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2016 Annual Minutes Locked Topic 0 R. Marlar Plasma Coagulation Inhibitors 26 June 2016 14:15 – 18:45   Chairman: Richard A. Marlar (USA) Co-Chair: Ian Jennings (UK), Jun Teruya (USA), Hiroko Tsuda (Japan), Cecilia Guillermo (Uruguay)   Working Session #1: On-going Projects Project: Investigation into discrepancies in Protein S activity assays results- I. Jennings (UK). The protein S (PS) activity assay has been shown in External Quality Control studies to have significant variation among laboratories and these issues extend to patient results as well. This study will attempt to elucidate the causes of the variation observed in the clinical PS activity assays. Up to 40 laboratories will participate in this study. The design of the study has been completed. The difficulty was obtaining the appropriate samples for distribution to the participating laboratories. However, normal and protein S deficient individuals have been identified and obtaining sufficient plasma for the proposed studies is in progress. Both lyophilized and frozen plasma will be distributed in the fall of 2016. The results will be analyzed during the spring of 2017. It is anticipated to have the data by the next meeting in Berlin. Project: Investigation into racial differences in genetic risk factor (AT, PC, PS) for venous thrombosis- H. Tsuda (Japan). The design for the study is developed and request for specimens is on-going. At this time, the East Asian samples and some European and South American samples have been received and tested. Preliminary data from both plasma and genetic polymorphisms have been analyzed. It is anticipated that the collection of specimens and the analysis of data will be continued and more countries and regions will be recruited. Enough data will be available for a final presentation at the next meeting. Working Session #2: Guidance Manuscripts for Clinical Testing. Project: Manuscript on Guidance for Clinical Testing for Antithrombin, Protein C, Protein S and APC-Resistance. Clinical laboratory assessment of plasma levels of AT, PC, PS and APC-Resistance is difficult and associated with many problems and pitfalls. The working groups are producing four manuscripts on the state-of-the-art clinical assessment of plasma levels of the common thrombophilic risk factors. The manuscript progress for these four thrombophilic factors was presented: Antithrombin: P. Meijer (The Netherlands)- Presented the preliminary outline and basic data to be included in the manuscript. The major AT assays types were discussed as to their sensitivity and specificity. There are three major types of AT deficiency which were discussed in relationship to the molecular mechanisms and effect on the various assay types. The manuscript is approximately half completed at this point. Protein C: P. Cooper (UK)- Recommendations for protein C (PC) assays for plasma assessment using either the clotting based assay or a chromogenic based assay and the antigenic assay was presented. In addition the genetic deficiencies of PC testing was summarized. The manuscript has been written and is in final editing for submission. Protein S: R. Marlar (USA)- The biochemistry and coagulation physiology for PS was presented as this molecule is difficult to assay because of the complexity of PS interaction with other plasma molecules. The pitfalls of the PS activity assays were discussed. The recommended initial or screening assay for PS is Free PS antigen assay using monoclonal antibody assay. The lack of useful information from the Total PS antigen assay was also discussed. Various parameters that affect the reference range such as gender differences was also discussed. The manuscript is about half completed (writing portion) but should be completed and edited by the December, 2016 deadline. APC-Resistance: G. Moore (UK) - The concept of APC Resistance was presented. The various modifications of the APC-R assay were reviewed. Discussions about the relationship with the genetic mutations and the assay results was also presented. Other causes (non-genetic) of APC-Resistance were reviewed. The initial writing of the manuscript has been done and final editing will be completed this summer and fall for the deadline of December, 2016. The writing of these manuscripts will be completed by December, 2016 and final review, editing, harmonization will be completed by spring, 2017 with submission to the Journal of Thrombosis and Hemostasis as SSC reports planned for June, 2017. Working Session 3: Thrombophilia Testing Schemes. Thrombophilia Testing Schemes- I. Jennings (UK), P. Meijer (The Netherlands), E. Favoloro (Australia), C. Guillermo (Uruguay): Thrombophilia testing schemes vary by country and/or region. This session reviewed the thrombophilia testing schemes from two different European perspectives (based on External QA studies), and Australia and South America. Testing variations occur for a variety of reasons, including mandated testing or restricted testing (from national health services), racial differences, and clinician decisions or test availability. The information presented showed that there are multiple approaches to thrombophilia testing with no unified approach to testing schemes. The criteria for who to test and when the patient should be tested are not consistent. Further studies into the variability are necessary. To round out the evaluation of thrombophilia testing schemes, other countries and regions will also be presented at the next meeting. The major areas of the world will be covered and a summation of the testing methods will be presented and possibly submitted for publication. Working Session #4: Coagulation Inhibitor Interference by DOACs and Protein S Cleavage. Effect of New Anticoagulants on Thrombophilia Assays- R. Gosselin (USA) The oral anticoagulant drugs pose significant issues with clinical testing of the plasma coagulation inhibitors. The DOAC drugs when present in the specimen appear to increase the thrombophilic factor values giving the potential for a false normal test. The impact of these inhibitors on the different testing methods was presented. Significant problems are associated with clotting assays for protein C, protein S and APC-Resistance. Some inhibitors can specifically affect antithrombin assays based on the enzyme used for the test. However some tests are not affected by the DOAC drugs (PC chromogenic assay or immunologic assays). Recommendations are not to test when a patient is on a DOAC drugs but rather wait until the patient has been off of the drug for at least 4-5 days. The genetic tests are not affected by the DOAC drugs. Protein S cleavage- H Brinkman (The Netherlands) Protein S is cleaved and inactivated in a region known as the thrombin sensitive loop. Thrombin and other enzymes can cleave protein S in this region and subsequently inactivate the PS activity. There at least 3 specific sites that can be cleaved in this specific loop by different enzymes. Work was presented showing the mechanism and parameters of this cleavage by the various enzymes. This work may have clinical significance for assay performance. 
