An international collaborative study to compare different von Willebrand factor glycoprotein Ib binding activity assays: the COMPASS-VWF study.

Essentials New VWF activity assays are increasingly used but information on their comparability is limited. This is an ISTH SSC-organized study (expert labs, 5 countries) to compare all available assays. VWF activity by six assays correlated well with each other. The new assays show improved characteristics - minor differences are noted. SUMMARY: Background Several new assays have become available to measure von Willebrand factor (VWF) activity. The new assays appear to have improved performance characteristics compared with the old reference standard, ristocetin cofactor activity (VWF:RCo), but information is limited about how they compare with VWF:RCo and each other. Methods The von Willebrand factor Subcommittee of the International Society for Thrombosis and Haemostasis (ISTH) Scientific and Standardization Committee (SSC) designed a collaborative study involving expert laboratories from several countries to compare available tests with each other and with VWF:RCo. Eight laboratories from five countries were provided with blinded samples from normal healthy individuals and well-characterized clinical cases. Laboratories measured VWF activity using all tests available to them; data from six laboratories, not affected by thawing during transportation, are included in this study. Results All tests correlated well with VWF:RCo activity (r-values ranged from 0.963 to 0.989). Slightly steeper regression lines for VWF:Ab and VWF:GPIbM were clinically insignificant. The new assays showed improved performance characteristics. Of the commercially available assays, the VWF:GPIbR using the AcuStar system was the most sensitive and could reliably detect VWF activity below 1 IU dL-1 . The lower limit of the measuring interval for the VWF:GPIbM and the VWF:GPIbR assays was in the 3-4 and 3-6 IU dL-1 range, respectively. Inter-laboratory variation was also improved for most new assays. Conclusion All VWF activity assays correlated well with each other and the VWF:RCo assay. The slight differences in characteristics found in the COMPASS-VWF study will assist the VWF community in interpreting and comparing activity results.

von Willebrand disease and platelet disorders.

The diagnosis and management of bleeding disorders is made difficult by the complexity and variety of disorders, clinical symptoms and bleeding type and severity. von Willebrand disease (VWD) and platelet disorders are disorders of primary haemostasis and together represent the most common inherited bleeding disorders. In this article, we describe the diagnosis of VWD and platelet disorders and the treatment options for VWD.

Possible genetic sign of heat stress adaptation in Hungarian Grey Bos taurus breed.

The synthesis of Heat Shock Protein 70.2 mRNA is also regulated by the Upper Promoter elements of the gene. This promoter region is polymorphic in cattle. These polymorphisms have a major effect on the activity of the mRNA transcription. In a comparison of quantity of transcribed mRNA from the wild type and AP2 mutant allele the wild type can produce 2-3-fold more transcripts.The Hungarian Grey Cattle (HG) and Norwegian Red (NFR) as control breed were genotyped with PCR-RFLP method. Our results showed that the frequencies of alleles in breeds (p(wt)HG = 0.859419, p(wt)NFR = 0.5) are different. The effective response to heat stress in the Norwegian Red seems to be less important than in the Hungarian Grey breed. The extensive keeping in hot and arid region during centuries could have been proved as selection pressure for the heat tolerance.Our results combined with the global climate forecasts emphasize the role of autochthonous, well adopted, heat tolerant breeds in the near future.

Common large partial VWF gene deletion does not cause alloantibody formation in the Hungarian type 3 von Willebrand disease population.

BACKGROUND: Type 3 von Willebrand disease (VWD) is an autosomal recessive bleeding disorder, characterized by virtually undetectable plasma von Willebrand factor (VWF) and consequently reduced plasma factor VIII levels. Genetic mutations responsible for type 3 VWD are very heterogeneous, scattered throughout the VWF gene and show high variability among different populations. METHODS: Twenty-five severe VWD patients were studied by direct sequencing of the 51 coding exons of the VWF gene. The total number of VWD type 3 families in Hungary is 24, of which 23 were investigated. RESULTS: Fifteen novel mutations were identified in 31 alleles, five being nonsense mutations (p.Q1238X, p.Q1898X, p.Q1931X, p.S2505X and p.S2568X), four small deletions and insertions resulting in frame shifts (c.1992insC, c.3622delT, c.5315insGA and c.7333delG), one a large partial deletion (delExon1-3) of the 5'-region, four candidate missense mutations (p.C35R, p.R81G, p.C295S, p.C623T) and one a candidate splice site mutation (c.1730-10C>A). Six previously described mutations were detected in 17 alleles, including the repeatedly found c.2435delC, p.R1659X and p.R1853X. Only one patient developed alloantibodies to VWF, carrying a homozygous c.3622delT. CONCLUSION: We report the genetic background of the entire Hungarian type 3 VWD population. A large novel deletion, most probably due to a founder effect, seems to be unique to Hungarian type 3 VWD patients with high allele frequency. In contrast to previous reports, none of the five patients homozygous for the large partial deletion developed inhibitors to VWF. This discrepancy raises the possibility of selection bias in some of the reports.

