ALBERT

Description: A patient with chronic myelogenous leukemia developed anemia with evidence of increased red cell destruction. After anemia had persisted for eleven months in spite of 49 blood transfusions, splenectomy was performed. The hemolysis subsided and her health remained good during the following thirty months.

Description: Von Willebrand Factor (VWF) is a large multimeric glycoprotein that mediates platelet adhesion to the damaged blood vessel wall and subsequent platelet aggregation at the site of vascular injury. The size of VWF multimers is regulated by the metalloprotease, ADAMTS13. Alterations in VWF sequence can lead to an increase or decrease in ADAMTS13-mediated cleavage, resulting in either a loss or increase in high molecular weight VWF multimers, respectively. With the availability of a VWF knockout mouse, variant forms of VWF can be evaluated in vivo in terms of their contribution to hemostasis and thrombosis. In these studies, we have taken into account the significant differences in VWF-GPIb binding and ADAMTS13 cleavage efficiency seen between mice and humans and have also assumed that functionally important residues are likely to be conserved between species. With these considerations in mind, the protocols described in this report utilize mouse-exclusive reagents. Previous reports of correction of the VWF KO phenotype using hydrodynamic gene delivery have shown contradictory results for correction of the bleeding time and blood volume loss as well as a grossly abnormal multimer structure of the rescued VWF protein, due initially to an inadvertent C799R mutation and latterly to factors possibly related to the site of VWF synthesis. In this study, we compare wild type VWF clearance to a cleavage site knockout, Y1605A/M1606A, a type 2A Von Willebrand Disease (VWD) mutation, R1597W, and the common type 1 VWD mutation, Y1584C. The murine R1597W variant exhibited increased ADAMTS13-mediated cleavage (0.36- fold ADAMTS13 concentration), and the Y1605A/M1606A variant greatly decreased cleavage in vitro (〉100-fold ADAMTS13 concentration). VWF KO mice, 7-10 weeks old, were injected with recombinant murine VWF (200U/kg) produced in HEK293 cells. VWF antigen levels (VWF:Ag), multimers, and complete blood counts (CBCs) were performed. Compared to the wild type infused protein (T1/2=33.2 minutes), the Y1605A/M1606A and R1597W mutant proteins show faster clearance (T1/2=17.7 minutes, p

Description: Abstract 22 Introduction: Type 1 von Willebrand disease (VWD) is caused by mutations that result in moderate decreases in VWF antigen (VWF:Ag is 5–50% of normal levels) and a mild bleeding phenotype. The common recurrent VWF missense mutation Y1584C is associated with mildly decreased VWF:Ag levels, increased ADAMTS13 cleavage, as well as a possible increase in clearance. The Vicenza mutation, R1205H, exhibits a more severe phenotype (VWF:Ag ∼10%) and accelerated clearance. Although well described in patients and through in vitro studies, extensive controlled in vivo investigation of these mutations has yet to be performed. In this study, we compared both Y1584C and R1205H to wild type VWF using hydrodynamic gene delivery of mouse VWF and ADAMTS13 transgenes in the VWF knockout mouse to determine the pathological mechanisms associated with these variants. Methods: Hydrodynamic injections were performed using 100 μ g wild type (WT) or mutant mouse Vwf cDNA in Ringer's solution in 7–9 week old C57Bl6 VWF knockout mice, replacing plasma VWF. Co-injections with mouse Adamts13 cDNA were also performed. Mice were sampled at days 2, 5, 8, and then weekly. Mouse plasma was analyzed for complete blood counts, VWF:Ag, VWF propeptide, and VWF multimer structure. Thrombotic injury was induced using ferric chloride injury to the arterioles of the cremaster in VWF knockout mice expressing VWF:Ag levels from 0.5–2 U/ml. Platelets were labeled with Rhodamine-6G to evaluate platelet accumulation. Time to stable vessel occlusion and platelet accumulation by relative fluorescence intensity were compared. Results: Hydrodynamic injection caused no adverse events