ALBERT

Description: Acute systemic painful vaso-occlusive crisis (VOC) often serves as an antecedent to acute chest syndrome (ACS), which is a type of acute lung injury and the leading cause of mortality among sickle cell disease (SCD) patients. Thrombocytopenia secondary to pulmonary thrombosis is major risk factor for ACS, however, only 20% of ACS patients are diagnosed with pulmonary thrombosis as an underlying cause of ACS. Although clinical evidence supports the presence of prothrombotic state in subset of SCD patients, the molecular, cellular and genetic mechanisms that selectively render subset of SCD patients at either higher or lower risk of developing pulmonary thrombosis remains elusive. Adenosine diphosphate (ADP) released from lysed erythrocytes can activate platelets by stimulating their purinergic P2Y1 and P2Y12 receptors, however, P2Y12 receptor antagonists have not shown any benefit in clinical trials, justifying the need for better understanding of purinergic signaling in SCD. Here, we use intravital lung microscopy in transgenic humanized SCD mice to show that intravenous administration of ADP triggered pulmonary thrombosis in control mice but failed to trigger pulmonary thrombosis in SCD mice. In contrast, collagen evoked pulmonary thrombosis identically in both control and SCD mice. Identical to intravital findings, IV ADP administration also evoked transient thrombocytopenia in control but not SCD mice, while, IV collagen led to comparable drop in platelet count in both SCD and control mice. ADP is metabolized by the ecto-nucleoside-tri-phosphate-diphosphohydrolase-1 (E-NTPDase1) CD39. IV administration of fluorescent analogue of ADP, N⁶- ethenoadenosine- 5'- O- diphosphate (ε-ADP) followed by invivo microdialysis and HPLC analysis revealed impaired ε-ADP degradation in SCD mice, suggestive of decreased CD39 activity. Our current findings suggest that loss of CD39 activity in SCD possibly prevents ADP-mediated pulmonary thrombosis. Currently, experiments are underway to identify pathways contributing to loss of CD39 activity in SCD, how that affects purinergic signaling and whether selective activation vs deactivation of this pathway is responsible for risk of pulmonary thrombosis in only 20% of ACS patients. Disclosures Gladwin: Globin Solutions, Inc: Patents & Royalties: Provisional patents for the use of recombinant neuroglobin and heme-based molecules as antidotes for CO poisoning; United Therapeutics: Patents & Royalties: Co-inventor on an NIH government patent for the use of nitrite salts in cardiovascular diseases ; Bayer Pharmaceuticals: Other: Co-investigator.

Description: Introduction: Sickle Cell Disease (SCD) is an autosomal-recessive-hemolytic disorder caused by a single point mutation in the β-globin gene that leads to sickling of RBCs under deoxygenated condition. Sickle RBCs (sRBCs) are not only rigid but also express adhesion molecules, which are not normally expressed on RBCs. The sticky and rigid sRBCs are believed to get trapped in blood vessels along with leukocytes to cause vaso-occlusion, which is the predominant pathophysiology underlying acute pain crisis in SCD patients. The process of sickling and vaso-occlusion leads to sRBC hemolysis, which releases hemoglobin, ADP and other RBC contents into the blood giving rise to a pro-inflammatory and pro-coagulant state, characterized by activated leukocytes, platelets, endothelial cells (ECs), tissue factor (TF) and enhanced adhesion of these cells to each other. Leukocyte–endothelium adhesion starts with leukocyte rolling mediated by P-selectin-glycoprotein-ligand (PSGL)-1 on leukocytes binding to P-selectin on endothelium. Rolling is followed by firm arrest, which is mediated by activated β­2-integrins (LFA-1 and Mac-1) on the leukocytes binding to inter-cellular-adhesion-molecule (ICAM)-1 on endothelium. Although neutrophils have been shown to play a role in the onset of vaso-occlusion by interacting with sRBCs and platelets in cremaster venules of SCD mice; the cellular, molecular and biophysical mechanisms that enable vaso-occlusion in SCD patients are not known. Materials and Methods: Freshly collected heparinized blood from SCD patients and race matched control subjects was perfused through a polydimethylsiloxane (PDMS) based microfluidic flow chamber with a glass bottom coated with either human micro-vascular endothelial cells or a cocktail of recombinant human P-selection, ICAM-1 and IL-8 at a venular/arteriolar wall shear stress. Fluorochrome conjugated Abs against CD16, CD235a and CD49b were added to the blood to stain neutrophils, sRBCs and platelets, respectively, and cellular interactions were recorded using multi-color Quantitative Dynamic Footprinting (qDF; Sundd et al Nature Methods 2010) or epifluorescence microscopy. Specificity of cellular interactions was tested using function blocking Abs against human Mac-1, LFA-1, P-selectin and PSGL-1. Results: SCD patients had much higher number of circulating neutrophils than control patients. Neutrophils rolled, arrested and then captured free flowing platelets in both SCD and control blood. However, significantly larger number of neutrophils rolled and arrested in SCD blood than control blood. As a result, much higher number of platelets was captured by arrested neutrophils in SCD blood than control blood, which led to the formation of neutrophil-platelet micro-emboli. The micro-emboli formation was mediated by a unique biophysical mechanism, which involved PSGL-1 and Mac-1 on neutrophils binding to P-selectin and GPIbα on platelets, respectively. Conclusion: Vaso-occlusion involves a cascade of adhesive events. First, neutrophils roll and arrest at the site of vaso-occlusion. Second, arrested neutrophils capture free flowing platelets and RBCs to form micro-emboli. Third, eventually these micro-emboli give rise to micro-thrombi, which cause stasis of blood flow. Acknowledgments: This study is supported by 11SDG7340005 from the American Heart Association (P.S.), VMI start-up funds (P.S.) and CBTP-T32 fellowship HL076124 (M.J). Disclosures No relevant conflicts of interest to declare.

