MECHANISM OF ACTIVATION OF BLOOD COAGULATION BY LEUKOCYTE PROCOAGULANT ACTIVITY

1987 ◽  
Author(s):  
N Narahara ◽  
H Sadakata ◽  
T Uchiyama ◽  
K Andoh ◽  
H Tanaka ◽  
...  
Keyword(s):  
Blood Coagulation ◽  
Calf Serum ◽  
Leukemia Cell ◽  
Specific Radioactivity ◽  
Activation Mechanism ◽  
Leukemia Cell Line ◽  
Coagulation Cascade ◽  
Factor Vii ◽  
Factor X ◽  
Viii And Ix

To investigate the process of activation mechanism of blood coagulation by leukocytes, binding of radiolabelled Factor X and the activation of Factor X on the cell surface of leukocytes were studied by using cultured leukemia cell line, Molt-4 cells. Cells were cultured in RPMI 1640 medium with 10% inactivated fetal, calf serum at a concentration of 1x106cells/ml. After 6 hours' stimulation with 1 ug/ml of endotoxin(LPS: Escherichia coli 026:B6), cells were separated by centrifugation, washed three times with Tris containing NaCl buffer(pH 7.5, TBS), and then suspended in TBS containing 0.5% bovine serum albumin(TBS-BSA) to the concentration of 5x106 cells/ml. Factors X, VII and IX were purified from fresh-frozen human plasma by the method of Bajaj in a modified version. Factor VIII was purified from cryoprecipitate as starting material. Factor X labelled with 1-125 by the method of McFarlane showed a single band on autoradiography. Specific radioactivity was 0.3 mCi/mg. For the study on binding of Factor X, TBS-BSA solution containing 4 mM of CaClp, various amounts of radiolabelled Factor X with/without purified Factors VII, VIII and IX were added to the LPS-stimulated washed cell suspension and mixed well. N-butyl-phtalate was layered over the reaction mixture after incubation at room temperature for various minutes. Total amount of bound Factor X was calculated from the radioactivity of the cell pellet separated by centrifugation of the reaction mixture. The Xa activity generated in the supernatants was assayed using S2222.Results: Factor X bound specifically to the LPS-stimulated Molt-4 cells. Amount of bound Factor X and the dissociation constant was 1.0 ng/5x10bcells (5.2x103sites/cell) and 5x106M, respectively. More amounts of Factor X bound when Factors VIII and IX were present in the reaction mixture than their absence. Five times more Factor Xa was generated when Factors VII, VIII and IX were present in the reaction mixture as compared with presence of Factor VII, alone. These results suggest that blood coagulation cascade proceeds on the LPS-stimulated leukocyte surface.

Self-Damping Mechanism in Blood Coagulation

1974 ◽  
Vol 32 (01) ◽  
pp. 057-064 ◽  
Author(s):  
Y Nemerson ◽  
S.A Silverberg ◽  
J Jesty
Keyword(s):  
Tissue Factor ◽  
Blood Coagulation ◽  
Calcium Ions ◽  
Control Mechanism ◽  
Factor Vii ◽  
Damping Factor ◽  
Factor V ◽  
Factor X ◽  
Extrinsic Pathway ◽  
Two Systems

SummaryTwo reactions of the extrinsic pathway of coagulation, the activations of Factor X and prothrombin, have been studied in purified systems and shown to be self-damping. Factor X was activated by the tissue factor - Factor VII complex, and prothrombin by two systems: the coagulant protein of Taipan venom, and the physiological complex of activated Factor X, Factor V, lipid, and calcium ions. In each case the yield of enzyme, activated Factor X or thrombin, is a function of the concentration of activator. These and other observations are considered as a basis for a control mechanism in coagulation.

scholarly journals The contributions of Ca2+, phospholipids and tissue-factor apoprotein to the activation of human blood-coagulation factor X by activated factor VII

1990 ◽  
Vol 265 (2) ◽  
pp. 327-336 ◽  
Author(s):  
V J J Bom ◽  
R M Bertina
Keyword(s):  
Tissue Factor ◽  
Blood Coagulation ◽  
Reaction Rate ◽  
Factor Viia ◽  
Coagulation Factor ◽  
Fluid Phase ◽  
Fold Increase ◽  
Factor Vii ◽  
Factor X ◽  
Extrinsic Pathway

