Factor XIII

7 August 2005
8:30 to 12:00
The Ballroom 1
Sydney Convention and Exhibition Centre

Chair: Robert Ariëns, UK

Co-chairs: Paul Bishop , USA , Akitada Ichinose , Japan , Hans Kohler, Switzerland ,Rainer Seitz , Germany

Active Members: Laszlo Muszbek , Hungary , Muriel Maurer, USA , Ivaskevicius , Germany

SSC approval sought for:

Ongoing activities:

The FXIII subcommittee held a joint meeting with the fibrinogen subcommittee this year. We had a very busy agenda for both subcommittees, due to which the total joint meeting exceeded its allocated timeslot of 3.5 hours by up to almost 1 hour. Apologies were received from Bishop and Maurer, all other chairs and active members were present. The meeting was attended by around 80-100 delegates.

The FXIII session was opened by Akitada Ichinose ( Japan ), who provided an overview of the diseases with which transglutaminases are associated. In addition to thrombosis, cardiovascular disease and bleeding, these include neurological disorders, cancer, celiac disease and Huntington disease. Ichinose gave an overview also of the activities of the FXIII standard working party, which has been active since 2002 and has developed the 1 st International Standard for FXIII, approved by the SSC and WHO in late 2004.

Ivaskevicius ( Germany ) presented data from a registry for FXIII deficiencies previously endorsed by ETRO. Currently the registry contains around 100 entries, but the estimated number of cases of FXIII deficiency worldwide is somewhere between the figures of 6,500-19,500. The registry provides important information for the management of FXIII deficient patients. It will also aid in our understanding of genotype-phenotype relationships for FXIII, and structure – function relationships. It is proposed that this registry should be expanded as a true international registry. The SSC is asked to endorse the development of this new, expanded registry for FXIII deficiency.

A new method measuring FXIII activity was presented by the group of Rainer Seitz ( Germany ), using a biotinylated selection peptide bound to a streptavidin coated microtiter plate. FXIII in a sample is activated outside the plate by thrombin; the reaction is stopped by hirudin. A fluorescence labelled detection peptide is incubated in the plate together with the mixture containing the activated FXIII. The reaction is stopped by EDTA and the plate washed with urea, before bound fluorescence is measured. Multiple variations of the detection peptide can be used as a powerful tool to study the enzymatic characteristics of FXIII.

The measurement of FXIII activity in concentrates was discussed by Laszlo Muszbek ( Hungary ). Different FXIII activity assays behave differently with regards to the diluent of the concentrate. Choice of diluent includes buffer, FXIII deficient plasma or FXIII free fibrinogen. The latter two appear to be the materials of choice for the accurate and consistent measurement of diluted FXIII concentrates. It is proposed to use FXIII free fibrinogen at a concentration of 2 mg/ml for future standardisation studies of concentrates.

FXIII nomenclature was discussed by Laszlo Muszbek. Nomenclature of FXIII had been previously considered at the SSC meeting in Florence , 1997. At that time a draft proposal was made and it was decided to test its use by researchers in the haemostasis and thrombosis field. The nomenclature proposal was revisited at this meeting (attached) and it was unanimously accepted in unmodified form by everyone present. It is proposed to seek endorsement of this FXIII nomenclature by the SSC, after which it is planned to submit an SSC brief communication outlining the details to the Journal of Thrombosis and Haemostasis on behalf of the FXIII subcommittee.

