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Sriram Krishnaswamy - One of the best experts on this subject based on the ideXlab platform.
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crystal structure of the Prothrombinase complex from the venom of pseudonaja textilis
Blood, 2013Co-Authors: Bernhard C Lechtenberg, Sriram Krishnaswamy, Thomas A Murrayrust, Daniel J D Johnson, Ty E Adams, Rodney M Camire, James A. HuntingtonAbstract:The Prothrombinase complex, composed of the protease factor (f)Xa and cofactor fVa, efficiently converts prothrombin to thrombin by specific sequential cleavage at 2 sites. How the complex assembles and its mechanism of prothrombin processing are of central importance to human health and disease, because insufficient thrombin generation is the root cause of hemophilia, and excessive thrombin production results in thrombosis. Efforts to determine the crystal structure of the Prothrombinase complex have been thwarted by the dependence of complex formation on phospholipid membrane association. Pseutarin C is an intrinsically stable Prothrombinase complex preassembled in the venom gland of the Australian Eastern Brown Snake (Pseudonaja textilis). Here we report the crystal structures of the fX-fV complex and of activated fXa from P textilis venom and the derived model of active pseutarin C. Structural analysis supports a single substrate binding channel on fVa, to which prothrombin and the intermediate meizothrombin bind in 2 different orientations, providing insight into the architecture and mechanism of the Prothrombinase complex—the molecular engine of blood coagulation.
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membrane binding by prothrombin mediates its constrained presentation to Prothrombinase for cleavage
Journal of Biological Chemistry, 2013Co-Authors: Harlan N Bradford, Sriram Krishnaswamy, Steven J OrcuttAbstract:Abstract Long-standing dogma proposes a profound contribution of membrane binding by prothrombin in determining the rate at which it is converted to thrombin by Prothrombinase. We have examined the action of Prothrombinase on full-length prothrombin variants lacking γ-carboxyglutamate modifications (desGla) with impaired membrane binding. We show an unexpectedly modest decrease in the rate of thrombin formation for desGla prothrombin but with a major effect on the pathway for substrate cleavage. Using desGla prothrombin variants in which the individual cleavage sites have been singly rendered uncleavable, we find that loss of membrane binding and other Gla-dependent functions in the substrate leads to a decrease in the rate of cleavage at Arg320 and a surprising increase in the rate of cleavage at Arg271. These compensating effects arise from a loss in the membrane component of exosite-dependent tethering of substrate to Prothrombinase and a relaxation in the constrained presentation of the individual cleavage sites for active site docking and catalysis. Loss of constraint is evident as a switch in the pathway for prothrombin cleavage and the intermediate produced but without the expected profound decrease in rate. Extension of these findings to the action of Prothrombinase assembled on platelets and endothelial cells on fully carboxylated prothrombin reveals new mechanistic insights into function on physiological membranes. Cell-dependent enzyme function is probably governed by a differential ability to support prothrombin binding and the variable accumulation of intermediates from the two possible pathways of prothrombin activation.
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Conformational Dynamics of Prothrombin Dictate Its Ordered Cleavage by Prothrombinase
Blood, 2012Co-Authors: Michelle Homsher, Erin Zekas, Sriram KrishnaswamyAbstract:Abstract 3359 The Prothrombinase complex (membranes/Xa/Va) binds prothrombin through exosite interactions which orient and present the substrate to the protease active site for cleavage. Although both cleavage sites within the substrate are accessible, prothrombin is preferentially activated by Prothrombinase through sequential cleavage at R320 followed by cleavage at R271. We investigated the exosite-dependent interaction between prothrombin and Prothrombinase using fluorescence resonance energy transfer (FRET). An acceptor fluorophore was placed onto the prothrombin cleavage site by introducing the mutation R271C and fluorescently modifying the introduced thiol (IIC271*). The active site of Xa was covalently modified with a chloromethyl ketone conjugated to a donor fluorescent compound. Titrations of the fluorescently labeled Prothrombinase complex with IIC271* revealed a maximum energy transfer efficiency of 70–80 % observed with two different donor probes. Resonance energy transfer arising from the substrate docking interaction was confirmed by equivalent changes in the excited state lifetime of the donors. The calculated distance between the probe at C271 and the probe tethered at the active site of Xa ranged between 34 and 40 A. Variation in the measured distance reflects the different lengths of the tethers used to connect the donor probes. The FRET measurements, validated with two donor-acceptor pairs, indicate that the 271 site within prothrombin docked to Prothrombinase is positioned distant from the active site of Xa within the enzyme complex. This distant constraint prevents R271 from effectively engaging the active site of Xa within Prothrombinase, thereby providing a physical explanation for the ordered action of Prothrombinase on prothrombin. It follows that initial cleavage at R320 must reorient the substrate to allow R271 access to the Xa active site. We pursued an intramolecular FRET approach in which both donor and acceptor probes were placed on the prothrombin molecule to investigate the putative conformational change of prothrombin as a result of initial cleavage. Orthogonal labeling of prothrombin was accomplished using a prothrombin variant containing C271 and a LPETG extension at its C terminus. The optimized C271 label was used for the first modification. For the second, the sequence-specific transpeptidation activity of bacterial Sortase A was used to incorporate a fluorophore at the C-terminus (IICterm*). To assess the new fluorescent modification, FRET studies of prothrombin binding to Prothrombinase were repeated with the new IICterm* construct. Titrations revealed a maximal energy transfer of 16 %, confirmed by corresponding changes in excited state lifetime and yielded a Kd = 39 nM indistinguishable from the Kd= 41 nM seen for IIC271* binding. Thus, two different approaches illustrate that the affinity of prothrombin for Prothrombinase is independent of active site docking by the substrate. To examine the intramolecular dynamics of prothrombin during activation, the C271 site was labeled with BADAN (donor) and the Sortase labeling method placed the Alexa532 (acceptor) at the C terminus. The orthogonal placement of BADAN and Alexa532 on the prothrombin molecule resulted in extensive energy transfer between the two probes. The cleavage of doubly-labeled prothrombin to produce meizothrombin was associated with a change in intramolecular energy transfer. The variation of the energy transfers observed between the zymogen and protease forms can be interpreted as global rearrangement of the prothrombin molecule renders the second cleavage site accessible of active site docking. The FRET studies provide a comprehensive physical explanation for the ordered cleavage of prothrombin by Prothrombinase. Initial cleavage occurs at the spatially accessible cleavage site R320. The prothrombin molecule then significantly re-orients itself making the second R271 cleavage site accessible to Prothrombinase in order for activation to proceed and produce thrombin. Disclosures: No relevant conflicts of interest to declare.
