Determination of the amino acid substitution in human prothrombin type 3 (157 Glu Lys) and the localization of a third thrombin cleavage site

1983 ◽  
Vol 54 (2) ◽  
pp. 245-254 ◽  
Author(s):  
P. G. Board ◽  
D. C. Shaw
Keyword(s):  
Amino Acid ◽  
Amino Acid Substitution ◽  
Cleavage Site ◽  
Thrombin Cleavage ◽  
Type 3

Determination of the amino acid substitution in prothrombin type 3

Pathology ◽  
1983 ◽  
Vol 15 (1) ◽  
pp. 101
Author(s):  
P.G. Board ◽  
D.C. Shaw
Keyword(s):  
Amino Acid ◽  
Amino Acid Substitution ◽  
Type 3

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.

FIBRINOGEN LONG BEACH

1987 ◽  
Author(s):  
H Pirkle ◽  
I Theodor ◽  
P Vukasin ◽  
B Nandi ◽  
D Miyada ◽  
...  
Keyword(s):  
Amino Acid ◽  
Gel Electrophoresis ◽  
Amino Acid Analysis ◽  
Cleavage Site ◽  
Fibrinopeptide A ◽  
Long Beach ◽  
Thrombin Cleavage ◽  
Complete Failure

We find that, in addition to a normal fibrinopeptide A (FPA), fibrinogen Long Beach releases an abnormal FPA whose HPLC behavior suggests and whose amino acid analysis confirms the substitution of His for Arg at position 16, the thrombin cleavage site. However, this dysfibrinogen displays properties strikingly different from those previously reported to be associated with Aa 16 Arg → His substitutions. These properties include a limited release of normal FPA (18-20%) by both thrombin and batroxobin, an essentially full release of abnormal FPA (40-50%) by thrombin (1.5 NIH u/ml) in contrast to a 7-8% release of abnormal FPA by batroxobin, and a complete failure of highly purified batroxobin to clot the dysfibrinogen while generating a fragment which migrates between the B8 and y chains on SDS gel electrophoresis. The basis for these differing properties is not yet clear.

scholarly journals Use of Engineered Thrombin Cleavage Site for Determination of Translational Reading Frames

BioTechniques ◽  
1996 ◽  
Vol 21 (5) ◽  
pp. 822-824 ◽  
Author(s):  
Gang Zeng ◽  
Timothy J. Larson
Keyword(s):  
Cleavage Site ◽  
Thrombin Cleavage ◽  
Reading Frames

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 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.

scholarly journals An arginine to cysteine amino acid substitution at a critical thrombin cleavage site in a dysfunctional factor VIII molecule

Blood ◽  
1989 ◽  
Vol 74 (5) ◽  
pp. 1612-1617 ◽  
Author(s):  
M Shima ◽  
J Ware ◽  
A Yoshioka ◽  
H Fukui ◽  
CA Fulcher
Keyword(s):  
Amino Acid ◽  
Factor Viii ◽  
Heavy Chain ◽  
Light Chain ◽  
Cleavage Site ◽  
Cleavage Sites ◽  
Amino Acid Residues ◽  
Thrombin Cleavage ◽  
Coagulant Activity ◽  
Polymerase Chain

We have analyzed the factor VIII (FVIII) protein and the nucleotide sequence around two thrombin cleavage sites, at arginine 372 in the FVIII heavy chain and arginine 1689 in the FVIII light chain in a naturally occurring dysfunctional FVIII variant, FVIII Okayama. The patient was a 42-year-old hemophiliac with a FVIII coagulant activity of 0.03 U/mL and a FVIII antigen level of 0.8 U/mL. The patient's FVIII was not thrombin activatable to levels seen in normal plasma. Immunoblotting of partially purified FVIII Okayama and normal FVIII showed that thrombin cleavage of the 92 kilodalton (Kd) heavy chain was impaired in the mutant protein. The patient's genomic DNA was amplified using the polymerase chain reaction with two sets of synthetic oligonucleotide primers spanning amino acid residues 319 to 400 and 1630 to 1720. Sequence analysis of the amplified DNA fragments revealed a cytosine to thymine transition, converting an arginine to a cysteine codon at residue 372. No abnormality was found in the FVIII light chain region analyzed. The patient's hemophilic brother and carrier mother revealed the same mutation. We conclude that the pathogenesis of hemophilia A in this patient is probably due to an arginine to cysteine substitution at a thrombin cleavage site in the FVIII heavy chain.

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