Binding of ADAMTS13 to von Willebrand factor

ADAMTS13, a metalloprotease, cleaves von Willebrand factor (VWF) in plasma to generate smaller, less thrombogenic fragments. The interaction of von Willebrand factor with specific ADAMTS13 domains was characterized with a binding assay employing von Willebrand factor immobilized on a plastic surface. ADAMTS13 binding was saturable and reversible. Equilibrium binding occurred within 2 h and the half-time for dissociation was approximately 4 h. Binding to von Willebrand factor was similar with either recombinant ADAMTS13 or normal plasma ADAMTS13; plasma from a patient who lacked ADAMTS13 activity showed no binding. The stoichiometry of binding was one ADAMTS13 per two von Willebrand factor monomers, and the K(d) was 14 nm. The ADAMTS13 metalloprotease and disintegrin domains did not bind VWF detectably. ADAMTS13 truncated after the first thrombospondin type 1 repeat bound VWF with a K(d) of 206 nm, whereas ADAMTS13 truncated after the spacer domain had a K(d) of 23 nm, which is comparable with that of full-length ADAMTS13. Truncation after the eighth thrombospondin type 1 repeat reduced the binding affinity by approximately 3-fold and truncation after the seventh thrombospondin type 1 repeat in addition to the CUB domains increased the affinity for von Willebrand factor by approximately 2-fold. Therefore, the spacer domain is required for ADAMTS13 binding to von Willebrand factor. The first thrombospondin repeat also affects binding, and the C-terminal thrombospondin type 1 and CUB domains of ADAMTS13 may modulate this interaction.     

Cleavage of von Willebrand factor requires the spacer domain of the metalloprotease ADAMTS13

ADAMTS13 consists of a reprolysin-type metalloprotease domain followed by a disintegrin domain, a thrombospondin type 1 motif (TSP1), Cys-rich and spacer domains, seven more TSP1 motifs, and two CUB domains. ADAMTS13 limits platelet accumulation in microvascular thrombi by cleaving the Tyr1605-Met1606 bond in von Willebrand factor, and ADAMTS13 deficiency causes a lethal syndrome, thrombotic thrombocytopenic purpura. ADAMTS13 domains required for substrate recognition were localized by the characterization of recombinant deletion mutants. Constructs with C-terminal His6 and V5 epitopes were expressed by transient transfection of COS-7 cells or in a baculovirus system. No association with extracellular matrix or cell surface was detected for any ADAMTS13 variant by immunofluorescence microscopy or chemical modification. Both plasma and recombinant full-length ADAMTS13 cleaved von Willebrand factor subunits into two fragments of 176 kDa and 140 kDa. Recombinant ADAMTS13 was divalent metal ion-dependent and was inhibited by IgG from a patient with idiopathic thrombotic thrombocytopenic purpura. ADAMTS13 that was truncated after the metalloprotease domain, the disintegrin domain, the first TSP1 repeat, or the Cys-rich domain was not able to cleave von Willebrand factor, whereas addition of the spacer region restored protease activity. Therefore, the spacer region is necessary for normal ADAMTS13 activity toward von Willebrand factor, and the more C-terminal TSP1 and CUB domains are dispensable in vitro.     

Analysis on the molecular species and concentration of circulating ADAMTS13 in Blood

