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.

The Role of Phospholipids and Factor Va in the Mechanism of Prothrombin Activation

1979 ◽  
Author(s):  
J. Rosing ◽  
G. Tans ◽  
J.W.P. Govers-Riemslag ◽  
R.F.A. Zwaal ◽  
H.C. Hemker
Keyword(s):  
Kinetic Parameters ◽  
Factor Xa ◽  
Clotting Factor ◽  
Factor V ◽  
Factor X ◽  
Factor Va ◽  
Prothrombin Activation ◽  
Thrombin Formation

The kinetic parameters of the conversion of prothrombin into thrombin by activated clotting factor X (factor Xa) have been determined in the absence and presence of Ca2+, phospholipid (phosphatidyl serine/phosphatidylcholine vesicles) and activated blood clotting factor V (factor Va). In free solution the Km for prothrombin is 298 μM which is well above its plasma concentration of 4μM. Under these conditions the Vmax of thrombin formation is 1.25 Moles min-1 Mole Xa -1. When phospholipid is present the km for prothrombin drops to 0.1μM while the Vmax is only slightly affected (3 Moles min-1 Mo Le Xa -1). For the complete prothrombin activating complex consisting of factor Xa, factor Va, Ca2+ and phospholipids the kinetic constants greatly favour thrombin formation. A for prothrombin of 0.26μM and a Vmax of 2130 Moles min-1 Mole xa -1 are measured under these conditions. These results help to elucidate the role of phospholipid and factor Va in prothrombin activation. The earlier observed rate enhancements caused by phospholipid and factor Va are explained as effects on the Km for prothrombin and the Vmax of thrombin formation, respectively. The changes of the kinetic parameters for prothrombinase complexes of various composition will be considered with respect to the function of the accessory components in the mechanism of prothrombin activation. Implications of these data for in vivo blood coagulation will be discussed.

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 Role of gamma-carboxyglutamic acid residues in the binding of factor IXa to platelets and in factor-X activation

Blood ◽  
1992 ◽  
Vol 79 (2) ◽  
pp. 398-405 ◽  
Author(s):  
R Rawala-Sheikh ◽  
SS Ahmad ◽  
DM Monroe ◽  
HR Roberts ◽  
PN Walsh
Keyword(s):  
Binding Sites ◽  
Factor Xa ◽  
Catalytic Efficiency ◽  
Factor Ix ◽  
Factor X ◽  
Apparent Dissociation Constant ◽  
Factor Ixa ◽  
Factor Viiia ◽  
Carboxyglutamic Acid ◽  
Factor X Activation

To study the requirements for factor-IXa binding to platelets and factor-X activation, we examined the consequences of chemical modification (factor IXMOD) or enzymatic removal (factor IXDES) of gamma-carboxyglutamic acid (Gla) residues. In the presence of factor VIIIa and factor X, there were 344 (+/- 52) binding sites/platelet for factor IXaMOD (apparent dissociation constant [kdapp] = 4.5 +/- 0.9 nmol/L) and 275 (+/- 35) sites/platelet for factor IXaDES (kdapp = 5.0 +/- 0.8 nmol/L) compared with 580 (+/-65) sites/platelet for normal factor IXa (factor IXaN) (kdapp = 0.61 +/- 0.1 nmol/L) and 300 (+/-62) sites/platelet for factor IX (kdapp = 2.9 +/- 0.29 nmol/L). The concentrations of factor IXaN, factor IXaMOD and factor IXaDES required for half-maximal rates of factor-Xa formation were 0.67 nmol/L, 3.5 nmol/L, and 6.7 nmol/L. Whereas maximal velocities (Vmax) of factor Xa formation by factor IXaMOD (approximately 0.8 nmol/L.min-1) and factor IXaN (approximately 10.5 nmol/L.min-1), turnover numbers (kcat expressed as moles of factor Xa formed per minute per mole of factor IXa bound), and values of catalytic efficiency (kcat/Km) were normal, indicating that the decreased rates of factor X activation observed with factor IXaMOD and factor IXaDES are solely a consequence of the abnormal binding of these proteins to thrombin-activated platelets in the presence of factor VIIIa and factor X. Thus, factor IXa binding to platelets is mediated in part, but not exclusively, by high-affinity Ca2+ binding sites in the Gla domain of factor IX.

