ALBERT

Description: Abstract 3306 Background. Von Willebrand factor (vWF) is involved in relevant biological functions such as i) platelet adhesion to the vascular endothelium, through interaction with the platelet membrane glycoprotein 1b-alpha (GpIbα), ii) transport of factor VIII in the circulation, preventing its proteolytic degradation by protein C and iii) regulation of angiogenesis and megakaryocytopoiesis Under the effect of hydrodynamic stress vWF changes its conformation from a globular to an elongated shape, which allows self-aggregation of vWF multimers and the formation of sticky grid, where blood platelets can adhere under high shear flow. Natural type 2B vW mutants (T2B vWD) are considered to have both an increased affinity for platelet GpIb and accelerated hydrolysis by ADAMTS-13. Methods. In this study, the ability of recombinant WT and R1306W vWF mutant to self associate and bind to platelets was investigated in a flow-chamber system under controlled shear stress ranging from 5 to 60 dyn/cm2. The recombinant proteins were produced in HEK-293 cells and purified by affinity and size-exclusion chromatography. The analysis of vWF self-association was performed by atomic force microscopy and dynamic light scattering spectroscopy. The interaction between immobilized recombinant GpIb and WT and R1306W vWF was studied with Surface Plasmon Resonance spectroscopy (SPR). Results. The SPR measurements showed that WT and mutant R1306W A1-A2-A3 domains bind to platelet GpIb with comparable affinity (Kd ≈ 20 nM). Full length WT vWF does not significantly interact with GpIb under static conditions, whereas the R1306W mutant showed a significant binding to GpIb. The process of vWF self-association evolved differently as a function of shear stress, whereby at values

Description: In a patient with apparently type 2A VWD (mean values: FVIII:C = 32 U/dL, VWF:Ag = 7 U/dL, VWF:RCo = 〈 6 U/dL, VWF:CB = 2.0 mg/ml, loss of high and intermediate molecular weight multimers in plasma and low platelet VWF content) a transient thrombocytopenia occurred after an infusion test with desmopressin. The propositus brother showed a less severe laboratory data (mean values: FVIII:C = 36 U/dL, VWF:Ag = 17 U/dL, VWF:RCo = 6 U/dL, VWF:CB=1, RIPA = 1.2 mg/ml). In order to better characterize VWD type diagnosis, the complete VWF gene was screened by SSCP analysis and direct DNA sequencing. The two brothers resulted to be compound heterozygous for mutation P1337L and a novel candidate defect C275R. In their children, mutations were associated with either type 2B (P1337L) or type 1 (C275R) VWD in heterozigous state. Both mutations were introduced separately into an individual vector pSV-VWF by site direct mutagenesis. rVWF-P1337L and rVWF-C275R, were transiently expressed in Cos 7 cells, solely, together and with the rVWF-WT. All the rVWFs in cells supernatants were tested by VWF:Ag and multimer analysis. Binding of different rVWFs to the GpIb platelet receptor was evaluated by an ELISA method (Federici et al. Haematologica89:77, 2004), at increasing concentrations of ristocetin (0, 0.125, 0.25, 0.5, 0.8 and 1 mg/mL), and the rVWF bound to GpIb was revealed by anti-VWF antibody-HRP reading O.D. 492 nm. Only the expressed rVWF-C275R showed a strongly reduced VWF:Ag, in comparison with the rVWF-WT, in cells medium and a complete absence of large and intermediate multimers, only dimers being present. The other rVWFs (P1337L, P1337L/WT and C275R/WT) showed only a slightly reduced VWF:Ag, in comparison to rVWF-WT, with the exception of rVWF C275S/WT that was about 40% of the rVWF-WT; all of them, however showed a full set of multimers. GpIb binding assay (see Table) showed that rVWF obtained by co-expression of mutation P1337L with C275R behave very similarly to rVWF-P1337L, suggesting that rVWF-C275R subunits cannot be assembled into intermediate and large molecular weight multimers. Therefore, in the propositus and his brother, mutation P1337L, present in all the VWF subunits, strongly enhanced the VWF interaction with GpIb receptor. As a consequence, in vivo, these patients showed a reduced RIPA (〉2 mg/ml), as typically observed in VWD type 2A, because of the loss of VWF high- and intermediate multimers due to the spontaneous interactions of such “hemizygous” type 2B VWF with circulating platelets. Our data suggest once more the importance of mutation analysis within family members and in vitro expression studies especially when phenotypic data are not certain. rVWFs Ristocetin 0 mg/mL Ristocetin 0.125 mg/mL Ristocetin 0.25 mg/mL Ristocetin 0.5 mg/mL Ristocetin 0.8 mg/mL Ristocetin 1 mg/mL WT 0.066 O.D. 0.071 O.D. 0.128 O.D. 0.154 O.D. 0.691 O.D. 0.924 O.D. P1337L 0.117 O.D. 0.310 O.D. 0.689 O.D. 0.984 O.D. 0.984 O.D. 1.039 O.D. P1337L/WT 0.063 O.D. 0.166 O.D. 0.409 O.D. 0.740 O.D. 0.894 O.D. 1.008 O.D. P1337L/C275R 0.084 O.D. 0.372 O.D. 0.635 O.D. 0.925 O.D. 1.105 O.D. 1.117 O.D.