Relationships between Podolic cattle breeds assessed by single nucleotide polymorphisms (SNPs) genotyping.

Italian Maremmana, Turkish Grey and Hungarian Grey breeds belong to the same Podolic group of cattle, have a similar conformation and recently experienced a similar demographic reduction. The aim of this study was to assess the relationship among the analysed Podolic breeds and to verify whether their genetic state reflects their history. To do so, approximately 100 single nucleotide polymorphisms (SNPs) were genotyped on individuals belonging to these breeds and compared to genotypes of individuals of two Italian beef breeds, Marchigiana and Piemontese, which underwent different selection and migration histories. Population genetic parameters such as allelic frequencies and heterozygosity values were assessed, genetic distances calculated and assignment test performed to evaluate the possibility of recent admixture between the populations. The data show that the physical similarity among the Podolic breeds examined, and particularly between Hungarian Grey and Maremmana cattle that experienced admixture in the recent past, is mainly morphological. The assignment of individuals from genotype data was achieved using Bayesian inference, confirming that the set of chosen SNPs is able to distinguish among the breeds and that the breeds are genetically distinct. Individuals of Turkish Grey breed were clearly assigned to their breed of origin for all clustering alternatives, showing that this breed can be differentiated from the others on the basis of the allelic frequencies. Remarkably, in the Turkish Grey there were differences observed between the population of Enez district, where in situ conservation studies are practised, and that of Bandirma district of Balikesir, where ex situ conservation studies are practised out of the original raising area. In conclusion, this study demonstrates that molecular data could be used to reveal an unbiased view of past events and provide the basis for a rational exploitation of livestock, suggesting appropriate cross-breeding plans based on genetic distance or breeding strategies that include the population structure.

Accelerated clearance alone explains ultra-large multimers in von Willebrand disease Vicenza.

BACKGROUND: von Willebrand disease (VWD) Vicenza is characterized by low plasma von Willebrand factor (VWF) levels, the presence of ultra-large (UL) VWF multimers and less prominent satellite bands on multimer gels, and the heterozygous amino acid substitution R1205H in the VWF gene. The pathogenesis of VWD Vicenza has been elusive. Accelerated clearance is implicated as a cause of low VWF level. OBJECTIVES: We addressed the question, whether the presence of ultra-large multimers is a cause, or a result of accelerated VWF clearance, or whether it is an unrelated phenomenon. PATIENTS/METHODS: We studied the detailed phenotype of three Hungarian patients with VWD Vicenza, expressed the mutant VWF-R1205H in 293T cells and developed a mathematical model to simulate VWF synthesis and catabolism. RESULTS: We found that the half-life of VWF after DDAVP was approximately one-tenth of that after the administration of Haemate P, a source of exogenous wild-type (WT) VWF (0.81 + or - 0.2 vs. 7.25 + or - 2.38 h). An analysis of recombinant mutant VWF-R1205H showed that the biosynthesis and multimer structure of WT and mutant VWF were indistinguishable. A mathematical model of the complex interplay of VWF synthesis, clearance and cleavage showed that decreasing VWF half-life to one-tenth of normal reproduced all features of VWD Vicenza including low VWF level, ultra-large multimers and a decrease of satellite band intensity. CONCLUSION: We conclude that accelerated clearance alone may explain all features of VWD Vicenza.

An Alu-mediated novel large deletion is the most frequent cause of type 3 von Willebrand disease in Hungary.

BACKGROUND: We studied 24 Hungarian patients from 23 unrelated families to identify the genetic background of the entire type 3 von Willebrand disease (VWD) population in this country. The current report focuses on the molecular characterization of a novel large deletion. RESULTS: A large partial deletion (delExon1-3) of the 5'-region of the von Willebrand factor gene (VWF) was detected in 12/48 alleles (25% of all type 3 alleles). The 5'-deletion breakpoint is located in the untranslated region between VWF and CD9, whereas the 3' breakpoint is in intron 3 of VWF. Analysis of the breakpoints showed Alu Y and Alu SP repetitive sequences at the ends of the deletion, suggesting that a recombination event caused the subsequent loss of the 35-kb fragment. DelExon1-3 was not found in any of the other screened populations. CONCLUSION: We report a large novel deletion including exons 1, 2 and 3 of VWF commonly causing type 3 VWD in the Hungarian population. This mutation, probably caused by an Alu-mediated recombination event, and subsequently distributed in Hungary by a founder effect, seems to be unique to Hungarian patients with a high allele frequency. Together, delExon1-3 and 2435delC make up 37.5% of the genetic defects in Hungarian patients with VWD type 3. This offers a rational approach to molecular testing of relevant families in Hungary.

