The C-Terminus Basic Region of TFPIα Dynamically Regulates FV(a) Function: Evaluation of FV-Short

Abstract Coagulation Factor V (FV) is present in plasma as an inactive procofactor. Following B-domain removal, the active cofactor FVa, enhances the catalytic efficiency of FXa by several orders of magnitude. Previous findings have established that evolutionary conserved regions within the B-domain play a key role in keeping FV in an inactive state by, in part, concealing FXa binding site(s). These regions of the FV B-domain consist of basic and acidic elements and define the minimal sequence necessary to maintain FV as a procofactor. Recent data have shown that removal of either one of these elements results in FVa-like activity and that B-domain fragments spanning the basic region act in-trans to suppress the activity of FV variants bearing only the acidic region (J Biol Chem. 287:26342-51, 2012, J Biol Chem. 288:30151-60, 2013). Physiologically, forms of FV that are missing a basic region but harbor an acidic region are released by activated platelets. Interestingly, another form of FV that only harbors an acidic region has been described. The variant FV-East Texas results in an alternatively spliced form of FV that has most of the B-domain removed but retains the acidic region (FV-short; J Clin. Invest. 123:3777-87; 2013). Together these forms of FV should be constitutively active. However, it is possible that physiologic ligands that mimic the basic region could inhibit their activity. Previous studies have identified tissue factor pathway inhibitor (TFPIα) as one of these potential ligands. Remarkably, the C-terminal segment of TFPIα shares substantial sequence homology with the FV basic region and binds forms of FV that only harbor the acidic region (PNAS, 110:17838-43; 2013). While there are several remaining unanswered questions, the FV(a)-TFPIα interaction has the potential to fundamentally alter our understanding of cofactor regulation at the site of injury. In order to investigate this fascinating prospect, we expressed and purified recombinant FV-short and a protein fragment containing the basic region of TFPIα. As anticipated, FV-short exhibited FVa-like activity, however this cofactor function in the prothrombinase complex was greatly impaired in the presence of TFPIα basic region. Similar results were obtained in clotting assays, supporting the idea of a trans-acting function of TFPIα on FV derivatives missing the acidic region. To better understand the mechanism of interaction, direct binding measurements by fluorescence were established using a labeled TFPIα basic region fragment. Changes in anisotropy were monitored as a function of the FV-short concentration. Analysis of the data revealed a high affinity interaction between FV-short and the TFPIα basic region (Kd = 3.42 ± 0.39 nM). Based on this high affinity, a proportion of FV-short should be largely bound to TFPIα; an observations consistent with the FV-East Texas family. Thus instead of FV-short being constitutively active, when bound to TFPIα it would effectively revert to a procofactor state. To investigate the implications of this circulating complex on function, we next evaluated whether FV-short could be normally converted to FVa by thrombin (IIa) through proteolysis at Arg709 and Arg1545 (Arg1018 is missing). Surprisingly, in the presence of TFPIα basic region, cleavage at Arg1545 was significantly delayed suggesting TFPIα modulates both the activity of FV and its interaction with IIa. TFPIα could either directly and/or allosterically interfere with IIa binding sites on FV. Based on previous unpublished work in our lab, we believe that the basic region of TFPIα provides a trans-acting sequence that causes FV-short to revert to a procofactor state both functionally and structurally. The absence of Arg1018 abrogates proteolysis necessary to alleviate these structural constraints, hence resulting in delayed cleavage at Arg1545. This study shows direct evidence of a high affinity interaction between the TFPIα c-terminal basic region and the naturally-occurring truncated form of FV responsible for the East Texas bleeding disorder. We now have unique tools to investigate the role of full length TPFIα not only in this interesting case, but also in relation to partially activated forms of FV released from platelets. This work sets the stage to achieve a better understanding of the early hemostatic events occurring at the site of injury, providing the bases for potential therapeutic regulation. Disclosures Camire: Pfizer: Consultancy, Patents & Royalties, Research Funding.

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Conserved Structural Motifs Cooperate to Maintain Blood Coagulation Factor V in An Inactive Procofactor State.

