Multimer Distribution Research Articles

Role of Von Willebrand Factor Triplet Bands in Glycoprotein Ib-Dependent Platelet Adhesion Under Flow Conditions

AbstractAbstract 3356 Objective:Multimeric glycoprotein von Willebrand factor (VWF) exhibits a unique triplet structure of individual oligomers, resulting from ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type 1 motifs 13) cleavage. The faster and slower migrating triplet bands of a given VWF multimer respectively have one shorter or longer N-terminal peptide sequence. Within this peptide sequence, the A1 domain regulates interaction of VWF with platelet glycoprotein (GP)Ib. Distribution of VWF triplet bands is significantly altered in some types of VWD, however, the impact of triplet structure on VWF function has not been investigated so far. Methods:Platelet-adhesive properties of two VWF preparations with similar multimeric distribution but different triplet composition obtained by size exclusion in addition to heparin affinity chromatography were investigated for differential functional activities. Preparation A was enriched in intermediate triplet bands, while preparation B predominantly contained larger triplet bands. Collagen- and GPIb-binding was determined by surface plasmon resonance (SPR). Platelet adhesion under flow was determined using flow-chamber models. Results:Binding studies revealed that preparation A displayed a reduced affinity for recombinant GPIb, but an unchanged affinity for collagen type III, when compared to preparation B. Under high-shear flow conditions, preparation A was less active in recruiting platelets to collagen type III. Furthermore, when added to blood from patients with von Willebrand disease (VWD), defective thrombus formation was less restored. Conclusion:Thus, VWF forms lacking larger size triplet bands appear to have a decreased potential to recruit platelets to collagen-bound VWF under arterial flow conditions. By implication, changes in triplet band distribution observed in patients with VWD may result in altered platelet adhesion at high-shear flow. Disclosures:Schwartz:Octapharma: Employment. Kannicht:Octapharma: Employment. Fuchs:octapharma: Employment. Kröning:octapharma: Employment. Solecka:Octapharma: Employment.

The structure of the von Willebrand factor is not altered in patients with colorectal carcinoma

Elevated levels of von Willebrand factor (VWF) are often observed in many diseases including colorectal cancer, but this finding is not definite. The aim of our study was to examine the change in VWF multimer distribution in patients with colorectal cancer. We randomly selected nine patients from each of the four Union for International Cancer Control (UICC) stages of colon cancer. VWF antigen (VWF:Ag), VWF-cleaving protease ADAMTS-13 level and factor VIII activity (FVIII:C) were determined. The multimer distribution of VWF was visualized using electrophoretic multimer analysis. The VWF multimer structure was normal with no difference between the four UICC stages. There was no significant increase in VWF:Ag and FVIII:C levels in the more advanced UICC stages. There was no significant difference in the ADAMTS-13 level according to the UICC stage. There was no change in the VWF multimer distribution to indicate acquired von Willebrand disease.

Characterisation of von Willebrand factor A1 domain mutants I1416N and I1416T: correlation of clinical phenotype with flow‐based platelet adhesion

Type 2M von Willebrand disease (VWD) results from mutations in the A1 domain of von Willebrand factor (VWF) that reduce its platelet-binding function. However, currently employed VWF functional static assays may not distinguish between clinical phenotype. Fifteen individuals from five kindreds with VWF-A1 domain mutations I1416T or I1416N, correlated with mild and moderate clinical phenotypes, respectively, were investigated. The mutations were reproduced by site-directed mutagenesis and expressed in HEK293T cells; functional studies of the recombinant mutants, including GPIbα binding using a flow-based assay, were performed. Plasma from all individuals demonstrated discordant reductions in VWF antigen and platelet-binding function in the presence of high-molecular-weight VWF multimers consistent with VWD type 2M. There was lowered expression and secretion of both mutants compared with wild type (WT) recombinant (r)VWF as well as a significant reduction in GPIbα binding. Binding to collagen was normal and electrophoretic analysis demonstrated a similar multimer distribution between the mutant proteins and wt-rVWF. GPIbα binding under flow was also significantly reduced for I1416N and I1416T rVWF. Impairment of GPIbα binding was more marked for I1416N rVWF than I1416T under both static and flow conditions: this was in spite of similar VWF:Ristocetin cofactor (RCo) activities in patient plasma and is consistent with a respective clinical phenotype. Our findings have established for the first time that I1416N and I1416T are responsible for a type 2M VWD phenotype and demonstrate that quantification of VWF function under shear stress may provide a more accurate measure of clinical severity than the static functional measurements in current diagnostic use.

