Fortunately, the factor XIII Subcommittee had more than 100 participants this year, probably because almost all of its members attended the ISTH congress, as usual. Thus, the chairman of this committee would like to suggest that the SSC meeting be associated certain closely related meetings, in order to discuss important issues in the factor XIII field with a large number of members.

1) Recent Progress in Factor XIII Science: Gene Targeting of Factor XIII.

CHARACTERISATION OF COAGULATION FACTOR XIIIA DEFICIENT MICE by Gerhard Dickneite (Germany).
A German group have established a transgenic factor XIIIA deficiency mouse model (FXIIIA knock-out (KO) mice) with an exon 7 deletion of the XIIIA gene by homologous recombination in embryonic stem cells. Transglutaminase activity in plasma was <5 % in homozygous XIIIA KO mice, no gamma-dimerization of fibrin in the plasma of the XIII KO mice could be detected. Mortality rate was higher in the XIII KO mice compared to normal mice because of bleeding episodes (one-year survival: 70 % in FXIII KO mice vs. 100 % in normal mice). When examined for the bleeding disorder in more depth, XIII deficient mice were found to have an increased bleeding time. Thrombelastography experiments demonstrated impaired clot formation in the XIII KO mice, the maximal amplitude was decreased and premature clot destruction was observed. It was concluded that these KO mice represent a good model to study the impact of factor XIII deficiency.

GENE TARGETING OF FACTOR XIII IN MICE by Akitada Ichinose, Shiori Koseki, Masayoshi Souri, Naoki Takeda, Gerhard Dickneite (Japan, Germany)
Prof. Ichinose's group have identified a number of mutations in the XIIIA and the XIIIB genes in patients' genomic DNA and also analyzed the molecular mechanisms using in vitro procedures. However, one cannot understand completely the clinical pathological mechanisms of this disease in vivo. To generate its disease model and ascertain the role of XIIIB in vivo, XIIIB knock-out (XIIIB KO) mice have been established. Both homozygous and heterozygous KO mice showed no marked difference from the wild-type mice in general appearance. Although XIIIB KO mice had somewhat prolonged bleeding time of their tail tips than the wild-type mice, this result should be reproduced by more standardized bleeding test.

As to XIIIA KO mice, three pairs of the homozygous XIIIA KO male and female mice were mated. All female mice became pregnant, and one of these mice died after 2 weeks because of massive bleeding from its vagina. Another female mouse also bled from its vagina and had abortion. These observations remind us the spontaneous absorption in human female patients with factor XIII deficiency. Further analysis of these XIII KO mice would lead to understanding the physiological and pathological functions of XIII in vivo.

2) Clinical Research on Factor XIII Deficiency

GEFFXIII: A FRENCH WORKING GROUP ON FXIII, LOOKING FOR EXTENSION AND CONNECTIONS by Dreyfus M. (GEFFXIII; Arnutti B, Barrois D, Beurrier P, Borg JY, Claeyssens S, Doki-Thonon M, Gaillard S, Garnier JM, Girardel JM, LeBerre D, Pautard B, Pernod G, pollet F, Pouzol P, Seaume , Torchet MF, Wibaut B)
A working group including all the French physicians in charge of FXIII severely deficient patients has been organized in 2000 and named GEFF XIII (Groupe d'Etudes Francophone du FXIII). This group of physicians from 16 centers conducted the first study to assess the tolerance and safety of the FXIII plasma concentrate Fibrogammin P (Centeon Aventis). Eight out of the 19 patients had previous histories of intra-cerebral hemorrhages (ICH). Seven out of 8 episodes of IHC occurred in non-treated children before the age of 11, indicating the need for a systematic prophylactic replacement therapy in these patients.

This group plans future studies on the retrospective collection of data on patients undergoing surgery, systematic characterization patients' genotype, prenatal diagnosis, standardization of the FXIII assays, etc.

3) Activation and Its Implication of "Variant A subunits" of Factor XIII.