Blood ◽

10.1182/blood.v114.22.850.850 ◽

2009 ◽

Vol 114 (22) ◽

pp. 850-850

Author(s):

Mettine H.A. Bos ◽

Rodney M. Camire

Keyword(s):

Amino Acids ◽

Blood Coagulation ◽

Coagulation Factor ◽

Factor V ◽

Vertebrate Lineage ◽

New Variant ◽

Coagulation Factor V ◽

Blood Coagulation Factor V ◽

Basic Region ◽

Constitutively Active

Abstract Abstract 850 Blood coagulation factor V (FV) is a multi-domain protein which circulates as an inactive procofactor and has high structural homology with factor VIII. To express procoagulant activity, FV must be proteolytically processed within its central B-domain (836 residues) with thrombin being considered the key physiological activator. Following liberation of the B-domain (residues 710-1545), activated FV (FVa) functions as a cofactor for factor Xa within the prothrombinase complex and dramatically enhances the rate of thrombin generation. The central role which FVa assumes in prothrombinase indicates that its activation must be a key regulatory step in hemostasis. Although the proteolytic events that lead to the activation of FV have been well studied, the molecular mechanism by which B-domain release facilitates the procofactor to cofactor transition is not well understood. Recently, we have shown that in the absence of intentional proteolysis, deletion or substitution of discrete B-domain sequences drives the expression of procoagulant function (JBC, 282, 15030-9, 2007). Conversion to the constitutively active cofactor state is related, at least in part, to a cluster of amino acids (963-1008) which is highly basic and well conserved, even though most of the B-domain has weak homology within the vertebrate lineage. In the current study, we examined if this basic B-domain region is sufficient to preserve FV as an inactive procofactor. To investigate this, the basic region (46 residues) was incorporated within the short B-domain of a previously characterized FV variant, FV-810. Factor V-810 has amino acids 811-1491 within the B-domain deleted and is a constitutively active cofactor, with functional properties equivalent to FVa. Using a PT-based clotting assay, purified prothrombinase assay, and direct fluorescent binding measurements with FXa-membranes we found that insertion of the basic region into FV-810 (inserted after residue 810) converted this cofactor-like species back to the procofactor-like state, despite >75% of the B-domain being absent. Next, using this new variant (FV+BR; B-domain of 201 residues), we assessed whether residual B-domain sequences within FV+BR contribute to maintaining FV in an inactive, procofactor state. Elimination of ∼100 residues on the N-terminal side of FV+BR was without functional consequence; that is, the procofactor state was maintained. In contrast, removal of B-domain sequences (∼50 residues, 30% of which are acidic) to the C-terminal side of the basic region shifted FV-810+BR from an inactive procofactor to an active cofactor. As expected, all purified FV derivatives exhibited full cofactor activity following treatment with thrombin. Together, these data show that B-domain sequences 963-1008 (basic region) appear to work in concert with the acidic C-terminal region of the B-domain (1492-1545) to keep FV in an inactive procofator state. These sequence elements appear to be necessary and sufficient as we were able to construct a FV variant with a B-domain length of only 103 amino acids that remarkably still had procofactor-like properties. Interestingly, these two regions of the B-domain (963-1008 and 1492-1545) are generally well conserved throughout the vertebrate lineage, while the remaining regions of the B-domain are not. We speculate that these B-domain sequences bind intramolecularly to heavy and/or light chain sequences thereby concealing critical binding sites on the FV molecule which govern the function of the active cofactor species. Disclosures: Camire: Wyeth: Patents & Royalties, Research Funding.

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The C-Terminus of Tfpiα Inhibits Factor V Activation By Protecting the Arg1545 Cleavage Site

Blood ◽

10.1182/blood.v128.22.257.257 ◽

2016 ◽

Vol 128 (22) ◽

pp. 257-257

Author(s):

Peter van Doorn ◽

Jan Rosing ◽

Simone Wielders ◽

Tilman M. Hackeng ◽

Elisabetta Castoldi

Keyword(s):