Thrombosis risk in systemic lupus erythematosus: the role of thrombophilic risk factors.

OBJECTIVES: Thromboembolic episodes are frequent manifestations of systemic lupus erythematosus (SLE). Although the presence of anti-phospholipid antibodies (aPL) is known to contribute to thromboembolism (TE), the relative contribution of other TE risk factors is unknown. The aim of this study was to determine the prevalence of TE in a Caucasian SLE population, to identify the risk factors of highest importance, and to assess the clinical value of thrombophilia screening among SLE patients. METHODS: Samples from 105 patients were analysed with a screen including aPL, activated protein C resistance, factor V Leiden (FVL) and prothrombin G20210A mutations; protein C, protein S and antithrombin activity; factor VIII (FVIII) and von Willebrand factor (vWF), and homocysteine (Hcy) levels. RESULTS: The annual incidence of arterial and venous TE events in our SLE population was 5.4 and 12.4 per 1000, respectively. The highest risk of thrombosis was carried by the simultaneous presence of lupus anticoagulant (LA) and anti-cardiolipin (aCL) [relative risk (RR) = 4.03, 95% confidence interval (CI) 2.06-7.86] or anti-beta2-glycoprotein I antibodies (abeta2-GPI) (RR = 5.10, 95% CI 2.58-10.1). Positivity for the individual aPL tests all carried an elevated TE risk. The presence of other risk factors seemed to be of less importance. CONCLUSIONS: In SLE patients, the presence of aPL is a more significant risk factor for the development of thrombosis than the known inherited deficiencies. Based on these data, routine screening for additional hereditary risk factors seems to be unwarranted.

Quantitative genetic aspects of coat color in horses.

The aim of this study was to estimate genetic parameters for coat color in horses. Besides defining coat color classes (gray, chestnut, bay, and black), the phenotypes were also measured quantitatively according to standardized international procedures (Commission Internationale de l'Eclairage L*, a*, b*), where L* describes lightness, a* describes color saturation from red to green, and b* describes color saturation from yellow to blue. The total color saturation was derived from a* and b* and referred to as Chroma. A total of 294 horses from the breeds Lipizzan, Nonius, Arabian Pure Bred, Shagya Arabian, and Gidran were measured at neck, shoulder, and belly. Heritabilities (within and between breeds or color classes) and repeatabilities were estimated using REML from univariate animal models defined separately for gray and nongray horses. For gray horses, the estimated within-breed heritabilities for L* ranged from 0.45 to 0.49 and for a*, b*, and Chroma from 0.09 to 0.52, indicating moderate polygenic effect. For nongray horses, between-color class heritabilities were high (0.70 to 0.85) and within-color class heritabilities were negligible (except for L* measured on neck and belly, 0.21 and 0.34, respectively). Additionally, the importance of L* was described by the relation with the total melanin content of horse coat hair; for gray and nongray horses, a strong negative linear relationship was detected (P < 0.01). The spectrometric measures and the results of this study demonstrate a possible approach to the estimation of the polygenic component involved in coat color inheritance.

Microsatellite diversity, population subdivision and gene flow in the Lipizzan horse.

Blood samples of 561 Lipizzan horses from subpopulations (studs) of seven European countries representing a large fraction of the breed's population were used to examine the genetic diversity, population subdivision and gene flow in the breed. DNA analysis based on 18 microsatellite loci revealed that genetic diversity (observed heterozygosity = 0.663, gene diversity = 0.675 and the mean number of alleles = 7.056) in the Lipizzan horse is similar to other horse breeds as well as to other domestic animal species. The genetic differentiation between Lipizzan horses from different studs, although moderate, was apparent (pairwise F(ST) coefficients ranged from 0.021 to 0.080). Complementary findings explaining the genetic relationship among studs were revealed by genetic distance and principal component analysis. One genetic cluster consisted of the subpopulations of Austria, Italy and Slovenia, which represent the classical pool of Lipizzan horse breeding. A second cluster was formed by the Croatian, Hungarian and Slovakian subpopulations. The Romanian subpopulation formed a separate unit. The largest genetic differentiation was found between the Romanian and Italian subpopulation. Genetic results are consistent with the known breeding history of the Lipizzan horse. Correct stud assignment was obtained for 80.9% and 92.1% of Lipizzan horses depending on the inclusion or exclusion of migrant horses, respectively. The results of the present study will be useful for the development of breeding strategies, which consider classical horse breeding as well as recent achievements of population and conservation genetics.

