scholarly journals The Critical Role of the 185–189-Loop in the Factor Xa Interaction with Na+and Factor Va in the Prothrombinase Complex

2004 ◽  
Vol 279 (46) ◽  
pp. 48262-48269 ◽  
Author(s):  
Alireza R. Rezaie ◽  
Farooqahmed S. Kittur
Keyword(s):  
Critical Role ◽  
Factor Xa ◽  
Prothrombinase Complex ◽  
Factor Va

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.

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.

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.

scholarly journals Factor Va Alters the Conformation of the Na+-Binding Loop of Factor Xa in the Prothrombinase Complex†

Biochemistry ◽  
2008 ◽  
Vol 47 (22) ◽  
pp. 5976-5985 ◽  
Author(s):  
Likui Yang ◽  
Chandrashekhara Manithody ◽  
Shabir H. Qureshi ◽  
Alireza R. Rezaie
Keyword(s):  
Factor Xa ◽  
Prothrombinase Complex ◽  
Factor Va ◽  
Binding Loop

Stressed Endothelial Cells Compartmentalize the Prothrombinase Complex on Filopodia and Other Convex Membrane Features near the Cell Margin

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 372-372
Author(s):  
Jialan Shi ◽  
Dessislava N. Nikova ◽  
Gary E. Gilbert
Keyword(s):  
Endothelial Cells ◽  
Binding Sites ◽  
Umbilical Vein ◽  
Factor Xa ◽  
Annexin V ◽  
Complex Assembly ◽  
Prothrombinase Complex ◽  
Factor Va ◽  
Tnf Α ◽  
Prothrombinase Activity

Abstract Abstract 372 The dependence of procoagulant activity on phosphatidylserine (PS) has been recognized for at least four decades but the location of physiologically relevant membranes with PS exposure remains uncertain. PS is exposed on apoptotic cells and cell microparticles but in vitro and in vivo studies have failed to demonstrate a clear relationship of microparticles or apoptotic cells to fibrin deposition. Exposure of endothelial cells to stimulants or toxins leads to retraction of cell margins, mounding of the central cell, and extension of filopodia. We have also found that cell stress also leads to limited, focal PS exposure. Furthermore, we found that binding sites for lactadherin, a PS-binding protein that shares homology with factor VIII and factor V, are concentrated on convex surfaces such as filopodia. In this study we ask whether the limited, focal PS exposure on stressed human umbilical vein endothelial cells is sufficient to support prothrombinase complex assembly and whether the prothrombinase complex assembly is restricted to the convex membrane features that bind lactadherin. We allowed Human Umbilical Vein Endothelial Cells (HUVEC) to grow to confluent monolayers prior to exposure to TNF-α, 10 ng/ml, for 5–24 hours. PS exposure was detected by simultaneous staining using 10 nM lactadherin–Alexa 488 and annexin V–Cy 3.18, both exhibiting high affinity for PS. Stressed cells withdrew from their prior borders, leaving residual fibrils connected to original attachment points. In addition, they extended filopodia that were up to several cell diameters in length. Confocal microscopy demonstrated focal staining of filopodia, fibrils and cell margins with lactadherin and patches near the nucleus with annexin A5. We asked whether the selective binding might be determined by the membrane topology. To mimic the curvature of a cell membrane we prepared nano-fabricated silica substrates with ridge radii of 10 nm. The AFM topographic and fluorescent images of synthetic membrane bilayers supported by the substrates showed that, over a PS content of 4–15%, lactadherin preferentially binds to the convex nano-ridges with a ridge: valley staining ratio >80:1, while annexin V selectively binds the concave areas of the nano-trenches with a ridge. Combined fluorescence/AFM imaging of TNF-α treated HUVEC's, demonstrated that the new thin filaments staining with lactadherin had radii of curvature of approx. 12 nm, similar to the ridges of our synthetic bilayers. We asked whether factor Va and factor Xa share preference for convex surfaces, analogous to lactadherin. Supported membranes of 4% PS had preferential ridge staining by factor Va-fluorescein-maleimide with a ridge/valley ratio > 10/1. Co-staining with factor Va and factor Xa-EGRck-biotin (complexed to Alexa 647-steptavidin) indicated that factor Va enhanced binding of factor Xa to ridges, thus the prothrombinase complex has highly preferential binding to convex ridges. TNF-α-treated endothelial cells bound factor Va, like lactadherin, selectively on filopodia and fibrils near the retracted edges of endothelial cells. Factor Xa also localized to these features in the presence of factor Va, indicating prothrombinase complex assembly. Stressed endothelial cells exhibited at least 8-fold higher support for thrombin production and prothrombinase activity. Prothrombinase activity was efficiently inhibited by lactadherin, demonstrating that the lactadherin-binding sites were the functional sites for prothrombinase activity. Together, these data indicate that stressed endothelial cells can support the prothrombinase complex and that prothrombinase activity is compartmentalized near the periphery of the cell and in the intracellular area through binding sites on highly convex membrane features with exposed PS. We have hypothesized that this compartment of procoagulant activity is relatively protected from anti-coagulant proteins that are localized elsewhere on the stimulated/stressed endothelial cell. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Endotoxin enhances the expression of monocyte prothrombinase activity

