scholarly journals Quantitative N-terminal analysis of fibrinogen-fibrin-related antigen [FR antigen] from human plasma

1979 ◽  
Vol 183 (3) ◽  
pp. 623-632 ◽  
Author(s):  
J U Harris ◽  
A J Johnson ◽  
C Merskey ◽  
M T Wang ◽  
D Robinson
Keyword(s):  
Human Plasma ◽  
Disseminated Intravascular Coagulation ◽  
Normal Subjects ◽  
Proteolytic Degradation ◽  
Fibrinopeptide A ◽  
Human Fibrinogen ◽  
Intravascular Coagulation ◽  
Thrombin Cleavage ◽  
Terminal Amino

Fibrinogen-fibrin-related antigen (FR antigen) was isolated from as little as 1 ml of human plasma by immuno-affinity chromatography with agarose-bound antibody to human fibrinogen. N-terminal analysis was performed to determine the nature and extent of proteolytic degradation of the FR antigen in patients with disseminated intravascular coagulation and in normal subjects. Thrombin cleavage of the A- and B-peptides from fibrinogen in vitro was monitored by the appearance of N-terminal glycine, and an increase in glycine was shown in the FR antigen of patients with disseminated intravascular coagulation. As plasmin progressively degraded fibrinogen, increases in N-terminal alanine, aspartic acid and lysine were observed, corresponding to the known plasmin-cleavage points of fibrinogen; increases in these N-terminal amino acids were also found in the patients' FR antigen. Thrombin treatment in vitro was used to remove fibrinopeptide A (N-terminal alanine) from the samples and to reflect specifically the N-terminal alanine at the plasmin-cleavage point (Arg-42-Ala-43) of the B beta-chain on assay; this alanine was increased progressively in the FR antigen of a patient during urokinase therapy, and was high in other patients when the FR antigen was examined by this procedure.

Fibrinopeptide A in Urine from Patients with Venous Thromboembolism, Disseminated Intravascular Coagulation and Rheumatoid Arthritis - Evidence for Dephosphorylation and Carboxyterminal Degradation of the Peptide by the Kidney

1985 ◽  
Vol 54 (04) ◽  
pp. 792-798 ◽  
Author(s):  
O C Leeksma ◽  
F Meijer-Huizinga ◽  
E A Stoepman-van Dalen ◽  
W G van Aken ◽  
J A van Mourik
Keyword(s):  
Rheumatoid Arthritis ◽  
Venous Thromboembolism ◽  
Disseminated Intravascular Coagulation ◽  
High Performance ◽  
Active Role ◽  
Study Data ◽  
Fibrinopeptide A ◽  
Intravascular Coagulation

SummaryUrinary fibrinopeptide A immunoreactivity was determined by radioimmunoassay using two anti-fibrinopeptide A sera with a different specificity in patients with venous thromboembolism, disseminated intravascular coagulation and rheumatoid arthritis. Elevated levels were frequently observed with both sera, and intravenous administration of heparin in patients with a thromboembolic disorder resulted in a decline of urinary fibrinopeptide A (FPA) concentrations to normal or nearly normal values. For both sera significant correlations with plasma levels were found although one of the sera reacted significantly better with the material in urine samples from these patients than the other (p <0.0001, n = 73). Analysis of urinary fibrinopeptide A immunoreactivity by high performance liquid chromatography (HPLC) provided evidence that A peptide material present in this body fluid was heterogeneous. In view of the characteristics of the antisera used in this study, data suggest that urinary FPA immunoreactivity consists to a large extent of carboxyterminally degraded FPA. Excretion of circulating FPA immunoreactive material through the kidneys apparently involves dephosphorylation and carboxyterminal breakdown of the A peptide. Since both synthetic and native phosphorylated or unphosphorylated fibrinopeptide A appeared to be stable in urine in vitro, an active role of the kidney in degrading the A peptide is likely.