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an anticoagulant rna aptamer that inhibits proteinase cofactor interactions within Prothrombinase
Journal of Biological Chemistry, 2010Co-Authors: Sai K Buddai, Juliana M Layzer, Genmin Lu, Christopher P Rusconi, Bruce A Sullenger, Dougald M Monroe, Sriram KrishnaswamyAbstract:Abstract The interaction of factor Xa with factor Va on membranes to form Prothrombinase profoundly increases the rate of the proteolytic conversion of prothrombin to thrombin. We present the characterization of an RNA aptamer (RNA11F7t) selected from a combinatorial library based on its ability to bind factor Xa. We show that RNA11F7t inhibits thrombin formation catalyzed by Prothrombinase without obscuring the active site of Xa within the enzyme complex. Selective inhibition of protein substrate cleavage arises from the ability of the aptamer to bind to factor Xa and exclude interactions between the proteinase and cofactor within Prothrombinase. Competition for enzyme complex assembly results from the binding of RNA11F7t to factor Xa with nanomolar affinity in a Ca2+-dependent interaction. RNA11F7t binds equivalently to the zymogen factor X as well as derivatives lacking γ-carboxyglutamic acid residues. We suggest that the ability of RNA11F7t to compete for the Xa-Va interaction with surprisingly high affinity likely reflects a significant contribution from its ability to indirectly impact regions of Xa that participate in the proteinase-cofactor interaction. Thus, despite the complexity of the macromolecular interactions that underlie the assembly of Prothrombinase, efficient inhibition of enzyme complex assembly and thrombin formation can be achieved by tight binding ligands that target factor Xa in a discrete manner.
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regulated cleavage of prothrombin by Prothrombinase repositioning a cleavage site reveals the unique kinetic behavior of the action of Prothrombinase on its compound substrate
Journal of Biological Chemistry, 2010Co-Authors: Harlan N Bradford, Joseph A Micucci, Sriram KrishnaswamyAbstract:Prothrombinase converts prothrombin to thrombin via cleavage at Arg320 followed by cleavage at Arg271. Exosite-dependent binding of prothrombin to Prothrombinase facilitates active site docking by Arg320 and initial cleavage at this site. Precise positioning of the Arg320 site for cleavage is implied by essentially normal cleavage at Arg320 in recombinant prothrombin variants bearing additional Arg side chains either one or two residues away. However, mutation of Arg320 to Gln reveals that Prothrombinase can cleave prothrombin following Arg side chains shifted by as many as two residues N-terminal to the 320 position at near normal rates. Further repositioning leads to a loss in cleavage at this region with an abrupt shift toward slow cleavage at Arg271. In contrast, the binding constant for the active site docking step is strongly dependent on the sequence preceding the scissile bond as well as position. Large effects on binding only yield minor changes in rate until the binding constant passes a threshold value. This behavior is expected for a substrate that can engage the enzyme through mutually exclusive active site docking reactions followed by cleavage to yield different products. Cleavage site specificity as well as the ordered action of Prothrombinase on its compound substrate is regulated by the thermodynamics of active site engagement of the individual sites as well as competition between alternate cleavage sites for active site docking.
Alan E Mast - One of the best experts on this subject based on the ideXlab platform.