In the extrinsic pathway of blood coagulation, Factor X is activated by a complex of tissue factor, factor VII(a) and Ca2+ ions. Using purified human coagulation factors and a sensitive spectrophotometric assay for Factor Xa, we could demonstrate activation of Factor X by Factor VIIa in the absence of tissue-factor apoprotein, phospholipids and Ca2+. This finding allowed a kinetic analysis of the contribution of each of the cofactors. Ca2+ stimulated the reaction rate 10-fold at an optimum of 6 mM (Vmax. of 1.1 x 10(-3) min-1) mainly by decreasing the Km of Factor X (to 11.4 microM). In the presence of Ca2+, 25 microM-phospholipid caused a 150-fold decrease of the apparent Km and a 2-fold increase of the apparent Vmax. of the reaction; however, both kinetic parameters increased with increasing phospholipid concentration. Tissue-factor apoprotein contributed to the reaction rate mainly by an increase of the Vmax., in both the presence (40,500-fold) and absence (4900-fold) of phospholipid. The formation of a ternary complex of Factor VIIa with tissue-factor apoprotein and phospholipid was responsible for a 15 million-fold increase in the catalytic efficiency of Factor X activation. The presence of Ca2+ was absolutely required for the stimulatory effects of phospholipid and apoprotein. The data fit a general model in which the Ca2(+)-dependent conformation allows Factor VIIa to bind tissue-factor apoprotein and/or a negatively charged phospholipid surface resulting into a decreased intrinsic Km and an increased Vmax. for the activation of fluid-phase Factor X.

Model of a Ternary Complex between Activated Factor VII, Tissue Factor and Factor IX

2002 ◽  
Vol 88 (07) ◽  
pp. 74-82 ◽  
Author(s):  
Shu-wen Chen ◽  
Jean-François Schved ◽  
Jean-Luc Pellequer ◽  
Muriel Giansily-Blaizot
Keyword(s):  
Tissue Factor ◽  
Ternary Complex ◽  
Search Algorithm ◽  
Factor Ix ◽  
Site Directed Mutagenesis ◽  
Coagulation Cascade ◽  
Factor Vii ◽  
Factor X ◽  
Three Dimensional Model ◽  
Inhibitory Peptide

SummaryUpon binding to tissue factor, FVIIa triggers coagulation by activating vitamin K-dependent zymogens, factor IX (FIX) and factor X (FX). To understand recognition mechanisms in the initiation step of the coagulation cascade, we present a three-dimensional model of the ternary complex between FVIIa:TF:FIX. This model was built using a full-space search algorithm in combination with computational graphics. With the known crystallographic complex FVIIa:TF kept fixed, the FIX docking was performed first with FIX Gla-EGF1 domains, followed by the FIX protease/EGF2 domains. Because the FIXa crystal structure lacks electron density for the Gla domain, we constructed a chimeric FIX molecule that contains the Gla-EGF1 domains of FVIIa and the EGF2-protease domains of FIXa. The FVIIa:TF:FIX complex has been extensively challenged against experimental data including site-directed mutagenesis, inhibitory peptide data, haemophilia B database mutations, inhibitor antibodies and a novel exosite binding inhibitor peptide. This FVIIa:TF:FIX complex provides a powerful tool to study the regulation of FVIIa production and presents new avenues for developing therapeutic inhibitory compounds of FVIIa:TF:substrate complex.

Clinical Implications of Crosstalk Between Coagulation and Inflammation: The Role of Anticoagulation in Treating Sepsis

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2874-2874
Author(s):  
Zhi Xu ◽  
Elizabeth Phillips ◽  
Prasanta Basak ◽  
Stephen Jesmajian
Keyword(s):  
Vitamin K ◽  
Inflammatory Responses ◽  
Conflicts Of Interest ◽  
Vitamin K Antagonist ◽  
Coagulation Cascade ◽  
Factor Vii ◽  
Clinical Implications ◽  
Factor X ◽  
All Cause Mortality

