Blood Cell Adhesion Research Articles

Serum thrombospondin 2 is a novel predictor for the severity in the patients with NAFLD.

Thrombospondins are a family of multidomain and secretory glycoproteins. Among them, thrombospondin 2 (TSP2) encoded by TSP2 gene has been reported to be involved in various functions such as collagen/fibrin formation, maintenance of normal blood vessel density and cell adhesion properties. Microarray analyses ranked TSP2 as one of the most highly up-regulated genes in the fibrotic liver in patients with non-alcoholic fatty liver disease (NAFLD). Since TSP2 possesses unique properties as a secretory protein, we hypothesized that hepatic TSP2 gene expression levels would be reflected in serum TSP2 levels. In this study, we examined the relationship between serum TSP2 concentrations and clinicopathological findings in NAFLD patients. One hundred and thirty NAFLD patients who had undergone liver biopsy between 2009 and 2015 were retrospectively enrolled. Serum samples were collected at the time of biopsy, and TSP2 was measured by enzyme immunoassays. Serum TSP2 levels moderately correlated with ballooning (r=0.56, P<.001) and fibrosis stage (r=0.53, P<.001). The AUC values of TSP2 for predicting mild fibrosis (≧F1), moderate fibrosis (≧F2) and severe fibrosis (≧F3) were 0.73, 0.76 and 0.82 respectively. Additionally, NAFLD activity score (NAS) correlated best with TSP2 (r=0.52, P<.001) compared to conventional NAFLD-related biomarkers, such as cytokeratin 18 M30, hyaluronic acid, type IV collagen 7S, APRI and FIB-4 index. Serum TSP2 levels reflected hepatocyte ballooning, fibrosis and NAS in NAFLD patients. For clinical application of serum TSP2 as a predictor of NAFLD histological activity, additional validation and mechanistic investigations are required.

Highly resilient, biocompatible, and antibacterial carbon nanotube/hydroxybutyl chitosan sponge dressing for rapid and effective hemostasis.

Uncontrolled hemorrhage is the leading cause of trauma death. The development of safe and efficient hemostatic agents that can rapidly and effectively control bleeding is of great significance to rescue the injured. However, the mechanical, absorptive, and antibacterial properties of conventional two-dimensional hemostatic agents are not satisfactory. Herein, a series of effective three-dimensional hemostatic dressings (JWCNT/HBC sponges) are developed by chemical modification of joint-welded carbon nanotube (JWCNT) sponges with hydroxybutyl chitosan (HBC) for hemorrhage hemostasis. The JWCNT/HBC sponges exhibit high elasticity, porous structure, and suitable blood-absorption and blood-maintaining performance. Moreover, the introduction of HBC endows the JWCNT/HBC sponges with favorable blood compatibility and good antibacterial activity. The sponge treated with 0.5% HBC (JWCNT/0.5%HBC sponge) displays better antiseptic capability, faster blood clotting ability in vitro and shorter hemostasis time in vivo than the commercial gelatin sponge. The JWCNT/HBC sponges combine the advantages of JWCNT sponges and HBC in the adhesion and activation of platelets and red blood cells, thus becoming a good medical material for trauma hemostasis.

Engineered multifunctional metal-phenolic nanocoatings for label-free capture and "self-release" of heterogeneous circulating tumor cells.

Immunomagnetic beads have been widely explored as an important analytical tool for the rapid and sensitive detection of circulating tumor cells (CTCs). However, their clinical application is seriously hindered by the tedious preparation procedures and heterogeneous nature of CTCs. To this end, a designed multifunctional platform named Fe3O4@TA/CuII superparamagnetic nanoparticles (SPMNPs) is expected to have the following features: (i) the formation of a tannic acid-copper (II) ion (TA/CuII) coating which could be accomplished by a one-step method is very simple; (ii) the TA/CuII coating shows high affinity for heterogeneous CTCs and good resistance to nonspecific adhesion of blood cells; (iii) "self-release" of the captured cells could be achieved as the TA/CuII coating gradually degrades in the cell culture environment without any additional interventions. Therefore, the resulting Fe3O4@TA/CuII SPMNPs could capture various CTCs (MCF-7, HepG2 and HeLa cells) with different expression levels of the epithelial cell adhesion molecule (EpCAM). And the capture efficiency and cell purity can reach 88% and 87%, respectively. In addition, 68% of the captured cells are self-released after 6 h of incubation and most of the released cells show high cell proliferation activity. In particular, Fe3O4@TA/CuII SPMNPs can successfully detect 1-13 CTCs from 1 mL of blood of 14 patients with 6 types of cancers. Hence, we expect that the as-prepared Fe3O4@TA/CuII SPMNPs with simple, efficient, and universal yet cost-efficient characteristics could act as a promising analytical tool for clinical CTC detection.

Capture and Kill: Selective Eradication of Target Bacteria by a Flexible Bacteria-Imprinted Chip.

This paper reports an antibacterial chip that can selectively capture bacteria and kill them using low-voltage DC electricity. We prepared a bacteria-imprinted, flexible PDMS chip that can separate target bacteria from suspensions with high selectivity. The chip contained integrated electrodes that can kill the captured bacteria within 10 min by applying a low DC voltage. The used chip could be easily regenerated by solution immersion. Meanwhile, the PDMS chip showed good biocompatibility and inhibited adhesion of human blood cells. Our work points to a new strategy to address pathogenic bacterial contamination and infection.

Insights Into Immunothrombosis: The Interplay Among Neutrophil Extracellular Trap, von Willebrand Factor, and ADAMTS13.