Inbreeding, microsatellite heterozygosity, and morphological traits in Lipizzan horses.

While the negative effects of inbreeding and reduced heterozygosity on fecundity and survival are well established, only a few investigations have been carried out concerning their influence on morphological traits. This topic is of particular interest for a small and closed population such as the Lipizzan horse. Thus, 27 morphological traits were measured in 360 Lipizzan mares and were regressed on the individual inbreeding coefficients, as well as on the individual heterozygosity and mean squared distances (mean d(2)) between microsatellite alleles within an individual. Both individual heterozygosity and mean d(2) were based on 17 microsatellite loci dispersed over 14 chromosomes. The results obtained by multivariate analysis reveal significant effects of stud (P <.0001), age at measurement (P <.0001), and mean d(2) (P =.0143). In univariate analyses, significant associations were obtained between length of pastern-hindlimbs and inbreeding coefficient (P <.01), length of cannons-hindlimb and mean d(2) (P <.01), and length of neck and mean d(2) (P <.001). After adjustment of single-test P values for multiple tests (Hochberg's step-up Bonferroni method), only the association of the length of neck and mean d(2) remained significant (P =.0213). Thus, no overall large effects of inbreeding, microsatellite heterozygosity, and mean d(2) on morphological traits were observed in the Lipizzan horse.

Type 1 von Willebrand disease mutation Cys1149Arg causes intracellular retention and degradation of heterodimers: a possible general mechanism for dominant mutations of oligomeric proteins.

Some families affected by von Willebrand disease type 1 show high penetrance with exceptionally low von Willebrand factor (VWF) levels. Previously, a mutation associated with this dominant phenotype, Cys1149Arg, was found to decrease the secretion of coexpressed normal VWF, and the mutation was proposed to cause intracellular retention of pro-VWF heterodimers. To demonstrate heterodimer formation, a model was developed in which subunits could be distinguished immunologically and by size. Recombinant VWF lacking domain A1 (dA1), A3 (dA3), or both (dA13) was secreted efficiently as a full range of multimers. Cotransfection of Cys1149Arg and dA13 resulted in the secretion of multimeric VWF containing about 250 kd (Cys1149Arg) and about 210 kd (dA13). Cell lysates contained pro-VWF forms of Cys1149Arg and dA13. Immunoprecipitation with an antidomain A1 antibody recovered both subunits in heterodimers, and subunit ratios were consistent with random dimerization. Similar results were obtained for cotransfection of Cys1149Arg and dA1. Normal VWF has a Cys1149-Cys1169 intrachain bond. When cotransfected with normal VWF, Cys1149Arg or the double mutant Cys1149Arg+Cys1169Ser caused a similar decrease in VWF secretion, suggesting that an unpaired Cys1169 does not explain the intracellular retention of Cys1149Arg. VWF Cys1149Arg was not secreted from BHK cells but was degraded intracellularly within about 4 hours, and the proteasome inhibitor lactacystin delayed its clearance more than 16 hours. Thus, dominant von Willebrand disease type 1 may be caused by heterodimerization of mutant and normal subunits in the endoplasmic reticulum followed by proteasomal degradation in the cytoplasm. A similar dominant negative mechanism could cause quantitative deficiencies of other multisubunit proteins.

Influence of environmental and genetic factors on allergen-specific immunoglobulin-E levels in sera from Lipizzan horses.

To investigate whether allergen-specific IgE production is influenced by environmental and genetic factors, IgE levels against 2 mould extracts (Alternaria alternata [Alt a] and Aspergillus fumigatus [Asp f]) and against recombinant (r) rAlt a 1, rAsp f 7 and rAsp f 8 were determined by ELISA in sera from 448 Lipizzan horses living in 6 studfarms. Statistical evaluation showed a significant effect of studfarm-specific environment on IgE levels against the different allergens, but genetic factors also influenced allergen-specific IgE production: an heritability of 0.33 was found for IgE levels against the 2 mould extracts and of 0.21 for rAsp f 8-specific IgE. Heritability estimates for rAlt a 1- and rAsp f 7-specific IgE were negligible. Investigations for a possible association between Major Histocompatibility Complex (MHC) class I antigens and specific IgE levels were carried out. The most consistent significant association was found between the equine leucocyte antigen (ELA) A8 and undetectable IgE titres against rAsp f 7 and rAsp f 8. Significant ELA associations were also demonstrated between ELA A1 and higher specific IgE levels, between ELA A14 and lower IgE levels against the mould extracts and in one studfarm between ELA Be27 and lower Aspergillus-specific IgE levels.