Description: Background: Type 2B von Willebrand disease (VWD) is an inherited bleeding disorder caused by abnormal von Willebrand factor (VWF) that displays increased affinity to the platelet glicoprotein 1b alpha (GpIba) and is due to a group of mutations clustered within VWF A1 domain. Such an enhanced 2BVWF-GpIba binding usually result in loss of large VWF multimers and moderate-mild thrombocytopenia. A llama-derived antibody fragment (AuVWFa11) recognizing the GpIba-binding conformation has been recently developed (Blood2005;106:3035). Aims and design of the study: to further explore the usefulness of AuVWFa11 in type 2B diagnosis, we have prospectively tested AuVWFa11 in our cohort of 16 patients previously characterized by platelet count, VWF multimers and mutations. Methods: Data of platelet count with mean platelet volume (MPV) and morphologic evaluation of the blood smear to search for giant platelets or aggregates were associated with the history of physiologic or pathologic stress conditions such as pregnancy, infections, surgery or use of DDAVP. All patients were diagnosed by ristocetin induced platelet agglutination (RIPA) in the Platelet Rich Plasma (PRP), ristocetin cofactor activity (VWF:RCo) with VWF antigen (VWF:Ag), multimeric structure of VWF. Mutations within VWF A1 domain were searched for and confirmed by sequencing exon 28. AuVWFa11 was tested in 40 normal individuals (expressed as % of active VWF in normal pool plasma =0.70±0.13) and in type 2B. Results: Data (mean ± SD) of the AuVWFa11 tested in the 16 patients with type 2B VWD are correlated with the main phenotypic data and genotype (Table1). Platelet count 〈 140,000 was found at baseline in only 3/16 (%), but was observed after stress conditions in 12/16 cases (%); no reduced platelet counts was found in 4/16 patients (%) from two different families (R1308L, R1341Q). An increased MPV was found in 12 cases but giant platelet and aggregates in only 1 case. Activated VWF as tested by AuVWFa11 was positive in all but 3 (R1308L) cases, with values ranging from 2 to 6 times higher than normal controls: values 〉 3 correlate with loss of large VWF multimers and mild-moderate thrombocytopenia. Conclusions: The AVWF11a can show activated VWF in most type 2B VWD patients, especially when 2B VWF mutants induce significant loss of large multimers and thrombocytopenia. Therefore AuVWF11a can be a useful additional tool in the diagnosis of type 2B VWD. Table 1 Mutation (n) RIPA (mg/ml) VWF.Ag (U/dL) Plat Count (×10^9/L) MPV (micron^3) Loss of HMW Mult AuVWFa11 (ratioNPP) R1306W (5) 0.65 40±9 165±39 10.3±2.3 YES 3.7±1.5 R1308C (3) 0.72 53±16 163±61 11.5±1.9 YES 3.3±2.3 R1308L (3) 0.50 48±13 341±104 8.1±3.1 NO 0.5±0.2 I1309V (1) 0.40 115 222 11.8 PARTIAL 2.1 V1316M (2) 0.50 32±7 119±30 9.2±2.4 YES 4.4±0.1 P1337L (1) 0.50 48 222 9.5 PARTIAL 1.3 R1341Q (1) 0.67 43 422 9.9 PARTIAL 2.9

Description: We analyzed the association of bleeding severity with eight candidate gene haplotypes within pedigrees of twelve index cases of VWD type 2 (six type 2M, three type 2A and three type 2B), using a covariance components model for multivariate traits (Mendel 5.5: QTL Association). In addition to the 12 index cases, these pedigrees included 58 affected and 55 unaffected relatives, as characterized by plasma Ristocetin cofactor (VWF:RCo), levels, VWF antigen (VWF:Ag) levels, ristocetin induced platelet agglutination (RIPA), VWF multimeric analysis and specific mutations of VWF. A bleeding severity score was derived from a detailed questionnaire. The severity of bleeding episodes was ranked from 0 to 5 in each of eleven bleeding categories: epistaxis, cutaneous symptomatic bleeding, bleeding from minor wounds, oral cavity bleeding, gastrointestinal bleeding, bleeding associated with tooth extraction, surgery, postpartum hematoma, muscle hematoma, hemarthrosis, and menorrhagia. Donors were genotyped using specific fluorescent-labeled oligonucleotides in a melting curve analysis (LightTyper, Roche, Inc.). VWF:RCo levels, followed closely by VWF:Ag levels, had the strongest influence on bleeding severity score. ITGA2 promoter haplotype -52T and ITGA2B haplotype 1 (Ile843) were each associated with increased bleeding severity scores (p=0.039 and p = 0.024, respectively), and the association of both remained significant when bleeding severity score was adjusted for age (p=0.012 and p = 0.010, respectively). These associations persisted even if menorrhagia, the only gender-associated bleeding category, was excluded from the