Soluble Fibrin Assays

2001 Dec;27(6):657-66.
Fibrin degradation products, fibrin monomer and soluble fibrin in disseminated intravascular
coagulation.
Horan JT, Francis CW.
Hematology Unit, Department of Pediatrics, University of Rochester School of Medicine and Dentistry,
Rochester, New York, USA. charles_francis@urmc.rochester.edu
Abstract
Disseminated intravascular coagulation (DIC) is characterized by activation of hemostasis and
fibrinolysis resulting in the formation of thrombin and plasmin, and the characteristic effects of these
enzymes on plasma fibrinogen can be useful in diagnosis. Thrombin cleaves fibrinopeptides from
fibrinogen, forming fibrin monomer that rapidly polymerizes to form a clot. Small amounts can circulate
in plasma as "soluble fibrin," which may have a complex composition and include fibrinogen and a
variable amount of cross-linking. Plasmic degradation of cross-linked fibrin forms a heterogeneous group
of degradation products reactive in assays for D-dimer, and their levels provide a measure of the amount
of fibrin formation and lysis. Caution should be exercised in comparing quantitative results using
different assays because of problems with standardization and variable reactivity with different molecular
forms. Marked elevations of fibrin(ogen) degradation products are a constant finding in experimental
animal models of DIC. In human models of DIC resulting from endotoxin infusion, D-dimer is elevated
early and high levels persist, reflecting lysis of microvascular fibrin deposits. Elevated levels of D-dimer
and soluble fibrin are very sensitive for the diagnosis of DIC, and a normal level has a high negative
predictive value. Serial monitoring of soluble fibrin or D-dimer assays may be of value in evaluating the
response to therapy and possibly in identifying at-risk patients.
PMID: 11740689 [PubMed - indexed for MEDLINE]
Assays for plasminogen activator inhibitor and soluble fibrin
TECHNICAL FIELD
The present invention relates to an improved method for measuring tissue plasminogen activator,
plasminogen activator inhibitor, and soluble fibrin. More particularly, the present invention relates to a
method for measuring plasminogen activator inhibitor and soluble fibrin which utilizes a genetically
modified tissue plasminogen activator protein as a reagent. The present invention also relates to improved
methods of collecting blood so that endogenous plasminogen activator is not inactivated.
BACKGROUND
The term "tPA" means "tissue plasminogen activator". The term "PAI" means plasminogen activator
inhibitor. "PAI 1" is plasminogen activator inhibitor one and is sometimes called endothelial plasminogen
activator inhibitor. "PAI 2" is plasminogen activator inhibitor two and is sometimes called placental
plasminogen activator inhibitor. The term "α 2 AP" means alpha 2 antiplasmin and is a protein found in
blood of normal individuals that inhibits the enzyme plasmin. The term "fibrinogen digests" includes the
products from digestion of fibrinogen or fibrin with proteolytic enzymes, such as plasmin.
Investigation of tissue plasminogen activator inactivation in plasma has been hampered by poor
methodology. A specific and sensitive method for measuring tPA in plasma samples where potential
fibrinolytic inhibitors were neutralized by controlled acidification was described. (See Wiman, B., et al.,
Clin. Chim Acta, 127, 279-288, 1982). tPA subsequently measured by this method exhibited a parabolic
rate assay. (See Rånby, M., et al, Thromb. Res. 27, 743-749, 1982). With this method it was also possible
to specifically determine inhibitory activity to tPA in plasma and kinetic evidence for a fast tPA inhibitor
in plasma was presented.
Assuming the formation of a stoichiometric 1:1 complex, a rate constant of about 10 7 M -1 s -1 was
calculated, and the plasma concentration of the new inhibitor in healthy individuals was determined as
8±2 unit/ml (1 unit=inhibition of 1 international unit of tPA). The tPA inhibitory content was also
determined in plasma from various patients. High inhibitory activity content was frequently found in
patients with deep venous thrombosis, hemostatic problems during late pregnancy, or severe coronary
heart disease. (See Chmielewska, J., et al., Thromb. Res. 31, 427-436 (1983)). The PAI activity observed
is now known to be the result of PAI1 activity.
There are several assay systems that are commercially available for the measurement of tPA and PAI
which utilize native tPA. However, the results concerning PAI levels obtained from these assays are not
always reliable because of the cleavage of tPA into the two chain form of the protein. Native one-chain
tPA is cleaved by plasmin or by trypsin after the Arg in the sequence -Gln-Phe-Arg-Ile-Lys- in the tPA
protein. This creates a problem when trying to measure the PAI1 level in a biological fluid by inhibition
of the tPA activity because two chain tPA also reacts rapidly with PAI 2 and reacts much faster than
single chain tPA with other protease inhibitors such as α 2 AP.
Thus, what is needed is a tPA that is resistant to cleavage by proteolytic enzymes to prevent formation of
two chain tPA in the preparation procedure or during the assay procedure. With such a tPA, levels of
PAI1 activity could be more accurately measured.
Another problem encountered in the prior art methods of measuring tPA and PAI is the fact that tPA and
PAI1 activity are unstable after blood is collected and the activity of the two proteins decreases after
blood is collected. In blood with high PAI1 levels, the tPA activity can decrease by 50% in about one
minute. The PAI1 activity typically has a half life of 4 hours at room temperature.
When tPA is assayed in blood, it has been found that polylysine is an effective stimulator of tPA activity.
However, it has also been found that polylysine is not an effective activator of tPA in biological fluids
other than blood and blood plasma. Thus, what is needed is to identify the preparation in plasma that,
together with polylysine, constitutes the effective tPA stimulator which will allow one to design better
methods to determine the tPA level. The improved methods will allow one to perform tPA activity assays
in non-plasma systems.
Another problem encountered in the prior art is that plasma samples must be acidified and incubated for
relatively long periods of time at low pH to destroy the plasmin inhibitory activity in the plasma sample
that interferes with the assay. What is needed is to identify the inhibitory activity and neutralize this with
specific antibodies. This will make both tPA activity and PAI activity more convenient to assay.
Yet another problem encountered in the prior art of determining tPA activity is that the tPA activity is
underestimated in biological fluids that also contain PAI1 activity. This is because PAI1 in this study is
found to be relatively stable during acidification and will react with tPA when the sample is neutralized
during the tPA assay procedure. What is needed is a method of inhibiting PAI1 activity in the assay
system when measuring tPA activity in biological fluids.
SUMMARY OF THE INVENTION
In accordance with the present invention, variants of one-chain tPA where the -Arg- amino acid was
replaced by a His (Arg to His) or by a Lys (Arg to Lys) or by a Thr (Arg to Thr) were made through
genetic modification of the native tPA protein. All the mutants, including the uncleavable Arg to Thr
mutant could be used in determination of PAI activity in plasma samples. The Arg to Thr mutant
represents an advantage in PAI1 activity determination. Preparations of the Arg to Thr mutant are sure to
be free of two chain tPA. The two chain tPA, in contrast to single chain tPA, reacts readily also with PAI
2. Thus, by using the mutant tPA one can accurately measure PAI1, an inhibitor that selectively reacts
with single chain tPA. One can measure PAI1 in samples containing both PAI1 and PAI 2, e.g., plasma
from pregnant women.
In addition, the present invention encompasses an improved method of collecting blood so that the tPA
present in the blood is stabilized and is not readily inactivated by PAI present in the blood. The improved
method of collecting blood comprises acidifying the blood from the physiological pH of about 7.3 to a pH
of between approximately 4.0 to 6.0. The preferred method of acidifying the blood for assaying tPA is
with citrate buffer although it is to be understood that other buffers can be used in practicing the present
invention. Buffer substances that also chelate calcium ions (e.g., citrate and EDTA) are particularly suited
since these buffer substances also inhibit blood coagulation. To reduce hemolysis of blood during the
acidification, it has been found advantageous to add Pluronic® F-68 to the blood citrate buffer mixture.
Another aspect of the present invention is the discovery that fibrinogen is the plasma component that
together with polylysine is the stimulator of tPA. Plasmin digests of fibrinogen or fibrin will also function
in this respect. Thus, when tPA is being measured in biological fluids other than blood, fibrinogen (or
fibrinogen digestion products) must be added along with polylysine to obtain maximum stimulation of
tPA activity.
Yet another aspect of the present invention is the verification that α 2 AP and PAI1 activites will interfere
with assay of tPA activity. Furthermore it was found that antibodies that inhibit these activites also
improve the performance of the tPA activity assay.
Accordingly, it is an object of the present invention to provide an improved method of assaying

