Thrombotic thrombocytopenic purpura is associated with acquired or congenital deficiency of a plasma von Willebrand factor-cleaving protease (VWFCP). Based on partial amino acid sequence, VWFCP was identified recently as a new member of the ADAMTS family of metalloproteases and designated ADAMTS13. The 4.6-kilobase pair cDNA sequence for VWFCP has now been determined. By Northern blotting, full-length VWFCP mRNA was detected only in liver. VWFCP consists of 1427 amino acid residues and has a signal peptide, a short propeptide terminating in the sequence RQRR, a reprolysin-like metalloprotease domain, a disintegrinlike domain, a thrombospondin-1 repeat, a Cys-rich domain, an ADAMTS spacer, seven additional thrombospondin-1 repeats, and two CUB domains. VWFCP apparently is made as a zymogen that requires proteolytic activation, possibly by furin intracellularly. Sites for Zn 2؉ 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, 2). TTP often strikes young adults, mainly females, suggesting an autoimmune etiology. If untreated, the mortality may exceed 90% (2), but plasma exchange therapy has reduced the mortality to less than 20% (3).The basis for the efficacy of plasma exchange remains unknown, although a plausible model has been suggested in which the proteolysis of von Willebrand factor (VWF) plays a central role. In 1982, patients with chronic relapsing TTP were reported to have "unusually large" VWF multimers (UL-VWF) because of the absence of a VWF depolymerase activity. Binding of UL-VWF to platelets could promote microvascular thrombosis, platelet consumption, and hemolysis. Therapeutic infusion of plasma was proposed to replace the missing depolymerase activity or other factors and thereby limit VWF-dependent platelet thrombosis (4). Later discoveries have strengthened the relationship between VWF proteolysis and TTP. A plasma metalloprotease was identified that requires both calcium and zinc ions and cleaves the Tyr 1605 -Met 1606 bond 2 in the central A2 domain of the VWF subunit (5, 6). Cleavage was stimulated by shear forces like those occurring at sites of arterial thrombosis or by low concentrations of urea or guanidine. Furthermore, most adult patients with TTP were found to have congenital deficiency or an acquired autoantibody inhibitor of this VWF-cleaving protease (VWFCP) (7-9). These findings are consistent with the UL-VWF model of TTP pathogenesis. Alternatively, it is possible that failure to cleave another unknown substrate contributes to the pathogenesis of TTP.VWFCP was recently purified from human plasma sources, and an N-terminal amino acid sequence was obtained (10,11). This information showed it to be a member of the ADAMTS family of metalloproteases, named for the characteristic combination of a disintegrin-like and metalloprotease (reprolysintype), with thrombospondin type 1 motif (12-14). Furthermore, the VWFCP gene wa...

Angiomyolipomata (AML) belong to a family of tumors known as perivascular epithelioid cell tumors (PEComas) that share a common immunophenotypic profile of muscle and melanocytic differentiation. These tumors are clonal in nature and have a strong association with tuberous sclerosis. Genetic analyses have reported allelic imbalance at the TSC2 locus on 16p13. In the context of non-TSC, non-LAM associated AMLs and non-renal PEComas, the functional status of the TSC2 signaling pathway has not been reported. Studies over the last several years have uncovered a critical role of the TSC1/2 genes in negatively regulating the Rheb/mTOR/p70S6K cascade. Here, we examined the activity of this pathway in sporadic AMLs and PEComas using immunohistochemical and biochemical analyses. We found increased levels of phospho-p70S6K, a marker of mTOR activity, in 15 of 15 non-TSC AMLs. This was accompanied by reduced phospho-AKT expression, a pattern that is consistent with the disruption of TSC1/2 function. Western blot analysis confirmed mTOR activation concurrent with the loss of TSC2 and not TSC1 in sporadic AMLs. Similarly, elevated phospho-p70S6K and reduced phospho-AKT expression was detected in 14/15 cases of extra-renal PEComas. These observations provide the first functional evidence that mTOR activation is common to sporadic, non-TSC-related AMLs and PEComas. This suggests the possibility that mTOR inhibitors such as rapamycin may be therapeutic for this class of disease.

hK4 (prostase, KLK4), a recently cloned prostate-specific serine protease and a member of the tissue kallikrein family, is a zymogen composed of 228 amino acid residues including an amino-terminal propiece, Ser-Cys-Ser-Gln-. A chimeric form of hK4 (ch-hK4) was constructed in which the propiece of hK4 was replaced by that of prostate-specific antigen (PSA) to create an activation site susceptible to trypsin-type proteases. ch-hK4 was expressed in Escherichia coli, isolated from inclusion bodies, refolded, and purified with an overall yield of 25%. The zymogen was readily self-activated during the refolding process to generate an active form (21 kDa) of hK4 (rhK4). rhK4 cleaved the chromogenic substrates Val-Leu-Arg-pNA (S-2266), Pro-Phe-Arg-pNA (S-2302), Ile-Glu-Gly-Arg-pNA (S-2222), and Val-Leu-Lys-pNA (S-2251), indicating that rhK4 has a trypsin-type substrate specificity. The rhK4 was inhibited by aprotinin (6 kDa), forming an equimolar 27 kDa complex. rhK4 readily activated both the precursor of PSA (pro-PSA) and single chain urokinase-type plasminogen activator (scuPA, pro-uPA). rhK4 also completely degraded prostatic acid phosphatase but failed to cleave serum albumin, another protein purified from human seminal plasma. These results indicate that hK4 may have a role in the physiologic processing of seminal plasma proteins such as pro-PSA, as well as in the pathogenesis of prostate cancer through its activation of pro-uPA.

Human kallikrein-related peptidase 4 (KLK4/prostase), a trypsin-like serine protease, is a potential target for prostate cancer treatment because of its proteolytic ability to activate many tumorigenic and metastatic pathways including the protease activated receptors (PARs). Currently there are no KLK4-specific small-molecule inhibitors available for therapeutic development. Here we re-engineer the naturally occurring sunflower trypsin inhibitor to selectively block the proteolytic activity of KLK4 and prevent stimulation of PAR activity in a cell-based system. The re-engineered inhibitor was designed using a combination of molecular modeling and sparse matrix substrate screening.