DIRECTED MUTAGENESIS OF HUMAN FIBRINOGEN: Aα CHAIN SUBSTITUTIONS THAT ALTER THROMBIN CLEAVAGE AND ANTIBODY RECOGNITION

1987 ◽  
Author(s):  
S T Lord
Keyword(s):  
Amino Acids ◽  
Blot Analysis ◽  
Fibrin Clot ◽  
Fibrinopeptide A ◽  
Human Fibrinogen ◽  
Antibody Recognition ◽  
Amino Terminal ◽  
Thrombin Cleavage ◽  
Initial Event ◽  
Bacterial Lysates

The initial event in fibrin clot formation is the thrombin catalized cleavage of the Aa chain of fibrinogen between Argl6 and Glyl7, releasing fibrinopeptide A. Previous data indicate that most of the information required for thrombin recognition and cleavage of the Aa chain lies within the amino terminal 51 residue CNBr fragment. In order to use protein engineering techniques to study the interaction of thrombin with the Aa chain, we have constructed a plasmid expression vector which encodes a tripartite protein consisting of amino acids 1-50 of the Aa chain of human fibrinogen followed by 60 amino acids of chicken collagen, and the beta-galactosidase protein from Escherichia coli. The codons for an initiator methionine and amino acids 1-50 were assembled from 7 oligonucleotides. Protein blot analysis of bacterial lysates of cells induced to synthesize this tribrid protein show a single band (MW = 125,000) crossreactive with a monoclonal antibody, Y-18, which recognizes the Aa chain of fibrinogen but not the products of thrombin cleavage. When these lysates are incubated with thrombin, fibrinopeptide A is released as demonstrated both by protein blot analysis and radioimmunoassay. By including one heterogeneous oligonucleotide in the assembly process, we have constructed plasmids which encode specific amino acid substitutions within residues 1-23. One of these substitutions, Glyl4 to val, significantly alters both cleavage by thrombin and recognition by Y-18. Substitution of ilu for Arg23 alters neither thrombin cleavage nor monoclonal recognition while substitution of leu for Argl6 alters thrombin cleavage, but not recognition by Y-18.

Disseminated Intravascular Coagulation and Acute Renal Failure

1971 ◽  
Vol 26 (02) ◽  
pp. 332-340 ◽  
Author(s):  
I Crîsnic ◽  
M Cucuianu ◽  
M Manasia ◽  
G Uza
Keyword(s):  
Renal Failure ◽  
Acute Renal Failure ◽  
Disseminated Intravascular Coagulation ◽  
Early Stage ◽  
Platelet Adhesiveness ◽  
Intravascular Coagulation ◽  
Clinical Observations ◽  
Lysis Time ◽  
Euglobulin Lysis Time

SummaryStarting from a hypothesis according to which disseminated intravascular coagulation might be an intermediary mechanism in the production of acute renal failure, investigations were made in 94 cases of anuria of different etiology, in order to detect signs of a consumption coagulopathy. After an average lapse of time of 48 h since the onset of anuria, the most frequently encountered hemostatic defect was a decreased platelet adhesiveness. In vitro experiments and clinical observations suggest that in the early stage of acute renal failure caused by a septic abortion, deficient platelet adhesiveness is due, mainly to platelet damage caused by intravascular coagulation or by bacterial toxins and not by the retention of metabolites. Euglobulin lysis time was prolonged, but a significant decrease of the plasminogen level indicates that an activation of fibrinolysis might have occured in the evolution of the process.

scholarly journals Comparison of 125I-fibrinogen kinetics and fibrinopeptide A in patients with disseminated neoplasias

Blood ◽  
1982 ◽  
Vol 60 (2) ◽  
pp. 381-388 ◽  
Author(s):  
G Mombelli ◽  
A Roux ◽  
A Haeberli ◽  
PW Straub
Keyword(s):  
Disseminated Intravascular Coagulation ◽  
Fibrinogen Level ◽  
Normal Values ◽  
Normal Subjects ◽  
The Other ◽  
Fibrinopeptide A ◽  
Intravenous Heparin ◽  
Catabolic Rate ◽  
Minor Part ◽  
Heparin Dosage

