Tandem P-selectin glycoprotein ligand immunoglobulin prevents lung vaso-occlusion in sickle cell disease mice
2020; Elsevier BV; Volume: 84; Linguagem: Inglês
10.1016/j.exphem.2020.03.002
ISSN1873-2399
AutoresRavi Vats, Egemen Tütüncüoğlu, Tirthadipa Pradhan‐Sundd, Jesús Tejero, Gray D. Shaw, Prithu Sundd,
Tópico(s)Blood disorders and treatments
Resumo•Intravenous administration of hemoglobin triggers lung vaso-occlusion in SCD mice.•Hemoglobin triggers sequestration of neutrophil–platelet aggregates in lung arterioles.•TSGL-Ig prevents lung vaso-occlusion in SCD mice.•Safety and efficacy of TSGL-Ig should be evaluated in SCD patients. Sickle cell disease (SCD) is a monogenic disorder estimated to affect more than three million people worldwide. Acute systemic painful vaso-occlusive episode (VOE) is the primary reason for emergency medical care among SCD patients. VOE may also progress to acute chest syndrome (ACS), a type of acute lung injury and one of the primary reasons for mortality among SCD patients. Recently, P-selectin monoclonal antibodies were found to attenuate VOE in SCD patients and lung vaso-occlusion in transgenic humanized SCD mice, highlighting the therapeutic benefit of P-selectin inhibition in SCD. Here, we use quantitative fluorescence intravital lung microscopy (qFILM) to illustrate that tandem P-selectin–glycoprotein ligand–immunoglobulin (TSGL-Ig) fusion molecule containing four P-selectin binding sites, significantly attenuated intravenous (IV) oxyhemoglobin triggered lung vaso-occlusion in SCD mice. These findings highlight the therapeutic potential of TSGL-Ig in preventing VOE and ACS in SCD. Sickle cell disease (SCD) is a monogenic disorder estimated to affect more than three million people worldwide. Acute systemic painful vaso-occlusive episode (VOE) is the primary reason for emergency medical care among SCD patients. VOE may also progress to acute chest syndrome (ACS), a type of acute lung injury and one of the primary reasons for mortality among SCD patients. Recently, P-selectin monoclonal antibodies were found to attenuate VOE in SCD patients and lung vaso-occlusion in transgenic humanized SCD mice, highlighting the therapeutic benefit of P-selectin inhibition in SCD. Here, we use quantitative fluorescence intravital lung microscopy (qFILM) to illustrate that tandem P-selectin–glycoprotein ligand–immunoglobulin (TSGL-Ig) fusion molecule containing four P-selectin binding sites, significantly attenuated intravenous (IV) oxyhemoglobin triggered lung vaso-occlusion in SCD mice. These findings highlight the therapeutic potential of TSGL-Ig in preventing VOE and ACS in SCD. Sickle cell anemia, the most common form of sickle cell disease (SCD), is caused by a homozygous mutation in the β-globin gene, which leads to erythrocyte sickling, impaired rheology, vaso-occlusion, and premature hemolysis [1Sundd P Gladwin MT Novelli EM Pathophysiology of sickle cell disease.Annu Rev Pathol. 2019; 14: 263-292Crossref PubMed Scopus (144) Google Scholar]. Vaso-occlusion and hemolysis are the two predominant pathophysiologic events in SCD that contribute to chronic organ damage and acute systemic painful vaso-occlusive episode (VOE) [1Sundd P Gladwin MT Novelli EM Pathophysiology of sickle cell disease.Annu Rev Pathol. 2019; 14: 263-292Crossref PubMed Scopus (144) Google Scholar,2Rees DC Williams TN Gladwin MT Sickle-cell disease.Lancet. 