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Data are available on request from the corresponding author, R. Pawlinski (rafal_pawlinski@med.unc.edu). The online version of this article contains a data supplement. The American Society of Hematology A hypercoagulable state, chronic inflammation, and increased risk of venous thrombosis and stroke are prominent features in patients with sickle cell disease (SCD). Coagulation factor XII (FXII) triggers activation of the contact system that is known to be involved in both thrombosis and inflammation, but not in physiological hemostasis. Therefore, we investigated whether FXII contributes to the prothrombotic and inflammatory complications associated with SCD. We found that when compared with healthy controls, patients with SCD exhibit increased circulating biomarkers of FXII activation that are associated with increased activation of the contact pathway. We also found that FXII, but not tissue factor, contributes to enhanced thrombin generation and systemic inflammation observed in sickle cell mice challenged with tumor necrosis factor α. In addition, FXII inhibition significantly reduced experimental venous thrombosis, congestion, and microvascular stasis in a mouse model of SCD. Moreover, inhibition of FXII attenuated brain damage and reduced neutrophil adhesion to the brain vasculature of sickle cell mice after ischemia/reperfusion induced by transient middle cerebral artery occlusion. Finally, we found higher FXII, urokinase plasminogen activator receptor, and αMβ2 integrin expression in neutrophils of patients with SCD compared with healthy controls. Our data indicate that targeting FXII effectively reduces experimental thromboinflammation and vascular complications in a mouse model of SCD, suggesting that FXII inhibition may provide a safe approach for interference with inflammation, thrombotic complications, and vaso-occlusion in patients with SCD. This work was funded in part by National Institutes of Health (NIH) R01s HL142604 (RP), HL157441 (RP, NK) and NIH U01 HL117659 (RP, NK). EMS is supported by NIH R01 HL 155193. MK was supported by R00HL144817 from NHLBI and UAB AMC21 MULTIPI6724 grant. GMV and JDB were supported by research funding from NIH 5R01 HL114567. TR acknowledges Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) grants P6 - KFO306, 80750187 - SFB841 and 470698011 -SFB877. DG was supported by NIH R35 HL140025. FG was supported by Royal Society Wolfson Foundation (RSWF\R3\183001). AG and MW were supported by NIH R44 HL126235. This work was supported by the NIH R01 HL137695 (E.X.S.), Merit Review Awards BX003851 (E.X.S.) and the Oscar D. Ratnoff Endowed Professorship (E.X.S.). The contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH, the U.S. Department of Veterans Affairs, or the United States Government.

P-selectin (Psel) is an adhesion molecule expressed on platelets and endothelial cells, which interacts with its ligand PSGL-1 on leukocytes and a PSGL-1-like molecule on sickle red blood cells (RBCs). Psel mediates the formation of the multicellular aggregates that promote vaso-occlusive crisis (VOC) and acute painful episodes in sickle cell disease (SCD). Crizanlizumab, a monoclonal antibody blocking Psel, reduces the frequency of VOC in SCD patients. In SCD mice, Psel contributes to heme-induced microvascular stasis, and thrombus and platelet-neutrophil aggregate formation in the lung and liver. Unexpectedly, Psel deficiency promotes liver senescence in a mouse model of SCD, suggesting a possible detrimental effect of Psel inhibition. In this study, we further investigated the long-term effects of Psel deficiency on thromboinflammation and end-organ damage in murine SCD. We used Townes wild type (AA) and sickle (SS) mice (n=12-27) that either express Psel (Psel +/+) or lack Psel (Psel -/-) at 11-12 months of age. Kidney damage was evaluated by histology and urine analysis. Heart function was determined using echocardiography. Complete blood counts and plasma biomarkers of thrombin generation (thrombin anti-thrombin [TAT] complexes) and inflammation (interleukin [IL]-6) were also analyzed. In addition, in a pilot experiment, we analyzed the effect of Psel deficiency on experimental venous