High Affinity Ca2+-binding Site in the Serine Protease Domain of Human Factor VIIa and Its Role in Tissue Factor Binding and Development of Catalytic Activity

SummaryFactor VII (FVII) is a four-domain glycoprotein that plays a critical role in the initiation of blood coagulation. Hereditary deficiencies of this plasma protein results in a bleeding diathesis that varies in severity amongst affected patients. We have analysed the FVII gene in 27 patients with FVII deficiency from 21 unrelated families predominantly of Middle-Eastern extraction. A total of 19 different mutations were identified, of which 12 were novel and 7 had been previously reported. Nine of the 12 novel mutations were missense mutations located in the Gla domain (Ser23Pro), the second epidermal growth factor domain (Cys135Arg) and the catalytic serine protease domain (Arg247Cys, Arg277Cys, Ser282Arg, Pro303Thr, Ser363Ile, Trp364Cys, Trp364Phe), of which five are homozygous. Three novel splice mutations were identified in intron 1a (IVS1a+5), intron 2 (IVS2+1) and intron 6 (IVS6+1). Of the seven previously reported mutations, five were missense mutations of which three are homozygous (Gln100Arg, Arg152Gln, Arg304Gln, Cys310Phe and Thr359Met), one was a 17 bp deletion (10585del17bp) and one was a splice site mutation within intron 7 (IVS7+7). This study has significantly extended the current database of FVII mutations, including the number of known homozygous mutations. Conformational analyses of crystal structures for FVIIa and the FVIIa-tissue factor complex provided likely explanations for the effect of the missense mutations on FVIIa secretion or function. In particular, since 23 missense mutations were located to the serine protease domain, mostly to the region between the catalytic triad and the contact surface with tissue factor, this showed that the orientation of the serine protease domain relative to bound tissue factor in the complex is crucial for functional activity.

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An Enzyme-Linked Immunosorbent Assay for Urokinase-Type Plasminogen Activator (u-PA) and Mutants and Chimeras Containing the Serine Protease Domain of u-PA

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648387 ◽

1992 ◽

Vol 67 (01) ◽

pp. 095-100 ◽

Cited By ~ 18

Author(s):

Paul J Declerck ◽

Leen Van Keer ◽

Maria Verstreken ◽

Désiré Collen

Keyword(s):

Serine Protease ◽

Plasminogen Activator ◽

Active Site ◽

Enzyme Linked Immunosorbent Assay ◽

Single Chain ◽

Protease Domain ◽

Serine Protease Domain ◽

Type Plasminogen Activator ◽

Urokinase Type Plasminogen Activator ◽

Normal Human

SummaryAn enzyme-linked immunosorbent assay (ELISA) for quantitation of natural and recombinant plasminogen activators containing the serine protease domain (B-chain) of urokinase-type plasminogen activator (u-PA) was developed, based on two murine monoclonal antibodies, MA-4D1E8 and MA-2L3, raised against u-PA and reacting with non-overlapping epitopes in the B-chain. MA-4D1E8 was coated on microtiter plates and bound antigen was quantitated with MA-2L3 conjugated with horseradish peroxidase. The intra-assay, inter-assay and inter-dilution coefficients of variation of the assay were 6%, 15% and 9%, respectively. Using recombinant single-chain u-PA (rscu-PA) as a standard, the u-PA-related antigen level in normal human plasma was 1.4 ± 0.6 ng/ml (mean ± SD, n = 27).The ELISA recognized the following compounds with comparable sensitivity: intact scu-PA (amino acids, AA, 1 to 411), scu-PA-32k (AA 144 to 411), a truncated (thrombin-derived) scu-PA comprising A A 157 to 411, and chimeric t-PA/u-PA molecules including t-PA(AA1-263)/scu-PA(AA144-411), t-PA(AA1-274)/scu-PA(AA138-411) and t-PA(AA87-274)/scu-PA(AA138-411). Conversion of single-chain to two-chain forms of u-PA or inhibition of active two-chain forms with plasminogen activator inhibitor-1 or with the active site serine inhibitor phenyl-methyl-sulfonyl fluoride, did not alter the reactivity in the assay. In contrast, inactivation with α2-antiplasmin or with the active site histidine inhibitor Glu-Gly-Arg-CH2Cl resulted in a 3- to 5-fold reduction of the reactivity. When purified scu-PA-32k was added to pooled normal human plasma at final concentrations ranging from 20 to 1,000 ng/ml, recoveries in the ELISA were between 84 and 110%.The assay was successfully applied for the quantitation of pharmacological levels of scu-PA and t-PA(AA87_274)/scu-PA(AA138-411) in plasma during experimental thrombolysis in baboons.Thus the present ELISA, which is specifically dependent on the presence of the serine protease part of u-PA, is useful for measurement of a wide variety of variants and chimeras of u-PA which are presently being developed for improved thrombolytic therapy.

