©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Hepsin, a Putative Membrane-associated Serine Protease, Activates Human Factor VII and Initiates a Pathway of Blood Coagulation on the Cell Surface Leading to Thrombin Formation (*)

(Received for publication, February 28, 1994; and in revised form, September 27, 1994)

Yoshiaki Kazama (1) Takayoshi Hamamoto (1) Donald C. Foster (2) Walter Kisiel (1)(§)

From the  (1)Department of Pathology, University of New Mexico School of Medicine, Albuquerque, New Mexico 87131 and (2)ZymoGenetics, Inc., Seattle, Washington 98102

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Previous studies have shown that hepsin is a putative membrane-associated serine protease that is required for cell growth (Torres-Rosado, A., O'Shea, K. S., Tsuji, A., Chou, S.-H., and Kurachi, K.(1993) Proc. Natl. Acad. Sci. U. S. A. 90, 7181 7185). In the present study, we have transfected baby hamster kidney (BHK) cells with a plasmid containing the cDNA for human hepsin and examined these cells for their ability to activate several blood coagulation factors including factors X, IX, VII, prothrombin, and protein C. Little, if any, proteolytic activation of factors X, IX, prothrombin, or protein C was observed when these clotting factors were incubated with hepsin-transfected cells. On the other hand, hepsin-transfected cells proteolytically activated significant concentrations of human factor VII in a time- and calcium-dependent manner, whereas essentially no activation of factor VII was observed in BHK cells transfected with plasmid lacking the cDNA for hepsin. The factor VII activating activity in the hepsin-transfected BHK cell line was confined exclusively to the total membrane fraction and was inhibited >95% by antibody raised against a fusion protein consisting of maltose-binding protein and the extracellular domain of human hepsin. An active site factor VII mutant, S344A factor VII, was cleaved as readily as plasma-derived factor VII by hepsin-transfected cells, indicating that factor VII was not converted to factor VIIa autocatalytically on the cell surface. In contrast, an activation cleavage site factor VII mutant, R152E factor VII, was not cleaved by hepsin-transfected cells, suggesting that factor VII and S344A factor VII were activated on these cells by cleavage of the Arg-Ile peptide bond. In the copresence of factor VII and factor X, hepsin-transfected BHK cells supported the formation of factor Xa. In addition, in the copresence of factor VII, factor X, and prothrombin, hepsin-transfected BHK cells supported the formation of thrombin. These results strongly suggest that membrane-associated hepsin converts zymogen factor VII to factor VIIa, which in turn, is capable of initiating a coagulation pathway on the cell surface that ultimately leads to thrombin formation.


INTRODUCTION

Considerable clinical and experimental evidence support the view that a variety of neoplastic cells activate the blood coagulation system, leading to hypercoagulability and intravascular thrombosis(1) . Although the mechanism whereby tumor cells promote blood coagulation is poorly understood, direct activation of the extrinsic pathway of blood coagulation is widely believed to be instrumental in fibrin formation around neoplastic cells. In this regard, several tumor cells synthesize and express cell-surface tissue factor(2) , which serves as a cell-surface receptor and cofactor for factor VIIa. The factor VIIa-tissue factor complex then rapidly activates factors IX and X by limited proteolysis(3, 4, 5, 6) . Precisely how factor VII is activated in the tumor microenvironment is unclear. In addition, several tumor cells generate thrombin and fibrin without detectable cell-surface tissue factor, and precisely how this happens is unknown although cancer procoagulant A synthesis by these cells has been implicated in this mechanism(7) .