Both neutrophil extracellular traps (NETs) and von Willebrand factor (VWF) are essential for thrombosis and inflammation. During these processes, a complex series of events, including endothelial activation, NET formation, VWF secretion, and blood cell adhesion, aggregation and activation, occurs in an ordered manner in the vasculature. The adhesive activity of VWF multimers is regulated by a specific metalloprotease ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type 1 motifs, member 13). Increasing evidence indicates that the interaction between NETs and VWF contributes to arterial and venous thrombosis as well as inflammation. Furthermore, contents released from activated neutrophils or NETs induce the reduction of ADAMTS13 activity, which may occur in both thrombotic microangiopathies (TMAs) and acute ischemic stroke (AIS). Recently, NET is considered as a driver of endothelial damage and immunothrombosis in COVID-19. In addition, the levels of VWF and ADAMTS13 can predict the mortality of COVID-19. In this review, we summarize the biological characteristics and interactions of NETs, VWF, and ADAMTS13, and discuss their roles in TMAs, AIS, and COVID-19. Targeting the NET-VWF axis may be a novel therapeutic strategy for inflammation-associated TMAs, AIS, and COVID-19.

Maternal proteomic profiling reveals alterations in lipid metabolism in late-onset fetal growth restriction

Fetal growth restriction defined as the failure to achieve the fetal genetic growth potential is a major cause of perinatal morbidity and mortality. The role of maternal adaptations to placental insufficiency in this disorder is still not fully understood. We aimed to investigate the biological processes and protein–protein interactions involved in late-onset fetal growth restriction in particular. We applied 2D nano LC–MS/MS proteomics analysis on maternal blood samples collected at the time of delivery from 5 singleton pregnancies with late-onset fetal growth restriction and 5 uncomplicated pregnancies. Data were analyzed using R package “limma” and Ingenuity Pathway Analysis. 25 proteins showed significant changes in their relative abundance in late-onset fetal growth restriction (p value < 0.05). Direct protein–protein interactions network demonstrated that Neurogenic locus notch homolog protein 1 (NOTCH1) was the most significant putative upstream regulator of the observed profile. Gene ontology analysis of these proteins revealed the involvement of 14 canonical pathways. The most significant biological processes were efflux of cholesterol, efflux of phospholipids, adhesion of blood cells, fatty acid metabolism and dyslipidemia. Future studies are warranted to validate the potential role of the detected altered proteins as potential therapeutic targets in the late-onset form of fetal growth restriction.

Synthesis of 2-Methacryloyloxyethyl Phosphorylcholine (MPC) Based P(2-hydroxyethyl methacrylate) P(HEMA) Cryogel Membranes

Synthesis of artificial/natural polymeric biomaterials having resistance to nonspecific protein adsorption, blood coagulation and bacterial adhesion has attracted great attention, so nonspecific adsorption of proteins and biomolecules causes unfavorable biological responses inluding blood clotting, inflammation, cell adhesion, cell differentiation, and biofilm formation. A zwitterionic phosphorylcholine (PC) group of 2-methacryloyloxyethyl phosphorylcholine (MPC) is employed for biologically inert functions, especially in resistance to protein adsorption. So, it is aimed to develop bio-inspired, efficient and environmentally friendly MPC containing cryogel membranes for polymeric scaffolds for promoting cell-biomaterial. Cryogel membranes were synthesized in semi-frozen medium by free radical polymerization in an ice bath and characterized by SEM/EDX, micro-CT, and swelling ratio measurements. In vitro biocompatibility was assessed from cell viability studies performed using cultured fibroblast cells.

A Deep Learning Framework for Sickle Cell Disease Microfluidic Biomarker Assays

Introduction: Vaso-occlusive crises (VOCs) are a leading cause of morbidity and early mortality in individuals with sickle cell disease (SCD). These crises are triggered by sickle red blood cell (sRBC) aggregation in blood vessels and are influenced by factors such as enhanced sRBC and white blood cell (WBC) adhesion to inflamed endothelium. Advances in microfluidic biomarker assays (i.e., SCD Biochip systems) have led to clinical studies of blood cell adhesion onto endothelial proteins, including, fibronectin, laminin, P-selectin, ICAM-1, functionalized in microchannels. These microfluidic assays allow mimicking the physiological aspects of human microvasculature and help characterize biomechanical properties of adhered sRBCs under flow. However, analysis of the microfluidic biomarker assay data has so far relied on manual cell counting and exhaustive visual morphological characterization of cells by trained personnel. Integrating deep learning algorithms with microscopic imaging of adhesion protein functionalized microfluidic channels can accelerate and standardize accurate classification of blood cells in microfluidic biomarker assays. Here we present a deep learning approach into a general-purpose analytical tool covering a wide range of conditions: channels functionalized with different proteins (laminin or P-selectin), with varying degrees of adhesion by both sRBCs and WBCs, and in both normoxic and hypoxic environments. Methods: Our neural networks were trained on a repository of manually labeled SCD Biochip microfluidic biomarker assay whole channel images. Each channel contained adhered cells pertaining to clinical whole blood under constant shear stress of 0.1 Pa, mimicking physiological levels in post-capillary venules. The machine learning (ML) framework consists of two phases: Phase I segments pixels belonging to blood cells adhered to the microfluidic channel surface, while Phase II associates pixel clusters with specific cell types (sRBCs or WBCs). Phase I is implemented through an ensemble of seven generative fully convolutional neural networks, and Phase II is an ensemble of five neural networks based on a Resnet50 backbone. Each pixel cluster is given a probability of belonging to one of three classes: adhered sRBC, adhered WBC, or non-adhered / other. Results and Discussion: We applied our trained ML framework to 107 novel whole channel images not used during training and compared the results against counts from human experts. As seen in Fig. 1A, there was excellent agreement in counts across all protein and cell types investigated: sRBCs adhered to laminin, sRBCs adhered to P-selectin, and WBCs adhered to P-selectin. Not only was the approach able to handle surfaces functionalized with different proteins, but it also performed well for high cell density images (up to 5000 cells per image) in both normoxic and hypoxic conditions (Fig. 1B). The average uncertainty for the ML counts, obtained from accuracy metrics on the test dataset, was 3%. This uncertainty is a significant improvement on the 20% average uncertainty of the human counts, estimated from the variance in repeated manual analyses of the images. Moreover, manual classification of each image may take up to 2 hours, versus about 6 minutes per image for the ML analysis. Thus, ML provides greater consistency in the classification at a fraction of the processing time. To assess which features the network used to distinguish adhered cells, we generated class activation maps (Fig. 1C-E). These heat maps indicate the regions of focus for the algorithm in making each classification decision. Intriguingly, the highlighted features were similar to those used by human experts: the dimple in partially sickled RBCs, the sharp endpoints for highly sickled RBCs, and the uniform curvature of the WBCs. Overall the robust performance of the ML approach in our study sets the stage for generalizing it to other endothelial proteins and experimental conditions, a first step toward a universal microfluidic ML framework targeting blood disorders. Such a framework would not only be able to integrate advanced biophysical characterization into fast, point-of-care diagnostic devices, but also provide a standardized and reliable way of monitoring patients undergoing targeted therapies and curative interventions, including, stem cell and gene-based therapies for SCD. Disclosures Gurkan: Dx Now Inc.: Patents &amp; Royalties; Xatek Inc.: Patents &amp; Royalties; BioChip Labs: Patents &amp; Royalties; Hemex Health, Inc.: Consultancy, Current Employment, Patents &amp; Royalties, Research Funding.