Blood ◽  
1992 ◽  
Vol 79 (2) ◽  
pp. 406-416 ◽  
Author(s):  
RA Robinson ◽  
L Worfolk ◽  
PB Tracy
Keyword(s):  
Mononuclear Cells ◽  
Membrane Surface ◽  
Factor Xa ◽  
Dependent Manner ◽  
Prothrombinase Complex ◽  
Factor Va ◽  
Flow Cytometric ◽  
And Function ◽  
Microscopy Techniques ◽  
Prothrombinase Activity

Thrombin is generated on the surface of mononuclear cells (MNCs) through the assembly and function of the prothrombinase complex consisting of the enzyme factor Xa, the cofactor/factor Va, calcium ions, and an appropriate membrane surface for proper assembly of the protein constituents. Assays performed in the presence of factors Va and Xa indicated that endotoxin significantly enhanced the prothrombinase activity (1.5- to 2.5-fold; P less than .001) expressed by MNCs in a dose- and time-dependent manner. Monocytes present in the MNC suspensions were responsible for this increased activity through processes resulting in both enhanced cellular activity and the enhanced release of membranous vesicles. Endotoxin was without effect on the expression of lymphocyte prothrombinase activity. Scanning electron microscopy techniques indicated that endotoxin resulted in extensive membrane blebbing of the monocytes present in the MNC suspensions with no effect on the morphology of the lymphocytes. Within 5 hours, endotoxin maximally enhanced the prothrombinase activity expressed by the monocyte membrane surface 2.8-fold, whereas 8 hours was required to maximally enhance the activity associated with the released vesicles by twofold. The observed increase in activity expressed by the monocyte membrane surface was due solely to endotoxin, since the activity expressed by the unstimulated monocyte membrane surface remained unaltered over time. In contrast, cell vesiculation, which occurred in the absence of any stimulus, was further enhanced by endotoxin. The increase in activity associated with the released vesicles from both stimulated and unstimulated cells paralleled an increase in the vesicle number as determined by flow cytometric analyses. The vesicle released from both unstimulated and stimulated monocytes were indistinguishable in size as determined by image analysis and ranged between 0.05 and 0.3 microns in diameter. 2-Deoxy-D-glucose (2DG) significantly enhanced the prothrombinase activity expressed by the monocyte membrane surface, as well as the released vesicle fraction, when used alone or in addition to endotoxin. The enhanced activity associated with the vesicle fraction again was attributed to the release of more vesicles. In contrast, cycloheximide decreased the prothrombinase activity expressed by the monocyte membrane surface, as well as the activity associated with vesicles released from both stimulated and unstimulated cells. These data suggest that the expression of monocyte prothrombinase activity can be significantly enhanced by endotoxin through processes that alter the monocyte membrane surface and augment the vesiculation process. Both processes appear to be regulated by protein synthesis and adenosine triphosphate (ATP)-dependent mechanisms.

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