Hemocompatibility of Silicon-Based Substrates for Biomedical Implant Applications

Silicon membranes with highly uniform nanopore sizes fabricated using microelectromechanical systems (MEMS) technology allow for the development of miniaturized implants such as those needed for renal replacement therapies. However, the blood compatibility of silicon has thus far been an unresolved issue in the use of these substrates in implantable biomedical devices. We report the results of hemocompatibility studies using bare silicon, polysilicon, and modified silicon substrates. The surface modifications tested have been shown to reduce protein and/or platelet adhesion, thus potentially improving biocompatibility of silicon. Hemocompatibility was evaluated under four categories—coagulation (thrombin–antithrombin complex, TAT generation), complement activation (complement protein, C3a production), platelet activation (P-selectin, CD62P expression), and platelet adhesion. Our tests revealed that all silicon substrates display low coagulation and complement activation, comparable to that of Teflon and stainless steel, two materials commonly used in medical implants, and significantly lower than that of diethylaminoethyl (DEAE) cellulose, a polymer used in dialysis membranes. Unmodified silicon and polysilicon showed significant platelet attachment; however, the surface modifications on silicon reduced platelet adhesion and activation to levels comparable to that on Teflon. These results suggest that surface-modified silicon substrates are viable for the development of miniaturized renal replacement systems

Platelet clearance via shear-induced unfolding of a membrane mechanoreceptor

Mechanisms by which blood cells sense shear stress are poorly characterized. In platelets, glycoprotein (GP)Ib-IX receptor complex has been long suggested to be a shear sensor and receptor. Recently, a relatively unstable and mechanosensitive domain in the GPIba subunit of GPIb-IX was identified. Here we show that binding of its ligand, von Willebrand factor, under physiological shear stress induces unfolding of this mechanosensory domain (MSD) on the platelet surface. The unfolded MSD, particularly the juxtamembrane € Trigger' sequence therein, leads to intracellular signalling and rapid platelet clearance. These results illustrate the initial molecular event underlying platelet shear sensing and provide a mechanism linking GPIb-IX to platelet clearance. Our results have implications on the mechanism of platelet activation, and on the pathophysiology of von Willebrand disease and related thrombocytopenic disorders. The mechanosensation via receptor unfolding may be applicable for many other cell adhesion receptors

Platelets disrupt vasculogenic mimicry by cancer cells

Tumour vasculature supports the growth and progression of solid cancers with both angiogenesis (endothelial cell proliferation) and vasculogenic mimicry (VM, the formation of vascular structures by cancer cells themselves) predictors of poor patient outcomes. Increased circulating platelet counts also predict poor outcome for cancer patients but the influence of platelets on tumour vasculature is incompletely understood. Herein, we show with in vitro assays that platelets did not influence angiogenesis but did actively inhibit VM formation by cancer cell lines. Both platelet sized beads and the releasates from platelets were partially effective at inhibiting VM formation suggesting that direct contact maximises the effect. Platelets also promoted cancer cell invasion in vitro. B16F10 melanomas in Bcl-xPlt20/Plt20 thrombocytopenic mice showed a higher content of VM than their wildtype counterparts while angiogenesis did not differ. In a xenograft mouse model of breast cancer with low-dose aspirin to inactivate the platelets, the burden of MDA-MB-231-LM2 breast cancer cells was reduced and the gene expression profile of the cancer cells was altered; but no effect on tumour vasculature was observed. Taken together, this study provides new insights into the action of platelets on VM formation and their involvement in cancer progression

Dual roles for hepatic lectin receptors in the clearance of chilled platelets

Rapid chilling causes glycoprotein-Ib (GPIb) receptors to cluster on blood platelets. Hepatic macrophage beta(2) integrin binding to beta-N-acetylglucosamine (beta-GlcNAc) residues in the clusters leads to rapid clearance of acutely chilled platelets after transfusion. Although capping the beta-GlcNAc moieties by galactosylation prevents clearance of short-term-cooled platelets, this strategy is ineffective after prolonged refrigeration. We report here that prolonged refrigeration increased the density and concentration of exposed galactose residues on platelets such that hepatocytes, through Ashwell-Morell receptor binding, become increasingly involved in platelet removal. Macrophages rapidly removed a large fraction of transfused platelets independent of their storage conditions. With prolonged platelet chilling, hepatocyte-dependent clearance further diminishes platelet recovery and survival after transfusion. Inhibition of chilled platelet clearance by both beta(2) integrin and Ashwell-Morell receptors may afford a potentially simple method for storing platelets in the cold

N-methyl-D-aspartate receptor mediated calcium influx supports in vitro differentiation of normal mouse megakaryocytes but proliferation of leukemic cell lines

