scholarly journals Potential role of activated factor VIII (FVIIIa) in FVIIa/tissue factor-dependent FXa generation in initiation phase of blood coagulation

2019 ◽  
Vol 109 (4) ◽  
pp. 390-401 ◽  
Author(s):  
Shoko Furukawa ◽  
Keiji Nogami ◽  
Kenichi Ogiwara ◽  
Midori Shima
Keyword(s):  
Factor Viii ◽  
Tissue Factor ◽  
Blood Coagulation ◽  
Potential Role ◽  
Initiation Phase

Tissue Factor-Dependent Activation of Factor VIII by Factor VIIa in the Early Phase of Blood Coagulation

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1036-1036
Author(s):  
Tetsuhiro Soeda ◽  
Keiji Nogami ◽  
Tomoko Matsumoto ◽  
Kenichi Ogiwara ◽  
Katsumi Nishiya ◽  
...  
Keyword(s):  
Factor Viii ◽  
Tissue Factor ◽  
Blood Coagulation ◽  
Heavy Chain ◽  
Light Chain ◽  
Early Phase ◽  
Factor Viia ◽  
Clotting Factors ◽  
Initiation Phase ◽  
A1 Domain

Abstract Factor VIIa (FVIIa), complexed with tissue factor (TF), is a trigger of blood coagulation through activation of factor X in the initiation phase. FVIIa can catalyze intrinsic clotting factors such as not only factor IX, but also factor VIII (FVIII). However the role and the mechanisms of the FVIIa-catalyzed FVIII are poorly understood. We first examined FVIIa-catalyzed FVIII activation in the presence of phospholipid (PL) using a one-stage clotting assay. The levels of FVIII activity elevated rapidly by ~4-fold within 30 sec after the addition of FVIIa (1 nM)-TF (1 nM)complex, and subsequently decreased to the initial level within 20 min. This time-dependent reaction was enhanced by the presence of TF and PL in dose-dependent manners, but was moderately inhibited (~50%) in the presence of von Willebrand factor at physiological concentration of 10 μg/mL. FVIII cleavage was evaluated using western blotting immediately after the addition of FVIIa-TF complex. The heavy chain of FVIII was proteolyzed more rapidly (at 15 sec) by cleavages at Arg740 (A2-B junction) and Arg372 (A1-A2 junction) by FVIIa-TF complex, whilst the cleavage at Arg336 in the A1 domain was appeared at ~2.5 min. However little cleavage of the light chain of FVIII was observed, supporting that cleavages at Arg740/Arg372 and Arg336 by FVIIa-TF complex contribute to the up- and down-regulation of FVIII(a) activity, respectively. Of interest, no proteolysis of isolated intact heavy chain was observed, indicating that the proteolysis of the heavy chain was governed by the presence of the light chain. Compared to FVIII activation by thrombin (0.1–1 nM), the activation by FVIIa (0.1–1 nM)-TF (1 nM) complex was observed more rapidly. The activation rate observed by the addition of FVIIa-TF complex was ~50-fold greater than that by thrombin. Kinetics by the chromogenic Xa generation assay showed the catalytic efficiency (kcat/Km; 8.9 min−1/32.8 nM) on FVIIa-TF complex-catalyzed FVIII activation showed ~4-fold greater than that on thrombin-catalyzed activation (kcat/Km; 7.5 min−1/86.4 nM). Furthermore, the catalytic efficiencies on cleavages at Arg740 and Arg372 of FVIII by FVIIa-TF complex were ~3- and ~20-fold greater compared to those by thrombin, respectively. These findings suggested that FVIIa-TF complex was a greater FVIII activator than thrombin in very early phase. In order to localize the binding region for FVIIa, we evaluated the interactions between FVIII subunit and Glu-Gly-Arg-active site modified FVIIa, lacking enzymatic activity, in a surface plasmon resonance-based assay. The heavy chain of FVIII bound to EGR-FVIIa with higher affinity than the light chain (Kd; 2.1 and 45 nM, respectively). Binding was particularly evident with the A2, A3, and C2 domains (Kd; 34, 37, and 44 nM, respectively), whilst the A1 domain failed to bind. In conclusion, we demonstrated that FVIIa-TF complex rapidly activated FVIII by proteolysis of the heavy chain and the activation was governed by the presence of the light chain. Furthermore, present results suggested the role of TF-dependent FVIII activation by FVIIa which is responsible for the initiation phase of blood coagulation.