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Bfpi: A Minimal Prothrombinase Inhibitor from the Saliva of Simulium Vittatum
Blood, 2018Co-Authors: Jeremy P Wood, Xiao H. Song, Alan E MastAbstract:Abstract Background: The black fly, Simulium Vittatum, has an anticoagulant protein in its saliva that allows it to feed on mammalian blood (black fly protease inhibitor; BFPI). Remarkably, BFPI is similar to the human anticoagulant tissue factor pathway inhibitor alpha (TFPIα). TFPIα contains three Kunitz-type protease inhibitor domains (K1, K2, K3), which inhibit factor VIIa (FVIIa) and factor Xa (FXa) and bind the co-factor protein S (PS), respectively; BFPI contains a single Kunitz domain that inhibits FXa. In addition, TFPIα and BFPI contain homologous basic regions (BRs) near their C-termini (252LIKTKRKRKK261 in human TFPIα, LIKTRKRKPKK in BFPI). The TFPIα BR binds a regulatory acidic region (AR) in factor Va (FVa). The AR is present in forms of FVa released by collagen-activated platelets and generated through limited proteolysis by FXa (FVaXa), and is removed by thrombin (FVaIIa). We hypothesized that BFPI, through its Kunitz domain and basic C-terminus, inhibits early forms of the Prothrombinase complex, but does not possess the other inhibitory functions of TFPIα: (1) K1-dependent inhibition of the tissue factor (TF)-FVIIa complex; and (2) PS/K3-dependent FXa inhibition. Results: Recombinant BFPI inhibited FXa in an amidolytic activity assay, and PS did not promote this inhibition. BFPI did not inhibit TF-FVIIa-mediated FX activation. As described with TFPIα, FV promoted FXa inhibition by BFPI but FVaIIa did not, suggesting that the BFPI BR is capable of binding the FVa AR. In a purified protein assay, BFPI inhibited Prothrombinase assembled with FVaXa (IC50=4.9nM), but not FVaIIa. Similarly, 5nM BFPI increased the lag time for FXa-initiated plasma thrombin generation by 10.4±1.5%. We next used BFPI as a backbone to evaluate a reported human mutation in the TFPIα BR, K254E. Every mammalian, avian, or reptilian TFPIα sequence available contains either a Lys or Arg residue at this position, suggesting that this residue is functionally important. In purified protein assays, BFPI-K254E inhibited FXa amidolytic activity identically to BFPI, but FV did not promote this inhibition, suggesting that BFPI-K254E has a specific defect in its interaction with FV. Consistent with this, BFPI-K254E was a weaker inhibitor of Prothrombinase assembled with FVaXa (IC50 = 15.8nM) and FXa-initiated plasma thrombin generation. The results obtained with BFPI-K254E were confirmed using peptides and full-length TFPIα proteins. First, a peptide mimicking the wild type TFPIα BR (LIKTKRKRKK) inhibited Prothrombinase assembled with FVaXa (IC50 = 1.0 µM), while the substituted peptide (LIETKRKRKK) was substantially weaker (20% inhibition observed with 340 µM peptide). Second, full-length TFPIα-K254E was a weaker inhibitor of Prothrombinase containing FXa-activated FVa (IC50 = 14.8 nM, vs. 1.8 nM for TFPIα) and had greatly reduced anticoagulant activity in plasma-based thrombin generation assays. Conclusions: In summary, the anticoagulant effect of BFPI is mediated through inhibition of early forms of Prothrombinase, independent of TF-FVIIa inhibition or PS-dependent FXa inhibition. The natural mutation TFPIα K254E disrupts Prothrombinase inhibition, despite the presence of six other conserved basic residues, and is thus procoagulant in human plasma. The absolute conservation of the TFPIα BR, and its usurpation to allow feeding by black flies, point to formation of the initial Prothrombinase complex as a key regulatory step in blood coagulation. Disclosures Mast: Novo Nordisk: Research Funding.
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tfpiα interacts with fva and fxa to inhibit Prothrombinase during the initiation of coagulation
Blood Advances, 2017Co-Authors: Jeremy P Wood, Helle Heibroch Petersen, Bingke Yu, Xiaoai Wu, Ida Hilden, Alan E MastAbstract:Tissue factor pathway inhibitor α (TFPIα) inhibits Prothrombinase, the thrombin-generating complex of factor Xa (FXa) and factor Va (FVa), during the initiation of coagulation. This inhibition requires binding of a conserved basic region within TFPIα to a conserved acidic region in FXa-activated and platelet-released FVa. In this study, the contribution of interactions between TFPIα and the FXa active site and FVa heavy chain to Prothrombinase inhibition were examined to further define the inhibitory biochemistry. Removal of FXa active site binding by mutation or by deletion of the second Kunitz domain (K2) of TFPIα produced 17- or 34-fold weaker Prothrombinase inhibition, respectively, establishing that K2 binding to the FXa active site is required for efficient inhibition. Substitution of the TFPIα basic region uncharged residues (Leu252, Ile253, Thr255) with Ala (TFPI-AAKA) produced 5.8-fold decreased inhibition. This finding was confirmed using a basic region peptide (Leu252-Lys261) and Ala substitution peptides, which established that the uncharged residues are required for Prothrombinase inhibitory activity but not for binding the FVa acidic region. This suggests that the uncharged residues mediate a secondary interaction with FVa subsequent to acidic region binding. This secondary interaction seems to be with the FVa heavy chain, because the FV Leiden mutation weakened Prothrombinase inhibition by TFPIα but did not alter TFPI-AAKA inhibitory activity. Thus, efficient inhibition of Prothrombinase by TFPIα requires at least 3 intermolecular interactions: (1) the TFPIα basic region binds the FVa acidic region, (2) K2 binds the FXa active site, and (3) Leu252-Thr255 binds the FVa heavy chain.