Cell-surface proteases of normal and malignant cells are generally thought to play roles in cell growth, chemotaxis, endocytosis, exocytosis, blood coagulation, fibrinolysis, and tissue invasion during metastasis(8) . Several plasma membrane-associated proteases have been purified and include endopeptidases, metalloproteases, neutral serine proteases, carboxypeptidases, aminopeptidases, and dipeptidases(8) . A novel zymogen of a trypsin-like serine protease, designated as hepsin, was recently identified by screening a human liver cDNA library with a synthetic oligonucleotide probe coding for a highly conserved amino acid sequence found in the serine protease family(9) . Hepsin (51 kDa) is composed of an amino-terminal transmembrane domain followed by a potential catalytic domain featuring the active site triad residues of His, Asp, and Ser that participate in enzyme catalysis. It is currently assumed that the transmembrane domain anchors hepsin to the cell membrane and that the carboxyl-terminal catalytic domain is extracellular. Hepsin is expressed in high levels in liver tissue but is also expressed at lower levels in other tissues including kidney, pancreas, lung, thyroid, pituitary gland, and testis(10) . Western blot analyses of solubilized fractions of a human hepatocellular carcinoma cell line (HepG2) indicated that hepsin is located on the membrane but not in the cytosol or conditioned media of these cells(10) . Although the precise biological role of hepsin is not clear, recent studies by Torres-Rosado and co-workers (11) indicate that hepsin may play a significant role in cell growth and maintenance of cell morphology. In the present study, we have transfected BHK (^1)cells with a plasmid containing the cDNA for human hepsin. We demonstrate herein that the hepsin-transfected BHK cells activate human factor VII by limited proteolytic cleavage of the Arg-Ile peptide bond. Furthermore, in the presence of plasma levels of factor X and prothrombin, newly formed factor VIIa can initiate a pathway that culminates in thrombin formation. Our results suggest that hepsin may be involved in the formation of thrombin on tumor cells that lack demonstrable immunoreactive tissue factor.


EXPERIMENTAL PROCEDURES

Materials

H-D-Phe-Pip-Arg-p-nitroanilide (S-2238), Bz-Ile-Glu-Gly-Arg-^p-nitroanilide (S-2222), and H-D-Ile-Pro-Arg-p-nitroanilide (S-2288) were obtained from Helena Laboratories. Glutamyl-glycyl-arginyl-chloromethyl ketone (EGRck) was obtained from Calbiochem. Bovine serum albumin, aprotinin, penicillin-streptomycin, methotrexate, heparin (grade 1), trypsin-EDTA solution, ampicillin, 4-amidinophenylmethylsulfonyl fluoride, and normal goat IgG were obtained from Sigma. Nitrocellulose membranes were purchased from Schleicher and Schuell. Protein dye reagent concentrate was obtained from Bio-Rad. Tissue culture T-75 flasks and 12-well plates were obtained from Corning. Dulbecco's modified Eagle's medium was a product of Mediatech. Fetal bovine serum was obtained from Hyclone Laboratories. I-Labeled protein A was purchased from DuPont NEN. N-Tosyl-L-phenylalanine chloromethyl ketone (TPCK)-treated bovine trypsin and soybean trypsin inhibitor were obtained from Worthington. Epicurian Coli® XL1-Blue supercompetent cells and Bsh1236I restriction endonuclease were from Stratagene. Amylose resin, pMAL-c2 vector, anti-MBP antiserum, XmnI, and EcoRI were products of New England Biolabs. Calf intestinal alkaline phosphatase was obtained from Boehringer Mannheim. Hamster citrated plasma and hamster brain tissue were obtained from Harlan Bioproducts for Science. Isopropyl-thiogalactopyranoside (IPTG) was a product of United States Biochemical Corp. The GENECLEAN(TM) kit was obtained from BIO 101 Inc. Protein A-Sepharose was purchased from Pharmacia Biotech Inc. All other reagents were the highest grade commercially available.

Proteins

-Human plasma-derived factor VII(12), factor X (12) , factor Xa(12) , factor IX(12) , protein C(13) , prothrombin(14) , thrombin(15) , prothrombin fragment 1(16) , factor VII gla-peptide (17) , and antithrombin III (18) were purified to homogeneity essentially as described. Recombinant preparations of human factor VIIa (19) , S344A factor VII(20) , R152E factor VII(21) , gla-domainless factor VII(22) , and full-length tissue factor pathway inhibitor (23) were prepared by published methods. A carboxyl-terminal truncated human tissue factor apoprotein consisting of the 219-amino-acid extracellular domain (TF), produced in human 293 cells(24) , was generously provided by Dr. Gordon Vehar, Genentech, Inc., South San Francisco. Factor X (1.1 mg/ml) and prothrombin (1.0 mg/ml) preparations contained no detectable factor VII antigen as determined by a specific enzyme-linked immunosorbent assay (limit of detection 0.1 ng/ml). Prior to use, factor VII and factor X preparations were treated with a 500-fold molar excess of EGRck for 2 h at 37 °C followed by extensive dialysis at 4 °C against Tris-buffered saline (TBS). Prothrombin preparations were also treated with a 50-fold molar excess of 4-amidinophenylmethylsulfonyl fluoride and dialyzed against TBS (50 mM Tris-HCl (pH 7.5) containing 100 mM NaCl) prior to use. Human brain cephalin (mixed phosphopholipids) (25) and factor VII-deficient plasma (<1% factor VII antigen) (26) were prepared as described. Hamster brain tissue factor (thromboplastin) was prepared from acetone-dehydrated hamster brain powder by saline extraction at 52 °C according to Quick(27) . Goat anti-rabbit tissue factor IgG was purified by DEAE-AffiGel Blue chromatography and kindly provided by Dr. L. V. M. Rao, University of Texas/Tyler. Protein concentrations were determined by the Coomassie Blue dye binding assay (28) calibrated with bovine serum albumin.