by R. Marlar
Thursday, June 23, 2016
2015 Annual Minutes Locked Topic 0 L. Schmeidler Chairman:  Richard A. Marlar Co-Chair:  Ian Jennings, Jun Teruya, Hiroko Tsuda Educational Session:  Plasma Coagulation Inhibitors: Relationship of Phenotype, Plasma Levels and Clinical Phenotype (Thrombosis). Pieter Reitsma (The Netherlands):  Molecular Defects in Protein S Protein S is a very complex protein. It is a vitamin K-dependent protein significantly different from the other vitamin K proteins in that it has a unique region that allows the interaction with C4B bp Binding Protein. This binding interaction creates very complex molecular and physiological interrelationships, hence difficulty in assessing the plasma levels and determining the laboratory phenotype of the deficiency.  Possible protein S deficient individuals were studied from the Dutch population using a multitude of genetic methodologies to assess genetic deficiencies. The majority of deficient patients did have identifiable genetic defects. Genetic defects span the complete gene and have all genetic types of defects. Anna Pavlova (Germany): Comparison of Phenotype and Genotype for Antithrombin, Protein C and Protein S. Clinical phenotype and laboratory values were compared to the genetic defects in AT, PC and PS deficient patients. Evidence presented demonstrated that genetic defects were more likely to be found in patients with lower plasma levels of the factor. In patients with only mildly abnormal levels the potential for finding a genetic defect was significantly decreased. Patients with 40-60% plasma levels of AT, PS or PS decreased the potential for detecting defects in only 20-30% of the patients.   Working Session #1: On-going Projects Project: Update and maintain genetic mutation databases for AT, PC and PS. The committee was mandated 6 years ago to update the mutation databases for AT, PC and PS. Over the last 6 years this has been attempted. However using only volunteer participants with no funding, there was no significant progress toward the completion of this project. With the creation of the Subcommittee on Genomic in Thrombosis and Hemostasis, the updating and maintenance of a database for AT, PC and PS will be transferred to the new Subcommittee on Genomics.   Project: Investigation into discrepancies in Protein S activity assays results.    The protein S activity assay was has been shown in External Quality Control studies to have very significant variation among laboratories and these issues extend to patient results as well. This study will attempt to elucidate the causes of the variation observed in the clinical PS activity assays. Up to 40 laboratories will participate in this study. The design of the study has been completed. The difficulty is obtaining the appropriate samples for distribution to the participating laboratories. Both lyophilized and frozen plasma will be distributed. The results will be analyzed. It is anticipated to have the data by the next meeting.   Project: Investigation into racial differences in genetic risk factor (AT, PC, PS) for venous thrombosis. The design for the study is developed and request and receipt of specimens is on-going. At this time, the East Asian samples and some European have been received and processed. It is anticipated that the collection of specimens and the analysis of data will be completed by the next meeting. Then the complete set of data will be presented.   Project: Manuscript on Guidance for Clinical Testing for AT, PC, PS and APC-Resistance. Clinical laboratory assessment of plasma levels of AT, PC, PS and APC-R is difficult and is associated with many problems and pitfalls. The committee is producing four manuscripts on the state-of-the-art in assessing plasma levels of the common thrombophilic risk factors. The project leaders for these four manuscripts are: Antithrombin:   Piet Meijer Protein C:        Peter Cooper Protein S:        Richard Marlar APC-R:           Gary Moore and Dorothy Adcock The manuscripts will be completed by December, 2015 with submission planned for May, 2016.   Working Session 2: Jun Teruya:   Other Plasma Coagulation Inhibitors The first part of the presentation dealt with all of the plasma inhibitors that might play a role in the coagulation process. Heparin Cofactor II data was presented to show the possibility of the clinical relevance of this inhibitor. Levels of this inhibitor were discussed in both adult and children. This inhibitor appears to not play a role as a risk factor for the development of thrombosis. It may play roles in other physiologic systems not related to coagulation. Thrombomodulin is the cofactor for thrombin activation of protein C. The soluble form of Thrombomodulin is being investigated as a potential drug for the treatment of sepsis. It appears that levels of Thrombomodulin may be of benefit for increasing the survival of septic patients. Since it is not a plasma protein, it is difficult to assess the naturally occurring protein. However plasma levels will probably need to be assessed if the soluble form is used as treatment of sepsis. Tissue Factor Pathway Inhibitor (TFPI) is a regulator of factor VII and factor X. The physiology and biochemistry of TFPI is complex which may lead to potential bleeding issues. No genetic deficiencies of this protein have been identified. Acquired decreases and increases have been seen in a number of clinical conditions. With this assessment, TFPI may play a role in causing bleeding or thrombotic pathology.   Thrombophilia Testing Schemes: Thrombophilia testing schemes vary by country and/or region. This session reviewed three different countries standards for thrombophilia testing schemes (United Kingdom, Japan and the United States). Testing variations occur for a variety of reasons, including mandated testing or restricted testing (from insurance companies or national health services), racial differences (no factor VLeiden or prothrombin 20210 testing in Japan), and clinician driven or testing availability. The information presented showed that there are multiple approaches to thrombophilia testing with no unified approach to testing schemes. The criteria for who to test and when the patient should be tested are not consistent. Further studies into the variability are necessary. Other countries and regions will also be presented at the next meeting to allow.    
by L. Schmeidler
Sunday, June 21, 2015
2014 Annual Minutes Locked Topic 0 R. Marlar Plasma Coagulation Inhibitors Chairman: Richard Marlar (USA) Co-Chairmen: Elisabetta Castoldi (the Netherlands), Steven Kitchen (UK), Jun Teruya (USA), Hiroko Tsuda (Japan) Tuesday, 24 June (14:00-18:00)   Session #1:  Welcome and Abstract Presentation    Welcome and Introduction             Speaker:  Richard Marlar (USA)     Top Rated Abstract             Speaker:  Vera Korneeva (Russia Federation): Corn trypsin inhibitor non-loop regions are required for the specific  inhibition of factor XIIa  V. Korneeva presented her research based on her abstract dealing with the molecular structure responsible for the molecular mechanism of corn trypsin inhibitor inhibition of the coagulation enzyme, factor XIIa.       Session #2:  Molecular Genetics                        Chairman: Jun Teruya (USA)   Update and Maintenance of the Antithrombin, Protein C and Protein S Mutation Databases              Speaker: Richard Marlar for Elisabetta Castoldi (The Netherlands)   In 2011, the Plasma Coagulation Inhibitor Subcommittee proposed developing and maintaining a locus-specific genetic mutation database for Antithrombin, protein C and protein S. However in the last three years only three people volunteered to help develop and maintain the database. This project would be a long term (continuous) undertaking requiring experienced curators and a significant time commitment to check the literature, clinical presentations and probably require long term funding. Since the SSC has a Working Party on Genomics, the Plasma Coagulation Inhibitor Subcommittee recommended contacting the Working Group on Genomics to transfer this project to this group.  Initial contact was made with Willem Ouwehand of the Working Group on Genomics.     Subcommittee Project Update: Racial differences in genetic risk factors for venous thromboembolism Speaker: Hiroko Tsuda (Japan)  Background: Factor V Leiden and prothrombin G20210A, well-known hereditary thrombophilia in Caucasians, are not found in Asian, Africans and Australoid. In contrast, protein S (PS) and protein C (PC) deficiencies are much more prevalent among Asians than non-Asians. PS Tokushima (K155E, K196E in HGVS nomenclature) and two PC gene mutations, PROC c.565C>T and PROC c.574_576del, all three representing type II deficiency, are identified as hereditary thrombophilia in Japanese and Chinese, respectively. In order to elucidate the racial differences in genetic risk factor for VTE, the worldwide distribution of these three mutations is investigated.  