Membrane Phosphatidylserine and Plasma Calcium Levels Switch Factor Xa From An Inactive Dimer to An Active Monomer.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3180-3180
Author(s):  
Tilen Koklic ◽  
Rinku Majumder ◽  
Barry Lentz
Keyword(s):  
Factor Xa ◽  
Coagulation Cascade ◽  
Concentration Addition ◽  
Main Role ◽  
Dimer Formation ◽  
Proteolytic Activation ◽  
Fixed Membrane ◽  
Short Period ◽  
Tight Association ◽  
Membrane Concentration

Abstract Abstract 3180 Poster Board III-103 Factor Xa has a prominent role in amplifying both inflammation and coagulation cascades. In the coagulation cascade, its main role is catalyzing the proteolytic activation of prothrombin to thrombin. Efficient proteolysis is well known to require phosphatidylserine (PS)-containing membranes that are provided by platelets in vivo. PS, in the presence of Ca2+, triggers tight association of factor Xa with its cofactor, factor Va. PS also triggers tight association of factor Xa with factor Xa, at least in solution (Majumder R, Wang JF, and Lentz BR. Biophys. J. 2003, 84:1238-1251) to form an inactive factor Xa dimer (Chattopadhyay et al. Biophys. J. 96(3) pp. 974 – 986, 2009). We report here that PS-triggered factor Xa dimer formation is a sharp sigmoidal function of Ca2+ concentration and that this Ca2+ switch occurs just below plasma Ca2+ concentrations. We have determined the proteolytic activity of human factor Xa towards human prethrombin2 as a substrate both at fixed membrane concentration and increasing factor Xa concentration, and at fixed factor Xa concentration and increasing membrane concentration. Neither of these experiments showed the expected behavior of an increase in activity as factor Xa bound to membranes. Factor Xa activity actually decreased as low concentrations of PS-containing membranes were added, and increased only at higher membrane concentrations. At fixed membrane concentrations, the total factor Xa activity did not increase proportionally with factor Xa concentration. These observations showed that membranes actually inhibited factor Xa activity under conditions of high factor Xa or low membrane concentrations, suggesting the existence of inactive membrane-bound oligomers of factor Xa. The binding of factor Xa to PS-containing membranes also appeared to be tighter at low than at high membrane concentration. Because factor Xa forms dimers in solution (Majumder R, Wang JF, and Lentz BR. Biophys. J. 2003, 84:1238-1251), we attempted to explain these observations in terms of a model that takes into account dimerization of factor Xa after binding to a membrane. This dimer model successfully described all our data, with the parameters of best fit being kcat/KMdimer = 0 M-1s-1, kcat/KMmonomer = 9000 M-1s-1, kcat/KMsolution = 94 M-1s-1, and Kd,surfacedimer = ( 40±25) · 10-15 mol/(dm)2. This surface dimerization constant corresponds to a solution-phase Kddimer = 1 nM at 10 mM lipid concentration, nearly what we observed for formation of bovine factor Xa dimer in the presence of short-chain PS (20 nM; Majumder et al. Biophys. J. 2003, 84:1238-1251). As we observed for soluble-PS-induced dimer formation in solution, dimer formation on a membrane was Ca2+ dependent. Unlike in solution, factor Xa was activated by membrane binding below 1.5 mM Ca2+, but inactivated above this Ca2+ concentration. The transition of factor Xa from monomer to inactive dimer state on PS-containing membranes is a sensitive function of Ca2+ concentration. Just below the normal range of plasma Ca2+ concentration, addition of PS-containing membranes promotes factor Xa activity, while just above this level PS-containing membranes inhibit factor Xa. This suggests that Ca2+-dependent formation of inactive factor Xa dimers might have an important role in factor Xa activity during the initial phase of the blood coagulation process when generation of a small amount of thrombin in a short period of time activates platelets. Supported by USPHS grant HL072827. Disclosures No relevant conflicts of interest to declare.

