Clinical and laboratory phenotype variability in type 2M von Willebrand disease.

Essentials The pathophysiology of type 2M von Willebrand disease (VWD) is poorly understood. Sequence variations in type 2M VWD subjects were characterized. A high degree of clinical and laboratory variability exists within type 2M VWD variants. Some type 2M variants may share features of type 2A VWD. SUMMARY: Background von Willebrand factor (VWF) is a multimeric coagulation factor that tethers platelets to injured subendothelium. Type 2M von Willebrand disease (VWD) is characterized by a qualitative defect in VWF with preserved multimer distribution. Objectives Through the Zimmerman Program for the Molecular and Clinical Biology for VWD, five VWF sequence variations were studied in subjects diagnosed with type 2M VWD. Methods Bleeding phenotype was assessed using the ISTH bleeding assessment tool. Full-length VWF gene sequencing was performed for each subject. Each variant was placed into a recombinant VWF vector using site-directed mutagenesis and expressed in HEK293T cells as homozygous or heterozygous VWF. Variant expression, collagen binding and platelet GPIbα binding were studied through ELISA assays. Multimer analysis was performed by gel electrophoresis. Results Bleeding scores were elevated for all subjects except for the p.P1162L and p.R1374C variants. Although all had reduced VWF ristocetin cofactor activity/VWF antigen ratios on plasma testing, recombinant VWF did not show a classic type 2M phenotype for any of the five variants. Homozygous expression of variants p.D1283Y, p.R1349C, p.R1374C and p.I1453N was consistent with type 2A VWD, although all had normal expression as heterozygous recombinant VWF. Variant p.P1162L had normal VWF expression and function, consistent with the lack of bleeding symptoms. Conclusions Although originally classified as type 2M VWD, these homozygous recombinant VWF variants do not fulfill complete 2M VWD diagnostic criteria. A better classification schema and improved testing for putative type 2M variants is needed in order to effectively diagnose and treat affected patients.

Normal range of bleeding scores for the ISTH-BAT: adult and pediatric data from the merging project.

Bleeding Assessment Tools (BATs) have been developed to aid in the standardized evaluation of bleeding symptoms. The Vicenza Bleeding Questionnaire (BQ), published in 2005, established a common framework and scoring key that has undergone subsequent modification over the years, culminating in the publication of the ISTH-BAT in 2010. Understanding the normal range of bleeding scores is critical when assessing the utility of a BAT. Within the context of The Merging Project, a bioinformatics system was created to facilitate the merging of legacy data derived from four different (but all Vicenza-based) BATs; the MCMDM1-VWD BQ, the Condensed MCMDM-1VWD BQ, the Pediatric Bleeding Questionnaire and the ISTH-BAT. Data from 1040 normal adults and 328 children were included in the final analysis, which showed that the normal range is 0-3 for adult males, 0-5 for adult females and 0-2 in children for both males and females. Therefore, the cut-off for a positive or abnormal BS is ≥4 in adult males, ≥6 in adult females and ≥3 in children. This information can now be used to objectively assess bleeding symptoms as normal or abnormal in future studies.

Critical von Willebrand factor A1 domain residues influence type VI collagen binding.

BACKGROUND: von Willebrand factor (VWF) binds to subendothelial collagen at sites of vascular injury. Laboratory testing for von Willebrand disease (VWD), however, does not always include collagen binding assays (VWF:CB) and standard VWF:CB assays use type I and/or type III collagen rather than type VI collagen. OBJECTIVES: We report here on several mutations that exclusively alter binding to type VI collagen. PATIENTS/METHODS: Healthy controls and index cases from the Zimmerman Program for the Molecular and Clinical Biology of VWD were analyzed for VWF antigen (VWF:Ag), VWF ristocetin cofactor activity and VWF:CB with types I, III and VI collagen. VWF gene sequencing was performed for all subjects. RESULTS: Two healthy controls and one type 1 VWD subject were heterozygous for an A1 domain sequence variation, R1399H, and displayed a selective decreased binding to type VI collagen but not types I and III. Expression of recombinant 1399H VWF resulted in absent binding to type VI collagen. Two other VWF A1 domain mutations, S1387I and Q1402P, displayed diminished binding to type VI collagen. An 11 amino acid deletion in the A1 domain also abrogated binding to type VI collagen. CONCLUSIONS: VWF:CB may be useful in diagnosis of VWD, as a decreased VWF:CB/VWF:Ag ratio may reflect specific loss of collagen binding ability. Mutations that exclusively affect type VI collagen binding may be associated with bleeding, yet missed by current VWF testing.

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

BACKGROUND: 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). OBJECTIVES: This study presents data on collagen binding for healthy controls and VWD subjects to compare three different collagens. PATIENTS/METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Calcium hydroxyapatite promotes mitogenesis and matrix metalloproteinase expression in human breast cancer cell lines.

