Intermolecular Interactions and Characterization of the Novel Factor Xa Exosite Involved in Macromolecular Recognition and Inhibition: Crystal Structure of Human Gla-domainless Factor Xa Complexed with the Anticoagulant Protein NAPc2 from the Hematophagous Nematode Ancylostoma caninum

Murakami, Mário Tyago; Rios-Steiner, J.; Weaver, Sidney; Tulinsky, A.; Geiger, James H.; Arni, R.K.

doi:10.1016/j.jmb.2006.11.040

Cited by 40 publications

(46 citation statements)

References 35 publications

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“…While these observations are consistent with the hypothesis that HSPGs may play a role in Ad.FX hepatocyte transduction in vivo, NAPc2 and Ixolaris have additional effects on coagulation that are distinct from blocking the heparin binding exosite of FX (44)(45)(46).…”

Section: Discussionmentioning

confidence: 99%

Hepatocyte Heparan Sulfate Is Required for Adeno-Associated Virus 2 but Dispensable for Adenovirus 5 Liver Transduction In Vivo

Zaiss

Foley

Lawrence

et al. 2016

J Virol

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Adeno-associated virus 2 (AAV2) and adenovirus 5 (Ad5) are promising gene therapy vectors. Both display liver tropism and are currently thought to enter hepatocytes in vivo through cell surface heparan sulfate proteoglycans (HSPGs). To test directly this hypothesis, we created mice that lack Ext1, an enzyme required for heparan sulfate biosynthesis, in hepatocytes. A better understanding of how viral vectors enter cells in vivo is critical to improve their therapeutic use. Adeno-associated virus 2 (AAV2) and adenovirus 5 (Ad5) vectors have shown promise in clinical trials for treatment of a wide variety of diseases (1, 2). Both vectors, when injected intravenously into mice, exhibit transgene expression in liver (3-5). Heparan sulfate proteoglycans (HSPGs) are the primary receptors currently thought to facilitate AAV2 and Ad5 entry into hepatocytes (6-8).HSPGs are present both on the cell surface and in the extracellular matrix (9, 10). They consist of a protein core posttranslationally modified to contain heparan sulfate (HS) chains (11). HS biosynthesis occurs by polymerization of alternating glucuronic acid and N-acetylglucosamine residues (12-14), catalyzed by an enzyme complex composed of EXT1 and EXT2 (15). EXT1 and EXT2 are essential molecules required for HS synthesis; cells lacking either molecule do not synthesize HS (16,49).AAV2 binds directly to cell surface HSPGs via an HS-binding motif on the virus capsid (3,17,18). AAV capsid modifications that alter the cluster of positive amino acids that constitute the HS binding motif abrogate liver transduction (3,19,20), suggesting that the ability of the capsid to bind to HS is critical for AAV2 liver transduction in vivo. In contrast, Ad5 binding to HSPGs requires the presence of blood coagulation factor X (FX), which binds to the Ad5 hexon when the virus comes in contact with blood (7,(21)(22)(23). The interaction of Ad.FX and HS is mediated by electrostatic interactions between the heparin binding exosite of the FX serine protease domain and the sulfate groups of HS (6,(23)(24)(25). FX is required for Ad5 transduction in vivo in wild-type mice. In the absence of FX, or when viruses with mutant hexon proteins unable to bind FX are used, Ad5 liver transduction is essentially completely abrogated (7, 21-23, 26, 27).

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Section: Discussionmentioning

confidence: 99%

Hepatocyte Heparan Sulfate Is Required for Adeno-Associated Virus 2 but Dispensable for Adenovirus 5 Liver Transduction In Vivo

Zaiss

Foley

Lawrence

et al. 2016

J Virol

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“…The crystal structure of the TF-fVIIa-fXa-rNAPc2 quaternary complex is not available as yet, however that for the fXa-NAPc2 binary complex has recently been resolved [112]. From a highly flexible form, NAPc2 undergoes significant structural changes in the C-terminus upon binding to the fXa exosite.…”

Section: Recombinant Nematode Anticoagulant Protein C2mentioning

confidence: 99%

Recent Research Developments in the Direct Inhibition of Coagulation Proteinases – Inhibitors of the Initiation Phase

