Tissue Factor Deficiency Decreases Sickle Cell-Induced Vascular Stasis in a Hematopoietic Stem Cell Transplant Model of Murine Sickle Cell Disease.
Authors: Hillery Cheryl A. , Foster Thomas D. , Holzhauer Sandra L. , Scott J. Paul , Panepinto Julie A. , Mohandas Narla , Mackman Nigel , Wandersee Nancy J. 
Published: April 2020

Abstract
There is considerable evidence for increased levels of procoagulant proteins, thrombin generation and tissue factor activity in patients with sickle cell disease (SCD). Both thrombin and tissue factor have potent proinflammatory effects. We found increased fibrin deposition in organs that develop SCD vaso-occlusive pathologies in the Berkeley murine model of SCD. In addition, we demonstrated that transplantation of sickle hematopoietic stem cells (HSC) into mice deficient in fibrinogen (SCD-Fib−/− mice) resulted in a decreased number and size of liver infarcts. These data suggest that fibrin clot formation is critical for the final irreversible stoppage of blood flow that can progress to focal tissue infarction. However, the SCD-Fib−/− mice still develop other typical organ pathologies associated with SCD, including focal sites of prominent vascular congestion that are likely due to persistent endothelial injury and inflammation. Because patients with afibrinogenemia have high levels of circulating thrombin, the persistent organ pathology in the SCD-Fib−/− mice may also be linked to the increased exposure to thrombin’s potent proinflammatory effects. In agreement, we now have evidence for increased circulating thrombin-antithrombin (TAT) complexes in SCD-Fib−/− mice compared to SCD-wt transplant control mice (54 ± 25 vs 9.9 ± 3.5 ng/mL, p=0.04) demonstrating increased thrombin generation in the SCD-Fib−/− mice. In addition, soluble VCAM-1, a marker of endothelia injury, was increased in SCD-Fib−/−mice compared to SCD-wt mice (843 ± 90 vs 628 ± 110). In order to determine whether the tissue factor pathway contributes significantly to proinflammatory pathologies observed in SCD, we transplanted fetal liver HSC from mice that predominately express human sickle hemoglobin (mα−/−, mβ−/−, Tg[HbS]+: “SCD mice” or hemizygous mα−/−, mβ+/−, Tg[HbS]x2: “hSCD2 mice,” Berkeley model) into mice deficient in tissue factor (mTF−/−, hTF+: “TFLow mice” that express ~1%TF levels) or control heterozygote mice (mTF+/−, hTF+) following lethal irradiation. Four months following transplant, we obtained blood samples for engraftment and hematologic studies (&gt;90% engraftment required for inclusion in this study), followed by perfusion of anesthetized animals. Perfused organs were divided with one part fixed for histologic study. Frozen nonfixed sections of the organs were homogenized and organ Hb content determined per gram organ weight. We found that tissue factor deficiency (SCD-TFLow and hSCD2-TFLow mice) resulted in a dramatic reduction of vascular congestion in liver histologic sections that was confirmed by quantitation of organ-specific trapped Hb: 1.2 ± 0.3 vs 6.3 ± 1.7 mg Hb/gm liver in hSCD2-TFLow vs hSCD2-TF+/− control mice (n = 8–9 per group, p&lt;0.001) with similar results in a smaller number of SCD-TFLow mice. These data suggest that the tissue factor pathway contributes importantly to vascular inflammation and cellular stasis. Thus, while the coagulation pathway appears to be a critical component of HbS-induced tissue infarction, endothelial injury and inflammation also significantly contribute to HbS-induced organ pathology. These data also suggest that targeted anticoagulation therapies may ameliorate the focal organ infarction and/or vascular inflammation that are prevalent in SCD.