Construction, Expression, and Purification of a Maltose-binding Protein/Hepsin Fusion Protein

A mal E::hepsin gene fusion was constructed as follows. Hepsin cDNA was cleaved with Bsh1236I to generate a 1-kilobase DNA fragment that was purified by gel electrophoresis(29) . This fragment, encoding hepsin residues 80-417, was ligated into vector pMAL-c2 that had previously been cleaved with XmnI and EcoRI, and subsequently treated with calf intestinal phosphatase prior to ligation (30) . Epicurian coli® XL-1 Blue supercompetent cells were transformed with 50 ng of pMAL-c2/hepsin DNA essentially as outlined by the manufacturer, and 5 µl of the transformation mixture spread on Luria-Bertani (LB)/ampicillin plates. Individual colonies were grown in LB/ampicillin medium and examined for fusion protein synthesis by SDS-PAGE (31) following induction with 0.3 mM IPTG. Cells containing plasmids that coded for a MBP/hepsin fusion protein were grown to 5 times 10^8 cells/ml at 37 °C with shaking in LB/glucose/ampicillin for 5 h. IPTG was then added to a final concentration of 0.3 mM, and the culture was incubated with vigorous shaking at 30 °C for an additional 5 h. The cells were harvested by low speed centrifugation, resuspended in 10 mM Tris-HCl, pH 7.5, 200 mM NaCl/1 mM EDTA (column buffer), and lysed by sonication. Cell debris was removed by high speed centrifugation and the supernatant applied directly to a column of amylose resin (1.6 times 8 cm) previously equilibrated at 4 °C with column buffer. Following sample application and wash, the MBP/hepsin fusion protein was eluted from the affinity resin in column buffer containing 10 mM maltose. Examination of the maltose eluent by SDS-PAGE indicated a prominent 84 kDa band (fusion protein) and two minor bands with apparent molecular masses of 67 and 43 kDa that presumably represent degradation products of the fusion protein and endogenously synthesized maltose-binding protein, respectively(32) . Antibodies against the MBP/hepsin fusion protein were generated in rabbits (33) and the IgG fraction purified by protein A-Sepharose column chromatography. Immunoblot analysis of a total membrane fraction derived from Hepsin-1/BHK cells indicated that the rabbit anti-MBP/hepsin IgG recognized a major protein with an apparent M(r) of 55,000 and two minor proteins with M(r) values of 48,000 and 33,000.

Expression Vector

A full-length human hepsin cDNA called HepG2UW7 (9) was ligated into a mammalian cell expression vector called Zem229R(34) . This vector permits insertion of EcoRI or BamHI fragments into a cloning site downstream of the mouse metallothionine promoter and upstream of the SV-40 polyadenylation sequence and also carries an expression unit for the dihydrofolate reductase gene under control of the SV-40 early promoter.

Cell Culture, Transfection, and Screening

BHK cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum. Subconfluent cells were transfected with the hepsin expression plasmid by the calcium phosphate-mediated transfection procedure(35) . Two days post-transfection, cells were treated with trypsin and diluted into selective medium containing 1 µM methotrexate. Resistant colonies were selected at random and cultured individually. Two colonies (Hepsin-1/BHK and Hepsin-4/BHK) was chosen for examination and cultured in T-75 flasks or 12-well (3.8 cm^2) plates containing DMEM supplemented with 10% fetal bovine serum, penicillin-streptomycin, and 200 nM methotrexate (DMEM/FCS/PS/MTX). After reaching confluence in 12-well plates, cells were washed twice with serum-free DMEM/PS/MTX, and cultured in serum-free DMEM/PS/MTX for 1 day prior to activation studies. BHK cells transfected with ``empty'' Zem229R expression plasmid (hepsin-free) served as control cells in this study and were cultured in T-75 flasks and 12-well plates in DMEM/FCS/PS/MTX followed by 1-day incubation with serum-free DMEM/PS/MTX prior to activation studies.