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Activation of Factor X by Factor VIIa Complexed with Human-mouse Tissue Factor Chimeras Requires Human Exon 3

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650584 ◽

1996 ◽

Vol 76 (03) ◽

pp. 361-368 ◽

Cited By ~ 13

Author(s):

Carrie H Fang ◽

T-C Lin ◽

Arabinda Guha ◽

Yale Nemerson ◽

William H Konigsberg

Keyword(s):

Tissue Factor ◽

Human Factor ◽

Factor Viia ◽

Mouse Tissue ◽

Factor X ◽

Lower Affinity ◽

E Coli ◽

Sequence Elements ◽

Specific Activities ◽

Human And Mouse

SummaryIn an attempt to define sequence elements in human and mouse tissue factor (TF) that are responsible for the species specificity observed in their interaction with human factor VIIa (HVIIa), we constructed human-mouse chimeric TF cDNAs, inserted them into plasmid vectors, and induced their expression in E.coli. Assays for procoagulant activity were carried out with the resulting E. coli lysates using (HVIIa) human and mouse (MVIIa). The ratio of the procoagulant activities, HVIIa/MVIIa, revealed that human TF exon 3 was essential for activity when the TF:VIIa complex was formed with HVIIa. By ligating the maltose binding protein (MBP) gene to TF cDNAs it was possible to construct, express and purify MBP-TF chimeras as well as to estimate their specific activities. With selected MBP-TF chimeras and HVIIa we determined kinetic parameters for the activation of human factor X. Replacement of exon 3 in human TF cDNA with the corresponding exon from mouse TF cDNA resulted in both lower affinity for HVIIa and failure to convert bound HVIIa into a potent protease

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Relations between factor VIIa binding and expression of factor VIIa/tissue factor catalytic activity on cell surfaces.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)49554-9 ◽

1992 ◽

Vol 267 (22) ◽

pp. 15447-15454

Author(s):

D.T. Le ◽

S.I. Rapaport ◽

L.V. Rao

Keyword(s):

Catalytic Activity ◽

Tissue Factor ◽

Factor Viia ◽

Cell Surfaces

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A 384-HTS for Human Factor VIIa: Comparison With 96- and 864-Well Formats

CrossRef Listing of Deleted DOIs ◽

10.1177/108705719700200307 ◽

1997 ◽

Vol 2 (3) ◽

pp. 171-178 ◽

Cited By ~ 15

Author(s):

John C.W. Comley ◽

Alastair Binnie ◽

Caroline Bonk ◽

John G. Houston

Keyword(s):

Tissue Factor ◽

Dose Response ◽

Human Factor ◽

Factor Viia ◽

Assay Development ◽

Fluorescent Detection ◽

Response Curves ◽

Liquid Handling ◽

Dose Response Curves ◽

Assay Volume

A homogenous fluorescent HTS for recombinant human factor VIIa (FVIIa) using soluble tissue factor has been developed in 384-well microplates. In this report we discuss our experiences with assay development, liquid handling using a Tomtec Quadra and Matrix PlateMate, fluorescent detection and screening of -200,000 compounds against FVIIa in 384-well plate format. Assays using the entire Helix 864-well plate were prototyped using contact dispensing with a modified Hamilton Microlab 2200. FVIIa was used as a model assay to compare between 96-, 384-, and 864-plate formats in a total assay volume of 100, 25, and 10 μl, respectively. FVIIa was assayed in 864 to the same degree of sensitivity as 384- and 96-well assays and dose-response curves for a standard inhibitor (benzamidine) in the FVIIa assay were identical in all plate formats. Finally, we review the prospects for HTS in 864-well microplates.

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