Sodium Binding Site of Factor Xa:  Role of Sodium in the Prothrombinase Complex†

Biochemistry ◽  
2000 ◽  
Vol 39 (7) ◽  
pp. 1817-1825 ◽  
Author(s):  
Alireza R. Rezaie ◽  
Xuhua He
Keyword(s):  
Binding Site ◽  
Factor Xa ◽  
Prothrombinase Complex ◽  
Sodium Binding

Modulation of Prothrombinse Assembly and Activity by Phosphotidylethanolamine.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4207-4207
Author(s):  
Rinku Majumder ◽  
Xiaoe Liang ◽  
Mary Ann Quinn-Allen ◽  
Barry R. Lentz ◽  
William H. Kane
Keyword(s):  
Binding Sites ◽  
Conflicts Of Interest ◽  
Membrane Surface ◽  
Factor Xa ◽  
Catalytic Efficiency ◽  
Prothrombinase Complex ◽  
Factor Va ◽  
Naturally Occurring ◽  
Early Phases

Abstract Abstract 4207 Constituents of naturally occurring phospholipid membranes regulate the activity of the prothrombinase complex. In the present study we demonstrate that membranes containing phosphatidylcholine and phosphatidylethanolamine (PC:PE) bind factor Va with high affinity (Kd ∼10 nM) in the absence of phosphatidylserine (PS). These membranes support formation of a functional prothrombinase complex though thrombin generation at saturating factor Va concentrations is reduced approximately 60-70% compared to membranes containing 5% or more PS. The presence of PE markedly enhances the catalytic efficiency of the prothrombinase complex on membranes containing 1% PS with only modest effects on membranes containing 5% or more PS. The effect of PE on factor Va membrane binding appears to be due to direct interactions between PE and factor Va rather than to changes in membrane surface packing. Finally, we find that soluble C6PE is able to bind to factor Va (Kd ∼6.5 uM) and factor Xa (Kd ∼ 91 uM). We also show that soluble C6PE is able to stimulate formation of a partially active factor Va-factor Xa complex capable of catalyzing conversion of prothrombin to thrombin in the absence of a membrane surface. We further demonstrate that C6PE and C6PS binding sites in factor Xa are linked, as binding of one lipid enhances the binding and activity of the other. These findings provide important new insights into the role of PE in assembly of the prothrombinase complex that are relevant to understanding the activity of factor Xa on the surface of platelets particularly in the early phases of hemostasis when the concentration of PS may be limiting. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The Action of a Synthetic Pentasaccharide on Thrombin Generation in Whole Plasma

1989 ◽  
Vol 61 (03) ◽  
pp. 397-401 ◽  
Author(s):  
S Béguin ◽  
J Choay ◽  
H C Hemker
Keyword(s):  
Binding Site ◽  
Blood Coagulation ◽  
Thrombin Generation ◽  
Platelet Rich Plasma ◽  
Factor Xa ◽  
Heparin Binding ◽  
Extrinsic Pathway ◽  
Prothrombinase Complex ◽  
Factor Va ◽  
Prothrombinase Activity

SummaryWe investigated the effect on thrombin generation in plasma of the pentasaccharide that represent the AT II/binding site in heparin. This compound has no effect on the breakdown of thrombin in plasma. It dose-dependently inhibits the formation of thrombin in both the intrinsic and the extrinsic pathway. If coagulation is triggered by the complete prothrombinase complex (phospholipid – factor Va – factor Xa) under conditions in which the large majority of factor Xa is bound to the complex, the inhibition of prothrombinase activity is only minor. If no factor Va is present or if the prothrombinase activity is triggered by adding complete tenase (PL-FVIIIa-FIXa) or incomplete tenase (PLFIXa) to the plasma the inhibition by pentasaccharide is of the same magnitude as that in the intrinsic or extrinsic system.We conclude that the pentasaccharide inhibits blood coagulation by katalysing the inactivation of free factor Xa. In contrast to classical heparin it does inhibit the peak of thrombin formation in platelet rich plasma, probably because it is less subject to inactivation by heparin binding proteins from platelets than classical heparin is.

Efficient Thrombin Generation Requires Molecular Phosphatidylserine, Not a Membrane Surface.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1022-1022
Author(s):  
Rinku Majumder ◽  
Gabriel E. Weinreb ◽  
Barry R. Lentz
Keyword(s):  
Reaction Pathway ◽  
Membrane Surface ◽  
Factor Xa ◽  
Elastic Light Scattering ◽  
Complex Assembly ◽  
Peptide Bonds ◽  
Prothrombinase Complex ◽  
Pyrene Fluorescence ◽  
Factor Va