ADAMTS13 is the metalloprotease responsible for the proteolytic degradation of von Willebrand factor (VWF). A severe deficiency of this VWF-cleaving protease activity causes thrombotic thrombocytopenic purpura. This protease, comprising 1,427 amino acid residues, is composed of multiple domains, i.e., a preproregion, a metalloprotease domain, a disintegrin-like domain, a thrombospondin type-1 motif (Tsp1), a cysteine-rich domain, a spacer domain, seven Tsp1 repeats, and two CUB domains. We prepared one polyclonal and seven monoclonal antibodies recognizing distinct epitopes spanning the entire ADAMTS13 molecule. Of these antibodies, two of the monoclonal ones, which recognize the disintegrin-like and cysteine-rich/spacer domains, respectively, abolished the hydrolytic activity of ADAMTS13 toward both a synthetic substrate, FRETS-VWF73, and the natural substrate, VWF. In addition, these antibodies blocked the binding of ADAMTS13 to VWF. These results revealed that the region between the disintegrin-like and cysteine-rich/spacer domains interacts with VWF. Employing these established polyclonal and monoclonal antibodies, we examined the molecular species of ADAMTS13 circulating in the blood by immunoprecipitation followed by Western blot analysis, and estimated the plasma concentration of ADAMTS13 by enzyme-linked immunosorbent assay. These studies indicated that the major fraction of ADAMTS13 in blood plasma consisted of the full-length form. The concentration of ADAMTS13 in normal plasma was approximately 0.5-1 microg/ml.     

The proximal carboxyl-terminal domains of ADAMTS13 determine substrate specificity and are all required for cleavage of von Willebrand factor

ADAMTS13 limits platelet-rich thrombosis by cleaving von Willebrand factor at the Tyr(1605)-Met(1606) bond. Previous studies showed that ADAMTS13 truncated after spacer domain remains proteolytically active or hyperactive. However, the relative contribution of each domain within the proximal carboxyl terminus of ADAMTS13 in substrate recognition and specificity is not known. We showed that a metalloprotease domain alone was unable to cleave the Tyr-Met bond of glutathione S-transferase (GST)-VWF73-H substrate in 3 h, but it did cleave the substrate at a site other than the Tyr-Met bond after 16-24 h of incubation. Remarkably, the addition of even one or several proximal carboxyl-terminal domains of ADAMTS13 restored substrate specificity. Full proteolytic activity, however, was not achieved until all of the proximal carboxyl-terminal domains were added. The addition of TSP1 2-8 repeats and two CUB domains did not further increase proteolytic activity. Furthermore, ADAMTS13 truncated after the spacer domain with or without metalloprotease domain bound GST-VWF73-H with a K(d) of approximately 7.0 or 13 nm, comparable with full-length ADAMTS13 (K(d) = 4.6 nm). Metalloprotease domain did not bind GST-VWF73-H detectably, but the disintegrin domain, first TSP1 repeat, Cys-rich domain, and spacer domain bound GST-VWF73-H with K(d) values of 489, 136, 121, and 108 nm, respectively. These proximal carboxyl-terminal domains dose-dependently inhibited cleavage of fluorescent resonance energy transfer (FRETS)-VWF73 by full-length ADAMTS13 and ADAMTS13 truncated after the spacer domain. These data demonstrated that the proximal carboxyl-terminal domains of ADAMTS13 determine substrate specificity and are all required for recognition and cleavage of von Willebrand factor between amino acid residues Asp(1595) and Arg(1668).     

Structure of von Willebrand factor-cleaving protease (ADAMTS13), a metalloprotease involved in thrombotic thrombocytopenic purpura

Thrombotic thrombocytopenic purpura is associated with acquired or congenital deficiency of a plasma von Willebrand factor-cleaving protease (VWFCP). Based on partial amino acid sequence, VWFCP was identified recently as a new member of the ADAMTS family of metalloproteases and designated ADAMTS13. The 4.6-kilobase pair cDNA sequence for VWFCP has now been determined. By Northern blotting, full-length VWFCP mRNA was detected only in liver. VWFCP consists of 1427 amino acid residues and has a signal peptide, a short propeptide terminating in the sequence RQRR, a reprolysin-like metalloprotease domain, a disintegrin-like domain, a thrombospondin-1 repeat, a Cys-rich domain, an ADAMTS spacer, seven additional thrombospondin-1 repeats, and two CUB domains. VWFCP apparently is made as a zymogen that requires proteolytic activation, possibly by furin intracellularly. Sites for Zn(2+) and Ca(2+) ions are conserved in the protease domain. The Cys-rich domain contains an RGDS sequence that could mediate integrin-dependent binding to platelets or other cells. Alternative splicing gives rise to at least seven potential variants that truncate the protein at different positions after the protease domain. Alternative splicing may have functional significance, producing proteins with distinct abilities to interact with cofactors, connective tissue, platelets, and von Willebrand factor.     