Background: N-methyl-d-aspartate receptors (NMDARs) contribute calcium influx in megakaryocytic cells but their roles remain unclear; both pro- and anti-differentiating effects have been shown in different contexts. Objectives: The aim of this study was to clarify NMDAR contribution to megakaryocytic differentiation in both normal and leukemic cells. Methods: Meg-01, Set-2, and K-562 leukemic cell lines were differentiated using phorbol-12-myristate-13-acetate (PMA, 10 nmol L-1) or valproic acid (VPA, 500 μmol L-1). Normal megakaryocytes were grown from mouse marrow-derived hematopoietic progenitors (lineage-negative and CD41a-enriched) in the presence of thrombopoietin (30-40 nmol L-1). Marrow explants were used to monitor proplatelet formation in the native bone marrow milieu. In all culture systems, NMDARs were inhibited using memantine and MK-801 (100 μmol L-1); their effects compared against appropriate controls. Results: The most striking observation from our studies was that NMDAR antagonists markedly inhibited proplatelet formation in all primary cultures employed. Proplatelets were either absent (in the presence of memantine) or short, broad and intertwined (with MK-801). Earlier steps of megakaryocytic differentiation (acquisition of CD41a and nuclear ploidy) were maintained, albeit reduced. In contrast, in leukemic Meg-01 cells, NMDAR antagonists inhibited differentiation in the presence of PMA and VPA but induced differentiation when applied by themselves. Conclusions: NMDAR-mediated calcium influx is required for normal megakaryocytic differentiation, in particular proplatelet formation. However, in leukemic cells, the main NMDAR role is to inhibit differentiation, suggesting diversion of NMDAR activity to support leukemia growth. Further elucidation of the NMDAR and calcium pathways in megakaryocytic cells may suggest novel ways to modulate abnormal megakaryopoiesis

The necroptotic cell death pathway operates in megakaryocytes, but not in platelet synthesis

Necroptosis is a pro-inflammatory cell death program executed by the terminal effector, mixed lineage kinase domain-like (MLKL). Previous studies suggested a role for the necroptotic machinery in platelets, where loss of MLKL or its upstream regulator, RIPK3 kinase, impacted thrombosis and haemostasis. However, it remains unknown whether necroptosis operates within megakaryocytes, the progenitors of platelets, and whether necroptotic cell death might contribute to or diminish platelet production. Here, we demonstrate that megakaryocytes possess a functional necroptosis signalling cascade. Necroptosis activation leads to phosphorylation of MLKL, loss of viability and cell swelling. Analyses at steady state and post antibody-mediated thrombocytopenia revealed that platelet production was normal in the absence of MLKL, however, platelet activation and haemostasis were impaired with prolonged tail re-bleeding times. We conclude that MLKL plays a role in regulating platelet function and haemostasis and that necroptosis signalling in megakaryocytes is dispensable for platelet production

Mpl expression on megakaryocytes and platelets is dispensable for thrombopoiesis but essential to prevent myeloproliferation

Thrombopoietin (TPO) acting via its receptor, the cellular homologue of the myeloproliferative leukemia virus oncogene (Mpl), is the major cytokine regulator of platelet number. To precisely define the role of specific hematopoietic cells in TPO-dependent hematopoiesis, we generated mice that express the Mpl receptor normally on stem/progenitor cells but lack expression on megakaryocytes and platelets (Mpl(PF4cre/PF4cre)). Mpl(PF4cre/PF4cre) mice displayed profound megakaryocytosis and thrombocytosis with a remarkable expansion of megakaryocyte-committed and multipotential progenitor cells, the latter displaying biological responses and a gene expression signature indicative of chronic TPO overstimulation as the underlying causative mechanism, despite a normal circulating TPO level. Thus, TPO signaling in megakaryocytes is dispensable for platelet production; its key role in control of platelet number is via generation and stimulation of the bipotential megakaryocyte precursors. Nevertheless, Mpl expression on megakaryocytes and platelets is essential to prevent megakaryocytosis and myeloproliferation by restricting the amount of TPO available to stimulate the production of megakaryocytes from the progenitor cell pool

Targeting platelets for improved outcome in KRAS-driven lung adenocarcinoma

Elevated platelet count is associated with poor survival in certain solid cancers, including lung cancer. In addition, experimental transplantation of cancer cell lines has uncovered a role for platelets in blood-borne metastasis. These studies, however, do not account for heterogeneity between lung cancer subtypes. Subsequently, the role of platelets in the major subtypes of non-small cell lung cancer (adenocarcinoma (ADC) and squamous cell carcinoma (SqCC)) is not fully understood. We utilised an autochthonous KrasLSL-G12D/+;p53flox/flox mouse model of lung ADC together with genetic models of thrombocytopenia to interrogate the role of platelets in lung cancer growth and progression. While thrombocytopenia failed to impact primary tumour growth, in experimental metastatic models however, thrombocytopenic mice displayed significantly extended survival. Utilising a novel thrombocytopenic immunocompromised mouse, the importance of platelets in metastatic dissemination was confirmed with human KRAS-mutant ADC cell lines. Finally, retrospective analysis of a NSCLC patient cohort revealed thrombocytosis was predictive of poor survival in ADC patients with metastatic disease. Interestingly, this association was not apparent in SqCC patients. Overall, these data highlight the possibility of patient stratification using thrombocytosis as a biomarker, and indicates opportunities for potential novel treatment strategies that combine anti-platelet and lung cancer therapies