Effect of factor VIII on tissue factor-initiated spatial clot growth

2003 ◽  
Vol 89 (02) ◽  
pp. 235-242 ◽  
Author(s):  
Mikhail Ovanesov ◽  
Elena Lopatina ◽  
Natalya Ananyeva ◽  
Ljudmila Ul’yanova ◽  
Olga Plyushch ◽  
...  
Keyword(s):  
Factor Viii ◽  
Tissue Factor ◽  
Coagulation Factor ◽  
Time Lapse ◽  
Initiation Phase ◽  
Healthy Donors ◽  
Growth Initiation ◽  
Two Phases ◽  
Elongation Phase

SummaryUsing time-lapse videomicroscopy, we studied the role of coagulation factor VIII (fVIII) in tissue factor-initiated spatial clot growth on fibroblast monolayers in a thin layer of non-stirred recalcified plasma from healthy donors or patients with severe Haemophilia A. Analysis of temporal evolution of light-scattering profiles from a growing clot revealed existence of two phases in the clot growth-initiation phase in a narrow (0.2 mm) zone adjacent to activator surface and elongation phase in plasma volume. While the initiation phase did not differ in normal and haemophilic plasmas, the rate of clot growth in the elongation phase in haemophilic plasma constituted only 30% of that in normal plasma. Supplementation of haemophilic plasma with 0.05 U/ml fVIII restored the normal clot growth rate (44.9 ± 2.5 μm/min) at high but not at low fibroblast density. Our results indicate that the functioning of the intrinsic tenase complex is critical for normal spatial clot growth.

scholarly journals In Hemophilia Α Plasma Treated with Emicizumab, Factor IX Activation By Factor VIIa Drives Thrombin Generation

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 17-17
Author(s):  
Dougald Monroe ◽  
Mirella Ezban ◽  
Maureane Hoffman
Keyword(s):  
Factor Viii ◽  
Tissue Factor ◽  
Plasma Levels ◽  
Thrombin Generation ◽  
Hemophilia A ◽  
Factor Viia ◽  
Factor Ix ◽  
Factor X ◽  
Dose Dependent

Background.Recently a novel bifunctional antibody (emicizumab) that binds both factor IXa (FIXa) and factor X (FX) has been used to treat hemophilia A. Emicizumab has proven remarkably effective as a prophylactic treatment for hemophilia A; however there are patients that still experience bleeding. An approach to safely and effectively treating this bleeding in hemophilia A patients with inhibitors is recombinant factor VIIa (rFVIIa). When given at therapeutic levels, rFVIIa can enhance tissue factor (TF) dependent activation of FX as well as activating FX independently of TF. At therapeutic levels rFVIIa can also activate FIX. The goal of this study was to assess the role of the FIXa activated by rFVIIa when emicizumab is added to hemophilia A plasma. Methods. Thrombin generation assays were done in plasma using 100 µM lipid and 420 µM Z-Gly-Gly-Arg-AMC with or without emicizumab at 55 µg/mL which is the clinical steady state level. The reactions were initiated with low (1 pM) tissue factor (TF). rFVIIa was added at concentrations of 25-100 nM with 25 nM corresponding to the plasma levels achieved by a single clinical dose of 90 µg/mL. To study to the role of factor IX in the absence of factor VIII, it was necessary to create a double deficient plasma (factors VIII and IX deficient). This was done by taking antigen negative hemophilia B plasma and adding a neutralizing antibody to factor VIII (Haematologic Technologies, Essex Junction, VT, USA). Now varying concentrations of factor IX could be reconstituted into the plasma to give hemophilia A plasma. Results. As expected, in the double deficient plasma with low TF there was essentially no thrombin generation. Also as expected from previous studies, addition of rFVIIa to double deficient plasma gave a dose dependent increase in thrombin generation through activation of FX. Interestingly addition of plasma levels of FIX to the rFVIIa did not increase thrombin generation. Starting from double deficient plasma, as expected emicizumab did not increase thrombin generation since no factor IX was present. Also, in double deficient plasma with rFVIIa, emicizumab did not increase thrombin generation. But in double deficient plasma with FIX and rFVIIa, emicizumab significantly increased thrombin generation. The levels of thrombin generation increased in a dose dependent fashion with higher concentrations of rFVIIa giving higher levels of thrombin generation. Conclusion. Since addition of FIX to the double deficient plasma with rFVIIa did not increase thrombin generation, it suggests that rFVIIa activation of FX is the only source of the FXa needed for thrombin generation. So in the absence of factor VIII (or emicizumab) FIX activation does not contribute to thrombin generation. However, in the presence of emicizumab, while rFVIIa can still activate FX, FIXa formed by rFVIIa can complex with emicizumab to provide an additional source of FX activation. Thus rFVIIa activation of FIX explains the synergistic effect in thrombin generation observed when combining rFVIIa with emicizumab. The generation of FIXa at a site of injury is consistent with the safety profile observed in clinical use. Disclosures Monroe: Novo Nordisk:Research Funding.Ezban:Novo Nordisk:Current Employment.Hoffman:Novo Nordisk:Research Funding.