Update: 1) We build up a global network of professional contacts, which includes Asian and European countries and USA, in collaboration with the members of JSTH-SSC, APSTH, and ISTH-SSC. 2) After the ethical approval, VTE patients and healthy individuals are recruited through the use of standardized protocol and registry sheets. Racial groups are divided into white (Caucasians), Hispanic, black, East Asian, South Asian, Other Asian, or Other. 3) Analysis of plasma and DNA samples are mainly performed in Japan. The total PS assay system (Tsuda T. et al. Blood Coag Fibrinolysis. 23: 56-63, 2012) has been improved, achieving a high sensitivity for screening of PS Tokushima by evaluating the specific activity of the APC cofactor function of plasma PS.    Session #3:  New Proteins           Chairman: Steve Kitchen (UK) & Hiroko Tsuda (Japan)  Tissue Factor Pathway Inhibitor- General Aspects             Speaker: Paul Ellery (USA)  Tissue factor pathway inhibitor (TFPI) inhibits factor VIIa and factor Xa to limit thrombin generation. It is produced by the endothelium of the microvasculature and platelets. Two isoforms, TFPIα and TFPIβ, arise from alternative splicing. While both isoforms inhibit factor VIIa and factor Xa, they have different C-termini which give them distinct anticoagulant activities. TFPIα is a soluble protein. Protein S is a co-factor for TFPIα that enhances its inhibition of factor Xa by localizing it to a membrane surface. TFPIα also tightly binds to an acidic region of the factor V B-domain providing a key exosite interaction that allows physiological inhibition of early forms of prothrombinase. TFPIβ is GPI-anchored at the endothelial surface, where it is optimally located to inhibit TF-mediated processes. Platelet TFPI is exclusively TFPIα. Platelet TFPIα modulates hemostasis in mice with hemophilia and limits thrombus development following murine vascular injury. Plasma TFPI is heterogeneous and exists in the full-length (TFPIα) and several C-terminally truncated forms that associate with plasma lipoproteins. It has been measured in numerous disease states. Low levels weakly correlate with thrombosis risk. Patients with factor V East Texas have 10-fold elevated plasma TFPIα, which is thought to be responsible for the moderately severe bleeding diathesis associated with this disorder. Plasma TFPI is affected by a number of variables, including gender, age, plasma LDL, glucose, factor V and protein S concentrations, as well as oral contraceptive use. Measurement of platelet TFPI may be a more informative hemostatic/thrombotic indicator in disease states because it is affected by fewer variables than plasma TFPI.    Tissue Factor Pathway Inhibitor- Clinical Assays             Speaker: Dorothy Adcock (USA)   A number of manufacturers produce TFPI assay kits to measure free TFPI, Total TFPI or TFPI activity.  All are labeled as research use only in the U.S.  TFPI is a complex glycoprotein that exists as two primary isoforms (these vary in composition) that can be found in multiple locations, specifically, free in plasma, bound to lipoprotein in plasma, bound to cell surfaces, as well as intracellular pools. TFPI in the plasma includes a full length (total) glycoprotein as well as truncated forms of varied sizes which are largely bound to lipoprotein. Antigen kits are based on quantitative sandwich enzyme immunoassay methodology.  The capture and detection antibodies are proprietary and are not standardized between kits.    These kits therefore likely measure at least slightly different plasma TFPI populations based on the epitopes of the capture and detection antibodies. Activity assays measure essentially only free TFPI and not TFPI bound to cell surfaces and therefore measures only a fraction of TFPI’s intravascular anticoagulant potential.   These assays are based on TFPI’s ability to inhibit the catalytic activity of TF/FVIIa and do not measure TFPI’s ability to inhibit prothrombinase. Plasma TFPI activity therefore is not a reliable indicator of intravascular TFPI anticoagulant activity. Calibrators for both antigen and activity assays consist of either normal plasma or recombinant TFPI and there is no TFPI standard.  Finally, both TFPI antigen and activity assays are likely affected by pre-analytic variables as platelet activation with sample procurement and processing may cause release of TFPI alpha and there is potentially an impact on levels of TFPI alpha related to the length of tourniquet when drawing the sample..  Following processing, the post centrifugation platelet count may also affect levels of TFPI alpha in those samples that have undergone a freeze-thaw cycle.  Tissue Factor Pathway Inhibitor- Clinical Relevance             Speaker: Jun Teruya (USA)  Total and free TFPI were measured for patients on ECMO who were receiving unfractionated heparin therapy. Heparin increases TFPI level. It is not clearly understood the mechanism, but some articles suggest heparin not only releases TFPI from endothelial cells, but also stimulates synthesis of TFPI in endothelial cells. Our data showed continuous increase of TFPI during ECMO therapy, suggesting heparin may be stimulating to synthesize more TFPI. The response of TFPI upon heparin administration is different between patients >1 year old and < 1year old. It was concluded that TFPI also plays a major role for heparin anticoagulation.    Session #4:  Laboratory Testing                        Chairman: Steve Kitchen (UK) & Richard Marlar (USA)  Subcommittee Project Update: Protein S activity assays             Speaker: Ian Jennings (UK)   External Quality Assessment data from multiple EQA programs has repeatedly demonstrated that protein S activity assays have significant statistical and clinical differences depending on the different assay methods, reagents and manufacturers. This occurs in normal samples and protein S deficient samples. The Plasma Coagulation Inhibitor Subcommittee is investigating the cause and extent of the discrepancies between these reagents and methods. A proposal for this study has been approved. The delay has been the inability to obtain appropriate samples for shipment to multiple laboratories for testing; however acquiring plasma has been reinstituted at the NEQAS site. This project will restart in the next few months and samples will be sent to participating laboratories. Preliminary results are expected to be presented at the next SSC meeting.    Planning for Preparing Guideline Manuscripts             Speaker: Richard Marlar for Piet Meijer (The Netherlands)   The Plasma Coagulation Inhibitor Subcommittee has proposed the development and submission of four manuscripts for publication in JTH detailing assay performance, acceptable methodology and significant pitfalls based on published literature (evidence based). The four manuscript topics are:  Antithrombin, Coordinator: P. Meijer (The Netherlands)             Protein C, Coordinator: S. Kitchen (UK)             Protein S, Coordinator: R. Marlar (USA)             APC-Resistance,  Coordinators: D. Adcock (USA) and G. Moore (UK) During the committee meeting, a request was made for volunteers to help with preparation of the manuscripts. Briefly, the manuscripts will contain: a short description of the component, the assay principles, review of the available clinical assay methods, pitfalls and problems, pre- and post-analytical issues and recommendations for testing methods. Submission for publication is expected by the next SSC meeting.      Session #5:  Discussion and Future Directions                    Chairman: Richard Marlar (USA)  Planned activities for the 2015 Subcommittee Meetings Speaker: Richard Marlar (USA)  For the 2015 Meeting of the ISTH-SSC Subcommittee on Plasma Coagulation Inhibitors, the committee proposed the following topics.   Resolution of the locus-specific Antithrombin, protein C and protein S databases Progress report and preliminary data on the racial differences project.Progress report and preliminary data on the protein S activity assays project.Presentation of the near-final drafts on the four proposed manuscripts on clinical assays for antithrombin, protein C, protein S and APC-Resistance.Proposed status of TFPI as an important inhibitor of coagulation.    Future and long term goals for SubcommitteeSpeaker: Richard Marlar (USA) For future studies, the Plasma Coagulation Inhibitor committee will consider TFPI, other plasma coagulation inhibitors, other components of plasma inhibitor systems. As well, the committee will evaluate the roles of plasma coagulation inhibitors in the development of thrombophilia.
