scholarly journals Conformational Plasticity-Rigidity Axis of the Coagulation Factor VII Zymogen Elucidated by Atomistic Simulations of the N-Terminally Truncated Factor VIIa Protease Domain

Biomolecules ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 549
Author(s):  
Jesper J. Madsen ◽  
Ole H. Olsen
Keyword(s):  
Factor Viia ◽  
Conformational Dynamics ◽  
Coagulation Factor ◽  
Factor Vii ◽  
Atomistic Simulations ◽  
Protease Domain ◽  
Proteolytic Activation ◽  
Coagulation Factor Vii ◽  
Conformational Plasticity ◽  
And Function

The vast majority of coagulation factor VII (FVII), a trypsin-like protease, circulates as the inactive zymogen. Activated FVII (FVIIa) is formed upon proteolytic activation of FVII, where it remains in a zymogen-like state and it is fully activated only when bound to tissue factor (TF). The catalytic domains of trypsin-like proteases adopt strikingly similar structures in their fully active forms. However, the dynamics and structures of the available corresponding zymogens reveal remarkable conformational plasticity of the protease domain prior to activation in many cases. Exactly how ligands and cofactors modulate the conformational dynamics and function of these proteases is not entirely understood. Here, we employ atomistic simulations of FVIIa (and variants hereof, including a TF-independent variant and N-terminally truncated variants) to provide fundamental insights with atomistic resolution into the plasticity-rigidity interplay of the protease domain conformations that appears to govern the functional response to proteolytic and allosteric activation. We argue that these findings are relevant to the FVII zymogen, whose structure has remained elusive despite substantial efforts. Our results shed light on the nature of FVII and demonstrate how conformational dynamics has played a crucial role in the evolutionary adaptation of regulatory mechanisms that were not present in the ancestral trypsin. Exploiting this knowledge could lead to engineering of protease variants for use as next-generation hemostatic therapeutics.

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 Funksionele beskrywing van ’n faktor VIIa inhiberende peptied, IP-7, geselekteer deur faagblootleggingstegnologie

2006 ◽  
Vol 25 (4) ◽  
pp. 209-220
Author(s):  
S.M. Meiring ◽  
C.E. Roets ◽  
P.N. Badenhorst
Keyword(s):  
Phage Display ◽  
Tissue Factor ◽  
Factor Viia ◽  
Coagulation Factor ◽  
Competitive Inhibitor ◽  
Factor Vii ◽  
Antithrombotic Agent ◽  
Phage Display Technology ◽  
Current Form ◽  
Coagulation Factor Vii

Die tegniek van faagblootlegging is gebruik om ’n sikliese heptapeptied te selekteer wat met weefselfaktor(WF) kompeteer vir binding aan stollingsfaktor VIIa. Die aminosuurvolgorde van die peptied is Cys-Ala- Trp-Pro-His-Thr-Pro-Asp-Cys (C-AWPHTPD-C) en dit verleng die protrombientyd (PT) op ’n konsentrasie-afhanklike wyse. Die peptied beperk plaatjieklewing aan beide menslike endoteelsel- en weefselfaktormatrikse in ’n vloeikamermodel onder arteriële vloeitoestande. Die peptied funksioneer as ’n volledig mededingende inhibeerder van faktor VIIa met ’n inhibisiekonstante (Ki) van 123,2 μM. In sy huidige vorm is die peptied waarskynlik nie sterk genoeg om verder as antitrombotiese middel ontwikkel te word nie, maar verskillende strategieë kan gevolg word om die werking daarvan te versterk. AbstractFunctional characterisation of a factor VIIa inhibiting peptide, IP-7 selected by phage display technology By using the technique of phage display, we selected a cyclic heptapeptide sequence Cys-Ala-Trp-Pro-His-Thr-Pro-Asp-Cys (C-AWPHTPD-C) that competes with tissue factor for binding to coagulation factor VII. This peptide prolongs the prothrombin time (PT) in a concentration dependent way. It also reduces platelet adhesion to both human endothelial cell and tissue factor matrixes in a flow chamber under arterial flow conditions. Furthermore, it acts as a full competitive inhibitor of factor VIIa with an inhibition constant (Ki) of 123,2 μM. In its current form the peptide is probably not sufficiently potent for development as an antithrombotic agent, but different strategies could be followed to reinforce its performance.