2010; 376: 2018-2031Abstract Full Text Full Text PDF PubMed Scopus (1182) Google Scholar]. Earlier, we reported that VOE involves entrapment of large neutrophil–platelet aggregates in lung arterioles of SCD mice, which is inhibited after administration of P-selectin function blocking antibody intravenously (IV) [3Bennewitz MF Jimenez MA Vats R et al.Lung vaso-occlusion in sickle cell disease mediated by arteriolar neutrophil-platelet microemboli.JCI Insight. 2017; 2: e89761Crossref PubMed Scopus (76) Google Scholar]. These findings were supported by the SUSTAIN clinical trial, which reported significant reduction in the frequency of VOE in SCD patients administered the humanized P-selectin monoclonal antibody crizanlizumab IV [4Ataga KI Kutlar A Kanter J et al.Crizanlizumab for the prevention of pain crises in sickle cell disease.N Engl J Med. 2017; 376: 429-439Crossref PubMed Scopus (339) Google Scholar]. P-Selectin–glycoprotein ligand 1 (PSGL-1), constitutively expressed on neutrophils, binds to P-selectin on activated platelets and both P- and E-selectin on activated endothelial cells to promote neutrophil–platelet aggregation and neutrophil adhesive rolling along vascular endothelium, respectively [4Ataga KI Kutlar A Kanter J et al.Crizanlizumab for the prevention of pain crises in sickle cell disease.N Engl J Med. 2017; 376: 429-439Crossref PubMed Scopus (339) Google Scholar, 5Sundd P Gutierrez E Koltsova EK et al.'Slings' enable neutrophil rolling at high shear.Nature. 2012; 488: 399-403Crossref PubMed Scopus (127) Google Scholar, 6Koltsova EK Sundd P Zarpellon A et al.Genetic deletion of platelet glycoprotein Ib alpha but not its extracellular domain protects from atherosclerosis.Thromb Haemost. 2014; 112: 1252-1263Crossref PubMed Scopus (18) Google Scholar]. Previously, a soluble form of recombinant PSGL-1 was reported to prevent ischemia–reperfusion injury of liver allografts in both mice and humans by competitively inhibiting E-/P-selectin-PSGL-1-dependent neutrophil–endothelium adhesion without increasing the risk of bleeding [7Dulkanchainun TS Goss JA Imagawa DK et al.Reduction of hepatic ischemia/reperfusion injury by a soluble P-selectin glycoprotein ligand-1.Ann Surg. 1998; 227: 832-840Crossref PubMed Scopus (110) Google Scholar, 8Amersi F Farmer DG Shaw GD et al.P-selectin glycoprotein ligand-1 (rPSGL-Ig)-mediated blockade of CD62 selectin molecules protects rat steatotic liver grafts from ischemia/reperfusion injury.Am J Transplant. 2002; 2: 600-608Crossref PubMed Scopus (63) Google Scholar, 9Busuttil RW Lipshutz GS Kupiec-Weglinski JW et al.rPSGL-Ig for improvement of early liver allograft function: a double-blind, placebo-controlled, single-center phase II study.Am J Transplant. 2011; 11: 786-797Crossref PubMed Scopus (53) Google Scholar]. Taken together, these findings suggest that biomolecules mimicking the binding domain of PSGL-1 can be therapeutically beneficial in preventing vaso-occlusion in SCD. TSGL-Ig is a recombinant fusion protein that carries two P-selectin sulfated glycopeptide-binding domains in a tandem configuration on a single polypeptide chain [10Zhang C Zhang Y Liu Y et al.A soluble form of P selectin glycoprotein ligand 1 requires signaling by nuclear factor erythroid 2-related factor 2 to protect liver transplant endothelial cells against ischemia-reperfusion injury.Am J Transplant. 2017; 17: 1462-1475Crossref PubMed Scopus (12) Google Scholar]. Two such polypeptide chains are fused to an inactivated Fc domain of human IgG1, resulting in a dimer with four P-selectin binding sites per molecule of TSGL-Ig (Supplementary Figure E1, online only, available at www.exphem.org) [10Zhang C Zhang Y Liu Y et al.A soluble form of P selectin glycoprotein ligand 1 requires signaling by nuclear factor erythroid 2-related factor 2 to protect liver transplant endothelial cells against ischemia-reperfusion injury.Am J Transplant. 