Lipid rafts are necessary for tonic inhibition of cellular tissue factor procoagulant activity

Blood ◽  
2004 ◽  
Vol 103 (8) ◽  
pp. 3038-3044 ◽  
Author(s):  
Dennis J. Dietzen ◽  
Keith L. Page ◽  
Tina A. Tetzloff
Keyword(s):  
Tissue Factor ◽  
Lipid Rafts ◽  
Factor Viia ◽  
Cellular Tissue ◽  
Procoagulant Activity ◽  
Hek293 Cells ◽  
Factor Activity ◽  
Tissue Factor Activity ◽  
Factor Antigen

Abstract A fraction of total cellular tissue factor procoagulant activity remains masked or “encrypted” in intact cells. Decryption of this activity partly involves the extracellular exposure of anionic phospholipids such as phosphatidylserine. Because of the potential association of tissue factor and phospholipid scramblase activity with lipid rafts, we have explored the role of lipid rafts in regulating factor VIIa/tissue factor activity. In HEK293 cells, tissue factor antigen was not stably associated with lipid rafts, yet disruption of rafts with methyl-β-cyclodextrin resulted in a 3-fold stimulation of tissue factor procoagulant activity. Treatment with methyl-β-cyclodextrin was not associated with cytotoxicity and did not result in the exposure of additional tissue factor antigen. Factor VIIa/tissue factor activity decrypted with methyl-β-cyclodextrin was quantitatively similar to that obtained by using lytic concentrations of octyl glucoside but more sensitive to inhibition by cell surface tissue factor pathway inhibitor and the phospholipid binding protein, annexin V. Partial decryption of tissue factor was achieved with methyl-β-cyclodextrin prior to complete disruption of lipid rafts, suggesting the role of an enzyme localized to lipid rafts in the transbilayer transport of phosphatidylserine. We conclude that lipid rafts are required for the maintenance of cellular tissue factor in an encrypted state. (Blood. 2004;103:3038-3044)

scholarly journals Evidence for the presence of tissue factor activity on subendothelium

Blood ◽  
1989 ◽  
Vol 73 (4) ◽  
pp. 968-975
Author(s):  
HJ Weiss ◽  
VT Turitto ◽  
HR Baumgartner ◽  
Y Nemerson ◽  
T Hoffmann
Keyword(s):  
Tissue Factor ◽  
Umbilical Artery ◽  
Factor Viia ◽  
Rabbit Aorta ◽  
Coagulation Factors ◽  
Factor Vii ◽  
Factor X ◽  
Factor Activity ◽  
Fibrin Deposition ◽  
Tissue Factor Activity

By a variety of methods, tissue factor activity was demonstrated in the subendothelium of rabbit aorta and human umbilical artery. In one method, everted segments of de-endothelialized vessels were mounted in an annular perfusion chamber and the subendothelial surface was exposed to nonanticoagulated human blood under controlled flow. Procoagulant activity was assessed by measuring fibrin deposition on subendothelium and fibrinopeptide A (FPA) levels in post chamber blood. Both fibrin deposition and FPA were decreased with rabbit vessel segments exposed (at a shear rate of 650 seconds-1) to blood from patients with factor VII deficiency and with umbilical artery segments (at shear rates of 90 to 180 seconds-1) that had been pretreated with a monoclonal antibody to human tissue factor. In a second method, everted umbilical artery segments were mounted on a stir bar and the subendothelial surface was exposed, with stirring, to plasma or purified coagulation factors. The capacity of the surface to clot plasma on addition of calcium was inhibited by the antibody to tissue factor. The surface also activated purified 3H-factor X in the presence of factor VIIa, but not in its absence, and this surface property was almost entirely eliminated by pretreating the vessel segments with antitissue factor. Tissue factor activity in subendothelium could play a role in both the arrest of bleeding and in promoting the formation of thrombi at sites of vascular injury.

scholarly journals Complement-mediated regulation of tissue factor activity in endothelium.