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relieving inhibition of Prothrombinase by tfpiα a procoagulant activity of unfractionated low molecular weight and nonanticoagulant heparins
Blood, 2014Co-Authors: Jeremy P Wood, Alan E Mast, Rodney M Camire, Lisa Baumann Kreuziger, Umesh R DesaiAbstract:Introduction: Prothrombinase, the complex of factor Xa (FXa) and factor Va (FVa), is inhibited by tissue factor pathway inhibitor (TFPI)α during the initiation of coagulation (Wood JP et al, PNAS 2013). Efficient inhibition of thrombin generation by Prothrombinase requires an interaction between the TFPIα basic C-terminus and an acidic region of the FVa B-domain. This acidic region is present in FXa-activated FVa and FVa released from activated platelets, but is rapidly removed by thrombin. Thus, Prothrombinase inhibition only occurs during the initiation phase of thrombin generation. As the exosite interaction is charge-dependent, large negatively charged molecules, including unfractionated heparin (UFH), block it, prevent Prothrombinase inhibition, and promote thrombin generation. Studies using the negatively charged molecule polyphosphate have suggested a size requirement for blocking this TFPIα activity (Smith SA et al, Blood 2010). A similar size-dependence may exist with heparins and could have clinical implications, as currently-used heparins range from long (unfractionated heparin; UFH) to medium (low molecular weight heparins; LMWHs) to short (the antithrombin-binding pentasaccharide fondaparinux). Studies were performed to assess the ability of the LMWHs enoxaparin and dalteparin, fondaparinux, and the nonanticoagulant heparin 2-O, 3-O desulfated heparin (ODSH) to block TFPIα and promote thrombin generation through this mechanism. Methods: TFPIα inhibition of thrombin generation by Prothrombinase, assembled with a form of FVa containing the acidic region of the B domain, was measured in the absence or presence of UFH, enoxaparin, dalteparin, fondaparinux, and ODSH. The effect of these compounds on the direct inhibition of FXa by TFPIα was measured using a FXa chromogenic substrate. The effect of these compounds on thrombin generation in plasma was measured by calibrated automated thrombography using human plasma immunodepleted of antithrombin III and heparin cofactor II (AT3/HCII-depleted plasma). Results: TFPIα inhibited Prothrombinase activity (IC50 = 6.8 nM), and UFH blocked this inhibition (IC50 = 12.5 nM or 14.9 nM at 0.5 or 1 U/mL, respectively). Enoxaparin (0.8 U/mL; IC50 = 30.3 nM) and dalteparin (1 U/mL; IC50 = 29.7 nM) appeared to be more effective at reversing TFPIα inhibition. The reason for this apparent enhanced effect of LMWHs compared to UFH is not clear, as UFH and the LMWHs similarly enhanced the direct inhibition of FXa by TFPIα, and the differential activity was also observed when heparins were normalized to saccharide concentration. The same pattern was observed when measuring thrombin generation in AT3/HCII-depleted plasma, with LMWHs being more procoagulant than UFH. Consistent with TFPIα inhibition being charge-dependent, ODSH promoted thrombin generation similarly to LMWHs in both purified systems and AT3/HCII-depleted plasma. In contrast, clinical doses of fondaparinux had no effect in any assay. In a purified system, ~1000 times the clinical dose of fondaparinux was required to promote thrombin generation. Conclusion: There is a size-dependence for blocking TFPIα inhibition of Prothrombinase using heparins, as the pentasaccharide has no effect. However, both LMWHs and UFH are sufficiently long to express this procoagulant activity at therapeutic doses. In addition, the nonanticoagulant heparin ODSH blocks Prothrombinase inhibition by TFPIα. This procoagulant activity is likely most clinically relevant under conditions of antithrombin deficiency, which may result from sepsis, liver failure, or administration of L-asparaginase. Under any of these conditions, UFH, LMWHs, and ODSH may have unanticipated procoagulant activity mediated by blocking TFPIα. Disclosures Camire: Pfizer: Consultancy, Patents & Royalties, Research Funding. Mast: Novo Nordisk: Research Funding.
Michael Kalafatis - One of the best experts on this subject based on the ideXlab platform.
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Spellbinding Effects of the Acidic COOH-Terminus of Factor Va Heavy Chain on Prothrombinase Activity and Function.
ACS omega, 2017Co-Authors: Jamila Hirbawi, Michael KalafatisAbstract:Human factor Va (hfVa) is the important regulatory subunit of Prothrombinase. Recent modeling data have suggested a critical role for amino acid Arg701 of hfVa for human prothrombin (hPro) activation by Prothrombinase. Furthermore, it has also been demonstrated that hfVa has a different effect than that of bovine fVa on prethrombin-1 activation by Prothrombinase. The difference between the two cofactor molecules was also found within the Asn700–Arg701 dipeptide in the human factor V (hfV) molecule, which is replaced by the Asp–Glu sequence in bfV. As a consequence, we produced a recombinant hfV (rhfV) molecule with the substitution 700NR701→DE. rhfVNR→DE together with the wild-type molecule (rhfVWT) were expressed in COS7 cells, purified, and tested for their capability to function within Prothrombinase. Kinetic studies showed that the Kd of rhfVaNR→DE for human fXa as well as the kcat and Km of Prothrombinase made with rhfVaNR→DE for hPro activation were similar to the values obtained following hPro acti...
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The Role of Amino Acids 659-663 of Factor Va Heavy Chain During Prothrombin Activation by Prothrombinase.
