Inhibition of Tissue Kallikrein by Protein C Inhibitor

We studied the inhibition of tissue kallikrein by protein C inhibitor (PCI), a relatively unspecific heparindependent serine protease inhibitor present in plasma and urine. PC1 inhibited the amidolytic activity (cleavage of H-D-vdyl-L-leucyl-arginine-p-nitroaniline) of urinary kallikrein with an apparent second order rate constant of 2.3 X lo4 M" s" and formed stable complexes (85 kDa) with urinary kallikrein as judged from silver-stained sodium dodecyl sulfate-polyacrylamide gels. Complex formation was time-dependent and was paralleled by a decrease in the intensity of the main PC1 protein band (M. = 57,000) and an increase in the intensity of the lower M, (54,000) PC1 form (cleaved inhibitor). Heparin interfered with the inhibition of tissue kallikrein by PC1 and with the formation of tissue kallikrein-PC1 complexes in a dose-dependent fashion and completely abolished PCI-tissue kallikrein interaction at 300 pg/ml. This is in contrast to findings on the interaction of PC1 with all other target proteases studied so far (i.e. stimulation of inhibition by heparin) but is similar to the reaction pattern of '261-labeled tissue kallikrein with so called kallikrein binding protein described in serum and other systems. To study a possible relationship between PC1 and this kallikrein binding protein we incubated '261-labeled urinary kallikrein in serum and in PCI-immunodepleted serum in the absence and presence of heparin and analyzed complex formation using sodium  dodecyl sulfate-polyacrylamide gel electrophoresis. In normal  serum, formed complexes co-migrated with complexes of purified PC1 and '261-kallikrein and were less intense in the presence of heparin. No complex formation at all was seen in PCI-depleted serum. Our data indicate that PC1 may be a physiologically important endogenous inhibitor of tissue kallikrein and provide evidence that PC1 may be identical to the previously described kallikrein binding protein.

We studied the inhibition of tissue kallikrein by protein C inhibitor (PCI), a relatively unspecific heparindependent serine protease inhibitor present in plasma and urine. PC1 inhibited the amidolytic activity (cleavage of H-D-vdyl-L-leucyl-arginine-p-nitroaniline) of urinary kallikrein with an apparent second order rate constant of 2.3 X lo4 M" s" and formed stable complexes (85 kDa) with urinary kallikrein as judged from silver-stained sodium dodecyl sulfate-polyacrylamide gels. Complex formation was time-dependent and was paralleled by a decrease in the intensity of the main PC1 protein band (M. = 57,000) and an increase in the intensity of the lower M, (54,000) PC1 form (cleaved inhibitor). Heparin interfered with the inhibition of tissue kallikrein by PC1 and with the formation of tissue kallikrein-PC1 complexes in a dose-dependent fashion and completely abolished PCI-tissue kallikrein interaction at 300 pg/ml. This is in contrast to findings on the interaction of PC1 with all other target proteases studied so far (i.e. stimulation of inhibition by heparin) but is similar to the reaction pattern of '261-labeled tissue kallikrein with so called kallikrein binding protein described in serum and other systems. To study a possible relationship between PC1 and this kallikrein binding protein we incubated '261-labeled urinary kallikrein in serum and in PCI-immunodepleted serum in the absence and presence of heparin and analyzed complex formation using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In normal serum, formed complexes co-migrated with complexes of purified PC1 and '261-kallikrein and were less intense in the presence of heparin. No complex formation at all was seen in PCI-depleted serum. Our data indicate that PC1 may be a physiologically important endogenous inhibitor of tissue kallikrein and provide evidence that PC1 may be identical to the previously described kallikrein binding protein.
Tissue/glandular kallikreins are trypsin-like serine proteases that have been isolated from several tissues, including kidney, salivary glands, and pancreas (1-6). They are also present in the exocrine secretions of the respective organs (2, * This work was supported in part by Grants P7579-M and P8108-M from the Austrian Funds for the Promotion of Scientific Research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.  6,7). The primary physiological function of tissue kallikreins seems to be kinin release from high and low M, kininogens by the cleavage of two peptide bonds within the kininogen molecules (2). Kinins have a variety of species and tissuespecific biological activities, including vasodilation and contraction of visceral smooth muscles (2). In addition to kinin generation, kallikrein may also be involved in renin release (8) and angiotensin I1 generation (9)(10)(11). Therefore, the regulation of kallikrein activity seems to be of major physiological importance. So far, no efficient endogenous inhibitor of tissue kallikrein has been identified. The only proteinase inhibitor exhibiting some inhibitory activity toward tissue kallikrein is al-protease inhibitor (12). However, the interaction of tissue kallikrein with al-protease inhibitor is slow, with a half-life of several hours (12,13). Recently, a tissue kallikrein binding protein has been described in several biological systems including serum (13)(14)(15). This binding protein interacts with tissue kallikrein by forming SDS'-stable complexes with a M, of 92,000. Complex formation is only observed with enzymatically active kallikrein (14) and is, at least in human systems, abolished in the presence of heparin (13)(14)(15). The identity of this kallikrein binding protein has not yet been defined. However, its reaction pattern suggests that it may be a serine protease inhibitor with a M, of approximately 50,000 and with affinity for heparin.
