Thrombotic thrombocytopenic purpura (TTP) is a life-threatening systemic illness of abrupt onset and unknown cause. Proteolysis of the blood-clotting protein von Willebrand factor (VWF) observed in normal plasma is decreased in TTP patients. However, the identity of the responsible protease and its role in the pathophysiology of TTP remain unknown. We performed genome-wide linkage analysis in four pedigrees of humans with congenital TTP and mapped the responsible genetic locus to chromosome 9q34. A predicted gene in the identified interval corresponds to a segment of a much larger transcript, identifying a new member of the ADAMTS family of zinc metalloproteinase genes (ADAMTS13). Analysis of patients' genomic DNA identified 12 mutations in the ADAMTS13 gene, accounting for 14 of the 15 disease alleles studied. We show that deficiency of ADAMTS13 is the molecular mechanism responsible for TTP, and suggest that physiologic proteolysis of VWF and/or other ADAMTS13 substrates is required for normal vascular homeostasis.

Thrombotic thrombocytopenic purpura can occur after the initiation of clopidogrel therapy, often within the first two weeks of treatment. Physicians should be aware of the possibility of this syndrome when initiating clopidogrel treatment.

ADAMTS13 is a circulating zinc metalloprotease that cleaves the hemostatic glycoprotein von Willebrand factor (VWF) in a shear-dependent manner. Deficiency in ADAMTS13, owing to genetic mutations or autoimmune inhibitors, causes thrombotic thrombocytopenic purpura (TPP). Northern blot analysis has shown that ADAMTS13 is expressed primarily in the liver. By using real-time RT-PCR, we confirmed that in mice the liver had the highest level of the ADAMTS13 transcript. To identify the liver cell-type-specific origin of ADAMTS13, we used in situ hybridization techniques to investigate the pattern of ADAMTS13 expression in the liver; analyzed the ADAMTS13 proteolytic activity in the culture media of fractionated liver cells; and confirmed ADAMTS13 expression with RT-PCR analysis and cloning of the mouse ADAMTS13 gene. The results revealed that ADAMTS13 was expressed primarily in cell fractions enriched in hepatic stellate cells. The mouse ADAMTS13 cloned from primary hepatic stellate cells was similar to its human counterpart in digesting VWF and was susceptible to suppression by EDTA or the IgG inhibitors of patients with TTP. Since hepatic stellate cells are believed to play a major role in the development of hepatic fibrosis and cirrhosis, the identification of the liver cell-type expressing ADAMTS13 will have important implications for understanding pathophysiological mechanisms regulating ADAMTS13 expression. The von Willebrand factor (VWF) is a multimeric glycoprotein that mediates adhesion and aggregation of platelets at sites of vascular injury. A metalloprotease cleaves VWF at the Tyr1605-Met1606 bond, generating homodimers of the 176 and 140 kDa fragments. 1,2 The VWF-cleaving protease is essential for preventing platelet aggregation in the circulation, and deficiency of the protease is associated with thrombotic thrombocytopenic purpura (TTP), a disease characterized by the development of VWF-platelet-rich thrombi in the arterioles and capillaries. 3 Recent studies identified this protease as ADAMTS13, a member of the reprolysin-type zinc metalloprotease family. [4][5][6] The human ADAMTS13 gene, spanning 37 kb on human chromosome 9q34, comprises 29 exons that encode a polypeptide of 1427-amino-acid residues and possibly several splicing isoforms. Although it shares with other members of the ADAMTS family a common domain architecture consisting of metalloprotease, disintegrin-like sequence, thrombospondin type 1 repeat, cysteine-rich and spacer regions, ADAMTS13 exhibits several distinct features, such as an RGDS sequence in the spacer domain and two copies of CUB domains at the carboxyl terminus. Substitution of the D residue in the RGDS sequence does not appear to diminish the proteolytic activity of ADAMTS13. 7 Unlike other ADAMTS proteases, pro-ADAMTS13 is proteolytically active. 8 These unique features of ADAMTS13 are consistent with