The Tissue Factor Pathway and Wound Healing

2017 ◽  
Vol 44 (02) ◽  
pp. 142-150 ◽  
Author(s):  
Maureane Hoffman
Keyword(s):  
Wound Healing ◽  
Growth Factor ◽  
Cell Signaling ◽  
Tissue Factor ◽  
Blood Coagulation ◽  
Host Response ◽  
Cellular Processes ◽  
Injured Tissue ◽  
Tissue Factor Pathway

AbstractThe role of tissue factor (TF) as the major initiator of hemostatic blood coagulation is well recognized. The ability to form an adequate hemostatic clot is essential to the normal healing of an injury by staunching bleeding, stabilizing the injured tissue, and serving as a scaffold for repair processes. Also, some molecules produced during hemostasis, particularly thrombin, have cytokine and growth factor-like activities that contribute to inflammation and repair. However, TF itself has activities as a regulator of cellular processes via direct signaling, as well as by facilitating activation of proteolytically activated receptors by activated factors VII and X. The importance of hemostasis in the host response to injury makes it very difficult to separate the hemostatic from nonhemostatic effects of TF on wound healing. The literature in this area remains sparse but suggests that TF influences the course and tempo of healing by cell signaling events that impact inflammation, epithelialization, and angiogenesis.

Role of Tissue Factor-FVIIa Blood Coagulation Initiation Complex in Cancer

2017 ◽  
pp. 101-119 ◽  
Author(s):  
Abhishek Roy ◽  
Ramesh Prasad ◽  
Anindita Bhattacharya ◽  
Kaushik Das ◽  
Prosenjit Sen
Keyword(s):  
Tissue Factor ◽  
Blood Coagulation ◽  
Initiation Complex

Thrombin functions during tissue factor–induced blood coagulation

Blood ◽  
2002 ◽  
Vol 100 (1) ◽  
pp. 148-152 ◽  
Author(s):  
Kathleen E. Brummel ◽  
Sara G. Paradis ◽  
Saulius Butenas ◽  
Kenneth G. Mann
Keyword(s):  
Complex Formation ◽  
Tissue Factor ◽  
Blood Coagulation ◽  
Platelet Activation ◽  
Factor Xiii ◽  
Average Rate ◽  
Product Formation ◽  
Initiation Phase ◽  
Factor Va ◽  
Contact Pathway

Abstract Tissue factor–induced blood coagulation was studied in 20 individuals, for varying periods of time during 54 months, in contact pathway–inhibited whole blood at 37°C and evaluated in terms of the activation of various substrates. After quenching over time with inhibitors, the soluble phases were analyzed for thrombin–antithrombin III (TAT) complex formation, prothrombin fragments, platelet activation (osteonectin release), factor Va generation, fibrinopeptide (FP) A and FPB release, and factor XIII activation. TAT complex formation, for 35 experiments, showed an initiation phase (up to 4.6 ± 0.6 minutes) in which thrombin was generated at an average rate of 0.93 ± 0.3 nM/min catalyzed by about 1.3 pM prothrombinase yielding approximately 26 nM thrombin. During a subsequent propagation phase, thrombin was generated at a rate of 83.9 ± 3.8 nM/min by about 120 pM prothrombinase, reaching ultimate levels of 851 ± 53 nM. Clot time, determined subjectively, occurred at 4.7 ± 0.2 minutes and correlated with the inception of the propagation phase. The thrombin concentrations associated with the transitions to rapid product formation are 510 ± 180 pM for platelet activation (1.9 ± 0.2 minutes), 840 ± 280 pM for factor XIII activation and factor Va generation (2.2 ± 0.6 minutes), 1.3 ± 0.4 nM for FPA release (2.5 ± 0.7 minutes), 1.7 ± 0.5 nM for FPB release and prethrombin 2 (2.8 ± 0.8 minutes), 7.0 ± 2.2 nM for thrombin B chain (3.6 ± 0.2 minutes), and 26 ± 6.2 nM for the propagation phase of TAT formation (4.6 ± 0.6 minutes). These results illustrate that the initial activation of thrombin substrates occurs during the initiation phase at less than 2 nM thrombin (0.2%). Most thrombin (96%) is formed well after clotting occurs.