scholarly journals A severe deficiency of coagulation factor VIIa results in attenuation of the asthmatic response in mice

2009 ◽  
Vol 296 (5) ◽  
pp. L763-L770 ◽  
Author(s):  
Kazuhiko Shinagawa ◽  
Victoria A. Ploplis ◽  
Francis J. Castellino
Keyword(s):  
Airway Hyperresponsiveness ◽  
Factor Viia ◽  
Coagulation Factor ◽  
Lavage Fluid ◽  
Factor Vii ◽  
Factor X ◽  
Asthmatic Response ◽  
Mucin Production ◽  
Coagulation Factor Vii ◽  
Lung Eosinophilia

Eosinophil counts in the bronchoalveolar lavage fluid of wild-type (WT) mice increased after ovalbumin (OVA) challenge, a response that was diminished in comparably challenged low-expressing coagulation factor VII (FVIItTA/tTA) mice. Levels of T helper type 2 (Th2) cytokines, IL-4, IL-5, and IL-13, and eosinophil-attracting chemokines, eotaxin and RANTES, were also lower in the OVA-challenged FVIItTA/tTAmice. Eosinophils purified from low-FVII mice underwent apoptosis at a faster rate compared with WT eosinophils, and eosinophil migration in response to eotaxin was reduced in eosinophils obtained from FVIItTA/tTAmice. Airway hyperresponsiveness and mucous layer thickness were reduced in OVA-treated FVIItTA/tTAmice, and addition of exogenous coagulation factor X (FX) enhanced mucin production in human epithelial NCI-H292 cells. Correspondingly, incubation of FX with NCI-H292 cells resulted in activated (a) FX production, suggesting that the components required for FX activation were present on NCI-H292 cells. These results demonstrate that FVIIa functions in the asthmatic response to an allergen by stimulating lung eosinophilia, airway hyperresponsiveness, and mucin production, this latter effect through its ability to activate FX in conjunction with tissue factor.

Generation of Transgenic Mice Expressing High Levels of Activated Murine Coagulation Factor VII.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 5253-5253
Author(s):  
Majed N. Aljamali ◽  
Paris Margaritis ◽  
Alexander Schlachterman ◽  
Katherine A. High
Keyword(s):  
Transgenic Mice ◽  
Factor Viia ◽  
Coagulation Factor ◽  
Factor Vii ◽  
Wild Type ◽  
Extrinsic Pathway ◽  
Coagulation Activity ◽  
Coagulation Factor Vii ◽  
Gene Therapy Approach

