Sequence analysis of the ADAMTS13 locus of 2 patients with hereditary thrombotic thrombocytopenic purpura (TTP) revealed the homozygous presence of 4 single nucleotide polymorphisms (SNPs) (R7W, Q448E, P618A, A732V) and a rare missense mutation (R1336W). Analysis of the individual effect of any amino acid exchanges showed that several sequence variations can interact with each other, thereby altering the phenotype of ADAMTS13 deficiency. Introduction of polymorphisms R7W, Q448E, and A732V had no or only minor effects on ADAMTS13 secretion. In contrast, P618A, R1336W, and the A732V-P618A combination strongly reduced ADAMTS13-specific activity and antigen levels. Surprisingly, R7W and Q448E were positive modifiers of ADAMTS13 secretion in the context of P618A and A732V but neither could rescue the severely reduced specific activity conferred by P618A. However, in the context of R1336W, polymorphisms R7W and Q448E enhanced the detrimental effect of the missense mutation and led to undetectable enzyme activity. We show that dependent on the sequence context, the same polymorphisms might be either positive or negative modifiers of gene expression. Our results might therefore be widely relevant to understanding the influence of polymorphisms on the phenotypic expression of complex diseases. Introductionvon Willebrand factor (VWF) is crucial for primary hemostasis by mediating extracellular matrix-platelet interactions and delivering factor VIII to the site of vascular injury. The large and heterogeneous glycoprotein circulates as a multimer composed of identical units, ranging in molecular weight from 500 kDa to more than 20 000 kDa. 1 Plasma VWF is predominately secreted as high molecular weight, "unusually large," VWF (ULVWF) multimers. Because ULVWF binds more avidly to components of the extracellular matrix 2 and to various platelet receptors 3,4 than low molecular weight VWF, the hemostatic activity of VWF is size dependent and much more pronounced for high molecular weight VWF than for smaller multimers. This intrinsic property of ULVWF allows for platelet attachment to sites of vascular injury under high shear stress conditions of fastflowing blood. 5 However, immediately downstream from the site of injury, the multimeric size of VWF needs to be physiologically regulated to prevent the formation of platelet and VWF-rich thrombi.A VWF-cleaving protease, now called ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type-1 motifs) has been identified. [6][7][8][9][10][11][12][13] ADAMTS13 cleaves VWF between Tyr1605 and Met1606 in the A2 domain of the VWF monomer, 6,7,14,15 yielding typical fragments of 176 kDa and 140 kDa and smaller VWF multimers. Severe deficiency of ADAMTS13 may result in ULVWF spontaneously interacting with platelet receptors 5 and cause the life-threatening disorder thrombotic thrombocytopenic purpura (TTP). 16,17 Acquired TTP has been associated with the inhibition of ADAMTS13 activity by autoimmune antibodies, [18][19][20][21] whereas mutations in the ADAMTS13 gene have been ...

T is not able to synthesize dTDP-L-rhamnose. These findings confirm that in each strain the enzymes act specifically on S-layer glycoprotein glycan formation.Crystalline surface layers (S-layers) represent the outermost cell envelope component of many archaea and bacteria (for reviews see references 41, 45, and 46). Distinct functions of S-layers have been reported only in a few cases (47). For pathogenic bacteria variation of surface antigens is important for evading the immune response of the host. Campylobacter fetus achieves antigenic variation by expression of different S-layer proteins from at least eight homologs of the structural S-layer gene (6). Variations in the expression of S-layer proteins are not limited to pathogenic species and have also been described for nonpathogenic bacteria, such as Bacillus sphaericus (3), Geobacillus (formerly Bacillus) stearothermophilus (24), and Lactobacillus acidophilus (2). These organisms seem to adapt to altered environmental conditions by changing the S-layer protein. Modification of the surface properties is also achieved by posttranslational modification of the S-layer proteins, particularly by glycosylation (for reviews see references 15, 31, 32, 34, 42, and 51). Few analyses have been carried out so far to elucidate the biosynthesis of bacterial S-layer glycoprotein glycans (12,44). The studies that have been performed confirmed that there are differences in activation of mono-and oligosaccharides for eukaryotic glycoprotein biosynthesis (20) and even for the formation of other bacterial glycoconjugates. Most of the nucleotide-activated monosaccharides used for biosynthesis of Slayer glycoproteins are identical to those used for biosynthesis of other glycoconjugates. However, in Thermoanaerobacter thermosaccharolyticum strain E207-71 N-acetylmannosamine is activated not as the common UDP-N-acetylmannosamine but as GDP-N-acetylmannosamine (44). These monosaccharide precursors are not used for the formation of lipid-activated oligosaccharides but as precursors for the synthesis of nucleotide-activated oligosaccharides.In our attempts to elucidate the formation of specific sugar components of bacterial S-layer glycans, we focused on the biosynthesis of L-rhamnose. This sugar has often been found in S-layer glycoproteins (for reviews see references 31, 32, 34, and 42), and its biosynthetic pathway in gram-negative organisms has been known since the 1960s (8,21,36). The reaction steps and enzymes and the corresponding gene loci are as follows (10,11,14,16,38): (i) dTTP ϩ D-Glc-1-P 3 dTDP-D-Glc ϩ PP i (glucose-1-phosphate thymidylyltransferase; EC 2.7.7.24; * Corresponding author. Mailing address: Zentrum für Ultrastrukturforschung,