Description: Introduction: Sickle Cell Disease (SCD) is an autosomal recessive genetic disorder that leads to sickling and hemolysis of RBCs under hypoxic conditions. As a result of chronic hemolysis, SCD is associated with a hyper-inflammatory and hyper-coagulation state, which accounts for enhanced adhesion of leukocytes, platelets, RBCs and vascular endothelial cells leading to vaso-occlusion. Acute vaso-occlusive pain crisis (VOC) is the primary reason for emergency medical care by SCD patients. Although neutrophils have been shown to play a role in the on-set of vaso-occlusion by interacting with sickle RBCs and platelets in cremaster venules of transgenic SCD mice, the cellular, molecular and biophysical mechanisms that promote vaso-occlusion in SCD patients is not completely understood. Materials and Methods: Freshly collected heparinized blood from steady-state SCD (SS) patients and race matched control subjects was perfused through polydimethylsiloxane (PDMS) based microfluidic flow channels (30 µm x 500 µm) with a glass bottom coated with either human microvascular endothelial cells or a cocktail of recombinant human P-selectin, ICAM-1 and IL-8 at a physiological shear stress (6 dyn cm-2). Fluorescent Abs against CD16 and CD49b were added to the blood for in-situ staining of neutrophils and platelets, respectively. Cellular interactions were recorded using quantitative microfluidic fluorescence microscopy (qMFM)1, which is a combination of quantitative dynamic footprinting1 and epifluorescence microscopy. Results and Discussion: Neutrophils in SS blood were observed to roll, arrest and then capture freely flowing platelets leading to the formation of vaso-occlusive aggregates. RBCs were observed getting trapped within the platelet-neutrophil aggregates. The number of platelet-neutrophil interactions, lifetime of these interactions and the extent of platelet-neutrophil aggregation were several folds higher in SS than control subject blood. Bacterial lipopolysaccharide (LPS; 500 ng/ml) pretreatment led to enhanced platelet-neutrophil aggregations in SS but not control blood. The enhanced platelet-neutrophil aggregations in SS blood (+/-LPS) was attenuated to the level observed in control blood by simultaneous blockage of P-selectin on platelets and Mac-1 on neutrophils with functional blocking Abs. Conclusion: Our data demonstrates that the vaso-occlusive pathophysiology in SCD involves sequential steps of neutrophil arrest, nucleation of platelets on arrested neutrophils, formation of platelet-neutrophil aggregates and trapping of RBCs in these aggregates. The inflammatory milieu of SS patient blood sets a lower threshold for bacterial endotoxin induced platelet-neutrophil aggregation than control blood. Vaso-occlusion can be ameliorated in SS blood by simultaneous inhibition of platelet P-selectin and neutrophil Mac-1. Understanding the molecular mechanism of vaso-occlusion will enable the development of therapies that can prevent VOC in SS patients. References: 1. Jimenez MA, Tutuncuoglu E, Barge S, Novelli EM, Sundd P. Quantitative microfluidic fluorescence microscopy to study vaso-occlusion in Sickle Cell Disease. Haematologica, 2015. 2 Sundd, P. et al. Quantitative dynamic footprinting microscopy reveals mechanisms of neutrophil rolling. Nat Methods 7, 821-824, doi:10.1038/nmeth.1508 (2010). Disclosures No relevant conflicts of interest to declare.