2017; 17: 1462-1475Crossref PubMed Scopus (12) Google Scholar]. Recently, TSGL-Ig was reported to prevent ischemia–reperfusion injury of orthotopic liver transplants in mice by attenuating leukocyte sequestration in the liver microcirculation [10Zhang C Zhang Y Liu Y et al.A soluble form of P selectin glycoprotein ligand 1 requires signaling by nuclear factor erythroid 2-related factor 2 to protect liver transplant endothelial cells against ischemia-reperfusion injury.Am J Transplant. 2017; 17: 1462-1475Crossref PubMed Scopus (12) Google Scholar]. The current study assesses the therapeutic efficacy of TSGL-Ig in preventing lung vaso-occlusion in SCD mice. Oxyhemoglobin (oxy-Hb) released during hemolysis is an erythrocyte-derived damage-associated molecular pattern (eDAMP) molecule, which promotes vaso-occlusion by causing endothelial dysfunction, nitric oxide depletion, oxidative stress, and sterile inflammation, leading to activation of leukocytes, platelets, and vascular endothelium [1Sundd P Gladwin MT Novelli EM Pathophysiology of sickle cell disease.Annu Rev Pathol. 2019; 14: 263-292Crossref PubMed Scopus (144) Google Scholar]. Therefore, a nonlethal dose of intravenous oxy-Hb (10 μmol/kg) was established after multiple titrations and administered IV to SCD mice to trigger VOE. On the basis of previous studies [11Raat NJ Tabima DM Specht PA et al.Direct sGC activation bypasses NO scavenging reactions of intravascular free oxy-hemoglobin and limits vasoconstriction.Antioxid Redox Signal. 2013; 19: 2232-2243Crossref PubMed Scopus (22) Google Scholar], intravenous VetStarch was used as a negative control (vehicle) to account for the changes in volume and viscosity caused by intravenous oxy-Hb administration. As illustrated in the experimental scheme (Figure 1A), Townes-SS (SCD) mice were administered IV with 10 μmol/kg oxy-Hb or 80 μL VetStarch or 10 μmol/kg oxy-Hb with 100 μg/mouse of TSGL-Ig or 10 μmol/kg oxy-Hb with 100 µg/mouse of recombinant human IgG1-Fc (rh IgG1-Fc). Two hours later, mice were IV administered fluorescein isothiocyanate (FITC)–dextran, AlexaFluor546 (AF546)-conjugated anti-mouse Ly6G mAb and Violet450 (V450)-conjugated anti-mouse CD49b mAb for visualization of blood vessels and in vivo staining of neutrophils and platelets, respectively. Lung microcirculation in live mice was assessed using the qFILM imaging approach described previously in detail [3Bennewitz MF Jimenez MA Vats R et al.Lung vaso-occlusion in sickle cell disease mediated by arteriolar neutrophil-platelet microemboli.JCI Insight. 2017; 2: e89761Crossref PubMed Scopus (76) Google Scholar,12Bennewitz MF Watkins SC Sundd P Quantitative intravital two-photon excitation microscopy reveals absence of pulmonary vaso-occlusion in unchallenged sickle cell disease mice.Intravital. 2014; 3: e29748Crossref PubMed Scopus (21) Google Scholar]. Refer to Methods for experimental details. TSGL-Ig did not result in excessive bleeding during the qFILM imaging in SCD mice. Intravenous oxy-Hb-triggered lung vaso-occlusion in SCD mice, which involved occlusion of pulmonary arteriole bottlenecks (junction of pulmonary arterioles and capillaries) by neutrophil–platelet aggregates (representative field of view [FOV] shown in Figure 1B and Supplementary Video E1 [online only, available at www.exphem.org]). Intravenous oxy-Hb did not promote vaso-occlusion in control mice (data not