scholarly journals Coagulation factor XII Locarno: the functional defect is caused by the amino acid substitution Arg 353-->Pro leading to loss of a kallikrein cleavage site

Blood ◽  
1994 ◽  
Vol 84 (4) ◽  
pp. 1173-1181 ◽  
Author(s):  
JK Hovinga ◽  
J Schaller ◽  
H Stricker ◽  
WA Wuillemin ◽  
M Furlan ◽  
...  
Keyword(s):  
Amino Acid ◽  
Proteolytic Activity ◽  
Amino Acid Substitution ◽  
Cleavage Site ◽  
Dextran Sulfate ◽  
Coagulation Factor ◽  
Molecular Defect ◽  
Factor Xii ◽  
Amino Acid Sequence Analysis ◽  
Functional Defect

Abstract The dysfunctional coagulation factor XII (FXII) Locarno was purified from 2 L of the proposita's plasma. Studies to identify the molecular defect responsible for the lack of amidolytic and proteolytic activity of this FXII variant were performed. Amino acid sequence analysis of peptides obtained from FXII Locarno on activation with either trypsin or plasma kallikrein and dextran sulfate showed an amino acid substitution of Arg 353 by Pro. Thereby, the kallikrein cleavage site at Arg 353-Val 354 is lost. Although trypsin-activated FXII Locarno was fully cleaved at Arg 334-Asn 335 and at Arg 343-Leu 344, neither amidolytic nor proteolytic activity was generated. We conclude that proteolytic cleavage at Arg 343 in the absence of cleavage at Arg 353 is not sufficient to expose the enzymatic active site in FXII Locarno.

scholarly journals Coagulation factor XII Locarno: the functional defect is caused by the amino acid substitution Arg 353-->Pro leading to loss of a kallikrein cleavage site

Blood ◽  
1994 ◽  
Vol 84 (4) ◽  
pp. 1173-1181 ◽  
Author(s):  
JK Hovinga ◽  
J Schaller ◽  
H Stricker ◽  
WA Wuillemin ◽  
M Furlan ◽  
...  
Keyword(s):  
Amino Acid ◽  
Proteolytic Activity ◽  
Amino Acid Substitution ◽  
Cleavage Site ◽  
Dextran Sulfate ◽  
Coagulation Factor ◽  
Molecular Defect ◽  
Factor Xii ◽  
Amino Acid Sequence Analysis ◽  
Functional Defect

The dysfunctional coagulation factor XII (FXII) Locarno was purified from 2 L of the proposita's plasma. Studies to identify the molecular defect responsible for the lack of amidolytic and proteolytic activity of this FXII variant were performed. Amino acid sequence analysis of peptides obtained from FXII Locarno on activation with either trypsin or plasma kallikrein and dextran sulfate showed an amino acid substitution of Arg 353 by Pro. Thereby, the kallikrein cleavage site at Arg 353-Val 354 is lost. Although trypsin-activated FXII Locarno was fully cleaved at Arg 334-Asn 335 and at Arg 343-Leu 344, neither amidolytic nor proteolytic activity was generated. We conclude that proteolytic cleavage at Arg 343 in the absence of cleavage at Arg 353 is not sufficient to expose the enzymatic active site in FXII Locarno.

Functional characterization of an abnormal factor XII molecule (F XII Bern)

Blood ◽  
1991 ◽  
Vol 78 (4) ◽  
pp. 997-1004
Author(s):  
WA Wuillemin ◽  
I Huber ◽  
M Furlan ◽  
B Lammle
Keyword(s):  
Dextran Sulfate ◽  
Functional Characterization ◽  
Healthy Woman ◽  
Chain Molecule ◽  
Molecular Defect ◽  
Factor Xii ◽  
Single Chain ◽  
Functional Studies ◽  
Functional Defect

An 18-year-old healthy woman was found to have cross-reacting material (CRM)-positive factor XII (F XII) deficiency, F XII clotting activity was less than 0.01 U/mL, whereas F XII antigen was 0.11 U/mL. An F XII inhibitor was excluded. To partially characterize the molecular defect of the abnormal F XII, immunologic and functional studies were performed on the proposita's plasma. The abnormal F XII was a single chain molecule with the same molecular weight (80 Kd) and the same isoelectric points (pl, 5.9 to 6.8) as normal F XII. Dextran sulfate activation of the proposita's plasma showed no proteolytic cleavage of F XII even after 120 minutes, whereas F XII in pooled normal plasma, diluted 1:10 with CRM-negative F XII-deficient plasma, was completely cleaved after 40 minutes. Adsorption to kaolin was identical for both abnormal and normal F XII. In the presence of dextran sulfate and exogenous plasma kallikrein, the abnormal F XII was cleaved with the same rate as normal F XII. However, kallikrein-cleaved abnormal F XII was not able to cleave factor XI and plasma prekallikrein, in contrast to activated normal F XII. Thus, these studies show that the functional defect of this abnormal F XII, denoted as F XII Bern, is due to the lack of protease activity of the kallikrein-cleaved molecule. Therefore, the structural defect is likely to be located in the light chain region of F XII, containing the enzymatic active site.

