Human Mast Cell Tryptase Activates Single-chain Urinary-type Plasminogen Activator ( Pro-urokinase ) *

Human lung mast cell tryptase is a trypsin-like serine proteinase that is stored in mast cell granules and released by activated mast cells. Here we report that mast cell tryptase is a potent activator of single-chain urinary-type  plasminogen  activator  (scu-PA, or prourokinase), the zymogen form of urinary-type plasminogen activator (u-PA). Activation was complete within 75 min using an enzyme:substrate molar ratio of 1:50 and was accompanied by cleavage of scu-PA at Lys1S8-Ile1Se, generating active two-chain u-PA. The reaction was dependent on enzyme concentration and obeyed Michaelis-Menten kinetics. Kinetic constants calculated for scu-PA activation by mast cell tryptase are K , = 34 p ~ , V, = 3.6 pmol of u-PNmin, and kc,, = 0.08 s-l. These data suggest that tryptase from tumor-associated mast cells may participate in the activation of scu-PA.

responsible for physiologic fibrinolysis. Alternatively, u-PA is synthesized by several cell types including kidney cells and numerous neoplastic cells (Dano et al., 1985). In contrast to t-PA, u-PA is active both in solution phase and when bound to its specific cell surface receptor, indicating that this activator is associated with processes involving cell migration, extracellular matrix degradation, and tissue invasion (Dano et al., 1985).
u-PA is secreted as a single-chain zymogen form (prou-PA, single-chain u-PA, or scu-PA) with a molecular mass of 55 kDa (Nolan et al., 1977;Skriver et al., 1982;Wun et al., 1982). The zymogen has low intrinsic activity against synthetic substrates and can catalyze conversion of plasminogen to plasmin at a significantly reduced rate (Gurewich et al., 1984;Lijnen et al., 1990;Ellis et al., 1989). The resulting plasmin then rapidly converts scu-PA t o the fully active two chain form (tcu-PA) by cleavage at the L y~'~~-I l e '~~ bond, leading to enhanced activation of plasminogen by tcu-PA (Lijnen et al., 1987a). In addition to plasmin, activation of scu-PA by kallikrein, trypsin, T cellassociated serine proteinase, and the thiol proteinase cathepsin B has been reported (Ichinose et al., 1986;Brunner et al., 1990;Kobayashi et al., 1991). In contrast, thrombin cleavage of scu-PA at an alternative site (A~-gl~~-Phe'~~) generates an inactive tcu-PA derivative with greatly reduced amidolytic and plasminogen-activating activity (Ichinose et al., 1986;Gurewich and Pannell, 1987).
Tryptases are trypsin-like serine proteinases found in the cytoplasmic granules of mammalian mast cells and are stored and released as active enzymes (Glenner and Cohen, 1960;Alter et al., 1987). All human mast cells contain tryptaseb), accounting for 23% of the total cellular protein (Irani et al., 1986;Schwartz et al., 1981a). Tryptases cleave peptide substrates on the carboxyl side of Lys and Arg residues, but differ from trypsin in that they have little or no activity on denatured proteins such as casein. Human tryptase activity is stabilized by heparin, an additional component of mast cell granules Schwartz and Bradford, 1986;Alter et al., 1987). Upon mast cell degranulation, tryptase is released along with other mediators, such as histamine, into the extracellular milieu (Schwartz et al., 1981b). There are no known inhibitors of tryptase in the human , and two insertion loops in tryptase, relative to trypsin, are thought to protect tryptase from blood plasma inhibitors such as cy1proteinase inhibitor (Johnson and Barton, 1992). Additionally, tryptase may remain near the site of release from mast cells because it binds heparin and forms tetramers (Schwartz et al., 1981a;Smith et al., 1984).
Although the physiologic function of tryptase remains unclear, tryptase has been found to degrade fibronectin, a component of the pericellular matrix that must be modified for cell migration (Lohi et al., 1992). In addition, increased levels of a tryptase-like enzyme have been found in rat mammary tumors, suggesting a role in tumor invasion (Eto and Grubbs, 1992). Mast cells have long been known to be associated with tumors (Folkman and Shing, 1992) and mast cell-deficient mice injected with tumor cells display a significantly reduced angiogenic response (Crowle and Starkey, 1986). Together these data suggest that tryptase may participate in the proteolysis associated with tumor invasion andor angiogenesis. As scu-PA is also found in association with numerous tumor cells, we have investigated the ability of tryptase to catalyze cleavage of scu-PA to tcu-PA. Here we report that mast cell tryptase is an efficient activator of scu-PA, suggesting a mechanism by which mast cells may initiate plasmin-dependent proteolysis.
EXPERIMENTAL PROCEDURES MuterialsSingle-chain u-PA and two-chain uPA were purchased from American Diagnostica, Greenwich, CT. scu-PA was purchased in 100-pg aliquots and diluted to a final concentration of 1 mg/ml. Molar concentrations were determined based on a molecular weight of 55,000 (Nolan et al., 1977).
Proteins-'Ikyptase was purified from human lung tissue obtained a t autopsy as previously described with minor modifications . Changes included the use of cetyl pyridinium chloride to reduce the viscosity of the extract and Toyopearl Butyl 650" hydrophobic chromatography media was substituted for octyl-Sepharose. Tryptase used in these studies corresponded to the major isoform present in preparations from lung (Little, 1993). Tryptase concentration was determined based on the 280-nm extinction coefficient of 28.1 for a 1% solution as reported by Smith et al. (1984). Plasminogen was purified from pooled human plasma by affinity chromatography as previously described (Deutsch and Mertz, 1970;Gonzalez-Gronow and Robbins, 1984).
Activation of scu-PA by lFyptuse-Because tryptase efficiently cleaves all synthetic peptide substrates normally used to monitor activation of scu-PA (data not shown), conversion of scu-PA to tcu-PA was analyzed by electrophoresis followed by densitometric scanning to quantitate reaction products. Tryptase was incubated with scu-PA at 37 "C in 100 mM Hepes, 0.05% Brij 35, pH 7.5, and the reaction was stopped by the addition of Laemmli sample dilution buffer and boiling (Laemmli, 1970). Samples were electrophoresed on 11% SDS-polyacrylamide gels under reducing conditions, gels were stained with Coomassie Blue R250, reaction products were quantitated by densitometry using an Hoefer GS300 scanning densitometer, and peak areas were integrated using Hoefer GS370 software. Amino-terminal sequence analysis of the reaction products was determined using an Applied Biosystems 477A pulse liquid phase sequenator with "on-line" phenylthiohydantoin analysis. Reaction products were separated by SDS-polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membranes (Immobilon), stained with Coomassie Blue, and the bands of interest were excised, placed on Porton sample support disks, and sequenced (Matsudaira, 1987). The net yields of amino acids were calculated by subtraction of the amount of phenylthiohydantoin derivatives in each cycle from the background in the previous cycle.

