scholarly journals How Na+ activates thrombin – a review of the functional and structural data

2008 ◽  
Vol 389 (8) ◽  
Author(s):  
James A. Huntington
Keyword(s):  
Crystal Structures ◽  
Blood Coagulation ◽  
Protein C ◽  
Coagulation Factor ◽  
Structural Data ◽  
Monovalent Cation ◽  
Coagulation Cascade ◽  
Structural Basis ◽  
Thrombin Activity ◽  
Great Relevance

Abstract Thrombin is the ultimate coagulation factor; it is the final protease generated in the blood coagulation cascade and is the effector of clot formation. Regulation of thrombin activity is thus of great relevance to determining the correct haemostatic balance, with dysregulation leading to bleeding or thrombosis. One of the most enigmatic and controversial regulators of thrombin activity is the monovalent cation Na+. When bound to Na+, thrombin adopts a ‘fast’ conformation which cleaves all procoagulant substrates more rapidly, and when free of Na+, thrombin reverts to a ‘slow’ state which preferentially activates the protein C anticoagulant pathway. Thus, Na+-binding allosterically modulates the activity of thrombin and helps determine the haemostatic balance. Over the last 30 years, there has been much research investigating the structural basis of thrombin allostery. Biochemical and mutagenesis studies established which regions and residues are involved in the slow→fast conformational change, and recently several crystal structures of the putative slow form have been solved. In this article, the biochemical and crystallographic data are reviewed to see if we are any closer to understanding the conformational basis of the Na+ activation of thrombin.

scholarly journals Quantitative measurement of APC-Resistant factor V clotting activity (FV-Leiden) and potential graduation of the associated thrombo-embolic risk

2021 ◽  
Vol 9 (1) ◽  
Author(s):  
PEYRAFITTE MARIE ◽  
VISSAC MARIE ◽  
AMIRAL JEAN
Keyword(s):  
Blood Coagulation ◽  
Protein C ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Factor V Leiden ◽  
Coagulation Factor ◽  
Protein S ◽  
Coagulation Cascade ◽  
Factor V ◽  
Thrombotic Risk

Coagulation Factor V (FV) is a key factor for regulating blood coagulation cascade, and it acts at the crossroads of the intrinsic and extrinsic pathways. It shows a dual activity as the procoagulant cofactor for Factor Xa in the prothrombinase complex, but it also supports an anticoagulant activity in combination with TFPI and Protein S. Its rapid cleavage by Activated Protein C (APC) complexed with Free Protein S (FPS), in presence of phospholipids and calcium, inhibits its activity and limits the propagation of blood coagulation, keeping it to where it is beneficial. Rapid inactivation of active FV by APC-FPS is essential for preventing the risk of thrombosis development. In 1993, Dahlbäck and coworkers reported an inherited disorder characterized by activated protein C resistance (APC-R) and associated to an increased occurrence of thromboembolic events in affected families. In 1994 Bertina demonstrated that this diathesis resulted from a Factor V mutation (R506Q), rendering this factor resistant to inactivation by APC. This mutated Factor V was called Factor V Leiden (FV-L). APTT based assays and molecular biology methods for detecting the mutation were developed, but these methods are only qualitative and classify tested individuals as normals, heterozygous or homozygous for the coagulation defect. Our group developed a quantitative assay for FV-L, which is described in this report, along with its performances. This assay allows to quantitate specifically FV-L coagulant activity, and to graduate its amount in heterozygous or homozygous patients. FV-L is absent in normal individuals and present in homozygous or heterozygous patients, accounting respectively for 100 % or 50 % of blood FV. Its amount is compared with FV clotting activity or antigenic concentration. Measured FV-L activities overlap between heterozygous patients with high FV and homozygous ones with low FV levels. This assay allows to better discriminate for the FV-L associated thrombotic risk, which depends on the effective FV-L concentration rather than on patients’ genetic status. This expectation is supported by literature review, which shows that FV-L concentrations correlate with presence of platelet released microparticles in patients carrying that mutation.