analysis. This is the first report of a statistically significant association of ITGA2 haplotype -52T with risk for bleeding in vivo, a finding that is consistent with the significant attenuation of ITGA2 transcription by this promoter haplotype in in vitro studies. No statistically significant association was observed with the major haplotypes of six other candidate genes, GP1BA, ITGB3, GP6, VWF, FGB, and IL6. Increased plasma VWF:Ag levels were associated with VWF haplotype 1 (−1793G) (p=0.04), although the impact of this VWF haplotype 1 association was not of itself sufficiently strong to directly influence bleeding severity score. These results establish that genetic differences in the expression or activity of integrin receptor subunits alpha-2 and alpha-IIb can influence the bleeding phenotype of VWD type 2. Together with the results of our previous studies, these findings demonstrate that platelet receptor gene haplotypes can influence bleeding tendency not only in VWD type 1 but also in VWD type 2. Interestingly, the influence of GP6 haplotype b (Pro219) on bleeding seen in VWD type 1 was not evident in this study of VWD type 2 pedigrees. Further studies on additional VWD type 1 and type 2 pedigrees, particularly pedigrees with diverse racial and/or ethnic backgrounds, will enable us to distinguish the variety and extent of genetic differences that can influence bleeding tendency in this disease.

Description: Abstract 3498 Poster Board III-435 Introduction Type 3 von Willebrand disease (VWD3) is a severe autosomal recessive inherited bleeding disorder caused by a virtually complete absence of von Willebrand Factor (VWF). Classically, patients are homozygous or compound heterozygous for null alleles due to nonsense mutations, small insertions/deletions, splice site defects or, more rarely, large gene deletions spread throughout the VWF gene. Nevertheless, several missense mutations have also been reported. Aims of the study, patients and methods The aim of this study was to investigate the molecular basis of VWD3 in 10 Italian patients using DNA direct sequencing, High Resolution Melting (HRM) analysis and duplex PCR. HRM is a simple, low-cost, and rapid PCR-based method for detecting sequence variation by measuring changes in the melting temperature of double stranded DNA. Duplex PCR was used to screen for the presence of some known large deletions causing VWD3: the 61-kb deletion encompassing exons 6-16 (Xie et al. Blood Cells Mol Dis. 2006; 36: 385), the 253-kb deletion involving the whole VWF gene (Schneppenheim et al. J Thromb Haemost. 2007; 5: 722), the exons 1-3 deletion (Mohl et al. J Thromb Haemost. 2008; 6: 1729), and the exons 4-5 deletion (Sutherland et al. Blood. 2009; 114: 1091). Results and discussion Twenty-four exons were analyzed by direct sequencing, 21 exons by HRM and, so far, 6 exons using both methods. The following mutations were identified in 8 of the 10 patients investigated: 2157delA/7729+7C〉T; C2184S*/undetermined; Q1526X*/C2325S*; del ex1-3/3940delG*; 8155+1G〉T*/8155+1G〉T*; E1549X*/undetermined; 658-2A〉G*/658-2A〉G*; del ex 1-3/undetermined. Direct sequencing revealed 7 mutations, HRM analysis could detect 2 defects (2157delA, C2325S) and duplex PCR identified one large deletion. Seven of these 11 mutations were novel (indicated with *). Two patients were found to carry mutations in the homozygous state. To confirm these findings, their parents will have to be investigated in order to exclude the presence of a large gene deletion in one of the alleles. Interestingly, the large deletion involving exons 1-3, which was previously reported in the Hungarian population, was also found in 2 unrelated patients. Two missense mutations were identified, both involving a cysteine residue, further suggesting the importance of these residues in the correct folding/processing/secretion of the neo-synthesized VWF. In those patients who still remain uncharacterized further analysis should be performed to search for intronic mutations or heterozygous large deletions responsible for aberrant splicing/post-transcriptional events. Conclusion Based on these preliminary data, HRM analysis, to our knowledge used for the first time in the molecular diagnosis of VWD3, in our hands seems to be an accurate and rapid method for mutational screening of VWF gene. However, so far, the presence of many polymorphic sites in the VWF coding region has strongly limited the use of this technique to 21 exons of the gene. Disclosures: Baronciani: Bayer Awards: Research Funding.