scholarly journals Functional characterization of an abnormal factor XII molecule (F XII Bern)

Blood ◽  
1991 ◽  
Vol 78 (4) ◽  
pp. 997-1004 ◽  
Author(s):  
WA Wuillemin ◽  
I Huber ◽  
M Furlan ◽  
B Lammle
Keyword(s):  
Dextran Sulfate ◽  
Functional Characterization ◽  
Healthy Woman ◽  
Chain Molecule ◽  
Molecular Defect ◽  
Factor Xii ◽  
Single Chain ◽  
Functional Studies ◽  
Functional Defect

Abstract An 18-year-old healthy woman was found to have cross-reacting material (CRM)-positive factor XII (F XII) deficiency, F XII clotting activity was less than 0.01 U/mL, whereas F XII antigen was 0.11 U/mL. An F XII inhibitor was excluded. To partially characterize the molecular defect of the abnormal F XII, immunologic and functional studies were performed on the proposita's plasma. The abnormal F XII was a single chain molecule with the same molecular weight (80 Kd) and the same isoelectric points (pl, 5.9 to 6.8) as normal F XII. Dextran sulfate activation of the proposita's plasma showed no proteolytic cleavage of F XII even after 120 minutes, whereas F XII in pooled normal plasma, diluted 1:10 with CRM-negative F XII-deficient plasma, was completely cleaved after 40 minutes. Adsorption to kaolin was identical for both abnormal and normal F XII. In the presence of dextran sulfate and exogenous plasma kallikrein, the abnormal F XII was cleaved with the same rate as normal F XII. However, kallikrein-cleaved abnormal F XII was not able to cleave factor XI and plasma prekallikrein, in contrast to activated normal F XII. Thus, these studies show that the functional defect of this abnormal F XII, denoted as F XII Bern, is due to the lack of protease activity of the kallikrein-cleaved molecule. Therefore, the structural defect is likely to be located in the light chain region of F XII, containing the enzymatic active site.

scholarly journals Molecular defect in factor IXHilo, a hemophilia Bm variant: Arg----Gln at the carboxyterminal cleavage site of the activation peptide

Blood ◽  
1989 ◽  
Vol 73 (3) ◽  
pp. 718-721 ◽  
Author(s):  
MN Huang ◽  
CK Kasper ◽  
HR Roberts ◽  
DW Stafford ◽  
KA High
Keyword(s):  
Amino Acid ◽  
Point Mutation ◽  
Cleavage Site ◽  
Human Factor ◽  
Dna Amplification ◽  
Factor Ix ◽  
Molecular Defect ◽  
Human Factor Ix ◽  
Amplification Technique ◽  
Activation Peptide

Abstract A genomic DNA library and the enzymatic DNA amplification technique were used to isolate human factor IX coding sequences of a hemophilia Bm variant, factor IXHilo. A point mutation that resulted in the substitution of a glutamine (CAG) for an arginine (CGG) at amino acid 180 was found in exon VI of the factor IX gene (G----A at nucleotide 20519). This mutation alters the carboxy terminal cleavage site for the activation peptide at Arg180-Val181. The arginine residue at the activation peptide cleavage site is conserved in mouse, canine, bovine, and human factor IX, suggesting that the arginine at amino acid 180 is important for normal cleavage. Sequencing of all of the coding regions of factor IXHilo revealed no other mutations. We have also shown that the point mutation in exon VI creates a new Dde I restriction site, which, in combination with the enzymatic DNA amplification technique, provides a quick, reliable, and sensitive method for carrier detection and antenatal diagnosis in affected kindreds. This is the first report of the molecular defect in a hemophilia Bm patient with a markedly prolonged ox brain prothrombin time.

scholarly journals Crystal structures of the recombinant β-factor XIIa protease with bound Thr-Arg and Pro-Arg substrate mimetics