Thrombin Generation ◽

Full Length ◽

Model Systems ◽

Factor V ◽

Dependent Manner ◽

High Affinity ◽

C Terminus ◽

Dose Dependent ◽

Acidic Region ◽

Basic Region

Abstract Coagulation factor V (FV) is the inactive precursor of FVa, which acts as an essential cofactor of factor Xa (FXa) in the prothrombinase complex. FV is maintained in the inactive state by the interaction between a basic and acidic region in the B-domain. The C-terminus of tissue factor pathway inhibitor-α (TFPIα) is highly homologous to the FV basic region and also binds to the acidic region of FV. In fact, a large fraction of plasma TFPIα circulates in complex with FV. Thanks to this interaction, FV acts as a cofactor of TFPIα in the inhibition of FXa and TFPIα inhibits prothrombinase complexes containing forms of FVa that retain the acidic region. However, when FV is activated through cleavage at Arg709, Arg1018 and Arg1545 by FXa or thrombin, it loses its anticoagulant properties and becomes a strong procoagulant. Recently, a FV splicing variant (FV-short) that lacks the basic region and binds TFPIα with high affinity has been described. FV-short is present in all individuals and represents ~5% of all plasma FV. To gain more insight in the functional implications of the FV-TFPIα interaction, we studied the effects of a peptide identical to the TFPIα C-terminus (TFPIα C-term) on thrombin generation in plasma and on FV activation in model systems. All major findings were confirmed with full-length TFPIα. TFPIα C-term (0-5 µM) prolonged the lag time and decreased the peak height of tissue factor- and FXa-triggered thrombin generation in a dose-dependent manner. These effects were more pronounced at low procoagulant stimuli and in the presence of plasma TFPIα. TFPIα C-term also inhibited thrombin generation in FV-depleted plasma reconstituted with FV, but not in FV-depleted plasma reconstituted with FVa, suggesting an effect on FV activation and/or prothrombinase. In model systems, TFPIα C-term inhibited the activation of purified FV by FXa and thrombin in a dose-dependent manner. This could be due to inhibition of FV proteolysis and/or to inhibition of prothrombinase in the assay used to quantify FVa activity. Therefore, FV activation was also followed by SDS-PAGE and Western blotting. This showed that TFPIα C-term (1 µM) interferes with FV proteolysis by both FXa and thrombin by selectively impairing cleavage of FV at Arg1545, which is located close to the FV acidic region (residues 1493-1537). The effect of TFPIα C-term on FV activation by thrombin was 3-fold stronger for FV-short than for full-length FV, in line with their respective affinities for the TFPIα C-terminus. Full-length TFPIα (10 nM) also inhibited FV cleavage at Arg1545 and delayed FV activation by thrombin. Its effect was also more pronounced on FV-short than on FV. In summary, binding of the TFPIα C-terminus to the acidic region of FV inhibits FV activation by FXa or thrombin by blocking access to the Arg1545 cleavage site. Since cleavage at this site marks the transition of FV from an anticoagulant form (TFPIα-cofactor) to a procoagulant form (FXa-cofactor), this may represent an important new anticoagulant function of TFPIα. The main target of this anticoagulant mechanism is presently unclear, but it is unlikely to be intact FV, whose plasma concentration is 100-fold higher than the TFPIα concentration. More likely candidates are low-abundance FV species that lack the basic region but retain the acidic region, and therefore bind TFPIα with high affinity, such as FV-short, early FV activation intermediates and/or platelet FV. Supported by grant nr. 2014-1 from the Dutch Thrombosis Foundation. Disclosures No relevant conflicts of interest to declare.

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Cryo-EM Structure of BIVV001 Reveals Coagulation Factor VIII-Von Willebrand Factor D'D3 Interaction Mode

Blood ◽

10.1182/blood-2019-126645 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 94-94

Author(s):

James Fuller ◽

Joseph Batchelor ◽

Kevin Knockenhauer ◽

Hans-Peter Biemann ◽

Robert Peters

Keyword(s):