Polymerase Chain Reaction

Untransfected BHK cells and Hepsin-1/BHK cells were grown in culture for RNA isolation. Total RNA was isolated from both cell types by the method of Chirgwin et al.(36) and poly(A) mRNA was prepared on oligo(dT)-cellulose. First strand cDNA was prepared from each mRNA preparation using reverse transcriptase primed with oligo(dT) primer. Twenty ng of each cDNA was used as template in polymerase chain reactions containing two 24-nucleotide primers with perfect complementarity to the human hepsin cDNA sequence (sense primer = GTG GCA GCT CTC ACT GCG GGG ACC; antisense primer = GAG CAG GGA TCC CCC ACA GAG GTG). In order to minimize the possibility of cross-priming non-human hepsin sequences, the polymerase chain reactions were carried out under ``hot start'' conditions in which the polymerase was added only after the reactions had reached the first cycle annealing temperature (72 °C).

Analysis of Factor VII Activation by Hepsin-1/BHK Cells

Confluent monolayers of Hepsin-1/BHK cells in 12-well plates were washed once with buffer A (10 mM HEPES (pH 7.45) containing 137 mM NaCl, 4 mM KCl and 11 mM glucose) and subsequently washed twice with buffer A supplemented with 0.1% bovine serum albumin and 5 mM CaCl(2) (buffer A). Cell monolayers were then incubated at 37 °C with 500 µl of factor VII (final concentration, 10 nM) in buffer A. At selected intervals, aliquots (30 µl) of the supernatant were removed and immediately assayed for factor VIIa by the TF assay essentially as described(37) . Briefly, 100 µl of the diluted sample was placed in a 10 times 75 glass culture tube followed by 100 µl of factor VII-deficient plasma, 50 µl of TF (final concentration, 1.25 µg/ml), 50 µl of human brain cephalin and 100 µl of 25 mM CaCl(2). A standard factor VIIa curve was constructed using various concentrations (2-200 pM) of recombinant factor VIIa. In some experiments, Hepsin-1/BHK cell monolayers were preincubated for 2 h at 37 °C with either preimmune rabbit IgG (4 mg/ml in buffer A) or rabbit anti-MBP/hepsin IgG (4 mg/ml in buffer A) prior to the addition of factor VII. To assess structural changes in factor VII during its incubation with Hepsin-1/BHK cells, 20-µl aliquots of the supernatant were removed at selected intervals and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) after reduction with 10% 2-mercaptoethanol. Following electrophoresis, proteins were electrophoretically transferred from the gel to nitrocellulose membranes and visualized with affinity-purified anti-human factor VII IgG (38) and I-labeled protein A.

Analysis of Factor X and Prothrombin Activation on Hepsin-1/BHK Cells

Confluent monolayers of either BHK cells or Hepsin-1/BHK cells in 12-well plates were incubated with either a mixture of factor VII (10 nM) and factor X (160 nM) or a mixture of factor VII (10 nM), factor X (160 nM), and prothrombin (1 µM) in 500 µl of buffer A at 37 °C. At selected intervals, 450 µl of the incubation mixture were removed and added to 20 µl of 0.5 M EDTA. An aliquot (280 µl) of this mixture was transferred to a 96-well microtitration plate containing either 40 µl of S-2222 (final concentration, 0.6 mM) for factor Xa assay or 30 µl of S-2238 (final concentration, 90 µM) for thrombin assay. After a preselected incubation time, substrate hydrolysis was terminated by the addition of 20 µl of 50% acetic acid, and the absorbance at 405 nm determined in a UV(max) kinetic microplate reader (Molecular Devices). The concentration of factor Xa or thrombin formed in each system was interpolated from a standard curve relating DeltaA/min versus factor Xa (0-50 pM) or thrombin (0-200 pM) concentration.