Comparison of type I, type III and type VI collagen binding assays in diagnosis of von Willebrand disease

von Willebrand factor (VWF) plays a key role in coagulation by tethering platelets to injured subendothelium through binding sites for collagen and platelet GPIb. Collagen binding assays (VWF:CB), however, are not part of the routine work-up for von Willebrand disease (VWD). This study presents data on collagen binding for healthy controls and VWD subjects to compare three different collagens. VWF antigen (VWF:Ag), VWF ristocetin cofactor activity and VWF:CB with types I, III and VI collagen were examined for samples obtained from the Zimmerman Program. Mean VWF:CB in healthy controls was similar and highly correlated for types I, III and VI collagen. The mean VWF:CB/VWF:Ag ratios for types I, III and VI collagen were 1.31, 1.19 and 1.21, respectively. In type 1 VWD subjects, VWF:CB was similar to VWF:Ag with mean VWF:CB/VWF:Ag ratios for types I, III and VI collagen of 1.32, 1.08 and 1.1, respectively. For type 2A and 2B subjects, VWF:CB was uniformly low, with mean ratios of 0.62 and 0.7 for type I collagen, 0.38 and 0.4 for type III collagen, and 0.5 and 0.47 for type VI collagen. Normal ranges for type I, III and VI collagen are correlated, but higher values were obtained with type I collagen as compared with types III and VI. The low VWF:CB in type 2A and 2B subjects suggests that VWF:CB may also supplement analysis of multimer distribution. However, these results reflect only one set of assay conditions per collagen type and therefore may not be generalizable to all collagen assays.

PK-sensitive PrPSc Is Infectious and Shares Basic Structural Features with PK-resistant PrPSc

One of the main characteristics of the transmissible isoform of the prion protein (PrPSc) is its partial resistance to proteinase K (PK) digestion. Diagnosis of prion disease typically relies upon immunodetection of PK-digested PrPSc following Western blot or ELISA. More recently, researchers determined that there is a sizeable fraction of PrPSc that is sensitive to PK hydrolysis (sPrPSc). Our group has previously reported a method to isolate this fraction by centrifugation and showed that it has protein misfolding cyclic amplification (PMCA) converting activity. We compared the infectivity of the sPrPSc versus the PK-resistant (rPrPSc) fractions of PrPSc and analyzed the biochemical characteristics of these fractions under conditions of limited proteolysis. Our results show that sPrPSc and rPrPSc fractions have comparable degrees of infectivity and that although they contain different sized multimers, these multimers share similar structural properties. Furthermore, the PK-sensitive fractions of two hamster strains, 263K and Drowsy (Dy), showed strain-dependent differences in the ratios of the sPrPSc to the rPrPSc forms of PrPSc. Although the sPrPSc and rPrPSc fractions have different resistance to PK-digestion, and have previously been shown to sediment differently, and have a different distribution of multimers, they share a common structure and phenotype.

The impact of glutathione transferase kappa deficiency on adiponectin multimerisation in vivo

Glutathione transferase Kappa (GSTK1-1) also termed disulfide bond-forming oxidoreductase A-like protein (DsbA-L) has been implicated in the post-translational multimerization of adiponectin and has been negatively correlated with obesity in mice and humans. We investigated adiponectin in Gstk1(-/-) mice and surprisingly found no difference in the levels of total serum adiponectin or the level of high molecular weight (HMW) multimers when compared with normal controls. Non-reducing SDS-polyacrylamide gel electrophoresis and western blotting also showed a similar distribution of low, middle and HMW multimers in normal and Gstk1(-/-) mice. Variation in adiponectin has been correlated with glucose tolerance and with the levels of phosphorylated AMP-kinase but we found similar glucose tolerance and similar levels of phospho 5-AMP-activated protein kinase in normal and Gstk1(-/-) mice. Consequently, our findings suggest that GSTK1-1 is not absolutely required for adiponectin multimerization in vivo and alternate pathways may be activated in GSTK1-1 deficiency.

Characterisation of adiponectin multimers and the IGF axis in humans with a heterozygote mutation in the tyrosine kinase domain of the insulin receptor gene

Low levels of adiponectin, IGF-binding protein 1 (IGFBP1) and IGFBP2 and high levels of leptin correlate with several indices of insulin resistance and risk of type 2 diabetes. However, in insulin receptoropathies, plasma adiponectin is paradoxically increased despite severe insulin resistance, whereas the IGF axis is sparsely described. Here, we aimed to characterise the multimeric distribution of adiponectin and the IGF axis in humans with a heterozygous INSR mutation (Arg1174Gln). Blood samples obtained from six Arg1174Gln carriers and ten lean, healthy controls before and after a euglycaemic-hyperinsulinaemic clamp were examined for plasma adiponectin multimers, leptin, total IGF1, IGF2, free IGF1, IGFBP1 and IGFBP2. Despite tenfold elevated fasting insulin and marked insulin resistance in Arg1174Gln carriers, the levels of total adiponectin, leptin, IGFBP1 and IGFBP2 were similar to those observed in controls, while total IGF1, IGF2 and free IGF1 levels were increased. The relative fraction of high-molecular weight adiponectin was increased, whereas both the absolute concentration and the fraction of low-molecular weight adiponectin were decreased in Arg1174Gln carriers. Interestingly, exogenous insulin failed to suppress total adiponectin in Arg1174Gln carriers, but reduced IGFBP1 and increased IGFBP2 as in controls. The normal levels of adiponectin, IGFBP1 and IGFBP2 in the face of highly elevated insulin levels suggest an impaired ability of insulin to suppress markers of common insulin resistance in carriers of a dominant-negative INSR mutation. However, together with the adaptive increases in IGF1 and IGF2 and a potentially improved distribution of adiponectin multimers, these changes may contribute to rescue insulin action in insulin receptor-deficient individuals.