Localization of disulfide bonds in the cystine knot domain of human von Willebrand factor.

von Willebrand factor (VWF) is a multimeric glycoprotein that is required for normal hemostasis. After translocation into the endoplasmic reticulum, proVWF subunits dimerize through disulfide bonds between their C-terminal cystine knot-like (CK) domains. CK domains are characterized by six conserved cysteines. Disulfide bonds between cysteines 2 and 5 and between cysteines 3 and 6 define a ring that is penetrated by a disulfide bond between cysteines 1 and 4. Dimerization often is mediated by additional cysteines that differ among CK domain subfamilies. When expressed in a baculovirus system, recombinant VWF CK domains (residues 1957-2050) were secreted as dimers that were converted to monomers by selective reduction and alkylation of three unconserved cysteine residues: Cys(2008), Cys(2010), and Cys(2048). By partial reduction and alkylation, chemical and proteolytic digestion, mass spectrometry, and amino acid sequencing, the remaining intrachain disulfide bonds were characterized: Cys(1961)-Cys(2011) (), Cys(1987)-Cys(2041) (), Cys(1991)-Cys(2043) (), and Cys(1976)-Cys(2025). The mutation C2008A or C2010A prevented dimerization, whereas the mutation C2048A did not. Symmetry considerations and molecular modeling based on the structure of transforming growth factor-beta suggest that one or three of residues Cys(2008), Cys(2010), and Cys(2048) in each subunit mediate the covalent dimerization of proVWF.

CD4 but not CD8 is comodulated with the T-cell antigen receptor (TCR) after activation of a CD4+ CD8+ human leukemia line with staphylococcal enterotoxin.

Recent studies support the possibility of an interaction between CD4/8 and the TCR complex. To determine if there is specificity in this interaction, we have studied the comodulation of CD4/8 with the CD3/TCR complex on a CD4+ CD8+ human leukemic T-cell line stimulated with staphylococcal enterotoxin A (SEA) bound to Raji cells. FACS analysis revealed that CD3 and the TCR were modulated from the surface. CD4 and not CD8 was comodulated with the T-cell receptor complex, supporting the existence of a docking site on the TCR with selectivity for CD4 or CD8 but not both. Fewer than 45 SEA molecules per presenting cell led to detectable comodulation. The ratio of %CD4/%TCR modulation varied with both time and the amount of SEA used for stimulation. ConA or PHA induced modulation of CD3 but, unlike SEA, failed to induce IL-2 secretion, suggesting multiple pathways and states of T-cell activation. Our findings also suggest that some human T leukemic lines can respond to antigen.

Anticardiolipin antibodies: association with anti-DNA antibodies, disease activity, renal involvement and a history of thrombosis in systemic lupus erythematosus.

A one-year study was conducted to evaluate the clinical significance of anticardiolipin antibody (ACA) whether it was a reliable predictor for thromboembolic events and related diseases in systemic lupus erythematosus (SLE) patients. The correlation between ACA and anti-ds-DNA antibodies and disease activity was also studied. Of particular importance was the question if any association could be found between ACA positivity and renal disorders in SLE patients. One hundred and eighty-seven serum samples from 88 SLE patients were assayed for ACA. Clinical records of these patients were reviewed for a history of thromboembolic events, related diseases and renal disorders, 80.7% of the 88 SLE patients were positive for ACA. The incidence of thrombosis and related diseases within this group was 35.1%. Since the correlation was not significant, it does not seem to be advisable to use elevated ACA values as predictive for thromboembolic events and related diseases. On the other hand, an apparent association between ACA levels, anti-DNA antibody levels and disease activity was found.

Effect of vitamin A treatment on the immune reactivity of patients with systemic lupus erythematosus.

Treatment of patients suffering from systemic lupus erythematosus with vitamin A (100,000 U daily for 2 weeks) resulted in an enhancement of antibody-dependent cell-mediated cytotoxicity, natural killer cell activity and blastogenic response to plant mitogens and interleukin-2 (IL-2).