EXPRESSION IN ONTOGENESIS OF HUMAN BLOOD COAGULATION FACTORS

1987 ◽  
Author(s):  
H J Hassan ◽  
A Leonardi ◽  
C Chelucci ◽  
R Guerriero ◽  
P M Mannucci ◽  
...  
Keyword(s):  
Blood Coagulation ◽  
Protein C ◽  
Antithrombin Iii ◽  
Liver Homogenate ◽  
Coagulation Factor ◽  
Coagulation Factors ◽  
Factor Ix ◽  
Adult Liver ◽  
Blood Coagulation Factors ◽  
Guanidine Isothiocyanate

We have analyzed the expression of several blood coagulation factors (IX, VIII, X, fibrinogen chains) and inhibitors (antithrombin III, protein C) in human embryonic and fetal livers, obtained from legal abortions at 6-11 week post-conception. The age was established by morphologic staging and particularly crown-rump lenght measurement.Total cellular RNA was isolated from partially purified hepatocytes or total liver homogenate using the guanidine isothiocyanate method. Poly(A)+ RNA was selected by oligodT cellulose chromatography. The size and the number of the embryonic and fetal transcripts are equivalent to those observed in adult liver, as evaluated by Northern blot analysis of total or poly(A)+ RNA hybridized to human cDNA probes.The level of coagulation factor transcripts in embryonic and fetal liver was evaluated by dot hybridization of total RNA (0.5-10 ug), as compared to RNA extracted from normal adult liver biopsies. The expression of blood coagulation factors in embryos is generally reduced for all factors, but at a different degree. In 5-11 wk liver, the level of factor IX is 5-10% of that observed in adults, while fibrinogen, protein C, antithrombin III RNA level rises from 25 to 50% and factor X is expressed at a level comparable to that observed in adult liver.We conclude that during these stages of development blood coagulation factors are expressed according to three different time, curves, possibly due to the effect of different types of regulatory mechanisms.

scholarly journals Cryo-EM structures of human coagulation factors V and Va

Blood ◽  
2021 ◽  
Author(s):  
Eliza A Ruben ◽  
Michael J Rau ◽  
James Fitzpatrick ◽  
Enrico Di Cera
Keyword(s):  
Protein C ◽  
Activated Protein C ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Coagulation Factors ◽  
Proteolytic Processing ◽  
Coagulation Cascade ◽  
Factor V ◽  
Prothrombinase Complex ◽  
A2 Domain

Coagulation factor V is the precursor of factor Va that, together with factor Xa, Ca2+ and phospholipids, defines the prothrombinase complex and activates prothrombin in the penultimate step of the coagulation cascade. Here we present cryo-EM structures of human factors V and Va at atomic (3.3 Å) and near-atomic (4.4 Å) resolution, respectively. The structure of fV reveals the entire A1-A2-B-A3-C1-C2 assembly but with a surprisingly disordered B domain. The C1 and C2 domains provide a platform for interaction with phospholipid membranes and support the A1 and A3 domains, with the A2 domain sitting on top of them. The B domain is highly dynamic and visible only for short segments connecting to the A2 and A3 domains. The A2 domain reveals all sites of proteolytic processing by thrombin and activated protein C, a partially buried epitope for binding factor Xa and fully exposed epitopes for binding activated protein C and prothrombin. Removal of the B domain and activation to fVa exposes the sites of cleavage by activated protein C at R306 and R506 and produces increased disorder in the A1-A2-A3-C1-C2 assembly, especially in the C-terminal acidic portion of the A2 domain responsible for prothrombin binding. Ordering of this region and full exposure of the factor Xa epitope emerge as a necessary step for the assembly of the prothrombin-prothrombinase complex. These structures offer molecular context for the function of factors V and Va and pioneer the analysis of coagulation factors by cryo-EM.

scholarly journals Role of the blood coagulation cascade in hepatic fibrosis

2018 ◽  
Vol 315 (2) ◽  
pp. G171-G176 ◽  
Author(s):  
Asmita Pant ◽  
Anna K. Kopec ◽  
James P. Luyendyk
Keyword(s):  
Blood Coagulation ◽  
Hepatic Fibrosis ◽  
Animal Studies ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Primary Source ◽  
Bleeding Risk ◽  
Normal Function ◽  
Coagulation Cascade ◽  
Rapid Progression