High Resolution ◽

Factor Viii ◽

Von Willebrand Factor ◽

Single Particle ◽

Half Life ◽

Coagulation Factor ◽

High Affinity ◽

Affinity Interaction ◽

Von Willebrand ◽

Willebrand Factor

Introduction Coagulation Factor VIII (FVIII) is a serine protease cofactor that directly interacts with coagulation factors IXa and X on activated platelets, and enhances FIXa activity toward FX by 105. von Willebrand Factor (VWF), via its D'D3 domains, interacts with FVIII and prevents premature deposition on phospholipids until activation by thrombin. Thrombin cleavage at Arg1689 of FVIII promotes VWF dissociation by disrupting the FVIII a3 high affinity interaction with the VWF D' domain. VWF extends the half-life of circulating FVIII from less than 3 hours to ~11 hours in humans. While crystal structures of FVIII and VWF D'D3 alone have been solved, the atomic details of a formed complex are unknown. We sought to determine the FVIII-VWF D'D3 complex structure by using BIVV001, our investigational new drug currently in clinical trials for the treatment of Hemophilia A. BIVV001 (rFVIIIFc-VWF-XTEN) is a novel fusion protein consisting of single chain B-domain deleted (BDD) human FVIII, the Fc domain of human immunoglobulin G1 (IgG1), the FVIII-binding D'D3 domain of human von Willebrand factor, and 2 XTEN polypeptide linkers. The Fc, VWF, and XTEN linker portions of the molecule are each designed to extend the half-life of FVIII. We anticipated that the tethering of FVIII to D'D3 through the Fc dimer in BIVV001 would stabilize the complex for structural studies. Given the large size of BIVV001, at 312 kDa, we thought it an ideal target for structure determination by single particle cryo-EM. Methods We collected a total of 3955 micrographs of BIVV001 embedded in vitreous ice at 81,000x magnification using a Titan Krios electron microscope equipped with a Gatan BioQuantum K3 energy filter and camera operating in super-resolution mode. Preferential particle orientation was a major challenge that was overcome through a variety of methods. Micrograph movies were motion-corrected and summed, and over 2 million candidate particle coordinates were extracted. Repeated rounds of reference-free 2D classification resulted in a set of 1.2 million particles that generated a reasonable ab initio/de novo 3D model. Initial full 3D refinements of this model produced a map at approximately 5 Å resolution, into which available crystal structures can be readily fit. Subsequent iterative 3D refinement and 3D classification resulted in a final map at high resolution, into which an atomic model was built. Results The structure of BIVV001 was solved by single particle cryo-EM. D' of VWF interacts with the front face of the C1 and A3 domains of FVIII, consistent with a lower resolution, negative stain EM map (Yee et al. 2015. Blood). Interface residues on FVIII identified in an HDX-MS dataset (Chiu et al. 2015. Blood.) largely correspond to this high affinity interaction. D' protrudes upward from the VWF D3 domain, which sits centrally located between the C1 and C2 domains of FVIII at a 45° tilt. By occupying this position, D3 likely sterically blocks the FVIII C domains from binding to membrane. The VWD3 module of the D3 domain contacts the base of the C1 domain, whereas C8-3 binds to the bottom of the C2 domain. The conserved Ca2+ site in VWD3 identified previously (Dong et al. 2019. Blood.) is in the interface with C1. This is consistent with Yee et al., where docking placed D3 below the C domains. In that study, a lack of density between FVIII and VWF D3 in the 3D reconstruction, due to flexibility, prevented the detailed analysis that is possible here. In this study, flexibility in this region is also apparent, as C2 is less well ordered than the rest of FVIII and VWF D3 is the least well-ordered portion of the resolved structure. The XTEN linkers are not visible in the final map and were not apparent in any 2D class averages. The Fc is absent in most 2D class averages, due to a lack of consistent positioning relative to FVIII. In the rare cases where the Fc is visible, it adopts a preferred position on the back side of FVIII below the A3 protrusion. Conclusions The structure of BIVV001 has been solved by cryo-electron microscopy to high resolution. Alignment with previous results and the averaging out of BIVV001 elaborations suggests the structure obtained here likely represents WT FVIII-D'D3. This structure demonstrates how VWF D'D3 prevents premature FVIII deposition on phospholipids. The structural basis of type 2N von Willebrand Disease mutations in D'D3 can be readily interpreted. Next steps include solving a FVIII-D'D3 dimer structure at high resolution. Disclosures Fuller: Sanofi: Employment. Batchelor:Sanofi: Employment. Knockenhauer:Sanofi: Employment. Biemann:Sanofi: Employment. Peters:Sanofi: Employment.

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Characterization of B-Domain Elements Which Maintain Factor V in a Procofactor Form.