in any animals. Complete blood count values were unaffected for both variants compared to WT. Initial high VWF:Ag values at day 2 were similar for WT VWF (25.4 ± 2.5 U/ml, n= 12, mean U/ml±SEM, n) and Y1584C (26.8 ± 5.5, n= 10), but R1205H levels were 36% lower (16.3 ± 2.1, n= 10). Lower VWF:Ag levels were demonstrated in both “homozygous” and “heterozygous” forms for both type 1 mutations from days 14–42, when VWF expression plateaus. R1205H VWF:Ag was 34.3 ± 5.9% of WT (P 〈 0.001) and “heterozygous” 1:1 ratio R1205H/WT co-delivery was 27.5 ± 4.7% (p 〈 0.001). Y1584C was 29.4 ± 7.5% of WT (P 〈 0.001), and Y1584C/WT was 51.1 ± 4.6% (p 〈 0.001). VWF propeptide to VWF:Ag ratios (days 2–42) demonstrate that R1205H mouse VWF had an increased clearance rate (165.4 ± 13.5%, p 〈 0.001), while Y1584C was normal (97.1 ± 6.8 %, P 〉 0.05) compared to WT (100.0 ± 10.0%). The R1205H mutation showed no significant difference in multimer structure by mean multimer band numbers (days 2 to 42, 93 ± 16%, n = 4, P 〉 0.05) to wild type VWF (100 ± 12%, n = 4). In contrast, Y1584C had a significant decrease (66 ± 18%, n = 4, P 〈 0.001). This effect was exaggerated by co-delivery of mouse ADAMTS13 for Y1584C, but not R1205H. Y1584C showed reduced thrombus formation in a ferric chloride injury model while R1205H demonstrated similar thrombogenic activity to wild type VWF. Mean occlusion times were WT = 29.9 ± 2.1 minutes, n = 8, R1205H = 29.1 ± 4.0, n = 8 (p 〉 0.05), and Y1584C = 38.7 ± 1.1, n = 9 (p = 0.001). Total platelet accumulation was decreased for Y1584C (83.6 ± 6.3%, p = 0.043), but was similar for R1205H (103 ± 6.3, P = 0.72) and WT (100 ± 5.3%). Conclusions: This study demonstrates that these two type 1 VWD mutations have a strong observable effect in the VWF knockout mouse model. R1205H exhibits a large decrease in VWF:Ag levels and evidence of accelerated clearance with R1205H. However, there is no alteration in multimer structure and apparently normal participation in a thrombosis model. Y1584C, in contrast, shows a loss of high molecular weight multimers that is exacerbated by the additional expression of ADAMTS13, indicating that ADAMTS13 cleavage is increased. Y1584C also has an initially high VWF:Ag level that was less than WT levels from day 14 onward, but shows no alteration in clearance, suggesting that there is a biosynthetic defect. Y1584C shows a significant defect in the arteriolar thrombosis model, presenting a Type 2A VWD-like phenotype in the mouse model, which is more severe than the human phenotype. This study has elucidated several novel mechanistic details for these two mutations and highlights that the pathogenic aspects of type 1 VWD can be recapitulated in the VWF knockout hydrodynamic injection model. Disclosures: No relevant conflicts of interest to declare.

Description: Type 1 VWD is the mild to moderate reduction of VWF levels. This study examined the mechanisms underlying 2 common type 1 VWD mutations, the severe R1205H and more moderate Y1584C. In vitro biosynthesis was reduced for both mutations in human and mouse VWF, with the effect being more severe in R1205H. VWF knockout mice received hydrodynamic injections of mouse Vwf cDNA. Lower VWF antigen levels were demonstrated in both homozygous and heterozygous forms for both type 1 mutations from days 14-42. Recombinant protein infusions and hydrodynamic-expressed VWF propeptide to antigen ratios demonstrate that R1205H mouse VWF has an increased clearance rate, while Y1584C is normal. Recombinant ADAMTS13 digestions of Y1584C demonstrated enhanced cleavage of both human and mouse VWF115 substrates. Hydrodynamic-expressed VWF shows a loss of high molecular weight multimers for Y1584C compared with wild-type and R1205H. At normal physiologic levels of VWF, Y1584C showed reduced thrombus formation in a ferric chloride injury model while R1205H demonstrated similar thrombogenic activity to wild-type VWF. This study has elucidated several novel mechanisms for these mutations and highlights that the type 1 VWD phenotype can be recapitulated in the VWF knockout hydrodynamic injection model.