Fractionation of Cell Components

The cytosol and total membrane fractions of Hepsin-1/BHK cells and untransfected BHK cells were prepared essentially as described(39) . Briefly, each cell line in a T-75 flask was washed twice with buffer A and dislodged from the flask in buffer A supplemented with 2 mM EDTA by gentle bumping. After centrifugation (1000 revolutions/min; 7 min; 25 °C), the cells (10^6 cells) were suspended in 450 µl of buffer A and kept at 0 °C (ice-water) for 20 min. Cells were then ruptured by six sequential passages though a 25-gauge 1.58-cm needle, and the needle effluent collected directly into thick-walled polycarbonate ultracentrifuge tubes (16 times 76 mm) placed on ice. Fractionation of cell lysates into total membrane and cytosolic fractions was accomplished by ultracentrifugation (100,000 times g; 30 min; 4 °C) in a Beckman model L5-75 ultracentrifuge equipped with a Beckman type 65 rotor. The cell pellet (total membrane fraction) was resuspended in 450 µl of buffer A and washed thee times with buffer A before testing for factor VII activating activity.

Effect of Trypsin on Cell-surface Hepsin Activity

In these studies, Hepsin-1/BHK cell suspension was prepared as described above. An aliquot (200 µl) of the cell suspension containing 2 times 10^6 cells in buffer A was incubated at 37 °C with various concentrations of TPCK-treated trypsin. At selected intervals, 200 µl of soybean trypsin inhibitor (10 mg/ml in buffer A) was added to the cell suspension. After a 15-min incubation, the cell suspension was centrifuged (1000 revolutions/min; 7 min) and the cells resuspended in 100 µl of buffer A containing 10 mg/ml soybean trypsin inhibitor. Following an additional 15 min of incubation at 37 °C, the cells were centrifuged, and washed six times in buffer A. The cells were then resuspended in 450 µl of buffer A, 50 µl of factor VII (10 nM final concentration) added, and the mixture incubated at 37 °C. At selected intervals, aliquots (30 µl) were removed from this incubation mixture and assayed for factor VIIa as described above. Control experiments indicated no hydrolysis of S-2288 in TPCK-treated trypsin and washed cells indicating that all trypsin activity had been neutralized and removed by the washing steps.


RESULTS

Activation of Factor VII by Hepsin-1/BHK Cells

In order to investigate the potential involvement of hepsin in the blood coagulation system, we have transfected BHK cells with a plasmid containing the cDNA of human hepsin and investigated the ability of cell-surface-expressed hepsin to activate selected members of the coagulation and anticoagulation pathways. Fig. 1demonstrates that Hepsin-1/BHK cells, but not untransfected BHK cells, contained mRNA for human hepsin. In order to avoid the possible side effects of adsorbed bovine serum components, Hepsin-1/BHK cells were cultured in serum-free medium for 1 day after reaching confluence and washed once with buffer A prior to the functional studies. Prothrombin (1 µM), protein C (100 nM), factor VII (10 nM), factor X (160 nM), and factor IX (100 nM), dissolved in buffer A, were incubated separately with Hepsin-1/BHK cells at 37 °C for 1 h, and the extent of their activation in the supernatant was measured using either a specific chromogenic substrate assay or a one-stage clotting assay. Of the above proteins, only factor VII was significantly activated under these conditions. Fig. 2A demonstrates that Hepsin-1/BHK cells, but not BHK cells transfected with empty Zem 229R plasmid, activated human factor VII in a time-dependent manner. In a 60-min incubation period, approximately 800 pM factor VIIa was generated in this system, in contrast to no factor VIIa formation on monolayers of BHK cells transfected with empty plasmid (Fig. 2A). SDS-PAGE and immunoblotting studies revealed that single-chain factor VII was progressively converted to two-chain factor VIIa resulting in 5-10% of factor VII being converted to factor VIIa in 60 min (Fig. 2B). The amount of factor VIIa formed on Hepsin-1/BHK cell monolayers was variable from experiment to experiment and ranged from 400-900 pM factor VIIa formed in a 60-min period. In addition, comparable factor VII activation rates were observed with another hepsin-transfected cell line designated as Hepsin-4/BHK (data not shown). Pretreatment of Hepsin-1/BHK cells with rabbit anti-maltose-binding protein/hepsin IgG inhibited the ability of the cells to activate factor VII greater than 95%, strongly suggesting that hepsin was in all likelihood responsible for the proteolytic activation of factor VII (Fig. 2A). Control experiments indicated that rabbit anti-MBP/hepsin IgG (0.1-3 mg/ml) had no effect on factor VIIa coagulant activity following incubation with human factor VIIa (10 µg/ml) for 2 h at 37 °C. While these results suggest that cell-surface-expressed hepsin is most likely responsible for the activation of factor VII, they do not rule out the possibility that Hepsin-1/BHK cells preferentially synthesize hamster tissue factor, which supports factor VII autoactivation by trace amounts of factor VIIa in the factor VII preparation. In order to address this possibility, we next incubated S344A factor VII, an active site mutant factor VII(20) , with Hepsin-1/BHK cells and examined structural changes in S344A factor VII during this incubation by SDS-PAGE and immunoblotting under reducing conditions. Under these conditions, 10% of the single-chain S344A factor VII was converted to a two-chain form in 60 min that migrated with authentic factor VIIa (data not shown), indicating that factor VII activation occurred on these cells independent of potential cell-surface hamster tissue factor. Furthermore, no cleavage of S344A factor VII was observed when this protein was incubated with BHK cells transfected with Zem 229R plasmid alone (data not shown). In order to demonstrate that hepsin cleaves factor VII at the Arg-Ile peptide bond(40) , R152E factor VII, an activation cleavage site factor VII mutant(21) , was incubated with Hepsin-1/BHK cells and the reaction monitored by clotting assays and SDS-PAGE-immunoblotting under reducing conditions. In these experiments, R152E factor VII was not cleaved under these conditions, suggesting that cell-surface hepsin cleaved and activated plasma-derived and S344A factor VII by specific cleavage of the Arg-Ile peptide bond.