Abstract BACKGROUND: Despite decades of active investigation, sepsis remains one of the leading causes of mortality worldwide. Multiple lines of evidence have illustrated that up-regulation of the activated Factor VII (FVIIa)/Tissue Factor (TF) complex, and its downstream extrinsic coagulation cascade, are major contributors to coagulopathies and inflammatory response during sepsis. For example, decreased mortality and inflammatory responses during sepsis were observed in mice with significantly reduced FVII expression. Another recent study demonstrated the association of increased mortality with higher levels of FVIIa in septic patients. Similar results have been demonstrated for Factor X (FX) and thrombin. In addition, several studies have been conducted to investigate the role of heparin in treating sepsis and have yielded promising results, however, the exact mechanisms remain elusive, and the clinical implications of crosstalk between coagulation pathways and sepsis are yet to be determined. Furthermore, the role of vitamin-K antagonist in sepsis has not been investigated. OBJECTIVE: To assess the effects of pre-existing anticoagulation with warfarin on the clinical course of septic patient. METHODS: This was a retrospective observational study undertaken in a community-based teaching hospital. Patients who were admitted with a primary diagnosis of sepsis from January 01 to June 30, 2012 were included in the study. The clinical characteristics between patient groups without and with prior anticoagulation were compared and analyzed. The primary outcomes include the severity of sepsis, length of hospitalization, and mortality rate during hospitalization. RESULTS: A total of 134 septic patients were included in the study. Among them, 105 patients were not anticoagulated, while 29 patients were anticoagulated, prior to admission (mean age: 76.0 + 1.2 vs. 77.5 + 2.6, p = 0.603). All of the patients with anticoagulation had been taking warfarin due to either pre-existing atrial fibrillation (79.3%) or deep vein thrombosis/pulmonary embolism (20.7%). There were significant differences in International Normalized Ratio (INR) of prothrombin time between groups without and with anticoagulation at the time of admission (1.28 + 0.04 vs. 4.59 + 0.83, p < 0.001). Septic patients who did not take warfarin prior to admission presented with higher Sepsis Indices (0.93 + 0.03 vs. 0.82 + 0.05, p < 0.05), resulting in longer hospitalizations (11.60 + 1.02 vs. 8.40 + 0.70, p < 0.001). The overall all-cause mortality rates during the hospitalization between patients without anticoagulation and those with anticoagulation were 23% vs. 14%, respectively. CONCLUSION: To our knowledge, this is the first study to demonstrate that septic patients with prior anticoagulation by a vitamin-K antagonist presented with less severity of sepsis, reduced length of hospital stay, and decreased all-cause mortality during hospitalization as compared with those without anticoagulation. In our study, prior administration of anticoagulation with warfarin may have had significant clinical implications in septic patients. This warrants further prospective studies. Disclosures No relevant conflicts of interest to declare.

scholarly journals The protein encoded by a growth arrest-specific gene (gas6) is a new member of the vitamin K-dependent proteins related to protein S, a negative coregulator in the blood coagulation cascade.

1993 ◽  
Vol 13 (8) ◽  
pp. 4976-4985 ◽  
Author(s):  
G Manfioletti ◽  
C Brancolini ◽  
G Avanzi ◽  
C Schneider
Keyword(s):  
Blood Coagulation ◽  
Vitamin K ◽  
Fetal Calf Serum ◽  
Human Fibroblasts ◽  
Calf Serum ◽  
Growth Arrest ◽  
Protein S ◽  
Coagulation Cascade ◽  
Specific Gene ◽  
Mouse Tissues

A set of growth arrest-specific genes (gas) whose expression is negatively regulated after serum induction has previously been described (C. Schneider, R. M. King, and L. Philipson, Cell 54:787-793, 1988). The detailed analysis of one of them, gas6, is reported here, gas6 mRNA (2.6 kb) is abundantly expressed in serum-starved (48 h in 0.5% fetal calf serum) NIH 3T3 cells but decreases dramatically after fetal calf serum or basic fibroblast growth factor stimulation. The human homolog of gas6 was also cloned and sequenced, revealing a high degree of homology and a similar pattern of expression in IMR90 human fibroblasts. Computer analysis of the protein encoded by murine and human gas6 cDNAs showed significant homology (43 and 44% amino acid identity, respectively) to human protein S, a negative coregulator in the blood coagulation pathway. By using an anti-human Gas6 monospecific affinity-purified antibody, we show that the biosynthetic level of human Gas6 fully reflects mRNA expression in IMR90 human fibroblasts. This finding thus defines a new member of vitamin K-dependent proteins that is expressed in many human and mouse tissues and may be involved in the regulation of a protease cascade relevant in growth regulation.

scholarly journals A candidate activation pathway for coagulation factor VII

2019 ◽  
Vol 476 (19) ◽  
pp. 2909-2926
Author(s):  
Tina M. Misenheimer ◽  
Kraig T. Kumfer ◽  
Barbara E. Bates ◽  
Emily R. Nettesheim ◽  
Bradford S. Schwartz
Keyword(s):  
Tissue Factor ◽  
Blood Coagulation ◽  
Factor Viia ◽  
Coagulation Factor ◽  
Factor Ix ◽  
Factor Vii ◽  
Factor X ◽  
Contact Activation ◽  
Coagulation Factor Vii ◽  
Factor Viiia

