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.

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Impaired fibrinolytic activity within the lung is a common manifestation of acute and chronic inflammatory lung diseases. Because the fibrinolytic system is active during repair processes that restore injured tissues to normal, reduced fibrinolytic activity may contribute to the subsequent development of pulmonary fibrosis. To examine the relationship between the fibrinolytic system and pulmonary fibrosis, lung inflammation was induced by bleomycin in transgenic mice that either overexpressed or were completely deficient in murine plasminogen activator inhibitor-1 (PAI-1). 2 wk after 0.075 U of bleomycin, the lungs of transgenic mice overexpressing PAI-1 contained significantly more hydroxyproline (118 Ϯ 8 g) than littermate controls (70.5 Ϯ 8 g, P Ͻ 0.005). 3 wk after administration of a higher dose of bleomycin (0.15 U), the lung hydroxyproline content of mice completely deficient in PAI-1 (49 Ϯ 8 g) was not significantly different ( P ϭ 0.63) than that of control animals receiving saline (37 Ϯ 1 g), while hydroxyproline content was significantly increased in heterozygote (77 Ϯ 12 g, P ϭ 0.06) and wild-type (124 Ϯ 19 g, P Ͻ 0.001) littermates. These data demonstrate a direct correlation between the genetically determined level of PAI-1 expression and the extent of collagen accumulation that follows inflammatory lung injury. These results strongly support the hypothesis that alterations in fibrinolytic activity influence the extent of pulmonary fibrosis that occurs after inflammatory injury.

The serine protease inhibitors of the serpin family are an unusual group of proteins thought to have metastable native structures. Functionally, they are unique among polypeptide protease inhibitors, although their precise mechanism of action remains controversial. Conflicting results from previous studies have suggested that the stable serpin-protease complex is trapped in either a tight Michaelis-like structure, a tetrahedral intermediate, or an acyl-enzyme. In this report we show that, upon association with a target protease, the serpin reactive-center loop (RCL) is cleaved resulting in formation of an acyl-enzyme intermediate. This cleavage is coupled to rapid movement of the RCL into the body of the protein bringing the inhibitor closer to its lowest free energy state. From these data we suggest a model for serpin action in which the drive toward the lowest free energy state results in trapping of the protease-inhibitor complex as an acyl-enzyme intermediate.The serpins are a large family of proteins which includes most of the protease inhibitors found in blood, as well as other proteins with unrelated or unknown functions (1). Serpins act as "suicide inhibitors" that react only once with their cognate protease, forming an SDS-stable complex. Current models of serpin structure suggest that, while their overall fold is generally homologous among family members, the RCL, 1 sometimes referred to as the strained or stressed loop, is capable of adopting markedly different conformations relative to the rest of the protein structure (2-5). This conformational flexibility appears to be necessary for function but can also lead to inactivation when the loop inserts into the main body of the inhibitor, becoming the central strand of the major serpin structural motif, ␤-sheet A (6 -8). This inactive conformation was first observed in RCL-cleaved ␣ 1 -antitrypsin (␣ 1 AT) (9) and more recently in the related structure of latent plasminogen activator inhibitor 1 (PAI-1) (10). Loop insertion leads to a large increase in thermal stability, presumably due to reorganization of the five-stranded ␤-sheet A from a mixed parallel-antiparallel arrangement to a six-stranded, predominantly antiparallel, ␤-sheet (11-14). This dramatic stabilization has led to the suggestion that native inhibitory serpins may be metastable structures, kinetically trapped in a state of higher free energy than their most stable thermodynamic state. Such an energetically unfavorable structure would almost certainly be subject to negative selection, and thus its retention in all inhibitory serpins implies that it has been conserved for functional reasons. Currently, the role of loop mobility in serpin function and the structure of the serpin-protease complex are controversial (15)(16)(17)(18)(19)(20)(21)28). In the late 1970s, it was reported that serpins were unlike other tight binding protease inhibitors and formed covalent ester linkages with enzymes (15). However, these conclusions were based on SDS-PAGE analysis of denatured complexes leaving the na...