by R. Marlar
Thursday, June 19, 2014
2013 Annual Minutes Locked Topic 0 S. Kitchen Plasma Coagulation Inhibitors Chairman: Steven Kitchen (UK) Co-Chairs: Elisabetta Castoldi (Netherlands), Tilman M. Hackeng (Netherlands), Richard A. Marlar (USA), P Meijer (Netherlands), Laurent O. Mosnier (USA), Jun Teruya (USA) June 29, 2013 Educational programme _ Chairs S Kitchen, L Mosnier 14:00-14:30 Laboratory Diagnosis and Characterisation of AT deficiency. Peter Cooper (UK) 14.30- 15:00 Structure-Function of activated protein C: deficiencies and endogenous anticoagulant activities. Laurent Burnier (USA) Minutes/Summaries   Laboratory Diagnosis and Characterisation of AT deficiency. Peter Cooper (UK)   Antithrombin (AT) deficiency may increase the risk of VTE by 5-50 folds, but risk depends upon AT level and type of deficiency. Most patients with inherited AT deficiency have type II defects where the AT activity level is significantly lower than the level of AT antigen. In functional AT assays, AT is activated by heparin, and an enzyme (thrombin or FXa) cleaves the arginine 425 serine 426 bond in AT (numbering according to the Human Genome Variation Society guidelines) and AT is inhibited, the residual enzyme is detected using a chromogenic substrate and optical density (405nm) is inversely proportional to AT level. On identifying a low or borderline AT activity level measurement of AT antigen establishes whether the defect is type I or type II, and the ratio of activity/antigen facilitates this, and can identify type II defects even when AT activity level is not reduced. AT antigen can be accurately measured by techniques such as latex-agglutination and the more labour-intensive ELISA assay, the radial immunodiffusion assay is widely available but can have poorer precision. Samples for AT level should be citrated plasma, and clotted samples have reduced AT activity. Testing the prothrombin time (PT) confirms that the sample is not clotted and can identify liver disease which can reduce AT level. PT, thrombin time and APTT can help identify presence of anticoagulation, especially important, as direct thrombin/FXa inhibitors cause overestimation of AT by thrombin and FXa-based assays, respectively. Antithrombin from healthy individuals is relatively robust, but sample storage in a self-defrosting freezer can greatly reduce functional AT level. Within run precision of AT activity when a pooled normal sample is tested can display a coefficient of variation of <2%, but instrumentation and reagent problems can cause drift and imprecision. AT activity assays should be sensitive to all AT defects but there is no guarantee that one assay will be sensitive to all type II defects. AT assay in the absence of heparin will not pick up type IIHBS (heparin binding site) defects, FXa-based assays do not detect AT Cambridge II (p.Ala416Ser) and we show a type IIRS (reactive site) defect, AT Denver (p.Ser426Leu) that is clearly detected by a thrombin-based assay but undetected by a FXa-based AT assay. We measured AT in a patient with AT Budapest 3 (p.Leu131Phe) AT levels were much lower in a human-FXa based AT assay (Innovance, Siemens) than three other assays and the very low results appeared more in keeping with heparin-resistance in two patients. Similarly, patients with AT Basel (p.Pro73Leu) had clearly low level by the Innovance AT assay but borderline levels with Berichrom assay even with 30s incubation and had normal AT levels using the Berichrom assay with the manufacturer’s recommended 180s incubation time. Interestingly, with AT Basel, the Coamatic AT assay (bovine FXa-based) had poor sensitivity to the deficiency, and it has been reported that the Liquid AT assay (Instrumentation Laboratory) is also insensitive to this defect. Shortened incubation time of enzyme with sample can increase sensitivity to type IIHBS defects and to at least one type IIRS defect (AT Glasgow, p.Arg425His). When investigating type II AT deficiency, more than one activity assay is required but there is little data on the sensitivities of different manufacturers’ kits to type II AT defects, however, EQA schemes have helped highlight differences between kit sensitivities as have some published studies. Structure-Function of activated protein C: deficiencies and endogenous anticoagulant activities. Laurent Burnier (USA) Activated protein C (APC) is a serine protease in blood that helps maintain a regulated balance between hemostasis and host defense systems in response to vascular injury. APC interacts with numerous cofactors and receptors on various cells. These interactions help APC express multiple anticoagulant and cytoprotective activities that contribute to maintaining the health and integrity of the vasculature. These different activities of APC will be reviewed in light of available structure-function information for APC and supported by experimental and clinical data. Recent advances and in vivo proof-of-principle data for activity selective APC mutants, targeting of APC activities by monoclonal antibodies, and studies of APC cofactors and receptors combined with insights from human congenital or acquired deficiencies and genetically modified murine models support an intricate system safeguarding vascular homeostasis. Although traditionally known as "anticoagulant APC” these novel activities put APC in a different spotlight with important implications for normal physiology, pathophysiology, and therapeutic applications in ischemic stroke and potentially other vascular diseases.  Subcommittee Business section   Laboratory testing Chairs : Jun Teruya (USA) and Richard Marlar (USA) Project proposal : Proposed Guideline for assays of AT, PC and PS. Piet Meijer (The Netherlands) Guideline for laboratory testing of natural coagulation inhibitors Person responsible (Chair / Principal Investigator): Piet Meijer, NL (other principal investigators: Steve Kitchen, UK; Richard Marlar, US) Current guideline for testing of heritable thrombophilia focuses mainly on clinical aspects and which tests are relevant to perform. No detailed information is provided how to measure thrombophilia parameters. Detailed information is on the technical aspects of the tests, test characteristics, interferences etc. is limited or lacking. Within the framework of the Scientific Standardization Committee on Plasma Coagulation Inhibitors a laboratory guideline for the measurement of coagulation inhibitors will be developed including the fore-mentioned analytical aspects. This projects starts in 2013 and will be finalized in 2014/201 Pre analytical issues for AT, PC and PS testing. Richard Marlar (USA) Pre-analytical variables (PAV) affect coagulation testing more often than tests in other areas of the clinical lab. These variables can cause an erroneous diagnosis if PAV issues are not recognized or taken into account when a patient’s sample is analyzed. This is especially true of the natural regulatory factors (AT, PC, PS). In its broadest sense, PAV issues include both patient status (food, general health and medications) and sample preparation (collection, transport, processing and storage of the blood specimen and plasma sample). The discussions covered some of the more common PAV affecting clinical testing of AT, PC and PS.   