Abstract Treatment of acute bleeding episodes in hemophilic patients with inhibitors can be successfully managed by the infusion of recombinant human factor VIIa (rhFVII, NovoSeven“). We have recently shown the efficacy of a gene transfer approach to treat hemophilia B (HB) mice by adeno-associated virus (AAV) expressing activated murine FVII (mFVIIa) (J Clin Invest. 2004 Apr; 113(7): 1025–31). To assess the consequences of long-term expression of different levels of mFVIIa, we generated transgenic mice expressing mFVIIa driven by a liver-restricted (transthyretin) promoter. Results from four founders have been analyzed. The levels of mFVII antigen in both founders and their offspring were 3.5–7.5 microgram/ml, about 2.5–5 fold the baseline compared to their non-transgenic littermates. Moreover, the expressed protein retains its coagulation activity in the extrinsic pathway as demonstrated by shortening of the prothrombin time (PT) from 22.3±0.6 sec in the non-transgenic mice to 12.3±1.6 sec in the transgenic littermates. We found two male HB mice that were also transgenic for mFVIIa, resulting from the breeding of one male founder and an HB heterozygote female. The high levels of mFVII antigen (7.5 and 5.5 microgram/ml) were accompanied by significantly shorter PTs (9.8 and 12.3, respectively) compared to wild-type baseline of 22 sec, and most importantly, by a shorter activated partial thromboblastin time (aPTT) compared to their two HB littermates (36.3 and 28.8 versus 61.3 and 61.8 sec, respectively), i.e., mice transgenic for mFVIIa show aPTT similar to their wild type littermates. Additionally, kinetics and general characteristics of in vivo clot formation after laser-induced injuries to the arteries of the cremaster muscle were similar in an HB-mFVIIa transgenic mouse (the only one tested) and in normal mice, while clots were absent in HB control mice. On the other hand, thrombin anti-thrombin (TAT) levels in the transgenic mice were comparable to their HB and wild type littermates. These findings support the efficacy and safety of a gene therapy approach for the expression of mFVIIa and should further allow us to assess the risk of continuous expression of elevated levels of mFVIIa in mouse plasma.

Two Independent Binding Sites on Monolayers of Human Endothelial Cells Are Responsible for Interaction with Coagulation Factor VII and Factor VIIa

1993 ◽  
Vol 69 (02) ◽  
pp. 197-204 ◽  
Author(s):  
Ute Reuning ◽  
Klaus T Preissner ◽  
Gert Müller-Berghaus
Keyword(s):  
Endothelial Cells ◽  
Vitamin K ◽  
Binding Sites ◽  
Radioligand Binding ◽  
Factor Viia ◽  
Coagulation Factor ◽  
Factor Vii ◽  
Factor X ◽  
Calcium Dependent ◽  
Coagulation Factor Vii

SummaryThe interaction of radiolabeled factor VII (FVII) and factor VIIa (FVIIa) with endotoxin-stimulated endothelial cells (EC), known to express tissue factor (TF), and unstimulated EC was studied. FVII/FVIIa binding to EC-monolayers was saturable within 4.5-6 h, reversible, temperature and calcium dependent on both, endotoxin-stimulated and on unstimulated EC. Upon 2 h of incubation on EC, FVII was partially converted to FVIIa in the absence of protease inhibitors. The affinity of this binding was K d = 45.4 ± 18.7 nM with a calculated number of binding sites B max = 3.75 ± 0.31 × 106 molecules/cell. In addition to unlabeled FVII and FVIIa, other vitamin K-dependent proteins reduced binding of [125I]-FVII/FVIIa to about 60-70%, and this type of common binding site for vitamin K-dependent proteins revealed a K d = 32.2 ± 5.6 nM and a B max = 3.03 ± 0.14 × 106 molecules/cell. Moreover, in the presence of 1 μM prothrombin to suppress common binding sites, only on endotoxin-stimulated EC additional inhibition of FVII/FVIIa binding was achieved by anti-TF antibodies. The characteristics of the FVII/FVIIa-TF interaction with a K d = 17.2 ± 5.2 nM and a B max = 342,000 ± 1,100 binding sites/cell revealed a similar saturation kinetics in radioligand binding and in functional factor X activation within 90-120 min. These data indicate the presence of at least two independent binding sites for FVII/FVIIa on stimulated EC of which about 10% are TF specific. The existence of binding sites common for vitamin K-dependent proteins on both types of EC may improve the availability of FVII/FVIIa once EC become stimulated and express TF on their surface.