CHARACTERIZATION OF ENHANCED CATALYTIC EFFICIENCY OF RABBIT FACTOR XIII IN COMPARISON WITH HUMAN ENZYME by Lee S.Y., Lee I.H., Oh J.T., Kim I.G. and Chung S.I. (Catholic Univ., Seoul National Univ., and Korea)
Dr. Chung purified rabbit and human enzyme and characterized their enzymatic, physicochemical and structural properties. Rabbit factor XIIIA (RXIIIA), like the human enzyme (HXIIIA), showed the same mechanism where the deamidation step (k3) during the acyl enzyme formation step was found to be rate-limiting. However, the kinetic efficiency measured by methylamine incorporation into acetylated oxidized B chain of insulin showed rabbit enzyme was significantly greater than human enzyme (V/K: 427.45 for RXIIIA; 62.12 for HXIIIA). Structure modeling of RXIIIA by a fit to the known HXIIIA 3D structure coordinates again showed a similar resemblance of active site pocket and Ca ion binding domains. In light of the report that Val34Leu variant retained greater catalytic activity, the N-terminal activation peptide domain of rabbit enzyme, which is quite heterogeneous from human, may contribute in the enhancement of catalytic efficiency in an unknown manner and correlates well with the previously reported rat enzyme catalytic efficiency and its amino acid sequence.

EFFECT OF VAL34LEU PENOTYPE ON THE ACTIVATION OF FACTOR XIII: HOW IMPORTANT IS IT? by Laszlo Muszbek (Hungary)
To address the problem regarding Val34Leu, Prof. Muszbek reported the following: As observed with purified proteins in the absence of plasma, the rate of FXIII-A cleavage and fibrin polymerization was higher with the homozygous Leu/Leu variant than with wild type (Val/Val) FXIII. However, when plasma or whole blood from patients of various genotypes were compared, Val34Leu polymorphism did not seem to be a major contributor to the speed of FXIII activation. The onset of fibrinogen clotting, i.e., the release of fibrinopeptide A and fibrin polymerization were of predominant importance in determining the time course of FXIII activation. The release of FXIII-A activation peptide always lagged behind the release of fibrinopeptide A and activated FXIII-A never appeared in the fluid phase. Prof. Muszbek concluded that the appearance of polymerizing fibrin and its accelerating effect on FXIII activation are the dominant factors that regulate the process of FXIII activation in whole blood.

FACTOR XIII VAL34LEU: RELATION TO THROMBOTIC DISORDERS by Peter J. Grant (UK)
Prof. Grant reviewed existing reports including his own on the implication of Val34Leu polymorphism of the XIIIA gene. The factor XIII genes are highly polymorphic. There are 5 common coding polymorphisms in the factor XIII A gene, of which a valine to leucine transition at residue 34 is of interest due to its vicinity to the thrombin cleavage site and its relation to thrombotic disorders. The relationship between Val34Leu and thrombosis has now been analyzed in approximately 18 epidemiological studies, of which 7 regard patients with cardiovascular disease, 6 venous thrombosis and 5 cerebrovascular disease. Several of these reports have shown that Val34Leu is protective against thrombosis in different vascular beds.

FACTOR XIII VAL34LEU: EFFECTS ON FIBRIN STRUCTURE AND FUNCTION by Robert A.S. Ariens (UK)
Factor XIIIA Val34Leu occurs three amino acids upstream of the thrombin cleavage site between arginine 37 and glycine 38. Dr. Ariens previously reported that the substitution of Val 34 with Leu accelerates the thrombin cleavage of the factor XIII activation peptide. He showed that early covalent cross-linking of the fibrin clot by factor XIII Leu34 reduced lateral aggregation of the fibrin fibers, leading to a reduction in fiber thickness from 121.0 +/- 23.9 nm to 75.7 +/- 11.3 nm and alteration in rates of fibrinolysis of the fibrin clot. The effect of Val34Leu on fibrin structure and function appeared to alter the interaction with platelets and was dependent on fibrinogen levels. Dr. Ariens concluded that Val34Leu is the first example of a mutation in the factor XIII activation peptide that alters Ttransglutaminase and fibrin structure and function.