Abstract 9379: Biomarkers of Aortic Stenosis Severity

Aortic stenosis (AS) requires repeat assessments to optimally time intervention. The utility of laboratory tests to complement clinical and hemodynamic assessments has not been established. We assessed clinical, echocardiographic, and laboratory data in 76 patients with AS or heart valve replacement (HVR), age 77 years, 37% women. Laboratory assessments included von Willebrand factor (VWF) antigen, activity, (Act/Ag, normal > 0.8), VWF multimer distribution (abnormal=loss of highest molecular weight multimers=Mult+), brain natriuretic peptide (BNP, abnormal > 155 pg/ml=BNP+), and platelet function analyzer 100 collagen-ADP closure time (PFA, normal < 121 seconds). Table: Coefficients of correlation, r, and F statistic. Chi-square, and probability. Correlation of BNP to mean gradient in patients with GFR>60 remained poor, r=0.29, while for PFA r = 0.65. Median (IQR) for PFA and BNP for mild, moderate, and severe AS and HVR are shown below. VWF multimers were abnormal in 3/14 mild AS, 6/14 moderate AS, 22/25 severe AS and 4/23 HVR patients. The severity of AS was most strongly associated with VWF multimer abnormality and prolonged PFA closure time, weakly with BNP, and not with VWF Act/Ag. Conclusion: Among various laboratory measures, PFA and VWF multimers are strongly associated with AS severity, and may merit assessment as complementary diagnostic and prognostic biomarkers in AS.

Von Willebrand Factor Collagen Binding Provides a Sensitive Screen for Identification of Type 2A and 2B Von Willebrand Disease

Abstract 379▪FN2▪This icon denotes a clinically relevant abstractCollagen binding is an easily performed test of von Willebrand factor (VWF) function but its role in clinical evaluation is still debated. Analysis of multimer distribution, on the other hand, is time-consuming and technically challenging. We hypothesized that VWF antigen (VWF:Ag), ristocetin cofactor activity (VWF:RCo), and collagen binding (VWF:CB) could identify the subset of von Willebrand disease (VWD) cases in which multimer analysis would be informative. Subjects from the Zimmerman Program for the Molecular and Clinical Biology of VWD were analyzed for VWF:Ag, VWF:RCo, VWF:CB (with type III human placental collagen), multimer distribution, and full VWF exon sequencing. Normal controls as well as patients with type 1, 2A, 2B, 2M, and 2N VWD were analyzed. The mean VWF:CB/VWF:Ag ratio for subjects with normal multimers was 1.10, while the mean ratio for subjects with abnormal multimers was 0.51 (p<0.001). When results were restricted to those subjects with confirmed type 2A or type 2B mutations, however, the mean ratio for subjects with abnormal multimers decreased to 0.41 (p<0.001 compared to those with normal multimers). For the 146 normal controls with multimer results available, 2 had absence of the highest molecular weight multimers, but normal collagen binding, normal bleeding scores, and no evidence of a VWF gene mutation, suggesting that the multimer results represented assay artifact. 354 type 1 subjects were examined; of those, 12 had abnormal multimer patterns. 7 had loss of the high molecular weight multimers. Of these, 5 had known type 1 VWD mutations and normal VWF:CB/VWF:Ag ratios, possibly representing sample artifacts rather than a true multimer abnormality, as no multimer issues have been previously reported for these mutations. One had no mutation found and one had a type 2A mutation. 2 had a full spectrum of multimers with relatively increased staining of the lower molecular weight bands; both with novel A1 domain mutations that are currently under investigation. 3 had larger than normal multimers observed, all with normal VWF:CB/VWF:Ag ratios. Of the 342 type 1 subjects with normal multimers, only one had a VWF:CB/VWF:Ag ratio of <0.7, likely due to very low values (VWF:CB of 2 and VWF:Ag of 4). There were 36 type 2A subjects available for analysis. 27 had loss of high molecular weight multimers. Only 3 of those had VWF:CB/VWF:Ag ratios >0.7, but none of those subjects had VWF mutations consistent with type 2A VWD. 7 subjects had a shift from high to low molecular weight multimers, 4 with VWF:CB/VWF:Ag ratios >0.7 and either known type 1 mutations or novel VWF gene mutations. 2 subjects had normal multimer distribution, one with a type1 VWD mutation and one with a novel mutation. Characterization of these novel mutations is in progress. All the 17 type 2B subjects had loss of high molecular weight multimers and abnormal collagen binding, with a VWF:CB/VWF:Ag ratio <0.7. Interestingly, however, not all had a reduced VWF:RCo/VWF:Ag ratio, suggesting VWF:CB would be required in addition to VWF:RCo if multimer distribution was omitted in initial evaluation of this type of VWD. Of 18 type 2M subjects, only one had an abnormal multimer distribution. That subject had no mutations in the VWF coding sequence and normal VWF:CB, although the VWF:RCo/VWF:Ag ratio was low at 0.53. Repeat analysis of a new sample from this subject is pending. All 7 type 2N VWD subjects had normal multimers and VWF:CB/VWF:Ag ratios >0.7. In our population, with the exception of mutations that are yet to be characterized, the combination of VWF:Ag, VWF:RCo and VWF:CB was sufficient to categorize patients as normal, type 1, type 2A, 2B or 2M in the before multimer analysis. These findings suggest that VWF:CB is a sensitive screen for detection of an abnormal multimer distribution. Collagen binding is technically much easier to perform, allowing multimer analysis to be reserved for those cases with low VWF:RCo/VWF:Ag or low VWF:CB/VWF:Ag ratios. Disclosures:No relevant conflicts of interest to declare.

