scholarly journals Macrophage factor X activator formation: metabolic requirements for synthesis of components

Blood ◽  
1985 ◽  
Vol 65 (1) ◽  
pp. 169-175 ◽  
Author(s):  
JW Jr Shands
Keyword(s):  
Tissue Factor ◽  
Actinomycin D ◽  
Factor Vii ◽  
Soluble Factor ◽  
Factor X ◽  
Factor Activity ◽  
In Vitro Production ◽  
Tissue Factor Activity ◽  
Culture Supernatants

Abstract The in vitro production of factor VII-like material and of tissue factor activity by murine thioglycollate exudate macrophages was measured by amidolytic assays. Tissue factor activity was inducible by endotoxin, and its induction was inhibited by 1 microgram/mL of actinomycin D, 10 micrograms/mL of cycloheximide, and 0.2 micrograms/mL of tunicamycin. Soluble factor VII-like material was secreted by macrophages into culture supernatants. The amount produced was not influenced by further activation of the cells by endotoxin, nor was its production inhibited significantly by 1 microgram/mL actinomycin D or 0.2 micrograms/mL tunicamycin. The production of the factor VII-like material was inhibited by 10 micrograms/mL of cycloheximide, and its appearance in culture supernatants was enhanced significantly by the addition of vitamin K1. When lysates of activated macrophages were suspended in ultracentrifuged culture supernatants, a particulate factor X activator was formed. Centrifugation at 100,000 g pelleted the factor X activator and left no factor VII-like material in the supernatant. The data indicate that thioglycollate-induced exudate macrophages make and excrete factor VII-like material, and this production is not modulated by further activation. However, activation of the macrophages induces tissue factor production. The factor X activator appears to result from the interaction and complexing of the soluble factor VII-like material and the membrane-bound tissue factor.

scholarly journals Macrophage factor X activator formation: metabolic requirements for synthesis of components

Blood ◽  
1985 ◽  
Vol 65 (1) ◽  
pp. 169-175
Author(s):  
JW Jr Shands
Keyword(s):  
Tissue Factor ◽  
Actinomycin D ◽  
Factor Vii ◽  
Soluble Factor ◽  
Factor X ◽  
Factor Activity ◽  
In Vitro Production ◽  
Tissue Factor Activity ◽  
Culture Supernatants

The in vitro production of factor VII-like material and of tissue factor activity by murine thioglycollate exudate macrophages was measured by amidolytic assays. Tissue factor activity was inducible by endotoxin, and its induction was inhibited by 1 microgram/mL of actinomycin D, 10 micrograms/mL of cycloheximide, and 0.2 micrograms/mL of tunicamycin. Soluble factor VII-like material was secreted by macrophages into culture supernatants. The amount produced was not influenced by further activation of the cells by endotoxin, nor was its production inhibited significantly by 1 microgram/mL actinomycin D or 0.2 micrograms/mL tunicamycin. The production of the factor VII-like material was inhibited by 10 micrograms/mL of cycloheximide, and its appearance in culture supernatants was enhanced significantly by the addition of vitamin K1. When lysates of activated macrophages were suspended in ultracentrifuged culture supernatants, a particulate factor X activator was formed. Centrifugation at 100,000 g pelleted the factor X activator and left no factor VII-like material in the supernatant. The data indicate that thioglycollate-induced exudate macrophages make and excrete factor VII-like material, and this production is not modulated by further activation. However, activation of the macrophages induces tissue factor production. The factor X activator appears to result from the interaction and complexing of the soluble factor VII-like material and the membrane-bound tissue factor.

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 The endotoxin-induced procoagulant of mouse exudate macrophages: a factor-X activator

Blood ◽  
1983 ◽  
Vol 62 (2) ◽  
pp. 333-340 ◽  
Author(s):  
JW Jr Shands
Keyword(s):  
Tissue Factor ◽  
Culture Supernatant ◽  
Factor Vii ◽  
Mouse Tissue ◽  
Factor X ◽  
Macrophage Culture ◽  
Cellular Factor ◽  
Culture Supernatants ◽  
Bovine Factor