Contribution of Red Blood Cell Derived Extracellular Vesicles to Sickle Red Blood Cell Adhesion Discerned Using an Endothelialized Microfluidic Assay

Introduction: Sickle cell disease (SCD) is an inherited hemoglobinopathy, in which the mutation of a single amino acid in the adult beta chain results in sickle hemoglobin (HbS). Upon deoxygenation, HbS polymerizes in red blood cells (RBCs) and provokes a complex pathophysiology of acute and chronic organ damage. Sickle RBCs have reduced deformability, increased adhesion to the endothelium, and are prone to hemolysis, which further contributes to endothelial dysfunction. Chronic inflammatory processes as well as hemostatic alterations and thrombotic events are common in SCD. Cumulatively, thromboinflammation plays a significant pathophysiologic role in SCD, contributing to venous thromboembolism, vaso-occlusion, ischemia-reperfusion, and chronic organ damage, which cumulatively lead to increased morbidity, health care utilization, and a reduced life expectancy. Several pathophysiological processes in SCD result in the activation of RBCs and in the release of sub-micron particles called extracellular vesicles (EVs). EVs are composed of a lipid bilayer, transmembrane proteins, and enclosing intracellular remnants, including cytosolic proteins, RNA, and micro-RNA (miRNA). EVs can serve as vehicles for cellular communication, in near and remote proximity, and can reflect the parent cell's activation state. In SCD, RBC-derived EVs (REVs) comprise the most prevalent (&amp;gt;50%) subtype. REVs express surface phosphatidylserine (PS), contain heme and miRNAs, and are capable of promoting blood coagulation and a pro-inflammatory/pro-adhesive endothelial phenotype. Despite the significant potential role of REVs as candidate biomarkers, REV associated proinflammatory effects are often evaluated using animal models, or by assessing white blood cell adhesion, and thus do not reflect the well-known clinical heterogeneity and the abnormal RBC adhesion amongst SCD patients. Methods: We have developed an in vitro microfluidic assay, the SCD-EV-BioChip, with which to assess RBC adhesion as a biomarker for REV-mediated lung microvascular endothelial dysfunction. The SCD-EV-BioChip contains microfluidic channels lined with human pulmonary microvascular endothelial cells (HPMECs) that are maintained under precise shear stress and oxygen tension at physiologically and clinically relevant levels. HPMECs were incubated with patient-specific or pooled REVs generated in vitro via exposure of RBCs to calcium ionophore (Fig. 1A&amp;B). We assessed RBC adhesion in 4 healthy subjects (HbAA), 10 homozygous SCD patients (HbSS), and 3 patients with HbSC disease for REV proinflammatory effects using HPMCs exposed to patient specific derived REVs. Results and Discussion: In non-patient-specific testing, adhesion assays were performed on samples from 12 individuals with HbSS, using HPMECs that had been exposed to pooled REVs derived from multiple patients, thus reflecting only patient-specific RBC intrinsic adhesion, rather than patient-specific contribution of RBC derived REVs. Patient specific REV activation of HPMCs (Fig. 1C&amp;D) showed that RBC adhesion was greater in HbSS-containing samples, compared with HbSC or HbAA (Fig. 1B&amp;C). In subjects with HbSS, RBC adhesion to HPMC, activated by patient-specific derived REV, was higher in those without, vs. those with a recent transfusion (non-TX vs. TX, Fig. 1D). However, non-TX samples showed intrinsically less adhesion to HPMECs activated by pooled REVs (Fig. 1E), compared with TX samples. Results suggest a paradoxical association between transfusion history and RBC adhesion in patient specific tests vs. non-patient-specific tests. This association suggests that the minority of RBCs containing HbSS in TX subjects generate fewer or less active patient specific REVs, but that these residual HbSS-RBCs are highly adherent when the endothelium is perturbed by pooled non-patient specific REVs. These data highlight that patient-specific contributions from both REVs and RBCs must be accounted for when describing abnormal RBC adhesion in individuals with SCD. Disclosures An: Hemex Health, Inc.: Patents &amp; Royalties. Little:NHLBI: Research Funding; GBT: Membership on an entity's Board of Directors or advisory committees; GBT: Research Funding; Bluebird Bio: Research Funding; BioChip Labs: Patents &amp; Royalties: SCD Biochip (patent, no royalties); Hemex Health, Inc.: Patents &amp; Royalties: Microfluidic electropheresis (patent, no royalties). Gurkan:Dx Now Inc.: Patents &amp; Royalties; Xatek Inc.: Patents &amp; Royalties; BioChip Labs: Patents &amp; Royalties; Hemex Health, Inc.: Consultancy, Current Employment, Patents &amp; Royalties, Research Funding.

