Cleavage of von Willebrand Factor Requires the Spacer Domain of the Metalloprotease ADAMTS13

Abstract

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...

Binding of platelet glycoprotein Ibα to von Willebrand factor domain A1 stimulates the cleavage of the adjacent domain A2 by ADAMTS13

“…In a previous study, ADAMTS13 truncated after the spacer domain (del2) and full-length ADAMTS13 had comparable activity toward plasma vWF, whereas ADAMTS13 truncated after the metalloprotease domain (del6) was inactive (23). Similar results were obtained when these truncated proteases were assayed for activity toward recombinant vWF fragments (Fig.…”

“…Nevertheless, these observations suggest an answer to the paradox that cleavage of vWF in vitro does not seem to depend on the C-terminal seven thrombospondin-1 repeats and two CUB domains of ADAMTS13 (23), even though these domains are conserved among fish, amphibians, birds, and mammals (ref. 41 and unpublished results).…”

“…ADAMTS13 mutants with C-terminal truncations after the metalloprotease domain (del6) or the spacer domain (del2) were expressed in Sf9 cells as reported (23). Recombinant GPIb␣ with the point mutations G233V and M239V and recombinant vWF A1 domain were expressed in High Five cells and Escherichia coli BL21 (DE3), respectively, and were purified as reported (24).…”

Conformational quiescence of ADAMTS‐13 prevents proteolytic promiscuity

“…Multiple VWF‐binding exosites have been identified across a number of ADAMTS‐13 domains 5, which have informed the development of a so‐called ‘molecular zipper’ model of interaction and proteolysis 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16. An interaction occurs between ADAMTS‐13 and globular VWF, in which the distal C‐terminal tail of ADAMTS‐13 and the C‐terminal D4‐CK domains of VWF make contact 17.…”

Production, crystallization and preliminary crystallographic analysis of an exosite-containing fragment of human von Willebrand factor-cleaving proteinase ADAMTS13

“…C-terminal truncation of ADAMTS13 after the C but not the S domain results in severe loss of proteolytic activity towards VWF (Soejima et al, 2003;Zheng et al, 2003). Therefore, in addition to the M domain, the ancillary domains including the D, T, C and S domains (DTCS) are necessary for normal ADAMTS13 activity, although the distal C-terminal domains are required for regulation of in vivo thrombus formation under high-shear conditions (Banno et al, 2009).…”