Blood, 2009Co-Authors: Jamila Hirbawi, Michael KalafatisAbstract:Abstract 2131 Poster Board II-108 The proteolytic conversion of prothrombin to thrombin is catalyzed by the Prothrombinase complex composed of the enzyme, factor Xa (fXa), and the cofactor, factor Va (fVa), assembled on a membrane surface in the presence of Ca2+. FXa alone can activate prothrombin following sequential cleavages at Arg271 and Arg320 yielding the transient inactive intermediate prethrombin 2. However, the interaction of fVa with fXa on a membrane/cell surface in the presence of divalent metal ions and formation of the Prothrombinase complex results in the reversal of the order of cleavages and a 300,000-fold increase in the catalytic efficiency of fXa for thrombin generation. A first cleavage of prothrombin by Prothrombinase at Arg320 produces the active intermediate meizothrombin, while the second cleavage at Arg271 produces thrombin. Thrombin and prothrombin contain two positively charged binding regions (anion binding exosite I, ABE-I and anion binding exosite II, ABE II), that are crucial for protein function. Initial cleavage of prothrombin at Arg320 by Prothrombinase which is absolutely factor Va dependent, entirely exposes (pro)exosite I. FVa is required for the specific recognition of Prothrombinase by (pro)exosite I of prothrombin. The COOH-terminal region of the heavy chain of fVa contains acidic amino acid clusters that are important for cofactor activity. We have investigated the role of amino acid region 659-663 that contains five consecutive acidic amino acid residues. To ascertain the function of this region, site-directed mutagenesis was performed. We have constructed a mutant molecule with this region deleted (fVD659-663) and a mutant molecule in which all five residues were mutated to lysine (fV5K, charge reversal). The recombinant molecules along with wild type fV (fVWT) were transiently expressed in COS7L cells, purified to homogeneity, and assessed for cofactor activity. Two-stage clotting assays revealed that the mutant molecules had reduced clotting activities compared to fVaWT. Kinetic analyses studying Prothrombinase assembled with the mutant molecules demonstrated diminished kcat values, while the affinity of all mutant molecules for factor plasma-derived fXa was similar to fVaWT. Gel electrophoresis analyzing plasma-derived and recombinant mutant prothrombin activation demonstrated delayed cleavage of prothrombin at both Arg320 and Arg271 by Prothrombinase assembled with the mutant molecules. Using recombinant prothrombin molecules we determined that cleavage at Arg271 by Prothrombinase assembled with either fVaD659-663 or fVa5K was severely impaired compared to cleavage at Arg320 by Prothrombinase assembled with the same recombinant cofactor molecules, resulting in lingering of meizothrombin throughout the activation process. To ascertain the effect of the mutations of the fVa heavy chain on the cleavage at Arg271 alone following the transition that occurs after cleavage at Arg320, we compared the rate of cleavage of active-site blocked meizothrombin (FPR-meizothrombin) by Prothrombinase assembled with either fVaWT or fVaD659-663. The data demonstrate a delay for cleavage of FPR-meizothrombin at Arg271 by Prothrombinase assembled with fVaD659-663 as compared to the same reaction catalyzed by Prothrombinase assembled with fVaWT. Quantitative scanning densitometry of fragment 1•2-A demonstrated a ∼4-fold delay in cleavage of FPR-meizothrombin at Arg271 by Prothrombinase assembled with fVaD659-663, compared to cleavage at Arg271 by Prothrombinase assembled with fVaWT. Direct comparison between the rates of cleavage of FPR-meizothrombin by membrane-bound fXa alone or by Prothrombinase assembled with fVaRVVD659-663 do not show any significant differences. Thus, deletion of amino acid region 659-663 virtually eliminates the acceleration in the rate of cleavage at Arg271 of meizothrombin attributed to the interaction of fVa with fXa. These data demonstrate that amino acid sequence 659DDDED663 from the factor Va heavy chain, regulates meizothrombin concentration during activation of prothrombin by Prothrombinase. Disclosures: No relevant conflicts of interest to declare.
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a control switch for Prothrombinase characterization of a hirudin like pentapeptide from the cooh terminus of factor va heavy chain that regulates the rate and pathway for prothrombin activation
Journal of Biological Chemistry, 2006Co-Authors: Michael A. Bukys, Michael E. Nesheim, Michael KalafatisAbstract:Abstract Membrane-bound factor Xa alone catalyzes prothrombin activation following initial cleavage at Arg271 and prethrombin 2 formation (pre2 pathway). Factor Va directs prothrombin activation by factor Xa through the meizothrombin pathway, characterized by initial cleavage at Arg320 (meizo pathway). We have shown previously that a pentapeptide encompassing amino acid sequence 695–699 from the COOH terminus of the heavy chain of factor Va (Asp-Tyr-Asp-Tyr-Gln, DYDYQ) inhibits prothrombin activation by Prothrombinase in a competitive manner with respect to substrate. To understand the mechanism of inhibition of thrombin formation by DYDYQ, we have studied prothrombin activation by gel electrophoresis. Titration of plasma-derived prothrombin activation by Prothrombinase, with increasing concentrations of peptide, resulted in complete inhibition of the meizo pathway. However, thrombin formation still occurred through the pre2 pathway. These data demonstrate that the peptide preferentially inhibits initial cleavage of prothrombin by Prothrombinase at Arg320. These findings were corroborated by studying the activation of recombinant mutant prothrombin molecules rMZ-II (R155A/R284A/R271A) and rP2-II (R155A/R284A/R320A) which can be only cleaved at Arg320 and Arg271, respectively. Cleavage of rMZ-II by Prothrombinase was completely inhibited by low concentrations of DYDYQ, whereas high concentrations of pentapeptide were required to inhibit cleavage of rP2-II. The pentapeptide also interfered with prothrombin cleavage by membrane-bound factor Xa alone in the absence of factor Va increasing the rate for cleavage at Arg271 of plasma-derived prothrombin or rP2-II. Our data demonstrate that pentapeptide DYDYQ has opposing effects on membrane-bound factor Xa for prothrombin cleavage, depending on the incorporation of factor Va in Prothrombinase.
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Controlling the Pathway for Prothrombin Activation by Prothrombinase.