plasminogen activator inhibitor activity.
It is an object of the present invention to provide a tPA that is resistant to cleavage by proteolytic
enzymes and is useful in the assay of PAI1.
It is another object of the present invention to provide a method of collecting blood or other biological
fluid that will stabilize tPA activity.
Another object of the present invention is to provide a method of immediately lowering the pH of the
blood with a minimum of hemolysis.
It is yet another object of the present invention to provide a method of using polylysine in combination
with fibrinogen or fibrinogen digests as a tPA cofactor.
Yet another object of the present invention is to provide an improved method of measuring fibrin in a
sample utilizing a single chain tPA that is resistant to cleavage by proteolytic enzymes.
Another object of the present invention is to improve the assay of tPA activity by including antibodies
that inhibit PAI1 and α 2 antiplasmin activities since these activities interfere with the assay of tPA
activity.
These and other objects, features and advantages of the present invention will become apparent after a
review of the following detailed description of the disclosed embodiment and the appended claims.
DETAILED DESCRIPTION OF THE DISCLOSED EMBODIMENT
Native one-chain tPA is cleaved by plasmin or by trypsin after the Arg in the sequence -Gln-Phe-Arg-Ile-
Lys- in the tPA protein. This creates a problem when trying to measure the PAI level in a biological fluid
by inhibition of the tPA activity. For example, tPA activity is known to be stimulated by fibrin or fibrin
fragments. The stimulation of single chain tPA by the presence of fibrin is much greater than that of the
two chain tPA.
The mutant tPAs in which the arginine residue in the sequence: -Gln-Pro-Gln-Phe-Arg-Ile-Lys-Gly-Gly-
had been replaced were enzymatically characterized. The specific activity expressed in IU/μg were as
follows: 810 (Arg to His), 640 (Arg to Lys), 290 (Arg to Thr) as compared to 810 for the wild type and
660 for Bowes melanoma tPA. The amidolytic activity against D-Ile-Pro-Arg-pNA at 37° C., pH 9.0
expressed in mOD per minute at 1 μg/ml of enzyme was 15.8 (Arg to His), 13.6 (Arg to Lys), 8.3 (Arg to
Thr), 10.0 (wild type), and 9.6 for melanoma one chain tPA as compared to 55.2 for two chain melanoma
tPA. Only the arginine to threonine mutant was resistant to plasmin and trypsin cleavage.
All mutant tPAs, including the uncleavable Arginine to Threonine mutant, could be used in determination
of PAI activity in plasma samples. The Arg to Thr mutant represents a advantage in PAI1 activity
determination. Preparations of the Arg to Thr mutant are sure to be free of two chain tPA which, in
contrast to the single chain tPA, also reacts readily with PAI 2.
It is to be understood that the genetically modified tPA that is described herein are readily made by those
of ordinary skill in the recombinant DNA art. In addition, it is also possible to chemically modify the tPA
molecule so that the molecule is more suitable for use in the aforementioned assays. For example, the
chemical modification can be performed so that the single chain tPA can no longer be cleaved by
proteolytic enzymes.
The plasminogen activation rate in the presence and absence of fibrin at 0.5 μm plasminogen and 37° C.
was measured and the stimulation factor calculated. This was about 950 fold for the Arg to Thr mutant
which was considerably higher than that of melanoma one chain tPA and the other mutant tPA which
were all about 550 fold. The stimulation factor for melanoma two chain tPA was about 120 fold. It is to
be understood that the extra fibrin sensitivity of the Arg to Thr mutant resulted in an improved soluble
fibrin assay according to the Wiman-Rånby protocol (See Wiman, B and Rånby, M, Thromb. Haemostas.
55, 189-183 (1986)). Thus, the plasmin insensitive protein-engineered mutant tPA is shown to be
advantageous over prior art methods when used in assays for PAI1 activity and soluble fibrin.
It is to be understood that the tPA activity measured according to the present invention can optionally be
determined using a reagent containing antibodies that inhibit anti-plasmin activity and PAI1 activity
present in the biological fluid to be analyzed.
The present invention also includes a method for determining the content of soluble fibrin in a sample
comprising the steps of mixing a fixed amount of sample with a fixed amount of reagent containing one-
chain tPA, plasminogen and a plasmin substrate, measuring plasmin substrate cleavage and correlating
this rate with the amount of soluble fibrin in the sample.
It was found that if during blood sample collecting, the pH of the blood is immediately lowered from the
physiological pH 7.3 to a pH of between approximately 4.0 and 6.0, several advantages for the stability of
the fibrinolytic components could be gained without introducing any disadvantages or inconveniences
when compared to commonly used Vacutainer®, Venaject® systems for collecting blood.
According to the present invention, nine parts of blood are drawn into a container that contains one part
citrate buffer with a pH of about 4 and a molarity of about 1 mol/l of citrate. The final pH of the blood
sample should preferably be between approximately 4.5 to 6.5. The citrate buffer should preferably be
between 0.5 to 2 mol/l of a sodium citrate buffer at a pH of approximately 4.0 to 5.5 to which 5 to 15
parts by volume of blood are added during collection.
To prevent hemolysis of the red blood cells and thrombocyte activation in the blood sample, Pluronic® F-
68 (BASF Corporation, Parsippany, N.J.) can optionally be included in the solution into which the blood
is being collected. The optimal final concentration of Pluronic® F-68 is between approximately 0.01% to
0.1% (0.1 to 1 mg/ml). It is to be understood that other Pluronic® surfactants can be used in the present
invention to prevent hemolysis.
The Pluronic® F-68 is a species within the following general formula: HO(C 2 H 4 O) b (C 3 H 6 O) a (C
2 H 4 O) b H
wherein a is an integer such that the hydrophobe represented by (C 3 H 6 O) has a molecular weight of
approximately 950 to 4000, preferably about 1750 to 3500, and b is an integer such that the hydrophile
portion represented by (C 2 H 4 O) constitutes approximately 50% to 90% by weight of the compound.
Pluronic® F-68 has the following specific formula: HO(C 2 H 4 O) b (C 3 H 6 O) a (C 2 H 4 O) b H
wherein the molecular weight of the hydrophobe (C 3 H 6 O) is approximately 1750 and the total
molecular weight of the compound is approximately 8400.
The advantages of the procedure are that the rates of the reactions between tPA and PAI1 and between
tPA and other inhibitors of blood are reduced at a pH of about 5 as compared to those at the physiological
pH of about 7.3. These reduced reaction rates will greatly increase the stability of tPA activity in plasma.
This is most important since it is relevant to determine the tPA activity at the time of sampling and not the
lower tPA activity found in stored blood samples collected according to the prior art. Thus, the present
invention will improve the value of measuring tPA activity to diagnose the etiology of thrombotic
disease, the risk of developing thrombotic disease, or the tPA activity obtained during tPA therapy.
It has been determined that the stability of PAI1 activity is increased significantly in blood samples
collected according to the present invention as compared to those collected in a conventional way. This is
of importance for this type of assay since the instability of PAI1 activity is limiting the spread of this
clinically important assay.
In addition, the method according to the present invention preserves soluble fibrin levels in blood and
plasma samples thereby improving the diagnostic importance of this important assay. High levels of
soluble fibrin are found in the blood of patients with malignancies, risk pregnancies and in patients
suffering from severe trauma. High levels of soluble fibrin is also a symptom of disseminated
intravascular coagulation.
The present invention is advantageous when monitoring fibrinogen and FVII levels during tPA therapy.
This is because plasmin generation is slow at the lower pH thereby eliminating much of the artifacts
caused in vitro by the action of in vitro generated plasmin.
Thus, the present invention of lowering the pH of blood during collection greatly improves the stability in
the blood sample and in the plasma sample derived thereof of several important fibrinolytic parameters
namely tPA activity, PAI1 activity and soluble fibrin as well as fibrinogen and other coagulation factors
such as FVIII and FV.
It has been determined that in the use of poly-D-lysine stimulation for tPA medicated plasminogen
activation, it was found the human plasma contained a factor which increased the stimulating effect of
poly-D-lysine. In this work, this factor was identified as fibrinogen and it was further found that the
plasmin digestion products of fibrinogen or fibrin also had the effect. This discovery is important because
it makes possible the formulation of practical and inexpensive reagents for determining tPA activity in
blood plasma and PAI1 activity in blood plasma. This is most important in the clinical routine.
EXAMPLE I
The following example describes measurement of tPA according to the present invention:
Citric acid monohydrate, M r =210, and Tri-sodium citrate dihydrate, M r =294, is obtained from Merck
Darnstadt, W. G. Pluronic® F-68 is obtained from BASF Corporation, Parsipanny, N.J. Blood is obtained
by vein puncture and collected on 0.13 mol/l trisodium citrate (1 part citrate buffer to 9 parts blood) in a
siliconized Venoject® collection tube.
0.5 mol/l solutions of citric acid and of tri-sodium citrate are mixed to give 0.5 mol/l sodium citrate
solutions with pH of 4.0, 4.5, 5.0 and 5.5. Each of these solutions is aliquoted and 25% F68 is added to
give final concentrations of 0, 0.1 or 1% by weight.
1.0 mol/l and 2.0 mol/l citrate buffers pH 4.0, 4.5, 5.0 and 5.5 containing 0, 0.1 or 1% F68 are made
accordingly.
Citrated blood is dispensed in 300 μl aliquoted to which 33 μl of each of the 36 different buffers are
added, mixed and incubated at room temperature (22° C.) for 20 hours. The samples are centrifuged six
minutes at 1500×g, diluted six fold in 0.15 mol/l NaCl and subjected to pH and absorptivity at 537 nm
determination. The absorption value (optical density at 1 cm path length) is multiplied by the dilution
factor of 6.
Thus, to a series of blood sample aliquots 1:9 volume of various citrate buffers are added. Plasma is
obtained some 20 hours later and the pH and 537 nm absorptivity is determined. The results are shown in
Table 1.
TABLE 1
______________________________________
pH and absorption at 537 nm for plasma obtained from blood samples to which 1:9 volume of citrate
buffers with various concentrations, pH and F68 content are added. 0.5 mol/l Citrate 1.0 mol/l Citrate 2.0
mol/l Citrate Conc. F68 Conc. F68 Conc. F68 (mg/ml) (mg/ml) (mg/ml) pH 0 1 10 0 1 10 0 1 10
______________________________________
4.0 pH
5.62 5.50 5.58 4.88 4.90 4.94 4.62
4.58
4.60
A 0.54 0.51 0.75 4.61 3.32 2.24 10.92
10.5 11.6
4.5 pH
6.07 6.04 6.09 5.48 5.44 5.41 5.08
5.10
5.10
A 0.62 0.56 0.55 0.61 0.49 0.46 5.24
1.09
1.40
5.0 pH
6.50 6.48 6.52 5.92 5.83 5.90 5.58
5.51
5.53
A 0.67 0.73 0.678
0.53 0.48 0.44 0.92
0.92
0.91
5.5 pH
6.78 6.83 6.92 6.34 6.33 6.35 6.02
5.93
5.99
A 0.72 0.68 0.65 0.84 0.70 --* 0.51
1.10
1.29
______________________________________
*not determined
As can be seen from Table 1, the addition of F68 to the freshly collected blood reduced the disruption of
the red blood cells as indicated by the absorbance at 537 nm. This is particularly clear with the addition of
1 mol/l citrate at a pH of 4.0 and with the addition of 2 mol/l citrate at a pH of 4.5. In both cases, there is
a dose dependent increase in the stability of the red blood cell membranes as shown by absorbance at 537
nm decreases as the Pluronic® F-68 concentration increases. It should be noted that this experiment is
performed with blood of an apparently healthy individual and problems with hemolysis are small. These
problems can be expected to be much greater when large numbers of patient plasmas are sampled.
EXAMPLE 2
Venoject®, Terumo Europe, Lewen, Belgium, are evacuated siliconized 4.5 ml of tubes containing 0.45
ml 0.13 mol/l sodium citrate and are, in the following, called "Venoject regular". Some "Venoject
regular" aer modified as an embodiment of the present invention. The citrate buffer in the "Venoject
regular" tubes is removed by suction with a hypodermic needle and 0.45 ml 1.0 mol/l citrate buffer pH 4.0
is introduced through the rubber stopper with a hypodermic needle. In this way, the citrate buffer content
is changed without disturbing the vacuum in the tube. The modified tubes are hereinafter called "Venoject
modified".
Blood is drawn by vein puncture from an apparently healthy individual into two "Venoject regular" and
into two "Venoject modified" tubes. To one "Venoject regular" and one "Venoject modified" tube, 45 μl
of 500 IU/ml single chain tPA dissolved in 1.0 mol/l KHCO 3 is added. This results in an increase in tPA
activity by about 8.9 IU/ml in the plasma (haematocrit of 0.45). The four tubes, "Venoject regular",
"Venoject regular+tPA", "Venoject modified" and "Venoject modified+tPA" are incubated at room
temperature (22° C.) and 1 ml aliquots are drawn after 0.25, 1, 2 and 3 hours. The aliquots are centrifuged
six minutes at 1500×g and 100 μl plasma is acidified by addition of 100 μl mol/l acetate buffer pH 3.9
and analyzed according to the protocol of Wiman, et al. Clin. Chem. Act. 127:279-288 (1983) using
Spectrolyse/fibrin reagents from Biopool AB, Umeå, Sweden. The results of the study are shown in Table
2.
In Table 2, tPA activity in blood plasma is measured after blood sample collection in "Venoject regular"
and "Venoject modified" with and without addition tPA. The blood is incubated at room temperature for
0.25, 1, 2 and 3 hours before separation of the blood cells. tPA activity found in the plasma is expressed
in IU/ml.
TABLE 2
______________________________________
Venoject Venoject Time Venoject Regular Venoject Modified Hours Regular and tPA Modified and tPA
______________________________________
0.25 0.51 3.8 1.3 8.4

