These data indicate that LPS-induced TF expression in hematopoietic cells is dramatically reduced in TFflox/flox,Tie-2Cre mice. we found that mouse platelets do not express TF pre-mRNA or mRNA. Our data demonstrate that in a mouse model of endotoxemia activation of the coagulation cascade is initiated by TF expressed by myeloid cells and an unidentified nonhematopoietic cell type(s). Introduction During endotoxemia and sepsis, activation of the coagulation cascade prospects to disseminated intravascular coagulation.1 Pharmacologic inhibition of tissue factor (TF) or a genetic reduction of TF expression has been shown to reduce coagulation and mortality in animal models of endotoxemia and sepsis.2C4 These results indicate that TF is the primary activator of coagulation in these models. However, the relative contribution of TF expression by different cell types to Pipemidic acid the activation of coagulation cascade is usually unknown. TF is usually constitutively expressed by cells within and surrounding the blood vessel wall, such pericytes and adventitial fibroblasts.5,6 It has been proposed that TF expressed by these cell types forms a hemostatic envelope that limits bleeding after vessel injury.5 Low levels of TF have also been observed in vascular easy muscle cells (VSMCs).7 In addition, in vitro studies demonstrated that lipopolysaccharide (LPS) activation of VSMCs increases TF expression.8 In vitro studies demonstrated that activated endothelial cells (ECs) express TF.9,10 However, only a limited number of studies were able to demonstrate TF expression by ECs in vivo. One study found colocalization of TF and the EC marker CD31 in the kidney of endotoxemic mice.11 Another study demonstrated the presence of TF protein only on ECs within the splenic microvasculature of septic baboons.12 More recently, TF protein was observed on ECs at aortic branch points of septic baboons.13 However, the TF associated with ECs was restricted to granular structures some of which were also positive for the leukocyte marker P-selectin glycoprotein ligand-1 (PSGL-1), suggesting that leukocyte-derived microparticles (MPs) may deliver TF to activated ECs in vivo.13 Early studies indicated that circulating blood cells do not normally express TF in healthy humans.5 However, a recent study found very low levels of TF antigen in a small subset of CD14-positive monocytes.14 LPS activation of monocytes and monocytic cells induces TF expression in vitro and in vivo.5,14C17 Furthermore, we as well as others have shown that TF expression by hematopoietic cells contributes to activation TSPAN11 of coagulation in endotoxemic mice.4,18 Other studies have reported TF expression by neutrophils and eosinophils.19,20 However, more recent studies found that neither neutrophils nor eosinophils express TF but can acquire TF by binding monocyte-derived MPs.21,22 TF expression by platelets is controversial. In 2001, Engelman and colleagues reported that activation of platelets results in translocation of TF from -granules to the cell surface.23 Other studies found that quiescent and stimulated platelets express variable levels of TF mRNA and protein.24,25 Recently, we exhibited that human platelets contained TF pre-mRNA that is spliced into TF mRNA and translated into protein.26 In contrast, Pipemidic acid other groups have failed to detect any TF protein or activity on resting platelets or calcium ionophore-stimulated platelets.27C29 Nevertheless, platelets may acquire TF by binding monocyte-derived MPs. For instance, it has been shown that monocyte-derived MPs can bind and fuse with activated platelets via an conversation between CD15 or PSGL-1 on MPs with P-selectin on platelets.29,30 In this study, we investigated the effect of either selective inhibition of TF or cell type-specific deletion of the TF gene around the activation of coagulation in a mouse model of endotoxemia. We exhibited that both hematopoietic and nonhematopoietic cell TF contributes to activation of coagulation in this model. Moreover, deletion of the TF gene in myeloid but not ECs is usually associated with a significant reduction in levels of plasma thrombin-antithrombin (TAT). Finally, deletion of the TF gene in either megakaryocytes/platelets or VSMCs did not reduce coagulation in endotoxemic mice. These results indicate that TF expression by myeloid cells and Pipemidic acid an unidentified nonhematopoietic cell type(s) prospects to activation of coagulation during endotoxemia. Methods Mice Generation of human TF (HTF) mice and TFflox/flox mice has been explained.31 Mice expressing Cre recombinase under control of lysozyme M (LysM), tyrosine kinase with immunoglobulin-like and EGF-like domains 2 (Tie-2), platelet factor 4 (PF4) and easy muscle 22 (SM22) promoters have been described.32C35 All mice utilized for the study were backcrossed to the C57BL/6 genetic background for 6 generations. Littermate controls were utilized for all mouse studies. All studies were approved by the Animal Care and Use Committee of the different institutions and comply with.