thrombosis in SS mice. SS Psel +/+ mice exhibited renal damage, urinary concentrating defect, and reduced creatinine clearance, which was not improved in SS Psel -/- mice. Psel deficiency attenuated glomerular (GLM) congestion (0.17±0.05 vs 0.39±0.06, p We observed cardiac hypertrophy (elevated heart weight to tibia length ratio) in SS mice, regardless of Psel expression. Echocardiography of left ventricle (LV) revealed that SS mice had increased LV mass and LV internal diameter with no change in ejection fraction or fractional shortening compared to AA mice. None of these parameters were affected by Psel expression. Furthermore, hypertrophy observed in other organs (kidney, liver, lung and spleen) of SS Psel +/+ mice was also not affected by Psel deficiency. Consistent with previous studies, SS Psel +/+ mice had elevated plasma levels of TAT and IL-6 compared to AA Psel +/+ mice. Psel deficiency significantly reduced IL-6 (47±15 vs 20±10 ng/mL, P As expected, SS Psel +/+ mice exhibited anemia as shown by reduced RBC number, hemoglobin (Hb) and hematocrit (Hct) compared to AA Psel +/+ animals. Interestingly, these parameters were further decreased in SS Psel -/- mice (p Our data suggest that despite improving acute vascular pathologies, including VOC and thromboinflammation, long term deficiency of Psel does not prevent end-organ damage. If fact, it may contribute to organ dysfunction and enhanced anemia. More mechanistic studies are needed to better understand the long-term effects of anti-Psel treatment in SCD patients. Disclosures Reeves: Incyte Corporation: Honoraria; Takeda: Honoraria; Bristol-Myers Squibb: Speakers Bureau; Pharma Essentia: Consultancy, Honoraria. Sundd: CSL Behring Inc: Research Funding; Bayer: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Introduction: Recent advances in preventive care, such as hydoxyurea and prophylactic antibiotics, have reduced the mortality of children with sickle cell disease (SCD) s in developed countries. Yet, the chronic hemolytic anemia and recurrent vaso-occlusive crises result in systemic inflammation and coagulopathy. Markers of coagulation activation correlate with painful crises, acute chest syndrome, stroke, venous thromboembolism, pulmonary hypertension, left ventricular diastolic heart disease, and sickle nephropathy. These complications result in end-organ failure that causes increased morbidity and mortality in adult SCD patients. We have shown that tissue factor (TF), the primary initiator of extrinsic coagulation, contributes to inflammation and coagulation in mouse models of SCD. (1,2). It has also been demonstrated that long-term reduction in thrombin protects from cardiopulmonary dysfunction and reduces mortality of sickle cell mice (3). Recent work from our laboratory demonstrates that high molecular weight kininogen (HK) promotes thrombin generation and inflammation in sickle mice. HK is proteolytically cleaved into bradykinin and cleaved HK fragments (HKf) by kallikrein and other proteases. HKf induces TF expression and activity on monocytes dependent on Mac-1 (CD11b/CD18). We found that Mac-1 inhibition attenuates thrombin generation and inflammation in sickle cell mice. Hypothesis: Long-term HK deficiency in sickle cell mice will attenuate TF-mediated coagulation and inflammation, and protect against end-organ damage and mortality. Methods and Results: To evaluate the effect of long-term HK deficiency on outcomes in sickle cell disease, we used bone marrow from Townes sickle (SS) and wild type (AA) mice to generate chimeras in lethally irradiated HK+/+ (WT) and HK-/- (KO) mice to create AA/WT, AA/KO, SS/WT, and SS/KO mice. Efficient reconstitution of bone marrow was confirmed by hemoglobin electrophoresis. Eight months after chimeras were generated, endpoints were assessed. SS/WT mice had early mortality (median survival 209 days, 6/23 mice survived to 250 days); HK deficiency significantly prolonged survival in SS mice (median survival 240 days, 24/29 mice survived to 250 days; p Conclusions: These data indicate that HK deficiency attenuates chronic inflammation, kidney failure, and heart hypertrophy, and improves survival of sickle cell mice. Disclosures No relevant conflicts of interest to declare.