Radiographic mammary microcalcifications are one of the most pertinent diagnostic markers of breast cancer. Breast tissue calcification in the form of calcium hydroxyapatite (HA) is strongly associated with malignant disease. We tested the hypothesis that calcium HA may exert biological effects on surrounding cells, thereby facilitating breast cancer progression. Our findings showed that HA crystals enhanced mitogenesis in breast cancer cell lines MCF-7 and Hs578T and also in normal human mammary epithelial cells. HA crystals were also found to upregulate the production of a variety of matrix metalloproteinases (MMPs), including MMP-2, -9, and -13 in MCF-7 and MMP-9 in human mammary epithelial cell lines. HA crystals were found to greatly augment prostaglandin E(2) levels in Hs578T cells, and treatment with a cyclooxygenase inhibitor, aspirin, abrogated the HA-induced mitogenesis. These results suggest that calcium HA crystals may play an active role in amplifying the pathological process involved in breast cancer.

Basic calcium phosphate crystals activate human osteoarthritic synovial fibroblasts and induce matrix metalloproteinase-13 (collagenase-3) in adult porcine articular chondrocytes.

OBJECTIVE: To determine the ability of basic calcium phosphate (BCP) crystals to induce (a) mitogenesis, matrix metalloproteinase (MMP)-1, and MMP-13 in human osteoarthritic synovial fibroblasts (HOAS) and (b) MMP-13 in cultured porcine articular chondrocytes. METHODS: Mitogenesis of HOAS was measured by [3H]thymidine incorporation assay and counts of cells in monolayer culture. MMP messenger RNA (mRNA) accumulation was determined either by northern blot analysis or reverse transcriptase-polymerase chain reaction (RT-PCR) of RNA from chondrocytes or HOAS treated with BCP crystals. MMP-13 secretion was identified by immunoprecipitation and MMP-1 secretion by western blot of conditioned media. RESULTS: BCP crystals caused a 4.5-fold increase in [3H]thymidine incorporation by HOAS within 20 hours compared with untreated control cultures (p< or =0.05). BCP crystals induced MMP-13 mRNA accumulation and MMP-13 protein secretion by articular chondrocytes. In contrast, in HOAS, MMP-13 mRNA induced by BCP crystals was detectable only by RT-PCR, and MMP-13 protein was undetectable. BCP crystals induced MMP-1 mRNA accumulation and MMP-1 protein secretion by HOAS. MMP-1 expression was further augmented when HOAS were co-incubated with either BCP and tumour necrosis factor alpha (TNFalpha; threefold) or BCP and interleukin 1alpha (IL1alpha; twofold). CONCLUSION: These data confirm the ability of BCP crystals to activate HOAS, leading to the induction of mitogenesis and MMP-1 production. MMP-13 production in response to BCP crystals is substantially more detectable in porcine articular chondrocytes than in HOAS. These data support the active role of BCP crystals in osteoarthritis and suggest that BCP crystals act synergistically with IL1alpha and TNFalpha to promote MMP production and subsequent joint degeneration.

Molecular cloning, expression, and characterization of CYP2D17 from cynomolgus monkey liver.

The cynomolgus monkey is a species used in drug-safety evaluation and biotransformation studies by the pharmaceutical industry. Relatively little is known, however, about the catalytic activities and specificities of cytochromes P450 (CYP) in this species. As a first step in characterizing monkey CYPs, a cDNA was cloned by reverse-transcriptase PCR from cynomolgus monkey liver mRNA using oligonucleotide primers based on the human CYP2D6 sequence. The full-length cDNA (called CYP2D17) encoded a 497-amino-acid protein that is 93% identical to human CYP2D6 and 90% identical to marmoset CYP2D19. The CYP2D17 cDNA was cloned into a baculovirus expression vector, and microsomes prepared from CYP2D17-infected insect cells were used to determine the catalytic properties of the recombinant enzyme. The recombinant CYP2D17 results were compared to data generated with monkey liver microsomes, human liver microsomes, and recombinant CYP2D6 and demonstrated catalytic similarity using probe substrates and inhibitors. Recombinant CYP2D17 catalyzed the oxidation of bufuralol to 1'-hydroxybufuralol and dextromethorphan to dextrorphan, reactions shown to be mediated by CYP2D6 in humans; the apparent K(m) values for bufuralol and dextromethorphan were 1 and 0.8 microM, respectively. Moreover, both of these reactions were more strongly inhibited by quinidine than by quinine. A more complete understanding of the substrate specificities and activities of monkey CYPs will be advantageous in delineating species differences in metabolite profiles and metabolic activation of new chemical entities in the pharmaceutical industry.

Molecular mechanism of basic calcium phosphate crystal-induced activation of human fibroblasts. Role of nuclear factor kappab, activator protein 1, and protein kinase c.