Blood ◽

10.1182/blood.v104.11.1726.1726 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1726-1726

Author(s):

Rodney M. Camire ◽

Hua Zhu

Keyword(s):

Amino Acids ◽

Functional Properties ◽

Tandem Repeats ◽

Coagulation Factor ◽

High Yield ◽

Factor V ◽

Single Chain ◽

High Affinity ◽

Single Chain Fv ◽

Cofactor Activity

Abstract Blood coagulation factor V (FV) has little or no procoagulant activity and is activated by thrombin (IIa) to FVa following proteolytic removal of a central glycosylated B-domain (residues 710-1545; 836 a.a.). Discrete proteolysis is necessary for activation; however, recent data indicates that it is incidental to the mechanism by which cofactor function is realized. It was found that shortening the B-domain in the absence of bond cleavage to 155 amino acids also yielded an active cofactor form that assembles and functions in prothrombinase. These observations support the conclusion that macromolecular binding sites, which govern the eventual function of FVa, are concealed on FV by B-domain sequences. At present, it is unknown which elements of the B-domain contribute to maintaining the procofactor form. To begin to investigate this, we have designed eight recombinant derivatives of FV with variable amounts of the B-domain: FV-810 (des811–1491), FV-866 (des867–1491), FV-902 (des903–1491), FV-956 (des957–1491), FV-1033 (des1034–1491), FV-1053 (des1054–1491), FV-1106 (des1107–1491), and FV-1152 (des1153–1491). These constructs were stably expressed in BHK cells and purified to homogeneity in high yield (1–2 mg/L of media). The proteins migrated as single bands on SDS-PAGE, and were completely processed to FVa following incubation with IIa. Using either a purified prothrombinase assay with limiting amounts of (pro)cofactor or a one-stage PT-based clotting assay, we found that most of the derivatives were grouped into two categories: procofactor-like and cofactor-like. Factor V-810, FV-866, and FV-902 exhibited activity profiles consistent with the cofactor-like form and, as expected, bound with high affinity to FXa-membranes (Kd = 0.2–0.4 nM; n = 1). In contrast, FV-1033, FV-1053, FV-1106, and FV-1152 had very little activity and exhibited functional properties consistent with the procofactor-like form. Direct binding fluorescent measurements revealed that unlike FV, these constructs did appear to bind FXa-membranes, albeit with a much lower affinity compared to FVa. Interestingly, an outlier to either of the groups was FV-956 (301 a.a. B-domain). This derivative bound FXa-membranes with moderate affinity (Kd = 5 nM; n = 1) and it exhibited functional properties intermediate between the procofactor and cofactor forms in both the PT-based clotting and purified component assays. Control experiments indicated that all of the derivatives exhibited full cofactor activity and high affinity binding to FXa-membranes following treatment with IIa. Collectively, these data demonstrate that the single-chain cofactor-like forms can be shifted to the procofactor-like form by relatively small, systematic increases in the length of the B-domain. This transition was accompanied by a marked decrease in functional activity and FXa binding. Surprisingly, these data also show that elimination of >50% of the B-domain (1034–1491; 458 out of 836 a.a. deleted), a region that contains 31, nine amino acid tandem repeats and 16 out of 25 potential N-linked carbohydrate sites, has little, if any, influence on maintaining the procofactor form. Taken together, our data are consistent with the interpretation that a length of at least 375 amino acids or specific sequences contained within residues 902–1033 is sufficient to suppress cofactor activity of single chain FV.

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Mapping the Location of Autoinhibitory B-Domain Motifs within Factor V: New Insights Into How Factor V Activation Unfolds

Blood ◽

10.1182/blood.v120.21.495.495 ◽

2012 ◽

Vol 120 (21) ◽

pp. 495-495

Author(s):

Raffaella Toso ◽

Matthew W. Bunce ◽

Rodney M. Camire

Keyword(s):