Quantitative Analysis of VWF Multimer Structure: Discrimination Between VWD Subtypes

Abstract 1215The diagnosis of von Willebrand disease (VWD) and discrimination between its subtypes includes analysis of VWF:Ag, VWF:RCo, and VWF multimer structure. VWF multimer analysis is qualitative, and therefore a subjective assessment open to interpretation. It is often difficult to assess subtle differences in multimer structure. To address these shortcomings we have developed a quantitative method for analysis of VWF multimers. We have analyzed multimer structure for VWD patients and healthy controls recruited through the Zimmerman Program for the Molecular and Clinical Biology of von Willebrand Disease (ZPMCB-VWD). The patient population includes type 1 and type 2 VWD with well-defined genotypes and phenotypes. Multimer analysis was performed using a 0.65% LiDS-agarose gel electrophoresis system and western blotting with chemilumiscent detection using the Fujifilm LAS-3000 luminescent image analyzer. Densitometry was performed and area-under-the-curve calculated using MultiGauge analysis software. We calculated the percentage of low molecular weight (LMW) multimers defined as bands 1 – 5, mid-molecular weight (MMW) multimers (bands 6 – 10) and high molecular weight (HMW) multimers (bands >10). For healthy controls, the distribution of multimer density (mean ± standard deviation) was 25.3 ± 2.7% HMW, 56.1 ± 4.9% MMW, and 18.6 ± 3.4% LMW. Type 1 VWD (including type 1C) patients had a similar distribution of multimers (22.5 ± 7.6% HMW, 48.5 ± 6.7% MMW, 29.0 ± 7.2 % LMW), although there was a slight shift in distribution to increased LMW. For some type 1C patients with mutations including C1130Y and W1144G, we observed a small loss of HMW multimers (14.2 ± 0.8% HMW, 51.1 ± 1.4% MMW, 34.7 ± 2.3% LMW), as has been previously reported in patients with a C1130F variation. In contrast, some patients with the type 1C “Vicenza” mutation, R1205H, demonstrated increased HMW multimers (32.6 ± 1.0% HMW, 42.2 ± 4.0% MMW, 25.2 ± 3.0% LMW) as previously reported. Although the multimers in the type 1 patients are essentially normal, quantitative analysis reveals subtle abnormalities in structure. In type 2B VWD patients with mutations including V1316M, R1306W, and R1341W, a loss of HMW and MMW multimers was observed (7.1 ± 3.2% HMW, 40.4 ± 8.3% MMW, and 52.5 ± 11.4% LMW). A greater loss of HMW and MMW multimers was observed in patients with type 2A VWD with mutations including Y1349C, R1597W, G1609R, I1628T, G1631D, and G1670S (3.5 ± 6.2% HMW, 19.7 ± 20.4% MMW, and 76.9 ± 26.3% LMW). The type 2A subjects consisted of two groups: those with a virtually complete loss of HMW and MMW (0.0 ± 0% HMW, 4.0 ± 1.0% MMW, and 96.0 ± 1.0% LMW), and those with loss of HMW and decreased MMW (8.7 ± 7.5% HMW, 41.0 ± 14.7% MMW, and 50.3 ± 20.9% LMW). The latter group had a similar multimer distribution to that of type 2B VWD subjects. While most type 2A patients with mutations associated with increased susceptibility to ADAMTS13 proteolysis had severe multimer abnormalities (>95% LMW), some had only moderate abnormalities. Our study demonstrates that quantitative analysis of VWF multimer patterns more clearly distinguishes patients with various subtypes of VWD than subjective analysis. Although one of the two groups of type 2A patients is similar to the type 2B group, the other group is clearly different and is associated with specific genotypes, perhaps eliminating the need for DNA sequence analysis to make a definitive diagnosis for this group. This technique provides an objective measure of VWF structure to better characterize subtle changes observed in the subtypes of VWD and may help to determine the nature of any additional clinical laboratory testing to reach a clear-cut diagnosis. Disclosures:No relevant conflicts of interest to declare.