1 0.14 1.7 1.6 9.6
2 0.07 0.6 1.4 9.6
3 0.04 0.4 1.4 9.9
______________________________________
As seen in Table 2, tPA is stable in Venoject modified tubes while tPA activity in unmodified tubes
decreased rapidly.
It should be understood that the foregoing relates only to a preferred embodiment of the present invention
and that numerous modifications or alterations may be made without departing from the spirit and scope
of the invention as set forth in the appended claims.
Inventors:
Ranby, Mats G. (Umea, SE)
Application Number:
07/391744
Publication Date:
05/19/1992
Filing Date:
08/09/1989
Export Citation:
Click for automatic bibliography generation
Assignee:
Biopool International, Inc. (New York, NY)
Primary Class:
435/13
Other Classes:
435/23, 530/328, 930/10, 930/240
International Classes:
C12Q1/56; G01N33/86; C12Q1/56; G01N33/86; (IPC1-7): C12Q1/56
Field of Search:
435/13, 435/23, 435/212, 435/217, 930/240, 530/381, 530/382
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Other References:
Wiman et al., "Determination of Soluble Fibrin in Plasma by a Rapid and Quantitative
Spectrophotometric Assay", Thrombosis and Haemestasis, 55(2), pp. 189-193 (1986).
Tate et al., "Functional Role of Proteolytic Cleavage at Arginine-275 of Human Tissue Plasminogen
Activator as Assessed by Site-Directed Mutagenesis", Biochemistry 26(2), pp. 338-343 (Jan. 27, 1987).
Wiman et al., "Plasminogen Activator Release During Venous Stasis and Exercise as Determined by a
New Specific Assay", Clin. Chem. Acta, vol. 127, pp. 279-288 (1983).
Allen, "An Enhancing Effect of Poly-Lysine on the Activation of Plasminogen", Thromb. Haemostas.,
vol. 47, No. 1, pp. 41-45 (1982).
Chmielewska et al., "Evidence for a Rapid Inhibitor to Tissue Plasminogen Activator in Plasma",
Thromb. Res., vol. 31, pp. 427-436 (1983).
Haeggroth et al., "Plasminogen Activator Inhibitors in Plasma and Platelets from Patients with Recurrent
Venous Thrombosis and Pregnant Women", Thromb. Res., vol. 42(5), pp. 585-594, (1986).
Eriksson et al., "Determination of Plasminogen Activator Inhibitor in Plasma Using t-PA and a
Chromogenic Single-Point Poly-D-Lysine Stimulated Assay", Thromb. Res., vol. 50, pp. 91-101 (1988).
Berne et al., editor, Physiology, pp. 382-385 (1988).
L. Lorand, editor, "Proteolytic Enzymes", Methods in Enzymology, vol. 80, Pt. C, Academic Press, Inc.,
pp. 365-378 (1981).
Wiman et al., "The Role of the Fibrinolytic System in Deep Vein Thrombosis", J. Lab. Clin. Med., pp.
265-270 (Feb. 1985).
Ranby et al., "Age Dependence of Tissue Plasminogen Activator Concentrations, Plasma, as Studied by
an Improved Enzyme-Linked Immunosorbent Assay", Clin. Chem., vol. 32, pp. 2160-2165 (1986).
Ranby et al., "A Sensitive Assay for Tissue Plaminogen Activator", Thromb. Res., vol. 27.27, pp. 743-
749 (1982).
Rijken et al., "Measurement of Human Tissue-Type Plasminogen Activator by a Two-Site
Immunoradiometric Assay", J. Lab. Clin. Med., vol. 101, No. 2, pp. 274-284 (1983).
Bergsdorf, "An Enzyme-Linked Immunosorbent Assay for Determination of Tissue Plasminogen
Activator Applied to Patients with Thromboembolic Disease", Thromb. Haemostas., vol. 50, No. 3, pp.
740-744 (1983).
Holvoet et al., "Assay of Human Tissue-Type Plasminogen Activator with an ELISA Based on Three
Murine Monoclonal Antibodies to Tissue Plasminogen Activator", Thromb. Haemostas., vol. 54, No. 3,
pp. 684-687 (1985).
Faulkner, W. R., edit., "Selected Methods for Small Chemistry Laboratory", Selected Methods of Clinical
Chemistry, vol. 9, pp. 6-7 and 328.
Primary Examiner:
Kepplinger, Esther L.
Assistant Examiner:
Bidwell, Carol E.
Attorney, Agent or Firm:
Jones, Askew & Lundsford
Parent Case Data:
This application is a division of application Ser. No. 070,068, filed Jul. 6, 1987 now abandoned.
Claims:
I claim:
1. A method for determining the amount of soluble fibrin in a sample comprising the steps of:
a. mixing a fixed amount of sample with a fixed amount of reagent containing single chain tPA, wherein
the arginine residue in the sequence -Gln-Pro-Gln-Phe-Arg-Ile-Lys-Gly-Gly of the tPA has been
substituted with a threonine, plasminogen and a plasmin substrate;
b. measuring the rate of plasmin substrate cleavage;
c. correlating this with the amount of soluble fibrin in the sample.
Fibrin Degradation Products