A Ca2+ Switch Triggers Formation of Inactive Factor Xa Dimers on Phosphatidylserine-Containing Membranes.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1708-1708
Author(s):  
Tilen Koklic ◽  
Gabriel E. Weinreb ◽  
Rinku Majumder ◽  
Barry R. Lentz
Keyword(s):  
Factor Xa ◽  
Coagulation Cascade ◽  
Main Role ◽  
Dimerization Constant ◽  
Dimer Formation ◽  
Proteolytic Activation ◽  
Fixed Membrane ◽  
Tight Association ◽  
Membrane Concentration

Abstract Factor Xa has a prominent role in amplifying both inflammation and coagulation cascades. In the coagulation cascade, its main role is catalyzing the proteolytic activation of prothrombin to thrombin. Efficient proteolysis is well known to require phosphatidylserine (PS)-containing membranes that are provided by platelets in vivo. PS, in the presence of Ca2+, triggers tight association of factor Xa with its cofactor, factor Va. An interesting complication is that PS also triggers tight association of factor Xa with factor Xa, at least in solution (Majumder R, Wang JF, and Lentz BR. Biophys. J. 2003, 84:1238–1251), to form an inactive factor Xa dimer (Sen S, and Lentz BR, unpublished). In this work, we ask whether Ca2+ and PS also trigger formation of an inactive factor Xa dimer on a membrane and explore the possible physiological significance of this. We have determined the proteolytic activity of human factor Xa towards human prethrombin2 as a substrate both at fixed membrane concentration (increasing factor Xa concentration) and at fixed factor Xa concentration (increasing membrane concentration). Neither of these experiments showed the expected behavior of an increase in activity as factor Xa bound to membranes. The total factor Xa activity actually decreased as low concentrations of PS-containing membranes were added, and increased at higher membrane concentrations. At fixed membrane concentrations, the total factor Xa activity did not increase proportionally with factor Xa concentration. Both observations suggested the existence of membrane-bound and inactive multimeric forms of factor Xa. Because we have observed factor Xa to form dimers in solution (Majumder R, Wang JF, and Lentz BR. Biophys. J. 2003, 84:1238–1251), we tried to fit globally four such data sets to a model that takes into account dimerization of factor Xa after binding to a membrane. This dimer model successfully described all our data, with the parameters of best fit being kcat/KMdimer = 0 M−1s−1, kcat/KMmonomer = 430 M−1s−1, kcat/KMsolution = 38 M−1s−1, and Kd,surfacedimer = 4·10−12 mol/(dm)2. This surface dimerization constant corresponds to a solution-phase Kddimer = 10 nM at 100 μM lipid concentration, nearly what we observed for formation of bovine factor Xa dimer in the presence of short-chain PS (20 nM; Majumder R, Wang JF, and Lentz BR. Biophys. J. 2003, 84:1238–1251). Also consistent with the dimer hypothesis, we observed that the binding of factor Xa to PS-containing membranes appears to be tighter at low than at high membrane concentration. As we observed for soluble-PS-induced dimer formation in solution, dimer formation on a membrane was Ca2+ dependent. Unlike in solution, factor Xa was activated by membrane binding below 1.5 mM Ca2+, but inactivated above this Ca2+ concentration. This suggests that factor Xa activity may be regulated by Ca2+ concentrations close to plasma Ca2+ levels. We conclude that:factor Xa dimerizes on PS-containing membranes;, factor Xa dimer is inactive; and, the transition from monomer to dimer state depends critically on Ca2+ concentration.

scholarly journals Inhibition of human coagulation factor VIII by monoclonal antibodies. Mapping of functional epitopes with the use of recombinant factor VIII fragments

1989 ◽  
Vol 263 (1) ◽  
pp. 187-194 ◽  
Author(s):  
A Leyte ◽  
K Mertens ◽  
B Distel ◽  
R F Evers ◽  
M J M De Keyzer-Nellen ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Factor Viii ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Procoagulant Activity ◽  
Coagulation Factor Viii ◽  
Coagulation Cascade ◽  
Restriction Enzyme Digestion ◽  
Proteolytic Activation

