A new name in thrombosis, ADAMTS13.

T thrombocytopenic purpura (TTP) is a rapidly fatal illness, first described by Moschowitz in 1924 (1), that is characterized by anemia, a low platelet count (thrombocytopenia), and microvascular thrombosis. The anemia is caused by hemolysis and is associated with sharply pointed red cell fragments, termed schistocytes, that look as though they might have been sliced with a knife. The thrombocytopenia is caused by platelet deposition in thrombi, which leads to tissue damage that can produce renal failure, stroke, myocardial infarction, and acute respiratory distress. TTP often attacks otherwise healthy adults, with a preference for females of childbearing age. Without treatment the mortality rate exceeds 90%, but plasma exchange therapy has reduced the death rate to approximately 25% (2, 3). Until recently, the pathogenesis of TTP was obscure and the efficacy of plasma exchange therapy was unexplained. The thrombi in TTP are rich in platelets and von Willebrand factor (VWF) but relatively poor in fibrin (4), reflecting minimal participation of the classical blood clotting cascades. VWF is required for normal platelet adhesion (Fig. 1), and exaggerated VWF adhesive activity has been proposed to cause TTP (5). Breakthroughs reported during the last year support this model. A new metalloprotease that cleaves VWF, ADAMTS13, was cloned (6–8), and mutations in the ADAMTS13 gene were shown to cause a congenital form of TTP (9). In this issue of PNAS, Kokame et al. (10) report new ADAMTS13 mutations and characterize the recombinant mutant proteases, giving us our first glimpses of ADAMTS13 structure– function relationships and discovering unexpected heterogeneity at the ADAMTS13 locus. The first hint of VWF involvement in TTP was reported 20 years ago and, as often is the case, the crucial insight was suggested by careful observation of rare patients. In 1982, Joel Moake et al. (5) found ‘‘unusually large’’ VWF multimers in blood plasma from four patients with chronic relapsing TTP, particularly when their disease was in remission. The plasma VWF multimers were similar to the very large multimers secreted by cultured endothelial cells, suggesting that unusually large VWF multimers persisted after secretion into the blood because the patients lacked a VWF depolymerase, possibly a protease or a disulfide reductase. Congenital or acquired deficiency of the depolymerase might cause TTP, and plasma exchange therapy might provide a fresh supply of depolymerase or remove an inhibitor. Recent discoveries are consistent with this early proposal. In back-to-back papers, Furlan et al. (11) and Tsai (12) described a plasma metalloprotease activity that shortens VWF multimers by cleaving a single Tyr–Met bond within domain A2 of the VWF subunit. The enzyme cleaved VWF very slowly in plasma (Fig. 1), but the reaction was greatly accelerated by fluid shear stress or by mild denaturation of VWF with urea or guanidine hydrochloride. Shortly thereafter, deficiency of this VWF cleaving protease, whether congenital or caused by an acquired antibody inhibitor, was shown to be strongly associated with TTP (13–16). Last year, the VWF cleaving protease was purified to homogeneity, sequenced, and recognized as a new member of the ADAMTS protease family, which is named for the combination of A Disintegrin-like and Metalloprotease with Thrombospondin-1 repeats (6, 17, 18). Full-length cDNA clones for ADAMTS13 were obtained rapidly (7, 8). Simultaneously, the ADAMTS13 gene was identified by genomewide linkage analysis in families affected by congenital TTP, and 12 distinct mutations were characterized in seven unrelated families (9). Interestingly, no patient had definitive null mutations on both alleles, suggesting that total ADAMST13 deficiency may be lethal. ADAMTS13 consists of a signal peptide, a propeptide, and a typical reprolysin-like or adamalysin-like metalloprotease domain, followed by a disintegrin-like motif, a thrombospondin-1 repeat, a characteristic cysteine-rich and spacer domain, seven additional thrombospondin-1 repeats, and two CUB domains. Aside from the metalloprotease domain, the purpose of the domains is not known. However, all are conserved among ADAMTS13 from human, mouse (7, 9), and Japanese pufferfish (Takifugu rubripes) (unpublished observations), suggesting that they are essential for some function. Based on the role of their homologues in other proteins, these domains are candidates to interact with cell surfaces, extracellular matrix, protein cofactors, or VWF. The mutations described so far are distributed from the metalloprotease domain through the first CUB domain with no obvious mutational hotspot (9) and therefore could illuminate the function of several structural domains. Until now, however, no mutation had been characterized sufficiently to establish the corresponding mechanism of disease. Kokame et al. (10) studied two unrelated patients from Japan with Upshaw– Schulman syndrome, which refers to inherited TTP with a chronic relapsing course that often begins in the neonatal period (19, 20). Neither patient had detectable plasma ADAMTS13 activity, suggesting the presence of inactivating mutations on both alleles. One patient was homozygous for a Q449X nonsense mutation. The second patient had three candidate mutations: Q448E and C508Y on the maternal allele and R268P on the paternal allele. Q448E was reported previously as a polymorphism, although the effect of the substitution on function was not determined. In addition, the asymptomatic father had the mutation P475S on his other ADAMTS13 allele and plasma ADAMTS13 activity of only 5.6%, suggesting that the substitution P475S also impairs ADAMTS13 function.