We identified a novel metalloprotease, which could be responsible for cleaving the Tyr842-Met843 peptide bond of von Willebrand factor (vWF). This metalloprotease was purified from Cohn Fraction-I precipitate of human pooled plasma by the combination of gel filtration, DEAE chromatography, and preparative polyacrylamide gel electrophoresis in the presence of SDS. The NH2-terminal amino acid sequence of the isolated protein was: AAGGILHLELLVAVGPDVFQAHQEDTRRY. Based on this sequence, we searched human genomic and EST databases, and identified compatible nucleotide sequences. These results suggested that this protein is a novel metalloprotease, a member of the family of a disintegrin and metalloprotease with thrombospondin type-1 motifs (ADAMTS), and its genomic DNA was mapped to human chromosome 9q34. Multiple human tissue northern blotting analysis indicated that the mRNA encoding this protease spanned approximately 5 kilobases and was uniquely expressed in the liver. Furthermore, we determined the cDNA sequence encoding this protease, and found that this protease was comprised of a signal peptide, a proregion followed by the putative furin cleavage site, a reprolysin-type zinc-metalloprotease domain, a disintegrin-like domain, a thrombospondin type-1 (TSP1) motif, a cysteine-rich region, a spacer domain, and COOH-terminal TSP1 motif repeats.

A severe lack of von Willebrand factorcleaving protease (VWF-CP) activity can cause thrombotic thrombocytopenic purpura (TTP). This protease was recently identified as a member of the ADAMTS family, ADAMTS-13. It consists of a preproregion, a metalloprotease domain, a disintegrin-like domain, a thrombospondin type-1 motif (Tsp1), a cysteine-rich domain, a spacer domain, additional Tsp1 repeats, and CUB domains. To explore the structural and functional relationships of ADAMTS-13, we prepared here 13 sequential COOH-terminal truncated mutants and a single-point mutant (ArgGlyAsp [RGD] to ArgGlyGlu [RGE] in the cysteine-rich domain) and compared the activity of each mutant with that of the wild-type protein. The results revealed that the truncation of the cysteine-rich/spacer domains caused a remarkable reduction in VWF-CP activity. We also prepared immunoglobulin G (IgG) fractions containing inhibitory autoantibodies against ADAMTS-13 from plasma from 3 patients with acquired TTP, and we performed mapping of their epitopes using the aforementioned mutants. The major epitopes of these antibodies were found to reside within the cysteine-rich/spacer domains. These results suggest that the ADAMTS-13 cysteine-rich/spacer domains are essential for VWF-CP activity. IntroductionVon Willebrand factor (VWF) functions as a molecular glue through its anchoring of platelets at sites of injured vessel walls under high shear stress. 1 Mature VWF contains 2050-amino acid residues, has a molecular weight of approximately 250 kDa, and is released from endothelial cells as an "unusually large" multimer (UL-VWFM) with a molecular weight of approximately 30 000 kDa. [2][3][4][5] In healthy individuals, UL-VWFM is converted rapidly in the circulation to smaller forms, a series of multimers with molecular weights ranging from approximately 500 to 20 000 kDa. 6 It is known that the larger multimers have more potent biologic activity. 7,8 Under shear stress conditions in the circulation, VWF becomes more susceptible to proteolysis, 9-12 resulting in the formation of small but consistent proportions of 176-kDa and 140-kDa fragments derived from the approximately 250-kDa VWF subunit. 13 On the basis of these findings, the existence of a specific VWF-cleaving protease (VWF-CP) has been proposed.UL-VWFM has been found in plasma from patients with thrombotic thrombocytopenic purpura (TTP). 14 TTP is characterized by thrombocytopenia, microangiopathic hemolytic anemia, renal failure, neurologic dysfunction, and fever. 15 In 1996, a metalloprotease that cleaves a peptide bond between amino acid residues Tyr842 and Met843 within the VWF A2 domain was partially purified. 16,17 It was also reported that a deficiency in VWF-CP activity was associated with TTP. 18 On the basis of these findings, it was proposed that the UL-VWFM produced as a result of the deficiency in VWF-CP activity promotes microvascular thrombus formation. Congenital or acquired deficiency of VWF-CP activity can cause TTP. Congenital TTP with neonatal onset and frequent relapse...

We investigated the effect of activated protein C (APC) on pulmonary vascular injury and the increase in tumor necrosis factor (TNF) levels in lipopolysaccharide (LPS)-treated rats to determine whether APC reduces LPS-induced endothelial damage by inhibiting cytokine production. Intravenously administered LPS (5 mg/kg) induced pulmonary vascular injury, as indicated by an increase in the lung wet-to-dry weight ratio. LPS-induced pulmonary vascular injury was prevented by APC but not by active site-blocked factor Xa [dansyl glutamyl-glycyl-arginyl chloromethyl detone-treated activated factor X (DEGR-Xa)], a selective inhibitor of thrombin generation, or inactivated APC [diisopropyl fluorophosphate-treated APC (DIP-APC)]. APC, but not DEGR-Xa or DIP-APC, significantly inhibited the LPS-induced increase in the plasma level of TNF. APC significantly inhibited the production of TNF by LPS-stimulated monocytes in a dose-dependent fashion in vitro, but DIP-APC did not. APC did not inhibit the functions of activated neutrophils in vitro. These findings suggest that APC prevented LPS-induced pulmonary vascular injury by inhibiting TNF production by monocytes and not via its anticoagulant activity. The serine protease activity of APC appears to be essential for inhibition of TNF production.

Background: Platelet collagen receptor GPVI likely functions as a dimer rather than a monomer. Results: Preformed GPVI dimers, but not monomers, in resting platelets bind specific collagen sequences and are essential for platelet adhesion and activation. Conclusion: Constitutive GPVI dimers on resting platelets support platelet adhesion to collagen and activation. Significance: Resting platelets bind collagen through GPVI dimers, allowing immediate initiation of thrombus formation.