Fibrinolysis
Subcommittee Minutes
24 July 2011
08:00-12:00
Room D
Chairman: Ann Gils (BE)
Co-chairmen: Jonathan Foley (US), Dirk Hendriks (BE), Colin Longstaff (UK), Osamu Matsuo (JP), Nicola Mutch (UK), Michael Nesheim (CA), Craig Thelwell (UK), Tetsumei Urano (JP)
Educational session:
The SSC meeting started off with an educational session on the clinical significance and therapeutic opportunities to target PAI-1, TAFI and α2-antiplasmin.
PAI-1: Clinical significance and therapeutic opportunities. Ann Gils (BE)
Ann Gils gave a short overview on the basic characteristics of Plasminogen Activator Inhibitor-1 (PAI-1), the different conformations of PAI-1 and its role in fibrinolysis. She cited a number of papers to demonstrate the physiological relevance of PAI-1. The focus of the presentation was on potential therapeutic molecules that target PAI-1. First, she showed a number of in vivo studies in which highly specific antibodies towards PAI-1 were used. Subsequently, she cited a number of in vivo studies in which either Tiplaxtinin, S35225 or TM5275 were used.
Is TAFI a target? John Morser (US)
Thrombin Activatable Fibrinolysis Inhibitor (TAI) deficient mouse in models of abdominal aortic aneurysm, airway hypersensitivity and rheumatoid arthritis have shown that lack of TAFI has significant deleterious effects. In the latter two models the substrate for TAFIa was shown to be complement factor 5a (C5a) and not fibrin. In rheumatoid arthritis patients progression of the disease correlates with the SNP at nucleotide 1040 that encodes amino acid 325. In mice that were deficient in both TAFI and apoE that had been treated with streptozotocin developed tumors while singly deficient mice did not. John Morser demonstrated that although TAFI has been validated as a target, chronic use of a TAFI inhibitor could be problematic because of the effects in these models. John also suggested to rename TAFI to CPB2. The SSC subcommittee will consider this suggestion.
Alpha2-antiplasmin: The role of Alpha2-antiplasmin on the development of fibrosis. Yosuke Kanno (JP)
Dr. Kanno first gave an introduction on the characteristics of fibrotic disease. Fibrotic disease represents one of the largest groups of disorders for which there is no effective therapy. Some studies have shown that levels of plasmin-α2AP complex in plasma are elevated in fibrotic diseases. However, the roles of α2AP in fibrotic diseases are still not understood. He examined the experimental fibrosis in mice with a deficiency of α2AP by using bleomycin and found that the α2AP deficiency attenuated bleomycin-induced profibrotic proteins, TGF- α production and the development of fibrosis. In addition, he found that α2AP induced TGF-α expression and the development of fibrosis through PEDF-R binding and iPLA2 activation. Regulation of the α2AP expression may be an effective antifibrotic therapy.
SSC session
EQA for point of care D-dimer measurements. Ian Jennings (UK)
Point of Care testing (POCt) is becoming widely used in many countries, including the UK, and over 3000 centres now participate in the UK NEQAS POCt programme for INR measurement. Ian Jennings has also seen increasing interest in, and use of, point of care systems for D-Dimer testing, and a pilot EQA programme has been established for two of the point of care methods. In house studies have shown good correlation of lyophilised EQA material between point of care and laboratory-based D-Dimer methods. To date, 4 pilot studies have shown centres using the same point of care method do not agree on the result or the interpretation obtained on the same sample. This has implications in the use of these methods for VTE exclusion, and highlights the need for D-Dimer standardisation and EQA for point of care testing.
Update on WHO international Standards: Urokinase, PAI-1 and D-dimer. Colin Longstaff (UK)
A project to replace the current WHO IS for High Molecular Weight Urokinase
The existing WHO IS for High Molecular Weight Urokinase (87/594) is becoming depleted and a replacement is needed. The project to make a replacement IS has been endorsed by the WHO Expert Committee on Biological Standardisation (ECBS) and introduced previously to the Fibrinolysis Subcommittee of the SSC. Material has now been sourced from a manufacturer, trial fills completed and methods validated. The schedule for replacement involves a definitive fill of around 5000 ampoules in September 2011 and a collaborative study beginning in November 2011. A report of the study will be presented to the SSC in 2012 and it is expected that the new IS will be approved at the WHO/ECBS in October 2012. Additional participants are urgently needed to take part in the collaborative study.
