Identification of D-Dimer-E Complex in Disseminated Intravascular Coagulation (DIC)

1979 ◽  
Author(s):  
A.N. Whitaker ◽  
E.A. Rowe ◽  
P.P. Masci ◽  
P.J. Gaffney
Keyword(s):  
Gel Electrophoresis ◽  
Degradation Products ◽  
Polyacrylamide Gel Electrophoresis ◽  
Stable Complex ◽  
Fibrin Degradation Products ◽  
Pneumococcal Sepsis ◽  
D Antigen ◽  
D Dimer

D-dimer (D2), a product of the plasmin lysis of cross-linked (XL) fibrin, but not of non-XL fibrin or fibrinogen, has been identified in the plasma of patients with DIC due to amniotic fluid embolism. In vitro, D is involved with fragment E as a stable complex (D2-E) but D2 -E has not been identified in vivo before. Fibrin degradation products (FDP) were studied in a patient having fulminant postsplenectomy pneumococcal sepsis and DIC, by immunoprecipitation with anti-fibrinogen (f) and anti-fragment E and characterization by SDS polyacrylamide gel electrophoresis (PAGE). With both antisera soluble HMW fibrin complexes, D2 and E but no X, Y or D were obtained from serum. D2 and E were identified in the supernatant after removing partially XL HMW complexes and fibrinogen from plasma with 2.5 M β-alanine. The presence D antigen in the D2-E complex precipitated by monospecific anti-E was confirmed by crossed Ag-Ab electrophoresis. Crossed Ag-Ab electrophoresis of serum in agarose gave E peaks of slow mobility and no fast-moving free E was found. Thus, D2-E complex exists in vivo and its easy identification, proving the lysis of XL fibrin, would be of value in studying thrombosis. D2-E complex has been identified in other patients with sepsis but at lower concentrations than described above.

The Fibrin Assay Comparison Trial (FACT)

2001 ◽  
Vol 85 (04) ◽  
pp. 671-678 ◽  
Author(s):  
Sybille Zips ◽  
Hanimsah Ergül ◽  
Dieter Heene ◽  
Carl-Erik Dempfle ◽  
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Degradation Products ◽  
Plasma Samples ◽  
Fibrin Degradation Products ◽  
Comparison Trial ◽  
Clinical Conditions ◽  
D Dimer

SummaryAlthough D-dimer has gained widespread clinical use as a parameter for detection of in vivo fibrin formation, the issue of standardization of D-dimer assays remains to be resolved. The FACT study was performed to generate basic data for development of calibrators and standard preparations.A set of 86 samples, including plasma samples from patients with DIC, DVT, and other clinical conditions, serial dilutions of pooled plasma samples, and plasma samples containing fibrinogen- and fibrin derivatives, were distributed to 12 manufacturers of D-dimer assays.D-dimer assays differ concerning specificity for crosslinked fibrin, and preference for either high molecular weight fibrin complexes, or low molecular weight fibrin degradation products. Terminal plasmin digests of fibrin clots for calibration produce aberrant results in some assays, especially those with preference for high molecular weight crosslinked fibrin derivatives. The best conformity is achieved by the use of pooled plasma samples from patients with high levels of D-dimer antigen in plasma. In vitro preparations containing a comparable composition of fibrin derivatives to clinical plasma samples may also serve as reference material.

Soluble High-Molecular-Weight E Fragments in the Plasmin-Induced Degradation Products of Cross-Linked Human Fibrin

1975 ◽  
Vol 49 (2) ◽  
pp. 149-156 ◽  
Author(s):  
P. J. Gaffney ◽  
D. A. Lane ◽  
M. Brasher
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Gel Electrophoresis ◽  
Factor Xiii ◽  
Degradation Products ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cross Linking ◽  
D Dimer

