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

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.

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.

Cholesterol and Phosphatidylethanolamine Enhance the Binding of Factor Va and Factor Xa to Phospholipid Vesicles and Promote Assembly of the Prothrombinase Complex on Membranes Containing Low Concentrations of Phosphatidylserine.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1712-1712
Author(s):  
Xiaoe Liang ◽  
Mary Ann Quinn-Allen ◽  
William H. Kane
Keyword(s):  
Phospholipid Composition ◽  
Factor Xa ◽  
Phospholipid Vesicles ◽  
Prothrombinase Complex ◽  
Factor Va ◽  
Activated Platelets ◽  
Low Concentrations ◽  
Kd Values ◽  
Direct Binding ◽  
Prothrombinase Activity

Abstract Platelet membranes are composed of cholesterol (CH) and phospholipids including phosphatidylethanolamine (PE), sphingomeylin (SM), phosphatidylcholine (PC) and phosphatidylserine (PS). Phospholipid vesicles containing 20–25% PS have been used extensively to characterize assembly and regulation of the prothrombinase complex. However, the concentration of PS on the surface of activated platelets has been determined to be only 4–13%. Smeets et al (Thromb. Res. 81:419, 1996) demonstrated that CH and PE in the presence of 0–10% PS have stimulating effects on prothrombinase activity, while SM has an inhibitory one. To further investigate the roles of CH, PE and SM in the binding of factor Va and Xa to phospholipid vesicles and assembly of the prothrombinase complex, vesicles consisting of 1–25% DOPS plus 30% CH, 30% DOPE and/or 20% SM, were prepared for prothrombinase assays as a function of DOPS concentration. Compared to individual effect of DOPS alone or DOPS plus CH, PE or SM on prothrombinase activity, a synergistic effect was observed on vesicles containing CH, DOPE and SM, which was significant in the range of 1–15% DOPS. The PS concentration required for half-maximal rates of thrombin generation was 5.3% for vesicles containing CH, DOPE and SM, compared to 10.6, 11.2, 8.3 and 12.6% for vesicles containing DOPS alone, plus CH, plus DOPE or plus SM, respectively. This demonstrates that the requirement for PS is substantially decreased in the presence of CH, PE and SM. In order to further define the mechanisms for this effect, direct binding of factor Va to vesicles containing 30% CH, 30% PE and 20% SM was investigated using a fluorescence resonance energy transfer assay. These binding experiments demonstrated that factor Va bound to vesicles containing 1, 5, 10, and 20% DOPS with Kd values of 2.88±0.43 nM, 1.15±0.17 nM, 1.16±0.13 nM, and 1.18±0.19 nM, respectively. At low concentrations of PS (≤ 5%), CH, DOPE and SM increased the binding affinity of factor Va 2 to 6-fold compared to vesicles containing only DOPS. When prothrombinase activity was measured as a function of factor Va concentration on vesicles containing 5, 10, and 20% DOPS, K1/2Va values of 0.63±0.08nM, 0.38±0.05nM, and 0.37±0.07nM were observed which were comparable to the Kd’s determined in direct factor Va binding experiments. These results indicate that factor Va binds tightly to phospholipid vesicles mimicking the phospholipid composition of activated platelets even at very low concentrations of PS. When direct binding of factor Xa to vesicles containing 5, 10, and 20% DOPS plus CH, DOPE and SM was examined, the Kd values were 70.5±6.8 nM, 62.6±6.6 nM, and 49.6±4.0 nM, respectively. When the interaction of factor Xa with membranes was investigated using prothrombinase assays, K1/2Xa values of 0.12±0.03nM, 0.04±0.01nM, and 0.01±0.01nM were observed. In conclusion, the presence of CH and PE increases the affinity of factor Va and factor Xa binding to phospholipid vesicles containing physiologic concentrations of PS. The binding of factor Va to vesicles containing 1% PS is not sufficient for assembly and function of the prothrombinase complex, suggesting that higher concentrations of PS are needed to support the binding of factor Xa and prothrombin.