1995 ◽  
Vol 182 (6) ◽  
pp. 1807-1814 ◽  
Author(s):  
S Saadi ◽  
R A Holzknecht ◽  
C P Patte ◽  
D M Stern ◽  
J L Platt
Keyword(s):  
Tissue Factor ◽  
Messenger Rna ◽  
Factor Viia ◽  
Interleukin 1 ◽  
Membrane Attack Complex ◽  
Secondary Response ◽  
Cell Surface Expression ◽  
Intermediate Step ◽  
Factor Activity ◽  
Tissue Factor Activity

Inflammation and immunity may be associated with endothelial cell (EC) injury and thrombus formation. We explored the mechanisms through which a humoral immune response directed against the endothelium might promote coagulation. Using the interaction of anti-EC antibodies and complement (C) with cultured EC as a model, we studied the expression and function of tissue factor, a cofactor for factor VIIa-mediated conversion of factor X to Xa. Exposure of EC to anti-EC antibodies and C in sublytic amounts stimulated the synthesis of tissue factor over a period of 16-42 h. Cell surface expression of tissue factor activity required activation of C and assembly of the membrane attack complex, because expression was inhibited by soluble CR1 and was not detected in the absence of C8. Elaboration of tissue factor messenger RNA was observed over a period of 8-30 h and required protein synthesis. Expression of tissue factor was not a direct consequence of the action of C on the EC but was a secondary response that required as an intermediate step the release of interleukin 1 alpha, an early product of the EC response to C activation. These findings suggest that, after the assembly of membrane attack complex on EC, the production of tissue factor and initiation of coagulation in a blood vessel depend on the production of interleukin 1 alpha and on its availability to stimulate affected EC.

scholarly journals Monocyte-associated tissue factor is suppressed by phorbol myristate acetate

Blood ◽  
1988 ◽  
Vol 72 (2) ◽  
pp. 456-462 ◽  
Author(s):  
JP Brozna ◽  
SD Carson
Keyword(s):  
Tissue Factor ◽  
Phorbol Myristate Acetate ◽  
Immunofluorescent Staining ◽  
Blood Monocytes ◽  
Factor Activity ◽  
Myristate Acetate ◽  
Tissue Factor Activity ◽  
Dose Dependent ◽  
Oxygen Metabolites ◽  
Factor Antigen

Abstract The monocyte is the only normal circulating cell type capable of initiating blood coagulation through the expression of tissue factor. Recently isolated peripheral blood monocytes that contain no demonstrable tissue factor activity can be induced to express tissue factor activity by a number of stimulatory agents. Monocyte-associated tissue factor activity transiently increases in response to adherence to tissue culture plates and, consistent with other reports, markedly increases after the isolated monocytes are treated with endotoxin. Phorbol myristate acetate (PMA) induced an increase in tissue factor activity at low doses (10(-11) to 10(-12) mol/L). Conversely, concentrations of PMA that stimulate release of oxygen metabolites or that cause the cytosol-to-membrane translocation of protein kinase C (PKC) (10(-9) to 10(-7) mol/L) resulted in a rapid decrease in both adherence-induced and endotoxin-induced monocyte tissue factor activity. The effects of PMA on monocytes were time- and dose-dependent with respect to PKC translocation, release of oxygen metabolites, and changes in tissue factor activity. Immunofluorescent staining of monocytes with monoclonal antibody (MoAb) HTF1–7B8, directed against human tissue factor, revealed that tissue factor antigen was induced concurrently with tissue factor activity by adherence and endotoxin and that tissue factor antigen decreased after PMA stimulation.

Localization of tissue factor pathway inhibitor to lipid rafts is not required for inhibition of factor VIIa/tissue factor activity

2003 ◽  
Vol 89 (01) ◽  
pp. 65-73 ◽  
Author(s):  
Garnet Jack ◽  
Keith Page ◽  
Tina Tetzloff ◽  
Connie Hall ◽  
Alan Mast ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Tissue Factor ◽  
Lipid Rafts ◽  
Tissue Factor Pathway Inhibitor ◽  
Factor Viia ◽  
Factor Xa ◽  
Hek293 Cells ◽  
Membrane Domains ◽  
Gpi Anchor ◽  
Tissue Factor Pathway

SummaryTissue factor pathway inhibitor (TFPI) abrogates coagulation initiated by the factor VIIa/tissue factor catalytic complex. While the gene structure of TFPI suggests that it is a secreted protein, a large pool of TFPI is associated with the vascular endothelium through its affinity for a glycosylphosphatidylinositol (GPI)-linked membrane protein. Inhibition of tissue factor by TFPI coincides with the translocation of quaternary complexes containing tissue factor, factor VIIa, factor Xa, and TFPI to detergent-insoluble plasma membrane domains rich in cholesterol, sphingomyelin, and GPI-linked proteins known as lipid rafts and caveolae. It is not known if localization of TFPI to these membrane domains is required for its inhibition of tissue factor procoagulant activity. We generated chimeric TFPI molecules linked directly to the plasma membrane via a GPI anchor or hydrophobic transmembrane domain and expressed these in HEK293 cells that produce tissue factor but not endogenous TFPI. The GPI-anchored chimera was exclusively enriched in detergent-insoluble membrane fractions while the transmembrane molecule was not. Transfectants expressing equal levels of the GPI-linked or transmembrane TFPI displayed equal anticoagulant potency as assessed by tissue factor-mediated conversion of factor X to factor Xa. Disruption of lipid rafts with cyclodextrin likewise had no effect on the inhibitory activity of the transmembrane or GPI-linked TFPI chimeras in HEK293 cells, nor on endogenous TFPI expressed by ECV304 cells. Thus, we conclude that the GPI anchor and membrane localization to lipid rafts does not enhance inhibition of factor VIIa/ tissue factor by cell-surface associated TFPI.