shown). Unlike intravenous oxy-Hb, intravenous administration of VetStarch did not promote lung vaso-occlusion in SCD mice (representative FOV shown in Figure 1C and Supplementary Video E2 [online only, available at www.exphem.org]). The majority of FOVs in the lungs of SCD mice administered both oxy-Hb and TSGL-Ig IV were free of vaso-occlusion (representative FOV shown in Figure 1D and Supplementary Video E3 [online only, available at www.exphem.org]), while intravenous administration of control rh IgG1-Fc had no effect of Oxy-Hb + rh IgG1-Fc on lung vaso-occlusion in SCD mice. (representative FOV shown in Figure 1E and Supplementary Video E4 [online only, available at www.exphem.org]). Figure 1E and Supplementary Video E4 show a neutrophil–platelet aggregate occluding the pulmonary arteriole bottleneck in SCD mice administered both oxy-Hb and rh IgG1-Fc IV. Next, pulmonary vaso-occlusions were analyzed in 12 to15 FOVs (FOV size ∼65,536 µm2) per mouse with three or four mice in each treatment group and quantified based on three parameters: (1) average number of pulmonary vaso-occlusions per FOV; (2) percentage of total FOVs with pulmonary vaso-occlusions; and (3) average number of large pulmonary vaso-occlusions (size >1000 µm2) per FOV. This analysis approach has been described previously [3Bennewitz MF Jimenez MA Vats R et al.Lung vaso-occlusion in sickle cell disease mediated by arteriolar neutrophil-platelet microemboli.JCI Insight. 2017; 2: e89761Crossref PubMed Scopus (76) Google Scholar,13Bennewitz MF Tutuncuoglu E Gudapati S et al.P-Selectin-deficient mice to study pathophysiology of sickle cell disease.Blood Adv. 2020; 4: 266-273Crossref PubMed Scopus (16) Google Scholar,14Vats R Brzoska T Bennewitz MF et al.Platelet extracellular vesicles drive inflammasome-IL-1beta-dependent lung injury in sickle cell disease.Am J Respir Crit Care Med. 2020; 201: 33-46Crossref PubMed Scopus (43) Google Scholar], and the same details are also included under Methods. Based on our previous studies[3,13,14], vaso-occlusions covering an area >1000 µm2 in the two-dimensional qFILM images were referred to as large pulmonary vaso-occlusions. Large vaso-occlusions are known to be present in the pulmonary arterioles of ACS patients [15Anea CB Lyon M Lee IA et al.Pulmonary platelet thrombi and vascular pathology in acute chest syndrome in patients with sickle cell disease.Am J Hematol. 2016; 91: 173-178Crossref PubMed Scopus (23) Google Scholar,16Mekontso Dessap A Deux JF Abidi N et al.Pulmonary artery thrombosis during acute chest syndrome in sickle cell disease.Am J Respir Crit Care Med. 2011; 184: 1022-1029Crossref PubMed Scopus (80) Google Scholar]. After treatment with oxy-Hb IV, SCD mice manifested an average of at least one pulmonary vaso-occlusion per FOV (black bar in Figure 2A), at least one pulmonary vaso-occlusion in 80% of all the FOVs examined (black bar in Figure 2B), and an average of 0.4 large pulmonary vaso-occlusion per FOV (black bar in Figure 2C, i.e., every third FOV in SCD mice had a large pulmonary vaso-occlusion). Although TSGL-Ig treatment led to significant reduction in all three parameters (Figure 2A-C), the largest reduction was observed in the number of large vaso-occlusions (Figure 2C). Average number of pulmonary vaso-occlusions per FOV was reduced by 40% (gray bar in Figure 2A), percentage of FOVs with pulmonary vaso-occlusion was reduced by 33% (gray bar in Figure 2B), and average number of large pulmonary vaso-occlusions per FOV was reduced by 50% (gray bar in Figure 2C) in SCD mice administered oxy-Hb + TSGL-Ig IV compared with SCD mice administered only oxy-Hb IV. In contrast, the average number of pulmonary vaso-occlusions per FOV was unchanged in SCD mice administered IV oxy-Hb + rh IgG1-Fc compared with SCD mice administered oxy-Hb only IV (Figure 2D). Taken together, our data suggest that intravenous oxy-Hb triggered lung vaso-occlusion in SCD mice by promoting the occlusion of pulmonary arterioles by neutrophil–platelet aggregates. TSGL-Ig attenuated lung vaso-occlusion in SCD mice by reducing both the number and the size of neutrophil–platelet aggregates in the pulmonary arterioles. These findings highlight the need for clinical studies to evaluate the safety and efficacy of TSGL-Ig in preventing VOE and/or ACS in SCD patients. Violet 450 (V450)-conjugated rat anti-mouse CD49b mAb (clone DX5) was purchased from BD Biosciences (San Jose, CA). Alexa Fluor 546 (AF546)-conjugated rat anti-mouse Ly-6G mAb (clone 1A8) was purchased from BioLegend (San Diego, CA). Fluorescein isothiocyanate (FITC) dextran (MW 70,000) was purchased from Molecular Probes Inc. (Eugene, OR). VetStarch was purchased from Henry Schein Animal Health (Dublin, OH). Recombinant human lgG1 Fc (rh IgG1 Fc) was purchased from BioCell (West Lebanon, NH, USA). Ketamine HCl was purchased from Henry Shein Animal Health, and xylazine was purchased from LLOYD Laboratories (Shenandoah, IA). Recombinant tandem P-selectin–glycoprotein ligand–immunoglobulin (TSGL-Ig) fusion molecule was provided by Quell Pharma Inc. (Plymouth, MA). TSGL-Ig was produced in HEK293 cells after transfection with plasmids encoding human FucT-VII cDNA and TSGL-Ig, essentially as described elsewhere [17Barbaux S Poirier O Pincet F Hermand P Tiret L Deterre P The adhesion mediated by the P-selectin glycoprotein ligand-1 (PSGL-1) couple is stronger for shorter PSGL-1 variants.J Leukoc Biol. 2010; 87: 727-734Crossref PubMed Scopus (11) Google Scholar]. Secreted TSGL-Ig was purified from conditioned medium using protein A–Sepharose, and P-selectin binding kinetics were subsequently confirmed via multiple Octet binding assays. Oxyhemoglobin was prepared from expired, leukocyte-reduced red blood cell units from healthy human donors. These red blood cell units were provided by the Institute for Transfusion Medicine (ITxM, Pittsburgh, PA). Oxy-Hb purification was performed as described previously [18Geraci G Parkhurst LJ Gibson QH Preparation and properties of alpha- and beta-chains from human hemoglobin.J Biol Chem. 1969; 244: 4664-4667PubMed Google Scholar, 19Huang Z Shiva S Kim-Shapiro DB et al.Enzymatic function of hemoglobin as a nitrite reductase that produces NO under allosteric control.J Clin Invest. 2005; 115: 2099-2107Crossref PubMed Scopus (424) Google Scholar, 20Tejero J Basu S Helms C et al.Low NO concentration dependence of reductive nitrosylation reaction of hemoglobin.J Biol Chem. 2012; 287: 18262-18274Crossref PubMed Scopus (34) Google Scholar], with minor modifications. Red blood cells were washed with phosphate-buffered saline (PBS) in a centrifuge at 5000g for 10 min to obtain a packed cell pellet. Cells were lysed hypotonically by resuspension in distilled water (3:1, v/v) and mixing for 30 min at room temperature. Cell debris was removed by centrifugation at 25,000g for 30 min. The supernatant was dialyzed against PBS, and the final product stored at –80°C. Male and female (age ∼12–16 weeks) Townes SCD mice (SS, homozygous for Hbatm1(HBA)Tow, homozygous for Hbbtm2(HBG1,HBB*)Tow) were used in this study [21Wu LC Sun CW Ryan TM Pawlik KM Ren J Townes TM Correction of sickle cell disease by homologous recombination in embryonic stem cells.Blood. 