Monitoring prothrombin activation in plasma through loss of FRET.

BACKGROUND Current assays that monitor thrombin generation in plasma rely on fluorogenic substrates to follow the kinetics of zymogen activation, which may be complicated by substrate cleavage from other proteases. In addition, these assays depend on activation following cleavage at the R320 site of prothrombin and fail to report cleavage at the alternative R271 site that leads to shedding of the auxiliary Gla and kringle domains of prothrombin. OBJECTIVE To develop a plasma assay that directly monitors prothrombin activation independent of fluorogenic substrate hydrolysis. METHODS Cleavage at the R271 site of prothrombin is monitored through loss of FRET in plasma coagulated along the extrinsic or intrinsic pathway. RESULTS The availability of factor V (fV) in plasma strongly influences the rate of prothrombin activation. The rate of thrombin formation is equally perturbed in fV or prothrombin depleted plasma, implicating that the thrombin catalyzed feedback reactions that amplify the coagulation response play an important role in generating sufficient amounts of fVa required for assembly of prothrombinase. Congenital deficiencies in fVIII and fIX significantly slow down cleavage at R271 in plasma coagulated along the extrinsic and intrinsic pathway. Prothrombin activation in fXI deficient plasma is only perturbed when coagulation is triggered along the intrinsic pathway. CONCLUSIONS The FRET assay enables direct monitoring of prothrombin activation through cleavage at R271, without the need for fluorogenic substrates. The assay is sensitive enough to assess how deficiencies in coagulation factors affect thrombin formation.

• Publication year

  2023

• Venue

  Journal of Thrombosis and Haemostasis

• Publication date

  2023-03-01

• Fields of study

  Medicine, Chemistry

• Identifiers
  DOI 10.1016/j.jtha.2023.03.008PMID 36931601
• External record

  Open on Semantic Scholar

• Source metadata

  Semantic Scholar, PubMed

• No claims are published for this paper.

• No concepts are published for this paper.

• Comparative sequence analysis of vitamin K‐dependent coagulation factors

  2022cited by this paper
• Regulation of coagulation by tissue factor pathway inhibitor: Implications for hemophilia therapy

  2022cited by this paper
• Cryo‐EM structures of coagulation factors

  2022cited by this paper
• Cryo-EM structure of the prothrombin-prothrombinase complex

  2022cited by this paper
• Cryo-EM structures of human coagulation factors V and Va.

  2021cited by this paper
• Role of sequence and position of the cleavage sites in prothrombin activation

  2021cited by this paper
• Role of the activation peptide in the mechanism of protein C activation

  2020cited by this paper
• A Comprehensive Overview of Coagulation Factor V and Congenital Factor V Deficiency

  2019cited by this paper
• Occlusion of anion-binding exosite 2 in meizothrombin explains its impaired ability to activate factor V

  2018cited by this paper
• Enhancing the anticoagulant profile of meizothrombin

  2018cited by this paper
• Structure of prothrombin in the closed form reveals new details on the mechanism of activation

  2018cited by this paper
• A review of commercially available thrombin generation assays

  2017cited by this paper
• Selective factor VIII activation by the tissue factor-factor VIIa-factor Xa complex.

  2017cited by this paper
• How the Linker Connecting the Two Kringles Influences Activation and Conformational Plasticity of Prothrombin*

  2016cited by this paper
• Structural Architecture of Prothrombin in Solution Revealed by Single Molecule Spectroscopy*

  2016cited by this paper
• Limiting prothrombin activation to meizothrombin is compatible with survival but significantly alters hemostasis in mice.

  2016cited by this paper
• Hemophilia B: molecular pathogenesis and mutation analysis

  2015cited by this paper
• The linker connecting the two kringles plays a key role in prothrombin activation

  2014cited by this paper
• The transition of prothrombin to thrombin

  2013cited by this paper
• Hemophilia A in the third millennium.

  2013cited by this paper
• Congenital Prothrombin Deficiency: An Update

  2013cited by this paper
• Factor Xa Activation of Factor V Is of Paramount Importance in Initiating the Coagulation System: Lessons From a Tick Salivary Protein

  2013cited by this paper
• Fluorogenic Peptide‐Based Substrates for Monitoring Thrombin Activity

  2012cited by this paper
• Prothrombin activation in blood coagulation: the erythrocyte contribution to thrombin generation.

  2012cited by this paper
• Thrombin generation: what have we learned?

  2012cited by this paper
• Prothrombin Activation by Platelet-associated Prothrombinase Proceeds through the Prethrombin-2 Pathway via a Concerted Mechanism*

  2012cited by this paper
• Residual platelet factor V ensures thrombin generation in patients with severe congenital factor V deficiency and mild bleeding symptoms.