Diagnosis of AT Budapest variants. Ingrid Hrachovinova ( Czech republic) Deficiency of AT is the most clinically severe defect of plasma coagulation inhibitors. In the last fifteen years, we documented 126 patients from 62 families with a suspected hereditary AT deficiency due to low or borderline AT activity of plasma levels. Mutational analysis of the AT gene had been performed. Mutations were found in all of patients – 54 patients had subtype I, 10 subtype II RS (reaction side), 58 subtype IIHBS (heparin binding side) and 4 had subtype IIPE mutations. Most common mutations in our group were Budapest variants (Leu99Phe and Arg47His). They represented 0.28 of all cases and about half patients with II subtype (35pts). Screening of AT activity in family members together with the genetic screening of the family mutation revealed that some FXa based assays may fail to detect these mutations and in some cases, even FIIa based assays measured AT activity results which were in the normal range. AT activity in plasma from 20 patients with AT Budapest variants (no significant difference was found between two mutations) was measured in parallel by reagents DG-Chrom AT(FXa), Biophen Antithrombin (FXa) and Stachrom ATIII (FIIa) with the results of 62±14(IU/dl), 80±32(IU/dl) and 73±20(IU/dl)(x±2SD). However, not only type of reagent can influence the result of AT activity. We have observed considerable variations in activity in one patient during several years measured with the same method. This could lead to additional factors which might affect the activity of antithrombin.   Molecular genetics Chairs : Elisabetta Castoldi ( The Netherlands), Tilman Hackeng (The Netherlands)   Subcommittee Project update : Update and maintenance of the antithrombin, protein C and protein S mutation databases. Elisabetta Castoldi (The Netherlands) Despite the availability of several general open-access genetic databases that are maintained by major institutions and regularly updated (OMIM, HGMD, LOVD, etc.), recent years have witnessed a revival of locus-specific mutation databases. These databases focus on single genes and diseases and report not only the details of each identified mutation, but also a wealth of additional information specifically targeted to researchers and clinicians studying that particular gene/protein or disease. Within the SSC Subcommittee on Plasma Coagulation Inhibitors, locus-specific mutation databases for antithrombin (AT, encoded by the SERPINC1 gene), protein C (PC, encoded by the PROC gene) and protein S (PS, encoded by the PROS1 gene) have been established and maintained until the Nineties, but have since been abandoned and become outdated. During the SSC Subcommittee Meeting in Kyoto, Japan (2011), a plenary discussion on the usefulness of these locus-specific databases triggered an SSC project proposal for the update and indefinite maintenance of the AT, PC and PS mutation databases, which was approved shortly before the SSC Subcommittee Meeting in Liverpool, UK (2012). This presentation discussed the challenges (recruitment of an expert committee for each gene, financial support, ethical issues, etc.) and opportunities (desirable information to be included in the database, relationships with other locus-specific databases, etc.) of this enterprise. The bottom line is that updating and maintaining the locus-specific AT, PC and PS mutation databases is going to be a major effort which requires the long-term commitment of all researchers in the plasma coagulation inhibitors field for the shared benefit of the whole scientific community.   Subcommittee Project proposal : Racial differences in genetic risk factors for venous thromboembolism. Hiroko Tsuda ( Japan) Aim/Mandate: Factor V Leiden and prothrombin G20210A are well-known hereditary thrombophilias in Caucasians, in contrast, protein S (PS) and protein C (PC) deficiencies are much more prevalent among Asians than non-Asians. PS Tokushima (K155E), a PS gene mutation with a phenotype of type II PS deficiency, is a genetic risk factor for venous thromboembolism (VTE) in Japanese. Two PC gene mutations with phenotypes of type II PC deficiency, PROC c.565C>T and PROC c.574_576del, are identified as genetic risk factors for VTE in Chinese. In order to elucidate the racial differences in genetic risk factor for VTE, we build up a global network and investigate worldwide distributions of PS and PC type II deficiencies. Methodology: 1) Build up a network of professional contacts for researching racial differences in genetic risk factors for VTE, which includes Asian and European countries, and USA. The latter two geographic regions consist of different racial groups. 2) Collaborative study to determine the racial differences in the frequencies of PS and PC type II deficiencies and their causal gene mutations, in both general populations and the VTE patients. Plasma PS is analyzed using a new quantitative total PS assay system for high performance screening for type II PS deficiency [1] as well as conventional PS assays. Plasma PC is also analyzed, using chromogenic and clotting assays for PS activity. Gene analysis is performed to find out race-specific genetic risk factors for VTE. This project starts in 2013 and will be finalized in 2015. Reference: 1. Tsuda T. et al. Blood Coagul Fibrinolysis. 23: 56-63, 2012Can plasma levels of PS, PC and AT predict the presence of an underlying genetic defect? Anna Pavlova ( Germany)   Interplay of inherited and environmental factors and life events contributes the pathogenesis of thromboembolism. Of all inherited factors, deficiencies of natural anticoagulant proteins, including antithrombin (AT), protein C (PC) and protein S (PS) are the most important causes for thrombophilia. Thus, the ultimate purpose of thrombophilia investigation in a clinical setting is to enable accurate prediction of the thrombotic risk in a particular individual. First-line diagnosis depends entirely on performing a plethora of functional and antigenic assays to measure the factors that predispose to thrombosis. Despite current standardisation procedures, all these assays show significant inter-laboratory variability and inconsistency in phenotype between individuals with apparently similar levels of functional protein. Therefore, plasma activity levels of natural anticoagulant proteins have not always been found to be a good prognostic indicator of clinical severity of the disorder. This observation raises the question if plasma levels of AT, PC and PS and can predict a causative genetic defect. The molecular bases of AT, PC and PS deficiencies are highly heterogeneous. The most frequent genetic defects are missense mutations, but other types of gene defects, such as nonsense mutations, splice-site mutations, deletions and insertions have also been reported. A review of literature reveals a significant discordance between laboratory diagnostic phenotype data and genotype data in the ranked order PS > PC>AT, the later with the highest concordance of phenotype and genotype. Our data have shown that the highest mutation detection rate (MDR) was found for the SERPINC1 gene (83.5%), followed by the PROC (69%) and PROS1 (43%) genes. Even at AT activities close to the normal range (75%), the MDR was 70%. In contrast, for PC and PS deficiencies, the MDR dropped significantly at mildly lowered to subnormal values. Additionally, evidence has been provided that genetic analysis of patients with PC levels above 70% and PS levels above 55% may not be indicated because of the very low likelihood of detecting a mutation associated with inherited PC or PS deficiencies. DNA-based testing has the advantage of detecting a mutation independent of phenotypic characterization and therefore, represents a useful diagnostic tool for confirming inherited AT, PC or PS deficiencies, especially for patients with borderline activities or patients on an anticoagulation therapy. Thus, genetic testing in thrombophilia patients can be helpful to address specific issues of patient management, to guide the duration of anticoagulant therapy, to quantify the need for primary or secondary thrombosis prophylaxis, or determine whether family members also need evaluation.   