2006; 108: 1183-1188Crossref PubMed Scopus (193) Google Scholar]. In Townes SCD mice, human α- and β-sickle (βS) genes are knocked into the locus where mouse α and β genes were knocked out [21Wu LC Sun CW Ryan TM Pawlik KM Ren J Townes TM Correction of sickle cell disease by homologous recombination in embryonic stem cells.Blood. 2006; 108: 1183-1188Crossref PubMed Scopus (193) Google Scholar]. Townes SS mice were bred and genotyped in-house. All animal experiments were approved by the Institutional Animal Care and Use Committee at the University of Pittsburgh. Quantitative fluorescence intravital lung microscopy (qFILM) was developed by our group to study pulmonary vaso-occlusion in SCD mice [12Bennewitz MF Watkins SC Sundd P Quantitative intravital two-photon excitation microscopy reveals absence of pulmonary vaso-occlusion in unchallenged sickle cell disease mice.Intravital. 2014; 3: e29748Crossref PubMed Scopus (21) Google Scholar]. In this study, qFILM was used to visualize the lung microcirculation of SCD mice. Details of the experimental setup of qFILM are provided elsewhere [3Bennewitz MF Jimenez MA Vats R et al.Lung vaso-occlusion in sickle cell disease mediated by arteriolar neutrophil-platelet microemboli.JCI Insight. 2017; 2: e89761Crossref PubMed Scopus (76) Google Scholar]. qFILM was performed using a Nikon A1R multiphoton excitation upright motorized microscope (Nikon Instruments, Tokyo, Japan). The two-dimensional time series of qFILM images were acquired with NIS-Elements software using a prechirped Chameleon Laser Vision (Coherent, Santa Clara, CA) emitting an excitation wavelength of 850 nm, an APO LWD 25 × water immersion objective with 1.1 NA, a high-speed resonant scanning mode capable of acquisition at 512 × 512 resolution with 2 × line averaging and bidirectional scanning (∼15 frames/s) and four GaAsP NDD detectors. The four detectors collected fluorescent light transmitted through 450/20-nm (detector 1; blue channel), 525/50-nm (detector 2; green channel), 576/26-nm (detector 3; red channel), and 685/70-nm (detector 4; far red channel) bandpass filters. In this study, we used detector 1 for V450, detector 2 for FITC, and detector 3 for AF546. The position of the microscope stage was selected in the z and x–y planes through a Nano-Drive (Mad City Labs Inc., Madison, WI) and a control pad (Prior Scientific Inc., Rockland, MA), respectively. Approximately 2–2.5 hours before qFILM, SCD mice were IV administered (via tail vein) 80 µL of VetStarch, or 10 µmol/kg oxy-Hb, or 10 µmol/kg oxy-Hb with 100 µg/mouse of TSGL-Ig or 10 µmol/kg oxy-Hb with 100 µg/mouse rh IgG1-Fc. Mice were anesthetized with an intraperitoneal injection of 100 mg/kg ketamine HCl and 20 mg/kg xylazine. Just before imaging, intravenous fluorescent dyes and mAbs against blood cell lineage markers were intravascularly administered through a carotid artery catheter to enable visualization of the lung microcirculation and in vivo staining of neutrophils and platelets. Intravenous fluorescent dyes included 75 μg/mouse FITC–dextran, 12 μg/mouse AF546-conjugated anti-mouse Ly6G mAb to stain neutrophils, and 7 μg/mouse V450-conjugated anti-mouse CD49b mAb to stain platelets. qFILM was performed on a mouse for a total of 30 min, and the presence or absence of pulmonary vaso-occlusion was assessed for 30 s in each FOV by using Nikon's NIS Elements 4.20 software. Pulmonary vaso-occlusions were assessed in ∼12–15 FOVs in each mouse and across multiple mice per test group (n = 3 or 4), as previously described [3Bennewitz MF Jimenez MA Vats R et al.Lung vaso-occlusion in sickle cell disease mediated by arteriolar neutrophil-platelet microemboli.JCI Insight. 