Inhibition of Factor Xa in Prothrombinase Is Enhanced by Covalent Linkage of Antithrombin to Heparin.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1613-1613
Author(s):  
Sanjay Patel ◽  
Leslie R. Berry ◽  
Lesley Smith ◽  
Anthony Chan
Keyword(s):  
Rate Constants ◽  
Thrombin Generation ◽  
Factor Xa ◽  
Complex Mixtures ◽  
Free State ◽  
Phospholipid Vesicles ◽  
Covalent Linkage ◽  
Rate Difference ◽  
Prothrombinase Complex ◽  
Factor Va

Abstract The rate of prothrombin to thrombin conversion by factor Xa (Xa) is enhanced when Xa is incorporated into the surface-bound prothrombinase complex. However, in comparison to the free state, Xa within the prothrombinase complex is afforded protection from antithrombin + heparin (AT+H) inactivation. We have shown that, unlike AT+H, a covalent conjugate of AT and H (ATH) can neutralize fibrin-bound thrombin. In this study, AT+H and ATH were compared in their reaction with Xa +/− prothrombinase complex. Mixtures of CaCl2, phospholipid vesicles, factor Va (Va) and prothrombin in TSP buffer, were combined with Xa. Following addition of either AT+H or ATH, time samples were neutralized with Na2EDTA + polybrene + substrate (S-2222) and residual Xa activity measured. Second order rate constants (k2) were calculated from plots of activity versus time. Results were compared to corresponding experiments with Xa alone. AT+H inactivation of Xa in prothrombinase occurred at a k2 (x 107 M−1min−1) of 2.34 +/− 0.09. In contrast, neutralization of free Xa by AT+H was significantly faster (k2 = 8.34 +/− 0.18, p = 0.03). Reaction with ATH showed no significant rate difference for Xa inhibition in either the complexed or free states (18.5 +/− 3.3 and 16.3 +/− 3.7, respectively). Intriguingly, the rates achieved for ATH inhibition of complexed and free Xa were significantly greater than that for AT+H with free Xa (p=0.03 and p=0.02, respectively). We conclude that covalent complexes of AT and H do not encounter resistance in the inhibition of Xa in prothrombinase, as seen for non-covalent AT+H mixtures. Thus, it is possible for ATH to effectively inhibit the propagation phase of thrombin generation and thus dampen thrombin production via neutralization of Xa in prothrombinase.

STOPPED-FLOW KINETICS OF PROTHROMBINASE COMPLEX ASSEMBLY

1987 ◽  
Author(s):  
S Krishnaswamy ◽  
K Jones ◽  
K Mann
Keyword(s):  
Complex Formation ◽  
Calcium Ion ◽  
Factor Xa ◽  
Binary Complex ◽  
Stopped Flow ◽  
Initial Formation ◽  
First Order ◽  
Prothrombinase Complex ◽  
Factor Va ◽  
Pseudo First Order

Prothrombinase is the multi -component enzyme composed of factor Xa and the cofactor, factor Va, that assemble on a phospholipid surface in the presence of calcium ion. Fluoresence stopped-flow kinetic studies of prothrombinase assembly in the absence of the substrate, prothrombin were undertaken at saturating concentrations of calcium ion, using phospholipid vesicles (PCPS), factor Va and factor Xa modified at the active-site with the fluorophore dansyl-glutamylglycinylarginylchloro methylketone (DEGR-Xa). The substantial fluorescence enhancement of DEGR-Xa in the presence of factor Va, PCPS and calcium ion was used as a continuous measure of prothrombinase complex formation. The rate of complex formation studied under pseudo-first order conditions was most rapid when factor Va was reacted with the DEGR-Xa-PCPS binary complex. Under these conditions, the rate increased linearly with increasing PCPS concentrations and was independent of the concentration of factor Va. The reaction between Va and PCPS assessed by stopped-flow light scattering produced identical rates. The data indicate that the rate-limiting step in the reaction between Va and PCPS-bound DEGR-Xa is the initial formation of a Va-PCPS binary complex (8.4×107M−1s−1) followed by the very rapid reaction (>3×109M−1s−1) between PCPS-bound DEGR-Xa and Va. When complex formation was initiated by mixing DEGR-Xa and Va with PCPS or by mixing DEGR-Xa with Va-PCPS, the pseudo-first order rates were substantially lower. Under these conditions, increasing concentrations of factor Va inhibited the rate of prothrombinase complex formation which could be overcome by increasing PCPS concentrations. The data are consistent with the interpretation that the initial formation of the DEGR-Xa-PCPS complex is a prerequisite for prothrombinase assembly. The second order rate constant for the reaction between DEGR-Xa and PCPS (9.5×109M−1s−1 ) was independently determined by stopped-flow light scattering. Collectively, the data indicate that prothrombinase assembly proceeds via the initial formation of DEGR-Xa-PCPS and Va-PCPS binary complexes. The lipid-bound constituents then rapidly react via diffusion over the vesicle surface to form prothrombinase.(Supported by NIH grants HL-35058 and HL-34575)