Figure 1: Agarose electrophoresis of polymerase cahin reaction-amplified cDNA derived from untransfected BHK cells and hepsin-transfected BHK cells. First strand cDNA from untransfected BHK cells (lane 1) and hepsin-transfected BHK cells (lane 2) was amplified with polymerase chain reaction primers directed against the human Hepsin cDNA as described under ``Experimental Procedures.'' A negative control amplification (lane 3) was included with no cDNA template to assure that reaction products were not due to nonspecific sample contamination with hepsin cDNA sequences. The reaction products were fractionated on 1% agarose and stained with ethidium bromide.




Figure 2: Activation of factor VII by Hepsin-1/BHK cells and BHK cells transfected with Zem 229R. Hepsin-1/BHK cell monolayers were preincubated with either rabbit anti-MBP/hepsin IgG (box) or preimmune rabbit IgG () for 2 h at 37 °C. Following aspiration of the IgG solutions, cells were treated with 10 nM factor VII in buffer A, incubated at 37 °C, and the supernatants assayed for the temporal production of factor VIIa as described under ``Experimental Procedures.'' Zem 229R-transfected BHK cells (Delta) were incubated at 37 °C with 10 nM factor VII in buffer A, and at selected intervals factor VIIa formed was measured as described under ``Experimental Procedures.'' B, immunoblots of factor VII following incubation with Hepsin-1/BHK. Aliquots (20 µl) of the supernatants from Hepsin-1/BHK monolayers pretreated with preimmune rabbit IgG described in A were reduced, electrophorized, and reacted with affinity-purified anti-human factor VII. Lanes 1-5 represent reaction mixtures after 0, 15, 30, 45, and 60 min of incubation, while lane 6 is 20 µl of 350 pM authentic recombinant factor VIIa.



Cellular Localization of Hepsin

Hepsin is reported to be associated with the plasma membrane fraction of a variety of cells (10) . Using our functional factor VII activation assay, we examined whether hepsin is membrane-associated in Hepsin-1/BHK cells or conceivably secreted into the medium. Total membrane and cytosolic fractions were prepared from Hepsin-1/BHK cells by hypotonic shear methods and centrifugation and each fraction compared with intact cells and conditioned medium for their ability to activate factor VII. As shown in Fig. 3, hepsin factor VII activating activity was confined to the total membrane fraction of Hepsin-1/BHK cells. Neither Hepsin-1/BHK cytosolic fraction (Fig. 3) nor conditioned media (data not shown) activated factor VII. In addition, the conditioned media failed to activate factor VII in the presence of exogenous mixed phospholipids (data not shown). Furthermore, total membrane fractions prepared from either untransfected BHK cells or BHK cells transfected with Zem 229R plasmid containing factor VII cDNA failed to activate factor VII (data not shown), providing additional evidence that 1) hepsin was specifically expressed on the surface of Hepsin-1/BHK cells and was membrane associated, and 2) that tissue factor, potentially expressed by transfected cells, but not untransfected cells, was not contributing to the observed factor VII activation in an autocatalytic manner.