2017; 2: e89761Crossref PubMed Scopus (76) Google Scholar,13Bennewitz MF Tutuncuoglu E Gudapati S et al.P-Selectin-deficient mice to study pathophysiology of sickle cell disease.Blood Adv. 2020; 4: 266-273Crossref PubMed Scopus (16) Google Scholar,14Vats R Brzoska T Bennewitz MF et al.Platelet extracellular vesicles drive inflammasome-IL-1beta-dependent lung injury in sickle cell disease.Am J Respir Crit Care Med. 2020; 201: 33-46Crossref PubMed Scopus (43) Google Scholar]. The time series of qFILM images were processed using an image subtraction, a median filter, a noise-reduction algorithm, and adjustment of intensity histograms as described previously using the Nikon NIS Elements 4.20 software[1,3,6,12,20]. The lung microcirculation was pseudo-colored as purple, neutrophils and platelets were red and green, respectively. Pulmonary vaso-occlusion was compared between treatment groups using the following parameters as described previously [3Bennewitz MF Jimenez MA Vats R et al.Lung vaso-occlusion in sickle cell disease mediated by arteriolar neutrophil-platelet microemboli.JCI Insight. 2017; 2: e89761Crossref PubMed Scopus (76) Google Scholar,13Bennewitz MF Tutuncuoglu E Gudapati S et al.P-Selectin-deficient mice to study pathophysiology of sickle cell disease.Blood Adv. 2020; 4: 266-273Crossref PubMed Scopus (16) Google Scholar,14Vats R Brzoska T Bennewitz MF et al.Platelet extracellular vesicles drive inflammasome-IL-1beta-dependent lung injury in sickle cell disease.Am J Respir Crit Care Med. 2020; 201: 33-46Crossref PubMed Scopus (43) Google Scholar]: average number of pulmonary vaso-occlusions per FOV, percentage of FOVs with pulmonary vaso-occlusions, and average number of pulmonary vaso-occlusions (with area >1000 µm2) per FOV. Statistical analyses were conducted using the approach described in our previous studies [3Bennewitz MF Jimenez MA Vats R et al.Lung vaso-occlusion in sickle cell disease mediated by arteriolar neutrophil-platelet microemboli.JCI Insight. 2017; 2: e89761Crossref PubMed Scopus (76) Google Scholar,13Bennewitz MF Tutuncuoglu E Gudapati S et al.P-Selectin-deficient mice to study pathophysiology of sickle cell disease.Blood Adv. 2020; 4: 266-273Crossref PubMed Scopus (16) Google Scholar,14Vats R Brzoska T Bennewitz MF et al.Platelet extracellular vesicles drive inflammasome-IL-1beta-dependent lung injury in sickle cell disease.Am J Respir Crit Care Med. 2020; 201: 33-46Crossref PubMed Scopus (43) Google Scholar]. The average number of pulmonary vaso-occlusions per FOV and the average number of large pulmonary vaso-occlusions (size >1000 µm2) per FOV were compared using the two-tailed unpaired Student t test. The percentages of FOVs with pulmonary vaso-occlusions were compared using the fourfold table analyses with χ2 statistics. Data are expressed as means ± SEM. A p value < 0.05 was used to determine statistical significance. This study was supported by National Institutes of Health (NIH)– National Heart Lung and Blood Institute Grants 1R01HL128297-01 (PS) , 1R01HL141080-01A1 (PS) , and 2RO1-HL125886 (JT) ; American Heart Association Grant 18TPA34170588 (PS) ; and funds from the Hemophilia Center of Western Pennsylvania and Vitalant (PS). RV was supported by American Heart Association Grant 19PRE34430188 . The Nikon multiphoton excitation microscope was funded by NIH Grant 1S10RR028478-01. GDS is an