Liver is the primary source of numerous proteins that are critical for normal function of the blood coagulation cascade. Because of this, diseases of the liver, particularly when affiliated with severe complications like cirrhosis, are associated with abnormalities of blood clotting. Although conventional interpretation has inferred cirrhosis as a disorder of uniform bleeding risk, it is now increasingly appreciated as a disease wherein the coagulation cascade is precariously rebalanced. Moreover, prothrombotic risk factors are also associated with a more rapid progression of fibrosis in humans, suggesting that coagulation proteases participate in disease pathogenesis. Indeed, strong evidence drawn from experimental animal studies indicates that components of the coagulation cascade, particularly coagulation factor Xa and thrombin, drive profibrogenic events, leading to hepatic fibrosis. Here, we concisely review the evidence supporting a pathologic role for coagulation in the development of liver fibrosis and the potential mechanisms involved. Further, we highlight how studies in experimental animals may shed light on emerging clinical evidence, suggesting that beneficial effects of anticoagulation could extend beyond preventing thrombotic complications to include reducing pathologies like fibrosis.

scholarly journals Expression of human blood coagulation factor XI: characterization of the defect in factor XI type III deficiency

Blood ◽  
1992 ◽  
Vol 79 (6) ◽  
pp. 1435-1440 ◽  
Author(s):  
JC Meijers ◽  
EW Davie ◽  
DW Chung
Keyword(s):  
Blood Coagulation ◽  
Messenger Rna ◽  
Coagulation Factor ◽  
Coagulation Cascade ◽  
Mutant Protein ◽  
Chain Molecule ◽  
Single Chain ◽  
Factor Xi ◽  
Bhk Cells ◽  
Type Iii

Human factor XI (FXI) is a blood coagulation factor participating in the early phase of the intrinsic pathway of blood coagulation. It circulates in blood as a glycoprotein composed of two identical chains held together by a single disulfide bond between the fourth apple domains. FXI has been expressed in baby hamster kidney (BHK) cells, where it was synthesized as a single-chain molecule that was converted to the dimer before secretion. The recombinant protein was fully active in a clotting assay, indicating that it interacted readily with other components of the coagulation cascade. A mutant FXI in which Phe283 was converted to Leu (Phe283Leu) was also expressed in BHK cells. This amino acid change occurs in the fourth apple domain of FXI and corresponds to the type III deficiency in Ashkenazi Jews. The mutant protein was secreted at reduced levels (about 8%) compared with normal FXI. This was due to a defect in the dimerization of the molecule rather than a decrease in the transcription of type III messenger RNA. Once secreted, however, the mutant protein consisted of a dimer with full biologic activity. The in vitro expression of FXI indicated that the impaired dimerization and secretion of the Phe283Leu mutant can account for the defect found in patients who are homozygous for the type III FXI deficiency.

Recombinant Thrombomodulin and Activated Protein C in the Treatment of Disseminated Intravascular Coagulation

1999 ◽  
Vol 82 (08) ◽  
pp. 718-721 ◽  
Author(s):  
Ikuro Maruyama
Keyword(s):  
Endothelial Cells ◽  
Tissue Factor ◽  
Blood Coagulation ◽  
Disseminated Intravascular Coagulation ◽  
Protein C ◽  
Activated Protein C ◽  
Coagulation Cascade ◽  
Regulation System ◽  
Luminal Surface ◽  
Intravascular Coagulation

IntroductionThe blood coagulation cascade is regulated by the luminal surface of the endothelial cell lining.1 Endothelial cells synthesize tissue factor pathway inhibitor (TFPI), which, in part, binds to the cell surface glycosaminoglycans and inhibits factors Xa, VIIa, and tissue factor.2 Endothelial cells also produce and exhibit thrombomodulin (TM) on their luminal surface.3 TM is a kind of thrombin receptor that forms a 1:1 complex with thrombin. In this complex, thrombin activates protein C (PC) more than 1,000-fold more than thrombin alone. TM then loses its procoagulant activities, which include fibrinogen clotting, activation of factors V and VIII, and platelet activation. Thus, TM converts thrombin from a procoagulant protease to an anticoagulant. Pathologic states, such as an endothelial injury or perturbation or continuous rapid coagulation cascade activation, overcomes the endothelial regulating activity, resulting in the development of intravascular coagulation and the induction of disseminated intravascular coagulation (DIC). Theoretically, then, supplementing soluble TM or activated PC (APC) to reconstitute the endothelial coagulation regulation system in the circulation and regulate pathologically-activated blood coagulation could be beneficial. In this chapter, application of soluble TM and APC in the treatment of DIC is reviewed.