The epitopes of four monoclonal antibodies against coagulation Factor VIII were mapped with the use of recombinant DNA techniques. Full-length Factor VIII cDNA and parts thereof were inserted into the vector pSP64, permitting transcription in vitro with the use of a promoter specific for SP6 RNA polymerase. Factor VIII DNA inserts were truncated from their 3′-ends by selective restriction-enzyme digestion and used as templates for ‘run-off’ mRNA synthesis. Translation in vitro with rabbit reticulocyte lysate provided defined radiolabelled Factor VIII fragments for immunoprecipitation studies. Two antibodies are shown to be directed against epitopes on the 90 kDa chain of Factor VIII, between residues 712 and 741. The 80 kDa chain appeared to contain the epitopes of the other two antibodies, within the sequences 1649-1778 and 1779-1840 respectively. The effect of antibody binding to these sequences was evaluated at two distinct levels within the coagulation cascade. Both Factor VIII procoagulant activity and Factor VIII cofactor function in Factor Xa generation were neutralized upon binding to the region 1779-1840. The antibodies recognizing the region 713-740 or 1649-1778, though interfering with Factor VIII procoagulant activity, did not inhibit in Factor Xa generation. These findings demonstrate that antibodies that virtually inhibit Factor VIII in coagulation in vitro are not necessarily directed against epitopes involved in Factor VIII cofactor function. Inhibition of procoagulant activity rather than of cofactor function itself may be explained by interference in proteolytic activation of Factor VIII. This hypothesis is in agreement with the localization of the epitopes in the proximity of thrombin-cleavage or Factor Xa-cleavage sites.

Coagulation cascade proteases and tissue fibrosis

2002 ◽  
Vol 30 (2) ◽  
pp. 194-200 ◽  
Author(s):  
R. C. Chambers ◽  
G. J. Laurent
Keyword(s):  
Growth Factor ◽  
Factor Xa ◽  
Tissue Growth ◽  
In Vivo Studies ◽  
Coagulation Cascade ◽  
Proteolytic Activation ◽  
Surface Receptors ◽  
Relative Contribution

Fibrotic disorders of the liver, kidney and lung are associated with excessive deposition of extracellular matrix proteins and ongoing coagulation-cascade activity. In addition to their critical roles in blood coagulation, thrombin and the immediate upstream coagulation proteases, Factors Xa and VIIa, influence numerous cellular responses that may play critical roles in subsequent inflammatory and tissue repair processes in vascular and extra-vascular compartments. The cellular effects of these proteases are mediated via proteolytic activation of a novel family of cell-surface receptors, the protease-activated receptors (PAR-1, −2, −3 and −4). Although thrombin is capable of activating PAR-1, −3 and −4, there is accumulating in vitro evidence that the profibrotic effects of thrombin are predominantly mediated via PAR-1. Factor Xa is capable of activating PAR-1 and PAR-2, but its mitogenic effects for fibroblasts are similarly mediated via PAR-1. These proteases do not exert their profibrotic effects directly, but act via the induction of potent fibrogenic mediators, such as platelet-derived growth factor and connective tissue growth factor. In vivo studies using proteolytic inhibitors, PAR-1 antagonists and PAR-1-deficient mice have provided evidence that coagulation proteases play a key role in tissue inflammation and in a number of vascular pathologies associated with hyperproliferation of smooth muscle cells. More recently, coagulation proteases have also been shown to play a role in the pathogenesis of fibrosis but the relative contribution of their cellular versus their procoagulant effects awaits urgent evaluation in vivo. These studies will be informative in determining the potential application of PAR-1 antagonists as antifibrotic agents.