Description: Physiological concentrations of NaCl inhibit the hydrolysis of von Willebrand Factor (VWF) by ADAMTS-13. This effect is linked to the specific binding of chloride ions to VWF. Urea-induced unfolding was measured in the presence of NaCl, CH3COONa and NaClO4 at pH 8.0, 25°C, for multimeric VWF, the recombinant A1-A2-A3 VWF domains and the A1 domain. Chloride stabilizes the folded conformation of the A1-A2-A3 and A1 domains more efficiently than acetate but less strongly than perchlorate. Spectrofluorometric studies showed that chloride binds to both the A1 and A1-A2 domain, but not to the isolated A2 domain. Binding of Cl− to both wild type (WT) and the natural mutant (2B VWD) p.R1306W A1-A2-A3 domains of VWF has a large heat capacity change equal to −1.0 and −0.4 Kcal mol-1 K-1 for WT, and p.R1306W A1-A2-A3 domains, respectively. This result implies that a burial of a vast apolar surface area is caused by conformational transitions linked to chloride binding. At any temperature, chloride affinity was higher for WT than for the mutant p.R1306W form. Chloride ions inhibit hydrolysis by ADAMTS-13 of the A1-A2-A3 and A1-A2 domains in the presence of either urea or high shear stress (40 dyn/cm2), while this effect was either absent or negligible in experiments using A2 and A2-A3 domains. Steady-state kinetic experiments showed that chloride ions inhibit allosterically the cleavage by ADAMTS-13 of the WT A1-A2-A3, whereas this effect was significantly reduced in p.R1306W form, as a consequence of the reduced chloride affinity (Figure 1). On the whole, these findings showed the existence of a conformational linkage between the A1 and A2 domains in the VWF molecule. The p.R1306W natural mutant, that has an higher affinity for GpIb, bears a mutation which stabilizes a conformation of the A1 domain in a GpIb-bound like state and reduces at the same time the A1 domain affinity for chloride. These findings suggest that in some type 2B VWD forms chloride ions bind with lower affinity and the rate of hydrolysis by ADAMTS-13 increases. This can contribute, along with the enhanced binding of high molecular weight VWF multimers to platelet GpIb molecules, to the depletion of these VWF forms and to the hemorrhagic diathesis usually observed in these patients. Figure 1 Linkage graph showing the kcat/Km values of hydrolysis of WT A1-A2-A3 (•), p. R1306 W(○) WT A1-A2 (□) isolated A2 domain (•), and A2-A3 domains (▿). The continuous lines were drawn to eq 4 with the best - fit parameter values k+ = 7.26 ± 0.2 × 104 M−1 sec−1 kCI = 1.23 ± 0.3 × 104M−1 sec −1, Kd = 35 ± 5 mM (WT A1-A2-A3); k+ = 1.36 ± 0.1 × 105 M−1 sec −1 kCI = 3 ± 0.5 × 104 M−1 sec−1 , kd = 158 ± 20 mM (p R1306W), k+ 7.47 ± 0.3 × 104 M−1 sec−1 kCI = 1.38 ± 0.3 × 104 M−1 sec−1, Kd = 36.2 ± 8 mM(A1-A2). No significative NaCl effect was observed for both isolated A2 and A2 -A3 domains Figure 1. Linkage graph showing the kcat/Km values of hydrolysis of WT A1-A2-A3 (•), p. R1306 W(○) WT A1-A2 (□) isolated A2 domain (•), and A2-A3 domains (▿). The continuous lines were drawn to eq 4 with the best - fit parameter values k+ = 7.26 ± 0.2 × 104 M−1 sec−1 kCI = 1.23 ± 0.3 × 104M−1 sec −1, Kd = 35 ± 5 mM (WT A1-A2-A3); k+ = 1.36 ± 0.1 × 105 M−1 sec −1 kCI = 3 ± 0.5 × 104 M−1 sec−1 , kd = 158 ± 20 mM (p R1306W), k+ 7.47 ± 0.3 × 104 M−1 sec−1 kCI = 1.38 ± 0.3 × 104 M−1 sec−1, Kd = 36.2 ± 8 mM(A1-A2). No significative NaCl effect was observed for both isolated A2 and A2 -A3 domains