2019 ◽  
Vol 75 (6) ◽  
pp. 578-591 ◽  
Author(s):  
Monika Pathak ◽  
Rosa Manna ◽  
Chan Li ◽  
Bubacarr G. Kaira ◽  
Badraldin Kareem Hamad ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Conformational Changes ◽  
Cleavage Site ◽  
Coagulation Factor ◽  
Factor Xii ◽  
Chloromethyl Ketone ◽  
Factor Xiia ◽  
Contact Pathway ◽  
Inhibitor Recognition ◽  
Insight Into

Coagulation factor XII (FXII) is a key initiator of the contact pathway, which contributes to inflammatory pathways. FXII circulates as a zymogen, which when auto-activated forms factor XIIa (FXIIa). Here, the production of the recombinant FXIIa protease domain (βFXIIaHis) with yields of ∼1–2 mg per litre of insect-cell culture is reported. A second construct utilized an N-terminal maltose-binding protein (MBP) fusion (MBP-βFXIIaHis). Crystal structures were determined of MBP-βFXIIaHisin complex with the inhibitor D-Phe-Pro-Arg chloromethyl ketone (PPACK) and of βFXIIaHisin isolation. The βFXIIaHisstructure revealed that the S2 and S1 pockets were occupied by Thr and Arg residues, respectively, from an adjacent molecule in the crystal. The Thr-Arg sequence mimics the P2–P1 FXIIa cleavage-site residues present in the natural substrates prekallikrein and FXII, and Pro-Arg (from PPACK) mimics the factor XI cleavage site. A comparison of the βFXIIaHisstructure with the available crystal structure of the zymogen-like FXII protease revealed large conformational changes centred around the S1 pocket and an alternate conformation for the 99-loop, Tyr99 and the S2 pocket. Further comparison with activated protease structures of factors IXa and Xa, which also have the Tyr99 residue, reveals that a more open form of the S2 pocket only occurs in the presence of a substrate mimetic. The FXIIa inhibitors EcTI and infestin-4 have Pro-Arg and Phe-Arg P2–P1 sequences, respectively, and the interactions that these inhibitors make with βFXIIa are also described. These structural studies of βFXIIa provide insight into substrate and inhibitor recognition and establish a scaffold for the structure-guided drug design of novel antithrombotic and anti-inflammatory agents.

scholarly journals Mutations of factor VIII cleavage sites in hemophilia A

Blood ◽  
1988 ◽  
Vol 72 (3) ◽  
pp. 1022-1028 ◽  
Author(s):  
J Gitschier ◽  
S Kogan ◽  
B Levinson ◽  
EG Tuddenham
Keyword(s):  
Amino Acid ◽  
Factor Viii ◽  
Cleavage Site ◽  
Hemophilia A ◽  
Activated Protein C ◽  
Gene Mutations ◽  
Coagulation Factor ◽  
Cleavage Sites ◽  
Severe Hemophilia ◽  
Thrombin Activation

Abstract Hemophilia A is caused by a defect in coagulation factor VIII, a protein that undergoes extensive proteolysis during its activation and inactivation. To determine whether some cases of hemophilia are caused by mutations in important cleavage sites, we screened patient DNA samples for mutations in these sites by a two-step process. Regions of interest were amplified from genomic DNA by repeated rounds of primer- directed DNA synthesis. The amplified DNAs were then screened for mutations by discriminant hybridization using oligonucleotide probes. Two cleavage site mutations were found in a survey of 215 patients. A nonsense mutation in the activated protein C cleavage site at amino acid 336 was discovered in a patient with severe hemophilia. In another severely affected patient, a mis-sense mutation results in a substitution of cysteine for arginine in the thrombin activation site at amino acid 1689. This defect is associated with no detectable factor VIII activity, but with normal levels of factor VIII antigen. The severe hemophilia in this patient was sporadic; analysis of the mother suggested that the mutation originated in her gametes or during her embryogenesis. The results demonstrate that this approach can be used to identify factor VIII gene mutations in regions of the molecule known to be important for function.