Henry¹,

Desai²

2008

CHAMC

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Physiologic clotting is a defensive action. The new cell-based model of hemostasis proposes three steps -initiation, amplification and propagation -occurring on specific cell surfaces to generate a thrombus in a tightly regulated manner. The initiation phase relies on key players including tissue factor (TF), factor VIIa (fVIIa), platelets, Ca 2+ , phospholipids, and factor X/Xa (fX/fXa). Exposure of TF on sub-endothelial and other blood cells triggers a coagulation response, which may have to be inhibited to prevent a deleterious thrombotic effect. Inhibiting TF-initiated coagulation, akin to 'nipping coagulation in the bud', is predicted to have major advantages, including a more efficient separation of the antithrombotic and hemorrhagic responses. The availability of crystal structures of TF, fVIIa and TFfVIIa complex makes structure-based drug design feasible. Although no initiation phase small molecule inhibitor has reached the clinic as yet, several molecules have displayed promise. We discuss recent results on the discovery of inhibitors of the initiation phase with special emphasis on peptides, peptidomimetics and organic small molecules.Key Words: Anticoagulants, coagulation, factor VIIa, tissue factor, thrombin, factor Xa, Enzyme Inhibition, small molecule inhibitors. CELL-BASED HEMOSTASISPhysiologic clotting is a defensive action preventing excessive loss of blood and infiltration of microbes. The physiologic mechanism that produces this defensive reaction is an enzymatic cascade ( Fig. 1), proposed nearly 50 years ago [1,2]. In vivo, the enzymatic reactions occur on specific cell surfaces. The cell-dependent coagulation cascade is proposed to proceed sequentially in three stepsinitiation, amplification and propagation -on specific cell surfaces to generate a thrombus in a tightly regulated manner [3][4][5]. InitiationTissue factor (TF) is an integral membrane protein present on several extravascular cells [6] and is the most important initiator of coagulation. Under normal conditions, it is sequestered from circulating proteinases, especially factor VII (fVII), thus preventing inappropriate initiation of clotting. Vascular injury exposes TF to blood, resulting in rapid complex formation with free activated fVII (fVIIa) on TF-bearing cells (Fig. 1). It is believed that some fVIIa is always present in blood (~1-2%), which can rapidly interact with TF to initiate clotting. The formation of TF/fVIIa complex activates the circulating factor X (fX) to fXa, the formation of which can induce further production of fVIIa and fXa [7][8][9]. The TF/fVIIa complex can also activate factor IX (fIX) to fIXa [10][11][12], which in turn can result in more fXa. The earliest fXa formed remains localized to the site of TF-bearing cells, yet is free enough to form the prothrombinase complex with factor Va (fVa). The small amount of the prothrombinase complex so formed is responsible for the initial production of thrombin from prothrombin [12]. The in vivo source of the initial fVa needed for the assembly of p...

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“…FX has been shown to bind to at least 14 other Ad serotypes, indicating that this interaction is conserved (15). These findings have highlighted the importance of understanding the precise mechanisms governing cellular infectivity and tropism in order for adenoviruses to be successfully used as therapeutic gene delivery vectors.Our previous studies demonstrated that the anticoagulants nematode anticoagulant peptide 2 (NAPc2) and Ixolaris can block FX-mediated Ad5 transduction in vitro (7,8,15). Ixolaris has been shown to bind residues within the heparin binding exosite of FXa, previously defined as R93, K96, R125, R165, K169, K236, and R240 (12).…”

mentioning

confidence: 99%

“…Our previous studies demonstrated that the anticoagulants nematode anticoagulant peptide 2 (NAPc2) and Ixolaris can block FX-mediated Ad5 transduction in vitro (7,8,15). Ixolaris has been shown to bind residues within the heparin binding exosite of FXa, previously defined as R93, K96, R125, R165, K169, K236, and R240 (12).…”

mentioning

confidence: 99%

“…Ixolaris has been shown to bind residues within the heparin binding exosite of FXa, previously defined as R93, K96, R125, R165, K169, K236, and R240 (12). The crystal structure of NAPc2 with FXa indicates it also binds to this exosite and residues R93, R125, K236, and R240 (8). Moreover, preincubation of FX with each anticoagulant prior to injection into coagulation factor-depleted mice resulted in a significant decrease in Ad5 hepatic gene transfer (15).…”