Phenotypic expression of ADAMTS13 in glomerular endothelial cells

Background

ADAMTS13 is the physiological von Willebrand factor (VWF)-cleaving protease. The aim of this study was to examine ADAMTS13 expression in kidneys from ADAMTS13 wild-type (Adamts13^+/+) and deficient (Adamts13^−/−) mice and to investigate the expression pattern and bioactivity in human glomerular endothelial cells.

Methodology/Principal Findings

Immunohistochemistry was performed on kidney sections from ADAMTS13 wild-type and ADAMTS13-deficient mice. Phenotypic differences were examined by ultramorphology. ADAMTS13 expression in human glomerular endothelial cells and dermal microvascular endothelial cells was investigated by real-time PCR, flow cytometry, immunofluorescence and immunoblotting. VWF cleavage was demonstrated by multimer structure analysis and immunoblotting. ADAMTS13 was demonstrated in glomerular endothelial cells in Adamts13^+/+ mice but no staining was visible in tissue from Adamts13^−/− mice. Thickening of glomerular capillaries with platelet deposition on the vessel wall was detected in Adamts13^−/− mice. ADAMTS13 mRNA and protein were detected in both human endothelial cells and the protease was secreted. ADAMTS13 activity was demonstrated in glomerular endothelial cells as cleavage of VWF.

Conclusions/Significance

Glomerular endothelial cells express and secrete ADAMTS13. The proteolytic activity could have a protective effect preventing deposition of platelets along capillary lumina under the conditions of high shear stress present in glomerular capillaries.     

Cleavage of the ADAMTS13 propeptide is not required for protease activity

ADAMTS13 belongs to the "a disintegrin and metalloprotease with thrombospondin repeats" family, and cleaves von Willebrand factor multimers into smaller forms. For several related proteases, normal folding and enzymatic latency depend on an NH2-terminal propeptide that is removed by proteolytic processing during biosynthesis. However, the ADAMTS13 propeptide is unusually short and poorly conserved, suggesting it may not perform these functions. ADAMTS13 was secreted from transfected HeLa cells with a half-time of 7 h and the rate-limiting step was exported from the endoplasmic reticulum. Deletion of the propeptide did not impair the secretion of active ADAMTS13, indicating that the propeptide is dispensable for folding. Furin was shown to be sufficient for ADAMTS13 propeptide processing in two ways. First, mutation of the furin consensus recognition site prevented propeptide cleavage in HeLa cells and resulted in secretion of pro-ADAMTS13. Second, furin-deficient LoVo cells secreted ADAMTS13 with the propeptide intact, and cotransfection with furin restored propeptide cleavage. In both cell lines, secreted pro-ADAMTS13 had normal proteolytic activity toward von Willebrand factor. In cells coexpressing both ADAMTS13 and von Willebrand factor, pro-ADAMTS13 cleaved pro-von Willebrand factor intracellularly. Therefore, the ADAMTS13 propeptide is not required for folding or secretion, and does not perform the common function of maintaining enzyme latency.     

Discovery and characterization of a novel, widely expressed metalloprotease, ADAMTS10, and its proteolytic activation

