Factor XIII and Fibrinogen
DRAFT Subcommittee Minutes
23 July 2011
14:00-18:00
Room D
Chairman: Hans P. Kohler (CH)
Co-Chairmen: Moniek P. M. de Maat (NL), Aida Inbal (IL), Muriel Maurer (US), Leonid Medved (US), Marguerite Neerman-Arbez (CH), John W. Weisel (US), Sanj Raut (UK)
Educational Program
Dr Moniek de Maat (NL) gave a comprehensive overview on fibrinogen variants which arise from either genetic mutations/polymorphisms or protein changes due to proteolytic degradation, alternative splicing or posttranslational modifications, and their implications for cardiovascular disease were emphasised.
Dr Hans Kohler (CH) reviewed congenital and acquired FXIII deficiencies. He summarised the available diagnostic tests and pointed out their pitfalls, especially in the low-activity range. The clinical relevance of low FXIII levels was also discussed, in particular the relevance of heterozygous FXIII deficiency which is not well understood.
SSC Program
Dr Sanj Raut (UK) presented the results from two international collaborative studies for the value assignments of the 3rd International Standard for fibrinogen, plasma, and the 2nd International Standard for fibrinogen, concentrate. Over 20 labs took part in these studies. Inter-lab variability was low with results showing tight distribution. Fibrinogen potencies for the candidate materials were proposed and the proposals were discussed.
Dr Shirley Uitte de Willige (NL) summarised the current knowledge on the alternative splicing variant of the fibrinogen γ-chain (γ’). Fibrinogen γ’ has been shown to influence thrombotic risk, bind thrombin and FXIII, inhibit platelet aggregation, and influence clot structure. Novel findings on γ’ ratio in arterial and venous plasma samples from patients with coronary artery disease and controls free from CAD, both proven by angiography, were also presented.
Dr Tatiana Ugarova (USA) presented new findings on non-adhesive properties of fibrinogen matrices. In contrast to fibrinogen monolayers, fibrinogen multilayers (6-7 layers) exhibit low adhesion of platelets and leukocytes due to weakened integrin-mediated mechanotransduction. This may have implications for thrombosis/haemostasis and biomaterial science.
Dr Barbara Cardinali (USA) presented new insight on the concerted action of fibrinogen and FXIII activation by thrombin. Release of fibrinopeptides A and B and FXIII activation peptide were monitored over time and the influence of fibrinogen variants was investigated. A surprising finding was that FXIII activation proceeded faster in the presence of γ’/ γ’ fibrinogen compared with γA/ γA fibrinogen.
Dr Aida Inbal (IL) showed the results from the first phase 3 clinical trial on recombinant FXIIIA in patients with congenital FXIII deficiency. Forty one patients aged 7-60 were enrolled and 33 completed the study. The monthly dose was 35 IU/kg. No spontaneous bleeding was observed and the bleeding rate due trauma was significantly reduced. Four patients developed non-neutralising transient antibodies without any clinical consequences. Thus, rFXIII proves to be efficacious and safe.
Dr Lene Hørlyck (DK) gave more information on the production and characterisation of rFXIIIA. It is expressed in yeast and purified by several chromatographic steps resulting in a highly purified protein without any blood or tissue contaminants. Its native conformation and intact structure and function have been confirmed. Non-clinical and clinical trials have been successfully conducted and approval by the authorities is expected soon. Thus, the long-awaited first rFXIII product will be available to patients.
Dr Hans Kohler (CH) and Dr Sanj Raut (UK) presented a proposal for value assignment for FXIII B-subunit (total and free B) to the WHO 1st International Standard FXIII Plasma (02/206). It is important to establish the level of total and free FXIIIB as the half-life of FXIIIA depends on the amount of available FXIIIB. In addition, free FXIIIB may have so far unknown functions in binding and regulating FXIII and other plasma proteins, and FXIII substitution therapy with rFXIIIA also relies on FXIIIB. It was proposed to proceed as follows: 1.) Set up of ELISA methods using antibodies specific for free and bound FXIIIB, 2.) collaborative study involving laboratories including manufacturers, clinical labs and control labs to compare potency values calculated against locally collected plasma pools for calibration of the current WHO 1st IS FXIII Plasma, 3.) to submit the report to FXIII and Fibrinogen SSC Subcommittee, WHO-ISTH/SSC Liaison Group, and WHO/ECBS for endorsement/approval and value assignment. This proposal was well received and FXIII experts strongly agreed on the importance of this project. In the discussion the following points were raised: Dr Diane Nugent recommended to confirm by immunoblot that all free B is aspirated when using the indirect assay principle with aspiration after binding to anti-tetramer antibody. However, this assay principle will only be used in case no antibody specific to free B is available. Dr Laszlo Muszbek emphasised the importance of expressing FXIIIB concentration as mass concentration, as this is vital to estimate the true amount of free B for complex formation. Dr Akitada Ichinose also pointed out the need for FXIIIB standardisation in regard to diagnosis and treatment of B-subunit deficiencies, as this condition is probably underdiagnosed. These important points will be taken into consideration for the final project outline.
Dr Sanj Raut (UK) presented the results of an international collaborative study to calibrate the ISTH/SSC secondary plasma standard lot 4. The aim of this study was to value assign functional FXIII activity and antigen (A2B2). Twenty labs participated in this study. The results showed good agreement between labs. Potencies of 0.76 IU/ml (activity) and 0.74 IU/ml (antigen) were proposed.
Dr Laszlo Muszbek (HU) presented novel findings on the interaction of FXIII subunits in plasma and other body fluids. The aims of this work were 1.) to establish binding parameters for the interaction of A and B subunits and 2.) to investigate whether there is free A in plasma and other body fluids. Binding studies were performed by ELISA and surface plasmon resonance. Results of both methods consistently gave KD values around 10-10 M. Furthermore, it was shown that a small percentage of plasma FXIII A-subunit exists in non-complexed free form, while in cerebrospinal fluid and tears most A-subunit is free. Free A2 in plasma is in inactive form but can be activated by thrombin. This work revealed important new insights into basic FXIII biochemistry and physiology.
Dr Akitada Ichinose (JP) gave an update on the nationwide study on acquired FXIII deficiency. He addressed issues regarding nomenclature of acquired FXIII deficiency and proposed to distinguish between acquired deficiencies due to inhibitors or due to other causes including consumption. In the Japanese study, 26 patients with acquired deficiency due to inhibitors have been enrolled so far. Most patients had developed antibodies against FXIII A-subunit, only 3 patients had antibodies against B-subunit. The latter condition presents difficulties in diagnosis, as it tends to be overlooked or misjudged as thrombocytopenia. Clearly, more work is needed on acquired FXIII deficiencies.