Blood, 2006Co-Authors: Michael A. Bukys, Michael E. Nesheim, Michael KalafatisAbstract:Prothrombinase is the enzymatic complex responsible for timely thrombin formation. Activation of human prothrombin is the consequence of two cleavages at Arg271 and Arg320 in prothrombin by factor Xa. Membrane-bound factor Xa alone catalyzes prothrombin activation following initial cleavage at Arg271 and prethrombin 2 formation (pre2 pathway). Factor Va directs prothrombin activation by factor Xa through the meizothrombin pathway, characterized by initial cleavage at Arg320 (meizo pathway). We have previously shown that a pentapeptide encompassing amino acid sequence 695–699 from the COOH-terminus of the heavy chain of factor Va (Asp-Tyr-Asp-Tyr-Gln, DYDYQ) interacts with anion binding exosite I (ABE-I) of thrombin and inhibits prothrombin activation by Prothrombinase. The peptide was found to be a competitive inhibitor of Prothrombinase with respect to substrate. According to the mode of inhibition, we postulated that the peptide binds prothrombin in competition with the binding of the substrate to the enzyme, and inhibits Prothrombinase activity by substrate depletion. This mode of DYDYQ inhibition of prothrombin activation by the factor Va-factor Xa complex is similar to that previously demonstrated for sulfated hirugen. To understand the mechanism of inhibition of thrombin formation by DYDYQ we have studied prothrombin activation by gel electrophoresis. Titration of plasma-derived prothrombin activation by fully assembled Prothrombinase, with increasing concentrations of peptide, resulted in complete inhibition of the meizo pathway. However, thrombin formation still occurred through the pre2 pathway. Higher peptide concentrations were required to impair thrombin formation through the latter pathway. These data demonstrate that the peptide preferentially inhibits initial cleavage of prothrombin by Prothrombinase at Arg320. These findings were corroborated by studying the kinetics of activation of recombinant mutant prothrombin molecules rMZ-II (R155A/R284A/R271A) and rP2-II (R155A/R284A/R320A) which can be only cleaved at Arg320 and Arg271 respectively. Cleavage of rMZ-II by Prothrombinase was completely inhibited by low concentrations of DYDYQ while high concentrations of pentapeptide were required to inhibit cleavage of rP2-II. The pentapeptide also interfered with thrombin formation by membrane-bound factor Xa alone in the absence of factor Va. Nonetheless, while the rate for cleavage at Arg271 of plasma-derived prothrombin or rP2-II by membrane-bound factor Xa alone was significantly accelerated in the presence of DYDYQ, resulting in accumulation of prethrombin 2, the rate for cleavage at Arg320 of plasma-derived prothrombin or rMZ-II by membrane-bound factor Xa alone was only moderately affected by the pentapeptide. Our data demonstrate that a pentapeptide mimicking amino acids 695–699 of the heavy chain of factor Va has opposing effects on membrane-bound factor Xa for prothrombin activation, depending on the incorporation of factor Va in Prothrombinase. In the presence of the cofactor the peptide inhibits the rate of thrombin generation by specifically interfering with initial cleavage of prothrombin at Arg320, while in the absence of factor Va the pentapeptide accelerates cleavage of prothrombin by factor Xa at Arg271. Thus, the amino acid region spatially surrounding proexosite I in prothrombin most likely has two interactive sites for the components of Prothrombinase, a factor Va interactive site and a factor Xa binding site.
John H Griffin - One of the best experts on this subject based on the ideXlab platform.
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prothrombin residues 473 487 contribute to factor va binding in the Prothrombinase complex
Journal of Biological Chemistry, 2004Co-Authors: Subramanian Yegneswaran, Rolf M Mesters, Jose A Fernandez, John H GriffinAbstract:Abstract To identify sequences in prothrombin (fII) involved in Prothrombinase complex (fXa·fVa·fII·phospholipids) assembly, synthetic peptides based on fII sequences were prepared and screened for their ability to inhibit factor Xa (fXa)-induced clotting of normal plasma. The fII peptide (PT473–487, homologous to chymotrypsin residues 149D-163) potently inhibited plasma clotting assays and Prothrombinase activity, with 50% inhibition of 12 and 10 μm peptide, respectively. Prothrombinase inhibition by PT473–487 was factor Va (fVa)-dependent and sequence-specific, because the peptide did not inhibit fII activation in the absence of fVa, and a scrambled sequence peptide, PT473–487SCR, was not inhibitory. Peptide PT473–487 did not inhibit the amidolytic activities of fXa and thrombin, suggesting that the peptide did not alter the integrity of their active sites. To determine whether PT473–487 interacted directly with fVa, fluorescein-labeled fVa (Fl-fVa) was prepared. When PT473–487 was titrated into samples containing phospholipid-bound Fl-fVa, the peptide increased fluorescein anisotropy (EC50 at 3 μm peptide), whereas the control peptide PT473–487SCR did not alter the anisotropy, suggesting a direct binding interaction between PT473–487 and Fl-fVa. These functional and spectroscopic data suggest that fII residues 473–487 provide fVa-binding sites and mediate interactions between fVa and fII in the Prothrombinase complex.
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Prothrombin Residues 473–487 Contribute to Factor Va Binding in the Prothrombinase Complex
Journal of Biological Chemistry, 2004Co-Authors: Subramanian Yegneswaran, Rolf M Mesters, Jose A Fernandez, John H GriffinAbstract:Abstract To identify sequences in prothrombin (fII) involved in Prothrombinase complex (fXa·fVa·fII·phospholipids) assembly, synthetic peptides based on fII sequences were prepared and screened for their ability to inhibit factor Xa (fXa)-induced clotting of normal plasma. The fII peptide (PT473–487, homologous to chymotrypsin residues 149D-163) potently inhibited plasma clotting assays and Prothrombinase activity, with 50% inhibition of 12 and 10 μm peptide, respectively. Prothrombinase inhibition by PT473–487 was factor Va (fVa)-dependent and sequence-specific, because the peptide did not inhibit fII activation in the absence of fVa, and a scrambled sequence peptide, PT473–487SCR, was not inhibitory. Peptide PT473–487 did not inhibit the amidolytic activities of fXa and thrombin, suggesting that the peptide did not alter the integrity of their active sites. To determine whether PT473–487 interacted directly with fVa, fluorescein-labeled fVa (Fl-fVa) was prepared. When PT473–487 was titrated into samples containing phospholipid-bound Fl-fVa, the peptide increased fluorescein anisotropy (EC50 at 3 μm peptide), whereas the control peptide PT473–487SCR did not alter the anisotropy, suggesting a direct binding interaction between PT473–487 and Fl-fVa. These functional and spectroscopic data suggest that fII residues 473–487 provide fVa-binding sites and mediate interactions between fVa and fII in the Prothrombinase complex.