We describe the discovery and characterization of ADAMTS10, a novel metalloprotease encoded by a locus on human chromosome 19 and mouse chromosome 17. ADAMTS10 has the typical modular organization of the ADAMTS family, with five thrombospondin type 1 repeats and a cysteine-rich PLAC (protease and lacunin) domain at the carboxyl terminus. Its domain organization and primary structure is similar to a novel long form of ADAMTS6. In contrast to many ADAMTS proteases, ADAMTS10 is widely expressed in adult tissues and throughout mouse embryo development. In situ hybridization analysis showed widespread expression of Adamts10 in the mouse embryo until 12.5 days of gestation, after which it is then expressed in a more restricted fashion, with especially strong expression in developing lung, bone, and craniofacial region. Mesenchymal, not epithelial, expression in the developing lung, kidney, gonad, salivary gland, and gastrointestinal tract is a consistent feature of Adamts10 regulation. N-terminal sequencing and treatment with decanoyl-Arg-Val-Lys-Arg-chloromethylketone indicate that the ADAMTS10 zymogen is processed by a subtilisin-like proprotein convertase at two sites (Arg64/Gly and Arg233/Ser). The widespread expression of ADAMTS10 suggests that furin, a ubiquitously expressed proprotein convertase, is the likely processing enzyme. ADAMTS10 expressed in HEK293F and COS-1 cells is N-glycosylated and is secreted into the medium, as well as sequestered at the cell surface and extracellular matrix, as demonstrated by cell surface biotinylation and immunolocalization in nonpermeabilized cells. ADAMTS10 is a functional metalloprotease as demonstrated by cleavage of alpha2-macroglobulin, although physiological substrates are presently unknown.     

Characterization of coding synonymous and non-synonymous variants in ADAMTS13 using ex vivo and in silico approaches

Synonymous variations, which are defined as codon substitutions that do not change the encoded amino acid, were previously thought to have no effect on the properties of the synthesized protein(s). However, mounting evidence shows that these "silent" variations can have a significant impact on protein expression and function and should no longer be considered "silent". Here, the effects of six synonymous and six non-synonymous variations, previously found in the gene of ADAMTS13, the von Willebrand Factor (VWF) cleaving hemostatic protease, have been investigated using a variety of approaches. The ADAMTS13 mRNA and protein expression levels, as well as the conformation and activity of the variants have been compared to that of wild-type ADAMTS13. Interestingly, not only the non-synonymous variants but also the synonymous variants have been found to change the protein expression levels, conformation and function. Bioinformatic analysis of ADAMTS13 mRNA structure, amino acid conservation and codon usage allowed us to establish correlations between mRNA stability, RSCU, and intracellular protein expression. This study demonstrates that variants and more specifically, synonymous variants can have a substantial and definite effect on ADAMTS13 function and that bioinformatic analysis may allow development of predictive tools to identify variants that will have significant effects on the encoded protein.     

ADAMTS proteinases: a multi-domain,multi-functional family with roles in extracellular matrix turnover and arthritis

Members of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family are known to influence development, angiogenesis, coagulation and progression of arthritis. As proteinases their substrates include the von Willebrand factor precursor and extracellular matrix components such as procollagen, hyalectans (hyaluronan-binding proteoglycans including aggrecan), decorin, fibromodulin and cartilage oligomeric matrix protein. ADAMTS levels and activities are regulated at multiple levels through the control of gene expression, mRNA splicing, protein processing and inhibition by TIMP (tissue inhibitor of metalloproteinases). A recent screen of human cartilage has shown that multiple members of the ADAMTS family may be important in connective tissue homeostasis and pathology.     

Enzymatically active ADAMTS13 variants are not inhibited by anti-ADAMTS13 autoantibodies: a novel therapeutic strategy?

ADAMTS13 (a disintegrin and metalloprotease with thrombospondin motifs), a circulating multidomain zinc metalloprotease of the reprolysin subfamily, is critical for preventing von Willebrand factor-platelet interaction under high shear stress conditions. A deficiency of the protease, due to mutations in the ADAMTS13 gene or the presence of antibodies that inhibit the activity of the protease, causes thrombotic thrombocytopenic purpura (TTP). Plasma therapy, the conventional therapy for TTP, may cause serious adverse reactions and is ineffective in some patients. In order to develop new strategies for improving the diagnosis and treatment of TTP, we produced a series of truncated ADAMTS13 proteins in mammalian cells and analyzed their binding with and suppression by the IgG derived from the TTP patients. The results revealed that truncation of the ADAMTS13 protein at its cysteine-rich region eliminated its recognition by the antibodies without abolishing its von Willebrand factor-cleaving activity. This raises the possibility that resistant ADAMTS13 variants may be exploited to circumvent inhibitory antibodies that cause TTP.     