A Review of Structures Found in Vitro and in Vivo
1. PATRICK J. GAFFNEY
Article first published online: 25 JAN 2006
DOI: 10.1111/j.1749-6632.2001.tb03547.x
Issue
Annals of the New York Academy of Sciences
Volume 936, FIBRINOGEN: XVIth INTERNATIONAL FIBRINOGEN WORKSHOP pages 594–610,
June 2001
Additional Information(Show All)
How to CiteAuthor InformationPublication History
How to Cite
GAFFNEY, P. J. (2001), Fibrin Degradation Products. Annals of the New York Academy of Sciences,
936: 594–610. doi: 10.1111/j.1749-6632.2001.tb03547.x
Author Information
1. Division of Hæmatology, National Institute for Biological Standards and Control, Blanche Lane,
South Mimms, Potters Bar, Hertfordshire, United Kingdom
*Correspondence: PATRICK J. GAFFNEY,
*Correspondence: Address for correspondence: Patrick J. Gaffney, D.Sc., Ph.D., Academic Department
of Surgery, St. Thomas' Hospital, Lambeth Palace Road, London SE1 7EH, UK;
pejaygaffney@yahoo.com.
Publication History
1. Issue published online: 25 JAN 2006
2. Article first published online: 25 JAN 2006
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Keywords:
• Fibrin degradation products;
• Structures in vitro;
• Structures in vivo
Abstract: This review attempts to relate subunit structures of fibrin degradation products (FnDP) made in
vitro with structures found in vivo. The domainally directed fragmentation in vitro of both fibrinogen and
fibrin is emphasized, all fragments being various associations of the two core fibrin fragments D and E.
The digestion of fibrinogen by plasmin in vivo is rare, and the crosslinking of fibrin in vivo takes place at
a very early stage in the clotting/polymerization process. The notion that as fibrin forms in vivo it
orchestrates its own destruction is developed. Plasmas from patients suffering from dessiminated
intravascular coagulation demonstrate very large crosslinked FnDP fragments in their plasmas which
seem to contain not alone fibrinopeptide A but also subunits with intact alpha chains. This is interpreted
to mean that many of the large soluble fragments found in vivo, and heretofore known as FnDP, are in
reality long fibrin polymers, random parts of whose structures have been converted to FnDP by lysis of
the carboxy terminal regions of the alpha chains in the polymer. The ratio of intact fibrin to FnDP in these
large soluble structures may be a useful clinical marker; however, such data can only be relied upon when
blood samples are taken into an anticoagulant mix that contains a fibrinolytic inhibitor. Some of the
biological effects of FnDP structures in vivo (fibrinogen synthesis, vasoactivity) are still quite ambiguous.
The effect of moderate alcohol ingestion on blood coagulation and fibrinolysis at rest and in
response to exercise
Journal of Sports Sciences
Volume 17, Issue 6, 1999, Pages 513 - 520
Authors: MAHMOUD S. EL-SAYED; PAUL EASTLAND; XIA LIN; ANGELHEART M.J. RATTU
DOI: 10.1080/026404199365821
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Abstract
The effect of alcohol ingestion before exercise on blood haemostasis is not known. The present study
examined the effects of moderate alcohol ingestion on blood haemostatic variables at rest and in response
to exercise. Eleven normal healthy individuals randomly performed two tests separated by 7 days. A
moderate dose of ethanol (0.5 g.kg-1) was administered before one test, whereas an equal volume of an
alcohol-free drink was administered before the other. Forty-five minutes after the ingestion of either
drink, the participants cycled at 65% VO2max for 30 min followed by a 5-min all-out performance.
Venous blood samples were obtained before and 45 min after the ingestion of both drinks, and also
immediately after exercise. Exercise induced a significant increase in tissue-type plasminogen activator
activity and antigen, and factor VIII procoagulant activity. The post-exercise data also showed a
significant decrease in plasminogen activator inhibitor activity and soluble fibrin, with a significant
shortening in prothrombin time and activated partial thromboplastin time, but not thrombin time. No
significant changes were observed in antithrombin III. Although no significant differences were found
between trials in the haemostatic and fibrinolytic variables at rest, a significant decrease in fibrinogen
concentration was observed after exercise in the alcohol trial. This suggests that ingesting a moderate
dose of alcohol does not alter blood coagulation and fibrinolysis at rest. Apart from fibrinogen
concentration, which was significantly decreased after exercise in the alcohol trial, most of the
haemostatic and fibrinolytic variables were not affected by alcohol. The mechanism responsible for the
decrease in fibrinogen following exercise in the alcohol trial remains unknown, but might be related to
inhibition of fibrinogen synthesis by the liver or an enhanced rate of its catabolism.
2003 May;89(5):832-6.
Measurement of soluble fibrin monomer-fibrinogen complex in plasmas derived from patients with
various underlying clinical situations.
Nakahara K, Kazahaya Y, Shintani Y, Yamazumi K, Eguchi Y, Koga S, Wada H, Matsuda M.
Iatron Laboratories, Inc., 1460-6 Mitodai, Mito, Tako-machi, Katori-gun, Chiba 289-2247, Japan.
nakahara@iatron.co.jp
Abstract
We previously reported a monoclonal antibody named IF-43 that specifically recognizes thrombin-
modified fibrinogen (desAA- and desAABB- fibrin monomer) bound with fibrinogen or other D(1)
domain-containing plasmic fragments such as fragments X,Y, and D(1), but not intact fibrinogen or cross-
linked fibrin degradation products (XDP). Here, we tentatively named such complexes, soluble fibrin
monomer (FM) -fibrinogen complex. By utilizing IF-43, we have developed a kit to measure soluble FM-
fibrinogen complex and compared the profiles with those of two established molecular markers for
thrombo-embolic disorders: i.e. the thrombin-antithrombin complex (TAT) and the D-dimer in plasma of
patients who underwent surgery without any thrombo-embolic complications. The result indicated that
soluble FM-fibrinogen complex is a distinct entity from the two established molecular markers. We have
also attempted to observe their profiles in patients with the disseminated intravascular coagulation
syndrome (DIC). Although the pro-files of soluble FM-fibrinogen complex in individual patients
appeared to vary from one patient to the other, the plasma level of soluble FM-fibrinogen complex was
found to be increased at the initial phase of disseminated intravascular coagulation syndrome. Thus, the
soluble FM-fibrinogen complex may serve as an independent molecular marker for the detection of
thrombin generation and the diagnosis of thrombosis. The soluble FM-fibrinogen complex may also serve
as a risk factor for thrombosis, because it may precipitate as insoluble complexes beyond its threshold in
plasma, or when it is modified by thrombin.
2004 Apr;52(4):355-61.
[A novel molecular marker for thrombus formation and life prognosis--clinical usefulness of
measurement of soluble fibrin monomer-fibrinogen complex (SF)].
[Article in Japanese]
Koga S.
Division of Blood Transfusion Medicine and Hematology (Hematology Medicine), Wakayama Medical
University, Wakayama 641-8510.
Abstract
For a long time fibrinopeptide A(FPA), fibrinopeptide B(FPB), D-dimer, FM test, serum FDP, and
thrombin anti-thrombin complex(TAT) are being used as molecular markers to for sure diagnose
hypercoagulable state and thrombus formation. Indeed these molecular markers are very useful for
diagnosing thrombus formation, disseminated intravascular coagulation(DIC), and the indicator of
treatment of DIC. But these molecular parameters are not enough and difficult for prognosis of the
disease or predicting the complication of patients as the most important subject for clinicians. The soluble
fibrin monomer-fibrinogen complex (SF) is a complex coupling fibrin monomer and fibrinogen
molecules to be formed in the early-activated state of blood coagulation. Thus such a molecular complex
is expected to serve as a parameter for the diagnosis of thrombus formation and DIC, in particular its
early stage. The aim of the present study is to evaluate a potential usefulness of a newly developed SF test
utilizing an SF specific monoclonal antibody (IF-43). We measured SF together with established other
parameters in 195 patients with DIC, subclinical DIC/hypercoagulable state, and non-DIC. The diagnosis
of DIC was made based on a modified version of the criteria established by the Ministry of Health, Labor
and Welfare of Japan. Underlying disease includes leukemia, malignant lymphoma, myelodysplastic
syndrome (MDS), multiple injury, giant ovarian tumor, prostatic cancer with multiple bone metastasis,
lung cancer, breast cancer with multiple lung and bone metastasis, severe pneumoniae, sepsis,
hemophagocytic syndrome (HPS), and rheumatoid arthritis. The SF levels in DIC patients were
significantly higher than those in the subclinical DIC/hypercoagulable state, and the non-DIC patients.
Receiver operating characteristic (ROC) analysis shows that the specificity and sensitivity of the SF assay
appears to be satisfactory. As the level of SF reflects the thrombin generation activity in plasma, it would
serve as a strong tool to selectively kick up the state of thrombin generation. These results indicate that
the SF could be a specific and reliable parameter for the diagnosis of DIC and contribute to legitimate
managements of patients with DIC. The excessive life response to serious clinical insults, such as sepsis,
severe pancreatitis, trauma and shock, is called systemic inflammatory response syndrome (SIRS). Once
SIRS occurs, people may often die from serious complications such as adult respiratory distress syndrome
(ARDS), acute lung injury (ALI), disseminated intravascular coagulation (DIC) and multiple organ
failure (MOF). Especially, ALI followed by pneumoniae associated with SIRS could depend on patient's
prognosis and life. That is to say, it seems to be urgent for clinicians to make differential diagnosis
between Pneumoniae associated with SIRS and Coagulopathy (PASC) and Simple Pneumoniae (SP).
Soluble fibrin monomer-fibrinogen complex(SF) is formed in the early-activated state of blood
coagulation. Thus such a molecular complex is expected to serve as a parameter for the diagnosis of
coagulopathy, in particular its early stage. The aim of the present study is to make differential diagnosis
between Pneumoniae associated with SIRS and Coagulopathy (PASC) and Simple Pneumoniae(SP) by
using a newly developed SF test utilizing an SF specific monoclonal antibody (IF-43). We measured SF
together with established other parameters, hemogram, blood laboratory items in 7 patients with PASC
and 17 patients with SP. The diagnosis of Pneumoniae was defined according to the criteria: clinical
symptoms abnormal shadow in both Chest X-p and Chest CT, increased level of CRP, number of WBC.
The diagnosis of SIRS was based on the criteria established by American College of Chest Physicians
(ACCP)/Society of Critical Care Medicine (SCCM) Consensus Conference held in August of 1991 in
Northbrook, IL (USA). Underlying disease includes leukemias, malignant lymphoma, myelodysplastic
syndrome (MDS), multiple myeloma, idiopathic thrombocytopenia purpura(ITP), multiple injury (bone
fracture), cerebral hemorrhage, enterocolitis, Appendicitis, lung cancer, larynx cancer, bronchiolitis
obliterans organizing pneumonia(BOOP), chronic obstructive pulmonary disease(COPD), sepsis. The SF
levels in PASC patients are significantly higher than those in SP patients (p < 0.001). Otherwise, there is
no significant difference of the CRP levels between in PASC group and SP group (p < ns). There is no co-
relationship between SF level and D-dimer level. Receiver operating characteristic (ROC) analysis shows
that the specificity and sensitivity of the SF assay appears to be quite satisfactory. As the level of SF
reflects the thrombin generation activity in plasma, it would serve as a strong tool to selectively kick up
the state of thrombin generation. These results indicate that the SF could be a specific and reliable
parameter for the diagnosis of PASC and contribute to legitimate managements of patients with PASC.
Clinical implication of plasma level of soluble fibrin monomer-fibrinogen complex in patients with
abdominal aortic aneurysm
Top of Form
yes platform+medline author author
• Akihiro Hosaka, MD
,
• Tetsuro Miyata, MD
Affiliations
○ Reprint requests: Tetsuro Miyata, MD, Division of Vascular Surgery, Department of
Surgery, Graduate School of Medicine, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku,
Tokyo 113-8655, Japan.
,
• Haruo Aramoto, MD
,
• Hiroshi Shigematsu, MD
,
• Tatsu Nakazawa, MD
,
• Hiroyuki Okamoto, MD
,
• Kunihiro Shigematsu, MD
,
• Hirokazu Nagawa, MD
Bottom of Form
• Abstract
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• References
Objective
We prospectively studied the clinical implication of plasma level of soluble fibrin monomer (FM)-
fibrinogen complex, a recently established molecular marker reflecting thrombin activity, in patients with
abdominal aortic aneurysm (AAA) undergoing elective aortic repair.
Methods
The study included 49 patients who underwent elective aneurysm repair using a gelatin-sealed or
nonimpregnated Dacron prosthesis. Plasma level of soluble FM-fibrinogen complex was measured before
surgery and on days 1, 3, 5, 7, and 10 postoperatively by latex agglutination assay utilizing monoclonal
antibody IF-43. Plasma levels of thrombin-antithrombin complex (TAT), D-dimer, α2-plasmin inhibitor-
plasmin complex (PIC), and fibrinogen were also evaluated.
Results
The preoperative level of soluble FM-fibrinogen complex showed variation in the degree of hemostatic
activation, with fair correlations with TAT (r = 0.509, P < .001), D-dimer (r = 0.521, P < .001), and PIC
(r = 0.579, P < .001). The patients with greater intraoperative blood loss (>= 800 mL) showed a
significantly elevated plasma level of soluble FM-fibrinogen complex preoperatively compared with
those with less intraoperative blood loss (P = .009). Its postoperative fluctuation showed a similar pattern
to that of TAT, reflecting the time course of coagulation activity. Gelatin impregnation of the Dacron
vascular graft did not seem to influence the postoperative systemic coagulation mechanism.
Conclusions
The results indicated that soluble FM-fibrinogen complex appears to be a useful diagnostic molecular
marker to assess the activity of the coagulation system, and that its preoperative level may serve as a
potential risk factor for intraoperative hemorrhagic diathesis in patients undergoing elective AAA repair.
Competition of interest: none.

PII: S0741-5214(05)00481-7
doi:10.1016/j.jvs.2005.03.042
© 2005 The Society for Vascular Surgery. Published by Elsevier Inc. All rights reserved.
Volume 35, Number 6, 465-479, DOI: 10.1007/BF00996693

A sensitive latex agglutination method for the detection of soluble fibrin in plasma
U. P. Müller, R. Largo and P. W. Straub
Abstract
A sensitive method is described for the rapid detection of soluble fibrin in human plasma. It is based on
the agglutination of Latex particles suspended in a solution of purified fibrin monomers and Blue Dextran
2000. Photometric registration of the decrease in optical density (OD) during agglutination allows the
results to be quantitated. The OD depends on the ratio of fibrin monomers to fibrinogen in the tested
plasma rather than on the absolute amount of fibrin monomers present.
Wir beschreiben eine empfindliche Methode für den schnellen Nachweis von löslichem Fibrin in
menschlichem Plasma. Sie beruht auf der Agglutination von Latexpartikeln, die in einer Lösung von
gereinigtem Fibrin und Blue Dextran 2000 suspendiert sind. Die photometrische Aufzeichnung des
Abfalls der optischen Dichte (OD) wÄhrend der Agglutination führt zu quantitativen Resultaten. OD ist
abhÄngig vom VerhÄltnis der Fibrin-Monomere zum Fibrinogen im getesteten Plasma und nicht vom
absoluten Gehalt an Fibrin-Monomeren.
Key words Soluble fibrin - Latex agglutination - Fibrin monomers - Fibrinogen - Blue Dextran
Schlüsselwörter Lösliches Fibrin - Agglutination von Latexpartikeln - Fibrin-
Monomere - Fibrinogen - Blue Dextran
(Circulation. 1998;97:544-552.)
© 1998 American Heart Association, Inc.
Clinical Investigation and

Reports
Thrombin Binds to Soluble Fibrin Degradation Products Where it Is Protected From Inhibition by
Heparin-Antithrombin but Susceptible to Inactivation by Antithrombin-Independent Inhibitors
Jeffrey I. Weitz, MD; Beverly Leslie, BSc; ; Monika Hudoba, MD
From the Department of Medicine, McMaster University and Hamilton Civic Hospitals Research Centre,
Hamilton, Ontario, Canada.
Correspondence to Dr Jeffrey Weitz, Hamilton Civic Hospitals Research Centre, 711 Concession St,
Hamilton, Ontario, L8V 1C3. E-mail weitzj@fhs.mcmaster.ca
Top
Abstract
Introduction
Abstract Methods
Results
Discussion
Background—Thrombolytic therapy induces a procoagulant state characterized by References
elevated plasma levels of fibrinopeptide A (FPA), but the responsible mechanism is
uncertain.
Methods and Results—Washed plasma clots were incubated in citrated plasma in the presence or absence
of tissue plasminogen activator (t-PA), and FPA generation was monitored as an index of unopposed
thrombin activity. FPA levels are almost twofold higher in the presence of t-PA than in its absence. This
primarily reflects the action of thrombin bound to soluble fibrin degradation products because (a) there is
progressive FPA generation even after clots are removed from t-PA–containing plasma, and (b) clot
lysates produce concentration-dependent FPA generation when incubated in citrated plasma. Using
thrombin-agarose affinity chromatography, (DD)E and fragment E but not D-dimer were identified as the
thrombin-binding fibrin fragments, indicating that the thrombin-binding site is located within the E
domain. Heparin inhibits thrombin bound to fibrin degradation products less effectively than free
thrombin. In contrast, D-Phe-Pro-ArgCH2Cl, hirudin and hirugen inhibit free thrombin and thrombin
bound to fibrin degradation products equally well.
Conclusions—Thrombin bound to soluble fibrin degradation products is primarily responsible for the
increase in FPA levels that occurs when a clot undergoes t-PA–induced lysis. Like clot-bound thrombin,
thrombin bound to fibrin derivatives is protected from inhibition by heparin but susceptible to inactivation
by direct thrombin inhibitors. These findings help to explain the superiority of direct thrombin inhibitors
over heparin as adjuncts to thrombolytic therapy.
Key Words: plasminogen activators • thrombolysis • thrombus • anticoagulants