Standardisation of Protein S activity assays   The problem : Lack of agreement in PS activity results by different methods. Steve Kitchen (UK)   Recent data form UK National External Quality Assessment Scheme (NEQAS) exercises related to protein S activity assays were presented. These show statistically different and clinically relevant differences between results obtained with different reagents. This occurs in normal samples and in the presence of protein S deficiency. Not all PS activity kits were well represented amongst cebtres who participated in the calibration of the SSC plasma standard which also complicates potency assignment to commercial standards. This short presentation formed the introduction to the final session in the subcommittee programme. Moderator : Piet Meijer (The Netherlands),   Panel : Steve Kitchen (UK), Ian Jennings (UK), Richard Marlar (USA). Manufacturers of Protein S activity assays that are used in external quality assessment programs or are otherwise known to the panel members had been invited to make a 5 min presentation describing p. Manufacturers were given some guidance and were invited to address interferences and the effects of test sample lyophilisation on assay results. This was considered important to facilitate interpretation of data form EQA programs where this a regular cross assessment of assays but which is almost always done using lyophilised samples. Not all manufacturers accepted the invitation. Those who did were allocated a slot in alphabetical order. The following assays were described Hyphen _ Jean Amiral Instrumentation laboratory – Ralph Bottenus R2 diagnostics – Marc Goldford Siemens – Jorgen Patze Stago – Francois Depasse There is a subcommittee project investigating the discrepancies between results with different assays which will involve a collaborative exercise led by Ian Jennings and including Pioet Meijer and Richard Marlar. The panel felt it would be important to invite manufacturers to participate in this in addition to expert laboratories.  
by S. Kitchen
Wednesday, June 19, 2013
2012 Annual Minutes Locked Topic 0 S. Kitchen Plasma Coagulation Inhibitors Subcommittee Minutes 28 June 2012 Chairman: Steve Kitchen (UK) Co-chairmen: Elisabetta Castoldi (The Netherlands), Tilman Hackeng (The Netherlands), Richard Marlar (USA), Piet Meijer (The Netherlands) , Laurent Mosnier (USA) Update on Subcommittee projects. S Kitchen (UK)  Two projects are now approved and are beginning. One is led by Elisabetta Castoldi ( and will update the existing mutation databases of Antithrombin, Protein C, and Protein S and willcreate the infrastructure for the indefinite maintenance/update of these databases. A ‘task-force’ of 4-5 people led by an expert in the field will be set up for each gene .New mutations reported since the last update of the respective database will be identified by searching public resources (HGMD, PubMed) and entered in the existing databases manually. Meanwhile, an electronic interface for the submission of all newly identified mutations directly by the discoverer(s) will be created and advertised. Submitted information will be checked by the competent task-force before being officially posted.  The second approved project is the Investigation into discrepancies in Protein S activity assay results. This aims to elucidate the cause of marked differences between PS activity results obtained with different manufacturers kits ( as reported by several external quality assessment programmes). This is being led by Ian Jennings ( with Piet Meijer and Richard Marlar. Frozen and lyophilised samples from normal and PS deficient subjects will be sent to participatingcentres employing different PS activity methods together with a common calibrator.  Session 1. Effects of New Oral anticoagulants on plasma coagulation inhibitor testing: Chairs Steve Kitchen (UK) , Piet Meijer (The Netherlands)   1.1 The Effect of Rivaroxaban on laboratory testing for coagulation inhibitors: experience form ECAT surveys Piet Meijer (The Netherlands)  Rivaroxaban is an oral anticoagulant acting as a direct factor Xa inhibitor. It is used for the prevention of venous thromboembolism (VTE) in patients who have undergone total hip replacement or total knee replacement surgery as well as for stroke prophylaxis in patients with non-valvular atrial fibrillation. Rivaroxaban may affect haemostasis assays because of the effect on Factor Xa. This was investigated in a number of different studies [1-5]. These studies have clearly shown that samples with rivaroxaban taken from volunteers as well as in-vitro spiked samples affect both global and specific haemostasis assays. In haemostasis laboratories samples could be presented from patients under treatment with Rivaroxaban without any information available about this treatment. It is therefore important that technical personnel as well as clinical chemists are aware of the potential effect of Rivaroxaban on several haemostasis assays. For that reason the ECAT has distributed in several of their surveys a normal pooled plasma spiked with approx. 200 ng/mL Rivaroxaban. This concentration is within the therapeutic range and shown to have impact on several haemostasis assays [1-5]. In the 2011-2 survey such a sample was used in the thrombophilia module. The effect of Rivaroxaban is expressed as the relative change in activity in comparison to a similar normal pooled plasma, except for the APC Resistance ratio results. The activities of haemostasis factors investigated in the spiked plasma were corrected for the dilution factor of the plasma as a result of the addition of Rivaroxaban. Antithrombin, protein C and protein S activity assays . It was observed that there is no effect of Rivaroxaban on the anti-IIa Antithrombin assay and the chromogenic protein C assay. A significant effect of Rivaroxaban can be observed for the anti-Xa antithrombin assay (+20%), the protein C clotting activity assay (+30%) and the protein S activity assays (+ 100%).Because both the protein C clotting activity assay and the protein S activity assay are APTT-based assays an effect could be expected. APC Resistance .Table 1 shows the effect of Rivaroxaban on APC Resistance testing for the most frequently used methods. It can be observed that for all methods, except the Chromogenix Coatest APC Resistance test, an increase in the APC ratio occurs in the presence of Rivaroxaban. Such an effect was also observed in the study of Hillarp and co-workers [5]. They show a concentration-dependent increase of the APC Resistance ratio. This implies that in principal a heterozygous Factor V Leiden patient under the treatment with Rivaroxaban could have a ratio close to normal. Conclusion:Data in the literature as well as observations from ECAT surveys using a sample enriched with Rivaroxaban show a significant effect on several haemostasis assays. Laboratories should be aware of this phenomenon to interpret appropriately results from samples of a patient under treatment with Rivaroxaban.  