Abstract The properties of mouse macrophage procoagulant induced by endotoxin in vitro were studied by the acceleration of clotting and by chromogenic assays using as substrates human plasma and bovine components, which are not activated by mouse tissue factor. Maximal macrophage procoagulant activity occurred when activated cells were lysed in culture supernatant fluids, suggesting the interaction of cellular and supernatant factors. This procoagulant was clearly able to activate bovine factor X. The procoagulant also appeared to have prothrombinase activity. However, additional experiments suggested that the bulk of this activity was due to the activation of factor X contaminating the prothrombin. The production of the procoagulant was inhibited by warfarin (5 microM). Its activity was inhibited by 1 mM diisopropylfluorophosphate and unaffected by iodoacetamide, indicating that the procoagulant is a serine protease. Macrophage culture supernatants contained factor-VII-like activity. Neither mouse tissue factor nor macrophage culture supernatants alone activated bovine factor X. The two combined served as an efficient factor-X activator. Active supernatant factor (factor-VII-like) was not produced by macrophages cultured in the presence of warfarin, while the production of the macrophage cellular factor was unaffected by the presence of warfarin. I conclude that exudate macrophages cultured in vitro make and secrete factor VII or a factor-VII-like substance into the culture supernatant. When activated macrophages are lysed in this supernatant, the interaction of a cellular factor (? tissue factor) and factor VII gives rise to a factor-X activator.

scholarly journals The endotoxin-induced procoagulant of mouse exudate macrophages: a factor-X activator

Blood ◽  
1983 ◽  
Vol 62 (2) ◽  
pp. 333-340
Author(s):  
JW Jr Shands
Keyword(s):  
Tissue Factor ◽  
Culture Supernatant ◽  
Factor Vii ◽  
Mouse Tissue ◽  
Factor X ◽  
Macrophage Culture ◽  
Cellular Factor ◽  
Culture Supernatants ◽  
Bovine Factor

The properties of mouse macrophage procoagulant induced by endotoxin in vitro were studied by the acceleration of clotting and by chromogenic assays using as substrates human plasma and bovine components, which are not activated by mouse tissue factor. Maximal macrophage procoagulant activity occurred when activated cells were lysed in culture supernatant fluids, suggesting the interaction of cellular and supernatant factors. This procoagulant was clearly able to activate bovine factor X. The procoagulant also appeared to have prothrombinase activity. However, additional experiments suggested that the bulk of this activity was due to the activation of factor X contaminating the prothrombin. The production of the procoagulant was inhibited by warfarin (5 microM). Its activity was inhibited by 1 mM diisopropylfluorophosphate and unaffected by iodoacetamide, indicating that the procoagulant is a serine protease. Macrophage culture supernatants contained factor-VII-like activity. Neither mouse tissue factor nor macrophage culture supernatants alone activated bovine factor X. The two combined served as an efficient factor-X activator. Active supernatant factor (factor-VII-like) was not produced by macrophages cultured in the presence of warfarin, while the production of the macrophage cellular factor was unaffected by the presence of warfarin. I conclude that exudate macrophages cultured in vitro make and secrete factor VII or a factor-VII-like substance into the culture supernatant. When activated macrophages are lysed in this supernatant, the interaction of a cellular factor (? tissue factor) and factor VII gives rise to a factor-X activator.

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

Abstract 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 Activation of factor X and its regulation by tissue factor pathway inhibitor in small-diameter capillaries lined with human endothelial cells

Blood ◽  
1992 ◽  
Vol 79 (11) ◽  
pp. 2909-2916 ◽  
Author(s):  
T Lindhout ◽  
R Blezer ◽  
P Schoen ◽  
O Nordfang ◽  
C Reutelingsperger ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Factor Viia ◽  
Factor Xa ◽  
Factor Vii ◽  
Factor X ◽  
Factor Activity ◽  
Tissue Factor Activity ◽  
Tissue Factor Pathway

Abstract The activation of factor X at the surface of endothelial cells was investigated under controlled flow conditions. A method is described for preparing polyethylene capillaries whose inner walls are covered with a confluent layer of human umbilical vein endothelial cells. To obtain a stable and unperturbed layer of endothelial cells it was essential to pre-perfuse the endothelialized capillaries with medium for about 18 hours. At this stage no tissue factor activity could be detected, but when the seeded cells were perfused with medium containing tumor necrosis factor (TNF) a maximum steady-state rate of factor Xa production (16 fmol factor Xa/min/cm2) was observed within 8 hours. Further experiments were performed with endothelial cells incubated for 4 hours with TNF. Factor Xa was produced at a rate of 7 fmol factor Xa/min/cm2 on perfusion of the capillaries with factor X (100 nmol/L) and factor VII (0.1 U/mL) at a shear rate of 34 s-1. The extracellular matrix preparations of these cells produced factor Xa at a 20-fold higher rate (150 fmol factor Xa/min/cm2). In both cases factor Xa formation was dependent on the presence of factor VII and was completely inhibited when the perfusate also contained 5 nmol/L recombinant tissue factor pathway inhibitor (rTFPI). Pre-perfusion with factor Xa-TFPI complex in the absence of factor VIIa caused a much lesser inhibitory effect, suggesting that TFPI-mediated neutralization of endothelial cell and matrix tissue factor activity requires the presence of factor VIIa in addition to the presence of factor Xa.

scholarly journals Activation of factor X and its regulation by tissue factor pathway inhibitor in small-diameter capillaries lined with human endothelial cells

Blood ◽  
1992 ◽  
Vol 79 (11) ◽  
pp. 2909-2916 ◽  
Author(s):  
T Lindhout ◽  
R Blezer ◽  
P Schoen ◽  
O Nordfang ◽  
C Reutelingsperger ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Factor Viia ◽  
Factor Xa ◽  
Factor Vii ◽  
Factor X ◽  
Factor Activity ◽  
Tissue Factor Activity ◽  
Tissue Factor Pathway

The activation of factor X at the surface of endothelial cells was investigated under controlled flow conditions. A method is described for preparing polyethylene capillaries whose inner walls are covered with a confluent layer of human umbilical vein endothelial cells. To obtain a stable and unperturbed layer of endothelial cells it was essential to pre-perfuse the endothelialized capillaries with medium for about 18 hours. At this stage no tissue factor activity could be detected, but when the seeded cells were perfused with medium containing tumor necrosis factor (TNF) a maximum steady-state rate of factor Xa production (16 fmol factor Xa/min/cm2) was observed within 8 hours. Further experiments were performed with endothelial cells incubated for 4 hours with TNF. Factor Xa was produced at a rate of 7 fmol factor Xa/min/cm2 on perfusion of the capillaries with factor X (100 nmol/L) and factor VII (0.1 U/mL) at a shear rate of 34 s-1. The extracellular matrix preparations of these cells produced factor Xa at a 20-fold higher rate (150 fmol factor Xa/min/cm2). In both cases factor Xa formation was dependent on the presence of factor VII and was completely inhibited when the perfusate also contained 5 nmol/L recombinant tissue factor pathway inhibitor (rTFPI). Pre-perfusion with factor Xa-TFPI complex in the absence of factor VIIa caused a much lesser inhibitory effect, suggesting that TFPI-mediated neutralization of endothelial cell and matrix tissue factor activity requires the presence of factor VIIa in addition to the presence of factor Xa.

scholarly journals Activation of human factor VII during clotting in vitro

Blood ◽  
1985 ◽  
Vol 65 (1) ◽  
pp. 218-226 ◽  
Author(s):  
LV Rao ◽  
SP Bajaj ◽  
SI Rapaport
Keyword(s):  
Tissue Factor ◽  
Glass Tube ◽  
Factor Vii ◽  
Clotting Factor ◽  
Activate Factor ◽  
Factor Xii ◽  
Factor X ◽  
Normal Plasma

Abstract We have studied factor VII activation by measuring the ratio of factor VII clotting to coupled amidolytic activity (VIIc/VIIam) and cleavage of 125I-factor VII. In purified systems, a low concentration of Xa or a higher concentration of IXa rapidly activated 125I-factor VII, yielding a VIIc/VIIam ratio of 25 and similar gel profiles of heavy and light chain peaks of VIIa. On further incubation, VIIa activity diminished and a third 125I-peak appeared. When normal blood containing added 125I- factor VII was clotted in a glass tube, the VIIc/VIIam ratio rose fivefold, and 20% of the 125I-factor VII was cleaved. Clotting normal plasma in an activated partial thromboplastin time (APTT) system yielded a VIIc/VIIam ratio of 25 and over 90% cleavage of 125I-factor VII. Clotting factor XII-deficient plasma preincubated with antibodies to factor X in an APTT system with added XIa yielded a VIIc/VIIam ratio of 19 and about 60% cleavage, which indicates that IXa, at a concentration achievable in plasma, can effectively activate factor VII. Clotting normal plasma with undiluted tissue factor yielded a VIIc/VIIam ratio of 15 to 20 and 60% cleavage of 125I-factor VII, whereas clotting plasma with diluted tissue factor activated factor VII only minimally. We conclude that both Xa and IXa can function as significant activators of factor VII in in vitro clotting mixtures but believe that only small amounts of factor VII may be activated in vivo during hemostasis.

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