Molecular Analysis of Type 1 VWD Mutations: Effects of Mutant:Wild-Type Transfection Ratio and Protein Degradation

Abstract 1210 Background:Von Willebrand disease (VWD), the most frequently diagnosed bleeding disorder, is characterized by variable expressivity and incomplete penetrance. Bleeding severity in type 1 VWD does not always correlate with plasma VWF levels, except in cases of severe deficiency. It is possible that the phenotypic variability observed in type 1 VWD is related to the final ratio of mutant vs. wild-type (WT) subunits in the mature VWF multimeric structure. The aim of this study was to determine the role of mutant:WT transfection ratio on von Willebrand factor (VWF) expression, secretion and degradation in VWD type 1 mutations. Methods:Type 1 VWD mutations with reported normal multimer distribution were chosen from the D'-D3 region of VWF. Mutations of cysteine residues were eliminated to avoid interference with inter- and intra-chain disulfide linkages. Mutations were generated by performing site-directed mutagenesis on full-length human VWF cDNA within the pcDNA3.1(-)A vector, which appends VWF with a Myc-His tag (denoted mH). The following mutations were generated: M771I, R782Q, R924W, I1094T and T1156M.Mutant VWF was co-transfected with WT VWF contained within the pCIneo vector (mutant mH:WT pCIneo ratios investigated were 1:3, 2:2, 3:1, 4:0). Recombinant (r)VWF expression was measured using ELISA and concentrations were determined by comparison to a standard curve generated with pooled normal plasma. Multimer composition was analyzed using SDS-agarose gel electrophoresis followed by Western blot. Statistical significance was determined using one-way ANOVA with post-hoc Tukey test. Results:Homozygous expression of R924W or I1094T had no effect on rVWF expression or secretion compared to WT, while M771I, R782Q and T1156M significantly increased intracellular protein retention. Co-expression of M771I or R782Q at varying ratios with WT was able to partially correct rVWF secretion, although intracellular retention remained significantly higher than WT at all ratios (n=3, * p<0.05, Figure 1). Co-expression with WT cDNA was also able to correct T1156M retention in a dose-dependent manner (n=3, Figure 1), as described previously [Lethagen, Thromb and Haemost, 2002]. Multimer analysis of co-transfection supernatants exhibited normal and full distribution of multimers, as expected for type 1 VWD mutations. [Display omitted] Others have shown previously that heterodimers of WT and C1149R VWF, a type 1 VWD mutation, are degraded by the proteasome [Bodo et al, Blood, 2001], presumably via recognition of a folding defect within the mutant subunit. In order to determine the role of proteasomal degradation in the decreased secretion levels of our mutants, we performed experiments in the presence of the proteasome inhibitor MG-132. Treatment of co-transfected cells (mutant:WT 2:2) with 1 mM MG-132 for 16 hours prior to harvesting did not significantly affect secretion or overall expression of rVWF, suggesting that this pathway is not involved in the regulation of the expression of our mutants. Discussion:Our data demonstrate that M771I, T1156M and R782Q induce a significant increase in intracellular retention compared to WT protein, which could contribute to a quantitative deficiency in type 1 VWD, while R924W and I1094T do not appear to interfere with VWF production or secretion. Variable levels of intracellular retention have been observed in a previous study of VWF mutations identified in type 1 VWD patients [Eikenboom, et al, J Thromb Haemost, 2009]. While one interpretation of these results is that R924W or I1094T may not be causative mutations in type 1 VWD, other mechanisms including protein clearance and function remain to be explored. Although type 1 VWD mutations variably affect expression and secretion levels in vitro, studying platelet rolling on these mutants at a range of physiological shear stresses will provide valuable information regarding whether the degree of incorporation of mutant subunits into VWF multimers can affect supramolecular structure, and ultimately, hemostatic function. Disclosures:No relevant conflicts of interest to declare.

ADAMTS13 Cleavage of Von Willebrand Factor (VWF) in Commercial Concentrates and Effect on VWF Function

Abstract 4354 Objective:The characteristic multimeric pattern of plasmatic VWF results from asymmetric cleavage by ADAMTS13. Regulation of VWF multimer distribution is critical for its physiological function. In human plasma, VWF multimer gels reveal species of various multimeric sizes with flanking satellite bands (triplets). The faster and slower migrating bands encompassing a VWF multimer lack one N-terminal fragment or possess an additional N-terminal fragment, respectively. Defects in VWF secretion, impaired assembly of multimers, or increased proteolysis can cause von Willebrand Disease (VWD). Distribution of VWF triplet bands is significantly altered in some plasma-derived VWF concentrates. The impact of triplet structure on VWF function has not been investigated so far. Methods:Four commercially available VWF concentrates were analyzed for ADAMTS13 content as well as VWF multimer- and triplet structure using agarose gel electrophoresis. ADAMTS13 activity was quantified by fluorescence resonance energy transfer (FRET) assay. Samples composed of different VWF triplet distribution but comparable multimers were obtained by heparin affinity chromatography. Platelet adhesion under flow was determined using a flow-chamber model. Results:VWF concentrates markedly differed with respect to their content of ADAMTS13 antigen and activity. A higher content of ADAMTS13 correlated with an altered triplet structure reflected by an increased presence of the faster migrating triplet band, indicating VWF proteolysis. VWF-mediated platelet adhesion under flow over time was increased using a VWF fraction predominantly containing the slower migrating triplet band. Conclusion:These findings suggest that an intact triplet structure has an impact on platelet adhesion at physiological high arterial shear rate conditions. The relevance of VWF N-terminal domains for platelet binding and potential clinical consequences of enhanced proteolysis in commercial concentrates has to be further evaluated. Disclosures:Fuchs:octapharma: Employment. Solecka:Octapharma: Employment. Kröning:Octapharma: Employment. Kannicht:Octapharma: Employment. Schwartz:Octapharma: Employment.