Amino Acids ◽

Binding Site ◽

Heavy Chain ◽

3D Structure ◽

Regulatory Region ◽

Spatial Relationship ◽

Coagulation Factor ◽

Factor V ◽

High Affinity ◽

Cofactor Activity

Abstract Abstract 495 Blood coagulation factor V (FV) circulates as a procofactor with little or no procoagulant activity. Proteolytic removal of a large central B-domain converts FV (domain organization of A1-A2-B-A3-C1-C2) to an active cofactor for membrane-bound FXa to form prothrombinase. Recently we found that discrete, evolutionarily conserved sequences within the B-domain serve an autoinhibitory function and are necessary to maintain FV as an inactive procofactor. The autoinhibitory region within the B-domain (termed the procofactor regulatory region or PRR) is sequence specific and remarkably short (∼100 amino acids out of 836 from B-domain) consisting of basic (963–1008; basic region or BR) and acidic (1493–1537; acidic region or AR) sequences. Dismantling this region appears to be the driving force to unveil a high affinity binding site(s) for FXa and thus underpins the procofactor to cofactor transition. While the BR and AR seem to work together to provide “on-site” repression of cofactor activity, there is no structural information about the FV B-domain that could provide clues into how this critical region functions. Here we use B-domain fragments tethered to an artificial protease in order to map the position of the PRR relative to the rest of the FV molecule. A BR peptide with an introduced free Cys at position 990 was stoichiometrically modified with FeBABE (Fe-p-bromoacetamidobenzyl-EDTA). FeBABE is a protein cutting reagent with a sulfhydryl-reactive moiety for attachment to Cys and an EDTA-chelated iron atom which, when triggered with ascorbic acid and peroxide, generates hydroxyl radicals that cut peptide bonds in a sequence-independent fashion. When a protein labeled with FeBABE binds its target, the Fe-BABE moiety facilitates cutting if it is orientated correctly and near (<12 Å) the contact site of the target protein. In our experimental system, we employed a constitutively active B-domainless form of FV (FV-810) that harbors the AR but lacks the BR. Initial studies revealed that the BR peptide, without or tethered with FeBABE, binds to FV-810 with high affinity (nM) and inhibits cofactor activity. Proteolysis of FV-810 by BR-FeBABE, was visualized by Western blot, using monoclonal antibodies recognizing either the heavy or light chains. Using this approach, we consistently found that BR-FeBABE cut FV-810 predominantly at two sites. The first is at the C-terminal end of the B-domain. This region is enriched in acidic residues and represents the AR defined above. The second site of cleavage is within the heavy chain. While we cannot exclude the possibility that the site of contact is at the N-terminus of the A1 domain, based on the proposed 3D structure of FVa, it is more likely that the BR is binding near the C-terminal end of the A2 domain which is also enriched in acidic amino acids. Compared to a panel of FVa derivatives with variably truncated heavy chains, we suggest that BR-FeBABE cuts FV-810 near residues 675–685. Overall the data suggest that the B-domain must be folded in such a way as to place the BR in close spatial proximity to the AR at the C-terminal end of the B-domain and also to the C-terminal heavy chain region. Consistent with the idea that these interacting motifs work to keep FV as a procofactor by suppressing FXa binding, adding saturating amounts of FXa, but not the zymogen FX, prevented the BR-FeBABE induced cleavage of FV-810. Similarly, activated protein C (APC) which is thought to share a related binding site with FXa, also prevented BR-FeBABE from cutting FV-810. Addition of excess unlabeled BR peptide substantially reduced cutting of FV-810 by BR-FeBABE, indicating that proteolysis is not due to non-specific cleavage. Additionally, removing the basic charge of BR-FeBABE via acetylation eliminated binding to FV-810 and also any signs of specific cutting. Further, FV-derivatives harboring both the AR and BR in cis and hence not binding the BR peptide, were not cut by excess BR-FeBABE added in trans. In conclusion, these findings provide new insights into the spatial relationship between key regulatory B-domain motifs and the core heavy/light chain region. These interacting autoinhibitory motifs directly or indirectly obscure FXa binding and hence stabilize the inactive procofactor state of FV. These studies also document a remarkably powerful way to map the spatial relationship between regions of proteins and provide new information on coagulation factor binding sites. Disclosures: Camire: Pfizer: Patents & Royalties, Research Funding.

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Coagulation Factor V Leiden Mutation Was Detected in the Patients with Activated Protein C Resistance in Thailand

Thrombosis and Haemostasis ◽

10.1055/s-0037-1615201 ◽

1998 ◽

Vol 80 (08) ◽

pp. 344-345 ◽

Cited By ~ 2

Author(s):

Pasra Arnutti ◽

Motofumi Hiyoshi ◽

Wichai Prayoonwiwat ◽

Oytip Nathalang ◽

Chamaiporn Suwanasophon ◽

...