Protein C inhibitor (PCI; M , = 57,000) is a relatively nonspecific heparin-dependent serine protease inhibitor (serpin) that inhibits a variety of proteases (16)(17)(18). The presence of PC1 has been described in plasma (05 pg/ml) (16) and urine (19)(20)(21), and the urinary concentration is higher than expected from plasma filtration (0200 ng/ml, 21). So far, PC1 has mainly been studied with respect to its interaction with enzymes involved in coagulation and fibrinolysis (16)(17)(18)(19)(20)(21)(22)(23). It has been shown that PC1 inhibits activated protein C, thrombin, factor Xa, factor XIa, plasma kallikrein, and urokinase by forming SDS-stable complexes (16)(17)(18). The reaction of PC1 with its target proteases is stimulated by heparin (16)(17)(18) and other glycosaminoglycans (24). Although activated protein C-PC1 complexes have been shown in plasma samples from patients with disseminated intravascular coagulation (25) and urokinase-PC1 complexes in plasma samples from patients receiving exogenous urokinase therapy (26), the physiological role of PC1 is still unclear. The presence of PC1 in urine combined with the fact that urine is a major source of tissue kallikrein prompted us to investigate the possible inhibition of tissue kallikrein by PCI.

EXPERIMENTAL PROCEDURES
Materials-Sepharose 4B, activated CH-Sepharose 4B, and heparin-Sepharose CL-GB (Pharmacia, Sweden); CNBr (Merck, Germany); heparin sodium salt (~1 4 0 units/mg; Fluka AG, Switzerland); acrylamide, bisacrylamide, SDS, and calibration proteins for SDS-PAGE (Bio-Rad); H-D-Val-Leu-Arg-pNA (S-2266) and <Glu-Pro-Arg-pNA (S-2366) (Kabi, Sweden); and activated protein C (Technoclone GmbH, Austria) were obtained as indicated. A monoclonal antibody against protein C inhibitor (4PCI) was obtained by immunizing Balb/c mice with purified urinary PC1 (24). Immunization of mice, fusion of mouse spleen cells, and isolation of single anti-PC1 IgG secreting clones were performed as described previously (27), except that X63-Ag8.653 mouse myeloma cells were used as fusion partners. One clone producing anti PCI-IgG (4PCI) was selected and injected intraperitoneally into Pristan-primed Balb/c mice. Ascitic fluid was collected and 4PCI-IgG was isolated by ammonium sulfate precipitation (50% saturation) of the ascites fluid. The precipitate was dissolved in and dialyzed against 0.1 M NaHC03 and coupled to CNBr-activated Sepharose 4B according to the manufacturer's instructions at a concentration of 1 mg/ml Sepharose. Aprotinin was coupled to activated CH-Sepharose 4B as described (7). Urinary kallikrein was either purified as described previously (7) or obtained commercially from Protogen AG, Switzerland. ' ' ' 1 labeling of urinary kallikrein (Protogen AG) was performed by the Iodogen method (28). After radiolabeling, active lZ5I-kallikrein was purified by affinity chromatography on aprotinin-Sepharose (7). The '251-kallikrein used had a specific activity of 9.6 pCi/nmol. PCI-immunodepleted serum was prepared by passing whole blood serum (4 ml) over a 4PCI IgG-Sepharose 4B column (5 ml) equilibrated in 0.01 M Tris-HC1, 0.1 M NaCl, 0.01% Tween 80, pH 7.4. The first flow-through fraction (protein content 2 mg/ml) was used as PC1 immunodepleted serum. Control serum (protein content 2.5 mg/ml) was prepared in the same way but a Sepharose 4B column was used instead of 4PCI IgG-Sepharose. PCI-immunodepleted serum contained less than 1% of PC1 antigen present in control serum as determined by enzymelinked immunosorbent assay using 4PCI IgG as catching antibody and peroxidase-labelled immunopurified rabbit anti-PC1 IgG as detecting antibody (lower detection limit = 3 ng/ml).' Purification of Urinary Protein C Inhibitor-The PC1 purification protocol described previously (24) was modified in such a way that affinity chromatography on 4PCI-IgG Sepharose 4B was used as a first step and that affinity chromatography on anti-urokinase-IgG Sepharose and ion exchange chromatography on Q-Sepharose fast flow were omitted. Briefly, freshly voided human urine (21) was collected on benzamidine (10 mM final concentration) and aprotinin (100 