Factor XIII
Saturday July 12th, 2003
9.00 to 13.00
Hall 9
Birmingham International Conference Centre
Chair: RAS Ariëns-UK
Co-Chairs: A Ichinose-Japan, P Bishop-USA
Active Members: CS Greenberg-USA, L Muszbek-Hungary
Apologies were received from Dr Greenberg. All other chairs and members were
present. The FXIII SSC meeting had a full programme, all speakers and approximately
130 people in the audience attended the meeting. The presentations were of
a high standard and ranged from novel methodology including gene knockout,
atomic force microscopy, site-directed mutagenesis to study protein interactions
and protein chemistry to development of the first international standard
for FXIII. The presentations of Dr Lim and Dr Raut were exchanged, as Dr
Raut had to be at another SSC meeting in the late morning.
The meeting was opened by Dr Ariëns, University of Leeds, UK,
who put forward for discussion the issue of whether the FXIII SSC should
continue as a separate committee or whether it could merge with that of fibrinogen
to a combined Fibrinogen and FXIII subcommittee. Dr Ariëns pointed out
that most subcommittees relate to work in areas of research that involve
many proteins and genes such as that of fibrinolysis, coagulation inhibitors
and contact activation, and that there are only three subcommittees out of
20 that are dealing with one coagulation factor in isolation: the SSC for
FXIII, fibrinogen and von Willebrand factor. Dr Ariëns suggested that
it might be a good idea to combine the FXIII and fibrinogen SSC in one subcommittee
as the two proteins are functionally closely involved in the final stages
of the coagulation cascade. It was also suggested that thrombin could be
included in this new SSC as well. Advantages of a possible merger would be
that there would be a larger platform to discuss novel ideas regarding fibrinogen,
FXIII and thrombin, with people present from various expertise. Work relating
to standardization would be easier to perform and to discuss. Issues regarding
standardization for FXIII, fibrinogen and thrombin are more often related
than not, such as in the case of fibrin glue for example, that contains both
FXIII and fibrinogen.
The idea for a merger with the fibrinogen SSC was met by a short but lively
discussion. Dr Lord, University of North Carolina, USA, Acting Chair of the
fibrinogen SSC, was supportive of a possible merger and mentioned that the
idea would also be discussed in the fibrinogen subcommittee to be held the
same afternoon. Dr Bishop, Zymogenetics, expressed concern that there would
not be enough space on the agenda of a merged subcommittee to discuss all
relevant issues. Dr Muszbek, Debrecen University, Hungary, said that the
SSC for FXIII and fibrinogen used to be combined several years ago and that
it was then decided to separate them. He expressed concern that issues relating
to fibrinogen would dominate meetings of a merged SSC. Dr Kohler, University
of Bern, Switzerland, argued the opposite, and suggested FXIII issues may
dominate those of fibrinogen. Overall, there seemed to be concern regarding
dominance of one of the factors over the other and a possible lack of agenda
space in meetings of a merged SSC. Dr Ariëns suggested that space issues
could possibly be solved with longer agendas if necessary. Dr Muszbek suggested
organizing a trial of a combined SSC, by holding a combined meeting for FXIII
and fibrinogen at the next SSC. This suggestion was welcomed by Dr Ariëns.
The agenda item was closed with a call to everyone in the audience to express
their opinion on the matter by filling in a questionnaire form, so that a
recommendation on this issue in some form or the other could be presented
by the chair at the SSC Business meeting.
The main programme of the FXIII SSC was opened with a presentation from Dr
Ichinose, Yamagata University, Japan, on abortion in FXIII gene-knockout
mice. Dr Ichinose started his presentation with two minutes silence in honour
of the work of Dr Peter Steinert who recently passed away. Dr Steinert has
characterised many of the transglutaminase genes. Dr Ichinose presented data
on the molecular biology of FXIII deficiency. There are approximately 500
cases of FXIII A-subunit deficiency known. Mutations occur over the whole
molecule. There do not appear to be any particular hotspots for mutations
in this gene. B-subunit deficiency is rare, currently only 4 cases are known
with a characterized mutation in the B-subunit gene. FXIII deficiency is
associated with severe bleeding diathesis, in particular umbilical cord,
intracraneal and peritoneal bleeding, with poor wound healing and with miscarriage
in pregnancy. Dr Ichinose has developed a FXIII A-subunit gene-knockout model
in mice for the study of miscarriage in FXIII deficiency. The main symptoms
in the A knockout mice were vaginal and intrauterine bleeding. Dr Ichinose
concluded from this mouse model that spontaneous abortion in FXIII deficiency
is caused mainly by bleeding.