Reduction of ARHGAP21 Alters Platelet Biogenesis in Vitro and Accelerates Hemostatic Response In Vivo

Megakaryocyte differentiation and platelet biogenesis require profound cytoskeleton rearrangement regulated by the Rho family of GTPases. ARHGAP21 is a RhoGAP protein that has been shown to negatively regulate the activity of RhoA, RhoC and Cdc42. We have previously demonstrated that ARHGAP21 knockdown in human common myeloid progenitors and in bipotent megakaryocyte-erythrocyte progenitors may bias the fate decision toward the megakaryocyte lineage. In addition, a mouse model with reduction of Arhgap21 expression (Arhgap21+/-) present a slight reduction in platelet number and increased platelet volume. However, the participation of ARHGAP21 in platelet biogenesis and hemostatic response has never been investigated. We studied the role of ARHGAP21 on cytoskeletal changes during megakaryocyte differentiation in HEL cell line. Differentiation was induced with 20ηM phorbol-13 myristate-12 acetate (PMA) for four days. ARHGAP21 protein expression was increased during the differentiation and was mostly detected in the protein cell fraction containing polymerized tubulin, in comparison with cell extracts containing soluble tubulin. ARHGAP21 co-localized (R ≥ 0.86 in all days of differentiation) and interacted with α-tubulin on day 2 of megakaryocyte differentiation, when ARHGAP21 expression was the highest. Silencing of ARHGAP21 with siRNA decreased the expression of Glu-tubulin and enhanced CDC42 activity on days 2 and 3 of differentiation. Increased cell size and spreading and alteration of the adhesion proteins p-p130Cas, vinculin, p-zyxin and p-FAK925 were also observed upon ARHGAP21 inhibition. There was no change in the acquisition of CD61, CD41 and CD42 megakaryocytic markers, neither in the polyploidy of HEL cells during differentiation. We further investigated the effects of ARHGAP21 inhibition on platelet morphology and on the hemostatic response in vivo, using the C57BL/6 Arhgap21 heterozygous mouse model (Arhgap21+/-). The study was approved by the Ethical Committee of our Institution. No differences were observed in CD61+CD41+ nor in CD41+CD42+ bone marrow cells from Arhgap21+/- and wild type (WT) mice. However, transmission electron microscopy revealed that Arhgap21+/- platelets presented increased alpha-granule size when compared to wild-type (WT). Tail bleeding time of Arhgap21+/- mice was decreased compared to WT (P= 0.0008). Intravital microscopy of carotid artery injured by FeCl3 showed increased adhesion of platelet and white blood cells on the vessel wall of Arhgap21+/-, which reflected in accelerated occlusion time (twice as fast) compared to WT (P= 0.0150). In conclusion, ARHGAP21 silencing may alter cell morphology and lead to increased microtubule dynamic instability during megakaryocyte differentiation in vitro, without compromising the acquisition of differentiation markers. In vivo, deficiency of Arhgap21 increases platelet granule size and accelerates hemostatic response. Together, these results indicate that ARHGAP21 may be a critical protein in the regulation of platelet production and function through the control of cytoskeletal rearrangement. This study was supported by São Paulo Research Foundation (FAPESP) National Council for Scientific and Technological Development (CNPq) and Coordination for the Improvement of Higher Education Personnel (CAPES). Disclosures No relevant conflicts of interest to declare.

Increased Adhesiveness of Red Blood Cells from Sickle Cell Trait Carriers after Exposure to Daunirubicin Breast Cancer Chemotherapeutic Agent

Introduction: African-Americans (AA) with breast cancer continue to experience worse morbidity and mortality than white patients. In addition to barriers to care, an active area of investigation has been the etiology of the increased risk adverse events (AEs) related to chemotherapy. An emerging question is if sickle cell trait (i.e., heterozygotes for hemoglobin beta gene), present in an estimated 1 in 12 of the AA population, confers increased susceptibility to chemotherapy-related complications. We posit that conditions of high physiological stress might be manifest during systemic treatment with toxic agents resulting in alterations of red blood cells (RBCs). Using an experimental biomechanical model system, we hypothesized that RBCs from SCT carriers would be prone to increased adhesiveness after exposure to a common systemic anti-cancer agent used in breast cancer. Increased adhesiveness of RBCs can be a precipitating factor of blocked vasculature and subsequent sickling crises, potentially leading to AEs during treatment. Methods: Our study testedex vivoRBCs from two groups of healthy female participants: 20 African-American sickle cell trait carriers (AA-SCT); and 15 white subjects with wild-type hemoglobin (W-WT). Isolated RBCs were treated with scaled Daunorubicin (DNR) doses. The unbinding forces between αvβ3 ligands and intercellular adhesion molecule-4 (ICAM-4) receptors are reported using the frequency distribution, which states the percentage of events whose unbinding forces are within each width of the bin. We then compared the median values of the forces measured in experiments without and with treatment for each drug. The collective frequency (CF%) is related to the population of active ICAM-4 receptors and is defined as the percentage of all unbinding events divided by the total number of measurements, which is 32 × 32 =1024 for each cell multiplied by the number of tested cells for each blood sample. Results: For AA-SCT RBCs, pre-treatment baseline CF of active ICAM-4 receptors was 4.88 ± 0.87%, which was significantly increased after administration of DNR (13.13 ± 2.30%, p&amp;lt;0.0001). In contrast, treatment of W-WT RBCs held the CF of active ICAM-4 receptors at a comparable level as untreated RBCs (9.54 ± 1.48%; 7.47 ± 1.07%, p=0.50, respectively). Conclusion: Our findings could support hypotheses for adhesion-related RBC clumping among AAs with SCT during systemic treatment with anti-neoplastic agents, and, putatively, resultant AEs. Given past studies showing African-American women with breast cancer have higher rates of self-withdrawal from treatment, further exploration with additional systemic agents are warranted. Our novel study is limited by a small sample size as well lack of potential confounding factors in analyses. Future studies are planned with additional agent(s) and AAs without SCT. Should it be confirmed that SCT carrier status predicts RBC alterations during systemic treatment for breast cancer, and are linked to subsequent adverse events, it could lead to precision treatment planning. Disclosures Andemariam: Novartis:Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding;Hemanext:Membership on an entity's Board of Directors or advisory committees;Global Blood Therapeutics:Consultancy, Membership on an entity's Board of Directors or advisory committees;Vertex:Honoraria;Imara:Research Funding;Emmaus:Membership on an entity's Board of Directors or advisory committees;bluebird bio:Consultancy, Membership on an entity's Board of Directors or advisory committees;NovoNordisk:Consultancy, Membership on an entity's Board of Directors or advisory committees;CRISPR/Vertex:Consultancy, Membership on an entity's Board of Directors or advisory committees;CHNCT:Consultancy;Accordant:Membership on an entity's Board of Directors or advisory committees;Guidepoint:Honoraria;Sanofi Genzyme:Consultancy, Membership on an entity's Board of Directors or advisory committees;Terumo BCT:Consultancy, Membership on an entity's Board of Directors or advisory committees;Roche:Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees;Cyclerion:Consultancy, Membership on an entity's Board of Directors or advisory committees;Pfizer:Consultancy, Membership on an entity's Board of Directors or advisory committees.