First Case of Platelet-Type Von Willebrand Disease (PT-VWD) Associated with Type 2B Von Willebrand Disease (2B VWD)

Abstract 5313 IntroductionVWD 2B and PT-VWD are rare diseases, due to mutations inducing a gain of function respectively of von Willebrand factor (VWF) and of its platelet receptor, Glycoprotein (GP)1bα Case historyWe report here the case of a young girl, born with an extensive purpura and a severe thrombocytopenia: platelet count: 16G/L. There was no associated biological nor clinical abnormality. A high dose of 1g/kg of immunoglobulin G infused on day 1 was unsuccessful, and a HPA-1a (−) platelet concentrate infusion led to a partial and transient increase of the platelet count up to 60G/L. Thrombocytopenia then resolved spontaneously.Biological study showed no sign of materno-fetal allo- or auto-immunity, parents were not consanguineous.The diagnosis of type 2B VWD was performed when she was 5 months old: VWF:RCo < 13 IU/dl, VWF:Ag 60 IU/dl, positive ristocetin induced platelet aggregation (RIPA) at a low ristocetin concentration (0.5 mg/ml). RIPA mixing studies were unconclusive.The same biological abnormalities were found in the father, whereas the mother had normal hemostasis tests. The biological phenotype also included a study of the multimeric VWF structure, showing a marked decrease in percentage of VWF high and intermediate molecular multimers. Genetic analysis performed on VWF gene showed the heterozygous p.Pro1266Leu missense mutation in the VWF A1 domain. This mutation ( o ) is only slightly deleterious, and induces usually a mild disease, without thrombocytopenia, even in stress situations, with normal VWF multimeric distribution; therefore, it could not explain the biological phenotype severity in this family.GPIBA was then analysed, and a candidate point mutation p.Met239Ile was evidenced. This mutation had not been described yet, but p.Met255Val had already been found in diagnosed cases of PT-VWD. ConclusionThis case underlines the utmost importance to characterize precisely neonatal thrombocytopenia mechanism. Furthermore, it points out the difficulties to performing PT-VWD diagnosis, which incidence is most probably underestimated. In our case, it was the systematic and extensive biological workout performed in this case of isolated neonatal thrombocytopenia, without any obvious cause, which led to the diagnosis of a PT-VWD, inducing a severe biological phenotype, associated with type 2B VWD characterized by a mild expression. It is, to our knowledge, the first case described to date of such a morbid association. Disclosures:No relevant conflicts of interest to declare.

Characterization of Prefibrillar Tau Oligomers in Vitro and in Alzheimer Disease

Neurofibrillary tangles, composed of insoluble aggregates of the microtubule-associated protein Tau, are a pathological hallmark of Alzheimer disease (AD) and other tauopathies. However, recent evidence indicates that neuronal dysfunction precedes the formation of these insoluble fibrillar deposits, suggesting that earlier prefibrillar Tau aggregates may be neurotoxic. To determine the composition of these aggregates, we have employed a photochemical cross-linking technique to examine intermolecular interactions of full-length Tau in vitro. Using this method, we demonstrate that dimerization is an early event in the Tau aggregation process and that these dimers self-associate to form larger oligomeric aggregates. Moreover, using these stabilized Tau aggregates as immunogens, we generated a monoclonal antibody that selectively recognizes Tau dimers and higher order oligomeric aggregates but shows little reactivity to Tau filaments in vitro. Immunostaining indicates that these dimers/oligomers are markedly elevated in AD, appearing in early pathological inclusions such as neuropil threads and pretangle neurons as well as colocalizing with other early markers of Tau pathogenesis. Taken as a whole, the work presented herein demonstrates the existence of alternative Tau aggregates that precede formation of fibrillar Tau pathologies and raises the possibility that these hierarchical oligomeric forms of Tau may contribute to neurodegeneration.

Food restriction improves glucose and lipid metabolism through Sirt1 expression: A study using a new rat model with obesity and severe hypertension

Aims To determine the effects of food restriction (FR) on the expression of Sirt1 and its down-stream factors related to lipid and glucose metabolism in obese and hypertensive rats (SHRSP/IDmcr- fa), as a model of human metabolic syndrome. Main methods Male, 10-week-old SHRSP/IDmcr- fa rats were treated with 85% FR for 2 weeks. Metabolic parameters, serum adipocytokines and distribution of serum adiponectin multimers were investigated. Sirt1 expression was determined in epididymal adipose tissue, liver and skeletal muscle. We also determined the expression of PPARα, γ and other adipocyte-related genes in epididymal adipose tissue, and glucose transporters (GLUT2 and GLUT4) in the liver and skeletal muscle. Key findings FR improved the general conditions as well as blood chemistry of SHRSP/IDmcr- fa rats. In the epididymal adipose tissue of the FR rats, Sirt1 expression was enhanced, as was adiponectin, whereas leptin was downregulation, findings that were paralleled by the serum protein levels. Furthermore, the serum ratio of high to total adiponectin was increased in the FR group. The mRNA expression of Sirt1 was upregulated in the adipose tissue in the FR group. Sirt1 mRNA expression was downregulated, while PPARα and GLUT2 expression was enhanced in the liver. No differences were found in terms of Sirt1, PPAR or GLUT4 expression in skeletal muscle. Significance These results indicate that FR corrects adipokine dysfunction by activating PPARγ via Sirt1 in adipose tissue. Furthermore, glucose and lipid metabolism are activated by upregulation of GLUT2 via the activation of PPARα in the liver.