Activated Recombinant Factor VIIa for Intractable Extra-Cranial Hemorrhage in 55 Consecutive Patients

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1264-1264
Author(s):  
Brynjar Vidarsson ◽  
Robert Palmason ◽  
Tomas Gudbjartsson ◽  
Pall T. Onundarson
Keyword(s):  
Clinical Response ◽  
Heart Surgery ◽  
Factor Viia ◽  
Post Partum ◽  
Open Heart Surgery ◽  
Coagulation Factor ◽  
Factor Vii ◽  
Off Label ◽  
Coagulation Factor Vii ◽  
Transfusion Requirements

Abstract Abstract 1264 Background: Recombinant activated coagulation factor VII (rFVIIa) has been increasingly used in non-hemophiliac patients for unapproved indications. We undertook a 10 year nation-wide retrospective survey in order to evaluate the use of rFVIIa in Iceland in patients with intractable extra-cranial hemorrhage. Material and methods: Hospital charts of all patients that received rFVIIa between 1999–2008 at Landspitali, the single institution administering rFVIIa in Iceland, were reviewed and indications, coagulation profiles and clinical outcome were evaluated. This analysis focuses on patients treated off-label for desperate (intractable) extra-cranial hemorrhage (IH), ie only patients in who all other measures were considered to have failed to stop hemorrhage. Results: rFVIIa was used for IH in 55 patients (median age 53 yrs, range; 0–84, 51% males). This included IH in open heart surgery (n=23), non-cardiac surgery (n=7), post-partum hemorrhage (n=9), multitrauma (n=8) and miscellaneous (n=8). A single rFVIIa dose was used in 86% of cases and hemorrhage was considered effectively contained by immediate clinical response to rFVIIa in 81% of the IH cases. Survival at 30 days for all patients was 68%. Out of 55 patients with IH, six died within 24 hours of administration of rFVIIa (five assessed clinically as rFVIIa non-responders) and 11 other patients (total 17 patients, 31%) died within 30 days (six non-responders). The 24-hour mortality in rFVIIa clinical responders and non-responders was 2% and 50% respectively (p=0.0004) and the 30 day mortality was 25% and 60% respectively (p=0.05). The need for transfusion of blood products was significantly decreased (p<0.01) and also the prothrombin time (20.0 to 13.3 sec, p<0.0001). Conclusion: The majority of unselected consecutive patients receiving rFVIIa as last resort treatment for IH were considered to have favorable immediate clinical response as well as reduced transfusion requirements and normalisation of coagulation parameters. Also, patients deemed with clinical response to rFVIIa may have had lower mortality. Disclosures: Off Label Use: Activated recombinant factor VII for use in desperately bleeding patients.

Blocking the Initiation of Coagulation by RNA Aptamers to Factor VIIa

2000 ◽  
Vol 84 (11) ◽  
pp. 841-848 ◽  
Author(s):  
H. Lyerly ◽  
Jeffrey Lawson ◽  
Christopher Rusconi ◽  
Alice Yeh ◽  
Bruce Sullenger
Keyword(s):  
Human Plasma ◽  
Tissue Factor ◽  
In Vitro Selection ◽  
Factor Viia ◽  
Coagulation Factor ◽  
Factor Vii ◽  
Dependent Manner ◽  
Factor X ◽  
Coagulation Factor Vii

SummaryThe tissue factor/factor VIIa complex is thought to be the primary initiator of most physiologic blood coagulation events. Because of its proximal role in this process, we sought to generate new inhibitors of tissue factor/factor VIIa activity by targeting factor VIIa. We employed a combinatorial RNA library and in vitro selection methods to isolate a high affinity, nuclease-resistant RNA ligand that binds specifically to coagulation factor VII/VIIa. This RNA inhibits the tissue factordependent activation of factor X by factor VIIa. Kinetic analyses of the mechanism of action of this RNA suggest that it antagonizes factor VIIa activity by preventing formation of a functional factor VII/tissue factor complex. Furthermore, this RNA significantly prolongs the prothrombin time of human plasma in a dose dependent manner, and has an in vitro half-life of ∼15 h in human plasma. Thus, this RNA ligand represents a novel class of anticoagulant agents directed against factor VIIa.

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