Description: Key PointsThe robustness of the VWF:collagen-binding assay is confirmed in a comprehensive evaluation of VWD collagen-binding defects. Collagen binding by VWF, GPVI, and α2β1 have major albeit overlapping functions in primary hemostasis.

Description: Abstract 23 Introduction: Type 2B von Willebrand disease (2B VWD), a qualitative variant of VWD, is characterized by thrombocytopenia and loss of high molecular weight multimers. 2B VWF has both an increased affinity for platelet glycoprotein Ibα (GPIbα) and increased susceptibility to ADAMTS13-mediated cleavage. In order to determine whether the loss of high molecular weight (HMW) multimers associated with 2B VWD occurs due to the increased ADAMTS13 susceptibility or due to the increased binding to platelet GPIbα, we created a modified type 2B mouse model, in which the mice expressed ADAMTS13-insensitive 2B VWF. Methods: Three common type 2B VWD mutations (R1306W, V1316M, and R1341Q) were independently introduced into the mouse Vwf (mVWF) cDNA sequence containing the ADAMTS13 cleavage site knockout, Y1605A/M1606A (CSKO). Recombinant mutant mVWF was produced using transient transfection of HEK293T cells. ADAMTS13 digestion of the recombinant mVWF was performed using a Tris-Urea system. Platelet binding affinity was assessed using a botrocetin-induced platelet-binding ELISA assay. 8–9 week old C57Bl6 VWF KO mice were hydrodynamically injected with 100 μ g of naked plasmid DNA containing the liver specific ET promoter and wildtype (WT) or mutant mouse Vwf DNA. The mice were sampled weekly; complete blood counts, VWF:Ag levels, VWFpp levels, and VWF multimer structure were examined. Results: Following transient transfections, mutant and wildtype mVWF were secreted at similar levels, with a full range of VWF multimer sizes. Recombinant 2B/CSKO mVWF is markedly ADAMTS13 cleavage insensitive, with 〉80 U mADAMTS13 required for 50% cleavage (WT mVWF requires 1 U mADAMTS13). Similar to 2B VWF, 2B/CSKO mVWF showed enhanced platelet binding affinity in the presence of botrocetin. Hydrodynamic injections of the naked expression plasmids did not result in adverse events in the mice. Mean 2B/CSKO platelet counts at day 7 were significantly reduced compared to WT platelet counts (149 and 370, respectively). At day 14, only the mice expressing V1316M/CSKO remained thrombocytopenic, with platelet counts similar to those seen with the V1316M mutation alone (170 and 171, respectively). By day 14, the mice expressing 2B/CSKO VWF had significantly lower mean VWF:Ag levels than WT mice or mice expressing the CSKO alone (see table below). The mean VWFpp/VWF:Ag ratios were significantly increased in the 2B/CSKO mice when compared to the WT mice (1.53 and 1.00, respectively). There was no loss of HMW multimers in the mice expressing R1306W/CSKO and R1341Q/CSKO and, indeed, these mice showed significantly more multimer bands than WT mice (days 7 to 21; 14.5±0.6 and 13.9±1.2 multimer bands versus 10.2±0.5 bands for WT). In marked contrast, the mice expressing V1316M/CSKO VWF showed complete loss of high molecular weight multimers, similar to mice expressing the same 2B VWF mutation alone (8.3±1.8 and 7.7±0.9 multimer bands, respectively). Conclusions: Mice expressing R1306W/CSKO and R1341Q/CSKO VWF have an absence of thrombocytopenia and show the presence of supranormal high molecular weight VWF multimer bands. These results suggest that enhanced ADAMTS13-mediated cleavage plays an important role in the type 2B phenotype associated with these mutations. In contrast, low V1316M/CSKO VWF:Ag was associated with both thrombocytopenia and loss of HMW multimers, demonstrating that enhanced GPIbα binding affinity is the predominant mechanism associated with this mutation. Thus, the importance of ADAMTS13 susceptibility in 2B VWD is mutation-dependent, and ADAMTS13-mediated cleavage plays an important role in enhancing the severity of the type 2B VWD phenotype associated with the R1306W and R1341Q mutations. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 26 Introduction: Type 1 von Willebrand disease (VWD) is caused by mutations that result in moderate decreases in VWF (5-50% of normal levels) and a mild bleeding phenotype. The VWF missense mutation Y1584C is associated with mildly decreased VWF:Ag levels, increased ADAMTS13 cleavage, as well as a possible increase in clearance. The Vicenza mutation, R1205H, exhibits a more severe phenotype (VWF:Ag ∼10%), as well as accelerated clearance. However, extensive controlled in vitro and in vivo investigation of these mutations has yet to be described. In this study, we examine both Y1584C and R1205H in comparison to wild type VWF using in vitro and in vivo strategies employing both human and mouse VWF. Methods: Recombinant murine and human VWF and ADAMTS13 were produced via transient transfection in HEK293T cells in serum free OPTIMEM for 72 hours. Full length ADAMTS13 digests were performed in a Tris-Urea system and analyzed via multimer pattern. VWF115 digests were performed in an ELISA based assay. Hydrodynamic injections were performed using 100 μg wild type (WT) or mutant ET-mVWF plasmid DNA in Ringer's solution in 7-9 week old C57Bl6 VWF knockout mice. Mice were sampled at days 2, 5, 8, and then weekly. Mouse plasma was analyzed for CBCs, VWF:Ag, and VWF multimer structure. Results: In the HEK293T transient transfection system, secreted mutant protein was similar to that of wild type recombinant protein, with high molecular weight material present. ADAMTS13 digestion of full length recombinant Y1584C versus wild type showed no statistical difference: 50% cleavage for hVWF WT 1.78U hADAMTS13, hVWF Y1584C 1.67 U, P=0.58; mVWF WT 0.32 U mADAMTS13; Y1584C 0.42 U, P=0.11. In contrast, the Y1584C substitution in the hVWF115 construct required 40% less hADAMTS13 to effect equivalent cleavage (WT 0.087±0.014, 4; Y1584C 0.052±0.005, 8, mean U/ml ADAMTS13±SEM, N, P=0.013). Mouse ADAMTS13 cleavage of mVWF115 was also increased 20% for Y1584C (WT: 0.20±0.06, 4; Y1584C: 0.16±0.01, 4, P=0.014). Hydrodynamic injection caused no adverse events in any animals. CBC values were not statistically significantly different between wild type and mutants. Initial high VWF:Ag values were similar for wild type VWF (25.6±2.9, 15, mean U/ml±SEM, n) and Y1584C (27.2±5.0, 11), but R1205H levels were 36% lower (16.3±2.1, 10). At 14 days, WT VWF:Ag was 5.33±1.13, 15, with R1205H (1.73±0.44, 12) and Y1584C (1.84±0.59, 11) VWF:Ag levels being 68% and 65% lower, respectively. R1205H continued to remain approximately 40% of WT values for the next three weeks, while Y1584C continued to decrease, dropping to 15% on day 21 and 8% on day 28, compared to the wild type values at these time points. The R1205H mutation showed no significant difference in multimer structure defined by observed number of bands (days 2 to 42, mean difference -0.49, P〉0.05) to wild type VWF. In contrast, Y1584C had a significant decrease in band number (3.38, P

