A molecular model of the serine protease domain of activated protein C: application to the study of missense mutations causing protein C deficiency

SummaryAnalysis of naturally occurring protein mutations yields valuable insights into functionally important sequences. Characterizing mutations responsible for protein C deficiency at the molecular level has been the subject of intensive investigation. In a previous study, a three-dimensional model of the serine protease domain of protein C was used to analyze the set of protease domain mutations previously available. The mutations were largely found to fall into a limited number of categories. A recently updated protein C mutation data base has provided a number of new mutations which have been analyzed for structural predictions.

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Protein C Deficiency caused by Homozygosity for a Novel PROC D180G Mutation – in vitro Expression and Structural Analysis of the Mutation

Thrombosis and Haemostasis ◽

10.1055/s-0037-1613267 ◽

2002 ◽

Vol 88 (10) ◽

pp. 632-638 ◽

Cited By ~ 6

Author(s):

Tobias Gedde-Dahl ◽

Geir Tjønnfjord ◽

Bruno Villoutreix ◽

Frank Brosstad ◽

Bent Lind

Keyword(s):

Protein C ◽

Late Onset ◽

Culture Media ◽

Activated Protein C ◽

Wild Type ◽

Protease Domain ◽

Protein C Deficiency ◽

293 Cells ◽

Protein C Activity

SummryHomozygosity for a novel D180G mutation in the protease domain of protein C, associated with plasma protein C activity and antigen levels of 8% of normal was identified in a thrombosis prone family. Transient expression of protein C in HK-293 cells and analysis of protein C antigen in culture media and cell lysates showed that the secretion of mutant protein as compared with wild-type protein was reduced by 79% while the intracellular contents were similar. Computer analysis of the X-ray structure of activated protein C and of a theoretical model of the zymogen predicts that the mutation destabilises the molecule locally. Our results are compatible with a relatively unstable mutant molecule that could be trapped inside the cell and degraded. However, if secreted the mutant molecule could have a relatively normal catalytic activity and structure consistent with the plasma levels of protein C activity and the late onset of thrombosis.

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Identification and Three-dimensional Structural Analysis of Nine Novel Mutations in Patients with Prothrombin Deficiency

Thrombosis and Haemostasis ◽

10.1055/s-0037-1614161 ◽

2000 ◽

Vol 84 (12) ◽

pp. 989-997 ◽

Cited By ~ 41

Author(s):

Pier Mannucci ◽

Manijeh Lak ◽

Giacomo Mancuso ◽

Maria Mazzucconi ◽

Angiola Rocino ◽

...

Keyword(s):

Amino Acid ◽

Serine Protease ◽

Close Association ◽

Autosomal Recessive Disorder ◽

Single Amino Acid ◽

Severe Bleeding ◽

Bleeding Tendency ◽

Missense Mutations ◽

Protease Domain ◽

Serine Protease Domain

SummaryProthrombin deficiency is an autosomal recessive disorder associated with a moderately severe bleeding tendency. In this study, 13 patients with prothrombin deficiency were screened for the presence of alterations in the prothrombin gene, and nine novel candidate mutations were identified. Of 11 patients with hypoprothrombinemia, ten are homozygous for five mutations and one patient is a compound heterozygote. The two patients with dysprothrombinemia are homozygous for two mutations. Eight of nine mutations are missense ones associated with single amino acid substitutions in the propeptide (Arg-1Gln, Arg-2Trp), the kringle-1 (Asp118Try) and kringle-2 (Arg220Cys) domains and the catalytic serine protease domain (Gly330Ser, Ser354Arg, Arg382His and Arg538Cys). The ninth mutation is an in-frame deletion of 3 bp that results in the omission of one amino acid (del Lys 301/302). The combination of these missense mutations with crystal structures for α-thrombin and the prothrombin fragments 1 and 2 resulted in new insight into the function of α-thrombin. The hypoprothrombinemia mutations were inferred to affect either the cleavage of the propeptide from the Gla domain, the stability of the kringle-1 and −2 domains, or the close association of the A and B chains of the serine protease domain. The dysprothrombinemia mutations were inferred to directly affect catalytic function through their location at the active site crevice or exosite 1 within the serine protease domain.

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Amino acids 225-235 of the protein C serine-protease domain are important for the interaction with the thrombin-thrombomodulin complex

FEBS Letters ◽

10.1016/0014-5793(95)00552-k ◽

1995 ◽

Vol 367 (2) ◽

pp. 153-157 ◽

Cited By ~ 11

Author(s):

A. Vincenot ◽

P. Gaussem ◽

J.L. Pittet ◽

S. Debost ◽

M. Aiach

Keyword(s):

Amino Acids ◽

Serine Protease ◽

Protein C ◽

Protease Domain ◽

Serine Protease Domain

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Molecular Characterisation and Three-Dimensional Structural Analysis of Mutations in 21 Unrelated Families with Inherited Factor VII Deficiency

Thrombosis and Haemostasis ◽

10.1055/s-0037-1614004 ◽

2000 ◽

Vol 84 (08) ◽

pp. 250-257 ◽

Cited By ~ 47

Author(s):

Flora Peyvandi ◽

Vincent Jenkins ◽

Pier Mannucci ◽

Atto Billio ◽

Sirous Zeinali ◽

...

Keyword(s):

Serine Protease ◽

Tissue Factor ◽

Middle Eastern ◽

Critical Role ◽

Factor Vii ◽

Missense Mutations ◽

Protease Domain ◽

Site Mutation ◽

Epidermal Growth Factor Domain ◽

Serine Protease Domain

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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Three Missense Mutations in the Protein C Heavy Chain Causing Type I and Type II Protein C Deficiency

Thrombosis and Haemostasis ◽

10.1055/s-0038-1642381 ◽

1994 ◽

Vol 71 (01) ◽

pp. 032-037 ◽

Cited By ~ 24

Author(s):

Toshiyuki Miyata ◽

Yan-Zhen Zheng ◽

Toshiyuki Sakata ◽

Nobuko Tsushima ◽

Hisao Kato

Keyword(s):

Amino Acid ◽

Dna Sequencing ◽

Amino Acid Substitution ◽

Protein C ◽

Type I ◽

Missense Mutations ◽

Type Ii ◽

Protease Domain ◽

Protein C Deficiency ◽

Thrombin Cleavage

SummaryWe have studied the molecular basis of protein C deficiency in three families with a history of thromboembolic disease. An approximately 50% reduction in both functional and immunologic levels of protein C was detected in the plasma from two unrelated patients, designated protein C Osaka 1 and protein C Osaka 2. An approximately 50% reduction in functional level but normal immunologic level of protein C was detected in plasma from a third patient, designated protein C Osaka 3. DNA sequencing of the amplified DNA revealed one missense mutation in each case. Additional mutations in the coding sequence were excluded by DNA sequencing of all protein C exons. We identified a C-to-T change at nucleotide number 6,218 of the protein C gene in protein C Osaka 1. This results in the amino acid substitution of Arg-169 by Trp at the a-thrombin cleavage site. In protein C Osaka 2, a G-to-A change at nucleotide number 8,807 was identified leading to the amino acid substitution of Met-364 by lie in the protease domain. This substitution may impair the synthesis or stability of protein C Osaka 2. In protein C Osaka 3, a G-to-C change at nucleotide number 8,868 was identified. This results in substitution of Gly-385 by Arg in the protease domain. Based on these, it was concluded that Arg-169-to-Trp mutation and Met-364-to-Ile mutation cause type I protein C deficiency and Gly-385-to-Arg mutation causes type II deficiency.

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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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