Phosphatidylserine (PS) Binding Sites in Kringle Modules Regulate the Domain Organization and Conformation of Bovine Prothrombin.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1952-1952
Author(s):  
Shalmali Sen ◽  
Mou Banerjee ◽  
Vishwanath Koppaka ◽  
Chaoming Zhou ◽  
Barry R. Lentz
Keyword(s):  
Binding Sites ◽  
Structural Changes ◽  
Factor Xa ◽  
Scanning Calorimetry ◽  
Kringle Domains ◽  
Factor Va ◽  
Trp Fluorescence ◽  
Gla Domain ◽  
Fluorescence Titrations ◽  
Induced Changes

Abstract Phosphatidylserine (PS) specifically regulates prothrombin activation during blood coagulation by binding to specific on factor Xa (Koppaka et al. Biochemistry, 7483, 1996) and its cofactor, factor Va (Zhai et al. Biochemistry, 5675, 2002). Binding to PS-containing membranes also alters prothrombin conformation (Lentz et al., Biochemistry, 5460, 1994; Chen et al. ibid. 4701, 1997). We ask here whether specific PS binding sites on prothrombin also control these structural changes, and, if so, where these sites are located. Four methods (Trp fluorescence, circular dichroism [CD], differential scanning calorimetry [DSC], and quasi-elastic light scattering [QELS]) were used to define the structural consequences of soluble 1,2-dicaproyl-sn-glycero-3-phospho-L-serine; (C6PS) binding both to whole prothrombin and to its proteolytically generated fragments. Intrinsic fluorescence titrations suggested the existence of two linked C6PS binding sites in fragment 1.2 (F1.2) and prethrombin 1, both of which contain prothrombin’s kringle pair. The existence of two sites was supported by direct equilibrium binding measurements with F1.2. CD measurements at increasing C6PS concentrations with both F1.2 and the kringle pair (F1.2 minus the γ-carboxy-glutamic acid [GLA] domain) were consistent with formation of a structure akin to an anti-parallel β sheet. Thermal denaturation profiles of F1.2 suggested calcium-independent, C6PS-induced domain reorganization within this fragment. Denaturation profiles and Trp fluorescence of the N-terminal, membrane binding domain (F1) and fragment 2 (F2) did not reveal any C6PS-induced changes. F2 consists mainly of the second cys-rich kringle module. The hydrodynamic radius of prothrombin was also found to decrease substantially (from 3.3 nm to 2.6 nm) in the presence of saturating (1mM) C6PS. Other lipids (phosphatidylglycerol and phosphatidyl-D-serine) did bind to prothrombin but did not produce comparable structural changes. The results show that C6PS 1) binds specifically to linked, calcium-independent sites within prothrombin’s two kringle domains and 2) induces thereby a conformational reorganization in the whole molecule. Supported by USPHS grant HL072827 to BRL.

Kinetic Analysis Of Prothrombin Activation By Des (l-44) Factor Xa

1981 ◽  
Author(s):  
W F Skogen ◽  
C T Esmon ◽  
A C Cox
Keyword(s):  
Binding Sites ◽  
Factor Xa ◽  
Factor X ◽  
Apparent Dissociation Constant ◽  
Phospholipid Analysis ◽  
Factor Va ◽  
Gla Domain ◽  
Dissociation Constant Kd ◽  
Prothrombin Activation

The region of factor X containing the gla residues was released by mild chymotryptic digestion and the resulting Des (1-44) factor X was converted to its activated form with the Russell’s viper venom factor X activator as described by others. The modified enzyme, factor Xa(-gd), retains its ability to activate prothrombin, the activation was still accelerated by factor Va, but the activation was no longer accelerated by phospholipid. The interaction between factor Va and either factor Xa or factor Xa(-gd) was studied kinetically. The rate of prothrombin activation was measured as a function of increased factor Va concentration in reaction mixtures where the factor Xa or factor Xa(-gd), prothrombin, and Ca2+ concentrations were constant. In the absence of phospholipid, analysis of the above data indicated an apparent dissociation constant (Kd’) of factor Xa for factor Va of 3.6 x 10-8 M. The removal of the gla domain lowered the apparent affinity of factor Xa for factor Va to 2.3 x 10-6 M indicating a role of the gla domain in the factor Xa-factor Va interaction even in the absence of phospholipid. Although the affinity was lowered, the V max of the reaction was identical for factdr Xa and factor Xa(-gd) in the presence of saturating concentrations of factor Va. With the addition of phospholipid, the Kd’ of factor Xa for factor Va was 4.7 x 10-10 M and V max increased some 297 fold. Phospholipid had no effect on the Kd’ of the factor Xa(-gd)-factor Va complex (Kd’ s 2.3 x 10-6 M) and V max was unaltered. These results demonstrate that phospholipid has little or no effect on factor Va function when factor Xa has lost its gla mediated Ca2+ binding sites.