FT-4202, an Allosteric Activator of Pyruvate Kinase-R, Demonstrates Proof of Mechanism and Proof of Concept after a Single Dose and after Multiple Daily Doses in a Phase 1 Study of Patients with Sickle Cell Disease

FT-4202, an Allosteric Activator of Pyruvate Kinase-R, Demonstrates Proof of Mechanism and Proof of Concept after a Single Dose and after Multiple Daily Doses in a Phase 1 Study of Patients with Sickle Cell Disease

An Adaptive, Randomized, Placebo-Controlled, Double-Blind, Multi-Center Study of Oral FT-4202, a Pyruvate Kinase Activator in Patients with Sickle Cell Disease (PRAISE)

An Adaptive, Randomized, Placebo-Controlled, Double-Blind, Multi-Center Study of Oral FT-4202, a Pyruvate Kinase Activator in Patients with Sickle Cell Disease (PRAISE)

Assessment of Red Blood Cell-Mediated Microvascular Occlusion in Sickle Cell Disease By a Novel Electrical Impedance-Based Microfluidic Device

Introduction: Sickle cell disease (SCD) is a recessive genetic disorder caused by the mutated sickle hemoglobin (HbS) in red blood cells (RBCs). HbS polymerizes in a hypoxic environment, which leads to increased adhesiveness and decreased deformability of RBCs, and ultimately contributes to microvascular occlusion in SCD. As RBC deformability and the associated microvascular occlusion are predictors of individual microcirculatory health, previous studies have developed microfluidic devices for deformability-based RBC sorting in microscale flow, albeit with relatively low throughput. An easy-to-use, point-of-care assay to rapidly assess RBC-mediated microvascular occlusion can be clinically useful in examining the outcome of novel targeted and curative therapies, such as anti-sickling drugs and gene therapies, for patients with SCD. Here, we present an electrical impedance-based microfluidic device and functional assessment of RBC-mediated microvascular occlusion in SCD. Methods: Venous blood samples were collected in EDTA from subjects with homozygous SCD (HbSS, N=12) and controls (HbAA, N=5) under consent in an IRB-approved protocol. Microfluidic devices were fabricated using standard photolithography and polydimethylsiloxane (PDMS) micro-molding protocols. The microchannel consisted of micropillar arrays forming microcapillaries from 3-12 μm, with each array coupled with a pair of gold electrodes on the channel bottom surface (Figure 1A). Two 40-μm-wide side passageways mimicking the anastomoses in the capillary bed were designed to prevent microchannel upstream clogging (Figure 1A inset). A macroscopic view of the device is shown in Figure 1B. The 12-μm array was designed to filter large-cell aggregates and was excluded from our analysis. An impedance analyzer coupled with a custom printed-circuit board was used to record the electrical impedance at a spot frequency of 10 kHz. Prior to the experiments, the microchannel was blocked and rinsed to prevent non-specific adhesion of blood cells. Thereafter, the initial electrical impedance reading of each array was obtained. RBCs suspended at 20% hematocrit in PBS were then perfused through the microchannel under the same inlet pressure for 20 min. Next, the microchannel was washed and a second electrical impedance reading was obtained. The microchannel was then imaged under an inverted microscope, and occlusions of each array were manually quantified. The electrical impedance and occlusion results are reported as percent changes. Data are reported as mean ± standard deviation (SD). Pearson's correlation coefficient (PCC) was used to derive correlation statistics. Results: We observed increased microcapillary occlusion caused by HbSS- vs. HbAA-containing RBCs (Figure 1C, mean microcapillary occlusion percentage ± SD (%) = 24.33 ± 16.88 vs. 5.01 ± 1.25 for 3-μm array, 6.05 ± 4.09 vs. 2.19 ± 0.59 for 4-μm array, 2.77 ± 2.59 vs. 0.82 ± 0.82 for 6-μm array, 1.08 ± 2.28 vs. 0 ± 0 for 8-μm array, and 0.42 ± 1.14 vs. 0 ± 0 for 10-μm array). Similarly, we observed elevated electrical impedance change induced by HbSS- vs. HbAA-containing RBCs (Figure 1D, mean electrical impedance change ± SD (%) = 12.03 ± 8.97 vs. 2.44 ± 0.84 for 3-μm array, 1.79 ± 1.65 vs. 0.91 ± 0.42 for 4-μm array, 0.88 ± 1.14 vs. 0.58 ± 0.67 for 6-μm array, 0.16 ± 0.31 vs. 0.32 ± 0.37 for 8-μm array, and 0.06 ± 0.16 vs. 0.05 ± 0.17 for 10-μm array). Moreover, we found that the electrical impedance changes of individual arrays exhibited a significant correlation to the occlusion percentage within the corresponding arrays (Figure 1E, PCC = 0.9817, N = 85, p &amp;lt; 0.001). Conclusions: Findings suggest that a novel microfluidic platform integrated with micropillar arrays and electrical impedance readout can be used for standardized in vitro functional assessment of RBC-mediated microvascular occlusion in SCD. Electrical impedance change due to RBC-mediated microcapillary occlusion may serve as a new parameter for monitoring RBC health and function without the need for high-resolution microscopic imaging. RBC mediated microcapillary occlusion may serve as a new parameter to assess the clinical efficacy of treatments that improve RBC deformability and rheology, such as hemoglobin modifying drugs, anti-sickling agents, and therapies with curative intent. Disclosures Maji: Xatek Inc.: Patents &amp; Royalties. An:Hemex Health, Inc.: Patents &amp; Royalties. Ahuja:Genentech: Consultancy, Honoraria; Sanofi Genzyme: Consultancy, Honoraria; XaTek, Inc.: Consultancy, Patents &amp; Royalties, Research Funding. Little:BioChip Labs: Patents &amp; Royalties: SCD Biochip (patent, no royalties); Hemex Health, Inc.: Patents &amp; Royalties: Microfluidic electropheresis (patent, no royalties); Bluebird Bio: Research Funding; GBT: Research Funding; GBT: Membership on an entity's Board of Directors or advisory committees; NHLBI: Research Funding. Mohseni:Xatek Inc.: Consultancy, Patents &amp; Royalties, Research Funding. Suster:Xatek Inc.: Consultancy, Patents &amp; Royalties, Research Funding. Gurkan:BioChip Labs: Patents &amp; Royalties; Xatek Inc.: Patents &amp; Royalties; Dx Now Inc.: Patents &amp; Royalties; Hemex Health, Inc.: Consultancy, Current Employment, Patents &amp; Royalties, Research Funding.

