Plasma Coagulation Inhibitors
6 August 2005
16:00 to 19:30
The Ballroom 1
Sydney Convention and Exhibition Centre
Chair: E. Gray, UK
Co-chairs: F. Bernardi, Italy; F. Church, USA; C.J. Jackson, USA; D. Lane, UK; K. Suzuki, Japan; H. Whinna, USA
WHO International Standards: Chair: F Bernardi
International Genetic Reference Panel for Prothrombin G20210A Mutation. E Gray
An international collaborative study to validate the WHO 1 st International Genetic Reference Panel for Prothrombin G20210A Mutation was presented. The panel included 3 preparations of human gDNA: wild type, homozygote and heterozygote for G20210A mutation. The study involved 45 laboratories from 23 different countries, employing a total of 22 different methods against in-house known patient or commercially available controls. The majority of the participants correctly genotyped (error rate 0.7%) and therefore confirmed the validity of the panel. It was therefore recommended that the panel (05/130) should be considered by the SSC and the ECBS of the Who to be established as the international genetic reference panel for prothrombin G20210A mutation. The subcommittee approved the recommendation.
International Standard for Protein C, Plasma and Concentrate. E Gray
E Gray announced the forthcoming international collaborative study to replace the 1 st International Standard for Protein C, Plasma. The study will also serve to establish the 1 st International Standard for Protein C, Concentrate. The same exercise will also calibrate SSC secondary plasma standard Lot #3 for Protein C activity and antigen. The samples and protocol will be dispatched to the participants in August 2005 and the results of the study will be presented at the next SSC meeting in June 2006.
International Standard for Protein S, Plasma. AR Hubbard
The current WHO 1st International Standard for Protein S, Plasma was established in 1995 and the stocks have fallen to approximately 700 ampoules. It is therefore necessary to calibrate a replacement preparation. The candidate WHO 2nd IS Protein S Plasma (03/228) has been prepared from a pool of 24 plasma units from normal healthy donors. The variability of the liquid fill into ampoules was extremely low with a CV of only 0.09%; the mean fill weight was 1.0063 g and the residual moisture was only 0.064 %. Preliminary tests have indicated a level of 0.92 IU total Protein S antigen per ml. These results indicate that the preparation (03/228) is suitable for calibration as the Proposed 2nd IS Protein S Plasma. The calibration exercise will involve comparison with the current WHO 1st IS and with locally collected normal plasma pools in order to check on the continuity of the International Unit and the possible drift of the IU from the original calibration of the WHO 1st IS. As with the WHO 1st IS three parameters will be measured: total antigen, free antigen and function. The collaborative study is planned to commence in October/November 2005 with a view to submission to WHO and establishment in November 2006.
Protein S Multimers Chair: F Church and F Bernardi
Another look at protein S monomers/multimers and their direct anticoagulant activity. MJ Heeb
Plasma protein S has activated protein C-independent, direct anticoagulant activity (PS-direct). It was reported that monomeric purified protein S has only weak PS-direct, that multimeric purified protein S has good PS-direct in the presence of limiting phospholipids (0.1 µM), but that plasma contains only monomeric protein S, leading to the possible conclusion that either plasma has no PS-direct or that active purified protein S containing multimers has artifactual PS-direct. Dr Heeb showed that conventionally-purified protein S prepared with MonoQ-Sepharose had poor PS-direct and poor phospholipid affinity. Monomers, dimers, trimers and higher forms of affinity-purified protein S were identified by analytical ultracentrifugation. Multimers were not dissociated by Ca 2+ or promoted by EDTA alone, but may be concentration-dependent.
On a mass basis, monomers and multimers separated from affinity-purified protein S had the same specific PS-direct in the presence of saturating phospholipids (25 µM) and the same ability to compete with prothrombinase components for limiting phospholipids (2 µM). she concluded that Protein S monomers and multimers were detected in citrated plasma that had PS-direct, and in whole and fractionated hirudin-plasma. Thus, protein S multimers are naturally-occurring and plasma PS-direct is represented by affinity-purified protein S but not by some conventionally-purified protein S.
Should we bother about protein S multimers? T Hackeng
Dr Hackeng addressed the history and current status of protein S multimers and concluded that these multimers are in vitro artefacts that can seriously affect results of experiments and conclusions drawn. The APC-independent activity observed in model systems using purified proteins at low phospholipid concentrations could be completely tracked back to the presence of protein S multimers. The multimers were not present in plasma although APC-independent activity of protein S was observed in plasma. This activity therefore must follow a different mechanism than the APC-independent activity of purified protein S in model systems using purified proteins.
Global Coagulation/ Haemostatic Tests. Chair: H Whinna
Pre-clinical validation of the Calibrated Automated thrombogram (CAT). HMH Spronk
Dr Spronk presented data on setting reference ranges for thrombin generation test performed on the CAT. He compared results obtained from blood samples collected by half and full draw and found that there was no significant difference between these samples. Reference ranges for female and male were presented. He found that there is no need for reference range for different age groups, but there was a correlation between age and endogenous thrombin potential (ETP).