KIU/ml final concentration), adjusted to a pH between 7.0 and 7.4 by addition of 1 M NaOH and incubated, and end-over-end rotated overnight with 4PCI-IgG-Sepharose 4B (40 ml settled volume) equilibrated in 0.01 M Tris-HC1, 1.0 M NaC1, 0.01% Tween 80, 10 mM benzamidine, 100 KIU/ml aprotinin, pH 7.4. Unbound material was separated from the Sepharose gel by means of a sintered glass filter. The 4PCI-IgG Sepharose was washed extensively with 0.01 M Tris-HC1, 1.0 M NaCl, 0.01% Tween 80, 10 mM benzamidine, 100 KIU/ml aprotinin, pH 9.0, on the same filter. Thereafter the gel suspension was filled into a column and washed with 0.01 M Tris-HC1, 1.0 M NaCl, 0.01% Tween 80, pH 9.0, until the Azso was below 0.01. Elution was performed with 0.05 M triethylamine buffer, pH 11.5, containing 0.5 M NaC1. Fractions (3-4 ml each) were collected into tubes containing 1 ml of 0.5 M Tris-HC1, pH 7.5, to neutralize pH immediately. Protein containing fractions (Azs0 2 0.03) eluted at pH 11.5 were pooled and stored at -70 "C until further use. Eluates obtained in this way from three to five 4PCI-IgG-Sepharose steps were pooled, dialyzed extensively against 0.05 M Tris-HC1, 0.2 M NaCl, 0.01% Tween 80, pH 7.4, and subjected to heparin-Sepharose CL-GB chromatography as described previously (24). Elution of the heparin-Sepharose column was performed with a linear gradient between 0.2 and 0.8 M NaCl in 0.05 M Tris-HC1, 0.01% Tween 80, pH 7.4, and fractions (2.5 ml) were collected. Fractions were screened for protein (Am) and inhibition of activated protein C (24). PC1 activity coeluted with a protein peak at 0.35 M NaC1. PCI-containing fractions were pooled and stored in aliquots at -70 "C until further use. PC1 purified by this procedure was homogeneous and migrated on silverstained SDS-PAGE gels as a doublet with a major band corresponding to M , of 57,000 and a minor band ( M , = 54,000). The yield of this purification procedure was =lo0 pg PCI/liter urine as compared with =35 pg PCI/liter urine obtained with the previously described purification protocol (24).
Analytical Methods-Molar concentrations of purified active kallikrein were evaluated from the cleavage of S-2266 using the change in absorbance at 405 nm/min and cm (AA,05/min/cm) of 2 . 7 5 / p~ enzyme as given by the manufacturer. The concentration of purified urinary PC1 was determined from its absorbance at 280 nm using a Ai;$1 of 14.1 (16), M, of 57,000 was used to calculate molar concentrations; SDS-PAGE (10% acrylamide) was performed according to Laemmli (29) using 1.5-mm slab gels. After electrophoresis gels were stained by the silver stain method (30). Each lane of the silver-stained gels was scanned densitometrically at 523 nm using a Hirschmann Elscript 400 densitometer and peaks corresponding to the stained protein bands were obtained. The relative intensity of each stained band, represented by the size of the area of the corresponding peak, was expressed as percent of the total intensity of all stained bands present in a lane. Gels containing lZ5I-kallikrein were fixed with acetic acid/methanol/water (10:50:40), dried, and exposed to Kodak X-Omat AR films. Amidolytic Assay for Inhibition of Tissue Kallikrein Actiuity by PCI-Inhibition of the amidolytic activity of purified urinary kallikrein by purified urinary PC1 was tested on 96-well microtiter plates. Bioassay for Kallikrein Actiuity-For control purposes, kinin generation by urinary kallikrein in the absence or presence of PC1 and in the absence or presence of heparin was tested using the rat uterus bioassay system (31). Urinary kallikrein (2.3 nM final concentration) was incubated at 37 "C with PC1 (230 nM final concentration) in the absence or presence of heparin (0.05-500 pg/ml final concentration) in a total volume of 30 pl of 0.01 M Tris-HC1, 0.1 M NaC1, 0.01% Tween 80, pH 7.4. After 2 h a 5-pl aliquot of this reaction mixture was transferred to 100 pl of heat-inactivated human plasma as a source of kininogens (31). After a 5-min incubation at 37 "C, the mixture was applied to the rat uterus bioassay system and kinin generated was quantified using known bradykinin standards.