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identification of distinct sequences in human blood coagulation factor xa and prothrombin essential for substrate and cofactor recognition in the Prothrombinase complex
Journal of Biological Chemistry, 2003Co-Authors: Subramanian Yegneswaran, Rolf M Mesters, John H GriffinAbstract:Abstract To identify amino acid sequences in factor Xa (fXa) and prothrombin (fII) that may be involved in Prothrombinase complex (fXa·factor Va·fII·phospholipids) assembly, synthetic peptides based on fXa and fII sequences were prepared and screened for their ability to inhibit fXa-induced clotting of normal plasma. One fII peptide (PT557–571 homologous to chymotrypsin (CHT) residues 225–239) and two fXa peptides (X404–418, CHT231–244, and X415–429, CHT241–252C) potently inhibited plasma clotting and Prothrombinase activity with 50% inhibition between 41 and 115 μm peptide. Inhibition of Prothrombinase by PT557–571 and X415–429 was fVa-independent, whereas the inhibition by X404–418 was fVa-dependent. X404–418 inhibited the binding of fVa to fluorescein-labeled, inhibited fXai in the presence of phosphatidylcholine/phosphatidylserine vesicles, whereas X415–429 inhibited binding of fII to phospholipid-bound fluorescein-labeled, inhibited fXai. PT557–571 altered the fluorescence emission of fluorescein-labeled fXai, showing that PT557–571 binds to fXai. These data suggest that residues 404–418 in fXa provide fVa binding sites, whereas residues 557–571 in fII and 415–429 in fXa mediate interactions between fXa and fII in the Prothrombinase complex.
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inhibition of Prothrombinase by human secretory phospholipase a2 involves binding to factor xa
Journal of Biological Chemistry, 1998Co-Authors: Carine M Mounier, Grazyna Faure, Tilman M Hackeng, Francis Schaeffer, John H GriffinAbstract:Abstract Human group II secretory phospholipase A2 (hsPLA2) exhibits significant anticoagulant activity that does not require its enzymatic activity. We examined which coagulation factor was targeted by hsPLA2and analyzed which region of the protein may be involved in this inhibition. Prothrombin time coagulation assays indicated that hsPLA2 did not inhibit activated factor V (FVa) activity, whereas activated factor X (FXa) one-stage coagulation assays suggested that FXa was inhibited. The inhibitory effect of hsPLA2 on Prothrombinase activity of FXa, FV, phospholipids, and Ca2+complex was markedly enhanced upon preincubation of hsPLA2with FXa but not with FV. Prothrombinase activity was also strongly inhibited by hsPLA2 in the absence of PL. High concentrations of FVa in the Prothrombinase generation assay reversed the inhibitory effect of hsPLA2. By using isothermal titration calorimetry, we demonstrated that hsPLA2 binds to FXa in solution with a 1:1 stoichiometry and a K dof 230 nm. By using surface plasmon resonance we determined the rate constants, k on andk off, of the FXa/hsPLA2 interaction and analyzed the Ca2+ effect on these constants. When preincubated with FXa, synthetic peptides comprising residues 51–74 and 51–62 of hsPLA2 inhibited Prothrombinase assays, providing evidence that this part of the molecule, which shares similarities with a region of FVa that binds to FXa, is likely involved in the anticoagulant interaction of hsPLA2 with FXa. In conclusion, we propose that residues 51–62 of hsPLA2 bind to FXa at a FVa-binding site and that hsPLA2 decreases the Prothrombinase generation by preventing FXa·FVa complex formation.
Rodney M Camire - One of the best experts on this subject based on the ideXlab platform.