Figure 3: Activation of factor VII by cell components of Hepsin-1/BHK cells. Total membrane fraction (Delta), cytosol (), or 10^6 intact cells () were incubated with 10 nM factor VII in a total volume of 500 µl buffer A. At selected times, factor VIIa was determined as described under ``Experimental Procedures.''



Properties of Cell-surface Hepsin

In these studies, the pH stability, pH optimum, calcium dependence, and inhibition of cell-surface hepsin activation of factor VII were examined. Hepsin activity toward factor VII was stable between pH 6-9 following treatment of cells at various pH values between 2.5 and 11.5 in buffers consisting of either 100 mM glycine, 50 mM acetic acid, 50 mM HEPES, 50 mM Tris or 50 mM CAPS. The pH optimum for activation of factor VII by cell-surface hepsin was found to be pH 7-8 (data not shown). Table 1shows the effect of various protease inhibitors on the ability of cell-surface hepsin to activate factor VII at pH 7.4. Hepsin activity was strongly inhibited by 4-amidinophenylmethylsulfonyl fluoride, aprotinin, and antithrombin III but was not inhibited by soybean trypsin inhibitor and tissue factor pathway inhibitor, in the presence and absence of equimolar amounts of factor Xa. The presence of 1 unit/ml heparin slightly enhanced the inhibition of cell-surface hepsin by antithrombin III at its plasma concentration (3 µM). Activation of factor VII by cell-surface hepsin was found to be calcium dependent, with maximum activation occurring at 5 mM calcium ion concentration. Furthermore, no cleavage of factor VII was observed on Hepsin-1/BHK cells in 60 min in the presence of 2 mM EDTA as judged by SDS-PAGE and immunoblotting (data not shown).



Effects of Trypsin on Hepsin Proteolytic Activity

Recent studies have shown that the catalytic domain of hepsin is extracellular (10, 11) . Accordingly, we investigated the effects of treating Hepsin-1/BHK cells with various concentrations of TPCK-treated trypsin with respect to their ability to activate factor VII to further confirm the orientation of hepsin on the cell-surface. Fig. 4illustrates the time course of residual hepsin factor VII activating activity following treatment of the Hepsin-1/BHK cells with TPCK-treated trypsin. Although the hepsin proteolytic activity toward factor VII decreased in a time- and dose-dependent manner, approximately 25% of hepsin activity still remained after a 30-min incubation with 500 µg/ml trypsin. These results support the topological evidence that the catalytic domain of hepsin exists on the cell-surface(10) , but also indicate that hepsin expressed on the cell-surface is markedly resistant to trypsin digestion.


Figure 4: Effect of trypsin on the ability of cell-surface hepsin to activate factor VII. A Hepsin-1/BHK cell suspension (2 times 10^6 cells) was incubated with TPCK-treated trypsin at a final concentration of 5 (circle), 50 (bullet), and 500 (down triangle) µg/ml. At selected times, soybean trypsin inhibitor (10 mg/ml) was added to the cell suspension to stop the reaction. The cells were then centrifuged, washed six times, and subsequently assessed for their ability to activate factor VII. The results are expressed as percent factor VIIa formed in each system relative to the amount of factor VIIa formed in an untreated cell suspension under identical conditions.