inventor of U.S. Patent 8,889,628 relevant to TSGL molecules and is a founder of Quell Pharma Inc. Remaining authors have declared that no conflict of interest exists. eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiIyMGViZTRlN2NhN2Y0NjE5MzFkNGQ2OWFlNmM0OWY3ZSIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjYyODg5MDg1fQ.JsjiCm_3F_X_5yXaoRKwzUrrY8gRDBWSkuGco_TC_lMvcwvEVBDzVucDCU_lY5T6btd9RuzuUlieGlWOptKxtMy77Tc6-u_P7RLbnHemLzqjJE9tymuA6BDYHkbMWvn0lUBWDTrmwm5jQE1xdBqY2iXNtfM5Na7rmLuTL9XZu9UCKwGe7GONUZbJRthz7sECvuGQYyDYM3ibnKfcJFb9VyLH3AiZmEJrylXJNM06-dqbquWLHURprqdGwfScAHcQcQQwwycdcp9OgvOyoB2stxcO4JrSI1CLsp1yzzGn1o_vG5KkylCORF-PuvhuvLD46TMvf8f3bfh_tLKhF1yX4Q Download .mp4 (0.68 MB) Help with .mp4 files Movie S1: A neutrophil-platelet aggregate (white ellipse) occluding the lung arteriole bottle-neck in an SCD mouse IV administered 10 µmole/kg oxy-Hb. Neutrophils (red), platelets (green), and microcirculation (purple). 2/3x original acquisition rate. Scale bar 20 µm. Arrow-flow direction.eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiIwZGM0M2VkYzVlZWJkYjE5ZWRkZDhiOWRkMTYwOGE3NyIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjYyODg5MDg1fQ.dZPXpYWGeyaZ-kPgm4tbDHvBdRyEJvGUc5S2RcFpt2cKhHPEjKm08SVlg5KFbGhFvJ9Oe4MIpZ67XMtXvH3KDABhN8oAg3PewnDKPBGSESKkGntT316d91mDzLhQi9bWGKD_VgbiumrDl-kifOQB9iowAz7ZK7Z6NzGQNJ8aVTROoF72bEDh4gEkm_-rVb63owkZqolM64vnV6Qs1mlXOFm4p0nVdUqA6AIuf4cIi_CjovrTR9UU-Htbfsoxj47LMccmfCzDCNlV3yKNca1CFeygfryA1BJUsUC4BTposrrwC18ZthRBPTPSjHdTZN-oJc92nT71iNCWWXFRsYxw4A Download .mp4 (0.97 MB) Help with .mp4 files Movie S2: Neutrophil-platelet aggregates absent in the lung arteriole of an SCD mouse IV administered 80 μl of vet-starch. Neutrophils (red), platelets (green), and microcirculation (purple). 2/3x original acquisition rate. Scale bar 20 µm. Arrow-flow direction.eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiIyY2RjZWYwMzQ4MDUwODg2NjM5NDNkNjJhNTJiOWRkMiIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjYyODg5MDg1fQ.jiafUpkQ7w0ncPCBWRDod3fN7TAwtLQKKJQGo6u4acLik2yZIAHtpC0LTmeIhL-Xyc5QHI55hJgHrnPQ9DAhbIxzYPQoOxC0DfJoHkl0BMGiRQoMy98GyT0ofKdoiH83zFyLT0QP6ArEqHMVtxgREoCalbXGOJJYIul9sHf83mbYdQ4tLqgU8uZoY66TecV4oAQ5WJXQVr6iu3FNVrLPfQ6CC4VOg-LCjWYX0dOr-glJcAelHB-H1RR6X3Ke4Fj5Y3dmywzcOtd0UikWrt4XMjglR5In55bFCXtk327znItI-yhsUukEQgAgBLyYN5c-iiRh0_hnzD2KA7ytihCybg Download .mp4 (0.56 MB) Help with .mp4 files Movie S3: Neutrophil-platelet aggregates absent in the lung arteriole of an SCD mouse IV administered 10 µmole/kg Oxy-Hb with 100 µg TSGL-Ig. Neutrophils (red), platelets (green), and microcirculation (purple). 2/3x original acquisition rate. Scale bar 20 µm. Arrow-flow direction.eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiIxNmJmZTIzZmVjZDEwY2RmNDE5NWJlMjk1NWU4NzAxZSIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjYyODg5MDg1fQ.eQV0GIsklvPFPvXmNetWjC3vBx5ufctCb6PC2HmNcJ59P37Gl4VAePQARCZeXU__xkm4vDt2nf0VFLgkCb8t1UBV3gQBuX45Oi910uJ_aW0I3P2-zZOTIWDJ-HjQ-zSj5fWFyrLQeyxosvDLp2WxBtfBagNoXy2WbzobsPgTVg9AS-_-l6bFQih5LFMf9VNBf8BU1gOaG9GkGFSpGCjOWrFmNrwLQcpFXuJC-ITeki8V4HE4fC1jCFRFtp7eKbPgJzt8rD5tWQqiZzf1y4vDCnXu9cZUaLVzUSClTYuUk7ei9Ytj20dJuGulKeVpz9FpeaoGcKvoamLDe1QDh10XgQ Download .mp4 (0.76 MB) Help with .mp4 files Movie S4: A neutrophil-platelet aggregate (white ellipse) occluding the lung arteriole bottle-neck in an SCD mouse IV administered 10 µmole/kg Oxy-Hb with 100 µg IgG1-Fc. Neutrophils (red), platelets (green), and microcirculation (purple). 2/3x original acquisition rate. Scale bar 20 µm. Arrow-flow direction.
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