Abstract To provide more information on the pathways of fibrinogen catabolism in generalized cancer, the effect of heparin on fibrinopeptide A (fpA) and on 125I-fibrinogen kinetics was studied in 15 patients with disseminated neoplasia. Three patients had evidence of venous thrombosis and in 2 additional patients a low fibrinogen level together with increased amounts of FDP/fdp and a positive ethanol test indicated disseminated intravascular coagulation (DIC). The plasma levels of fpA were grossly elevated (4.6--20, mean 11.4 ng/ml, normal values 1.01 +/- 0.45 ng/ml) in patients with thrombosis or DIC, and normal to grossly elevated (0.4--10.4, mean 6.1 ng/ml) in the other patients. Intravenous heparin bolus lowered the fpA level in 11/11 patients, and continuous heparin treatment led to an impressive suppression or complete normalization of the plasma fpA in 5/6 patients. This finding is thought to reflect heparin suppression of thrombin activity on fibrinogen. In some cases, the fpA fall after heparin bolus was slow and/or incomplete, suggesting fpA generation at sites not easily accessible to heparin or insufficient heparin dosage. The 125I- fibrinogen kinetics were characterized by a significantly shorter half- life (t1/2: 2.5 days), increased catabolic rate constant (j: 0.44 days- 1), and increased absolute turnover (68.9 mg fibrinogen/kg/day) as compared to 4 normal subjects (t1/2: 4.2 days; j: 0.26 days-1; turnover 21.7 mg fibrinogen/kg/day). As estimated from the fpA generation rates, intravascular thrombin action on fibrinogen contributed only in minor part to increase the turnover of 125I-fibrinogen. In particular, the turnover was greatly accelerated in heparin-treated patients despite impressive suppression or normalization of the fpA levels in 5/6 cases.

scholarly journals Disseminated intravascular coagulation in rabbits induced by administration of endotoxin or tissue factor: effect of anti-tissue factor antibodies and measurement of plasma extrinsic pathway inhibitor activity

Blood ◽  
1990 ◽  
Vol 75 (7) ◽  
pp. 1481-1489 ◽  
Author(s):  
TA Warr ◽  
LV Rao ◽  
SI Rapaport
Keyword(s):  
Tissue Factor ◽  
Disseminated Intravascular Coagulation ◽  
Factor Viia ◽  
Factor Vii ◽  
Rabbit Brain ◽  
Extrinsic Pathway ◽  
Intravascular Coagulation ◽  
Activated Factor Vii ◽  
Factor Effect

Abstract Rabbits were given polyclonal anti-tissue factor (TF) immunoglobulin G (IgG) before an injection of endotoxin to test the hypothesis that TF triggers disseminated intravascular coagulation (DIC) after endotoxin. The rabbits had been prepared with cortisone to develop DIC after one injection of endotoxin. Anti-TF IgG substantially reduced the falls in fibrinogen, factors V and VIII, and platelets noted in control rabbits given preimmune IgG before endotoxin. At autopsy 24 hours later, fibrin was present in glomerular capillaries of 4 of 5 control rabbits, but in none of 11 rabbits given anti-TF IgG. DIC was also induced in a second group of rabbits by the infusion, over 4 hours, of 1 microgram/kg of purified, reconstituted rabbit brain TF. This resulted in striking falls in plasma fibrinogen, factors V, and VIII that were diminished, but not prevented by prior treatment with anti-TF IgG. Circulating activated factor VII, induced by either TF infusion or endotoxin, could not be detected after DIC. Mean plasma extrinsic pathway inhibitor (EPI) activity did not fall significantly after endotoxin, and only to about 65% of the preinfusion after infusion of TF. Thus, DIC induced by both agents proceeded despite nearly normal plasma EPI levels. Because EPI neutralizes factor VIIa/TF in vitro only after a short lag period, the DIC that persisted for up to 6 hours after injection of endotoxin suggests that TF activity continued to be generated during this period on cells to which the circulating blood was exposed. All animals given endotoxin became ill with cyanosis, tachypnea, cold ears, and diarrhea, regardless of whether they had received anti-TF IgG to attenuate DIC. Infusion of TF caused some animals to die acutely with pulmonary arterial thromboses, but surviving animals did not appear ill. The findings support the hypothesis that exposure of blood to TF triggers DIC after endotoxin, but is not important for the pathogenesis of endotoxin-induced shock.

