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 disintegrinlike 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؉ Thrombotic thrombocytopenic purpura (TTP)1 is a syndrome characterized by microangiopathic hemolytic anemia and thrombocytopenia, and it may be accompanied by neurological dysfunction, renal failure, and fever (1, 2). TTP often strikes young adults, mainly females, suggesting an autoimmune etiology. If untreated, the mortality may exceed 90% (2), but plasma exchange therapy has reduced the mortality to less than 20% (3).The basis for the efficacy of plasma exchange remains unknown, although a plausible model has been suggested in which the proteolysis of von Willebrand factor (VWF) plays a central role. In 1982, patients with chronic relapsing TTP were reported to have "unusually large" VWF multimers (UL-VWF) because of the absence of a VWF depolymerase activity. Binding of UL-VWF to platelets could promote microvascular thrombosis, platelet consumption, and hemolysis. Therapeutic infusion of plasma was proposed to replace the missing depolymerase activity or other factors and thereby limit VWF-dependent platelet thrombosis (4). Later discoveries have strengthened the relationship between VWF proteolysis and TTP. A plasma metalloprotease was identified that requires both calcium and zinc ions and cleaves the Tyr 1605 -Met 1606 bond 2 in the central A2 domain of the VWF subunit (5, 6). Cleavage was stimulated by shear forces like those occurring at sites of arterial thrombosis or by low concentrations of urea or guanidine. Furthermore, most adult patients with TTP were found to have congenital deficiency or an acquired autoantibody inhibitor of this VWF-cleaving protease (VWFCP) (7-9). These findings are consistent with the UL-VWF model of TTP pathogenesis. Alternatively, it is possible that failure to cleave another unknown substrate contributes to the pathogenesis of TTP.VWFCP was recently purified from human plasma sources, and an N-terminal amino acid sequence was obtained (10,11). This information showed it to be a member of the ADAMTS family of metalloproteases, named for the characteristic combination of a disintegrin-like and metalloprotease (reprolysintype), with thrombospondin type 1 motif (12-14). Furthermore, the VWFCP gene wa...

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 Tyr 1605 -Met 1606 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 His 6 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 Cterminal TSP1 and CUB domains are dispensable in vitro.Thrombotic thrombocytopenic purpura (TTP) 1 is a syndrome characterized by microangiopathic hemolytic anemia and thrombocytopenia, and it may be accompanied by neurological dysfunction, renal failure, and fever (1-3). If untreated, the mortality can exceed 90%, but plasma-exchange therapy has reduced the mortality to less than 20% (4). Although the pathophysiology of TTP is not fully understood, a plausible model has been proposed in which the proteolysis of von Willebrand factor (VWF) plays a central role (5). VWF is a multimeric protein that binds receptors on the surface of platelets and in connective tissue, thereby mediating the adhesion of platelets to sites of vascular injury. If unchecked, the process can lead to microvascular thrombosis. A plasma VWF-cleaving protease has been described that acts on the Tyr 1605 -Met 1606 bond in the central A2 domain of the VWF subunit, and cleavage is stimulated by shear forces like those occurring at sites of thrombosis or by low concentrations of urea or guanidine (6, 7). This proteolytic reaction limits VWF-dependent platelet adhesion, and most adults with idiopathic TTP have an acquired autoantibody that inhibits the VWF-cleaving protease (8, 9). Therefore, therapeutic plasma exchange may ameliorate TTP by replacing the missing protease and removing the inhibitory antibody.The VWF-cleaving protease was recently purified and identified as a new member of the ADAMTS family of metalloproteases (10, 11), so named for the combination of a disintegrin-like and metalloprote...

Von Willebrand factor (VWF) is a multimeric protein that mediates platelet adhesion at sites of vascular injury, and ADAMTS13 (a disintegrin and metalloprotease with thrombospondin)is a multidomain metalloprotease that limits platelet adhesion by a feedback mechanism in which fluid shear stress induces proteolysis of VWF and prevents disseminated microvascular thrombosis. Cleavage of the Tyr 1605 -Met 1606 scissile bond in the VWF A2 domain depends on a Glu 1660 -Arg 1668 segment in the same domain and on the noncatalytic spacer domain of ADAMTS13, suggesting that extensive enzyme-substrate interactions facilitate substrate recognition. Based on mutagenesis and kinetic analysis, we find that the ADAMTS13 spacer domain binds to an exosite near the C terminus of the VWF A2 domain. Deleting the spacer domain from ADAMTS13 or deleting the exosite from the VWF substrate reduced the rate of cleavage Ϸ20-fold. A cleavage product containing the exosite was a hyperbolic mixed-type inhibitor of ADAMTS13 proteolysis of either VWF multimers or model peptide substrates but only if the ADAMTS13 enzyme contained the spacer domain. The specificity of this unique mechanism depends on tension-induced unfolding of the VWF A2 domain, which exposes the scissile bond and exosite for interaction with complementary sites on ADAMTS13.enzyme kinetics ͉ thrombotic thrombocytopenic purpura ͉ fluid shear stress