Top
Abstract
Introduction
Introduction Methods
Results
Discussion
Coronary thrombolysis is the treatment of choice for patients presenting early after References
the onset of symptoms of acute myocardial infarction. Thrombolytic therapy with
either streptokinase or t-PA induces a procoagulant state characterized by elevated
plasma levels of FPA consistent with increased thrombin activity.1 2 3 4 5 This
procoagulant state may explain why reinfarction occurs in 3% to 6% of patients despite successful
coronary thrombolysis.6 A number of mechanisms have been proposed to explain the procoagulant state
induced by thrombolytic therapy. These include free plasmin formed as a result of the systemic lytic state,
which can trigger the coagulation mechanism by activating contact factors,7 factor V,8 and possibly
prothrombin.9 10 Another possibility is that thrombin bound to fibrin is progressively exposed as the clot
undergoes lysis.11 Since fibrin-bound thrombin is protected from inactivation by fluid-phase inhibitors,12 13
it has the potential to locally activate platelets14 and accelerate coagulation.15 Alternatively, thrombin
released from the lysing thrombus could induce a local procoagulant state.16 Finally, the increased FPA
levels in patients treated with t-PA may not reflect thrombin activity because like thrombin, t-PA can also
release FPA from fibrinogen.17 In this study we demonstrate a different mechanism. Thus we have shown
that as a clot undergoes lysis, thrombin remains bound to soluble fibrin degradation products, where it still
is protected from inactivation by fluid-phase inhibitors and hence has the potential to induce a systemic
procoagulant state.
Top
Abstract
Introduction
Methods Methods
Results
Discussion
Reagents References
Predominantly single-chain recombinant human t-PA (lot L9135A2) was obtained
from Genentech, Inc, San Francisco. Human -thrombin (sp act 2850 U/mg) was
kindly provided by Dr J. Fenton II, New York State Department of Health, Albany,
NY. PPACK was purchased from Hematologic Technologies, Inc, Essex Junction, Vt, and streptavidin-
agarose was obtained from Sigma Chemical Co. Fragment E was purchased from Diagnostica Stago,
Asnieres, France. Glu-plasminogen and a mouse monoclonal antibody against t-PA (PAM-2) were
obtained from American Diagnostica, New York, NY. A mouse monoclonal antibody against D-dimer
(DD) was generously provided by Dr D. Collen, University of Leuven, Belgium, whereas a monospecific
rabbit antibody against fragment E was purchased from Diagnostica Stago. Standard heparin from porcine
intestinal mucosa was obtained from Sigma Chemical Co, and PPACK was from Calbiochem Corp.
Recombinant hirudin with a Val-Val amino-terminal sequence was purchased from American
Diagnostica. The synthetic tyrosine-sulfated dodecapeptide known as hirugen,18 which is comprised of
residues 53 to 64 of hirudin (H-peptide, BG8865), was generously provided by Dr J. Maraganore, Biogen
Inc, Cambridge, Mass.
Preparation of 125I-Labeled Fibrinogen
Fibrinogen was precipitated from barium sulfate–adsorbed plasma with 2 mol/L ß-alanine as described in
detail elsewhere.19 The isolated fibrinogen was then trace labeled with 125I to a specific activity of 100±5
µCi/mg.20
Preparation of 125I-Labeled Cross-Linked Plasma Clots
Blood was collected from the antecubital veins of healthy volunteers into plastic syringes prefilled with
1/10 vol of 3.8% trisodium citrate. After thorough mixing with the anticoagulant, the red cells were
sedimented by centrifugation at 1700g for 15 minutes at 4°C. The harvested platelet-poor plasma was
supplemented with 125I-fibrinogen ( 120 000 cpm/mL) and 500-µL aliquots were then transferred to
polypropylene Eppendorf tubes. Labeled cross-linked fibrin clots were formed around wire hooks by the
addition of CaCl2 (final concentration, 25 mmol/L). The clots were aged for 60 minutes at 37°C with
constant agitation and then washed six times with 1-mL aliquots of 0.1 mol/L NaCl buffered with 0.05
mol/L Tris-HCl, pH 7.4 (TBS) over the course of 18 hours to eliminate FPA trapped within the clots.13
The washed clots were then counted for radioactivity for 1 minute using a Clinigamma counter (LKB
Instruments, Inc).
Clots formed in this fashion are cross-linked because they remain intact after 24 hours of incubation in 2%
acetic acid. Further, SDS-PAGE analysis under reducing conditions of clots solubilized in SDS as
described by Francis et al21 demonstrates bands corresponding to the ß-chains, - dimers, and -polymers
(data not shown). Non–cross-linked - or -chains are not present, thus indicating complete cross-linking.
Clot-Induced FPA Generation in the Presence or Absence of t-PA
Washed 125I-labeled plasma clots were incubated with 1-mL aliquots of fresh citrated plasma for 90
minutes at 37°C in the presence or absence of 1 µg/mL t-PA, and clot lysis and FPA generation were
monitored. As a control, citrated plasma was incubated without plasma clots for 90 minutes at 37°C in the
presence or absence of 1 µg/mL t-PA. The extent of t-PA induced clot lysis was quantified (1) by
monitoring the time course of release of 125I-labeled fibrin degradation products and (2) by removing the
clots from the plasma at intervals and counting their residual radioactivity for 1 minute after the clots were
briefly washed three times with 500-µL aliquots of TBS. To calculate the percentage clot lysis, the
difference between the radioactivity originally incorporated into the clot and the residual radioactivity was
calculated and expressed as a percentage of the original radioactivity.
In parallel, clot-induced FPA generation also was monitored. To accomplish this, 100-µL aliquots of
plasma were removed at intervals, and unreacted fibrinogen was precipitated by the addition of 300 µL of
chilled ethanol followed by centrifugation at 15 000g for 5 minutes. The ethanol supernatants were then
evaporated to dryness in a Speed-Vac concentrator (Savant Instruments, Inc), reconstituted to original
volume with distilled water, and assayed for FPA using a previously described radioimmunoassay.22 This
assay utilizes antiserum R2, which is specific for FPA and cross-reacts poorly with fibrinogen or larger
FPA-containing fragments.17
Preparation of Clot Lysates
Lysates of extensively washed 125I-labeled plasma clots were prepared as we have previously described
with only minor modifications.23 Briefly, the clots were incubated for 60 minutes at 37°C in 500-µL
aliquots of TBS containing 4 µg/mL t-PA, 1.7 µmol/L glu-plasminogen, and 0.02% Tween 80. At the end
of the incubation period, the residual clots were removed and the clot lysates were then pooled and
immunodepleted of t-PA by affinity chromatography. A mouse monoclonal antibody against the kringle-1
region24 of human t-PA (PAM-2, American Diagnostica) was coupled to cyanogen bromide activated CH
Sepharose 4B (Pharmacia Fine Chemicals) at a concentration of 20 mg/mL. The clot lysate (10 mL) and
anti–t-PA IgG coupled to CH Sepharose 4B (750 µL of a 50% suspension) were mixed in a tube and
agitated for 1 hour at 23°C. After centrifugation at 10 000g for 5 minutes, the supernatants were carefully
removed, and the immunodepletion procedure was then repeated for two additional cycles. The final
material, which contained <50 ng of t-PA as measured antigenically using an enzyme-linked
immunosorbent assay kit from American Diagnostica, was then concentrated 10-fold using an
ultrafiltration cell (series 8050, Amicon Division, W.R. Grace & Co) fitted with a 5000-mw cutoff
membrane (YM5 disk membrane, Amicon), and stored in aliquots at -70°C.
Clot Lysate-Induced FPA Generation in Plasma
To examine the ability of soluble fibrin degradation products to generate FPA, varying amounts of clot
lysate were incubated in 500-µL aliquots of citrated plasma for 60 minutes at 37°C. At intervals, 100-µL
aliquots were removed, and the unreacted fibrinogen was precipitated with 300 µL of chilled ethanol
followed by centrifugation at 15 000g for 5 minutes. The ethanol supernatants were then evaporated to
dryness, reconstituted to original volume with distilled water, and assayed for FPA.
Effect of Thrombin Inhibitors on FPA Generation in Plasma Induced by Fluid-Phase Thrombin
and Clot Lysates
To compare the ability of various thrombin inhibitors to block clot lysate-induced FPA generation with
their activity against FPA generation produced by fluid-phase thrombin, clot lysates (in amounts varying
from 2.5 to 10 µL) or human -thrombin (in concentrations ranging from 0.2 to 4.0 nmol/L) were
incubated with 500-µL aliquots of citrated plasma for 60 minutes at 37°C in the presence or absence of
heparin (at concentrations ranging from 0.1 to 2.0 U/mL), hirudin (at concentrations ranging from 1.0 to
5.0 nmol/L), hirugen (at concentrations ranging from 1.0 to 3.8 µmol/L), or PPACK (at concentrations
ranging from 1.0 to 6.0 nmol/L). At the end of the incubation period, 100-µL aliquots of plasma were
removed, and the unreacted fibrinogen was precipitated with 300-µL of chilled ethanol followed by
centrifugation at 15 000g for 5 minutes. The ethanol supernatants were then evaporated to dryness,
reconstituted to original volume with distilled water, and assayed for FPA. The percent inhibition of FPA
generation produced by each concentration of thrombin inhibitor was then calculated.
Adsorption of 125I-Labeled Fibrin Degradation Products to Thrombin-Agarose
To identify those fibrin derivatives that bind thrombin, aliquots of the lysates of 125I-labeled cross-linked
fibrin clots were subjected to chromatography on thrombin-agarose, and the bound and unbound fractions
were then isolated and characterized by SDS-PAGE and autoradiography. To couple thrombin to agarose,
human -thrombin (2 mg) was incubated for 30 minutes at 37°C with a 10-fold molar excess of
biotinylated PPACK. The mixture was then dialyzed against TBS containing 6 mg/mL polyethylene
glycol (PEG) to removed uncomplexed PPACK. After packing a column (5x1.2 cm) with 6 mL of
streptavidin-agarose (50% suspension), which was then equilibrated with TBS containing 6 mg/mL PEG,
the biotinylated PPACK-thrombin complexes were passed over the column. Based on the absorbance of
the effluent at 280 nm, over 98% of the active site-blocked thrombin bound to the column. After blocking
unreacted streptavidin with 10 mL of TBS containing 1.2 mg/mL biotin, the column was extensively
washed with 0.05 mol/L NaCl buffered with 0.05 mol/L Tris-HCl and containing 1 mg/ml PEG (TBS-
PEG).
Aliquots of the lysate of 125I-labeled fibrin clots, suspended in TBS-PEG, were then subjected to affinity
chromatography at 23°C on the thrombin-agarose column. 0.5-mL fractions were collected and their
radioactivity and absorbance at 280 nm were determined. After extensive washing of the column with
TBS-PEG, the bound material was eluted with 0.3 mol/L NaCl buffered with 0.05 mol/L HCl to pH 7.4
and containing 1 mg/mL PEG. Again, 0.5 mL fractions were collected, and their radioactivity and
absorbance at 280 nm were determined. After confirming the protein concentrations using the method of
Lowry,25 the peak protein-containing fractions were pooled, and then concentrated using Centricon-10
ultracentrifugation cartridges (Amicon Division, W.R. Grace & Co).
Gel Filtration of Clot Lysates
125
I-labeled fibrin degradation products eluted from thrombin-agarose were subjected to chromatography
at 23°C on a column (90x1.6 cm) of Sephacryl-S300 HR (Pharmacia Fine Chemicals) using TBS at a rate
of 50 mL/h. For preparative procedures, dextran blue (Sigma Chemical Co) was used to determine the
void volume, and the column was calibrated with thyroglobulin, gamma-globulin, ovalbumin, myoglobin,
and cyanocobalamin with molecular weights of 670 000, 158 000, 44 000, 17 000, and 1350 D,
respectively. Fractions (2 mL) were collected and their radioactivity and absorbance at 280 nm were
measured. After confirming the protein concentrations using the method of Lowry,25 peak protein-
containing fractions were pooled and concentrated using Centricon-10 ultrafiltration cartridges.
PAGE and Immunoblot Analysis of Fibrin Degradation Products
The fibrin degradation products that bind and do not bind to thrombin-agarose were characterized using a
combination of PAGE and immunoblot analysis. Samples were diluted in an equal volume of 60 mmol/L
Tris-HCl containing 0.001% bromophenol blue with or without 2% SDS and 5% glycerol. Two
electrophoretic systems were used: a 7.5% polyacrylamide slab gel (4% stacking gel) containing 0.1%
SDS using a modified Laemmli discontinuous buffer system26 under nonreducing conditions, and a 7.5%
polyacrylamide gel (4% stacking gel) under nondissociating conditions according to the method of
Davis.27 The gels were either fixed in 40% methanol/10% acetic acid and stained with Coomasie blue or
the separated proteins were electrophoretically transferred onto nitrocellulose membranes. The membranes
were then probed with either a monoclonal antibody against DD (8D3) or with a monospecific antibody
against fragment E as we have previously described.23
Statistical Analyses
Where appropriate, means and 95% confidence intervals (CI) were calculated. Significance of differences
was determined by one-way, two-way, or repeated-measures ANOVA,28 and a value of P<.05 was
considered statistically significant.
Top
Abstract
Introduction
Results Methods
Results
Discussion
Formation of 125I-Labeled Fibrin Clots References
The recalcification of 500-µL aliquots of plasma containing 125I-labeled fibrinogen
results in the formation of clots of standard size as determined by their radioactivity.
More than 95% of the radiolabeled fibrinogen is incorporated into the clots so that
each clot contains 57 215±1380 cpm (mean±SD).
Increased FPA Generation in the Presence of t-PA
When washed 125I-labeled fibrin clots are incubated in citrated plasma, there is time-dependent generation
of FPA (Fig 1 ). Almost twice as much FPA is generated when the clot is incubated in plasma containing
1 µg/mL t-PA, a concentration of plasminogen activator that produces 56% clot lysis at 90 minutes.
Repeated-measures ANOVA indicates a highly significant (P<.0001) interaction between treatment and
time with significantly more clot-induced FPA generation occurring in the presence of t-PA than in its
absence (P<.0001). In contrast, when plasma is incubated with 1 µg/mL of t-PA in the absence of a
plasma clot, only very small amounts of FPA are generated.
Figure 1. Clot-induced fibrinopeptide A generation in the