RESULTS
Incubation of scu-PA with catalytic amounts of tryptase resulted in time-dependent cleavage of the proenzyme (Fig. lA ), with 50% conversion occurring within approximately 30-40 min (Fig. 1B). The molecular weights of the reaction products were consistent with the generation of characteristic A ( M , = 22,000) and B (M, = 33,000) chains of the active form of u-PA (Skriver et al., 1982). Amino-terminal sequence analysis of the B chain indicated a sequence of Ile-Ile-Gly-Gly-Glu-Phe-Thr-Thr-Ile-Glu-Asn-Gln, corresponding to amino acids 159-170 of u-PA (Holmes et al., 1985). The resulting active tcu-PA catalyzed the conversion of plasminogen to two-chain plasmin (data not shown). Under identical conditions, no direct activation of plasminogen by tryptase was observed (data not shown). At low concentrations of tryptase (greater than 25-fold molar excess of the substrate scu-PA), scu-PA activation was a linear function of tryptase concentration (Fig. 2, A and B ) . The kinetics of scu-PA activation were determined by incubating increasing concentrations of scu-PA (2.5-17.5 PM) with tryptase (0.04 w) (Fig. 3A). The Michaelis-Menten plot shows that the activation reaction is concentration dependent and saturable (Fig. 3B). Analysis of the data using a double reciprocal plot to determine kinetic constants (Fig. 3C) gave a K, of 34 and a V,, of 3.6 pmol of tcu-PA generatedmin. The catalytic rate constant for the reaction (kcat) was 0.08 SI.

DISCUSSION
Previous studies have shown that cleavage of scu-PA at the L y~'~~-I l e '~~ bond by serine proteinases including plasmin and kallikrein generates the proteolytically active two-chain form of the molecule, which is a potent plasminogen activator (Lijnen et al., 1987b;Ichinose et al., 1986). The identical activation cleavage is also catalyzed by the thiol proteinase cathepsin B (Kobayashi et al., 1991). Alternatively, cleavage a t A~-g '~~-P h e '~~ by thrombin generates a catalytically inactive tcu-PA derivative (Ichinose et al., 1986;Gurewich and Pannell, 1987). The present study demonstrates that activation of scu-PA can also be initiated by human mast cell tryptase. Comparison of the kinetic constants for scu-PA activation by tryptase and plasmin indicates a decreased catalytic efficiency for the tryptasecatalyzed reaction, resulting from both an increase in K, and a decrease in kc,, (tryptase K, = 34 w, kc,, = 0.08 s-'; plasmin K, = 7.1 PM, kc,, = 1.3 s-'1 (Lijnen et al., 1987a). Although no kinetic data are available for the kallikrein-catalyzed reaction, the activation follows a similar time course as observed for tryptase, with activation complete within 2 h (using an enzyme: substrate ratio of 1:30 (w/w)) (Ichinose et al., 1986).
Many studies have demonstrated an increase in mast cells, both intact and degranulated, in the vicinity of tumors (Crowle and Starkey, 1989). While mast cell degranulation resulting from antigen binding by cell surface-IgE is well recognized, other factors such as hypoxia, neurotransmitters, opiates, factors derived from other cells, and even physical stimuli can serve as mast cell secretagogues (Friedman and Kaliner, 1987;Wasserman, 1990). Thus, it is likely that tumor-associated mast cells release tryptase in response to various stimuli. Plasminogen activators, particularly u-PA, have also been found in association with numerous invasive and metastatic cells and in a variety of tumor tissues (Dano et al., 1985). In addition, recent data indicate that scu-PA is the predominant molecular form in cultures of adherent tumor cells (Tapiovaara et al., 1993). The present data demonstrate that human mast cell tryptase is a potent activator of scu-PA. In contrast to other activators of scu-PA such as plasmin, kallikrein, and cathepsin B, which are rapidly inactivated by endogenous proteinase inhibitors, tryptase is remarkably resistant to inhibition by plasma and other proteinaceous inhibitors . Although the significance of our findings relative to other activation pathways will require additional study, the possibility that tryptase retains activity in the extracellular milieu, coupled with its ability to activate scu-PA, suggests a previously undescribed role for tryptase in the initiation and/or amplification of tumor-associated proteolytic activity.