Inhibition of Factor X Activation at Extracellular Matrix of Fibroblasts During Flow Conditions: A Comparison Between Tissue Factor Pathway Inhibitor and Inactive Factor VIIa

1995 ◽  
Vol 74 (06) ◽  
pp. 1478-1485 ◽  
Author(s):  
Sanne Valentin ◽  
Chris P M Reutlingsperger ◽  
Ole Nordfang ◽  
Theo Lindhout
Keyword(s):  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Factor Viia ◽  
Factor Xa ◽  
Flow Chamber ◽  
Factor X ◽  
Factor Activity ◽  
Tissue Factor Activity ◽  
Tissue Factor Pathway ◽  
Factor X Activation

SummaryTissue factor pathway inhibitor (TFPI) is a naturally occurring factor Xa-dependent inhibitor of factor VIIa/tissue factor activity. In the present study, we examined the importance of the TFPI C-terminus and 3rd Kunitz-like domain for the inhibitory capacity of TFPI towards factor VIIa/tissue factor-catalyzed factor X activation and compared the inhibition with that of inactivated factor VIIa (factor VIIai). The extracellular matrix of fibroblasts, mounted in a parallel-plate flow chamber, were perfused with reaction mixtures that contained factors X, VIIa, and varying amounts of TFPI or factor VIIai. Inhibition was evaluated from the time course of factor Xa production at the outlet of the flow chamber. The factor VIIa/tissue factor-catalyzed factor Xa production was inhibited by factor VIIai and compatible with a direct competition between factor VIIai and factor VIIa for tissue factor. In contrast, TFPI showed a progressive inhibition of factor Xa production; the initial rate of factor X activation, however, was not inhibited by TFPI. Inhibition of factor Xa generation already in progress was seen for TFPI but not factor VIIai. In both cases we found that the truncated TFPI variants were as potent as full length TFPI. As to the stability of the enzyme- inhibitor complexes, TFPI/Xa/VIIa/tissue factor and factor VIIai/tissue factor, marked differences were observed. About 60% of the factor VIIa/tissue factor activity was recovered from the truncated TFPI/Xa/VIIa/tissue factor complex after 150 min of perfusion with reaction mixtures that contained factors X and VIIa. In contrast, full length TFPI did not dissociate from the complex, nor could factor VIIai be displaced by a large excess of factor VIIa.

Selective Increases of Procoagulant Activity in Rabbit Lymphoid Populations In Vitro Following Stimulation with Endotoxin: Dependence on Anatomic Source

1984 ◽  
Vol 51 (02) ◽  
pp. 228-231 ◽  
Author(s):  
Maria P McGee ◽  
Henry Rothberger ◽  
Tung-Kuang Lee
Keyword(s):  
Lymph Node ◽  
Tissue Factor ◽  
Mononuclear Cells ◽  
Procoagulant Activity ◽  
Mononuclear Leukocytes ◽  
Cell Populations ◽  
Factor Activity ◽  
Tissue Factor Activity

SummaryRabbit mononuclear leukocytes isolated from a variety of anatomic sites were examined for ability to generate procoagulant activity in vitro. Marrow, blood and spleen mononuclear cell populations were found to differ functionally from lymph node, thymus and alveolar populations by having much greater ability to increase in tissue factor activity in response to an endotoxin stimulus. Thus, after incubation in the presence of endotoxin, leukocytes obtained from marrow, blood, and spleen were found to increase in procoagulant activity characterized as tissue factor by 832, 1942 and 12.6 fold, respectively. In contrast, pulmonary alveolar macrophages increased in tissue factor activity only by 2.8 fold, and lymph node and thymus mononuclear cells showed little or no increases. These functional differences, demonstrated by exposing the six cell populations to endotoxin under controlled conditions in vitro, likely explain the similar pattern of anatomic selectivity of leukocyte tissue factor increases reported to occur in vivo during endotoxemia and Shwartzman reactions (1).

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