Description: Background: Vaso-occlusive crisis (VOC) is the primary reason for emergency medical care by sickle cell disease (SCD) patients. SCD patients hospitalized with VOC often develop acute chest syndrome (ACS), a form of acute lung injury, suggesting a role for pulmonary vaso-occlusion in the onset of ACS. However, the cellular, molecular and biophysical mechanism of pulmonary vaso-occlusion is unknown. Methods: SCD transgenic or non-sickle control mice were intravenously (IV) challenged with 2 to 3 ng of bacterial lipopolysaccharide (LPS). Fluorescent anti-mouse Ly-6G and CD49b mAbs were administered IV for in vivo staining of circulating neutrophils and platelets, respectively. Multiphoton excitation enabled quantitative fluorescence intravital lung microscopy (qFILM) was used to determine the molecular mechanism of pulmonary vaso-occlusion in live mice. Function-blocking anti-mouse P-selectin mAb (Fab fragments) was administered IV to assess the role of platelet P-selectin in promoting pulmonary vaso-occlusion. Results: A nanogram dose of IV LPS selectively triggered pulmonary vaso-occlusion in SCD but not control mice. Remarkably, pulmonary vaso-occlusion involved occlusion of the pre-capillary pulmonary arteriole bottle-necks (junction of an arteriole and capillaries) by large neutrophil-platelet embolic aggregates. IV administration of Fab fragments of function blocking anti-P-selectin mAb led to the resolution of pulmonary vaso-occlusion, which was primarily mediated by the attenuation of large neutrophil-platelet aggregates into smaller aggregates that are not stopped by the arteriolar bottle-necks. Conclusion: These results establish the relevance of neutrophil-platelet aggregation in pulmonary arterioles in promoting pulmonary vaso-occlusion in SCD and also highlight the therapeutic potential of inhibiting platelet P-selectin to prevent ACS in SCD patients hospitalized with VOC. Acknowledgments: This study was supported by 1R01HL128297-01 (P.S.), AHA 11SDG7340005 (P.S.), VMI startup funds (P.S.). M.F.B. was supported by NIH-NHLBI training grant T32HL110849 and NIH-NHLBI F32 NRSA 1F32HL131216-01. Disclosures No relevant conflicts of interest to declare.

Description: Introduction: Sickle Cell Disease (SCD) is an autosomal-recessive-genetic disorder that leads to sickling and hemolysis of red blood cells (RBCs). Acute vaso-occlusive pain crisis (VOC) is the predominant pathophysiology faced by SCD patients and the primary reason for emergency medical care. Although neutrophils have been shown to play a role in vaso-occlusion by interacting with sickle RBCs in the cremaster venules of transgenic SCD mice, the cellular, molecular and biophysical mechanisms that promote vaso-occlusion in SS patients is not completely understood. Materials and Methods: Freshly collected heparinized blood from steady-state SS patients and race matched control (AA) subjects was perfused through silicone based microfluidic flow channels with a glass bottom coated a cocktail of recombinant human P-selectin, ICAM-1 and IL-8 at a physiological wall shear stress (6 dyn cm-2). Fluorescent Abs against CD16 and CD49b were added to the blood for in-situ staining of neutrophils and platelets, respectively. Cellular interactions were recorded at a single cell-resolution using quantitative microfluidic fluorescence microscopy (qMFM)1, which allows quantitative assessment of vaso-occlusive events at an unprecedented single cell resolution2. Results: Vaso-occlusion in the microfluidic channel involved neutrophil arrest followed by nucleation of platelets on arrested neutrophils, formation of neutrophil-platelet-aggregates (NPA) and partial occlusion of the microfluidic flow channel. Remarkably, the number of platelet-neutrophil interactions and the lifetime of these interactions were several folds higher in SS patient than control AA blood. Surprisingly, preincubation with 250 ng/ml of bacterial lipopolysaccharide (LPS) led to a significant increase in the number and lifetime of platelet-neutrophil interactions in SS but not AA blood. This enhanced NPA formation in SS patient blood was attenuated to the level observed in AA blood by simultaneous blockage of P-selectin on platelets and Mac-1 on neutrophils as well as pretreatment with a small molecule inhibitor of toll-like-receptor-4 (TLR4) signaling pathway. Conclusion: Our data shows that the vaso-occlusive pathophysiology in SCD involves sequential steps of neutrophil arrest, nucleation of platelets on arrested neutrophils, formation of large NPAs and obstruction of blood flow. Platelet-neutrophil aggregation can be ameliorated by the simultaneous blockage of P-selectin on platelets and Mac-1 on neutrophils. The inflammatory milieu of SS patient blood sets a lower threshold for bacterial endotoxin induced neutrophil-platelet aggregation than control blood. The enhanced platelet-neutrophil aggregation in SS blood is dependent on activation of TLR-4 pathway. Understanding the molecular mechanism of vaso-occlusion will enable the development of therapeutics to prevent VOC in SS patients. References: 1 Jimenez MA, Tutuncuoglu E, Barge S, Novelli EM, Sundd P. Quantitative microfluidic fluorescence microscopy to study vaso-occlusion in sickle cell disease. Haematologica. 2015;100(10):e390-e393. doi:10.3324/haematol.2015.126631. 2 Sundd, P. et al. Quantitative dynamic footprinting microscopy reveals mechanisms of neutrophil rolling. Nat Methods7, 821-824, doi:10.1038/nmeth.1508 (2010). Disclosures Kato: Mast Therapeutics: Consultancy; Bayer: Research Funding.