  2010cited by this paper
• Novel biomarkers predict liver fibrosis in hepatitis C patients: alpha 2 macroglobulin, vitamin D binding protein and apolipoprotein AI

  2010cited by this paper
• The molecular basis of factor V and VIII procofactor activation

  2009cited by this paper
• Factor XI deficiency in humans

  2009cited by this paper
• The interaction of fragment 1 of prothrombin with the membrane surface is a prerequisite for optimum expression of factor Va cofactor activity within prothrombinase

  2008cited by this paper
• Role of the A chain in thrombin function

  2008cited by this paper
• Low plasma levels of tissue factor pathway inhibitor in patients with congenital factor V deficiency.

  2008cited by this paper
• Thrombin generation: the functional role of alpha‐2‐macroglobulin and influence of developmental haemostasis

  2007cited by this paper
• The intrinsic pathway of coagulation: a target for treating thromboembolic disease?

  2007cited by this paper
• Immunologic quantitation of tissue factors

  2006cited by this paper
• The Structural Integrity of Anion Binding Exosite I of Thrombin Is Required and Sufficient for Timely Cleavage and Activation of Factor V and Factor VIII*

  2006cited by this paper
• Calibrated Automated Thrombin Generation Measurement in Clotting Plasma

  2003cited by this paper
• Factor V: a combination of Dr Jekyll and Mr Hyde.

  2003cited by this paper
• Analysis of the Kinetics of Prothrombin Activation and Evidence That Two Equilibrating Forms of Prothrombinase Are Involved in the Process*

  2003cited by this paper
• Thrombin formation.

  2003cited by this paper
• The dynamics of thrombin formation.

  2003cited by this paper
• Thrombin functions during tissue factor-induced blood coagulation.

  2002cited by this paper
• The Thrombogram in Rare Inherited Coagulation Disorders: Its Relation to Clinical Bleeding

  2002cited by this paper
• The Role of the Factor X Activation Peptide: A Deletion Mutagenesis Approach

  2002cited by this paper
• The Role of Factor XI in Thrombin Generation Induced by Low Concentrations of Tissue Factor

  2001cited by this paper
• Role of Proexosite I in Factor Va-dependent Substrate Interactions of Prothrombin Activation*

  2000cited by this paper
• The protein C pathway

  2000cited by this paper
• Blood coagulation in hemophilia A and hemophilia C.

  1998cited by this paper
• Fatal haemorrhage and incomplete block to embryogenesis in mice lacking coagulation factor V

  1996cited by this paper
• The Influence of Fibrinogen and Fibrin on Thrombin Generation - Evidence for Feedback Activation of the Clotting System by Clot Bound Thrombin

  1994cited by this paper
• Characterization of a stable form of human meizothrombin derived from recombinant prothrombin (R155A, R271A, and R284A).

  1994cited by this paper
• Activation of human factor V by meizothrombin.

  1994cited by this paper
• Factor XI activation in a revised model of blood coagulation

  1991cited by this paper
• Meizothrombin formation during factor Xa-catalyzed prothrombin activation. Formation in a purified system and in plasma.

  1991cited by this paper
• The coagulation cascade: initiation, maintenance, and regulation.

  1991cited by this paper
• Alpha 2 macroglobulin state in acute pancreatitis. Raised values of alpha 2 macroglobulin-protease complexes in severe and mild attacks.

  1991cited by this paper
• Studies of the role of factor Va in the factor Xa-catalyzed activation of prothrombin, fragment 1.2-prethrombin-2, and dansyl-L-glutamyl-glycyl-L-arginine-meizothrombin in the absence of phospholipid.

  1990cited by this paper
• In situ-generated thrombin is the only enzyme that effectively activates factor VIII and factor V in thromboplastin-activated plasma.

  1989cited by this paper
• The prothrombinase-catalyzed activation of prothrombin proceeds through the intermediate meizothrombin in an ordered, sequential reaction.

  1986cited by this paper
• Formation of meizothrombin as intermediate in factor Xa-catalyzed prothrombin activation.

  1986cited by this paper
• The behavior of antithrombin III, alpha 2 macroglobulin, and alpha 1 antitrypsin during cardiopulmonary bypass.

  1983cited by this paper
• Activation of Human Coagulation Factor VIII by Activated Factor X, the Common Product of the Intrinsic and the Extrinsic Pathway of Blood Coagulation

  1982cited by this paper
• An alternative extrinsic pathway of human blood coagulation.

  1982cited by this paper
• The role of phospholipids and factor Va in the prothrombinase complex.

  1980cited by this paper
• Normal and pathological serum levels of alpha2-macroglobulins in men and mice.

  1975cited by this paper
• The conversion of prothrombin to thrombin. V. The activation of prothrombin by factor Xa in the presence of phospholipid.

  1974cited by this paper
• A Thrombin Generation Test

  1953cited by this paper
• The Reaction of Haemophilic Plasma to Thromboplastin

  1951cited by this paper

• Research Progress in In Vitro Screening Techniques for Natural Antithrombotic Medicines

  2025cites this paper
• Conformation of factor Xa in solution revealed by single molecule spectroscopy.

  2024cites this paper