Abstract Activation of prothrombin to thrombin is catalyzed by “prothrombinase” complex, traditionally viewed as factor Xa (FXa) in complex with factor Va (FVa) on a phosphatidylserine (PS)-containing membrane surface, which is widely regarded as required for efficient activation. Activation involves cleavage of two peptide bonds and proceeds via one of two released intermediates or through “channeling” (activation without release of an intermediate). We ask here whether the PS molecule itself, not the membrane surface, is sufficient to produce fully active human “prothrombinase” complex in solution. Both FXa and FVa bind soluble dicaproyl-phosphatidylserine (C6PS). In the presence of sufficient C6PS to saturate both FXa and FVa2 (light isoform of FVa), these proteins form a tight (Kd = 0.6 ±0.09 nM at 37°C) soluble complex. Complex assembly occurs well below the critical micelle concentration of C6PS, as established in the presence of the proteins by quasi-elastic light scattering and pyrene fluorescence. Ferguson analysis of native gels show that the complex migrates with an apparent molecular mass only slightly larger than that expected for one FXa and one FVa2, further ruling out complex assembly on C6PS micelles. Human prothrombin activation by this complex occurs at nearly the same overall rate (2.2x108 M− 1sec− 1) and via the same reaction pathway (50–60% channeling, rest via meizothrombin intermediate) as activation catalyzed by a complex assembled on PS-containing membranes (4.4x108 M− 1sec− 1). These results question the accepted role of PS-membranes as providing “dimensionality-reduction” and favor a regulatory role for platelet-membrane-exposed PS.

Abstract 30: Tissue Factor Pathway Inhibitor α Inhibits Prothrombinase via a Three-Step Mechanism

2017 ◽  
Vol 37 (suppl_1) ◽  
Author(s):  
Jeremy P Wood ◽  
Alan E Mast
Keyword(s):  
Active Site ◽  
Inhibitory Activity ◽  
Thrombin Generation ◽  
Platelet Rich Plasma ◽  
Factor Xa ◽  
Prothrombinase Complex ◽  
Factor Va ◽  
Direct Binding ◽  
Affinity Interaction

TFPIα inhibits early forms of the prothrombinase complex (factor Xa (FXa), factor Va (FVa)), though the inhibitory mechanism is not entirely understood. One step of inhibition is a high affinity interaction between a TFPIα C-terminal basic region (BR) (252-LIKTKRKRKK-261) and an acidic region (AR) present in FXa-activated and platelet-released forms of FVa. We investigated two additional potential mechanistic steps: (1) binding of the second Kunitz-type inhibitory domain (K2) of TFPIα to the FXa active site; and (2) the function of uncharged residues L252, I253, and T255 within the BR, which are evolutionarily conserved, suggesting they have activity. Direct inhibition of FXa was investigated using TFPIα with an altered K2 (TFPI-R107A) incapable of binding FXa. TFPI-R107A inhibited purified prothrombinase 17-fold weaker than TFPIα (IC50 = 30.6nM vs. 1.8nM) and did not inhibit FXa-initiated thrombin generation in platelet-rich plasma (PRP). Therefore, direct binding of FXa and K2 is required for efficient inhibition of prothrombinase under physiological conditions. Similarly, the role of L252, I253, and T255 was investigated by substituting them with alanine (TFPI-AAKA). The IC50 for prothrombinase inhibition by TFPI-AAKA was 10.4nM, and it had reduced inhibitory activity in PRP, revealing that these residues are also required for efficient prothrombinase inhibition. The role of L252, I253, and T255 was further probed using the peptide LIKTKRKRKK, which inhibited purified prothrombinase (IC50 = 1.0μM) and thrombin generation in PRP at 1μM. AAKAKRKRKK had very little activity in either assay (~20% prothrombinase inhibition with 225μM peptide), but bound the FVa AR equivalently to LIKTKRKRKK (K d = 5.9nM and 6.0nM, respectively). Thus, the basic residues are responsible for AR binding, while a second step, mediated by L252-T255, is necessary for inhibitory activity. These residues may be necessary for displacement of FXa from FVa, as proposed by Bunce et al. We propose that prothrombinase inhibition by TFPIα involves three steps: (1) the TFPIα BR basic residues bind the FVa AR; (2) residues L252-T255 block prothrombinase assembly; and (3) K2 binds the FXa active site. All three steps are required for physiologic inhibition of prothrombinase by TFPIα.

Directing thrombin

Blood ◽  
2005 ◽  
Vol 106 (8) ◽  
pp. 2605-2612 ◽  
Author(s):  
David A. Lane ◽  
Helen Philippou ◽  
James A. Huntington
Keyword(s):  
Crystal Structure ◽  
Structure Function ◽  
Factor Xa ◽  
Multiple Roles ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Dominant Mechanism ◽  
Prothrombinase Complex ◽  
The Past