Persistance of Inflammatory Phenotype of Neutrophils in Sickle Cell Disease Children Despite Chronic Exchange Transfusion Program: A Cause of the Progression of Cerebral Vasculopathy?

Sickle cell disease (SCD) is a severe hemoglobinopathy due to the production of abnormal hemoglobin S (HbS). Although red blood cell (RBC) dysfunction is the major contributor to disease, several studies highlighted the important role of polymorphonuclear neutrophils (PMNs), both during acute and chronic complications. One of the most severe complication of SCD is ischemic stroke due to large cerebral artery occlusion. In 1998, the Stroke Prevention (STOP) trial demonstrated that monthly blood transfusions could reduce the risk of stroke by 90% in SCD children with cerebral vasculopathy (CV). However, there is a wide heterogeneity in the course of CV in patients receiving chronic transfusions, since only about half of them improved their CV under transfusion program, while 25% are only stabilized and 29% continue to get worse despite a percentage of HbS permanently below 30%. The aim of our study is to investigate the impact of transfusion programs on neutrophils activation and ageing, in order to identify if inflammation could contribute to the persistence of SCD complications despite red cell transfusion. We performed a prospective study including 58 homozygous SCD children and 10 healthy donors. Of these, 12 had no specific treatment, 11 were on Hydroxyurea (HU) treatment, 21 were on an exchange transfusion program, and 14 were on both an exchange transfusion program and HU treatment for an average of 4.9 years due to persistent CV. Monthly exchange transfusion are carried out either by erythrapheresis or by manual exchanges, consisting of the continuous bleeding of whole blood compensated by simultaneous transfusion of packed red blood cells. Neutrophils were isolated from fresh blood samples before exchange transfusion session and labelled with 8 markers specific of adhesion, activation and ageing. We quantified by flow cytometry the expression of 3 integrins (CD18, CD11a, CD11b), 3 ageing markers (CD182, CD184, CD62L) and 2 adhesion molecules (CD162 and CD66a). We also measured the plasmatic level of elastase, which reflects the NETose activity of PMNs As previously reported, we observed a high leukocytosis and an activated profile of PMNs in the 12 non-transfused SCD patients compared to healthy controls (Figure 1), characterized by an overexpression of the integrin CD18/CD11b (p=0,03) and CD18/CD11a (p=0,02), a higher level of circulating aged PMNs CD184 high/CD62Llow (p=0,04), a higher expression of CD162 (p=0,01) and CD66a (0,01) as well as a higher plasmatic level of elastase (p=0.01). Interestingly, in the PMNs of the 21 patients receiving monthly exchange transfusion, we found an identical expression pattern of integrins, selectins, ageing markers and elastase level compared to those of the PMNs from non-transfused patients. Furthermore, we also observed a persistence of high neutrophilic leukocytosis. This activation pattern was the same for patients on manual exchange or erythrapheresis, even with a tendency towards a more inflammatory profile in patients on erythrapheresis (Figure 1). In the PMNs from the 11 patients receiving HU compared to untreated SCD patients, we found an expected decrease in high leukocytosis and membrane integrin expression CD18/CD11b and CD18/CD11a. The addition of HU therapy in 14 patients in exchange transfusion program allows to alleviate neutrophilic leukocytosis and membrane integrin expression. Our study shows for the first time that replacing sickle RBCs with healthy RBCs is not sufficient to reverse the pathological phenotype of PMNs in SCD. A persistence of the PMNs activation pattern is observed both despite erythrapheresis, where plasma and white blood cells go back to the patient, and in manual exchanges, where the patient is bled from a large volume of whole blood. Given the major role of inflammation in endothelial damage and vasculopathy in SCD, our data could explain the incomplete efficacy of transfusion exchange programs to treat CV. This raises the question to systematically combine anti-inflammatory and anti-white blood cell adhesion treatments such as Hydroxyurea or P-Selectin inhibitors for these patients. Disclosures No relevant conflicts of interest to declare.