1. The factor XIII-mediated cross-linked α chains in fibrin have no effect on the nature of the fragments released during the solubilization of fibrin by plasmin. 2. Besides the known D dimer and E fragments solubilized during the lysis of cross-linked fibrin, other fragments have been observed on sodium dodecyl sulphate-polyacrylamide gel electrophoresis which have a molecular weight of about 135 000. After prolonged plasmin digestion, these fragments (U fragments) were no longer evident on the gels and the high-molecular-weight E antigen was absent. It is assumed that the E antigen was associated with the U fragments. These fragments also cross-reacted with an anti-D serum. 3. The U fragments have been tentatively presumed to be a factor XIII-mediated cross-linked D–E complex since they degrade only after prolonged degradation with plasmin. Whereas it is known that the fibrin D dimer fragment contains the cross-linked γ chain residues of the originating fibrin, the presumed covalent cross-linking of the D–E fragments has not been proved. 4. The presence of these high-molecular-weight fragments, containing the E antigen, in cross-linked human fibrin digests should be taken into account in the development of D dimer assays to monitor fibrin lysis in vivo.

scholarly journals Plasma crosslinked fibrin polymers: quantitation based on tissue plasminogen activator conversion to D-dimer and measurement in normal and patients with acute thrombotic disorders

Blood ◽  
1992 ◽  
Vol 80 (3) ◽  
pp. 709-717 ◽  
Author(s):  
A Kornberg ◽  
CW Francis ◽  
VJ Marder
Keyword(s):  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Western Blotting ◽  
Gel Electrophoresis ◽  
Degradation Products ◽  
Thrombotic Disease ◽  
Tissue Plasminogen ◽  
D Dimer

Abstract Plasma crosslinked fibrin polymers (XLFP) are formed as a result of in vivo hemostatic activation and are elevated in thrombotic disease. We have investigated the plasmic degradation of plasma XLFP in vitro to provide information regarding the pattern of crosslinking and the composition of degradation products. Plasma XLFP were identified by sodium dodecyl sulfate (SDS)-agarose electrophoresis and Western blotting and quantitated by gel scanning. D-dimer was measured by enzyme-linked immunosorbent assay and the results were verified by SDS- polyacrylamide gel electrophoresis and Western blotting of the digests. Complete degradation of XLFP occurred only after supplementation of plasma with plasminogen (5 U/mL) and incubation with recombinant tissue plasminogen activator (rt-PA), indicating that the normal plasma plasminogen concentration limits plasmic degradation in vitro. Gel electrophoresis showed that the principal terminal degradation products of XLDP were fragments D, DD, and E, indicating that crosslinking occurred primarily through gamma chain dimers. After adding a low concentration of thrombin to plasma in vitro, XLFP increased progressively before clotting, and the concentration correlated with the increase in the D-dimer concentration after degradation (r = .98). Plasma XLFP and D-dimer concentrations in plasmic digests were significantly elevated in patients with stroke (150 +/- 83 micrograms/mL and 88 +/- 32 micrograms/mL), myocardial infarction (217 +/- 110 micrograms/mL and 84 +/- 30 micrograms/mL), and venous thrombosis (187 +/- 80 micrograms/mL and 86 +/- 19 micrograms/mL) compared with normals (28 +/- 12 micrograms/mL and 25 +/- 7 micrograms/mL). There was a strong correlation between the plasma concentration of XLFP and the D-dimer immunoreactivity of plasma after plasmic degradation (r = .87). The results indicate that XLFP in plasma are crosslinked primarily through gamma chains and degrade to fragment DD with plasminogen activation. Also, the immunoreactivity of in vitro plasmic digests of plasma reflects the concentration of XLFP and may provide a useful indirect measure of in vivo hemostatic activation in patients with thrombotic disease.

scholarly journals Plasma crosslinked fibrin polymers: quantitation based on tissue plasminogen activator conversion to D-dimer and measurement in normal and patients with acute thrombotic disorders

Blood ◽  
1992 ◽  
Vol 80 (3) ◽  
pp. 709-717
Author(s):  
A Kornberg ◽  
CW Francis ◽  
VJ Marder
Keyword(s):  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Western Blotting ◽  
Gel Electrophoresis ◽  
Degradation Products ◽  
Thrombotic Disease ◽  
Tissue Plasminogen ◽  
D Dimer