Description: Hemarthrosis is a chronic arthropathy responsible for irreversible joint damage, disability, and acute joint pain in hemophilia patients. Hemarthrosis is caused by spontaneous or traumatic bleeding into joints, which when recurrent, leads to synovial inflammation and cartilage degeneration. Although release of erythrocyte-derived damage associated molecular pattern molecules (eDAMPs) is believed to promote sterile inflammation in the synovium, the innate immune mechanism of hemarthrosis remains poorly understood and the current therapy is limited to factor replacement and pain management. Here, we use factor 8 total knock-out (F8TKO) hemophilia A mice (C57BL/6J background) that manifest a complete deletion of the F8 coding region, expressing no detectable F8 mRNA and exhibiting a severe hemophilia phenotype. Right knee joint capsules of F8TKO mice were punctured with a 30-g needle below the patella between the anterior portions of the femur and tibia, followed by assessment of bleeding severity score and histological analysis one-week post injury. Intravital multiphoton excitation fluorescence microscopy of injured synovium was performed to assess the role of thrombo-inflammatory events in promoting hemarthrosis. Neutrophil, platelets, and blood vessels were visualized by intravenous administration of fluorescent anti-Ly6G mAb, anti-CD49b mAb, and dextran, respectively. Protein and mRNA levels of proinflammatory cytokines were measured in plasma, joint, synovium, and cartilage tissue using Real-Time PCR and ELISA, respectively. F8TKO but not wild-type (WT) control (C57BL/6J) mice manifested unresolved joint bleeding, cartilage degeneration and synovitis leading to impaired mobility and high bleeding severity scores. Significantly more recruitment of neutrophils and neutrophil-platelet aggregates as well as elevated IL-1b levels were observed in the synovium of F8TKO compared to WT mice. These results are the first to suggest that sterile inflammation contributing to hemarthrosis in hemophilia involves enhanced neutrophil-platelet recruitment to the synovium. Currently, experiments are underway to identify the role of eDAMPs (heme and hemoglobin) mediated activation of TLR4 and inflammasome pathway in promoting IL-1b generation, neutrophil-platelet aggregation, and progression of joint injury in F8TKO mice Disclosures Ragni: OPKO: Research Funding; Sangamo: Research Funding; Shire/Takeda: Other: Advisory Board, Research Funding; Spark Therapeutics: Other: Advisory Board, Research Funding; Alnylam/Sanofi: Other: Advisory Board, Research Funding; BioMarin: Other: Advisory Board, Research Funding; Bioverativ/Sanofi: Other: Advisory Board, Research Funding. Gladwin:Globin Solutions, Inc: Patents & Royalties: Provisional patents for the use of recombinant neuroglobin and heme-based molecules as antidotes for CO poisoning; Bayer Pharmaceuticals: Other: Co-investigator; United Therapeutics: Patents & Royalties: Co-inventor on an NIH government patent for the use of nitrite salts in cardiovascular diseases .

Description: Vaso-occlusive crisis (VOC) is the primary cause of morbidity and hospitalization in sickle cell disease (SCD); however, only 4 therapies (hydroxyurea, l-glutamine, crizanlizumab, and voxeletor) are currently approved in SCD. These agents limit the duration, severity, and frequency of crises. Activation of coagulation is a hallmark of SCD. Studies in animal models of SCD have shown that coagulation contributes to the chronic inflammation and end-organ damage associated with the disease; however, it is unknown whether coagulation directly contributes to the microvascular stasis that causes VOC. Herein, we demonstrate that inhibition of tissue factor (TF) and the downstream coagulation proteases factor Xa and thrombin significantly attenuates heme-induced microvascular stasis in mouse models of VOC. Pharmacologic inhibition of the principal thrombin receptor, protease activated receptor-1 (PAR-1), as well as deficiency of PAR-1 in all nonhematopoietic cells, also reduces stasis in sickle mice. PAR-1 deficiency was associated with reduced endothelial von Willebrand factor expression, which has been shown to mediate microvascular stasis. In addition, TF inhibition reduces lung vaso-occlusion in sickle mice mediated by arteriolar neutrophil-platelet microemboli. In sum, these results suggest that prophylactic anticoagulation might attenuate the incidence of VOC.