Anti-Inflammatory Activities of Sulfated Non-Anticoagulant Heparin Derivative Beyond Its Anti-Sickling and Anti-Selectin for the Effective Management of Sickle Cell Disease

Sickle cell disease (SCD) is an autosomal recessive genetic disease caused by the inheritance of a single point mutation, resulting in abnormal sickle hemoglobin (HbS). During hypoxia or dehydration, HbS polymerizes to form insoluble aggregates and induces sickling of red blood cells (RBCs). RBC sickling increases the adhesiveness of RBCs to alter the rheological properties of the blood and trigger inflammatory responses, leading to hemolysis, vasoocclusive crisis, pulmonary complications, plethora, and other pathological sequelae. Glycosaminoglycans, such as low molecular weight heparin (LMWH), have been suggested as treatments to relieve coagulation complications in SCD because of their ability to decrease thrombin generation and sickle cell adhesion. However, they are associated with bleeding complications after repeated dosing. An alternative, sulfated non-anticoagulant LMWH derivative (S-NACH) was previously reported to have none to low systemic anticoagulant activity and no bleeding side effects, and it interfered with P-selectin-dependent binding of sickle cells to endothelial cells, with concomitant decrease in the levels of adhesion biomarkers in SCD mice (1,2). S-NACH has been further engineered to possess an aldehyde moiety, which confers anti-sickling properties primarily due to specific interactions with HbS to increase its affinity for oxygen. Our in vitro sickling assay under hypoxic conditions using S-NACH at 0.5 - 2 mM demonstrated that S-NACH significantly reduced the sickling of SS cells and in a concentration-dependent manner, with comparable to that of 1 mM GBT440 (Figure 1 A-C). A similar concentration dependent effect on increasing HbS affinity for oxygen using oxygen equilibrium study was documented. In an vivo animal model using Townes' SCD animals plasma levels of pro-inflammatory cytokines IL-1β, IL-6, IFN-γ, MCP-1, TNF-α, M-CSF, and VEGF were increased in SCD untreated samples in contrast to a significant decrease (*P&amp;lt; 0.001) in S-NACH-treated animals, at both 2 and 6 h. In addition, S-NACH was able to increase the decreased levels of the endogenous anti-inflammatory IL-10 (Figure 1 D). The above set of novel findings about S-NACH established it to be effective for the management of SCD via the modulation of thromboinflammatory pathways, involved in thromboembolism and end organ damage, beside anti-sickling, anti-selectin and without causing any bleeding risk. Reference 1. Alshaiban A, Muralidharan-Chari V, Nepo A, Mousa SA. Modulation of Sickle Red Blood Cell Adhesion and its Associated Changes in Biomarkers by Sulfated Non-anticoagulant Heparin Derivative. Clin Appl Thromb Hemost. 2016 ;22(3):230-8. 2. Mousa SA: Compositions and method for anti-sickling of red blood cells in sickle cell disease. US Patent 9,822,190, November 2017. Disclosures Mousa: Vascular Vision Pharma Co.: Patents &amp; Royalties.

Thrombin-Induced Endothelial Cell Damage Is Mitigated By Human Anti-Thrombin III in a Microfluidic Device

Introduction Sickle cell disease (SCD) is a pleiotropic disease that results in a hypercoagulable state due to the depletion of natural anticoagulants and abnormal activation of the coagulation cascade. Vascular inflammation is a characteristic of SCD and can lead to endothelial cell activation and abnormally enhanced adhesion of hematopoietic cells. These adhesive events can lead to vaso-occlusive crises and are thus a target of pharmaceutical exploration. The complex pathophysiology of SCD suggests that there are many potential therapeutic targets, particularly during activation of the coagulation cascade. Presently, we describe the use of a microfluidic device to investigate the effectiveness of anti-thrombin III (AT-III) in mitigating thrombin-mediated effects on and red blood cell adhesion (RBC) to endothelial cells. Our microfluidic device, endothelium-on-a-chip (Fig. 1A), incorporates microchannels functionalized with endothelial cells (ECs), which allows for investigation of SCD pathophysiology under physiological shear stress. Here, we examine the extent to which thrombin causes cellular contraction in human umbilical vein endothelial cells (HUVECs) and the capacity of AT-III to mitigate this contraction. Similarly, we quantify the adhesion of sickle RBCs to thrombin-activated HUVECs and the adhesion to HUVECs pre-treated with AT-III. Methods Microchannels seeded with human umbilical vein endothelial cells (HUVECs) were cultured to confluency under flow for 48 - 72 hrs at 37 °C in a 5% CO2 atmosphere. Culture media containing 10% fetal bovine serum (FBS) was perfused through the microchannels at a shear stress of 1 dyne/cm2 which corresponds to a typical value seen in human post-capillary venules. Approximately 18 hours prior to the start of the experiment, perfused media was exchanged for ITS+1 media in order to condition cells to a low-serum environment and to avoid neutralization of thrombin by residual serum factors. At the start of each experiment, devices were disconnected from the dynamic flow culturing system and incubated with experimental reagents under static conditions. Endothelialized microchannels were treated with one of three conditions: Control channels were incubated with ITS+1 media for 5 hrs; Thrombin channels were incubated with ITS+1 media for 1 hr, followed by 5 IU/mL human alpha thrombin for 4 hrs; Anti-thrombin III channels were incubated with 7 IU/mL human anti-thrombin III (AT-III) for 1 hr, followed by 5 IU/mL human alpha thrombin for 4 hrs. AT-III was donated by Grifols USA (Los Angeles, California). Following each experiment, the endothelialized microchannels were fixed with 3.7% formalin. Microchannels were then imaged and confluency was measured using the FIJI plug-in PHANTAST with default settings. For adhesion experiments, whole blood samples were collected from 6 patients with homozygous SCD (HbSS). RBCs were isolated and suspended at 20% hematocrit in basal media, then perfused through treated, endothelialized microchannels. Microchannels were then imaged and adherent RBCs were quantified. Results The results of contraction and adhesion experiments are shown in Figures 1B&amp;C, respectively. Control channels had an average confluency of 97.9% ± 1.7 (Fig. 1B&amp;D), while thrombin-treated channels resulted in HUVEC contraction causing an average confluency of 63.7% ± 8.9 (Fig. 1B&amp;E). Microchannels treated with AT-III prior to thrombin incubation had an average confluency of 92.5% ± 5.3 (Fig. 1B&amp;F). Adhesion of sickled RBCs followed a similar trend: control channels had an average of 16.0 ± 22.7 RBCs per analyzed region (Fig. 1C&amp;G), thrombin channels had an average of 222.8 ± 86.7 RBCs per analyzed region (Fig. 1C&amp;H), and AT-III channels had an average of 19.6 ± 12.3 RBCs per analyzed region (Fig. 1C&amp;I). Discussion We observed that thrombin treatment resulted in significant endothelial cell contraction. This thrombin induced cell contraction could be mitigated by pre-treating the microchannels with AT-III. Similarly, RBC adhesion significantly increased in thrombin-treated microchannels, and could be similarly mitigated by pre-treatment of the endothelium with AT-III. Taken together, our in vitro model suggests that AT-III may be worthy of investigation as a treatment for SCD associated thromboinflammation and vaso-occlusive crises. Disclosures An: Hemex Health, Inc.: Patents &amp; Royalties. Little:NHLBI: Research Funding; GBT: Membership on an entity's Board of Directors or advisory committees; GBT: Research Funding; Bluebird Bio: Research Funding; BioChip Labs: Patents &amp; Royalties: SCD Biochip (patent, no royalties); Hemex Health, Inc.: Patents &amp; Royalties: Microfluidic electropheresis (patent, no royalties). Key:Novo Nordisk: Other: Chair of Grants Committee; Takeda: Research Funding; Grifols: Research Funding; Uniqure: Consultancy. Gurkan:Hemex Health, Inc.: Consultancy, Current Employment, Patents &amp; Royalties, Research Funding; BioChip Labs: Patents &amp; Royalties; Dx Now Inc.: Patents &amp; Royalties; Xatek Inc.: Patents &amp; Royalties.