Plasma crosslinked fibrin polymers (XLFP) are formed as a result of in vivo hemostatic activation and are elevated in thrombotic disease. We have investigated the plasmic degradation of plasma XLFP in vitro to provide information regarding the pattern of crosslinking and the composition of degradation products. Plasma XLFP were identified by sodium dodecyl sulfate (SDS)-agarose electrophoresis and Western blotting and quantitated by gel scanning. D-dimer was measured by enzyme-linked immunosorbent assay and the results were verified by SDS- polyacrylamide gel electrophoresis and Western blotting of the digests. Complete degradation of XLFP occurred only after supplementation of plasma with plasminogen (5 U/mL) and incubation with recombinant tissue plasminogen activator (rt-PA), indicating that the normal plasma plasminogen concentration limits plasmic degradation in vitro. Gel electrophoresis showed that the principal terminal degradation products of XLDP were fragments D, DD, and E, indicating that crosslinking occurred primarily through gamma chain dimers. After adding a low concentration of thrombin to plasma in vitro, XLFP increased progressively before clotting, and the concentration correlated with the increase in the D-dimer concentration after degradation (r = .98). Plasma XLFP and D-dimer concentrations in plasmic digests were significantly elevated in patients with stroke (150 +/- 83 micrograms/mL and 88 +/- 32 micrograms/mL), myocardial infarction (217 +/- 110 micrograms/mL and 84 +/- 30 micrograms/mL), and venous thrombosis (187 +/- 80 micrograms/mL and 86 +/- 19 micrograms/mL) compared with normals (28 +/- 12 micrograms/mL and 25 +/- 7 micrograms/mL). There was a strong correlation between the plasma concentration of XLFP and the D-dimer immunoreactivity of plasma after plasmic degradation (r = .87). The results indicate that XLFP in plasma are crosslinked primarily through gamma chains and degrade to fragment DD with plasminogen activation. Also, the immunoreactivity of in vitro plasmic digests of plasma reflects the concentration of XLFP and may provide a useful indirect measure of in vivo hemostatic activation in patients with thrombotic disease.

In-vivo and in-vitro secretion of prolactin by lactating rat adenohypophyses as a function of intracellular age

1984 ◽  
Vol 101 (1) ◽  
pp. 27-32 ◽  
Author(s):  
F. Mena ◽  
G. Martínez-Escalera ◽  
C. Clapp ◽  
C. E. Grosvenor
Keyword(s):  
Pituitary Gland ◽  
Incubation Period ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Pituitary Glands ◽  
H 26

ABSTRACT Adenohypophysial prolactin of lactating rats was pulse-labelled by [3H]leucine injected i.v. at the time of removal of the pups. The [3H]prolactin concentration in the pituitary gland, analysed by polyacrylamide-gel electrophoresis, progressively fell as the time from labelling to removal of the pituitary gland increased from 8 to 24 h, which suggests that there was a loss of hormone as it aged within the gland. Suckling effectively provoked the depletion–transformation of total and [3H]prolactin (extracted at pH 7·2) when applied after 8 h but not when applied after either 16 or 24 h after removing the pups. In rats whose pups were removed for 8 h, suckling also depleted–transformed [3H]prolactin labelled 4 h, but not that labelled 1 h before suckling. The pituitary glands of other lactating rats were labelled with [3H]leucine injected i.v. at various times before removing the glands and incubating them in medium 199. The secretion into the medium of [3H]prolactin labelled either 4, 8, 16 or 24 h beforehand was maximal during the first 30 min then declined from 30 to 240 min of incubation. However, secretion of prolactin labelled 1 h and 10 min beforehand reached a maximum after 0·5–1 h and 2 h of incubation respectively, then remained constant during the remainder of the 4-h incubation period. The total 4-h secretion of [3H]prolactin was greatest (65% of preincubation concentration) from those glands labelled 4 h before in contrast to those labelled 10 min (15%) or 1 (38%), 8 (34%), 16 (18%) or 24 h (26%) before incubation. Taken together, these data suggest that prolactin synthesized 4 h earlier is more likely to be released in response to physiological stimuli than is more recently formed prolactin or prolactin which has remained in the pituitary gland for 16 h or longer. J. Endocr. (1984) 101, 27–32