presence ( ) and absence ( ) of t-PA. Washed plasma clots were
incubated in citrated plasma for 90 minutes at 37°C in the
presence ( ) or absence ( ) of 1 µg/mL t-PA. At the times
indicated, aliquots of plasma were removed, and after
precipitation of unreacted fibrinogen with ethanol, the ethanol
supernatants were assayed for fibrinopeptide A. In a control
experiment, 1 µg/mL t-PA was incubated with citrated plasma
in the absence of a plasma clot, and fibrinopeptide A levels were
quantified ( ). Each point represents the mean of three different
experiments, each done in duplicate.
View larger version (15K):
[in this window]
[in a new window]
Clot-induced FPA generation does not result from release of trapped peptide because <5 nmol/L FPA is
recovered when clots are incubated in buffer in place of plasma or when clots are completely lysed by t-
PA in buffer. Thrombin is responsible for clot-induced FPA generation since no FPA is generated if the
clots are preincubated for 30 minutes at 37°C in buffer containing 10 µmol/L hirudin or 6 µmol/L PPACK
prior to incubation in plasma in the presence or absence of t-PA (data not shown).
Mechanism for Increased Clot-Induced FPA Generation in the Presence of a Lysing Thrombus
To investigate the mechanism responsible for the increased FPA generation in the presence of a lysing
thrombus, 125I-labeled fibrin clots were incubated for 2 hours at 37°C in 500-µL aliquots of plasma
containing 1 µg/mL t-PA. At varying intervals, the clots were removed, and the time-course of clot lysis
was monitored by measuring the release of 125I-labeled fibrin degradation products (Fig 2A ), while clot-
induced FPA generation was followed in parallel (Fig 2B ). The concentration of released 125I-labeled
fibrin degradation products depends on the duration of exposure of the clots to t-PA. Once the clots are
removed from the t-PA-containing plasma however, release of 125I-labeled fibrin degradation products
ceases (Fig 2A ). In contrast, there is ongoing FPA generation after the clots are removed (Fig 2B ).
Figure 2. Time courses of concomitant t-PA-induced release of 125I-labeled

fibrin degradation products (A) and fibrinopeptide A generation (B) when
125
I-labeled plasma clots are incubated in t-PA-containing plasma. Washed
125
I-labeled plasma clots were incubated in citrated plasma containing 1
µg/mL t-PA for 120 minutes at 37°C. At the times indicated, aliquots of
plasma were removed and the concentrations of 125I-labeled fibrin
degradation products and fibrinopeptide A were determined ( ). To examine
the effect of early removal of the clot on clot lysis and fibrinopeptide A
generation, 125I-labeled plasma clots incubating in plasma containing 1 µg/mL
t-PA were removed at the times indicated by the arrows, and the time-courses
of release of 125I-labeled fibrin degradation products and FPA generation were
then determined. The effect of clot removal at 10 minutes. ( ), 20 minutes (
View larger version ), or 30 minutes ( ) incubation is illustrated. Each point represents the mean
(15K): of two different experiments, each done in duplicate.
[in this window]
[in a new window]
These data were analyzed by first calculating the slope of the relationships between the levels of 125I-
labeled fibrin degradation products or FPA and time from the points where the clots were removed from t-
PA–containing plasma. Using one-way ANOVA, the mean slopes did not differ as a function of the time
that the clots were removed from plasma. Whereas the mean slope for the time courses of 125I-labeled
fibrin degradation product generation (Fig 2A ) was 0.0078 (95% CI: -0.0049, 0.0051), a value by one-
sample t test not significantly different from zero (P=.95), the mean slope for the time courses of FPA
generation (Fig 2B ) was 0.84 (95% CI: 0.73, 0.95); a value that was significantly different from zero by t
test (P<.0001). The continuing FPA generation after clot removal excludes the possibility that clot-bound
thrombin is responsible for this phenomenon, and suggests that a soluble factor also contributes.
To examine the possibility that thrombin bound to soluble fibrin degradation products is responsible, at
least in part, for ongoing FPA generation after clots are removed from t-PA–containing plasma, varying
amounts of clot lysate were incubated in plasma and the levels of FPA were measured. As illustrated in
Fig 3A , with each concentration of lysate added there is a slow, progressive increase in FPA levels. In
contrast, when free thrombin is incubated in plasma, FPA generation reaches a plateau in minutes as the
thrombin is rapidly complexed to inhibitors (Fig 3B ). Analysis of these data by repeated-measures
ANOVA indicates a highly significant (P<.0001) interaction between lysate-induced FPA generation and
time (Fig 3A ). In contrast, with free thrombin (Fig 3B ), there is no significant time effect (P=.98) once
maximum FPA release has been achieved at 5 minutes. The slow, time-dependent increase in FPA levels
produced by the lysates indicates that the thrombin associated with soluble fibrin degradation products is
enzymatically active, and is able to cleave FPA from fibrinogen despite the presence of physiological
concentrations of antiproteinases.
Figure 3. Comparison of the time-courses of FPA generation produced

by incubating clot lysates (A) or free thrombin (B) with citrated plasma.
A, Citrated plasma was incubated for 60 minutes at 37°C in the absence (
) or presence of 2.5 µL ( ), 5 µL ( ), or 10 µL ( ) of clot lysate. At the
times indicated, aliquots of plasma were removed, unreacted fibrinogen
was precipitated with ethanol, and the ethanol supernatants were assayed
for fibrinopeptide A. B, Citrated plasma was incubated for 60 minutes at
37°C with 12.5 pmol/L ( ), 25 pmol/L ( ), or 50 pmol/L ( ) human -
thrombin. At the times indicated, aliquots were removed and assayed for
fibrinopeptide A. Each point represents the mean of two experiments,
each done in triplicate.
View larger version

(15K):
[in this window]
[in a new window]
Comparison of the Activity of Thrombin Inhibitors Against Thrombin Bound to Fibrin