Complex Formation of Urinary Kallikrein with PCI-Purified urinary kallikrein (7.7 nM final concentration) was incubated with purified urinary PC1 (200 nM final concentration) in the absence or presence of heparin (0.03-300 pg/ml final concentration) in a total volume of 70 pl of 0.01 M Tris-HC1,O.l M NaCl, 0.01% Tween 80, pH 7.4. Incubations were stopped at various times (0-4 h) by the addition of 35 p1 of 0.26 M Tris-HC1 buffer, pH 6.8, containing 2.5% SDS and 25% glycerol and heating the samples in a boiling water bath for 10 min. Thereafter the samples were applied to SDS-PAGE gels (10% acrylamide) prepared as described above.
Complex Formation of 1z51-Urino~ Kallikrein in Serum and PCIimmunodepleted Serum-"z61-Labeled urinary kallikrein (4.5 nM, 6000 cpm) was incubated with control serum (3 pl), with PCIimmunodepleted serum (3 pl), or with purified PC1 (270 nM final concentration) in the absence or presence of heparin (3 and 300 pg/ ml final concentrations) in a total volume of 63 pl 0.01 M Tris-HC1, 0.1 M NaC1, 0.01% Tween 80, pH 7.4. After 1 h the reactions were stopped by the addition of 40 pl 0.26 M Tris-HC1 buffer, pH 6.8, containing 2.5% SDS and 25% glycerol and heating the samples in a boiling water bath for 10 min. SDS-PAGE and autoradiography were done as described above. Fig. 1, purified urinary PC1 inhibited the amidolytic activity of urinary kallikrein and heparin decreased this inhibition in a dose-dependent manner. Furthermore, no concentration of heparin studied (0.03-300 pg/ml) had an effect on the amidolytic activity of kallikrein itself. In control experiments the effect of PC1 on the kinin-generating activity of urinary kallikrein was also tested using the rat uterus bioassay. In an assay system using 0.11 nM urinary kallikrein in 100 pl of heat-inactivated plasma as a source of kininogen, PC1 (10.9 nM final concentration) caused a decrease in kinin formed over 5 min from 10 to 5 ng/sample. When heparin was present in the preincubation mixture of PC1 and urinary kallikrein at concentrations 5 pg/ml, PC1 had no effect on kinin generation by urinary kallikrein.

As shown in
Since high concentrations of heparin completely abolished the interaction between PC1 and urinary kallikrein (Fig. l), the time course of kallikrein inhibition by PC1 was.analyzed using heparin (3 mg/ml final concentration) to stop the enzyme-inhibitor interactions at defined intervals. Fig. 2   The interaction of purified urinary PC1 and purified urinary kallikrein was analyzed using silver-stained SDS-PAGE gels. As can be seen from Fig. 3 (panel A) the time-dependent appearance of a new band (Mr = 85,000) was observed when purified urinary kallikrein (7.7 nM) and purified urinary PC1 (200 nM) were incubated together. As determined densitometrically, the maximal relative intensity of this new band, observed after incubation periods of 2 2 h, was about 6-7% of the total intensity of all stained bands. Complex formation was paralleled by a decrease in the relative intensity of the main (MI = 57,000) PC1 band and an increase in the relative intensity,of the MI = 54,000 PC1 band. The effect of heparin on kallikrein-PC1 complex formation was also studied. Fig. 3   (panel B ) shows the effect of increasing heparin concentrations on the interaction of PC1 with urinary kallikrein using an incubation period of 4 h. It can be seen from this figure that heparin inhibited in a dose-dependent way the formation of the 85-kDa band and diminished the generation of the 54-kDa PC1 band. 300 pg/ml heparin completely abolished the PCI-kallikrein interaction as judged from the fact that there was no 85-kDa band present and that the relative intensities of the 57-(81.5%) and the 54-kDa bands (18.5%) were comparable with those seen in the original PC1 preparation (Fig.  3A, inset, lane 1 ).