scholarly journals The Potential Role of Emicizuamb Prophylaxis in Severe Von Willebrand Disease

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 20-20
Author(s):  
Assaf Arie Barg ◽  
Gili Kenet ◽  
Tami Livnat ◽  
Einat Avishai ◽  
Ivan Budnik ◽  
...  
Keyword(s):  
Factor Viii ◽  
Potential Role ◽  
Prophylactic Treatment ◽  
Ex Vivo ◽  
Peak Height ◽  
Von Willebrand Disease ◽  
Type 3 ◽  
Von Willebrand

Severe Von Willebrand's disease (VWD) may be associated with chronic joint damage and may require prophylactic therapy. In severe VWD, factor VIII (FVIII) levels are low due to rapid clearness. Emicizumab is a humanized bispecific antibody which mimics the function of coagulation factor VIII (FVIII). It has been approved for prophylaxis in hemophilia A. This is the first study assessing the potential role of emicizumab as an alternative prophylactic treatment in a cohort of patients with severe VWD. We present a TG model evaluating patients' hemostasis following ex vivo spiking of their plasma samples with emicizumab. We also report 24 weeks of successful emicizumab prophylaxis in a child with severe VWD and repeated hemarthroses. A cohort of twenty-four VWD patients were included in the study. Fifty-four percent of our patients were males and the cohort consisted of 14 children (≤18 years) and 10 adults. The majority of patients (96%) were of Caucasian origin. Hemarthrosis was encountered in most type 3 VWD patients, whereas none of the type 2 VWD patients had any joint bleeds. Prophylactic treatment was administered in the majority of type 3 VWD patients, whereas type 2 VWD patients largely required only intermittent on demand therapy applied for bleeding episodes or any surgical interventions. Thrombin generation analysis was carried out blindly in plasma obtained from thirteen type 3 VWD patients and eleven type 2 VWD patients. Seventeen healthy volunteers served as a control group. In plasma from type 3 VWD patients, TG was substantially lower than in plasma from type 2 VWD patients, with ETP of 765 nM×min (596-962) vs. 1954 nM×min (1483-2008) (P = 0•001) and peak height of 47 nM (36-65) vs. 262 nM (142-318) (P = 0•002) In order to examine the potential use of emicizumab as an alternative treatment option for type 3 VWD patients, an ex vivo spiking analysis comparing the effect of Haemate P and emicizumab on TG was performed. An improvement in peak height was demonstrated following spiking with both Haemate P concentrations (P = 0•001 for both) and with the higher emicizumab concentration (P = 0•011). Notably, whereas spiking with both Haemate P concentrations increased peak height to near-normal level, spiking with higher emicizumab concentration increased it to a lesser extent (the median was still lower than in normal controls (P = 0•005). Following the decision to treat our impetus patient with emicizumab prophylaxis, TG analyses were performed in the patient's plasma before and during emicizumab loading and maintenance (Figure 3). As expected, patient's initial TG was extremely low and improved following the first administration of emicizumab loading dose (at week 2 after therapy initiation), at which time emicizumab level was 21 µg/mL. Further significant improvement of TG was noted following loading period completion while emicizumab level was 62 µg/mL. Our patient has been treated with emicizumab for more than six months altogether and did not encounter any joint bleeds since the commencement of therapy. During this period, a single dose of Haemate P was administered following tooth exfoliation. Our study contributes towards a better understanding of TG as a surrogate marker of VWD patients' hemostasis. Our data suggests that some severe VWD patients could be safely and efficiently treated with emicizumab. The successful prophylaxis of our patient and our ex vivo laboratory findings should set the ground for further collaborative multicenter studies to examine the efficacy and safety of emicizumab prophylaxis in type 3 VWD patients. Disclosures Barg: roshe: Honoraria, Speakers Bureau. Kenet:PI Healthcare, CSL Behring: Honoraria; Bayer, Pfizer, Takeda, BioMarin, Novo Nordisk: Speakers Bureau; Bayer, Pfizer, Roche, Alnylam (Sanofi), Shire: Research Funding; Bayer, Pfizer, BioMarin, Takeda, Roche, Novo Nordisk, Sanofi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. OffLabel Disclosure: The use of Emicizumab in sever Von Willebrand disease

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