Degradation Products With Their Activity Against Fluid-Phase Thrombin
To compare the ability of different thrombin inhibitors to inactivate thrombin bound to fibrin degradation
products, varying amounts of clot lysate were incubated in plasma in the presence or absence of the
inhibitors and FPA generation was determined. These results were then compared with the ability of the
thrombin inhibitors to block FPA generation induced by varying concentrations of free thrombin. PPACK
(Fig 4 ), hirugen (Fig 5 ), and hirudin (Fig 6 ) are equally effective inhibitors of free and bound
thrombin because they block FPA generation mediated by free thrombin and thrombin bound to soluble
fibrin degradation products to a similar extent. Thus two-way ANOVA indicate nonsignificant differences
in the extents to which PPACK, hirugen, and hirudin inhibit FPA generation triggered by free thrombin
and clot lysates, respectively (P=.75, .14, and .85, respectively). In contrast to direct thrombin inhibitors,
heparin is less effective at inhibiting thrombin bound to fibrin degradation products than it is at blocking
the fluid-phase enzyme (Fig 7 ). Thus, two-way analysis of variance shows highly significant (P<.0001)
differences between its effect on free thrombin and thrombin bound to fibrin degradation products. For
example, 0.1 U/mL of heparin produces 70% inhibition of FPA release mediated by fluid-phase thrombin
but only inhibits thrombin bound to fibrin degradation products by 30%. A concentration of heparin of 0.5
U/mL totally inhibits FPA generation by fluid-phase thrombin. In contrast, this concentration of heparin
only blocks lysate-induced FPA generation by 70%.
Figure 4. Comparison of the inhibitory effects of PPACK
against fluid-phase thrombin (open bars) and thrombin bound to
soluble fibrin degradation products (closed bars). To determine
the inhibitory effect against fluid-phase thrombin activity, -
thrombin (0.2 to 4.0 nmol/L) was incubated with citrated plasma
for 60 minutes at 37°C in the absence or presence of PPACK at
the concentrations indicated. At the end of the incubation
period, the plasma levels of fibrinopeptide A were determined,
and the percent inhibition of fibrinopeptide A generation was
then calculated for each inhibitor concentration. To determine
the inhibitory effect against thrombin bound to soluble fibrin
degradation products, different volumes of clot lysate (2.5 to 25
View larger version (17K): µL) were incubated in citrated plasma for 60 minutes at 37°C in
[in this window] the absence or presence of varying concentrations of PPACK.
[in a new window] At the end of the incubation period, the plasma levels of
fibrinopeptide A were determined, and the percent inhibition of
clot lysate-induced fibrinopeptide A generation was then
calculated for each inhibitor concentration. Each bar represents
the mean of three separate experiments (each done in duplicate),
while the lines above the bars represent the 95% CI.
Figure 5. Comparison of the inhibitory effect of hirugen against

fluid-phase thrombin (open bars) and thrombin bound to soluble
fibrin degradation products (closed bars). The inhibitory activity
was determined as described in the legend to Fig 4 . Each bar
represents the mean of three separate experiments (each done in
duplicate), while the lines above the bars represent the 95% CI.

[in this window]
[in a new window]
Figure 6. Comparison of the inhibitory effect of hirudin against

[in this window]
[in a new window]
Figure 7. Comparison of the inhibitory effect of heparin against


[in this window]
[in a new window]
Adsorption of 125I-Labeled Fibrin Degradation Products to Thrombin-Agarose

To isolate those soluble fibrin degradation products that bind thrombin, the adsorption of these derivatives
to thrombin-agarose was examined. Approximately 60% of the 125I-labeled fibrin degradation products in
the crude clot lysates bound to thrombin-agarose (data not shown), and the bound and unbound fractions
were analyzed using SDS-PAGE followed by autoradiography (Fig 8 ). In the material that binds to
thrombin-agarose, two major bands are visualized at 195 000 D and 60 000 D, respectively. In contrast,
only the 195 000 D band is seen when the material that does not bind to thrombin-agarose is analyzed.
Figure 8. Autoradiographic analysis of 125I-labeled clot lysates. The
crude clot lysate is shown in lane 1. Lane 2 illustrates the fraction
that does not bind to thrombin-agarose, whereas lane 3 shows the
material eluted from thrombin-agarose.

[in this window]
[in a new window]
Gel Filtration of Fibrin Degradation Products That Bind to Thrombin-Agarose

To further characterize the fibrin degradation products that bind and do not bind to thrombin-agarose, the
material that bound to this absorbant was subjected to chromatography on Sephacryl S300 HR to separate
the products according to their molecular weight. As illustrated in Fig 9 , the major protein peak (peak A)
has a molecular weight of 225 000 D, based on its elution profile, while the second smaller peak (peak
B) has a molecular weight of 60 000 D. The peak protein-containing fractions from the gel filtration
column were collected, pooled as indicated, and then further characterized by PAGE and immunoblot
analysis.
Figure 9. Gel filtration profile of clot lysate separated on a

Sephacryl S300R column. The elution positions of standard
proteins with known molecular weights are shown by the
vertical lines. Two-milliliter fractions were collected and pooled
as indicated.

[in this window]
[in a new window]
Characterization of Fibrin Degradation Products

Those fibrin derivatives that bind to thrombin-agarose were identified using a combination of PAGE and
immunoblot analysis. Electrophoresis was performed under both dissociating and nondissociating
conditions. PAGE analysis under nondissociating conditions indicates that the peak eluting at 225 000 D
(Fig 9 , peak A) consists mainly of (DD)E complex because there is a 255 000 D band (Fig 10A , lane 2)
that is recognized both by the antibody against DD (Fig 10B , lane 2) and by the antibody against
fragment E (Fig 10C , lane 2). In contrast, in the presence of SDS, this material separates into two bands
at 195 000 and 60 000 D, respectively (data not shown). The higher molecular weight material is
recognized by the monoclonal antibody against DD, whereas the antibody against fragment E primarily
reacts with the lower molecular weight band.
Figure 10. PAGE and immunoblot analysis of the fibrin