Rearranging Exosites in Noncatalytic Domains Can Redirect the Substrate Specificity of ADAMTS Proteases

ADAMTS proteases typically employ some combination of ancillary C-terminal disintegrin-like, thrombospondin-1, cysteine-rich, and spacer domains to bind substrates and facilitate proteolysis by an N-terminal metalloprotease domain. We constructed chimeric proteases and substrates to examine the role of C-terminal domains of ADAMTS13 and ADAMTS5 in the recognition of their physiological cleavage sites in von Willebrand factor (VWF) and aggrecan, respectively. ADAMTS5 cleaves Glu(373)-Ala(374) and Glu(1480)-Gly(1481) bonds in bovine aggrecan but does not cleave VWF. Conversely, ADAMTS13 cleaves the Tyr(1605)-Met(1606) bond of VWF, which is exposed by fluid shear stress but cannot cleave aggrecan. Replacing the thrombospondin-1/cysteine-rich/spacer domains of ADAMTS5 with those of ADAMTS13 conferred the ability to cleave the Glu(1615)-Ile(1616) bond of VWF domain A2 in peptide substrates or VWF multimers that had been sheared; native (unsheared) VWF multimers were resistant. Thus, by recombining exosites, we engineered ADAMTS5 to cleave a new bond in VWF, preserving physiological regulation by fluid shear stress. The results demonstrate that noncatalytic thrombospondin-1/cysteine-rich/spacer domains are principal modifiers of substrate recognition and cleavage by both ADAMTS5 and ADAMTS13. Noncatalytic domains may perform similar functions in other ADAMTS family members.     

Selective Decline of Synaptic Protein Levels in the Frontal Cortex of Female Mice Deficient in the Extracellular Metalloproteinase ADAMTS1

The chondroitin sulfate-bearing proteoglycans, also known as lecticans, are a major component of the extracellular matrix (ECM) in the central nervous system and regulate neural plasticity. Growing evidence indicates that endogenous, extracellular metalloproteinases that cleave lecticans mediate neural plasticity by altering the structure of ECM aggregates. The bulk of this in vivo data examined the matrix metalloproteinases, but another metalloproteinase family that cleaves lecticans, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS), modulates structural plasticity in vitro, although few in vivo studies have tested this concept. Thus, the purpose of this study was to examine the neurological phenotype of a mouse deficient in ADAMTS1. Adamts1 mRNA was absent in the ADAMTS1 null mouse frontal cortex, but there was no change in the abundance or proteolytic processing of the prominent lecticans brevican and versican V2. However, there was a marked increase in the perinatal lectican neurocan in juvenile ADAMTS1 null female frontal cortex. More prominently, there were declines in synaptic protein levels in the ADAMTS1 null female, but not male, frontal cortex beginning at postnatal day 28. These synaptic marker declines did not affect learning or memory in the adult female ADAMTS1 null mice when tested with the radial-arm water maze. These results indicate that in vivo Adamts1 knockout leads to sexual dimorphism in frontal cortex synaptic protein levels. Since changes in lectican abundance and proteolytic processing did not accompany the synaptic protein declines, ADAMTS1 may play a nonproteolytic role in regulating neural plasticity.     

The globular domains of type VI collagen are related to the collagen-binding domains of cartilage matrix protein and von Willebrand factor.