AbstractFollowing initiation of coagulation as part of the hemostatic response to injury, thrombin is generated from its inactive precursor prothrombin by factor Xa as part of the prothrombinase complex. Thrombin then has multiple roles. The way in which thrombin interacts with its many substrates has been carefully scrutinized in the past decades, but until recently there has been little consideration of how its many functions are coordinated or directed. Any understanding of how it is directed requires knowledge of its structure, how it interacts with its substrates, and the role of any cofactors for its interaction with substrates. Recently, many of the interactions of thrombin have been clarified by crystal structure and site-directed mutagenesis analyses. These analyses have revealed common residues used for recognition of some substrates and overlapping surface exosites used for recognition by cofactors. As many of its downstream reactions are cofactor driven, competition between cofactors for exosites must be a dominant mechanism that determines the fate of thrombin. This review draws together much recent work that has helped clarify structure function relationships of thrombin. It then attempts to provide a cogent proposal to explain how thrombin activity is directed during the hemostatic response.

scholarly journals Refinement of the Central Steps of Substrate Transport by the Aspartate Transporter GltPh: Elucidating the Role of the Na2 Sodium Binding Site

2015 ◽  
Vol 11 (10) ◽  
pp. e1004551 ◽  
Author(s):  
SanthoshKannan Venkatesan ◽  
Kusumika Saha ◽  
Azmat Sohail ◽  
Walter Sandtner ◽  
Michael Freissmuth ◽  
...  
Keyword(s):  
Binding Site ◽  
Substrate Transport ◽  
Sodium Binding

scholarly journals Prothrombin kringle 1 domain interacts with factor Va during the assembly of prothrombinase complex

1997 ◽  
Vol 321 (3) ◽  
pp. 729-735 ◽  
Author(s):  
Hiroshi DEGUCHI ◽  
Hiroyuki TAKEYA ◽  
Esteban C. GABAZZA ◽  
Junji NISHIOKA ◽  
Koji SUZUKI
Keyword(s):  
Dissociation Constants ◽  
Solid Phase ◽  
Factor Xa ◽  
Complex Activity ◽  
Prothrombinase Complex ◽  
Factor Va ◽  
Direct Binding ◽  
Kringle 2 ◽  
Prothrombin Activation

The kringle 2 domain of prothrombin has been shown to interact with factor Va during the activation of prothrombin by the prothrombinase complex composed of factor Xa, factor Va, negatively charged phospholipids and Ca2+ ions. However, contradictory results have been reported about the role of the kringle 1 domain of prothrombin during the assembly of the prothrombinase complex. In an attempt to clarify the role of the kringle 1 domain of prothrombin, its effect on the activation of prothrombin by the prothrombinase complex and its direct binding to human factor Va were assessed. Comparative evaluation with the effects caused by other prothrombin structural components [a fragment 1 (γ-carboxyglutamic acid and kringle 1 domains), a kringle 2 domain and a catalytic protease domain] was also performed. In the presence of factor Va, each kringle 1 and kringle 2 fragment significantly inhibited the factor Xa-catalysed prothrombin activation in the absence of phospholipids. However, in the absence of both factor Va and phospholipids, kringle 2 fragment, but not kringle 1 fragment, inhibited prothrombin activation. Evaluation of the molecular interaction of the kringle domains with factor Va in assays with solid-phase phospholipid vesicles showed that each kringle 1 and kringle 2 fragment inhibited the prothrombinase complex activity. Assessment of the direct binding of prothrombin and each kringle domain of prothrombin with factor Va by fluorescence polarization showed that prothrombin, kringle 1 and kringle 2 fragments bind directly to factor Va with dissociation constants of 1.9ŷ0.1, 2.3ŷ0.1 and 2.0ŷ0.4 ƁM (meansŷS.D.) respectively. These findings suggest that both kringle 1 and 2 domains of prothrombin interact with factor Va during the assembly of the prothrombinase complex.

Factor Xa Enhances the Binding of Tissue Factor Pathway Inhibitor to Acidic Phospholipids

1996 ◽  
Vol 75 (05) ◽  
pp. 796-800 ◽  
Author(s):  
Sanne Valentin ◽  
Inger Schousboe
Keyword(s):  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Factor Xa ◽  
Microtitre Plate ◽  
C Terminus ◽  
Microtitre Plate Assay ◽  
Kunitz Domain ◽  
Tissue Factor Pathway ◽  
Acidic Phospholipids

SummaryIn the present study, the interaction between tissue factor pathway inhibitor (TFPI) and phospholipids has been characterized using a microtitre plate assay. TFPI was shown to bind calcium-independently to an acidic phospholipid surface composed of phosphatidylserine, but not a surface composed of the neutral phosphatidylcholine. The interaction was demonstrated to be dependent on the presence of the TFPI C-terminus. The presence of heparin (1 U/ml, unfractionated) was able to significantly reduce the binding of TFPI to phospholipid. The interaction of TFPI with phosphatidylserine was significantly decreased in the presence of calcium, but this was counteracted, and even enhanced, following complex formation of TFPI with factor Xa prior to incubation with the phospholipid surface. Moreover, a TFPI variant, not containing the third Kunitz domain and the C-terminus, was unable to bind to phospholipid. However, following the formation of a TFPI/factor Xa-complex this TFPI variant was capable of interacting with the phospholipid surface. This indicates that the role of factor Xa as a TFPI cofactor, at least in part, is to mediate the binding of TFPI to the phospholipid surface.

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