degradation products done under nondissociating conditions. A,
Gel was stained with Coomassie blue. The separated proteins
also were electrophoretically transferred to nitrocellulose
View larger version (16K): membranes, which were then reacted with a monoclonal
[in this window] antibody against DD (B) or a monospecific antibody against
[in a new window] fragment E (C). Lane 1 shows the whole lysate, whereas lanes 2
and 3 represent the gel filtration peaks A and B, respectively,
illustrated in Fig 9 .
The lower molecular weight peak recovered from the gel filtration column (Fig 9 , peak B) migrates as a
major band at 55 000 D (Fig 10A , lane 3), which is recognized by the antibody against fragment E (Fig
10C , lane 3) and not by the antibody against DD (Fig 10B , lane 3), indicating that this is fragment E.
The fibrin degradation products that do not bind to thrombin-agarose (Fig 8 , lane 2) also were
characterized by immunoblot analysis (data not shown). This material consists of DD because the 195 000
D band is recognized by the monoclonal antibody against DD. Little or no fragment E is present since a
60 000 D band is not seen in Fig 8 , nor is a band of this molecular weight visualized on immunoblot
analysis using the antibody against fragment E.
Top
Abstract
Introduction
Discussion Methods
Results
Discussion
It is well established that thrombolytic therapy induces a procoagulant state References
characterized by elevated plasma levels of FPA consistent with increased thrombin
activity.1 2 3 4 5 Using plasma levels of FPA as an index of unopposed thrombin
activity, like Mirshahi and colleagues11 we have demonstrated that a lysing clot
generates more FPA than an intact thrombus (Fig 1 ). This is not due to direct, t-PA–mediated release of
FPA from fibrinogen because only small amounts of FPA are generated when plasma is incubated with t-
PA in the absence of a clot (Fig 1 ). Furthermore, our observation that there is ongoing FPA generation
after a clot is removed from t-PA–containing plasma (Fig 2 ) indicates that a soluble factor is a more
important mediator of this phenomenon than the progressive exposure of clot-bound thrombin as has been
proposed in the past.11 This concept is supported by the finding that clot lysates produce concentration-
dependent generation of FPA when incubated in plasma (Fig 3 ).
The pattern of FPA generation produced by clot lysates (Fig 3A ) is different from that seen with
solution-phase thrombin (Fig 3B ). Thus FPA release from fibrinogen reaches a plateau in minutes when
free thrombin is added to plasma because the enzyme is rapidly inhibited by plasma antithrombins (Fig
3B ). In contrast, the lysates produce slow progressive FPA generation throughout the incubation period
(Fig 3A ). These findings indicate that thrombin associated with the fibrin degradation products is not
only enzymatically active but also is protected from inactivation by plasma antiproteinases. Like the time
course of clot lysate-induced FPA generation, there is ongoing FPA generation when clots are removed
from t-PA–containing plasma indicating that release of free thrombin from the degrading clot is not
responsible for the increased FPA generation produced by a lysing thrombus. This concept is further
supported by the observation that heparin is considerably less effective at inhibiting lysate-induced FPA
generation than it is at blocking FPA release mediated by fluid-phase thrombin (Fig 7 ).
Thrombin binds to the E domain of the degradation products of cross-linked fibrin because (DD)E
complex and fragment E bind to thrombin-agarose, whereas D-dimer does not (Fig 8 ). These findings
indicate that like its interaction with fibrinogen, thrombin binds to a site within the N-terminal disulfide
knot of fibrin. Our results are consistent with those of Meh et al,29 who identified a low affinity thrombin
binding site on fibrin fragment E. The amino-terminal ß15 to 42 sequence appears to be an important
component of this site because fibrin molecules lacking this sequence showed diminished binding of
thrombin.29 However, the -chain may also be involved because a synthetic analogue of fibrinogen
residues 27 to 50 inhibits thrombin-induced clotting of fibrinogen,30 presumably by competing with
fibrinogen for thrombin binding. In addition to the low affinity thrombin binding site on the E domain,
Meh and colleagues29 also identified a high affinity thrombin binding site on the D-domain of '-chains, -
chain variants with highly anionic carboxyl-terminal sequences. Our observation that D-dimer does not
bind to thrombin-agarose raises the possibility that the high affinity binding sites on the '-chain of fibrin
is released early during the course of plasmic-mediated degradation of fibrin. The thrombin binding site
may be particularly susceptible to plasmic cleavage because of the Arg residue at its amino-terminal.29
Further support for the concept that thrombin binds to (DD)E comes from the studies of Francis and
colleagues,31 who demonstrated that thrombin found in plasmic digests of cross-linked clots was
associated with (DD)E and larger (DD)E-containing fragments. In keeping with our findings, these
authors also showed that thrombin bound to these fragments is enzymatically active. However, their
studies of thrombin activity were done in buffer systems, whereas ours were done in plasma. By
performing the experiments in a plasma system, we have demonstrated that thrombin bound to fibrin
degradation products is not only enzymatically active, but also is protected from inactivation by fluid-
phase inhibitors. Just as thrombin bound to fibrin degradation products is protected from inactivation by
circulating antithrombins, it also is less likely to be complexed by thrombomodulin lining the
microcirculation.
Whereas rate-limited diffusion could explain why circulating antithrombins are unable to inactivate
thrombin bound to fibrin within the interstices of an intact clot, this cannot account for their inability to
inhibit thrombin bound to soluble fibrin fragments. Instead, our findings suggest that the active site of
thrombin undergoes a conformational change when the enzyme binds to fibrin thereby limiting its
reactivity with its naturally occurring macromolecular inhibitors. This allosteric change may explain why,
Ro 46-6240, a small molecule that interacts with the active site of thrombin, appears to inhibit clot-bound
thrombin to a greater extent than free thrombin.32 However, the conformational change induced by fibrin
binding does not limit thrombin's reactivity with all active-site inhibitors because PPACK inhibits FPA
generation produced by clot lysates and free thrombin equally well (Fig 4 ).
Like thrombin bound to an intact fibrin clot, thrombin bound to soluble fibrin degradation products is
relatively protected from inhibition by heparin (Fig 7 ). Thus, 0.1 U/mL heparin produces 70%
inhibition of FPA release mediated by fluid-phase thrombin but only inhibits lysate-induced FPA
generation by about 30%. Even when heparin is used in a concentration of 0.5 U/mL, which is above the
therapeutic range of 0.2 U/mL to 0.4 U/mL,33 it only produces about 70% inhibition of lysate-induced
FPA generation. At a heparin concentration of 1 U/mL, a level which may be achieved after a 5000 U
bolus of heparin is given to patients,34 there still is incomplete inhibition of lysate-induced FPA
generation. These findings may explain why FPA levels remain elevated in some patients given
thrombolytic therapy, even in the face of therapeutic doses of heparin.5 35 36 In contrast, any free thrombin
generated as a result of plasmin-mediated activation of coagulation would be readily inhibited by heparin
(Fig 7 ) making this a less plausible explanation for the heparin-resistant increase in FPA levels in the
setting of thrombolytic therapy.
Comparison of the results of this study with our previous work13 indicates that thrombin bound to soluble
fibrin degradation products is less protected from inhibition by heparin than thrombin bound to intact
fibrin clots. For example, 0.2 U/mL heparin, a concentration that totally blocks FPA release by fluid-
phase thrombin, inhibits lysate- and clot-induced FPA generation by 52% and 20%, respectively. These
findings are consistent with the results of Prager and colleagues,37 who demonstrated that FPA generation
triggered by whole blood clots was more readily suppressed by heparin when the clots were partially
degraded by t-PA than when they were intact.
To catalyze thrombin inhibition by antithrombin, heparin must bind to both thrombin and antithrombin.33
38 39
The relative inability of heparin to inactivate thrombin bound to intact clots11 13 and to fibrin
degradation products (Fig 7 ) suggests that the heparin binding site on thrombin is masked when the
enzyme is bound to fibrin or its soluble fragments. In contrast to heparin, the direct thrombin inhibitors
inactivate free thrombin and thrombin bound to fibrin degradation products equally well (Figs 4 to 6 ),
suggesting that their sites of interaction remain accessible when the enzyme is bound to fibrin derivatives.
It is well established that thrombin's interaction with fibrin involves a site distinct from the catalytic
center.40 41 42 43 44 Since PPACK binds to the active site of thrombin, wherein it alkylates the active center
histidine,45 it is not surprising that it is an effective inhibitor of thrombin-bound to fibrin degradation
products. In contrast to PPACK however, hirudin not only interacts with the active site of thrombin but
also binds to the noncatalytic substrate-binding domain on thrombin known as anion-binding exosite 1,46
whereas hirugen interacts exclusively with exosite 1.18 If thrombin binds to fibrin or fibrin degradation
products via exosite 1,40 41 42 43 44 the observation that hirudin and hirugen inhibit free thrombin and
thrombin bound to soluble fibrin fragments equally well suggests that the affinity of these agents for
exosite 1 on thrombin is greater than thrombin's affinity for fibrin degradation products.
Hirudin inactivates free thrombin and thrombin bound to fibrin degradation products equally well (Fig 6
). In contrast, we previously reported that hirudin is somewhat limited in its ability to inhibit thrombin
bound to plasma clots.13 The limited ability of hirudin to inactivate clot-bound thrombin could reflect rate-
limited diffusion of hirudin into the interstices of the clot. Thus with a molecular mass of 7000, hirudin
may enter the clot less readily than PPACK whose molecular mass is about 500.
The finding that, like their superior activity against clot-bound thrombin,13 the direct thrombin inhibitors
also are better than heparin at inactivating thrombin bound to fibrin degradation products (Figs. 4 to 7
) provides a plausible explanation for the observation that these agents have advantages over heparin
when used as adjuncts to thrombolytic therapy in experimental animals or in humans. For example, in a rat
thrombolysis model, hirulog was better than heparin at accelerating t-PA–induced thrombolysis and
preventing thrombotic reocclusion.47 Similar results have also been obtained in humans. Thus when
hirulog was compared with heparin as adjunctive therapy to streptokinase in patients with acute
myocardial infarction, coronary artery patency rates at 90 and 120 minutes were significantly higher in
hirulog-treated subjects than in those given heparin.48 49 Likewise, early patency rates also appear to be
higher when hirudin was used in conjunction with t-PA than when heparin was given.50 51
Selected Abbreviations and Acronyms
FPA = fibrinopeptide A
PPACK = D-Phe-Pro-ArgCH2Cl
t-PA = tissue plasminogen activator
Acknowledgments
The authors thank Dr J. Hirsh for helpful discussion and critical review of this paper, and S. Crnic for
typing the manuscript. We are indebted to Prof R. Roberts and J. Roberts for their help with the statistical
analyses. This work was supported by grants from the Medical Research Council of Canada and the Heart
and Stroke Foundation of Ontario. Dr Weitz is a Career Investigator of the Heart and Stroke Foundation of
Ontario.
Received July 9, 1997; revision received October 8, 1997; accepted October 13,
1997.
Top
Abstract
Introduction
References Methods
Results
Discussion
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Thrombin converts singlet oxygen (1O2)-oxidized fibrinogen into a soluble t-PA cofactor
A new method for preparing a stimulator for functional t-PA assays
Thomas W. Stief, Volker Kretschmer, Britta Kosche, Manfred O. Doss and Harald Renz
Abstract
Activated phagocytes, particularly polymorphonuclear leukocytes (neutrophils), by means of oxidative
photonic burst, i.e., the combined activation of NADPH-oxidase and myeloperoxidase, generate large
amounts of oxidants of the hypochlorite/chloramine type that are an important physiologic source for the
nonradical, photon-emitting oxidant singlet oxygen (1O2), which (in the dark blood stream) is both a
signal and an agent of defense against bacteria or fibrin. 1O2-oxidized fibrinogen or oxidized fibrin
monomer has previously been shown to be unpolymerizable, and methionine to methionine sulfoxide-
oxidized fibrinogen occurs in circulating blood. The present study demonstrates that thrombin converts
oxidized fibrinogen into a soluble stimulator of tissue-type plasminogen activator (t-PA). After addition
of 0.1 IU thrombin to 25 µl oxidized normal human plasma and an incubation time of 10 min (room
temperature), t-PA activity increases about 20-fold when compared with oxidized plasma without the
addition of thrombin. Thus, since oxidized fibrin monomer is a t-PA cofactor, thrombin-degraded
oxidized fibrinogen can be used as a stimulator in functional t-PA assays.
Plasma Fibrinogen Levels in 1,016 Regular Blood Donors
I. The Influence of Age and Sex on Mean Values and Percentiles
1. O. Weisert,
2. M. Jeremic
Article first published online: 5 MAR 2009
DOI: 10.1111/j.1423-0410.1974.tb02405.x
© 1974 Blackwell Publishing Ltd
Issue
Vox Sanguinis
Volume 27, Issue 2, pages 176–185, August 1974
Additional Information(Show All)
How to CiteAuthor InformationPublication History
How to Cite
Weisert, O. and Jeremic, M. (1974), Plasma Fibrinogen Levels in 1,016 Regular Blood Donors. Vox
Sanguinis, 27: 176–185. doi: 10.1111/j.1423-0410.1974.tb02405.x
Author Information
1. State Laboratory of Microbiology, Department of Blood Transfusion and Immunohaematology,
Lillehammer
*Correspondence: O. Weisert,
*Correspondence: State Laboratory of Microbiology, N-2600 Lillehammer (Norway)
Publication History
1. Issue published online: 5 MAR 2009
2. Article first published online: 5 MAR 2009
3. Received: October 8, 1973; accepted: February 5, 1974.
Top of Form
Abstract. Plasma fibrinogen levels were measured in 676 male and 340 female regular blood doncrs by a
highly reproducible and accurate assay method. No sex differences were found for any age group
compared. Fibrinogen levels rose markedly with increasing age. The mean values in males rose from
271.6 in the youngest (18–29 years) to 347.8 mg% in the oldest (60–69 years) age group (p < 0.001), and
the values for corresponding female groups were 276.7 and 337.9 mg% (p < 0.001). The data indicate the
need to refer to age specific level whenever fibrinogen is used as a parameter in medicine. The problems
of ‘normal range’ and percentiles are discussed.
European Journal of Applied Physiology and Occupational Physiology

Volume 55, Number 4, 374-380, DOI: 10.1007/BF00422736
Blood platelet activation and increase in

thrombin activity following a marathon race
L. Röcker, W. K. Drygas and B. Heyduck
Abstract
To see whether strenuous prolonged exertion increases blood platelet activation and thrombin activity in
healthy well-trained men, 16 male amateur runners (mean age 31,8) were studied. A marathon race (mean
time 2 h 44 min 30 s) caused a significant increase in plasma -thromboglobulin ( -TG), platelet factor
4 (PF4), fibrinopetide A (FPA) and factor VIII (F VIII) activity. Sixty min after exertion -TG and F VIII
activity were still significantly elevated. FPA continued to rise, reaching peak values 60 min after the run.
22 h after finishing the race F VIII activity was still significantly elevated. The study has demonstrated
the great inter-individual variability of marathon race-induced haemostatic changes. The elevation of -
TG varied from 42% to 156%, F VIII from 112% to 625%, and in three runners FPA reached more than
900% of its pre-exercise value. In some individuals the haemostatic changes observed could be
potentially unfavourable for coronary heart disease prevention.
Key words Prolonged physical exercise - Coagulation - Fibrinopeptide A - Platelet specific

proteins ( -TG, PF4) - Coronary heart disease
Supported by Bundesinstitut für Sportwissenschaft, Köln

Soluble Fibrin Assays

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2001 Dec;27(6):657-66.

SUMMARY OF THE INVENTION

Accordingly, it is an object of the present invention to provide an improved method of assaying

DETAILED DESCRIPTION OF THE DISCLOSED EMBODIMENT

Pluronic® F-68 has the following specific formula: HO(C 2 H 4 O) b (C 3 H 6 O) a (C 2 H 4 O) b H

0.25 0.51 3.8 1.3 8.4

b. measuring the rate of plasmin substrate cleavage;

c. correlating this with the amount of soluble fibrin in the sample.

Fibrin Degradation Products

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Competition of interest: none.

Volume 35, Number 6, 465-479, DOI: 10.1007/BF00996693

Clinical Investigation and

Key Words: plasminogen activators • thrombolysis • thrombus • anticoagulants

Figure 1. Clot-induced fibrinopeptide A generation in the

Figure 2. Time courses of concomitant t-PA-induced release of 125I-labeled

Figure 3. Comparison of the time-courses of FPA generation produced

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Comparison of the Activity of Thrombin Inhibitors Against Thrombin Bound to Fibrin

Figure 5. Comparison of the inhibitory effect of hirugen against

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Figure 7. Comparison of the inhibitory effect of heparin against

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Adsorption of 125I-Labeled Fibrin Degradation Products to Thrombin-Agarose

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Gel Filtration of Fibrin Degradation Products That Bind to Thrombin-Agarose

Figure 9. Gel filtration profile of clot lysate separated on a

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Characterization of Fibrin Degradation Products

Figure 10. PAGE and immunoblot analysis of the fibrin

Selected Abbreviations and Acronyms

European Journal of Applied Physiology and Occupational Physiology

Blood platelet activation and increase in

Key words Prolonged physical exercise - Coagulation - Fibrinopeptide A - Platelet specific

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