Reconstitution of Recombinant Factor VIII In fVIII-/- mice Restores Von Willebrand Factor Homeostasis

AbstractAbstract 2213Coagulation factor VIII (FVIII) binds von Willebrand factor (VWF) at the D′D3 domain with high affinity. FVIII almost always circulates as a FVIII-VWF complex in blood. Our recent study has demonstrated that FVIII accelerates proteolytic cleavage of VWF by ADAMTS13 under mechanically induced fluid shear stress (Cao et al, PNAS, 2008). In this study, we sought to determine: 1) the domain of FVIII required for the enhancing effect on VWF proteolysis by ADAMTS13 under these conditions; 2) the physiological relevance of this enhancing effect on plasma VWF homeostasis in a murine model. First, we employed the novel vortex-based shear assay developed in the laboratory to assess the rate enhancing effect of various recombinant FVIII variants on VWF proteolysis by ADAMTS13. In these experiments, purified plasma VWF (150 nM) was incubated for 10 min with recombinant ADAMTS13 (50 nM) in the presence of various concentrations of recombinant FVIII variants (0-20 nM) in a total volume of 20 μl under constant fluid shear generated with a bench top mini-vortexer. This maneuver appears to produce ∼75 dyn/cm2 of fluid shear stress. Proteolytic cleavage product (∼350 kDa, the dimer of two C-terminal fragments linked by a disulfide bond) was then determined by 5% SDS-polyacrylamide gel electrophoresis and Western blotting. We showed that addition of a recombinant light chain of FVIII (FVIII-LC) increased the formation of proteolytic cleavage product as a function of increasing FVIII-LC concentrations. This rate enhancing effect was similar to that of full-length recombinant FVIII and B-domainless FVIII (FVIII-SQ). However, addition of a heavy chain of FVIII (FVIII-HC) or a light chain variant lacking the acidic (a3) region (FVIII-Δa3LC) did not increase the formation of cleavage product under the same conditions. These results suggest that FVIII-light chain is sufficient for accelerating VWF proteolysis by ADAMTS13 under physiological conditions. The rate enhancing effect of FVIII-LC depends on its high affinity interaction with VWF. Second, we determined plasma VWF antigen and multimer distribution in fVIII-/- and fVIII+/+ (WT) mice with same genetic background (C57BL/6) prior to and after reconstitution with recombinant FVIII-SQ and variants via a hydrodynamic approach. Plasma VWF antigen was determined by a sandwich ELISA assay and plasma VWF multimers were determined by 1% agarose gel electrophoresis and Western blotting. We showed that plasma VWF antigen levels in the fVIII-/- mice (n=18) were increased by ∼2.0 fold as compared with those in the WT mice (n=14). No difference in the ratio of ultra large (UL)-VWF to dimer was observed between the fVIII-/- mice and the WT mice (p>0.05). These data suggest that lack of FVIII may impair plasma VWF homeostasis. To assess whether plasma FVIII affects VWF proteolysis in vivo, plasma VWF multimer distribution was determined by agarose (1%) gel electrophoresis and Western blotting in the fVIII-/- mice 48 hours after injection of a series of endotoxin-free expression plasmids encoding various FVIII variants/fragments (i.e. plasmids diluted in 2 ml normal saline and injected into tail vein within 5 seconds). We showed that the ratio of the UL-VWF multimers to the dimer in plasma of fVIII-/- mice receiving normal saline alone was 1.70 ± 0.59 (means ± SD) (n=10). However, the ratios in plasma of fVIII-/- mice receiving plasmids encoding canine FVIII-SQ, human FVIII-SQ, human FVIII-HC+LC, and FVIII-LC were 0.44 ± 0.37 (n=20), 0.88 ± 0.18 (n=9), 0.40 ± 0.20 (n=5), and 0.97 ± 0.29 (n=9), respectively. These ratios were dramatically reduced compared with that in fVIII-/- mice receiving normal saline alone (p values<0.05∼0.001). Our hydrodynamic injection approach resulted in plasma levels of FVIII-SQ, FVIII-LC+HC and FVIII-LC between 150% and 200% of the WT. In contrast, a hydrodynamic injection of plasmid encoding human FVIII-HC or FVIII-Δa3LC did not prevent the accumulation of plasma UL-VWF multimers in fVIII-/- mice. We therefore conclude that FVIII, through the high affinity binding interaction between its light chain and D′D3 domain of VWF, may play a critical role in maintaining VWF homeostasis under (patho) physiological conditions. Disclosures:No relevant conflicts of interest to declare.