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 Group D prothrombin activators from snake venom are structural homologues of mammalian blood coagulation factor Xa

2003 ◽  
Vol 369 (3) ◽  
pp. 635-642 ◽  
Author(s):  
Veena S. RAO ◽  
Jeremiah S. JOSEPH ◽  
R. Manjunatha KINI
Keyword(s):  
Heavy Chain ◽  
Structural Information ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Serine Proteinase ◽  
Single Step ◽  
Step Method ◽  
Factor Va ◽  
Gla Domain ◽  
Group D

Procoagulant venoms of several Australian elapids contain proteinases that specifically activate prothrombin; among these, Group D activators are functionally similar to coagulation factor Xa (FXa). Structural information on this class of prothrombin activators will contribute significantly towards understanding the mechanism of FXa-mediated prothrombin activation. Here we present the purification of Group D prothrombin activators from three Australian snake venoms (Hoplocephalus stephensi, Notechis scutatus scutatus and Notechis ater niger) using a single-step method, and their N-terminal sequences. The N-terminal sequence of the heavy chain of hopsarin D (H. stephensi) revealed that a fully conserved Cys-7 was substituted with a Ser residue. We therefore determined the complete amino acid sequence of hopsarin D. Hopsarin D shows 70% similarity with FXa and 98% similarity with trocarin D, a Group D prothrombin activator from Tropidechis carinatus. It possesses the characteristic Gla domain, two epidermal growth factor-like domains and a serine proteinase domain. All residues important for catalysis are conserved, as are most regions involved in interactions with factor Va and prothrombin. However, there are some structural differences. Unlike FXa, hopsarin D is glycosylated in both its chains: in light-chain residue 52 and heavy-chain residue 45. The glycosylation on the heavy chain is a large carbohydrate moiety adjacent to the active-site pocket. Overall, hopsarin D is structurally and functionally similar to mammalian coagulation FXa.

scholarly journals Deficiency of factor Xa-factor Va binding sites on the platelets of a patient with a bleeding disorder

Blood ◽  
1979 ◽  
Vol 54 (5) ◽  
pp. 1015-1022 ◽  
Author(s):  
JP Miletich ◽  
WH Kane ◽  
SL Hofmann ◽  
N Stanford ◽  
PW Majerus
Keyword(s):  
Binding Sites ◽  
Factor Xa ◽  
Coagulation Factors ◽  
Severe Bleeding ◽  
Factor V ◽  
Platelet Factor ◽  
Platelet Surface ◽  
Factor Va ◽  
Number Of Binding Sites ◽  
Stimulation Of

Factor V (Va) is essential for binding of factor Xa to the surface of platelets. After thrombin treatment, normal platelets release at least five times more factor Va activity than is required for maximal factor Xa binding. The concentration of factor V activity obtained after thrombin stimulation of 10(7) normal platelets is sufficient to allow half-maximal factor Xa binding to 10(8) platelets (10% normal, 90% factor-V deficient). Therefore, factor Va activity is not limiting in platelet-surface factor Xa binding and prothrombin activation in normal platelets; some other components limit the number of binding sites. We report studies of a patient (M.S.) with a moderate to severe bleeding abnormality whose platelets are deficient in the platelet-surface component required for the factor Va-factor Xa binding. The patient's platelet factor Va activity released after thrombin treatment is normal, but factor Xa binding is 20%-25% of control values at saturation. Abnormal prothrombin consumption in a patient with normal plasma coagulation factors and platelet function suggests a disorder in platelet-surface thrombin formation.

scholarly journals Binding sites for blood coagulation factor Xa and protein S involving residues 493-506 in factor Va

1996 ◽  
Vol 5 (9) ◽  
pp. 1883-1889 ◽  
Author(s):  
Mary J. Heeb ◽  
Yumi Kojima ◽  
Tilman M. Hackeng ◽  
John H. Griffin
Keyword(s):  
Blood Coagulation ◽  
Binding Sites ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Protein S ◽  
Factor Va

scholarly journals Acylcarnitines Are Novel Anticoagulant Lipids That Target Factor Xa and That Are Reduced in Plasma of Venous Thrombosis Patients Based on Untargeted and Targeted Metabolomics

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2797-2797
Author(s):  
Hiroshi Deguchi ◽  
Yajnavalka Banerjee ◽  
Gary Siuzdak ◽  
Sunia Trauger ◽  
Ewa Kalisiak ◽  
...  
Keyword(s):  
Anticoagulant Activity ◽  
Acyl Chain ◽  
Factor Xa ◽  
Targeted Metabolomics ◽  
Metabolomics Data ◽  
Factor Va ◽  
Acyl Chains ◽  
Gla Domain ◽  
Long Chain ◽  
Prothrombin Activation