Quantitation of PAI-1 Antigen in Plasma
A collaborative study was performed in 2007 which compared a number of different methods, both commercial and non-commercial ELISAs, for the determination of PAI-1 antigen in 5 plasma pools sent to a group of 12 laboratories. Results showed that the different methods gave widely different values for PAI-1 antigen although ranking of the pools was generally good and a method was proposed which allowed harmonisation of results from different methods onto a common scale. However, consensus values for ng/ml PAI-1 antigen in plasma are not acceptable and work since the original study has focussed on establishing a real gravimetric value for PAI-1 antigen in 1 or more of the original plasma pools in the study. Biacore methods have been explored and seem to have the required sensitivity but have shown up some problems that may help explain the variability in ELISA methods. Recent work has focussed on a proteomics approach which involves trypsin digestion of proteins in plasma and quantitation of peptides unique to PAI-1 and using synthetic C-13 labelled peptides as calibrators. Initial results are promising.
A project to develop an IS for D-dimer in plasma
A project group has been established, connected with the Fibrinolysis Subcommittee of the SSC with the aim of developing an IS for D-dimer in plasma. The core project group is Colin Longstaff (UK), Carl-Erik Dempfle (De), Ian Jennings (UK), Steve Kitchen (UK), Piet Meijer (NL). Several other SSC subcommittees have also expressed interest in the project. The project to develop an IS has also been approved by the ECBS at WHO. A pool of 280 ml of small pools of patient plasma with high levels of D-dimer has been collected and a trial fill performed where 0.5 ml aliquots were freeze dried to give 519 ampoules. Some of these are currently being investigated in accelerated degradation trials to assess the long term stability of this type of preparation. A further collaborative study is planned in 2011/12 to explore the usefulness of this batch of trial filled material as a standard in assays performed in a number of laboratories along with some patient samples. If this study is successful more D-dimer plasma will be collected to prepare a larger volume and several thousand ampoules of a candidate IS. Further studies are also envisaged to characterise the material in this preparation to help with the future replacement strategy of an IS for D-dimer in plasma.
PAI-1 deficiency-Eugloblin clot lysis assay. Tetsu Urano (JP)
Dr. Urano reported the identification of two independent PAI-1 deficient patients. The two patients had distinct gene abnormalities. Their ECLT were very short, and more importantly, he did not see a calcium-dependent shortening of ECLT, in which inactivation of PAI-1 plays important role, in their plasma. Since undetectable plasma levels of PAI-1 may not be enough to suspect PAI-1 deficiency, such supportive data obtained by functional assay were helpful to move on to genetic analysis.
Microplasmin degrades fibrinogen like plasmin but fibrin degradation is impaired. Paul Y. Kim and Jeffrey I. Weitz (Ca)
Plasmin (Lys?Pn) and microplasmin (Micro?Pn) are being used for catheter?directed fibrinolytic therapy. Unlike Lys?Pn, however, Micro?Pn consists only of the protease domain and lacks all five kringle domains. Micro?Pn has been a molecule of interest for fibrinolytic therapy as its rate of inhibition by α2-AP is 2 orders of magnitude slower than Lys?Pn, leading to an increased half?life. In addition to degrading fibrin (Fn), both agents degrade fibrinogen (Fg). This suggests that kringle?mediated interactions with Fg or Fn are not essential for efficient degradation. Paul Kims data suggests that kringle domains are important for efficient Pn?mediated degradation of Fn, but not Fg. Additionally, kringle domains are more important for Pn?mediated degradation of the β? and γ?chains of Fn or Fg than the α?chain. He concluded that if used for catheter directed fibrinolytic therapy, Micro?Pn may produce significant Fg degradation, which could decrease hemostatic potential.
The kinetics of TAFIa catalyzed cleavage of C-terminal lysine residues from fibrin degradation products and removal of plasminogen binding sites. Jonathan Foley (CA)
Partial digestion of fibrin by plasmin exposes C-terminal lysine residues, which comprise new binding sites for both plasminogen and tissue-type plasminogen activator (tPA). This binding increases the catalytic efficiency of plasminogen activation by 3000-fold compared with tPA alone. The activated thrombin-activatable fibrinolysis inhibitor (TAFIa) attenuates fibrinolysis by removing these residues, which causes a 97% reduction in tPA catalytic efficiency. Jonathan Foley studied the kinetics of TAFIa-catalyzed lysine cleavage from fibrin degradation products and the kinetics of loss of plasminogen-binding sites. He showed that TAFIa binds both Glu-Pg and Lys-Pg.