ADAMTS13 is a plasma metalloproteinase that cleaves von Willebrand factor to smaller, less thrombogenic forms. Deficiency of ADAMTS13 activity in plasma leads to thrombotic thrombocytopenic purpura. ADAMTS13 contains eight thrombospondin type 1 repeats (TSR), seven of which contain a consensus sequence for the direct addition of fucose to the hydroxyl group of serine or threonine. Mass spectral analysis of tryptic peptides derived from human ADAMTS13 indicate that at least six of the TSRs are modified with an O-fucose disaccharide. Analysis of [3 H]fucose metabolically incorporated into ADAMTS13 demonstrated that the disaccharide has the structure glucose-␤1,3-fucose. Mutation of the modified serine to alanine in TSR2, TSR5, TSR7, and TSR8 reduced the secretion of ADAMTS13. Mutation of more than one site dramatically reduced secretion regardless of the sites mutated. When the expression of protein O-fucosyltransferase 2 (POFUT2), the enzyme that transfers fucose to serines in TSRs, was reduced using siRNA, the secretion of ADAMTS13 decreased. A similar outcome was observed when ADAMTS13 was expressed in a cell line unable to synthesize the donor for fucose addition, GDPfucose. Although overexpression of POFUT2 did not affect the secretion of wild-type ADAMTS13, it did increase the secretion of the ADAMTS13 TSR1,2 double mutant but not that of ADAMTS13 TSR1-8 mutant. Together these findings indicate that O-fucosylation is functionally significant for secretion of ADAMTS13.ADAMTS13 is a plasma metalloprotease that cleaves von Willebrand factor to smaller, less thrombogenic fragments. ADAMTS13 is a member of the ADAMTS family of metalloproteases that are characterized by a conserved domain structure. These include a metalloprotease domain, a disintegrin domain, a thrombospondin type 1 repeat (TSR), 2 a cysteinerich domain, and a spacer domain and conclude with a variable number of additional TSRs (1). ADAMTS13 uniquely contains seven additional TSRs and two CUB1 domains at its carboxyl end (2-4) (Fig. 1A). TSRs contain ϳ60 amino acids with conserved tryptophans and cysteines. They were first described in thrombospondin type 1 and are homologous to the properdin repeat found in many components of the complement system. Thrombospondin type 1 is a protein that is thought to play a role in angiogenesis, cell adhesion, and motility (5, 6). Many interactions of thrombospondin-1 are thought to be mediated through amino acids within the TSRs (7). For example, binding of thrombospondin-1 to the endothelial cell protein, CD36, can be inhibited by the peptide CSVTCG, which is found in the TSRs of thrombospondin-1 (8). The three TSRs of thrombospondin-1 have been shown to contain a fucose directly linked to a serine or threonine within a putative consensus sequence of C 1 XX(S/T)C 2 G (where C 1 and C 2 are the 1st and 2nd conserved cysteines in the TSR (9)), which is the putative CD36 binding region. The fucose on the TSRs was further modified with a glucose in ␤1-3 linkage to form a disaccharide (9, 10). Subsequent analysis ...

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. AD-AMTS13 binding was saturable and reversible. Equilibrium binding occurred within 2 h and the half-time for dissociation was ϳ4 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 ϳ3-fold and truncation after the seventh thrombospondin type 1 repeat in addition to the CUB domains increased the affinity for von Willebrand factor by ϳ2-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.von Willebrand factor (VWF) 1 is a large multimeric protein that promotes hemostasis by tethering platelets at sites of vascular injury (1). The largest multimers of VWF are most active in platelet interactions, and thrombosis can result if large multimers accumulate as a result of ADAMTS13 deficiency. VWF is secreted into plasma as ultra-large multimers that are proteolysed to smaller forms by the metalloprotease, ADAMTS13 (1-3). Deficiency of ADAMTS13 activity because of a congenital absence of the protein or because of the production of inhibitory anti-ADAMTS13 antibodies leads to thrombotic thrombocytopenic purpura (TTP) (4, 5). TTP is characterized by uncontrolled microvascular thrombosis that especially affects the cerebral and renal circulation (6).ADAMTS13 is a member of the ADAMTS family of metalloproteinases that are frequently involved in proteolysis of extracellular matrix proteins (7-11). The ADAMTS family is characterized by a modular domain structure consisting of a metalloproteinase domain, a disintegrin-like domain, a thrombospondin type 1 (TSP1) repeat, a cysteine-rich region, and a spacer domain (12). Most ADAMTS family members have a variable number of additional C-terminal TSP1 repeats. AD-AMTS13 contains seven additional TSP1 repeats and two CUB domains at its C terminus and is the most divergent member of the family (13-15).The CUB domains are unique to ADAMTS13 among the ADAMTS family members. CUB domains were first identified in the complement proteins C1r and C1s and have since been identified in ma...