4) Improvement of Screening Tests for Fzactor XIII.

SCREENING FOR FXIII ACTIVITY IN PLASMA by Lewis K.B., Heffernan J., Khuu Kien, Bishop P.D. (USA)
Although a variety of assays exist for screening FXIII levels in plasma samples, in many cases the reagents are not readily available and the protocols are not easily transferred to different laboratories. Rather than develop a new assay, an American has chosen to evaluate the Berichrom assay (not commercially available in the US). Where necessary, they have made simple modifications using readily available reagents. The principal modification has been to increase the thrombin concentration in the assay. A second modification has been the use of an absolute rFXIII [A2] standard rather than a pooled human plasma standard. With these modifications, FXIII activity levels have been measured in plasma from humans and other animal species.

5) Search for Standard Materials for Factor XIII.

SEEKING STANDARDIZATION MATERIALS FOR FACTOR XIII by Paul Bishop (USA)
Dr. Bishop proposed the following objectives: 1) Comparing the accuracy of factor XIII assays in common use for clinical evaluation. 2) Establishing an international standard for factor XIII activity. 3) Defining a specific activity for factor XIII. He also proposed to establish study participants and a work plan:

1) Solicit laboratories interested and willing to participate in such study.
2) Identify and designate reference plasma of normal FXIII activity (a lot of pooled normal plasma) and a FXIII deficient reference plasma.
3) Determine which assays will be employed by the various participating laboratories
4) Establish a protocol for testing the accuracy and availability and uniformity of FXIII assays in common use.
5) Discuss the possibility of providing FXIII (A2B2) plasma concentrate and & preparation of pure recombinant FXIII (A2) as an external reference.

FACTOR XIII AS A COMPONENT OF THE FIBRIN SEALANT, "BOLHEAL(R)," AND AN ASSAY FOR FACTOR XIII ACTIVITY by Hiroshi Kaetsu (Japan)
Factor XIII, one of the active ingredients of the fibrin sealant, "Bolheal(R)," is included in the fibrinogen component. Fibrinogen and factor XIII are purified individually in the production process of the fibrinogen component of Bolheal(R). Dr. Kaetsu uses the amine incorporation method as a routine assay for factor XIII activity. This method gave the dilution linearity in samples of "standard human plasma," "purified factor XIII," and "the fibrinogen component of Bolheal(R)." The linear range of the assay system was 0.05-2 units/ml for both factor XIII concentrate and fibrinogen concentrate including factor XIII. Attention needs to be paid as to whether or not the values of factor XIII activity obtained with different assay systems are comparable.

Dr. Kaetsu proposed a process to establish the reference preparations of factor XIII in which "standard plasma" is established as the first step, and thereafter "purified factor XIII standards" are evaluated, based on the value of activity in "standard plasma."

PROGRESS REPORT ON STANDARD FACTOR XIII MATERIAL by Trevor W Barrowcliffe (UK)
Last year, Dr. Barrowcliffe reported that he developed two XIII concentrates which are considerably stable at both 4 and 20 degrees when measured by a chromogenic assay. He also extended this study at higher temperatures, such as 37 and 45 degrees, where the two concentrates showed much less stability of 40-60%. Since these concentrates showed more than 90% stability, these should be used as one of candidates of XIII concentrates in the standardization study.

Dr. MacIntosh, co-chairman of the Fibrinogen Subcommittee, raised several issues regarding factor XIII materials and assay method; some fibrinogen preparations and clot solubility tests need to be?included in the future study.

6) General Discussion and Topics for 2002

Since the meeting was behind the schedule, and passed 10 min over 12:00, it was strongly suggested by a congress personnel to conclude the discussion. Accordingly, the chairman of this subcommittee just announced that factor XIII assay and material standardization activities would be continued during the year with reports at next year's meeting in Boston. Laboratories, institutes, and companies which are interested in this collaborative task force were requested to send an E-mail to Akitada Ichinose, <aichinos@med.id.yamagata-u.ac.jp>.