Proteolytic Degradation of the Aα Chain of Human Fibrinogen

1979 ◽  
Author(s):  
J.A. Conkie ◽  
Isobel D. Walker ◽  
J.F. Davidson
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Amino Acid Composition ◽  
Proteolytic Degradation ◽  
Composition Analysis ◽  
Terminal Part ◽  
High Solubility ◽  
Human Fibrinogen

On plasmin degradation of human fibrinogen, a number of polypeptides are released from the COOH-terminal part of the Aα chain. One of these fragments has been previously named by us as Aα-RA(26,000). By comparison of its molecular weight and amino acid composition analysis, this fragment is similar to the fragments A,H and Hi2-Met. Aα-RA(26,00O) appears to be derived from a precursor polypeptide of Mw 44,000, while in vitro and in vivo activation of the plasma fibrinolytic system also gives rise to an Aα-related antigen which is immunologically similar to the 44,000 MW polypeptide. On immunodiffusion with antiserum to the carboxymethylated Aα chain, Aα-RA(26 000) revealed a reaction of identity with the high solubility fibrinogen fraction 1-9 (major NH2-terminal Aα remnant, MW 34,000) and a reaction of non-identity with the ancrod-proauced COOH-terminal Aα polypeptides (MW 27,000-31,000). These immunodiffusion results provide evidence that the sequence of bond cleavages in the Aα chain leading to the formaron of fibrinogen 1-9 is different from that leading to the formation of Aα-Ra (126,000).

scholarly journals Degradation by cultured fibroblasts and macrophages of unmodified and 1,2-cyclohexanedione-modified low-density lipoprotein from normal and homozygous familial hypercholesterolaemic subjects

1982 ◽  
Vol 202 (1) ◽  
pp. 145-152 ◽  
Author(s):  
B L Knight ◽  
A K Soutar
Keyword(s):  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Normal Subjects ◽  
Proteolytic Degradation ◽  
Low Density ◽  
Human Skin Fibroblasts ◽  
Cultured Fibroblasts ◽  
Apparent Affinity ◽  
Monolayer Cultures

Monolayer cultures of human skin fibroblasts and monocyte-derived macrophages were used to examine the effect of cyclohexane-1,2-dione modification on the proteolytic degradation of 125I-labelled low-density lipoprotein (LDL) from normal subjects (NLDL) and homozygous familial hypercholesterolaemic subjects (FHLDL). Normal fibroblasts, pre-incubated in lipoprotein-deficient serum, and macrophages, pre-incubated in whole serum, exhibited both saturable and non-saturable degradation of LDL. In fibroblasts, the saturable receptor-mediated degradation of FHLDL was similar to that of NLDL and was abolished if the lipoproteins were modified with cyclohexanedione. The rate of non-saturable degradation of FHLDL was at least 3-fold higher than that of NLDL and each was decreased by approx. 60% after modification. In macrophages, saturable degradation was decreased but not abolished by modification. The apparent affinity for unmodified LDL was lower than that of the fibroblast receptor and was greater for NLDL than for FHLDL. Non-saturable degradation of FHLDL by macrophages was only slightly higher than that of NLDL. Modification with cyclohexanedione decreased the rate of non-saturable degradation of NLDL by 30%, but increased that of FHLDL by 75%. These experiments show differences between the degradation of 125I-labelled NLDL and FHLDL. They suggest that macrophages can degrade LDL by a saturable process with different properties from that mediated by the fibroblast receptor and that, in vitro, the rate of degradation of the modified LDL is not the same as the rate of non-receptor-mediated degradation of unmodified LDL.

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