Type VI collagen is a transformation-sensitive glycoprotein of the extracellular matrix of fibroblasts. We have isolated and sequenced several overlapping cDNA clones (4153 bp) which encode the entire alpha 2 subunit of chicken type VI collagen. The deduced amino acid sequence predicts that the alpha 2(VI) polypeptide consists of 1015 amino acid residues that are arranged in four domains: a hydrophobic signal peptide of 20 residues, an amino-terminal globular domain of 228 residues, a collagenous segment of 335 residues and a carboxy-terminal globular domain of 432 residues. The collagenous domain contains seven Arg-Gly-Asp tripeptide units, some of which are likely to be used as cell-binding sites. The globular domains contain three homologous repeats with an average length of 180 amino acid residues. These repeats show a striking similarity to the collagen-binding motifs found in von Willebrand factor and cartilage matrix protein. We therefore speculate that the globular domains of the alpha 2(VI) polypeptide may interact with collagenous structures.     

Detection of a secreted metalloprotease within the nuclei of liver cells

ADAMTS13 is a secreted zinc metalloprotease expressed by various cell types. Here, we investigate its cellular pathway in endogenously expressing liver cell lines and after transient transfection with ADAMTS13. Besides compartmentalizations of the cellular secretory system, we detected an appreciable level of endogenous ADAMTS13 within the nucleus. A positively charged amino acid cluster (R-Q-R-Q-R-Q-R-R) present in the ADAMTS13 propeptide may act as a nuclear localization signal (NLS). Fusing this NLS-containing region to eGFP greatly potentiated its nuclear localization. Bioinformatics analysis suggests that the ADAMTS13 CUB-2 domain has a double-stranded beta helix (DSBH) structural architecture characteristic of various protein-protein interaction modules like nucleoplasmins, class I collagenase, tumor necrosis factor ligand superfamily, supernatant protein factor (SPF) and the B1 domain of neuropilin-2. Based on this contextual evidence and that largely conserved polar residues could be mapped on to a template CUB domain homolog, we hypothesize that a region in the ADAMTS13 CUB-2 domain with conserved polar residues might be involved in protein-protein interaction within the nucleus.     

Novel mutations in ADAMTS13 CUB domains cause abnormal pre-mRNA splicing and defective secretion of ADAMTS13

Hereditary thrombotic thrombocytopenic purpura (TTP) is an autosomal recessive thrombosis disorder, caused by loss-of-function mutations in ADAMTS13. Mutations in the CUB domains of ADAMTS13 are rare, and the exact mechanisms through which these mutations result in the development of TTP have not yet been fully elucidated. In this study, we identified two novel mutations in the CUB domains in a TTP family with an acceptor splice-site mutation (c.3569−1, G>A, intron 25) and a point missense mutation (c.3923, G>A, exon 28), resulting in a glycine to aspartic acid substitution (p.G1308D). In vitro splicing analysis revealed that the intronic mutation resulted in abnormal pre-mRNA splicing, and an in vitro expression assay revealed that the missense mutation significantly impaired ADAMTS13 secretion. Although both the patient and her brother displayed significantly reduced ADAMTS13 activity and increased levels of ultra-large VWF (ULVWF) multimers in plasma, only the female developed acute episodes of TTP. Our findings indicate the importance of the CUB domains for the protein stability and extracellular secretion of ADAMTS13.     

