Arterial thrombosis is considered to arise from the interaction of tissue factor (TF) in the vascular wall with platelets and coagulation factors in circulating blood. According to this paradigm, coagulation is initiated after a vessel is damaged and blood is exposed to vessel-wall TF. We have examined thrombus formation on pig arterial media (which contains no stainable TF) and on collagen-coated glass slides (which are devoid of TF) exposed to f lowing native human blood. In both systems the thrombi that formed during a 5-min perfusion stained intensely for TF, much of which was not associated with cells. Antibodies against TF caused Ϸ70% reduction in the amount of thrombus formed on the pig arterial media and also reduced thrombi on the collagencoated glass slides. TF deposited on the slides was active, as there was abundant fibrin in the thrombi. Factor VII ai , a potent inhibitor of TF, essentially abolished fibrin production and markedly reduced the mass of the thrombi. Immunoelectron microscopy revealed TF-positive membrane vesicles that we frequently observed in large clusters near the surface of platelets. TF, measured by factor X a formation, was extracted from whole blood and plasma of healthy subjects. By using immunostaining, TF-containing neutrophils and monocytes were identified in peripheral blood; our data raise the possibility that leukocytes are the main source of blood TF. We suggest that blood-borne TF is inherently thrombogenic and may be involved in thrombus propagation at the site of vascular injury.Tissue factor (TF) present in the arterial wall has been considered to be responsible for the initiation of the coagulation cascade and thrombus formation (1). The role of vascular TF in acute thrombosis and atherosclerosis has been proposed based on our previous studies (2-5). To investigate the role of circulating TF in thrombogenesis, we have used a system in which pig aortic media or collagen-coated slides were mounted in a laminar flow chamber and perfused with native human blood. We noted that when stained either with derivatized factor VII a (FVII a ) or with specific anti-TF antibodies, the thrombi contained large amounts of TF staining, whereas the media and collagen-coated slides were essentially negative. Thus, we surmised that the TF came from the blood; accordingly, we examined whole blood and plasma for TF activity that we have extracted and assayed. We conclude that there is circulating, potentially active TF in normal subjects. We present evidence that this pool is thrombogenic in model flow systems. We also present evidence suggesting the TF comes from leukocytes and hypothesize that the cell-surface TF is completely encrypted (6-8) but becomes available during thrombosis. METHODSReagents. Human recombinant FVII a was a gift from NovoNordisk, Copenhagen. Factor X was purified from human plasma (9). Affigel-15 was purchased from Bio-Rad. The phospholipids used for relipidation of TF consisted of 30% 1,2-dioleoyl-sn-glycero-3-phosphatidylserine and 70% 1,2-dioleoyl-sn-gl...

The use of human pluripotent stem cells for in vitro disease modeling and clinical applications requires protocols that convert these cells into relevant adult cell types. Here, we report the rapid and efficient differentiation of human pluripotent stem cells into vascular endothelial and smooth muscle cells. We found that GSK3 inhibition and BMP4 treatment rapidly committed pluripotent cells to a mesodermal fate and subsequent exposure to VEGF or PDGF-BB resulted in the differentiation of either endothelial or vascular smooth muscle cells, respectively. Both protocols produced mature cells with efficiencies over 80% within six days. Upon purification to 99% via surface markers, endothelial cells maintained their identity, as assessed by marker gene expression, and showed relevant in vitro and in vivo functionality. Global transcriptional and metabolomic analyses confirmed that the cells closely resembled their in vivo counterparts. Our results suggest that these cells could be used to faithfully model human disease.

Abstract-Raised levels of soluble P-selectin (sP-selectin) have been reported in the plasma of patients with vascular diseases; however, the functional importance of this ligand remains unclear. In this study we have examined a potential role for plasma sP-selectin in regulating neutrophil adhesion in patients with peripheral arterial occlusive disease (PAOD). Patients with PAOD had significantly higher levels of sP-selectin (meanϮSD: 73.3Ϯ13.0 versus 16.7Ϯ6.4 ng/mL) and enhanced whole blood leukocyte adhesion to platelets under shear. To examine whether the raised sP-selectin levels can directly influence leukocyte adhesion, isolated neutrophils were incubated with plasma from PAOD patients before and after immunodepletion of sP-selectin. Neutrophil adhesion to fibrinogen increased 2-fold following incubation with PAOD plasma, which was abrogated on sP-selectin immunodepletion. We subsequently demonstrated that recombinant sP-selectin dose-dependently (75 to 250 ng/mL) increased leukocyte adhesion to fibrinogen and platelet monolayers. This increase was PSGL-1 and Src kinase-dependent and correlated with an increase in sP-selectin-mediated Mac-1 activation. sP-selectin-stimulated neutrophil adhesion to platelet monolayers was inversely correlated with shear, such that at low shear (50 s Ϫ1 ) a 92.7%Ϯ15.7 increase in adhesion was observed decreasing to 38.5%Ϯ11.9 at 150 s Ϫ1 and 10.1%Ϯ7.4 at 300 s Ϫ1 . These studies suggest a potentially important role for sP-selectin in modulating neutrophil adhesion in patients with PAOD, particularly at sites of low shear, where it raises the possibility that raised plasma sP-selectin levels may enhance leukocyte recruitment to vascular injury and promote disease progression.