Plasma von Willebrand factor multimer quantitative analysis by in-gel immunostaining and infrared fluorescent imaging

Introduction Electrophoretic analysis of plasma von Willebrand factor (VWF) multimer distribution and infrastructure is essential for subtyping von Willebrand disease. To improve the sensitivity, precision and efficiency of this assay, we developed and validated a new in-gel infrared fluorescent VWF multimer imaging method to visualize and quantify VWF multimers directly in the agarose gel, thus eliminating electroblotting or autoradiographic steps. Materials/Methods VWF multimer analyses of plasma samples from 34 patients with known von Willebrand disease or acquired von Willebrand syndrome, 9 patients with acquired VWF abnormalities, 26 normal volunteer donors and 49 patient samples referred for von Willebrand factor multimer analysis were performed by both traditional autoradiographic and the new infra-red imaging methods and compared. VWF multimer image data were electronically acquired, archived and analyzed. Results The in-gel infrared method has a sensitivity of detecting VWF antigen as low as approximately 1.6 IU/dL, a reliable fluorescent intensity with intra- and inter-day variability (CV) of 5% and 6% respectively, and provides superior imaging resolution and shortened test turnaround time. Using intermediate resolution agarose gel electrophoresis, the infra-red method sensitively detects subtle loss of highest molecular weight von Willebrand factor multimers in plasmas with acquired VWF abnormalities and in commercial normal reference plasmas, and provides evidence of increased proteolysis of ultralarge multimers in some type 2 VWD plasmas. Conclusions The in-gel infrared fluorescent VWF multimer imaging method provides a sensitive, reliable, efficient and robust system to improve laboratory testing for von Willebrand disease classification.

Structure and Function of a Recombinant von Willebrand Factor Drug Candidate

The complex structure, large size, and multiple posttranslational modifications of von Willebrand factor (VWF) presented a technological challenge for the production of recombinant VWF (rVWF). Nonetheless, we developed an rVWF product for treating von Willebrand disease, whereupon rVWF is coexpressed with recombinant factor VIII (rFVIII) in Chinese hamster ovary cells used to produce rFVIII for the treatment of hemophilia A. Here we describe the characterization of the structure and function of the rVWF drug product, with a focus on its in vitro platelet aggregation and matrix protein binding functions. Electron microscopy and multimer analysis revealed a highly organized structure for the rVWF protein, with a homogeneous multimer distribution including ultrahigh molecular weight multimers. The specific activity for binding to collagen and platelets mediated by ristocetin is higher in rVWF than in commercial plasma-derived VWF-FVIII complex products. The affinity and binding capacity of rVWF to FVIII is comparable to VWF in plasma. rVWF effectively binds to platelets and promotes platelet adhesion under shear stress similar to VWF in human plasma.

Analysis of von Willebrand factor structure by multimer analysis

Analysis of von Willebrand factor (VWF) structure is achieved by performing a highly specialized procedure, VWF multimer analysis. The test is reserved for the reference or specialized laboratory environment. The assay is qualitative (though under some circumstances multimers may be quantified) in that it assesses the overall size distribution of VWF multimers as well as their individual internal structure. The test is used predominantly to type or subtype von Willebrand disease. The analysis of VWF multimers generally consists of four steps: (1) electrophoresis of plasma in an agarose gel, (2) either gel fixation or transfer of the electrophoretic protein product to a membrane, (3) immunodetection of the protein, and (4) evaluation of the protein in the gel or membrane. The assay is complex, time consuming, requires specialized equipment and technical expertise, and is not standardized.

Distribution of adiponectin multimeric forms in Chinese women with polycystic ovary syndrome and their relation to insulin resistance

Adiponectin, an abundant adipokine with insulin-sensitizing properties, exists in different multimeric forms, including low-molecular weight, medium-molecular weight, and high-molecular weight (HMW) species. Alterations in the distribution of adiponectin multimers and the relationship between adiponectin multimers and insulin resistance (IR) in women with polycystic ovary syndrome (PCOS) remain unclear. The objective of this study was to compare adiponectin multimerization status and estimate insulin sensitivity in Chinese women with PCOS compared with age- and body mass index (BMI)-matched controls. Cross-sectional study involving 64 Chinese women with PCOS and 59 normal women. Circulating total adiponectin and its multimeric forms were determined by ELISA, and IR was estimated using the homeostasis model assessment IR index (HOMA-IR). After controlling for BMI status, levels of both total and HMW adiponectin were significantly lower in women with PCOS compared with normal women (P<0.05). Furthermore, HMW adiponectin provided a stronger contribution to models predicting IR than total adiponectin. Lastly, decreased HMW adiponectin was associated with increased HOMA-IR in both normal and PCOS women, and this association was independent of both overall adiposity and visceral adiposity. Levels of both total and HMW adiponectin were decreased in Chinese women with PCOS compared with normal control women, and the differences in HMW adiponectin persisted after controlling for BMI. Furthermore, HMW adiponectin is a stronger predictor of IR than total adiponectin in both women with PCOS and normal women.