We analyzed a possible relationship of PC1 and the kallikrein binding protein present in serum (13-15) by incubating lZ5I-labeled tissue kallikrein with serum and with PCI-immunodepleted serum in the absence and presence of heparin. As can be seen from Fig. 4, lZ6I-kallikrein formed a complex with a component present in serum; this complex co-migrated with the complex formed between '2SI-kallikrein and purified PCI, and its intensity was decreased by the presence of heparin. No 'z51-kallikrein complex formation was observed in PCI-immunodepleted serum.

DISCUSSION
PC1 is a relatively nonspecific heparin-binding serine protease inhibitor (16)(17)(18). It inhibits not only activated protein C, but also several other enzymes involved in coagulation (thrombin, factor Xa, factor XIa, plasma kallikrein) and fibrinolysis (urokinase) (16-18, 23). Its inhibitory activity is  lanes 1-4) or presence of heparin (lanes 5-10). Lanes 5, 7, and 9 contained 300 pg/ml heparin (final concentration) lanes 6,8, and 10 contained 3 pg/ml heparin (final concentrations). SDS-PAGE and autoradiography were performed as described under "Experimental Procedures." stimulated by heparin (16)(17)(18) and other glycosaminoglycans (24). The presence of naturally occurring stimulatory glycosaminoglycans might therefore play a crucial role for the in uiuo regulation of PC1 activity and possibly also enzyme specificity. However, so far the physiological role of PC1 is still unclear. PC1 is present in urine in a surprisingly high concentration and could therefore be an important protease inhibitor in the urinary tract. We have shown that PC1 is also present in saliva and intestinal fluid (32). Laurel1 et al. (33) have demonstrated recently that very high concentrations of PC1 immunoreactivity can be found in seminal plasma, Graaf follicle fluid, and synovial fluid. These findings may indicate a more general role for this inhibitor in secretions. These secretions are also major sources of tissue kallikrein, an enzyme for which no efficient endogenous serpin-type inhibitor has been described. We therefore investigated a possible inhibition of tissue kallikrein by PCI. In fact, using purified urinary kallikrein and purified urinary PCI, we were able to show that PC1 inhibited kallikrein activity in a dose-and time-dependent fashion; furthermore, this interaction was accompanied by the formation of SDSstable complexes (Mr = 85,000). As judged from densitometrically scanned silver-stained gels, the maximal amount of complex formed was somewhat higher (6-7% of total PCI) than expected using a %-fold excess of PC1 over kallikrein and assuming 1:l complex formation. However, estimation of the amount of protein present in a silver-stained band is difficult, since the intensity of staining is not necessarily the same for different proteins (30).
Complex formation of PC1 with urinary kallikrein was paralleled by the degradation of PC1 to a lower M , form. This is not uncommon for a serpin-type inhibitor, and cleavage of PC1 in the reactive site region by activated protein C and other target enzymes has already been described (16,17,34). PC1 preparations used as shown in Fig. 3A, indicating that PC1 might have been cleaved either in vivo or during the purification procedure. Kinetics for the inhibition of urinary kallikrein by PC1 were determined using a high heparin concentration to stop the enzyme-inhibitor interaction after defined intervals, assuming that heparin has no effect on the stability of the kallikrein-PC1 complex. Although the apparent second order rate constant ( k J calculated under these conditions for the inhibition of urinary kallikrein by PC1 (2.3 X lo4 M-' s-') does not seem to be very high, it is in the same order of magnitude (18) or higher (24) than the kl calculated for the reaction of PC1 with its hitherto known target proteases. This indicates that PC1 may, in fact, be a physiologically important inhibitor of tissue kallikrein.
Analysis of the effect of heparin on the PCI-tissue kallikrein interaction revealed an inhibitory effect of heparin. This is in contrast to findings on the interaction of PC1 with all other target proteases studied so far, where heparin enhanced the enzyme-inhibitor interaction. Such a reaction pattern, however, has been described by the group of Chao for the interaction of tissue kallikrein with the so called kallikrein binding protein (13-15); this peculiar heparin effect even led those authors to exclude PC1 as a possible candidate to be the kallikrein binding protein (13). Observing such a heparin effect during the interaction of PC1 with kallikrein, we, however, were prompted to investigate the possible identity of PC1 with this binding protein. Data obtained show that when '261-kallikrein was incubated either with serum or with purified PCI, the formed complexes displayed identical mobilities on SDS-PAGE. Furthermore, the fact that serum depleted of PC1 by monoclonal antibody affinity chromatography no longer reacted with '251-kallikrein to form detectable complexes strongly supports the concept that PC1 is the kallikrein binding protein.