STRUCTURE-FUNCTION RELATIONSHIPS IN ABNORMAL FIBRINOGEN WITH Bβ14 ARG→CYS SUBSTITUTION: FIBRINOGENS SEATTLE I AND CHRISTCHURCH II

1987 ◽  
Author(s):  
H Kaudewitz ◽  
A Henschen ◽  
H Pirkle ◽  
D Heaton ◽  
J Soria ◽  
...  
Keyword(s):  
Amino Acid ◽  
Structure Function ◽  
Amino Acid Substitution ◽  
Cleavage Site ◽  
Disulfide Bridge ◽  
Thrombin Cleavage ◽  
Parallel Arrangement ◽  
Fibrinogen Molecule

Genetically abnormal, dysfunctional fibrinogen variants may be used as unique models for studies of structure-function relationships both in vitro and in vivo. Out of the over kO so far structurally elucidated abnormal fibrinogens only 4 have an amino acid substitution in the Bg-chain. These variants are named Fibrinogen Pontoise, Nev York I, Christchurch II and Seattle I.Fibrinogens Seattle I and Christchurch II are slow-clotting fibrinogens which on thrombin-treatment release only half the normal amount of fibrinopeptide B and therefore were expected to contain an amino acid substitution close to the thrombin cleavage site in the Bβ-chain. In order to sequence the abnormal Bg-chains the fibrinogens were cleaved with thrombin and cyanogen bromide. The abnormal Bβ-chain components were isolated from the mercapto-lysed-pyridylethylated N-terminal disulfide knots. After pyroglu-tamyl-peptidase digestion the Bβ 14 Arg→Cys substitutions could be demonstrated for both variants by direct N-terminal sequence analysis. The form of the cyst(e)ine residue was determined by amino acid analysis of the alkylated native fibrinogen. As no alkylated cysteine was detected it was concluded that Bβ 14 Cys participates in a disulfide bridge.Fibrinogens Seattle I and Christchurch II are the first two elucidated fibrinogens with substitutions at the Bβ-chain thrombin cleavage site. Surprisingly, both the thrombin and Reptilase times are prolonged. It may be assumed that the half-cystine residues in position 14 of the two Bβ-chains within one fibrinogen molecule are disulfide-linked to each other, in an analogous way to that already established for the half-cystine residues in position 16 of the Aα-chains of several abnormal fibrinogens.This additional disulfide bridge might change the conformation and charge in the N-terminal region sufficiently to explain the prolonged clotting times. Furthermore, this bridge would provide the evidence for the parallel arrangement of the Bβ-chains at the fibrinogen N-terminus in a similar way as previously shown for the Aα-chains.

scholarly journals Abrogation of CTL Epitope Processing by Single Amino Acid Substitution Flanking the C-Terminal Proteasome Cleavage Site

2000 ◽  
Vol 164 (4) ◽  
pp. 1898-1905 ◽  
Author(s):  
Nico J. Beekman ◽  
Peter A. van Veelen ◽  
Thorbald van Hall ◽  
Anne Neisig ◽  
Alice Sijts ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Substitution ◽  
Cleavage Site ◽  
Single Amino Acid Substitution ◽  
Single Amino Acid ◽  
Ctl Epitope

scholarly journals Mutations of factor VIII cleavage sites in hemophilia A

Blood ◽  
1988 ◽  
Vol 72 (3) ◽  
pp. 1022-1028
Author(s):  
J Gitschier ◽  
S Kogan ◽  
B Levinson ◽  
EG Tuddenham
Keyword(s):  
Amino Acid ◽  
Factor Viii ◽  
Cleavage Site ◽  
Hemophilia A ◽  
Activated Protein C ◽  
Gene Mutations ◽  
Coagulation Factor ◽  
Cleavage Sites ◽  
Severe Hemophilia ◽  
Thrombin Activation

Hemophilia A is caused by a defect in coagulation factor VIII, a protein that undergoes extensive proteolysis during its activation and inactivation. To determine whether some cases of hemophilia are caused by mutations in important cleavage sites, we screened patient DNA samples for mutations in these sites by a two-step process. Regions of interest were amplified from genomic DNA by repeated rounds of primer- directed DNA synthesis. The amplified DNAs were then screened for mutations by discriminant hybridization using oligonucleotide probes. Two cleavage site mutations were found in a survey of 215 patients. A nonsense mutation in the activated protein C cleavage site at amino acid 336 was discovered in a patient with severe hemophilia. In another severely affected patient, a mis-sense mutation results in a substitution of cysteine for arginine in the thrombin activation site at amino acid 1689. This defect is associated with no detectable factor VIII activity, but with normal levels of factor VIII antigen. The severe hemophilia in this patient was sporadic; analysis of the mother suggested that the mutation originated in her gametes or during her embryogenesis. The results demonstrate that this approach can be used to identify factor VIII gene mutations in regions of the molecule known to be important for function.

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