Table 1: The effect of Rivaroxaban (≈ 200 ng/mL) on the measurement of APC Resistance. Method APC ratio - Rivaroxaban APC ratio + Rivaroxaban Chromogenix Coatest APC Resistance (global test) 3.58 3.15 Chromogenix APCR-V / I.L. HemosIL FVL (specific test) 2.58 3.03 Siemens ProC AcR (global test) 2.29 2.66 Siemens PC Global/FV (specific test) 2.14 2.74 Pentapharm Pefakit APC-R FVL (specific test) 3.83 5.17  References 1. Samama MM, Martinoli JL, LeFlem L, Guinet C, Plu-Bureau G, Depasse F, et al. Assessment of laboratory assays to measure rivaroxaban--an oral, direct factor Xa inhibitor. Thromb Haemost, 2010; 103: 815-25. 2. Mani H, Hesse C, Stratmann G, Lindhoff-Last E. Rivaroxaban differentially influences ex vivo global coagulation assays based on the administration time. Thromb Haemost, 2011; 106: 156-64. 3. Tichelaar V, de Jong H, Nijland H, Kluin-Nelemans H, Meijer KMulder A. Interference of rivaroxaban in one-stage and chromogenic factor VIII:C assays. Thromb Haemost, 2011; 106: 990-2. 4. Asmis LM, Alberio L, Angelillo-Scherrer A, Korte W, Mendez A, Reber G, et al. Rivaroxaban: Quantification by anti-FXa assay and influence on coagulation tests A study in 9 Swiss laboratories. Thromb Res, 2012; 129: 492 - 498. 5. Hillarp A, Baghaei F, Fagerberg Blixter I, Gustafsson KM, Stigendal L, Sten-Linder M, et al. Effects of the oral, direct factor Xa inhibitor rivaroxaban on commonly used coagulation assays. J Thromb Haemost, 2011; 9: 133-9.  1.2 Effects of dabigatran on PC PS and AT assays Bob Gosselin1, Dot Adcock2, Steve Kitchen3, Denis Dwyre1 1UC Davis Health System, Sacramento CA, 2Esoterix Coagulation Englewood, CO. 3Royal Hallamshire Hospital, Sheffield, UK.  Dabigatran etexilate, an inactive precursor, is rapidly converted in the blood and liver by esterase-catalyzed hydrolysis to its active form, dabigatran.Dabigatran is a rapidly acting, non-peptidic inhibitor of both free and bound thrombin used to anticoagulate patients with atrial fibrillation, hip and knee arthroplasty, or venous thromboembolism.We sought to assess the effect of dabigatran on various coagulation assays using plasma was spiked with this drug.Laboratories in United States, Canada and United Kingdom with expertise in coagulation laboratory testing volunteered to participate in our blinded study.Four laboratories performed antithrombin [AT], three performed protein C [PC], and three performed protein S [PS] testing.Thrombin-based AT assays overestimated AT concentrations in the presence of dabigatran at ~125ng/mL resulting in about 10% increase in concentration compared to baseline, with ~ 20-30% fictitious increase of AT with drug concentrations of ~400-500ng/ml.Falsely increased PC activity was observed in clot-based methods at dabigatran concentrations of >150 ng/mL, and results fell outside test linearity at drug concentration of 400-500ng/mL.Chromogenic PC methods demonstrated no significant effect to any concentration of dabigatran.Clot-based PS activity assays, whether APTT or RVV based, were sensitive to dabigatran with significantly elevated results at 25ng/mL of dabigatran.Free PS antigen by LIA methods, did not show any significant effect to any concentration of dabigatran.  1.3 Effects of rivaroxaban and dabigatran on plasma inhibitor testing.I Fagerberg Blixter , TL Lindahl , F Baghaei , M Berndtsson, K Strandberg, A Hillarp. On behalf of the expert group on coagulation of the External Quality Assurance in Laboratory Medicine in Sweden.  Rivaroxaban, an oral direct factor Xa inhibitor, and dabigatran, an oral direct thrombin inhibitor, are developed for prophylactic treatment of thromboembolic disorders. Laboratory monitoring is not needed but the effects on coagulation assays, including tests for natural plasma inhibitor, are incompletely known. The objectives were to investigate the effect of rivaroxaban and dabigatran on coagulation assays used to evaluate natural plasma inhibitors.Rivaroxaban and dabigatran were added to normal plasma in the concentration range 0–1000 μg/L and analysed using different reagents for antithrombin, protein C, protein C and activated protein C (APC) resistance.The results were highly variable, depending on the assay type and drug. Not surprisingly, antithrombin assays based on Xa was sensitive for rivaroxaban whereas thrombin-based antithrombin assays over-estimate the antithrombin in samples containing dabigatran. Chromogenic protein C assays and latex enhanced immunoassays for protein S were not affected but coagulation-based functional assays for protein C or protein S were compromised by both drugs. The APC resistance assay based on the activated partial thromboplastin time is unreliable during treatment with the new anticoagulants. However, the APC resistance test that is based on the activation of coagulation at the level of prothrombinase are unaffected by rivaroxaban. Different assays, and even different reagents within an assay group, display variable effects by therapeutic concentrations of rivaroxaban and dabigatran. Without detailed knowledge about the specific effects on locally used assays for plasma inhibitors it is advised against to perform these assays during treatment with the new anticoagulant drugs.   Session 2 Chairs : Richard Marlar, Steve Kitchen  2.1 The effect of CRP and factor VIII on clot based protein C and protein S assays. Jun Teruya, MD, DSc, Kim Nguyen, MLS(ASCP), Vadim Kostousov, MD, Sridevi Devaraj, PhD, DABCC,Texas Children’s Hospital and Baylor College of Medicine, Houston, TX, USA  PTT based protein C (PC) and protein S (PS) assays are known to be affected by heparin of >1 unit/mL and factor VIII (FVIII) of >250%. PTT is used to monitor heparin therapy, but is known to be affected by the presence of C-reactive protein (CRP) in the presence of heparin. CRP binds phosphocholine, thus the phospholipids in a PTT reagent may be partially neutralized by CRP. It is not known if an increased CRP level affects PTT-based PC and PS assays. This study investigated if CRP affects PC and PS assays in the presence of heparin. We also studied if PC and PS assays are not affected by FVIII up to 250%. Normal plasma was spiked with CRP purified from ascites fluid (endotoxin and azide-free) in the absence and presence of heparin (0, 0.6, and 1.2 units/mL). CRP did not clearly affect PC or PS level up to 25 mg/dL. Since the plasma specimens for PC and PS assay are diluted by Owren-Koller buffer, we assume that 10 times diluted CRP may not be affecting both assays. Higher heparin of 1.2 units/mL affected PC assay but not PS assay. Increased FVIII activity from 115 to 428% lowered both PC and PS levels in a linear manner that accompanied by ≈5.5% decrease PC and PS activity with each +100% FVIII increment.  2.2 Stability of Antithrombin, Protein C and Activated Protein C Resistance in Whole Blood . Kieron Hickey, PeterCooper andSteve Kitchen. Royal Hallamshire hospital, Shieffeild UK. Whole blood samples were drawn from healthy donors (0.109M, BD Vacutainer).One sample was processed (centrifuged 2000g for 10 minutes, frozen at -80oC) within 1 hour of venepuncture and theremaining samples stored at room temperature for 24, 48, or 72 hours prior to processing.Antithrombin, tested by FIIa and FXa assays demonstrated stability in normal donors (n=24) for up to 72 hours, with a mean 2% loss in activity for both FIIa and FXa methods.Protein C was assayed using an in house venom-chromogenic (n=23) and clot-based functional assay (n=13).Samples demonstrated stability for 24 hours (mean loss -3%, range +3 % to -11%) and less than 24 hours (mean loss -5%, range +5 to-15%) for the chromogenic and functional assay respectively .Samples tested using an activated protein C resistance assay (n=10) showed stability for up to 72 hours with a mean loss in activity of 9%.  2.3 Total protein S assay system: Clinical significance and pre-analytical quality control. Hiroko Tsuda1, Takao Kobayashi2, Tomohide Tsuda3 1Nakamura Gakuen University, 2Hamamatsu Medical Center, 3Shino-Test Corporation, Japan. Activated protein C (APC) cofactor activity of protein S (PS) has been measured on free PS using clotting assays. However, marked variations in results obtained with different kits make PS activity assays to be problematic for diagnosing type II PS deficiency. Recently, we developed a novel chromogenic total PS activity assay for automated analyzers, enabling a simultaneous measurement of total PS antigen using latex-based assay (Blood Coagul Fibrinolysis 2012, 23:56-63). By using our new total PS assay system, the specific activity of PS in the plasma samples of five out of twelve Japanese patients suffering from venous thromboembolism were found to be less than 0.70, and thus supposing type II PS deficiency. The storage of whole blood samples at room temperature for less than 4 hours resulted in a 3 to 5% reduction of total PS activity, the reduction rising to 20-40% after 24 hours. However, total PS antigen was unchanged after 24 hours sample storage. Western blotting analysis of the plasma with reduced total PS activity revealed the presence of an additional PS band with a slightly faster electrophoretic mobility, probably being the PS molecule cleaved at thrombin-sensitive region.  2.4 Genotype and laboratory and clinical phenotypes of protein s deficiency. Michael Spannagl, Munich, Germany,Hemostasis Outpatient Clinics, Munich University Hospital The diagnosis of hereditary thrombophilia caused by protein S deficiency remains laborious. From 2005 to 2010, we documented 135 patients with suspected hereditary protein S deficiency due to low PS activity and/or antigen plasma levels for whom mutational analysis of the PROS1 gene had been performed by direct double-stranded sequencing of the amplified 15 exons including splice sites. Multiplex ligation-dependent probe amplification was performed on 12 of 15 exons in cases with no mutation found but a large deletion in the PROS1 gene was suspected. Mutations were identified in 49 patients, 9 by familial screening. Altogether, 17 new and 11 previously described mutations of PROS1 were identified among the 49 patients. Lower protein S plasma levelshave been confirmed in women in the hormonally active age. After the exclusion of acquired protein S deficiency due to pregnancy or hormonal contraceptives, there remained only 1 case with protein S activity levels less than 40% that could not be explained by sequence variations or deletions in the examined regions of the PROS1 gene. In conclusion after the exclusion of conditions associated with acquired protein S deficiency, persistently low protein S activity levels are highly indicative of a genetic alteration in PROS1. We observed a clear correlation between the laboratory phenotype and the type of mutation. This could not be demonstrated comparing clinical presentation and laboratory phenotype, compatible with themultifactorial etiology of venous thrombosis.  Session3 Chairs : Laurent Mosnier (USA), Tilman Hackeng (The Netherlands)  3.1Novel assays for EPCR encryption and cellular APC resistance Eveline A. Bouwens and Laurent O. Mosnier (USA) . Scripps Research Institute, LA jolla, USA. Activated protein C (APC) is a plasma coagulation inhibitor with potent anticoagulant and cytoprotective activities. Activation of protein C zymogen to generate APC occurs on the endothelial cell surface by the thrombin-thrombomodulin complex and is greatly accelerated in the presence of the endothelial protein C receptor (EPCR). After activation, APC bound to negatively charged phospholipids mediates anticoagulant activity via the proteolytic inactivation of coagulation factors Va and VIIIa. Alternatively, APC in complex with EPCR mediates direct anti-apoptotic and anti-inflammatory cytoprotective effects on cells via EPCR-assisted activation of protease activated receptor 1 (PAR1). Thus, EPCR plays a central role in both APC’s anticoagulant and cytoprotective activities. During inflammation, inflammatory mediators and various cytokines severely reduce EPCR-dependent binding of (A)PC to cells, thereby negatively affecting protein C activation and APC’s cytoprotective effects. Depletion of EPCR-dependent binding of (A)PC to cells conceptualizes a novel phenomenon of "cellular APC resistance” similar to the well-known "anticoagulant APC resistance” associated with an increased risk for venous thrombosis. According to the current paradigm, inflammatory mediators induce EPCR shedding by the induction of the metalloproteinase TACE/ADAM17. Recently a second mechanism was proposed according to which a phospholipase can modify or "encrypt” EPCR to lose its ability to bind APC, thereby rendering EPCR dysfunctional. These observations may have important implications for thrombotic and inflammatory vascular disease since EPCR inactivation in vivo, either genetically or induced by blocking antibodies, aggravates and increases susceptibility to thrombotic and inflammatory disease.  3.2 Thrombin generation as an intermediate phenotype for genetic studies on venous thrombosisElisabetta. Castoldi (Maastricht University, The Netherlands)  Venous thrombosis is a multifactorial disease with a strong genetic component. Although several genetic risk factors for venous thrombosis have been identified in the last 50 years, recent progress has been slow. This may be due, at least in part, to the fact that classifying study subjects as cases (=individuals with the disease) and controls (=individuals without the disease) does not discriminate well between carriers and non-carriers of the risk alleles, leading to a dramatic loss of statistical power, especially for low-risk variants. This problem can be circumvented by taking an intermediate phenotype, i.e. a measurable quantitative trait that closely correlates with the disease risk, instead of disease status itself, as the end-point of linkage or association studies. In fact, measuring an appropriate intermediate phenotype makes it possible to place every study subject on a continuous scale of disease risk, thereby improving the correlation between genotype (=carriership of risk alleles) and phenotype (=position on the risk scale). Intermediate phenotypes are already successfully exploited in the genetics of several complex disorders, from type II diabetes to asthma. In this lecture, I present evidence that thrombin generation, which measures the overall tendency of a plasma sample to form thrombin, may be a suitable intermediate phenotype for genetic studies on venous thrombosis.
by S. Kitchen
Friday, September 14, 2012
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