Abstract Deep vein thrombosis and pulmonary embolism (VTE) are common causes of morbidity and mortality. No genetic or acquired biomarkers or risk factors can be identified in many VTE patients. Thus, there is a major unmet need to identify new biomarkers and new causal risk factors in VTE patients. Metabolomics is an unexplored frontier for VTE research. Thus, to discover novel plasma metabolite biomarkers for VTE risk, we performed liquid chromatography-mass spectrometry (LC-MS)-based untargeted metabolomics in a pilot study to analyze plasma metabolites without any specific targeting of known metabolites. Untargeted metabolomics data for 40 male idiopathic adult VTE cases and 40 age-matched male controls recorded 9,400 metabolic features for each plasma sample. Data analysis revealed two plasma long-chain acylcarnitines (ACs) (10:1 and 16:1) to be significantly decreased in VTE patients compared to controls, suggesting the association of plasma long-chain ACs levels with the risk of VTE. To validate the association of reduced plasma levels of long-chain ACs with the VTE risk, LC-MS-based targeted metabolomics was used to determine long-chain ACs levels. Data from targeted metabolomics additionally showed that several long-chain ACs (10:1, 12:0, 12:2 and 18:2) were lower in VTE patient plasmas than in control plasmas for subjects in our pilot VTE case-control study. ACs, which heretofore have no known function in coagulation reactions, consist of a hydrophobic side chain of an acyl moiety which is linked to the carnitine moiety. ACs circulate in plasma and also play key roles in mitochondrial energy metabolism. Studies were initiated to define any procoagulant or anticoagulant properties of ACs. Remarkably, ACs inhibited factor Xa-initiated clotting assays which were triggered by adding either purified factor Xa or RVV-X to plasma. The anticoagulant activities of various ACs were both dose-dependent and acyl chain length-dependent, where ACs with longer acyl chains were more potent procoagulants than ACs with shorter acyl chains (18, 16 > 14, 10 > 6 acyl chain carbons). However, thrombin-induced clotting was not inhibited by ACs, suggesting that ACs were acting on the prothrombinase complex. For purified prothrombinase reactant systems containing prothrombin, factor Xa, factor Va, and phospholipid vesicles [phosphatidylcholine:phosphatidylserine (PC/PS) 90:10 w/w], thrombin generation was dose-dependently inhibited by 16:0-AC (IC50 = 13 µM). For reactant mixtures lacking either phospholipids or factor Va, prothrombin activation by factor Xa was still dose-dependently inhibited by 16:0-AC (IC50 = 5.6 µM and 5.8 µM, respectively). Prothrombin activation by factor Xa in the absence of both factor Va and phospholipids was still inhibited by 16:0-AC (IC50 = 11 µM). In controls, 16:0-AC did not inhibit the amidolytic activity of either factor Xa or thrombin. These findings indicate that neither factor Va nor phospholipid was required for the anticoagulant property of 16:0-AC and that this lipid did not inhibit the enzyme active sites, suggesting that this lipid disrupts interactions between factor Xa and prothrombin. Although canonical coagulation paradigms emphasize key roles for lipid binding sites that are localized in the amino terminal Gla domain of vitamin K dependent clotting factors, Gla-domainless (DG)-prothrombin activation by factor Xa and prothrombin activation by DG-factor Xa were each similarly inhibited by 16:0-AC with IC50values of 11 and 7.0 μM, respectively. For surface plasmon resonance (SPR) binding studies, biotinylated-Glutamyl-Glycyl-Arginyl-chloromethyl ketone (BEGR) was used to label the active site of factors Xa, IXa, and VIIa. BEGR-factor Xa and BEGR-DG-factor Xa bound with similar affinities to 16:0-AC (10 µM and 23 µM, respectively) whereas no binding of 16:0-AC (60 μM) to BEGR-factor VIIa or BEGR-factor IXa was observed. Thus, these data suggest that a 16:0-AC binding site on factor Xa that is located outside the Gla domain mediates this lipid’s anticoagulant activity. In summary, first, untargeted and targeted metabolomics data for a pilot VTE case-control study identified ACs as potential biomarkers for VTE, and, second, detailed mechanistic studies show that the AC, 16:0 acyl-carnitine, has anticoagulant activity in the absence of factor Va or phospholipids that is related to its ability to bind factor Xa outside the Gla domain. Disclosures No relevant conflicts of interest to declare.

scholarly journals Modulation of Prothrombinase Assembly and Activity by Phosphatidylethanolamine