Synovial fluid basic calcium phosphate (BCP) crystals are markers of severe joint degeneration in osteoarthritis. BCP crystals cause mitogenesis of articular cells and stimulate matrix metalloprotease production, thus promoting degradation of articular tissues. Previous work suggested that BCP crystal-induced cell activation required intracellular crystal dissolution, induction of proto-oncogene expression, and activation of signal transduction pathways involving protein kinase C and mitogen-activated protein kinases. Here we further elucidate the mechanisms of BCP crystal-induced cell activation as BCP crystals activate transcription factors nuclear factor kappaB and activator protein 1 in human fibroblasts. We confirm the role of protein kinase C in BCP crystal-induced mitogenesis in human fibroblasts. In contrast, we demonstrate that BCP crystals do not activate signal transduction pathways involving protein tyrosine kinases or phosphatidylinositol 3-kinase. These data further define the mechanism of cell activation by BCP crystals and confirm its selectivity, an observation that may have therapeutic implications.

Basic calcium phosphate crystals induce synthesis and secretion of 92 kDa gelatinase (gelatinase B/matrix metalloprotease 9) in human fibroblasts.

OBJECTIVE: Synovial fluid basic calcium phosphate (BCP) crystals are associated with severe joint degeneration accompanied by synovial hypertrophy. The metalloprotease 92 kDa gelatinase (MMP-9) has been implicated in the degradation of extracellular matrix in osteoarthritis, but the ability of BCP crystals to induce gelatinase in human fibroblasts or in adult porcine chondrocytes has not previously been studied. The hypothesis that the mitogenic response to BCP crystals is accompanied by induction and secretion of MMP-9 was studied. METHODS: MMP-9 messenger RNA (mRNA) was detected by northern blot and reverse transcription-polymerase chain reaction (RT-PCR). Gelatinase secretion was identified by western blot and zymography of conditioned media. RESULTS: BCP crystals caused a concentration dependent induction of MMP-9 mRNA accumulation and protein secretion in human fibroblasts but not in adult porcine chondrocytes. CONCLUSION: BCP crystals induce MMP-9 production by HF but not adult porcine chondrocytes. Fibroblast MMP-9 may be an important mediator of the joint destruction associated with synovial fluid BCP crystals.

Type 2M von Willebrand disease: F606I and I662F mutations in the glycoprotein Ib binding domain selectively impair ristocetin- but not botrocetin-mediated binding of von Willebrand factor to platelets.

von Willebrand disease (vWD) is a common, autosomally inherited, bleeding disorder caused by quantitative and/or qualitative deficiency of von Willebrand factor (vWF). We describe two families with a variant form of vWD where affected members of both families have borderline or low vWF antigen levels, normal vWF multimer patterns, disproportionately low ristocetin cofactor activity, and significant bleeding symptoms. Whereas ristocetin-induced binding of plasma vWF from affected members of both families to fixed platelets was reduced, botrocetin-induced platelet binding was normal. The sequencing of genomic DNA identified unique missense mutations in each family in the vWF exon 28. In Family A, a missense mutation at nucleotide 4105T --> A resulted in a Phe606Ile amino acid substitution (F606I) and in Family B, a missense mutation at nucleotide 4273A --> T resulted in an Ile662Phe amino acid substitution (I662F). Both mutations are within the large disulfide loop between Cys509 and Cys695 in the A1 domain that mediates vWF interaction with platelet glycoprotein Ib. Expression of recombinant vWF containing either F606I or I662F mutations resulted in mutant recombinant vWF with decreased ristocetin-induced platelet binding, but normal multimer structure, botrocetin-induced platelet binding, collagen binding, and binding to the conformation-sensitive monoclonal antibody, AvW-3. Both mutations are phenotypically distinct from the previously reported variant type 2MMilwaukee-1 because of the presence of normal botrocetin-induced platelet binding, collagen binding, and AvW-3 binding, as well as the greater frequency and intensity of clinical bleeding. When the reported type 2M mutations are mapped on the predicted three-dimensional structure of the A1 loop of vWF, the mutations cluster in one region that is distinct from the region in which the type 2B mutations cluster.

Type 2M:Milwaukee-1 von Willebrand disease: an in-frame deletion in the Cys509-Cys695 loop of the von Willebrand factor A1 domain causes deficient binding of von Willebrand factor to platelets.

This report examines the genetic basis of a variant form of moderately severe von Willebrand disease (vWD) characterized by low plasma von Willebrand factor antigen (vWF:Ag) levels and normal multimerization, typical of type 1 vWD, but disproportionately-low agonist-mediated platelet-binding activity. We identified an in-frame deletion in vWF exon 28 in three generations of affected family members, who are heterozygous for this mutation. The deletion of nucleotides 4,173-4,205 results in the loss of amino acids Arg629-Gln639 in the Cys509-Cys695 loop of the A1 domain in mature vWF. The secreted mutant vWF showed a normal multimeric profile but did not bind to platelets in the presence of optimal concentrations of either ristocetin or botrocetin. The mutant vWF also failed to interact with heparin, and with vWF monoclonal antibody AvW3, which blocks the binding of vWF to GPlb. In addition, mutant vWF showed reduced secretion from transfected cells concomitant with increased intracellular levels. These results confirm that the deletion is the genetic defect responsible for the reduced interaction of vWF with platelets. We have designated this new variant type 2M:Milwaukee-1 vWD. Our analysis suggests that the potential frequency of this phenotype in individuals diagnosed with type 1 vWD is about 0.5%.