Dr Kohler, University of Bern, Switzerland, presented data on the
role of FXIII in vascular disease. Dr Kohler mentioned the importance of
distinguishing between chronic and acute vascular disease. The first interest
in the role of FXIII in vascular disease was developed by findings of an
association between FXIII Val34Leu and vascular disease. Recent studies suggest
that this polymorphism affects fibrin structure function through an interaction
of FXIII Val34Leu and fibrinogen levels. Other studies have shown an interaction
between insulin resistance and FXIII subunit levels, B-subunit levels in
particular. Factor XIII levels have been shown to relate to the extent of
coronary artery disease. In the Northwich Park Heart Study II, FXIII A-subunit
levels were reduced in the acute phase of the disease, with no difference
in B-subunit levels. The reduction in FXIII A is possibly due to activation
and increased consumption. This is in agreement with a study in acute stroke,
where FXIII A-subunit was reduced in patients with poor survival and the
level inversely correlated with F1+2. In another study of acute pulmonary
embolism, FXIII A-subunit levels were also reduced, with normal B-subunit
levels. The relationship was linear with occlusion rate, and Dr Kohler suggested
that FXIII consumption is related to the thrombus size. The relationship
between FXIII and vascular disease indicate the importance of standardization
of the assays to measure FXIII levels (activity, A-subunit and B-subunit).
Results from a pilot study for the standardization of FXIII activity assays
were reported by Dr Raut, National Institute for Biological Standards
and Control, UK. Phase 4 (out of 7) of the study was performed. Data were
obtained on two FXIII concentrates and 1 fibrinogen concentrate. The aims
of were to compare the Berichrom FXIII activity assay and the new Pentapharm
FXIII activity assay. The two assays use different principles to measure
FXIII activity, in the Pentapharm assay incorporation of pentylamine into
fibrinogen by FXIII is measured, whereas in the Berichrom assay the amount
of ammonia released during the cross-linking reaction is measured. Another
aim was to analyse assay differences in the presence or absence of fibrinogen.
Collaborating laboratories were that of Dr Muszbek, Dr Seitz and Dr Barrowcliffe.
It was found that differences between the two assays were large when buffer
was used as diluent, whereas differences were small with FXIII deficient
plasma as diluent. There was a high variability of measurements in fibrinogen
concentrates. Dr Raut ended with an update on the development of the first
international standard for FXIII. Recombinant FXIII was suggested as possible
additional standard material, and a resource laboratory for this material
would need to come forward. To establish an International standard for FXIII,
12-20 laboratories will have to participate and laboratories willing to participate
are asked to come forward. Please contact the chair of this SSC or Dr Raut
if you are interested. In the discussion, Dr Muszbek suggested that the difference
between variability between buffer and deficient plasma as diluent is due
to the presence of fibrinogen in plasma, which greatly enhances activation
rates of FXIII. This may lead to differences between assays that employ different
thrombin concentrations and have different sensitivity to the activation
step.
Dr Weisel, University of Pennsylvania, USA, presented data on the
effect of fibrin a- and g-chain cross-linking by activated FXIII on fibrin
structure and function. Recombinant Aa251 fibrinogen lacks the aC domain
and its connector polypeptide, and is therefore deficient of the a-chain
FXIII cross-linking sites. Dr Weisel showed data using this fibrinogen to
quantify the relative effects of a- and g-chain cross-linking on fibrin structure
and elastic properties. Experiments were performed with purified FXIII from
pooled plasma, which was fully activated to avoid any effects of the Val34Leu
polymorphism on fibrin structure and stiffness. Recombinant Aa251 fibrinogen
produced clots with smaller pores, thinner fibers and increased number of
branchpoints when compared with normal fibrinogen. Cross-linking by pooled
plasma FXIII had relatively little effect on overall fibrin structure, but
there was a dramatic effect on stiffness of the clot. Cross-linked Aa251
fibrin was much less stiff and showed more non-elastic slippage of the protofibrils
than cross-linked normal fibrin. This effect was due to the absence of a-chain
cross-linking in Aa251 fibrin. Gamma chain cross-linking also contributed
to clot stiffness as Aa251 fibrin showed reduced stiffness in the absence
of FXIIIa when compared to Aa251 fibrin in the presence of FXIIIa, the latter
of which is expected to show normal -gamma chain cross-linking. The effects
of alpha and gamma-chain cross-linking were also noted on the lysis speeds
of the fibrin clots as analysed by laser scanning confocal microscopy.
After a short coffee break of 15-20 minutes, the meeting was resumed with
a presentation on the use of FXIII peptides to investigate thrombin-FXIII
interaction by Dr Maurer, University of Louisville, USA. Using a variety
of novel methods, including 2D NOESY spectra, MALDI-TOF and pulsed alkylation
mass spectrometry, Dr Maurer investigated the relative roles of P1-4 of the
FXIII activation peptide in the interaction with thrombin. It was found that
the P4 residue, which is the site of a common Val34Leu polymorphism, plays
an important role in the interaction with thrombin. A change of Val to Leu
change at this position dramatically improved the kcat as well as Km. Substitution
of P4 with Ile or Ala improved the Km but the kcat worsened. The future plans
are to apply the same methodology to study interaction with the whole FXIII
protein, possibly as a recombinant. The aim is to obtain a fuller understanding
of the conformational changes that occur in FXIII upon binding to thrombin.