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.

scholarly journals Role of gamma-carboxyglutamic acid residues in the binding of factor IXa to platelets and in factor-X activation

Blood ◽  
1992 ◽  
Vol 79 (2) ◽  
pp. 398-405 ◽  
Author(s):  
R Rawala-Sheikh ◽  
SS Ahmad ◽  
DM Monroe ◽  
HR Roberts ◽  
PN Walsh
Keyword(s):  
Binding Sites ◽  
Factor Xa ◽  
Catalytic Efficiency ◽  
Factor Ix ◽  
Factor X ◽  
Apparent Dissociation Constant ◽  
Factor Ixa ◽  
Factor Viiia ◽  
Carboxyglutamic Acid ◽  
Factor X Activation

Abstract To study the requirements for factor-IXa binding to platelets and factor-X activation, we examined the consequences of chemical modification (factor IXMOD) or enzymatic removal (factor IXDES) of gamma-carboxyglutamic acid (Gla) residues. In the presence of factor VIIIa and factor X, there were 344 (+/- 52) binding sites/platelet for factor IXaMOD (apparent dissociation constant [kdapp] = 4.5 +/- 0.9 nmol/L) and 275 (+/- 35) sites/platelet for factor IXaDES (kdapp = 5.0 +/- 0.8 nmol/L) compared with 580 (+/-65) sites/platelet for normal factor IXa (factor IXaN) (kdapp = 0.61 +/- 0.1 nmol/L) and 300 (+/-62) sites/platelet for factor IX (kdapp = 2.9 +/- 0.29 nmol/L). The concentrations of factor IXaN, factor IXaMOD and factor IXaDES required for half-maximal rates of factor-Xa formation were 0.67 nmol/L, 3.5 nmol/L, and 6.7 nmol/L. Whereas maximal velocities (Vmax) of factor Xa formation by factor IXaMOD (approximately 0.8 nmol/L.min-1) and factor IXaN (approximately 10.5 nmol/L.min-1), turnover numbers (kcat expressed as moles of factor Xa formed per minute per mole of factor IXa bound), and values of catalytic efficiency (kcat/Km) were normal, indicating that the decreased rates of factor X activation observed with factor IXaMOD and factor IXaDES are solely a consequence of the abnormal binding of these proteins to thrombin-activated platelets in the presence of factor VIIIa and factor X. Thus, factor IXa binding to platelets is mediated in part, but not exclusively, by high-affinity Ca2+ binding sites in the Gla domain of factor IX.

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.

The Regulatory Function of Amino Acid Region 659–663 of Factor Va on Prothrombinase during Thrombin Formation.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2012-2012
Author(s):  
Jamila Hirbawi ◽  
Michael Kalafatis
Keyword(s):  
Amino Acids ◽  
Heavy Chain ◽  
Binding Sites ◽  
Factor Xa ◽  
Prothrombinase Complex ◽  
Amino Acid Region ◽  
Factor Va ◽  
Acidic Amino Acids ◽  
Prothrombin Activation ◽  
Acid Region