scholarly journals Expression of human blood coagulation factor XI: characterization of the defect in factor XI type III deficiency

Blood ◽  
1992 ◽  
Vol 79 (6) ◽  
pp. 1435-1440 ◽  
Author(s):  
JC Meijers ◽  
EW Davie ◽  
DW Chung
Keyword(s):  
Blood Coagulation ◽  
Messenger Rna ◽  
Coagulation Factor ◽  
Coagulation Cascade ◽  
Mutant Protein ◽  
Chain Molecule ◽  
Single Chain ◽  
Factor Xi ◽  
Bhk Cells ◽  
Type Iii

Abstract Human factor XI (FXI) is a blood coagulation factor participating in the early phase of the intrinsic pathway of blood coagulation. It circulates in blood as a glycoprotein composed of two identical chains held together by a single disulfide bond between the fourth apple domains. FXI has been expressed in baby hamster kidney (BHK) cells, where it was synthesized as a single-chain molecule that was converted to the dimer before secretion. The recombinant protein was fully active in a clotting assay, indicating that it interacted readily with other components of the coagulation cascade. A mutant FXI in which Phe283 was converted to Leu (Phe283Leu) was also expressed in BHK cells. This amino acid change occurs in the fourth apple domain of FXI and corresponds to the type III deficiency in Ashkenazi Jews. The mutant protein was secreted at reduced levels (about 8%) compared with normal FXI. This was due to a defect in the dimerization of the molecule rather than a decrease in the transcription of type III messenger RNA. Once secreted, however, the mutant protein consisted of a dimer with full biologic activity. The in vitro expression of FXI indicated that the impaired dimerization and secretion of the Phe283Leu mutant can account for the defect found in patients who are homozygous for the type III FXI deficiency.

Mutation in blood coagulation factor V associated with resistance to activated protein C

Nature ◽  
1994 ◽  
Vol 369 (6475) ◽  
pp. 64-67 ◽  
Author(s):  
Rogier M. Bertina ◽  
Bobby P. C. Koeleman ◽  
Ted Koster ◽  
Frits R. Rosendaal ◽  
Richard J. Dirven ◽  
...  
Keyword(s):  
Blood Coagulation ◽  
Protein C ◽  
Activated Protein C ◽  
Coagulation Factor ◽  
Factor V ◽  
Coagulation Factor V ◽  
Blood Coagulation Factor V

scholarly journals Exposure of R169 controls protein C activation and autoactivation

Blood ◽  
2012 ◽  
Vol 120 (3) ◽  
pp. 664-670 ◽  
Author(s):  
Nicola Pozzi ◽  
Sergio Barranco-Medina ◽  
Zhiwei Chen ◽  
Enrico Di Cera
Keyword(s):  
Protein C ◽  
Feedback Loop ◽  
Activated Protein C ◽  
Structural Data ◽  
Coagulation Cascade ◽  
Wild Type ◽  
Ionic Interaction ◽  
New Paradigm ◽  
A Value ◽  
The Poor

Abstract Protein C is activated by thrombin with a value of kcat/Km = 0.11mM−1s−1 that increases 1700-fold in the presence of the cofactor thrombomodulin. The molecular origin of this effect triggering an important feedback loop in the coagulation cascade remains elusive. Acidic residues in the activation domain of protein C are thought to electrostatically clash with the active site of thrombin. However, functional and structural data reported here support an alternative scenario. The thrombin precursor prethrombin-2 has R15 at the site of activation in ionic interaction with E14e, D14l, and E18, instead of being exposed to solvent for proteolytic attack. Residues E160, D167, and D172 around the site of activation at R169 of protein C occupy the same positions as E14e, D14l, and E18 in prethrombin-2. Caging of R169 by E160, D167, and D172 is responsible for much of the poor activity of thrombin toward protein C. The E160A/D167A/D172A mutant is activated by thrombin 63-fold faster than wild-type in the absence of thrombomodulin and, over a slower time scale, spontaneously converts to activated protein C. These findings establish a new paradigm for cofactor-assisted reactions in the coagulation cascade.

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