In the next presentation by Dr Philippou, Imperial College, UK, FXIII
and thrombin interactions were investigated from the thrombin point of view.
Dr Philippou used a library of 58 thrombin mutants encompassing a total of
78 mutated surface exposed residues, to study their interaction with FXIII.
The mutants were screened with a pentylamine incorporation assay using casein,
fibrinogen or fibrin as substrate, and the results were confirmed by kinetic
analysis of the activation peptide release by HPLC. The results showed that
residues R78/R180/D183, W50, E229, R233 of thrombin were involved in direct
interaction with FXIII during its activation. Additional thrombin residues
H66, Y71, N74 were involved in binding to fibrin to cause the fibrin-enhancement
effect of FXIII activation. Thrombin has many substrates, and general implications
were drawn from these findings for the mechanism by which thrombin chooses
its substrate. It was concluded that thrombin’s fate is directed primarily
by competition between its cofactors. In the specific case for FXIII for
example, activation is directed by competition between fibrin and thrombomodulin
for their overlapping binding site on thrombin. A model was proposed to explain
the enhancement effect of fibrin on FXIII activation.
Dr Reynolds from Zymogenetics, USA, presented the first data regarding
pharmacokinetics and safety of a recombinant FXIII preparation in healthy
human volunteers. Recombinant FXIII A-subunit was produced in yeast, and
administered at doses of 2, 5, 10, 25 and 50 u/kg to 8 healthy subjects.
Placebo was administered in 2 subjects. The highest dose of Rec FXIII was
chosen such that it would increase the baseline level of FXIII by 100%. Levels
were monitored using the Berichrom FXIII activity assay. Halflife of FXIII
after injection was 9-11 days, which is in good agreement with previous findings
using plasma substitution. There was a specific increase of FXIII of 1.77%
per 1U/kg. The product was found to be safe as adverse effects such as headache,
cramp, pain in limb, prolongation of thrombin time, abdominal pain or cough
were all absent. There was an increase in D-dimer and a slight drop in FXIII
B-subunit levels at high concentrations of recombinant FXIII.
Dr Lim, University of Leeds, UK, presented data using novel technology
of atomic force microscopy (AFM) to investigate the structure of fibrin(ogen)
and FXIII. AFM was developed in 1986 for applications in physics, but it
only since recently that it is being used more often in lifesciences. AFM
allows real-time visualization under physiological aqueous conditions at
almost unlimited resolution potential. Topographic images are generated by
a piezo-electric scanner that moves or taps a microtip over the sample. The
resolution is determined by the size of the tip with which the microscope
scans or ‘taps’ the surface. Current tips used are around 10-30 nm in diameter,
but future usage of tips of around 1 nm using carbon nanotubes should improve
resolution. For comparison, length of a fibrinogen molecule is around 45
nm. Further advantages of AFM are that it can be used to measure attractive
and repulsive forces on a single molecule level. Potential limitations of
AFM are that the sample can be altered or damaged by the tip movement. AFM
images were shown of fibrinogen molecules, showing their characteristic three-nodular
appearance. Images were also shown of fibrin polymerization at various time-frames
after the addition of thrombin, showing growing protofibrils that aggregate
laterally and branch out. The first AFM images of FXIII were produced, showing
a tetrameric structure that appears to be in agreement with the models of
tetramer association based on transmission electron microscopic images of
FXIII published by Carrell et al in 1989.
An update of the ETRO FXIII deficiency registry was presented by Dr Ivaskevicius,
Frankfurt Red Cross Blood Center, Germany. A background was given about the
structure of the FXIII A- and B-subunit genes and FXIII deficiency, which
is an autosomal recessive disorder that affects around 1 subject in every
1-3 million. The registry had 72 entries in 1996, 96 in 2000 and 109 in 2003,
with entries from a total of 20 different countries. Around 45% of the entries
have also been genetically characterized. A particular mutation in intron
E (IVS5-1 G>A) was most common, possibly due to an ancient founder effect,
as it was found in several countries. Dr Ivaskevicius continued the presentation
with data showing the usage of Denaturing HPLC (Wave technology) to screen
DNA for mutations. DHPLC proves to be a useful and rapid methodology for
screening, although some mutations are not detected. The average detection
rate is reported to be around 95%. Further characterization in healthy subjects
is required to exclude the possibility that a certain change found in the
DNA of the FXIII deficient patients is a common genetic polymorphism.
The meeting was closed at 12.55.
The questionnaire slip was returned by 48 people, of whom
17 or 35% voted in favor of a merger of the FXIII and fibrinogen SSC,
29 or 61% against and
2 or 4% said they were indifferent.