Keyword(s):

Protein C ◽

Activated Protein C ◽

Factor V Leiden ◽

Coagulation Factor ◽

Factor V ◽

Activated Protein C Resistance ◽

Factor V Leiden Mutation ◽

Coagulation Factor V

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Ancylostoma caninum Anticoagulant Peptide Blocks Metastasis In Vivo and Inhibits Factor Xa Binding to Melanoma Cells In Vitro

Thrombosis and Haemostasis ◽

10.1055/s-0037-1615117 ◽

1998 ◽

Vol 79 (05) ◽

pp. 1041-1047 ◽

Cited By ~ 20

Author(s):

Kathleen M. Donnelly ◽

Michael E. Bromberg ◽

Aaron Milstone ◽

Jennifer Madison McNiff ◽

Gordon Terwilliger ◽

...

Keyword(s):

Active Site ◽

Thrombin Generation ◽

Factor Xa ◽

Coagulation Factor ◽

Melanoma Cells ◽

Factor V ◽

Ancylostoma Caninum ◽

Metastatic Activity

SummaryWe evaluated the in vivo anti-metastatic activity of recombinant Ancylostoma caninum Anticoagulant Peptide (rAcAP), a potent (Ki = 265 pM) and specific active site inhibitor of human coagulation factor Xa originally isolated from bloodfeeding hookworms. Subcutaneous injection of SCID mice with rAcAP (0.01-0.2 mg/mouse) prior to tail vein injection of LOX human melanoma cells resulted in a dose dependent reduction in pulmonary metastases. In order to elucidate potential mechanisms of rAcAP’s anti-metastatic activity, experiments were carried out to identify specific interactions between factor Xa and LOX. Binding of biotinylated factor Xa to LOX monolayers was both specific and saturable (Kd = 15 nM). Competition experiments using antibodies to previously identified factor Xa binding proteins, including factor V/Va, effector cell protease receptor-1, and tissue factor pathway inhibitor failed to implicate any of these molecules as significant binding sites for Factor Xa. Functional prothrombinase activity was also supported by LOX, with a half maximal rate of thrombin generation detected at a factor Xa concentration of 2.4 nM. Additional competition experiments using an excess of either rAcAP or active site blocked factor Xa (EGR-Xa) revealed that most of the total factor Xa binding to LOX is mediated via interaction with the enzyme’s active site, predicting that the vast majority of cell-associated factor Xa does not participate directly in thrombin generation. In addition to establishing two distinct mechanisms of factor Xa binding to melanoma, these data raise the possibility that rAcAP’s antimetastatic effect in vivo might involve novel non-coagulant pathways, perhaps via inhibition of active-site mediated interactions between factor Xa and tumor cells.

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Phenotyping and Genotyping of Coagulation Factor V Leiden

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650258 ◽

1996 ◽

Vol 75 (02) ◽

pp. 267-269 ◽

Cited By ~ 27

Author(s):

H Engel ◽

L Zwang ◽

H H D M van Vliet ◽

J J Michiles ◽

J Stibbe ◽

...

Keyword(s):

Factor V Leiden ◽

Coagulation Factor ◽

Diagnostic Value ◽

Resistance Test ◽

Amplification Refractory Mutation System ◽

Factor V ◽

Chain Reactions ◽

Apc Resistance ◽

Specificity And Sensitivity ◽

Coagulation Factor V

SummaryThe currently used activated Protein C resistance test demonstrated to be of limited diagnostic value for the detection of the mutant Factor V Leiden. Moreover, this assay is not useful for patients under anticoagulant therapy. A modification of the APC resistance test, applying Factor V deficient plasma is described which demonstrates a specificity and sensitivity of 1.0. The superiority of the modified APC resistance test over the existing APC resistance test was verified by genotyping.For that purpose, the Amplification Refractory Mutation System (ARMS) was applied to the detection of the G to A mutation at position 1691 in the gene encoding coagulation Factor V. The mutation at that position could be easily detected by using each of two allele-specific oligonucleotide primers concomitantly with one common primer in two separate polymerase chain reactions, thereby amplifying a fragment of 186 base-pairs of the Factor V gene.

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