Studies of the Metabolism of Asialotransferrins: Evidence that Transferrin Does not Undergo Desialylation in Vivo

1974 ◽  
Vol 46 (6) ◽  
pp. 763-774
Author(s):  
K.-L. Wong ◽  
P. A. Charlwood ◽  
M. W. C. Hatton ◽  
E. Regoeczi
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Biological Significance ◽  
Polyacrylamide Gel ◽  
Fractional Catabolic Rate ◽  
Sialic Acid Content ◽  
Catabolic Rate ◽  
Bovine Transferrin

1. Experiments are reported which aimed at determining whether transferrin loses sialyl residues from the carbohydrate side-chains during the biological lifetime of the molecule. To explore this possibility, transferrin fractions of relatively high sialic acid content (referred to as sialotransferrin) were prepared from purified rabbit and bovine transferrin by preparative polyacrylamide-gel electrophoresis. After labelling with 125I, the preparations were injected into a group of three rabbits each. From the plasma samples obtained between 1 h and 6–8 days after injection, transferrin was partially purified, mixed with 131I-labelled asialotransferrin of the corresponding species and run in preparative polyacrylamide-gel electrophoresis. In each specimen examined, the 125I radioactivity migrated ahead of the marker asialotransferrin, and no portion of the dose was detected with the electrophoretic mobility of asialotransferrin. 2. Evidence is presented that bovine transferrin desialylated in vitro remains detectable in the plasma of rabbits for intervals which are comparable with those found in previous studies with rabbit asialotransferrin. 3. A mathematical model is described for the computation of asialo- to sialotransferrin radioactivity ratios in the plasma, continuous desialylation of pulse-injected sialotransferrin being assumed. Calculations were made at various hypothetical rates of desialylation. 4. On the basis of the experimental data and the model it is concluded that transferrin (both rabbit and bovine) is not subjected to systematic desialylation in rabbits. Random desialylation of some transferrin could take place at rates less than 5% of the fractional catabolic rate of transferrin, which would be without any biological significance.

EFFECTS OF GONADOTROPHINS AND CYCLIC 3′,5′-AMP ON IN VITRO INCORPORATION OF [3H]URIDINE INTO RNA OF THE PREPUBERTAL RAT OVARY

1973 ◽  
Vol 72 (4) ◽  
pp. 771-785 ◽  
Author(s):  
Jan Jarlstedt ◽  
Lars Nilsson ◽  
Lars Hamberger ◽  
Kurt Ahrén
Keyword(s):  
Cyclic Nucleotide ◽  
Gel Electrophoresis ◽  
Incubation Medium ◽  
Polyacrylamide Gel Electrophoresis ◽  
Labelling Pattern ◽  
Total Rna ◽  
Rat Ovary ◽  
Prepubertal Rats

ABSTRACT In vivo and in vitro effects of FSH and LH on in vitro incorporation of [3H]uridine into RNA of the prepubertal rat ovary have been studied. RNA was fractionated with composite agarose-polyacrylamide gel electrophoresis. When FSH was injected into the prepubertal rats 4 h before incubation of the ovaries, the incorporation of labelled uridine into total RNA was decreased showing relatively more radioactivity concentrated to the RNA fractions lighter than 28S as compared to the controls. These effects were not seen when FSH was added to the incubation medium in vitro. When LH was added in vitro to the isolated ovaries a higher percentage of incorporated radioactivity was concentrated in the RNA fractions heavier than 28S without any change in the incorporation of [3H]uridine into total RNA. LH administered in vivo 30 min before incubation of the ovaries gave the same change in the labelling pattern between the RNA fractions as LH in vitro but in addition showed a decreased incorporation of radioactivity into total RNA. The in vitro effects of cyclic 3′,5′-AMP were also studied. When prepubertal rat ovaries were incubated in 10 mmol/l of this cyclic nucleotide, the incorporation of [3H] uridine into total RNA was decreased without any change in the labelling pattern.