Sanj Raut ( UK ) presented data from a collaborative pilot study by the FXIII standard working party (SWG) for the measurement of FXIII antigen in the 1 st international standard for FXIII. During the international collaborative study in 2003-2004 to develop the 1 st IS for FXIII, antigen levels had already been determined at 0.93 (GCV 14%). At that time, however, it was decided not to assign this value as yet, as different methods for FXIII antigen determination (anti-A/anti-A, anti-A2B2/anti-A, anti-B/anti-A sandwich ELISA, and A2B2 Laurell) had been employed. As the tetrameric form of FXIII (A2B2) is the prevalent and (potentially) active form of FXIII in plasma, it was decided that this should provide the ‘gold’ standard for FXIII antigen determination. The current SWG pilot study is based on the use of one A2B2 ELISA kit, performed in 5 laboratories, using a protocol developed by the NIBSC. Preliminary data showed an antigen potency estimate of 0.91 (GCV 1.8%), which is in close agreement with the previous study using various antigen assays. The FXIII SWG proposes to await full analysis of the data and, if confirmatory, to pool the estimates from both studies for the assignment of a FXIII antigen potency estimate for the 1 st IS for FXIII. Approval for this antigen estimate by the SSC and WHO will be requested in due course.

The effect of polymorphic variants on kinetic activity assays for FXIII was discussed by Robert Ariens ( UK ). Assays that are based on the kinetic measurement of early pentylamine substrate incorporation into fibrin, can be sensitive to differences in activation rate and hence to FXIII Val34Leu, a common polymorphism in certain populations (25% allele frequency in Caucasians). Ariens presented data of the modification of one such kinetic pentylamine assays into an end-stage, total activity assay for FXIII. The reaction mixture was incubated for 60 minutes rather than 5-10 minutes, and the measurement principle was based on dose-response curves of absorbency against plasma dilution rather than kinetic curves of absorbency against time. The modified assay proved sensitive to FXIII in the ng range with high specificity as demonstrated by parallelism of dose response curves for plasma, purified FXIII, recombinant FXIII-A and a FXIII concentrate for clinical use. Identical dose-reponse curves were found for FXIII VV, VL and LL samples with similar A2B2 antigen concentrations. It is proposed that this end-stage assay can be used as alternative to kinetic assays when it is desirable to measure total activatable FXIII.

TERMINOLOGY TO DESIGNATE DIFFERENT FORMS OF BLOOD COAGULATION FACTOR XIII
(Laszlo Muszbek)

Factor XIII normally present in the plasma (tetramer; A2B2):

Recommended term:
Plasma coagulation factor XIII (Plasma factor XIII)
Not recommended terms:
Fibrin stabilizing factor, Plasma protransglutaminase,
Fibrinoligase, Fibrinase, Laki-Lorand factor, Plasma transamidase.

Factor XIII of intracellular localization present in platelets, megakaryocytes, monocytes, macrophages(dimer; A2):

Recommended term:
Cellular coagulation factor XIII, (Cellular factor XIII).
Not recommended terms:
Platelet, monocyte, placenta, etc. factor XIII,
Cellular protransglutaminase.

PROPOSED FXIII TERMINOLOGY AND ABBREVIATIONS I.

Blood coagulation factor XIII: FXIII
Plasma factor XIII (A2B2): pFXIII
Cellular factor XIII (A2): cFXIII
Recombinant factor XIII (A2): rFXIII

Potentially active FXIII subunit:

recommended term: A subunit of factor XIII (FXIII-A) not recommended terms: factor XIII a or a subunit, (FXIIIa, FXIIIa, FXIII a, FXIII a, FXIII-a, FXIII-a)

Inhibitory/carrier FXIII subunit:

recommended term: B subunit of factor XIII (FXIII-B) not recommended terms: factor XIII b, b or S subunit, (FXIIIb, FXIIIb, FXIIIS, FXIII b, FXIII b, FXIII S, FXIII-b, FXIII-b, FXIII-S)

 

 

 

 

 PROPOSED FXIII TERMINOLOGY AND

ABBREVIATIONS II.

Activated form of blood coagulation factor XIII in general:

activated factor XIII (FXIIIa)

Activation peptide cleaved off from the A subunit by thrombin: factor XIII activation peptide (AP-FXIII)

Intermediates and endproducts of the activation process:
Thrombin cleaved inactive form of plasma and cellular factor XIII:

Thrombin-cleaved active factor XIII:

Non-cleaved active factor XIII: