The meeting this year was sub-divided into 5 different categories: (1) Protein S genotype, phenotype and deficiency; (2) Factor V gene and mechanisms of APC resistance, protein C deficiency and prothrombin G20210A; (3) Serpins (serine protease inhibitors); (4) Thrombophilic risk factors; and (5) Assay performance results for testing for familial thrombophilia.
 
1. Protein S genotype, phenotype and deficiency- consisted of talks by Drs. Walker, Soria, Bates and Faioni. Dr. Walker described her work of the West Scotland Donor Study (~3800 people), and it was stated initially that identifying protein S deficiencies has always been a difficult problem. She found that men had more total protein S than women, and a trend for more free protein S than women. They also found in women that with increasing age there was more total protein S, and when adjusting for age, post-menopause women had the same amount of protein S. Dr. Soria described work on the GAIT Project focusing on protein S, and his work focused on identifying candidate genes for hemostatic defects, and his work found a linkage with the gene region D1S194 to the factor V gene. Genome-wide linkage analysis found that C4B-binding protein was near, and they concluded that 1q32 could influence protein S levels and that this may be related to expression of C4b-BP. Dr. Bates talked about levels of evidence and the link with hemostatic disorders, asking questions about: a) methodology, accuracy, verification and variability?; b) evidence that the abnormality is associated with venous thrombosis?; c) potential confounders?; d) magnitude of abnormal significance; and e) link between abnormality and venous thrombosis and does it make biologic sense? She used warfarin therapy, pregnancy and antiphospholipid antibodies to study their link with protein S deficiency and disease phenotype. Her studies found no clear evidence with these 3 different states (inherited or acquired) and venous thromboembolic disease linked to protein S deficiency. A primary problem seems to be a lack of statistical power in these analyses. Without a doubt her studies are suggesting that we need more powerful tools to appreciate protein S deficiency, and again it reinforces the notion that describing protein S deficiency is difficult and is problematic for clinicians and laboratory workers. Dr. Faoini presented data exploring the possibility that a dimorphism, A2148G (Pro 626) influence protein S levels - she studied the effect of phenotype, effect of risk, and effects on diagnosis. She used the PROSIT patient group, and she found that Pro 626 modulates free protein S in healthy women independent of a protein S alpha gene mutation; and that Pro 626 had little effect on total protein S levels in healthy or protein S-deficient individuals.

2. Factor V gene and mechanisms of APC resistance, protein C deficiency and prothrombin G20210A consisted of talks from Drs. Spek, Bovill, Bernardi, and Magdelaine. Dr. Spek reviewed data about protein C polymophisms of the promoter region, and the 3 strudies found an overall risk that ranged from 1.00-2.00; and the results suggest that protein C promoter polymorphisms do not increase thrombotic risk in factor V Leiden patients, and that homozygous or compound heterozygous states of protein C deficiency have no association between clinical severity and polymorphisms of the promoter region. It is not clear how promoter polymorphisms in protein C affect protein C antigen levels. Additional studies found no clear candidate for different transcription factor binding sites, but there is a moderate thrombotic risk associated with these promoter polymorphisms, but there is no biological explanation yet for the differences in plasma levels. Dr. Bovill described a large pedigree (>700 live individuals) in Northern Vermont U.S.A., Quebec Canada and now some studies in collaboration with others in Europe. They are characterizing the founder effect of the 3363 C mutation gene insertion found in this family, and he has obtained funds to fully sequence the genome of 300 family members. Dr. Bernardi presented an overview of the R2 factor V, and reported that up to 10% of western-European persons are carriers of the 2 polymorphisms found in R2 factor V. The studies described to date are not in agreement in that some report decrease in factor V levels, some do not, and others report an increase in APC resistance, while some do not. The risk in some studies reaches 2.0 while in others it is <1.4. There is much evidence to further describe the molecular basis for this phenotype, correlation of biochemistry versus in vivo finding, and the basis for the discrepancies between clinical phenotype and laboratory values. Dr. Bernardi suggests the formation of a Working Group in this SSC to further define the clinical and biological significance of R2 factor V. The audience and the Chair/co-Chairís were in agreement with this suggestion. Dr. Magdelaine described an interesting single case of a double thrombomodulin mutation (Ala 25® Thr and Ala 455® Val) with homozygous factor V Leiden. Her results highlight that we should continue to study multi-genetic defects in thrombotic-related genes, since these single thrombomodulin mutations are not strongly associated to thromboembolic disease.

3. Serpins (Serine protease inhibitors) consisted of talks by Drs. Church, Hayashi, and Rezaie. Dr. Church reviewed a landmark paper of Dr. Kenneth Brinkhous (in memory about) from 1939, where he described the "heparin cofactor" responsible for the biologic activity of heparin. Dr. Church also mentioned a paper in press (Gary A. Silverman, Phillip I. Bird, Robin W. Carrell, Paul B. Coughlin, Peter G.W. Gettins, James I. Irving, David A. Lomas, Cliff J. Luke, Richard W. Moyer, Philip A. Pemberton, Eileen Remold-O'Donnell, Guy S. Salvesen, James Travis, and James C. Whisstock, 2001, "The serpins are an expanding superfamily of structurally similar but funtionally diverse proteins: Evolution, mechanism of inhibition, novel functions, and a revised nomenclature", published in J.Biol. Chem., published July 2, 2001 as 10.1074/jbc.R100016200) where the suggestions for nomenclature of serpins, new and old, are summarized. Dr. Hayashi described his work with protein C inhibitor in renal cell carcinoma (RCC), and he reported that in RCC there was an absence of protein C inhibitor antigen, with no change in urokinase levels. Adding back protein C inhibitor to a RCC cell line resulted in the inhibition of invasion; thus, suggesting that protein C inhibitor might function to regulate tumor cell invasion. Dr. Rezaie summarized his work about vitronectin interacting with plasminogen activator inhibitor type-1, which then becames a potent inhibitor of activated protein C. This work suggests that the potential pro-fibrinolytic effect of activated protein C might be due to a process involving PAI-1/vitronectin inhibiting activated protein C.

4. Thrombophilic risk factors included talks by Dr. Miyata and Dr. Moll. Dr. Miyata described the prevalence of protein C, antithrombin and plasminogen deficiency in the general population of Japan. The study used blood donors/patients seeking normal check-ups from the ages of 30-90. This work identified that 0.2%, 0.18% and ~4% of the population had protein C, antithrombin and plasminogen deficiency, respectively. The odds ratio for patients with deep vein thrombosis, compared to Western man, was higher for protein C and antithrombin deficiency, yet the Japanese have an occurrence of 0% with factor V Leiden. Dr. Moll proposed an International Collaboration Group through this SSC to study "combined thrombophila" focusing on three rare compound thrombophilias: homozygous Factor V Leiden/heterozygous Factor II G20210A; heterozygous Factor V Leiden/ homozygous Factor II G20210A; and homozygous Factor V Leiden/homozygous Factor II G20210A. The proposal is based on the occurrence of these defects ranging from 1/1,000,000 3/100,000,000 for these combined thrombophilias. It was proposed that an international collaboration be established, genetic labs contacted to inquire about their database; and patients could respond to a questionnaire posted on the Internet. Discussion from the audience was enthusiastic and the co-Chairs found the idea worthy of further discussion, except Dr. Aiach raised a point about should we include other combined thrombophilic defects along with these suggestions. Dr. Moll commented that he thought this was complicated enough to do as proposed.

5. Assay performance results for testing for familial thrombophilia consisted of talks by Dr. Jennings and Dr. Siegert. Dr. Jennings reviewed the laboratory pitfalls of their experience in the "UK NEQAS/EQUALS Experience", and he went on to describe the frequency of how often incorrect diagnosis was made for factor V Leiden, antithrombin, protein C, and protein S. He summarized most of the finding as differences in assay methods, assay kits used, reference plasmas, methodology details, and expression of the results using normal ranges. Dr. Siegert reported on the influence of low molecular weight heparin (LMWH) on various assays including protein S, RVVDT, and TFPI. In all cases, LMWH had a global effect on clotting assay measurements, which differs from un-fractionated heparin, especially when LMWH is used at >1 U/mL. These results caution us that even when neutralizing LMWH with protamine sulfate, there may be differences in the properties of the plasma samples when analyzed.

 

 

In conclusion, Dr. Church thanked the co-chairís for help in organizing this SSC meeting and for help in chairing individual sessions, he thanked the speakers for keeping within the given time constraints of this many presentations, he thanked the attendants provided by the congress for their help with the speakers, he reiterated the call for formation of 2 Working Groups (from Drs. Bernardi and Moll), he thanked the audience for their participation in asking questions, and encouraged them to communicate with the subcommittee chairman. The meeting was then adjourned by Dr. ChurchÖAu revoir ëtil next year!

(It was estimated by those in attendance that ~300 people were in this session)