Recombinant tissue factor (rTF) and tissue plasmingon activator (t-PA) used in a new global assay to determine the combined effect of coagulation and fibrinolysis in plasma. S He
A global assay involving the addition of r-TF and tPA to platelet poor plasma was discussed. Overall coagulation potential (OCP) was obtained by the addition of rTF to platelet poor plasma (PPP), while the addition of t-PA gave results on overall fibrinolytic potential (OFP). The balance of OCP and OFP yielded the overall haemostatic potential (OHP). Results on application of this assay to haemophilic plasmas and patient samples ( coronary heart disease and DVT) were discussed.
The Nijmegen Haemostasis Assay (NHA). W van Heerde
Dr van Heerde presnted an assay that measures thrombin and plasmin generation simultaneously in a single well, using two different fluorogenic substrates. To validate the NHA several conditions were tested. Titration of TF varied the thrombin generation lagtime, the total thrombin generation (thrombin potential) and the plasmin generation lagtime. Total plasmin generation was not affected. Corn trypsin inhibitor did not interfere in the NHA indicating that initiation via the intrinsic pathway does not occur. Dilution of cephalin resulted in a diminished thrombin potential. Hirudin completely blocks thrombin and plasmin generation suggesting the requirement of fibrin for plasmin generation. Addition of carboxy-peptidase inhibitor diminishes clot lysis time indicating thrombin-activatable-fibrinolysis inhibitor (TAFI) activity. TAFI activity is increased by low thrombomodulin (TM) concentrations. High concentrations of TM and active protein C affect thrombin potential. e -aminocaproic-acid inhibits plasmin generation. Ultimately, increased concentrations of t-PA decrease thrombin generation lagtime suggesting interplay between fibrinolysis and coagulation. Titration of plasmin proved that an increased fibrinolytic activity results in an increased thrombin generation. In conclusion, the NHA allows the detection of coagulation and fibrinolysis and the interplay between both and may be suitable for screening haemostatic disorders.
Thrombin Dynamics Test (TDT) and ROTEM whole blood coagulation analysis potential value for the assessment of inhibitors and thrombophilic states. A Calatzis
The principle of ROTEM was described and it is based on the kinetic analysis of clot formation using whole blood. It allows the assessment of factors affecting thrombin generation, fibrin formation and polymerization as well as fibrinolysis. The principle of TDT was also described and it is an assay for the kinetic measurement of thrombin formation based on platelet poor plasma and optical detection on routine coagulation instrument. Results on the sensitivity of TDT to factor deficiencies and anticoagulants as well as procoagulant treatment were shown.
The possible use of the TDT in monitoring hypercoagulability. B Woodhams
Dr Woodhams reported results from a preliminary study to evaluate the use TDT to discriminate between normal subjects and hypocoagulable samples. Thrombin activity in patient citrated plasma was monitored using a 'fast' thrombin chromogenic substrate with added Gly-Pro-Arg-HydroxyPro to inhibit fibrin polymerisation. The assay can be performed easily on most automated routine haemostasis analyser. The data showed that there were some overlap between normal ranges and hypercoagulable plasma samples, but was useful for plasma samples from hypocoagulable patients.
Working Party on Thrombin Generation Test (TGT): Results on current practices survey and progress on working party activity. T Henckle
Dr Henckle reported on the activity of the Working Party. The survey results on current TGT practices is now available. The most widely used methods are the non-sampling chromogenic and fluorogenic assays. A mini review on TGT was published by the Working Party. A pilot study on a chromogenic non-sampling method has been initiated and an international collaborative study is now being planned to assess the comparability of current fluorogenic method for TGT.
Reference Materials and Methods for Antithrombin. Chair: E Gray
Structure and activities of the ISTH/IFCC Joint Committee on Standardisation of Coagulation Tests (C-SCT). E Gray
The structure and activities of the C-SCT is still to be decided. Activity such as reference methods and materials for antithrombin will continue to be carried out and reports on the activity of the C-SCT will continue to be presented at this subcommittee.
Assays for detection of antithrombin Type I and Type II deficiencies. S Kitchen
Dr Kitchen presented important differences between results of antithrombin (AT) assays in which human thrombin, bovine thrombin or factor Xa are used as the target enzyme for inhibition. In the case of AT Cambridge II the median results obtained by groups of UK NEQAS participants were 77 IU/dl , 81 IU//dl and 87 IU/dl for bovine thrombin human thrombin and factor Xa respectively. Both AT Cambridge II and AT Denver appear as normal or type I defects if Xa is used, but are phenotypically type II if bovine thrombin is employed. Furthermore the results of AT assays in some subjects are critically influenced by the incubation in the assays. It was concluded that there are a number of issues related to AT assays which must be taken into account in the standardisation of AT testing.
Working Party on reference materials and methods for antithrombin: Pilot study on primary reference methods for antithrombin. E Gray
The remit of the Working Party was reported and they are to develop a primary reference method for measurement of antithrombin with SI traceability, to establish reference materials using the primary reference method and to subsequently establish secondary reference materials for antithrombin type I and II deficiencies. A pilot study has now been set up to identify matrix effects and influence of different concentrations of reactants on the proposed primary reference method.