Abstract The mechanism of generation of factor VIIa, considered the initiating protease in the tissue factor-initiated extrinsic limb of blood coagulation, is obscure. Decreased levels of plasma VIIa in individuals with congenital factor IX deficiency suggest that generation of VIIa is dependent on an activation product of factor IX. Factor VIIa activates IX to IXa by a two-step removal of the activation peptide with cleavages occurring after R191 and R226. Factor IXaα, however, is IX cleaved only after R226, and not after R191. We tested the hypothesis that IXaα activates VII with mutant IX that could be cleaved only at R226 and thus generate only IXaα upon activation. Factor IXaα demonstrated 1.6% the coagulant activity of IXa in a contact activation-based assay of the intrinsic activation limb and was less efficient than IXa at activating factor X in the presence of factor VIIIa. However, IXaα and IXa had indistinguishable amidolytic activity, and, strikingly, both catalyzed the cleavage required to convert VII to VIIa with indistinguishable kinetic parameters that were augmented by phospholipids, but not by factor VIIIa or tissue factor. We propose that IXa and IXaα participate in a pathway of reciprocal activation of VII and IX that does not require a protein cofactor. Since both VIIa and activated IX are equally plausible as the initiating protease for the extrinsic limb of blood coagulation, it might be appropriate to illustrate this key step of hemostasis as currently being unknown.

scholarly journals Bridging the Missing Link with Emicizumab: A Bispecific Antibody for Treatment of Hemophilia A

2020 ◽  
Vol 120 (10) ◽  
pp. 1357-1370
Author(s):  
Georg Gelbenegger ◽  
Christian Schoergenhofer ◽  
Paul Knoebl ◽  
Bernd Jilma
Keyword(s):  
Clinical Trials ◽  
Hemophilia A ◽  
Coagulation Factor ◽  
Factor Ix ◽  
Coagulation Cascade ◽  
Factor Vii ◽  
Factor X ◽  
Current Standard ◽  
Bleeding Events ◽  
Recombinant Activated Factor Vii

AbstractHemophilia A, characterized by absent or ineffective coagulation factor VIII (FVIII), is a serious bleeding disorder that entails severe and potentially life-threatening bleeding events. Current standard therapy still involves replacement of FVIII, but is often complicated by the occurrence of neutralizing alloantibodies (inhibitors). Management of patients with inhibitors is challenging and necessitates immune tolerance induction for inhibitor eradication and the use of bypassing agents (activated prothrombin complex concentrates or recombinant activated factor VII), which are expensive and not always effective. Emicizumab is the first humanized bispecific monoclonal therapeutic antibody designed to replace the hemostatic function of activated FVIII by bridging activated factor IX and factor X (FX) to activate FX and allow the coagulation cascade to continue. In the majority of hemophilic patients with and without inhibitors, emicizumab reduced the annualized bleeding rate to almost zero in several clinical trials and demonstrated a good safety profile. However, the concurrent use of emicizumab and activated prothrombin complex concentrate imposes a high risk of thrombotic microangiopathy and thromboembolic events on patients and should be avoided. Yet, the management of breakthrough bleeds and surgery remains challenging with only limited evidence-based recommendations being available. This review summarizes published clinical trials and preliminary reports of emicizumab and discusses the clinical implications of emicizumab in treatment of hemophilia A.

The protein encoded by a growth arrest-specific gene (gas6) is a new member of the vitamin K-dependent proteins related to protein S, a negative coregulator in the blood coagulation cascade

1993 ◽  
Vol 13 (8) ◽  
pp. 4976-4985
Author(s):  
G Manfioletti ◽  
C Brancolini ◽  
G Avanzi ◽  
C Schneider
Keyword(s):  
Blood Coagulation ◽  
Vitamin K ◽  
Fetal Calf Serum ◽  
Human Fibroblasts ◽  
Calf Serum ◽  
Growth Arrest ◽  
Protein S ◽  
Coagulation Cascade ◽  
Specific Gene ◽  
Mouse Tissues

A set of growth arrest-specific genes (gas) whose expression is negatively regulated after serum induction has previously been described (C. Schneider, R. M. King, and L. Philipson, Cell 54:787-793, 1988). The detailed analysis of one of them, gas6, is reported here, gas6 mRNA (2.6 kb) is abundantly expressed in serum-starved (48 h in 0.5% fetal calf serum) NIH 3T3 cells but decreases dramatically after fetal calf serum or basic fibroblast growth factor stimulation. The human homolog of gas6 was also cloned and sequenced, revealing a high degree of homology and a similar pattern of expression in IMR90 human fibroblasts. Computer analysis of the protein encoded by murine and human gas6 cDNAs showed significant homology (43 and 44% amino acid identity, respectively) to human protein S, a negative coregulator in the blood coagulation pathway. By using an anti-human Gas6 monospecific affinity-purified antibody, we show that the biosynthetic level of human Gas6 fully reflects mRNA expression in IMR90 human fibroblasts. This finding thus defines a new member of vitamin K-dependent proteins that is expressed in many human and mouse tissues and may be involved in the regulation of a protease cascade relevant in growth regulation.