Abstract Following vascular injury, the process of hemostasis facilitates the generation of thrombin, which in turn allows the formation of a fibrin clot. Without the proper regulation of this process, serious life threatening conditions, such as DVT (deep vein thrombosis), can occur. The proteolytic conversion of prothrombin to thrombin is catalyzed by the prothrombinase complex composed of the enzyme, factor Xa (fXa), the cofactor, factor Va (fVa), assembled on a membrane surface in the presence of divalent metal ions. The incorporation of factor Va (fVa) into the prothrombinase complex results in a 300,000-fold increase in the catalytic efficiency of factor Xa (fXa) for thrombin generation. Factor Va is composed of heavy and light chains. The light chain of the cofactor contains the binding sites of the cofactor to the membrane surface while the heavy chain contains binding sites for the other components of prothrombinase. Portions of the fVa heavy chain have been found to act as fXa binding sites. It has been demonstrated that the COOH-terminal region of factor Va contains cluster of acidic amino acids that are crucial for its cofactor activity. More specifically, amino acid region 695–698 from fVa heavy chain regulates the rate of cleavage of prothrombin at Arg271 by prothrombinase. The COOH-terminal portion of the heavy chain also contains another cluster of acidic amino acids (encompassing residues 659–663). Site-directed mutagenesis was performed to generate a factor V (fV) molecule with region Asp659-Asp663 (fV663) deleted. We have also constructed mutant molecules with regions Lys680-Arg709 and Asp659-Asp663 (fV663+709)) deleted from the COOH-terminal region of the heavy chain. Finally, a mutant molecule containing point mutations in region Asp659-Asp663 where the five amino acids in this sequence are mutated to all lysines (fV5K), was also constructed. These recombinant molecules along with wild type fV (fVWT) were transiently expressed in COS7L cells and assessed for their capability to promote prothrombin activation following activation by thrombin. Prothrombin activation by prothrombinase assembled with the mutant molecules was evaluated by SDS-PAGE and the kinetic parameters of the reactions were determined. SDS-PAGE analyses of prothrombin activation time courses revealed that the overall cleavage of prothrombin by prothrombinase assembled with the recombinant mutant molecules was slower. Two-stage clotting assays revealed that FV663+709, fVa5K, and fVa663 all had reduced clotting activities compared to fVaWT and plasma-derived fVa. Kinetic analyses demonstrated that Kd values for fXa of all the mutants were similar to fVaWT. However, kcat values for the various molecules varied. The kcat values for prothrombinase assembled with fVa5K, and fVa663 were 10-fold reduced when compared to the values obtained with prothrombinase assembled with fVaWT, while prothrombinase assembled with fVa663+709 had a kcat value that was sligtly lower than that of fVaWT. Our data suggest that amino acid region 659–663 from fV plays a crucial role for fVa cofactor acivity and overall the data demonstrate that acidic amino acids from the COOH-terminus of the factor Va heavy chain play a preeminent role in proper prothrombinase complex assembly and function, resulting in competent thrombin formation. These data assign an important regulatory role of the acidic COOH-terminal region of fVa to the activity of factor Xa within prothrombinase. Finally, our data aid in further studies that may lead to the development of small synthetic molecules that could be used as anticoagulants in individuals with thrombotic tendencies.

scholarly journals An inhibitory monoclonal antibody to factor X that blocks prothrombin activation but not prothrombinase enzyme assembly

Blood ◽  
1988 ◽  
Vol 72 (6) ◽  
pp. 1911-1921
Author(s):  
WR Church ◽  
TL Messier ◽  
MM Tucker ◽  
KG Mann
Keyword(s):  
Monoclonal Antibody ◽  
Heavy Chain ◽  
Sodium Dodecyl ◽  
Factor Xa ◽  
Thrombin Inhibitor ◽  
Gel Chromatography ◽  
Factor X ◽  
Apparent Dissociation Constant ◽  
Factor Va ◽  
Canine Plasma

A monoclonal antibody (designated alpha BFX-2b) prepared against bovine factor X inhibited factor X activity in human, bovine, porcine, rabbit, and canine plasma. In assays using purified prothrombinase components, factor Xa, factor Va, phospholipid vesicles, and calcium ion with the fluorescent active site thrombin inhibitor dansylarginyl-N-(3-ethyl-1,5- pentanediyl)amide, the antibody inhibited the conversion of prothrombin to thrombin. Antibody alpha BFX-2b also blocked prothrombinase cleavage of the macromolecular substrates prethrombin 1 and prethrombin 2 but did not inhibit factor Xa hydrolysis of the synthetic substrate benzoyl- Ile-Glu-Gly-Arg-p-nitroanilide. The antibody also prevented the inactivation of factor Xa by antithrombin III but did not prevent the inactivation by soybean trypsin inhibitor. Antibody alpha BFX-2b bound factor Xa with a stoichiometry of 1:1 and an apparent dissociation constant of 9.0 x 10(-11) mol/L as estimated from its inhibition of prothrombinase activity. Antibody alpha BFX-2b did not prevent binding of factor Xa to factor Va-phospholipid as measured by using fluorescence polarization or high-pressure liquid gel chromatography with the fluorescent Factor Xa analogue dansyl-glutamyl-glycyl-arginyl- Xa. Immunoblotting of factor X following electrophoresis on sodium dodecyl sulphate-polyacrylamide gels and transfer to nitrocellulose indicated that the antigenic determinant recognized by antibody alpha BFX-2b was found on the heavy chain of factors X and Xa. From these observations it can be concluded that antibody alpha BFX-2b recognizes a highly conserved epitope on the factor X heavy chain that is remote from the topographic sites required for prothrombinase complex assembly and substrate hydrolysis but may be located at or near a portion of the macromolecular substrate binding site.

scholarly journals An inhibitory monoclonal antibody to factor X that blocks prothrombin activation but not prothrombinase enzyme assembly

Blood ◽  
1988 ◽  
Vol 72 (6) ◽  
pp. 1911-1921 ◽  
Author(s):  
WR Church ◽  
TL Messier ◽  
MM Tucker ◽  
KG Mann
Keyword(s):  
Monoclonal Antibody ◽  
Heavy Chain ◽  
Sodium Dodecyl ◽  
Factor Xa ◽  
Thrombin Inhibitor ◽  
Gel Chromatography ◽  
Factor X ◽  
Apparent Dissociation Constant ◽  
Factor Va ◽  
Canine Plasma

Abstract A monoclonal antibody (designated alpha BFX-2b) prepared against bovine factor X inhibited factor X activity in human, bovine, porcine, rabbit, and canine plasma. In assays using purified prothrombinase components, factor Xa, factor Va, phospholipid vesicles, and calcium ion with the fluorescent active site thrombin inhibitor dansylarginyl-N-(3-ethyl-1,5- pentanediyl)amide, the antibody inhibited the conversion of prothrombin to thrombin. Antibody alpha BFX-2b also blocked prothrombinase cleavage of the macromolecular substrates prethrombin 1 and prethrombin 2 but did not inhibit factor Xa hydrolysis of the synthetic substrate benzoyl- Ile-Glu-Gly-Arg-p-nitroanilide. The antibody also prevented the inactivation of factor Xa by antithrombin III but did not prevent the inactivation by soybean trypsin inhibitor. Antibody alpha BFX-2b bound factor Xa with a stoichiometry of 1:1 and an apparent dissociation constant of 9.0 x 10(-11) mol/L as estimated from its inhibition of prothrombinase activity. Antibody alpha BFX-2b did not prevent binding of factor Xa to factor Va-phospholipid as measured by using fluorescence polarization or high-pressure liquid gel chromatography with the fluorescent Factor Xa analogue dansyl-glutamyl-glycyl-arginyl- Xa. Immunoblotting of factor X following electrophoresis on sodium dodecyl sulphate-polyacrylamide gels and transfer to nitrocellulose indicated that the antigenic determinant recognized by antibody alpha BFX-2b was found on the heavy chain of factors X and Xa. From these observations it can be concluded that antibody alpha BFX-2b recognizes a highly conserved epitope on the factor X heavy chain that is remote from the topographic sites required for prothrombinase complex assembly and substrate hydrolysis but may be located at or near a portion of the macromolecular substrate binding site.

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.

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