The secreted metalloprotease ADAMTS20 is required for melanoblast survival

ADAMTS20 (Adisintegrin-like and metalloprotease domain with thrombospondin type-1 motifs) is a member of a family of secreted metalloproteases that can process a variety of extracellular matrix (ECM) components and secreted molecules. Adamts20 mutations in belted (bt) mice cause white spotting of the dorsal and ventral torso, indicative of defective neural crest (NC)-derived melanoblast development. The expression pattern of Adamts20 in dermal mesenchymal cells adjacent to migrating melanoblasts led us to initially propose that Adamts20 regulated melanoblast migration. However, using a Dct-LacZ transgene to track melanoblast development, we determined that melanoblasts were distributed normally in whole mount E12.5 bt/bt embryos, but were specifically reduced in the trunk of E13.5 bt/bt embryos due to a seven-fold higher rate of apoptosis. The melanoblast defect was exacerbated in newborn skin and embryos from bt/bt animals that were also haploinsufficient for Adamts9, a close homolog of Adamts20, indicating that these metalloproteases functionally overlap in melanoblast development. We identified two potential mechanisms by which Adamts20 may regulate melanoblast survival. First, skin explant cultures demonstrated that Adamts20 was required for melanoblasts to respond to soluble Kit ligand (sKitl). In support of this requirement, bt/bt;Kit(tm1Alf)/+ and bt/bt;Kitl(Sl)/+ mice exhibited synergistically increased spotting. Second, ADAMTS20 cleaved the aggregating proteoglycan versican in vitro and was necessary for versican processing in vivo, raising the possibility that versican can participate in melanoblast development. These findings reveal previously unrecognized roles for Adamts proteases in cell survival and in mediating Kit signaling during melanoblast colonization of the skin. Our results have implications not only for understanding mechanisms of NC-derived melanoblast development but also provide insights on novel biological functions of secreted metalloproteases.     

Complete primary structure and genomic organization of the mouse Col14a1 gene.

The entire mouse cDNA sequence for type XIV collagen was determined using overlapping PCR products. The 6456 nucleotide (nt) cDNA sequence contains a 5391-nt open reading frame encoding 1797 amino acid residues. The amino terminus has a 28-residue signal peptide that is followed by the mature polypeptide of 1769 amino acid residues with a calculated molecular mass of 193.2 kDa. The mouse alpha1(XIV) collagen chain is predicted to contain all the structural domains described for the polypeptide in chicken and human. These include fibronectin type III repeats, von Willebrand factor A domains, thrombospondin-N-terminal-like domains and two triple-helical domains similar to those of other collagen family members. The amino acid residue sequence of human alpha1(XIV) collagen showed an overall identity of 74% to the chicken sequence and 88% to the human sequence. The entire mouse genomic structure has been determined and is made up of 48 exons. Alternatively spliced forms of mouse type XIV, collagen were not identified corresponding to the findings for the human form.     

Complete primary structure and genomic organization of the mouse Col14a1 gene.

The entire mouse cDNA sequence for type XIV collagen was determined using overlapping PCR products. The 6456 nucleotide (nt) cDNA sequence contains a 5391-nt open reading frame encoding 1797 amino acid residues. The amino terminus has a 28-residue signal peptide that is followed by the mature polypeptide of 1769 amino acid residues with a calculated molecular mass of 193.2 kDa. The mouse alpha1(XIV) collagen chain is predicted to contain all the structural domains described for the polypeptide in chicken and human. These include fibronectin type III repeats, von Willebrand factor A domains, thrombospondin-N-terminal-like domains and two triple-helical domains similar to those of other collagen family members. The amino acid residue sequence of human alpha1(XIV) collagen showed an overall identity of 74% to the chicken sequence and 88% to the human sequence. The entire mouse genomic structure has been determined and is made up of 48 exons. Alternatively spliced forms of mouse type XIV, collagen were not identified corresponding to the findings for the human form.     

ADAMTS13 Bound to Endothelial Cells Exhibits Enhanced Cleavage of von Willebrand Factor

ADAMTS13 is a plasma metalloprotease that cleaves ultralarge von Willebrand factor multimers to generate less thrombogenic fragments. Although this cleavage can occur at the surface of endothelial cells, it is currently unknown whether this process involves binding of the ADAMTS13 to the endothelial cell plasma membrane. Using different assay systems, we present evidence that ADAMTS13 binds to endothelial cells in a specific, reversible, and time-dependent manner with a K_d of 58 nm. This binding requires the COOH-terminal thrombospondin type 1 repeats of the protease. Binding is inhibited in the presence of heparin and by trypsin treatment of the cells. ADAMTS13 that was prebound to endothelial cells exhibited increased proteolysis of VWF as compared with ADAMTS13 present only in solution. These data support the notion that cleavage of VWF occurs mainly at the endothelial cell surface.