scholarly journals Airway tissue factor-dependent coagulation activity in response to sulfur mustard analog 2-chloroethyl ethyl sulfide

2012 ◽  
Vol 302 (1) ◽  
pp. L82-L92 ◽  
Author(s):  
Raymond C. Rancourt ◽  
Livia A. Veress ◽  
XiaoLing Guo ◽  
Tara N. Jones ◽  
Tara B. Hendry-Hofer ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Tissue Factor ◽  
Sulfur Mustard ◽  
Culture Media ◽  
Active Form ◽  
Coagulation Cascade ◽  
Factor Vii ◽  
Factor X ◽  
Clotting Factors ◽  
Airway Injury

Acute lung injury is a principal cause of morbidity and mortality in response to mustard gas (SM) inhalation. Obstructive, fibrin-containing airway casts have recently been reported in a rat inhalation model employing the SM analog 2-chloroethyl ethyl sulfide (CEES). The present study was designed to identify the mechanism(s) causing activation of the coagulation cascade after CEES-induced airway injury. Here we report that CEES inhalation elevates tissue factor (TF) activity and numbers of detached epithelial cells present in lavage fluid (BALF) from rats after exposure (18 h). In vitro studies using 16HBE cells, or with rat BALF, indicated that detached epithelial cells could convert factor X (FX) to the active form FXa when incubated with factor VII and could elicit rapid clotting of plasma. In addition, immunocytochemical analysis demonstrated elevated cell surface (TF) expression on CEES-exposed 16HBE cells as a function of time. However, total cell TF expression did not increase. Since membrane surfaces bearing TF are important determinants of clot initiation, anticoagulants directed against these entities were tested for ability to limit plasma clotting or FX activation capacity of BALF or culture media. Addition of tifacogin, a TF pathway inhibitor, effectively blocked either activity, demonstrating that the procoagulant actions of CEES were TF pathway dependent. Lactadherin, a protein capable of competing with clotting factors for phospholipid-binding sites, was partially effective in limiting these procoagulant actions. These findings indicate that TF pathway inhibition could be an effective strategy to prevent airway obstruction after SM or CEES inhalation.

Molecular evolution of the vertebrate blood coagulation network

2003 ◽  
Vol 89 (03) ◽  
pp. 420-428 ◽  
Author(s):  
Colin Davidson ◽  
Robert Hirt ◽  
Kalpana Lal ◽  
Philip Snell ◽  
Greg Elgar ◽  
...  
Keyword(s):  
Blood Coagulation ◽  
Serine Proteases ◽  
Large Scale ◽  
Factor Ix ◽  
Supplementary Information ◽  
Factor Vii ◽  
Factor V ◽  
Factor X ◽  
Sequence Alignments ◽  
Comparative Sequence

SummaryIn mammalian blood coagulation 5 proteases, factor VII (FVII), factor IX (FIX), factor X (FX), protein C (PC) and prothrombin act with two cofactors factor V and factor VIII to control the generation of fibrin. Biochemical evidence and molecular cloning data have previously indicated that blood coagulation involving tissue factor, prothrombin and fibrinogen is present in all vertebrates. Using degenerate RT-PCR we have isolated and characterized novel cDNAs with sequence identity to the blood coagulation serine proteases and cofactors from chicken and the puffer fish (Fugu rubripes). Sequence alignments, phylogenetic and comparative sequence analysis all support the existence of the Gla-EGF1-EGF2-SP domain serine proteases FVII, FIX, FX, PC and the A1-A2-B-A3-C1-C2 domain protein cofactors FV and FVIII in these species. These results strongly suggest that the blood coagulation network is present in all jawed vertebrates and evolved before the divergence of tetrapods and teleosts over 430 million years ago; and that vertebrate blood coagulation may have benefited from two rounds of gene or whole genome duplication. Sequences identified in Fugu coding for additional FVII-like, FIX-like and PC-like sequences support the possibility of further tandem and large-scale duplications in teleosts. Comparative sequence analyses of amino acid residues in the active site region suggest these additional sequences have evolved new and as yet unknown functions.Supplementary information to this article available at both http://europium.csc.mrc.ac.uk and www.thrombosis-online.com

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