The uncalibrated prothrombinase-induced clotting time test

2010 ◽  
Vol 30 (04) ◽  
pp. 212-216 ◽  
Author(s):  
R. Jovic ◽  
M. Hollenstein ◽  
P. Degiacomi ◽  
M. Gautschi ◽  
A. Ferrández ◽  
...  
Keyword(s):  
Factor Xa ◽  
Heparin Therapy ◽  
Thrombin Inhibitors ◽  
Coagulation Cascade ◽  
Diagnostic Tools ◽  
Direct Thrombin Inhibitors ◽  
Functional Assay ◽  
Clotting Time ◽  
Coagulation Time ◽  
Time Test

SummaryThe activated partial thromboplastin time test (aPTT) represents one of the most commonly used diagnostic tools in order to monitor patients undergoing heparin therapy. Expression of aPTT coagulation time in seconds represents common practice in order to evaluate the integrity of the coagulation cascade. The prolongation of the aPTT thus can indicate whether or not the heparin level is likely to be within therapeutic range. Unfortunately aPTT results are highly variable depending on patient properties, manufacturer, different reagents and instruments among others but most importantly aPTT’s dose response curve to heparin often lacks linearity. Furthermore, aPTT assays are insensitive to drugs such as, for example, low molecular weight heparin (LMWH) and direct factor Xa (FXa) inhibitors among others. On the other hand, the protrombinase-induced clotting time assay (PiCT®) has been show to be a reliable functional assay sensitive to all heparinoids as well as direct thrombin inhibitors (DTIs). So far, the commercially available PiCT assay (Pefakit®-PiCT®, DSM Nutritional Products Ltd. Branch Pentapharm, Basel, Switzerland) is designed to express results in terms of units with the help of specific calibrators, while aPTT results are most commonly expressed as coagulation time in seconds. In this report, we describe the results of a pilot study indicating that the Pefakit PiCT UC assay is superior to the aPTT for the efficient monitoring of patients undergoing UFH therapy; it is also suitable to determine and quantitate the effect of LMWH therapy. This indicates a distinct benefit when using this new approach over the use of aPPT for heparin monitoring.

scholarly journals Reversal Strategies for Intracranial Hemorrhage Related to Direct Oral Anticoagulant Medications

2018 ◽  
Vol 2018 ◽  
pp. 1-11 ◽  
Author(s):  
Alok Dabi ◽  
Aristides P. Koutrouvelis
Keyword(s):  
Side Effects ◽  
Intracranial Hemorrhage ◽  
Vitamin K Antagonists ◽  
Oral Anticoagulants ◽  
Direct Oral Anticoagulants ◽  
Factor Xa ◽  
Coagulation Cascade ◽  
Reversal Agents ◽  
United States Food ◽  
Life Threatening

Direct oral anticoagulants (DOACs) are a new class of anticoagulants that directly inhibit either thrombin or factor Xa in the coagulation cascade. They are being increasingly used instead of warfarin or other vitamin K antagonists (VKAs). Adverse side effects of DOACs may result in hemorrhagic complications, including life-threatening intracranial hemorrhage (ICH), though to a much lesser degree than VKAs. Currently there are relatively limited indications for DOACS but their usage is certain to expand with the availability of their respective specific reversal agents. Currently, only idarucizumab (antidote for dabigatran) has been United States Food and Drug Administration- (FDA-) approved, but others (andexanet-α and ciraparantag) may be approved in near future, and the development and availability of such reversal agents have the potential to dramatically change the current anticoagulant use by providing reversal of multiple oral anticoagulants. Until all the DOACs have FDA-approved reversal agents, the treatment of the dreaded side effects of bleeding is challenging. This article is an attempt to provide an overview of the management of hemorrhage, especially ICH, related to DOAC use.

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.

Anticoagulation Treatment in Venous Thromboembolism: Options and Optimal Duration

2021 ◽  
Vol 27 ◽  
Author(s):  
Stavrianna Diavati ◽  
Marios Sagris ◽  
Dimitrios Terentes-Printzios ◽  
Charalambos Vlachopoulos
Keyword(s):  
Risk Factors ◽  
Venous Thromboembolism ◽  
Anticoagulation Therapy ◽  
Factor Xa ◽  
Coagulation Cascade ◽  
Clinical Genetic ◽  
Vein Thrombosis ◽  
Anticoagulation Treatment ◽  
Molecular Pathophysiology ◽  
Deep Vein