Description: Von Willebrand Factor (VWF) is a large multimeric glycoprotein that mediates platelet adhesion to the damaged blood vessel wall and subsequent platelet aggregation at the site of vascular injury. The size of VWF multimers in plasma is regulated by the specific VWF cleaving protease, ADAMTS13, that cleaves VWF at the Y1605-M1606 bond in the VWF A2 domain. The adhesive properties of VWF is directly related to the multimer size, with loss of high molecular weight VWF leading to the bleeding phenotype in Type 2A von Willebrand disease (VWD) and ultra high molecular weight VWF multimers observed in the prothrombotic state of thrombotic thrombocytopenic purpura (TTP). VWF mutations leading to multimer changes have not been examined in an animal model. Although the human and mouse VWF genes are highly conserved, mutations in the two genes have not been compared experimentally. To that end, we have made mutations in the mouse VWF (mVWF) cDNA to compare the ADAMTS13 cleavage patterns seen with human VWF mutations in two in vitro assays. Recombinant multimerized full-length mVWF was digested with different concentrations of recombinant mouse ADAMTS13 (mADAMTS13) and 1.5M urea, and analyzed via multimer migration distance. The GST and histidine-tagged G1554-T1668 A2 domain region of mVWF (mVWF115) was assayed via ELISA as follows: the mVWF115 was bound to anti-GST coated plates, digested with mADAMTS13, and intact mVWF115 detected via HRP-labeled anti-histidine tag antibody. We examined R1597W (VWD Type 2A), R1306W, (VWD Type 2B), Y1584C (VWD Type 1), as well as two previously described changes that drastically lower VWF cleavage, Y1605A/M1606A, and D1614A/E1615A/K1617A. Y1584C showed a statistically insignificant 41% decrease in cleavage in the full-length assay, but a 20% increase in the mVWF115 assay. Conversely, R1597W had a 64% decrease in the full-length assay, but a 28% increase in the mVWF115 assay. Y1605A/M1606A showed a dramatic loss of cleavage, with no observable loss of high molecular weight multimers at 32 units/ml of mADAMTS13, and a 126-fold higher mADAMTS13 concentration than wild type in the mVWF115 assay. This contrasted with the 8-fold increase observed with D1614A/E1615A/K1617A. The R1306W A1 domain mutation showed a significant increase in cleavage, with 63% less ADAMTS13 necessary in the full-length assay. The triple mutant R1306W/Y1605A/M1606A did not cleave at the highest concentration, 32 U/ml, similar to that of Y1605A/M1606A, showing that the R1306W mutation was not able to increase cleavage of the Y1605A/M1606A change. These results show a strong correlation between the mouse mutations described here and previously reported human VWF mutations in their susceptibility to ADAMTS13 cleavage. These results provide a rationale for the development of mouse models of type 1, 2A, and 2B VWD, as well as mVWF mutations that might lead to a prothrombotic state similar to TTP in humans. mADAMTS13 Concentration for 50% mVWF Cleavage ADAMTS13 Concentration (Units/ml) Relative mADAMTS13 Concentration (to Wild Type) mVWF Multimer (n=1) mVWF115 (n=4) mVWF Multimer (n=1) mVWF115 (n=4) *: P value 32** 24.8** 〉100** 126** D1614A/E1615A/K1617A (−) 1.58** (−) 8.0** R1306W 0.112** (−) 0.37** (−) R1306W/Y1605A/M1606A 〉32** (−) 〉100** (−)

Description: Autoimmune phenomena may precede or accompany lymphoid malignancies, especially B-chronic lymphocytic leukemia (B-CLL). We report a patient with a 7-year history of primary (idiopathic) cold agglutinin (CA) disease in whom B-CLL subsequently developed. Immunophenotyping and single-cell reverse transcription–polymerase chain reaction (RT-PCR) were applied to investigate the origin and diversification of leukemic B cells. The obtained data indicate a memory cell-type origin of the B-CLL cells. Remarkably, theIgVκ genes of the B-CLL cells showed intraclonal diversity, whereas the mutational pattern of their paired IgVH genes were invariant. Thus, the light-chain–restricted intraclonal diversity in individual leukemic B cells in this patient strongly indicates a differential regulation or selection of the ongoing mutational process. Of note, our findings suggest that this B-CLL had developed from the patient's CA-producing B-cell population.