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.

Mutation of the Hydrophobic Residues in Factor Va2 C1 Domain Affects the Phosphatidylserine Mediated Prothrombin Activation.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1733-1733
Author(s):  
Rinku Majumder ◽  
Mary Ann Quinn-Allen ◽  
William H. Kane ◽  
Barry R. Lentz
Keyword(s):  
Factor Xa ◽  
Binding Pocket ◽  
Regulatory Site ◽  
Wild Type ◽  
Prothrombinase Complex ◽  
Factor Va ◽  
Essentially Normal ◽  
Tryptophan Residues ◽  
C1 Domain ◽  
Prothrombin Activation

Abstract Tightly associated factors Va and Xa serve as the essential prothrombin-activating complex that assembles on phosphatidylserine (PS)-containing platelet membranes during blood coagulation. The binding of factor Va to PS membranes regulates assembly of the prothrombinase complex. The C-terminal domain (C2) of factor Va (residues 2037–2196 in human factor Va) contains a soluble phosphatidylserine (C6PS) binding pocket flanked by a pair of tryptophan residues, Trp2063/Trp2064 (Srivastava A, Quinn-Allen MA, Kim SW, Kane WH, Lentz BR. Biochemistry, 2001, 40(28): 8246–55). Our recent results have shown that mutating these Trp residues abolishes FVa2-membrane binding, but does not affect the assembly and activity of the prothrombinase in the presence of 25% PS membranes or soluble C6PS. Our data also indicates that there is another site on factor Va2 that might be specific for PS or C6PS and might serve as a regulatory site for assembly or activity of the FVa2-FXa complex. A pair of solvent exposed amino acids, Tyr1956/Leu1957 in the C1 domain is located analogously to the critical Trp residues in C2. Recently, we showed that prothrombin activation in the presence of the factor Va mutant (Y1956, L1957) A was markedly impaired on phospholipid vesicles containing 10% or less PS but was essentially normal on vesicles containing 25% PS (Saleh, M., Peng, W., Quinn-Allen, MA., Macedo-Ribeiro, S., Fuentes-Prior, P., Bode,W. and Kane, WH. Thromb. Haemost. 2004,91:16–27). Our work aims to test the hypothesis that the PS regulatory site in Va2 is located analogously to the C6PS binding site in the C2 domain. We have used factor Va mutants with mutations in either the C1 domain, (Y1956, L1957) A or in both the C1 and C2 domains, (Y1956, L1957, W2063, W2064) A. We determine the rate of thrombin formation in the presence of 400 mM C6PS and wild type, C1 and C1C2 mutated factor Va2 to be 170, 12 and 11 nM/S−1/M−1, respectively. Mutations in the C1 and C1C2 domains of factor Va2 reduced the rate of activation of prothrombin to thrombin by 14–15 fold. We have also determined the effect of these mutations on the assembly of factors Xa–Va2 complex by monitoring the change in fluorescence of dansyl-glutamyl-glycyl-arginyl-chloromethylketone (DEGR-CK)-Xa with the addition of wild type, C1 and C1C2 mutated factor Va2 in the presence of 400 mM C6PS. Our data shows that the Kd’s of factor Xa with factor Va2 (wild type, C1 mutant and C1C2 mutant) are 3, 564, 624 nM respectively. Our results support the hypothesis that a PS regulatory site is located in the C1 domain of factor Va and includes residues Tyr1956 and Leu1957.

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