: Venous thromboembolism (VTE), clinically presenting as deep-vein thrombosis (DVT) or pulmonary embolism (PE), constitutes a major global healthcare concern with severe complications, long-term morbidity and mortality. Although several clinical, genetic and acquired risk factors for VTE have been identified, the molecular pathophysiology and mechanisms of disease progression remain poorly understood. Anticoagulation has been the cornerstone of therapy for decades, but there still are uncertainties regarding primary and secondary VTE prevention, as well as optimal therapy duration. In this review we discuss the role of factor Xa in coagulation cascade and the different choices of anticoagulation therapy based on patients’ predisposing risk factors and risk of event recurrence. Further, we compare newer agents to traditional anticoagulation treatment, based on most recent studies and guidelines.

scholarly journals Enhancing the anticoagulant profile of meizothrombin

2018 ◽  
Vol 9 (1) ◽  
pp. 169-175 ◽  
Author(s):  
Bosko M. Stojanovski ◽  
Leslie A. Pelc ◽  
Xiaobing Zuo ◽  
Nicola Pozzi ◽  
Enrico Di Cera
Keyword(s):  
Single Molecule ◽  
Protein C ◽  
Coagulation Cascade ◽  
Proteolytic Activation ◽  
Single Molecule Fret ◽  
Functional Aspects ◽  
Active Intermediate ◽  
Thrombotic Complications ◽  
Gla Domain ◽  
Structural Architecture

AbstractMeizothrombin is an active intermediate generated during the proteolytic activation of prothrombin to thrombin in the penultimate step of the coagulation cascade. Structurally, meizothrombin differs from thrombin because it retains the auxiliary Gla domain and two kringles. Functionally, meizothrombin shares with thrombin the ability to cleave procoagulant (fibrinogen), prothrombotic (PAR1) and anticoagulant (protein C) substrates, although its specificity toward fibrinogen and PAR1 is less pronounced. In this study we report information on the structural architecture of meizothrombin resolved by SAXS and single molecule FRET as an elongated arrangement of its individual domains. In addition, we show the properties of a meizothrombin construct analogous to the anticoagulant thrombin mutant W215A/E217A currently in Phase I for the treatment of thrombotic complications and stroke. The findings reveal new structural and functional aspects of meizothrombin that advance our understanding of a key intermediate of the prothrombin activation pathway.

Effects of Tannins fromGeumjaponicumon the Catalytic Activity of Thrombin and Factor Xa of Blood Coagulation Cascade

1998 ◽  
Vol 61 (11) ◽  
pp. 1356-1360 ◽  
Author(s):  
Hui Dong ◽  
Shao-Xing Chen ◽  
R. Manjunatha Kini ◽  
Hong-Xi Xu
Keyword(s):  
Catalytic Activity ◽  
Blood Coagulation ◽  
Factor Xa ◽  
Coagulation Cascade

scholarly journals The Rivaroxaban Program and the Management of Unmet Needs in Thromboembolic Disease

2018 ◽  
Vol 118 (S 01) ◽  
pp. S2-S11
Author(s):  
A. Camm
Keyword(s):  
Potential Role ◽  
Unmet Needs ◽  
Antiplatelet Agent ◽  
Factor Xa ◽  
Vitamin K Antagonist ◽  
Thromboembolic Disease ◽  
Coagulation Cascade ◽  
Vascular Protection ◽  
Randomized Controlled

AbstractRivaroxaban is a non-vitamin K antagonist oral anticoagulant that acts as a direct factor Xa inhibitor, and is widely used for the prevention and treatment of thromboembolic disorders. As further knowledge gaps are identified in thrombosis management, the rivaroxaban research program has expanded in an attempt to elucidate the wider benefits of rivaroxaban. An increased understanding of the interactions taking place within the coagulation cascade may support a broader role for rivaroxaban (2.5 mg twice daily [bid] or 5 mg bid) in the setting of vascular protection, either alone or in combination with an antiplatelet agent. The aim of this article is to describe the potential role of rivaroxaban in the context of vascular protection and provide an overview of recently completed and ongoing randomized controlled trials of rivaroxaban in the areas of stroke prevention, venous protection and vascular protection.

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