2011 ◽  
Vol 286 (41) ◽  
pp. 35535-35542 ◽  
Author(s):  
Rinku Majumder ◽  
Xiaoe Liang ◽  
Mary Ann Quinn-Allen ◽  
William H. Kane ◽  
Barry R. Lentz
Keyword(s):  
Binding Sites ◽  
Factor Xa ◽  
Specific Protein ◽  
The Other ◽  
Prothrombinase Complex ◽  
High Affinity ◽  
Factor Va ◽  
Platelet Membranes ◽  
Enhanced Activity ◽  
Thrombin Formation

Constituents of platelet membranes regulate the activity of the prothrombinase complex. We demonstrate that membranes containing phosphatidylcholine and phosphatidylethanolamine (PE) bind factor Va with high affinity (Kd = ∼10 nm) in the absence of phosphatidylserine (PS). These membranes support formation of a 60–70% functional prothrombinase complex at saturating factor Va concentrations. Although reduced interfacial packing does contribute to factor Va binding in the absence of PS, it does not correlate with the enhanced activity of the Xa-Va complex assembled on PE-containing membranes. Instead, specific protein-PE interactions appear to contribute to the effects of PE. In support of this, soluble C6PE binds to recombinant factor Va2 (Kd = ∼6.5 μm) and to factor Xa (Kd = ∼91 μm). C6PE and C6PS binding sites of factor Xa are specific, distinct, and linked, because binding of one lipid enhances the binding and activity effects of the other. C6PE triggers assembly (Kdapp = ∼40 nm) of a partially active prothrombinase complex between factor Xa and factor Va2, compared with Kdapp for C6PS ∼2 nm. These findings provide new insights into the possible synergistic roles of platelet PE and PS in regulating thrombin formation, particularly when exposed membrane PS may be limiting.

Factor Xa Binding Sites on Factor Va in the Prothrombinase Complex.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1713-1713
Author(s):  
Andrew J. Gale ◽  
John H. Griffin
Keyword(s):  
Conformational Changes ◽  
Binding Sites ◽  
Factor Xa ◽  
Sequence Alignments ◽  
Proteolytic Activation ◽  
Apparent Affinity ◽  
Prothrombinase Complex ◽  
Factor Va ◽  
Kd Values ◽  
A2 Domain

Factor Va (FVa), a non-enzymatic cofactor for proteolytic activation of prothrombin by factor Xa (FXa), interacts with FXa via both its heavy and light chains. Phospholipids can bind both FVa and FXa and enhance prothrombin activation. The action of the prothrombinase complex (FXa:FVa) on its substrate, prothrombin, involves stepwise binding and enzymatic interactions, including FVa interactions with exosites on FXa that are topologically distinct from its active site. Synthetic peptides used as inhibitors of the prothrombinase complex implicated FVa residues 311–335 and 493–506 as probable FXa binding sites. Analysis of sequence alignments of FV with factor FVIII and of our 3-dimensional FVa model lead us to hypothesize that residues 312–315, 319–323, and 499–505, which delineate a remarkable compact triangular surface on the A2 domain are involved in FXa binding. Hence, we used homologous loop mutagenesis and substituted these three FV sequences with FVIII sequences, replacing 312-TREQ-315 with RSVA, 319-MKRWE-323 with PKTWV, and 499-KSRSLDR-505 with YKESVDQ. Each recombinant FV mutant was purified, characterized and compared to control wild type (wt)-FVa using ELISA and functional assays. Prothrombinase assays using thrombin-activated FVa and purified components were used to determine functional apparent Kd values of FVa for FXa by varying FXa concentrations in the absence or presence of saturating phospholipid (PL) vesicles (20%PS/80%PC). In the absence of PL vesicles, the kinetically determined apparent Kd values of each FVa mutant were greatly increased, such that mutation of either 312–315 or 319–323 in FVa decreased the apparent affinity for FXa by greater than 80-fold while mutation of 499–505 resulted in a 20-fold lower apparent affinity for FXa. However, in the presence of PL vesicles, these three FVa mutants each showed only a moderately reduced affinity for FXa (2.0–2.5 fold increased apparent Kd). None of the three FVa mutations significantly altered the Km for prothrombin. Therefore, each of the three mutated sequences in FVa is critical for normal functional interactions with FXa. Furthermore, these results illustrate the importance of each different kind of interactions, namely protein-protein and protein-PL, for the overall formation of the prothrombinase complex. Binding of both FXa and FVa to PL surfaces can help optimize the relative orientation of FVa to FXa to facilitate enzyme:cofactor exosite interactions and/or can promote conformational changes in one or both of FXa and FVa that affects exosite interactions. In the absence of PL, the effects of the A2 domain mutations on protein-protein interactions are magnified whereas when FXa and FVa are PL-bound, the observable effects of these mutations are greatly reduced. In summary, these mutagenesis studies support the hypothesis that three, closely located sequences on the surface of the FVa A2 domain comprising residues 312–315, 319–323, and 499–505 contribute significantly to FXa binding.

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