In vitro evaluation of profibrinolytic properties of TAFI and PAI-1 inhibitors. Tine Wyseure, Ann Gils, Paul Declerck (BE)
Ann Gils gave an overview of methods for evaluating the profibrinolytic effect of TAFI and PAI-1 inhibitors in vitro and elaborates specifically on the experimental setup of the in vitro clot lysis assay and thromboelastographic measurements using citrated plasma and whole blood, respectively. Based on examples, the challenges of the in vitro clot lysis assay are discussed, such as buffer effects, species-dependency and pool-to-pool variability. This presentation also shows our experience on the evaluation of TAFI or PAI-1 inhibitors using thromboelastographic measurements .
The role of TAFI in the regulation of growth and apoptosis in the hepatocyte. Taiichiro Seki (JP)
Taiichiro Seki investigated the role of TAFI in liver regeneration focusing on hepatocyte survival and death using TAFI-gene silenced primary hepatocytes. TAFI siRNA suppressed TAFI mRNA expression in primary hepatocytes by 90% of that in control hepatocytes. Apoptosis was induced in control hepatocytes during primary culture; however, it was reduced in TAFI-silenced hepatocytes. Cleaved caspase-3, which is an executioner at the down stream of apoptosis, was decreased in TAFI-silenced hepatocytes in comparison with control-hepatocytes. Conversely, phosphorylated-Akt suppressing apoptosis and promoting cell survival and proliferation was increased in TAFI- silenced hepatocytes.
Fibrinolysis in whole blood model thrombi formed under flow. Nuala Booth (UK)
Different models reveal different aspects of the fibrinolytic system. Most studies are performed on plasma or fractions such as euglobulin. Plasma, either free of or containing platelets, can be studied in clot lysis assays. These systems show sensitivity to PAI-1, a2-antiplasmin and TAFIa and they give some insight into the relative contribution of each of the inhibitors. The Booth laboratory has used the Chandler loop to make model thrombi from whole blood, which show similar contributions of the major inhibitors. This system shows clearly that the protease activity is strongest at the thrombus head; where no proteases are added the main contributor is uPA. Using this system, even for plasma rather than whole blood, gives remarkable sensitivity to cross-linking by factor XIIIa, which cross-links active a2-antiplasmin to forming fibrin. The Chandler whole blood system has been extended by making thrombi on strips of porcine aorta, in the Badimon chamber, allowing a marked effect of shear to be observed.
In vivo evaluation of TAFI inhibitors. Ellen Vercauteren (BE)
Ellen Vercauteren and colleagues set up a mouse thromboembolism model to evaluate TAFI inhibitors in vivo. In this well-characterized mouse thromboembolism model tissue factor injection led to (1) an increase of mice displaying abnormal physical activity or death, (2) obstruction of lung vessels by platelet-fibrin thrombi as shown in histological analyses, (3) fibrin deposition in the lungs as quantified by ELISA and (4) a massive systemic activation of coagulation that could not be counterbalanced by the fibrinolytic system as demonstrated by coagulation and fibrinolysis markers. However, administration of a TAFI inhibitor in this model resulted in (1) a decrease of mice displaying abnormal physical activity or death, (2) a reduced amount of platelet-fibrin thrombi, (3) a decreased fibrin deposition in the lungs and (4) an increase in plasmin generation indicative for an acceleration of fibrinolysis. In conclusion, this mouse thromboembolism model is an appropriate model to assess the profibrinolytic effect of TAFI inhibitors in vivo.
Alternative pathway for fibrinolysis: clinical significance and therapeutic opportunities, leukocyte elastase. Seiji Madoiwa (JP)
Seiji Madoiwa reported that plasma levels of cross-linked fibrin degradation products by leukocyte elastase (e-XDP) were significantly increased in patients with sepsis induced DIC.
Proteolytic and genetic variation of the alpha-2-antiplasmin C-terminus in myocardial infarction. Shirley Uitte de Willige (NL)
Shirley Uitte de Willige developed 2 new ELISAs to measure the antigen levels of free total α 2AP and free C-terminally intact α 2AP to investigate whether α 2AP antigen levels or α 2AP C-terminal cleavage were associated with myocardial infarction (MI) in 320 male MI survivors and 169 age-matched controls. Her data show that levels of free full-length α 2AP were decreased in MI, that the percentage of C-terminally cleaved α 2AP was unaltered, and that Arg407Lys did not influence α 2AP levels or MI risk.