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relieving inhibition of Prothrombinase by tfpiα a procoagulant activity of unfractionated low molecular weight and nonanticoagulant heparins
Blood, 2014Co-Authors: Jeremy P Wood, Alan E Mast, Rodney M Camire, Lisa Baumann Kreuziger, Umesh R DesaiAbstract:Introduction: Prothrombinase, the complex of factor Xa (FXa) and factor Va (FVa), is inhibited by tissue factor pathway inhibitor (TFPI)α during the initiation of coagulation (Wood JP et al, PNAS 2013). Efficient inhibition of thrombin generation by Prothrombinase requires an interaction between the TFPIα basic C-terminus and an acidic region of the FVa B-domain. This acidic region is present in FXa-activated FVa and FVa released from activated platelets, but is rapidly removed by thrombin. Thus, Prothrombinase inhibition only occurs during the initiation phase of thrombin generation. As the exosite interaction is charge-dependent, large negatively charged molecules, including unfractionated heparin (UFH), block it, prevent Prothrombinase inhibition, and promote thrombin generation. Studies using the negatively charged molecule polyphosphate have suggested a size requirement for blocking this TFPIα activity (Smith SA et al, Blood 2010). A similar size-dependence may exist with heparins and could have clinical implications, as currently-used heparins range from long (unfractionated heparin; UFH) to medium (low molecular weight heparins; LMWHs) to short (the antithrombin-binding pentasaccharide fondaparinux). Studies were performed to assess the ability of the LMWHs enoxaparin and dalteparin, fondaparinux, and the nonanticoagulant heparin 2-O, 3-O desulfated heparin (ODSH) to block TFPIα and promote thrombin generation through this mechanism. Methods: TFPIα inhibition of thrombin generation by Prothrombinase, assembled with a form of FVa containing the acidic region of the B domain, was measured in the absence or presence of UFH, enoxaparin, dalteparin, fondaparinux, and ODSH. The effect of these compounds on the direct inhibition of FXa by TFPIα was measured using a FXa chromogenic substrate. The effect of these compounds on thrombin generation in plasma was measured by calibrated automated thrombography using human plasma immunodepleted of antithrombin III and heparin cofactor II (AT3/HCII-depleted plasma). Results: TFPIα inhibited Prothrombinase activity (IC50 = 6.8 nM), and UFH blocked this inhibition (IC50 = 12.5 nM or 14.9 nM at 0.5 or 1 U/mL, respectively). Enoxaparin (0.8 U/mL; IC50 = 30.3 nM) and dalteparin (1 U/mL; IC50 = 29.7 nM) appeared to be more effective at reversing TFPIα inhibition. The reason for this apparent enhanced effect of LMWHs compared to UFH is not clear, as UFH and the LMWHs similarly enhanced the direct inhibition of FXa by TFPIα, and the differential activity was also observed when heparins were normalized to saccharide concentration. The same pattern was observed when measuring thrombin generation in AT3/HCII-depleted plasma, with LMWHs being more procoagulant than UFH. Consistent with TFPIα inhibition being charge-dependent, ODSH promoted thrombin generation similarly to LMWHs in both purified systems and AT3/HCII-depleted plasma. In contrast, clinical doses of fondaparinux had no effect in any assay. In a purified system, ~1000 times the clinical dose of fondaparinux was required to promote thrombin generation. Conclusion: There is a size-dependence for blocking TFPIα inhibition of Prothrombinase using heparins, as the pentasaccharide has no effect. However, both LMWHs and UFH are sufficiently long to express this procoagulant activity at therapeutic doses. In addition, the nonanticoagulant heparin ODSH blocks Prothrombinase inhibition by TFPIα. This procoagulant activity is likely most clinically relevant under conditions of antithrombin deficiency, which may result from sepsis, liver failure, or administration of L-asparaginase. Under any of these conditions, UFH, LMWHs, and ODSH may have unanticipated procoagulant activity mediated by blocking TFPIα. Disclosures Camire: Pfizer: Consultancy, Patents & Royalties, Research Funding. Mast: Novo Nordisk: Research Funding.
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crystal structure of the Prothrombinase complex from the venom of pseudonaja textilis
Blood, 2013Co-Authors: Bernhard C Lechtenberg, Sriram Krishnaswamy, Thomas A Murrayrust, Daniel J D Johnson, Ty E Adams, Rodney M Camire, James A. HuntingtonAbstract:The Prothrombinase complex, composed of the protease factor (f)Xa and cofactor fVa, efficiently converts prothrombin to thrombin by specific sequential cleavage at 2 sites. How the complex assembles and its mechanism of prothrombin processing are of central importance to human health and disease, because insufficient thrombin generation is the root cause of hemophilia, and excessive thrombin production results in thrombosis. Efforts to determine the crystal structure of the Prothrombinase complex have been thwarted by the dependence of complex formation on phospholipid membrane association. Pseutarin C is an intrinsically stable Prothrombinase complex preassembled in the venom gland of the Australian Eastern Brown Snake (Pseudonaja textilis). Here we report the crystal structures of the fX-fV complex and of activated fXa from P textilis venom and the derived model of active pseutarin C. Structural analysis supports a single substrate binding channel on fVa, to which prothrombin and the intermediate meizothrombin bind in 2 different orientations, providing insight into the architecture and mechanism of the Prothrombinase complex—the molecular engine of blood coagulation.
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restricted active site docking by enzyme bound substrate enforces the ordered cleavage of prothrombin by Prothrombinase
Journal of Biological Chemistry, 2007Co-Authors: Ayse Hacisalihoglu, Rodney M Camire, Paul E Bock, Peter Panizzi, Sriram KrishnaswamyAbstract:Abstract The preferred pathway for prothrombin activation by Prothrombinase involves initial cleavage at Arg320 to produce meizothrombin, which is then cleaved at Arg271 to liberate thrombin. Exosite binding drives substrate affinity and is independent of the bond being cleaved. The pathway for cleavage is determined by large differences in Vmax for cleavage at the two sites within intact prothrombin. By fluorescence binding studies in the absence of catalysis, we have assessed the ability of the individual cleavage sites to engage the active site of Xa within Prothrombinase at equilibrium. Using a panel of recombinant cleavage site mutants, we show that in intact prothrombin, the Arg320 site effectively engages the active site in a 1:1 interaction between substrate and enzyme. In contrast, the Arg271 site binds to the active site poorly in an interaction that is ∼600-fold weaker. Perceived substrate affinity is independent of active site engagement by either cleavage site. We further show that prior cleavage at the 320 site or the stabilization of the uncleaved zymogen in a proteinase-like state facilitates efficient docking of Arg271 at the active site of Prothrombinase. Therefore, we establish direct relationships between docking of either cleavage site at the active site of the catalyst, the Vmax for cleavage at that site, substrate conformation, and the resulting pathway for prothrombin cleavage. Exosite tethering of the substrate in either the zymogen or proteinase conformation dictates which cleavage site can engage the active site of the catalyst and enforces the sequential cleavage of prothrombin by Prothrombinase.