scholarly journals Role of plasmin in the degradation of the stroma-derived fibrin in human ovarian carcinoma

Blood ◽  
1990 ◽  
Vol 75 (8) ◽  
pp. 1673-1678 ◽  
Author(s):  
O Wilhelm ◽  
R Hafter ◽  
A Henschen ◽  
M Schmitt ◽  
H Graeff
Keyword(s):  
Ovarian Cancer ◽  
High Performance ◽  
Degradation Products ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Fibrin Degradation Products ◽  
Sds Page ◽  
Human Ovarian Carcinoma

Abstract The aim of this study was to evaluate the type of enzymes involved in tumor-associated fibrinolysis of the stroma component fibrin in ovarian cancer patients. For this purpose, the high-molecular-mass fibrin degradation products (HMM-XDP) were isolated from malignant ascitic fluid by protamine sulfate precipitation and further purified by gel filtration and acid precipitation. After reduction with 2- mercaptoethanol, the peptide chain components were separated by reverse- phase high-performance liquid chromatography (RP-HPLC). The nature of these components was elucidated by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) and N-terminal amino acid sequence analysis and compared with fibrin-derived fragments formed in vitro. The results indicate that plasmin is the essential protease involved in the degradation of the stroma-derived fibrin portion found in ovarian cancer ascites.

scholarly journals Gel-electrophoretic identification of hen brain neurotoxic esterase, labelled with tritiated di-isopropyl phosphorofluoridate

1981 ◽  
Vol 199 (2) ◽  
pp. 323-333 ◽  
Author(s):  
D G Williams ◽  
M K Johnson
Keyword(s):  
Active Site ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
The Other ◽  
Particulate Fraction ◽  
Molecular Weights ◽  
The Brain ◽  
Neurotoxic Esterase

The particulate fraction from hen brain was labelled with [3H]di-isopropyl phosphorofluoridate (DiPF) and separated by polyacrylamide-gel electrophoresis. Four radioactive protein bands (1--4) of molecular weights 155000, 92000, 60000, and 30000 were resolved. Most of the labelling of bands 2, 3 and 4 was inhibited by preincubation with Paraoxon. The residue in band 4 was sensitive to pH 5.2. Successive treatments with Paraoxon and pH 5.2 resulted in the abolition of bands 3 and 4. Bands 1 and 2 contained one and two polypeptides respectively, whose labelling was sensitive to Mipafox, but one, in band 2, was sensitive to higher concentrations of Paraoxon. The concentrations of the other two polypeptides were 6.7 and 1.95 pmol of DiPF bound/g of brain in bands 1 and 2 respectively. Both were as sensitive to Mipafox as neurotoxic esterase and were also sensitive to phenyl benzylcarbamate. 4-Nitrophenyl di-n-pentylphosphinate given in vivo inhibited neurotoxic esterase and the labelling of the band-1 polypeptide by 82% and 84% respectively, but inhibited the labelling of the band 2 polypeptide by 51%. The phosphinate in vitro produced 98% inhibition of the labelling of the band-1 polypeptide, with only 26% inhibition of the band-2 polypeptide, under conditions sufficient to inhibit neurotoxic esterase totally. Both neurotoxic esterase and the band-1 polypeptide were found in the forebrain at 1.74-fold their concentration in the rest of the brain, whereas the band-2 polypeptide was uniformly distributed. The evidence indicates that the Mipafox-sensitive polypeptide in band 1 is the [3H]DiPF-labelled active-site subunit of neurotoxic esterase. The catalytic-centre activity of the enzyme for phenyl valerate hydrolysis was found to be 2.6 x 10(5) min-1.

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