The procoagulatory serine protease, thrombin, is known to induce invasion and metastasis in various cancers, but the mechanisms by which it promotes tumorigenesis are poorly understood. Because the 92-kDa gelatinase (MMP-9) is a known mediator of tumor cell invasion, we sought to determine whether and how thrombin regulates MMP-9. The thrombin receptor, PAR-1, and MMP-9 are expressed in osteosarcomas, as determined by immunohistochemistry. Stimulation of U2-OS osteosarcoma cells with thrombin and a thrombin receptor-activating peptide induced pro-MMP-9 secretion as well as cell surface-associated pro-MMP-9 expression and proteolytic activity. This was paralleled by an increase in MMP-9 mRNA and MMP-9 promoter activity. Thrombin-induced invasion of U2-OS cells through Matrigel was mediated by the phosphatidylinositol 3-kinase signaling pathway and could be inhibited with an MMP-9 antibody. The stimulation of MMP-9 by thrombin was paralleled by an increase in ␤1-integrin mRNA and ␤1-integrin expression on the cell surface, which was also mediated by phosphatidylinositol 3-kinase and was required for invasion. Thrombin activation induced and co-localized both ␤1-integrin and pro-MMP-9 on the cell membrane, as evidenced by co-immunoprecipitation, confocal microscopy, and a protein binding assay. The thrombin-mediated association of these two proteins, as well as thrombin-mediated invasion of U2-OS cells, could be blocked with a cyclic peptide and with an antibody preventing binding of the MMP-9 hemopexin domain to ␤1-integrin. These results suggest that thrombin induces expression and association of ␤1-integrin with MMP-9 and that the cell surface localization of the protease by the integrin promotes tumor cell invasion.

Summary. Functional deficiency or absence of the human von Willebrand factor (VWF)-cleaving protease (VWF-cp), recently termed ADAMTS-13, has been shown to cause acquired and congenital thrombotic thrombocytopenic purpura (TTP), respectively. As a first step towards developing a small animal model of TTP, we have cloned the complete (non-truncated) murine Adamts-13 gene from BALB/c mice liver poly A + mRNA. Murine ADAMTS-13 is a 1426-amino-acid protein with a high homology and similar structural organization to the human ortholog. Transient expression of the murine Adamts-13 cDNA in HEK 293 cells yielded a protein with a molecular weight of approximately 180 kDa which degraded recombinant murine VWF (rVWF) in a dose-dependent manner. The cleavage products of murine rVWF had the expected size of 140 and 170 kDa. Murine ADAMTS-13 was inhibited by EDTA and the plasma from a TTP patient.

ADAMTS13 cleaves plasmatic von Willebrand factor (VWF) between Tyr1605 and Met1606 and regulates thereby the hemostatic activity of VWF. Mutations in the ADAMTS13 gene leading to severe ADAMTS13 deficiency have been found in patients with congenital thrombotic thrombocytopenic purpura (TTP). We have analyzed the ADAMTS13 gene defects in two brothers with hereditary TTP [Antoine et al, Brit. J. Hematol., 2003] where we detected a total of six nucleotide exchanges causing point mutations. On the maternal allele we found an accumulation of five amino acid substitutions (R7W, Q448E, P618A, A732V, R1336W) and on the paternal allele a stop mutation (Q44X) leading to premature protein termination in the propeptide region. Both brothers were double heterozygotes with < 3% of ADAMTS13 activity, whereas their asymptomatic parents have ~ 50% activity. Four (R7W, Q448E, P618A, A732V) of the five maternal mutations constitute single nucleotide polymorphisms (SNP) but R1336W was identified as novel rare mutation in the second cub domain. To evaluate the biologic phenotype of a given haplotype, e.g. the functional significance of the presence of the various SNPs, we analyzed the functional impact of the individual mutations on ADAMTS13 antigen levels and ADAMTS13 activity. A series of mutant ADAMTS13 molecules was expressed which contained either single amino acid substitutions or combinations of mutations with each other. We found that the common SNPs R7W, Q448E and A732V, as single mutations, had either no or only a minor impact on ADAMTS13 secretion and ADAMTS13 activity, whereas P618A and R1336W conferred a dominant adverse effect on ADAMTS13 secretion levels. Co-expression of SNPs R7W or Q448E with SNP P618A lead to improved ADAMTS13 secretion levels and could therefore partly attenuate the detrimental effect of P618A. Concomitant expression of all four SNPs reconstituted secretion levels similar to wild-type implicating a complex synergistically interaction of SNPs located in different ADAMTS13 domain regions, however, functional activity was impaired to 50%. Mutation R1336W was shown to be, as a single amino acid exchange, responsible for reduced ADAMTS13 antigen levels, but in contrast to P618A, the negative effect of R1336W was rather enhanced by the co-expression of R7W and Q448E, than rescued, leading to the total absence of ADAMTS13 secretion from the maternal allele. Our findings provide for the first time evidence that fairly common SNPs, dependent on the presence or absence of other mutations, may differently modulate functional ADAMTS13 protease levels.