Fibrinolysis
July 13, 2003
08:00 to 12:00
Hall 9
The International Convention Center, Birmingham
Chair: P. Declerck, Belgium
Co-chairs: N. Booth, UK; C. Dempfle, Germany; Dirk Hendriks, Belgium;
O. Matsuo, Japan; M. Nesheim, Canada
Procarboxypeptidase U / TAFI
Dr. Nesheim discussed the development and characteristics of a functional
assay for activated TAFI (TAFIa) . It is based on the down regulation
by TAFIa of the cofactor activity of high molecular weight, soluble fibrin
degradation products in the activation of a fluorescent plasminogen derivative
by vampire bat plasminogen activator. The standard curve of the assay spans
the concentration range from 0 to 200 picomolar; thus the assay is
very sensitive. The basal level of TAFIa was measured in a group of healthy
young volunteers and was found on average to be 11 picomolar. TAFIa levels
as high as 2000 picomolar were measured in the plasmas of baboons infused
with thrombin at low levels. With the cessation of thrombin infusion,
TAFIa levels decayed with a half life of about 20 minutes. A corresponding
prolongaton of in vitro clot lysis time was observed. These baboon
experiments show that TAFI can be activated in vivo in a primate to levels
that substantially effect fibrinolysis.
Two polymorphisms i.e. Ala/Thr at position 147 and Thr/Ile at position 325
in the coding region of TAFI have been reported. Dr. Gils described
the development and characteristics of two monoclonal antibody based ELISAs
for measurement of TAFI/proCPU antigen in plasma. One (MA-T12D11/MA-T28G6-HRP)
appeared to be genotype independent whereas the other (MA-T32F6/MA-T9G12-HRP)
revealed to be highly genotype 325 dependent, i.e. it does not recognize
the Ile(325) variant. A polyclonal based commercial assay appeared to be
partially genotype dependent (i.e. a decreased sensitivity towards the Ile(325)
variant). It is concluded that the interpretation of currently available
data on TAFI antigen should be interpreted with caution and should be done
in conjunction with the genotyping. Dr. Alessi presented additional data
using these assays in the analysis of 1000 patient samples confirming the
genotype dependency.
Dr. Guimaraes reported the determination of TAFI levels in plasma from 92
healthy individuals making use of 3 distinct assays, namely, a commercial
chromogenic assay – Actichrome® TAFI Activity assay (American Diagnostica),
an in-house developed rocket immunoelectrophoresis – (electroimmunoassay)
using rabbit polyclonal antibodies (van Tilburg et al. 2000) and an ELISA
using commercial sheep polyclonal antibodies against TAFI (Affinity Biologicals
Inc.). Each individual was also genotyped for the – 438 A/G and 1040 C/T
polymorphisms. The association between TAFI levels and genotype in each assay
was evaluated and a linear regression and Bland-Altman agreement analysis
in the whole sample group and in genotype sub-groups was performed. The
results demonstrate that artefacts may arise when measuring TAFI antigen
levels by ELISA with a lower response for the Ile-325 variant (T allele).
No artefacts were found with the Actichrome“ TAFI assay and with the electroimmunoassay.
The latter two assays support, however, a genotype-related variation of TAFI
concentration.
The overall problems on genotype dependency of TAFI assays was
further discussed. It was concluded that this raises problems in interpretation
of data and that there is a need to further explore these observations. It
was concluded that in an intial phase approximately 100 genotyped plasma
samples should be analysed by various expert laboratories using various methods
(coordination Paul Declerck). The outcome of these data will be reported
at the 2004 meeting.
D-dimer
Dr. Dempfle showed data on a new method for standardization of D-dimer antigen
assays, which is not based on consensus values or pooled plasma samples.
He discussed the possibility of using a well-characterized purified
DD-domain containing model peptide, generated from fibrin by using a novel
endoproteinase, in the calibration process. The model peptide would be used
to calibrate existing standards with regard to their content of this dimerized
D-domain. The meeting agreed that expressing all calibrators in ‘dimerized
D-domain’ may be a first step in harmonization of D-dimer levels as measured
by different assays. Therefore all interested attendants were asked to contact
Dr. Dempfle and to provide him with their currently used calibrators for
analysis. These data and the progress in the harmonization will be reported
at the 2004 meeting.
Dr. Dempfle also reported on his study of the measurement of D-dimer in animal
samples. It could be concluded that very few of the available commercial
assays tested recognized to a certain extent D-dimer from animal origin (
mainly horse, rat and bovine ). Again interested scientists are urged to
contact Dr. Dempfle in order to look for assays/reagents that may recognize
D-dimer in rabbit, sheep, pig, cat or dog and to provide material (plasma)
to generate calibrators for D-dimer from rat, horse and bovine origin.
Dr. Wada reported on an assay that reacts preferentially with granulocyte
elastase-digested XDP (GE-XDP). Increased levels of GE-XDP were observed
in severe trauma, burn and infections, which were not associated with disseminated
intravascular coagulation (DIC). There were good correlations between the
levels of D-dimer and plasmin plasmin inhibitor complex (PPIC) in patents
with DIC, indicating that the plasmin mediated fibrinolysis predominantly
functioned in these patients. On the contrary, no good correlations were
found between GE-XDP and PPIC in patients with severe infections, trauma
and burn without DIC. Since GE-XDP is a distinct criteria from the D-dimer
(or p-XDP), measurement of GE-XDP may provide a new tool for the analysis
of intravascular fibrin degradation.
Standardization and Methodology
Standardisation of bioassay methods is a means of reducing interlaboratory
variability when measuring biological parameters. Dr. Longstaff discussed
the development of a method for measuring potencies of plasminogen
activators which has many advantages over current methods and would make
a good reference method. The method is precise and accurate (it has
been field tested in 2 international collaborative studies with excellent
results). The assay system includes fibrin and can be compared to the
physiological situation during thrombolysis. However, the inclusion
of a chromogenic substrate means that good quality data can be collected
and results can be expressed in SI units (molar plasmin concentration generated
per second). This allows comparison of different plasminogen activators,
which is a particular advantage in fibrinolytic standardisation due to the
proliferation of molecular variants and the history of arbitrary units. The
meeting agrees that this method may well suit the requirements of a reference
method. Before making a final decision this method will be evaluated by different
labs. Dr. Longstaff will coordinate this study and report the findings at
the 2004 meeting.
Dr. Babu discussed current methods of plasminogen-based fibrinolysis methods
for the quantitation of plasminogen activators. He reported the
discovery of a bacterial enzyme with a direct fibrinolytic action and with
potential clinical application. He raised the problem of expressing
the activity of such compounds in defined units.
Dr. Antovic presented data on an assay for the evaluation of overall fibrinolytic
potential (OFP) derived from the overall hemostatic potential ( OHP). The
meeting expressed its concerns on the overall applicability of such a test
since, dependent on the reagents used to perform this test and dependent
on the possible presence, in (patient) plasma, of drugs that affect coagulation
as well as fibrinolysis, the outcome may not necessarily reflect the
real physiological fibrinolytic potential. More detailed characterization
of this assay would be needed.
General Discussion and topics for 2004
The meeting agreed that its activities on the methods for measurement of
proCPU/TAFI and D-dimer should be continued. The progress on the planned
proCPU/TAFI study and on the D-dimer standardization should be included.
In addition an update on the evaluation by various laboratories, of the method
described by Dr. Longstaff will be presented. Any of the (co-)chairs can
be contacted for further suggestions concerning the meeting in 2004.
The meeting was attended by 100-130 people including all (co-)chairs.
The meeting was closed at 11.30 pm.