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confidence: 99%

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A Cluster of Basic Amino Acids in the Factor X Serine Protease Mediates Surface Attachment of Adenovirus/FX Complexes

Duffy

Bradshaw

Parker

et al. 2011

J Virol

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Hepatocyte transduction following intravenous administration of adenovirus 5 (Ad5) is mediated by interaction between coagulation factor X (FX) and the hexon. The FX serine protease (SP) domain tethers the Ad5/FX complex to hepatocytes through binding heparan sulfate proteoglycans (HSPGs). Here, we identify the critical HSPG-interacting residues of FX. We generated an FX mutant by modifying seven residues in the SP domain. Surface plasmon resonance demonstrated that mutations did not affect binding to Ad5. FX-mediated, HSPG-associated cell binding and transduction were abolished. A cluster of basic amino acids in the SP domain therefore mediates surface interaction of the Ad/FX complex.Adenoviruses are nonenveloped, double-stranded DNA viruses belonging to the Adenoviridae family, of which there are 54 different serotypes divided into species based on their phylogenic origin and hemagglutination properties. Adenoviruses can infect a wide variety of cell types and are extensively used as vectors for gene transfer applications, from use in vitro or in vivo in the laboratory setting to gene therapy clinical protocols. Species C adenovirus serotype 5 (Ad5) is the most commonly used vector for clinical gene therapy and vaccination applications. However, liver sequestration after intravenous administration of Ad5 results in high-level gene transfer in hepatocytes, which can greatly reduce the efficacy of the vector for the target organ or disease site. Recent studies have shown that hepatocyte transduction by Ad5 is mediated by a high-affinity interaction between coagulation factor X (FX) and the Ad5 trimeric hexon protein (4,11,(13)(14)(15), which is the most abundant protein in the adenovirus capsid. FX is a zymogen of a vitamin K-dependent serine protease that is primarily synthesized in the liver and circulates in the bloodstream at 8 g/ml. It is composed of a light chain consisting of a ␥-carboxylated glutamic acid (Gla) and two epidermal growth factor (EGF)-like domains that are disulfide linked to the serine protease (SP) heavy chain. While the FX Gla domain has been shown to bind hexon proteins in the Ad5 capsid, the FX SP domain bridges the Ad5/FX complex to the hepatocyte surface (2,11,15). Previous studies have shown soluble heparin or heparan sulfate can completely ablate FX-mediated Ad5 uptake in vitro and in vivo, indicating the critical role of heparan sulfate proteogylcans (HSPGs) for Ad5 cellular binding (2). FX has been shown to bind to at least 14 other Ad serotypes, indicating that this interaction is conserved (15). These findings have highlighted the importance of understanding the precise mechanisms governing cellular infectivity and tropism in order for adenoviruses to be successfully used as therapeutic gene delivery vectors.Our previous studies demonstrated that the anticoagulants nematode anticoagulant peptide 2 (NAPc2) and Ixolaris can block FX-mediated Ad5 transduction in vitro (7,8,15). Ixolaris has been shown to bind residues within the heparin binding exosite of FXa, previously defined ...

show abstract

Intermolecular Interactions and Characterization of the Novel Factor Xa Exosite Involved in Macromolecular Recognition and Inhibition: Crystal Structure of Human Gla-domainless Factor Xa Complexed with the Anticoagulant Protein NAPc2 from the Hematophagous Nematode Ancylostoma caninum

Cited by 40 publications

References 35 publications

Hepatocyte Heparan Sulfate Is Required for Adeno-Associated Virus 2 but Dispensable for Adenovirus 5 Liver Transduction In Vivo

Hepatocyte Heparan Sulfate Is Required for Adeno-Associated Virus 2 but Dispensable for Adenovirus 5 Liver Transduction In Vivo

Recent Research Developments in the Direct Inhibition of Coagulation Proteinases – Inhibitors of the Initiation Phase

A Cluster of Basic Amino Acids in the Factor X Serine Protease Mediates Surface Attachment of Adenovirus/FX Complexes

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