Effects in Mouse Model of Lipopolysaccharide-Induced Acute Lung Injury.

Background:Acute lung injury (ALI) is a severe disease with high mortality and poor prognosis. Protectin DX (PDX), a pro-resolving lipid mediator, exhibits protective effects in ALI. Our experiment aimed to explore the effects and related mechanisms of PDX in mice with ALI induced by lipopolysaccharide (LPS).Methods:BALB/c mice were randomly divided into five groups: sham, LPS, LPS plus 1 ng of PDX (LPS + PDX-1 ng), LPS plus 10 ng of PDX (LPS + PDX-10 ng), and LPS plus 100 ng of PDX (LPS + PDX-100 ng). Bronchoalveolar lavage fluids (BALFs) were collected after 24 h, and total cells, polymorphonuclear leukocytes, monocyte-macrophages, and lymphocytes in BALF were enumerated. The concentration of interleukin (IL)-1β, IL-6, IL-10, tumor necrosis factor-alpha (TNF-α), macrophage inflammatory protein (MIP)-1α, and MIP-2 in BALF was determined, and histopathological changes of the lung were observed. The concentration of protein in BALF and lung wet/dry weight ratios were detected to evaluate pulmonary edema. After determining the optimal dose of PDX, neutrophil–platelet interactions in whole blood were evaluated by flow cytometry.Results:The highest dose of PDX (100 ng/mouse) failed to provide pulmonary protective effects, whereas lower doses of PDX (1 ng/mouse and 10 ng/mouse), especially 1 ng PDX, alleviated pulmonary histopathological changes, mitigated LPS-induced ALI and pulmonary edema, inhibited neutrophil infiltration, and reduced pro-inflammatory mediator (IL-1β, IL-6, TNF-α, and MIP-1α) levels. Meanwhile, 1 ng PDX exhibited pro-resolving functions in ALI including upregulation of monocyte-macrophage numbers and anti-inflammatory mediator IL-10 levels. The flow cytometry results showed that PDX could inhibit neutrophil–platelet interactions in ALI.Conclusion:PDX exerts protective effects in LPS-induced ALI by mitigating pulmonary inflammation and abrogating neutrophil–platelet interactions.