Letters to Blood To the editor: Thrombin-independent contribution of tissue factor to inflammation and cardiac hypertrophy in a mouse model of sickle cell disease Erica M. Sparkenbaugh, 1 Pichika Chantrathammachart, 1,2 Kasemsiri Chandarajoti, 1,3 Nigel Mackman, 1,4 Nigel S. Key, 1,4 and Rafal Pawlinski 1,4 1 McAllister Heart Institute, University of North Carolina at Chapel Hill, Chapel Hill, NC; 2 Department of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand; 3 Department of Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, Prince of Songkla University, Hat Yai, Songkhla, Thailand; and 4 Division of Hematology and Oncology, Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC Sickle cell disease (SCD) is caused by a single nucleotide mutation in the b-globin gene resulting in abnormal hemoglobin polymerization and formation of sickle red blood cells. Although vaso-occlusive crises and hemolytic anemia are the primary pathologies, SCD is also associated with chronic vascular inflammation and activation of coagulation. 1-3 This hypercoagulable state is characterized by increased tissue factor (TF) expression and elevated levels of thrombin generation measured by thrombin-antithrombin (TAT) complexes. 4 We have recently shown that short-term inhibition of all sources of TF in sickle mice attenuates activation of coagulation and reduces endothelial cell (EC) activation and systemic inflammation measured by plasma levels of soluble vascular cell adhesion molecule 1 (sVCAM-1) and interleukin-6 (IL-6), respectively. 3 Furthermore, SCD is 1 of a few pathological conditions in which increased TF expression is observed not only on leukocytes but also on ECs, 3,5-7 in particular on the pulmonary endothelium. 8 Interestingly, we showed that EC-specific deletion of TF significantly attenuated plasma levels of IL-6, but had no effect on thrombin generation or EC activation. 3 This surprising result suggests that EC TF is primarily involved in inflammation rather than coagulation in SCD. TF is constitutively expressed by perivascular cells surrounding blood vessels and parenchymal cells in certain organs. 9 During pathologic conditions that result in increased vascular permeability, this nonhematopoietic cellular source of TF will be exposed to circulating clotting factors and could activate the coagulation cascade, as we previously demonstrated in a mouse model of endotoxemia. 10 Because increased vascular permeability has been reported in mouse models of SCD, 11 we investigated if perivascular cell TF expression contributes to the activation of coagulation in sickle mice. To test this hypothesis, we used so-called “low TF mice” that are completely deficient in mouse TF but express human TF (mTF 2/2 , hTF 1 ) from a transgene at about 1% of normal levels; heterozygous littermates (mTF 1/2 , hTF 1 ) were used as controls. 12 These mice were transplanted with bone marrow (BM) from either Berkley (BERK) or wild-type (WT) mice, as previously described. 3 Importantly, transplantation of both types of BM into low TF mice resulted in the reconstitution of TF expression on all hema- topoietic cells and generated mice that express low levels of TF only on nonhematopoietic cells. Engraftment of the BM was determined by electrophoretic analysis of hemoglobin, as previously described. 3 Red blood cell counts, hemoglo- bin levels, and hematocrit levels were significantly lower, whereas white blood cell counts and plasma levels of lactate dehydrogenase were higher in heterozygous (Het) mice transplanted with BM from BERK mice (BERK BM /Het) compared with these parameters observed in Het mice transplanted with BM from non-sickle controls (WT BM /Het). This dem- onstrated successful reconstitution with sickle BM. Low level of TF on nonhematopoietic cells did not affect these parameters, indicating no effect on anemia or leukocytosis in sickle mice (Table 1). Consistent with our previous studies, plasma levels of TAT, sVCAM-1, and IL-6 as well as levels of myeloperoxidase (MPO) in the lung were significantly elevated in BERK BM /Het mice compared with these parameters observed in WT BM /Het mice (Figure 1A-D); however, low TF expression by nonhematopoietic cells had no effect on TAT or sVCAM-1 in sickle mice (BERK BM /low TF). This suggests that TF expression by nonhematopoietic cells does not contribute to either thrombin generation or EC activation in sickle mice, and, by deduction, these parameters must be driven by TF expressed by hematopoietic cells. Although this hypothesis has to be yet experimentally proven, we speculate that approaches specifically targeting pathological TF expression on hematopoietic cells could attenuate the prothrombotic state in SCD without interfering with hemostasis and increasing the risk of bleeding. The slight attenuation Table 1. Hematologic parameters from Het or low TF mice transplanted with BM from WT or BERK (sickle) mice Variable WT BM /Het WT BM /low TF BERK BM /Het BERK BM /low TF RBC, 10 6 /mL 8.1 6 0.2 7.8 6 0.7 5.9 6 0.2*** 5.9 6 0.3*** Hemoglobin, g/dL 8.9 6 0.6 8.2 6 1.1 5.8 6 0.5*** 5.9 6 0.4* Hematocrit, % 32.1 6 0.3 33.1 6 0.9 26.9 6 0.8*** 27.5 6 0.8*** MCV, fL 39.9 6 0.9 40.4 6 1.8 45.5 6 0.6*** 46.8 6 0.9*** LDH, IU/L 51.7 6 4.3 54.9 6 6.2 79.4 6 7.2* 75.9 6 8.1 WBC, 10 3 /mL 11.8 6 1.0 8.6 6 0.8 19.7 6 1.7*** 16.7 6 1.3*** Platelets, 10 3 /mL 699.4 6 24.5 682.4 6 48.8 754.4 6 41.13 812.4 6 34.2 Asterisks indicate statistical significance compared with WT BM within the same TF genotype. Data were analyzed by 2-way analysis of variance and Bonferroni post-hoc analysis. LDH, lactate dehydrogenase; MCV, mean corpuscular volume; RBC, red blood cells; WBC, white blood cells. *P . .05; ***P . .001. 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