Induced Stress on Red Blood Cell Promotes Red Blood Cell-Endothelial Adhesion

Besides disease condition, very few stress stimulants were determined to provoke red blood cell (RBC) adhesion to endothelial cells (EC). However, the possible role of other stress factors which disrupt RBCs anti-adhesive property is still unknown. To resolve this, we studied in vitro static adherence of RBC-EC after RBC exposure to physical (osmotic and shear stress) and chemical stress stimulants (cholesterol depletion and nitric oxide modulation). In support with earlier studies, RBC under hypertonic shock demonstrated a significant increase in RBC-EC adherence as a result of prominent structural modification. Besides, our study shows that shear stress, cholesterol depletion and nitric oxide inhibition in RBC increases RBC-EC adhesiveness which elucidates cholesterol, nitric oxide and shear stress importance in preventing RBC-EC adhesion. Thus, present study shows that assessment of RBC-EC interaction after exerting to external stress is critical in understanding pathophysiological conditions.

Investigation of the potency of leukodepletion filter membranes immobilized with bovine serum albumin via polydopamine spacer

Conjugation of biomolecules over membrane surfaces intended for blood contacting applications has been proven worthwhile for the improvement of their hemocompatibility. In conjunction with this factuality, this manuscript for the first time unveils the feasibility of bovine serum albumin (BSA) immobilization via polydopamine precursors on electrospun poly(ethylene-co-vinyl alcohol) (EVAL) leukodepletion filter membranes. The successful immobilization of different extents of BSA on EVAL membranes was endorsed by the Attenuated Total Reflectance—Fourier Transform Infrared (ATR-FTIR) Spectrum, variations in the wettability, electron microscopic images revealing the alterations in the fiber diameter and porousness of membranes. An interesting backlash was encountered in the in vitro plasma protein adsorption and in vitro platelet adhesion on the BSA-immobilized membranes as they exhibited a higher protein adsorption and lower platelet adhesion in comparison with neat EVAL. The infiltration of immobilized BSA to the leukodepletion capacity of those membranes was preliminarily studied by in vitro blood-material interactions followed by typical whole blood filtration of selected membranes. The data presented highlight that BSA moieties do not particularly affect the white blood cell (WBC) adhesion and platelet adhesion of EVAL membranes whereas resulted in a significantly higher red blood cell (RBC) recovery and decreased hemolysis under in vitro conditions. Accordingly, BSA-immobilized EVAL membranes were made to asymmetric leukodepletion filters; however, their further evaluation by whole blood filtration ensued that immobilization of BSA could not particularly enhance the overall whole blood leukodepletion efficacy of EVAL membranes.

Effect of the mechanical properties of carbon-based coatings on the mechanics of cell–material interactions

The paper presents an influence of the surface mechanical properties of thin-film materials on blood cell adhesion under shear stress conditions. Physical vapour deposited (PVD) coatings i.e. hydrogenated amorphous carbon (a-C:H) doped with nitrogen or silicon have been investigated. The mechanical properties of materials, namely their microhardness and Young’s modulus were measured using indentation test with Rockwell indenter. The adhesion efficiency of blood cells in dynamic conditions were analysed using a radial flow chamber. Red blood cells (RBC) were used as representative cells to analyse cell–material interactions. The biomaterial examinations were performed under physiological flow conditions at the single-cell level. The 3D FVM (finite volume method) model of multi-phase radial flow test was developed to reproduce the physical test and to predict distributions of shear stresses and velocity during blood washout with PBS. Cell–material interactions were found to be strongly associated with the mechanical properties of the thin-film material. The decrease in the hardness of the coatings translated into a weaker cell – material interactions.