Activation of Factor X and Prothrombin on Hepsin-1/BHK Cells

Additional studies were performed to assess the functional consequences of factor VIIa generation on the Hepsin-1/BHK cell-surface. Fig. 5demonstrates factor X activation on Hepsin-1/BHK and Zem 229R-transfected BHK cell-surfaces in the presence of factors VII and X. In the presence of factor X only, Hepsin-1/BHK cells generated extremely low levels of factor Xa (approximately 3 pM/60 min), while no measurable factor Xa was generated on Zem 229R-transfected BHK cell-surfaces (data not shown). However, in the presence of factors VII and X, significant amounts of factor Xa were generated on the Hepsin-1/BHK cell surface in 60 min. In the presence of factors VII and X, Zem 229R-transfected BHK cells generated 5% factor Xa relative to Hepsin-1/BHK cells. Furthermore, trace amounts of factor Xa is observed when Hepsin-1/BHK cells were incubated with factor X and S344A factor VII, and these levels were essentially that observed when Hepsin-1/BHK cells were incubated with factor X alone.


Figure 5: Activation of factor X by Hepsin-1/BHK cells and untransfected BHK cells in the presence of either factor VII or S344A factor VII. Hepsin-1/BHK cells (Delta, ) and untransfected cells () were incubated with either 160 nM factor X plus 10 nM factor VII (Delta, ) or 160 nM factor X plus 10 nM S344A factor VII (). At selected times, factor Xa formation was assessed in the supernatants as described under ``Experimental Procedures.''



In subsequent studies, we addressed the possibility that factor VIIa generated on Hepsin-1/BHK forms a complex with potential cell-surface hamster tissue factor and subsequently activates factor X in part or totally by a tissue factor-dependent reaction. In these studies, Hepsin-1/BHK cell monolayers were incubated for 2 h at 37 °C with either buffer A, preimmune goat IgG (6 mg/ml in buffer A) or purified goat anti-rabbit tissue factor IgG (6 mg/ml in buffer A) previously shown to cross-react with hamster tissue factor and inhibit the prothrombin time of hamster plasma and hamster brain thromboplastin in a concentration-dependent manner. In the latter studies, goat anti-rabbit tissue factor IgG prolonged the hamster prothrombin time 15 s at 3 mg/ml and 25 s at 6 mg/ml final concentration. In this system, a 25-s prolongation of the clotting time was equivalent to the clotting time observed when the hamster thromboplastin was diluted 5-fold. Thus, the goat anti-rabbit tissue factor IgG, at 6 mg/ml final concentration, inhibited roughly 80% of the hamster tissue factor in the thromboplastin preparation. In contrast, rabbit anti-human tissue factor IgG, at 0.2-6 mg/ml final concentration, had no effect on the hamster prothrombin time. Following incubation with either buffer A or the above IgG preparations dissolved in buffer A, the supernatant of each system was supplemented with CaCl(2) (5 mM), factor VIIa (10 nM), and factor X (180 nM) and allowed to incubate at 37 °C for 1 additional h. Aliquots (200 µl) of each reaction mixture were removed at 15, 30, 45, and 60 min and assayed for factor Xa using S-2222. The results of these studies indicated that the rate of factor Xa formation in all three systems was essentially identical, providing strong evidence that cell-surface hamster tissue factor was not participating in the factor VIIa-mediated activation of factor X on these cells.

Having demonstrated that the Hepsin-1/BHK cells support the activations of factors VII and X in presumably a tissue factor-independent reaction, we next examined whether Hepsin-1/BHK cells support the conversion of prothrombin to thrombin in the presence of plasma levels of factor VII, factor X and prothrombin. In the presence of prothrombin alone, Hepsin-1/BHK cells generated small amounts of thrombin (10 pM in 60 min), while no thrombin formation was observed in this time period on Zem 229R-transfected BHK cells (data not shown). With the addition of factors VII and X, significant amounts of thrombin (150 pM in 60 min) were generated on Hepsin-1/BHK cell monolayers (Fig. 6), while Zem-transfected BHK cells under these conditions generated approximately 10 pM thrombin in 60 min. When factor VII was deleted from the Hepsin-1/BHK cell system containing factor X and prothrombin, or replaced with an equal concentration of S344A factor VII, thrombin formation still occurred, but only 20% of that seen in the presence of functional factor VII (Fig. 6). Thus, while hepsin apparently can generate low levels of factor Xa and thrombin in the absence of factor VII, our findings indicate that the activation of factor VII by cell-surface hepsin markedly enhances subsequent factor Xa and thrombin formation on these cells.


Figure 6: Activation of prothrombin by Hepsin-1/BHK cells and untransfected BHK cells in the presence of various combinations of prothrombin, factor X, factor VII, and S344A factor VII. Hepsin-1/BHK cells (, , ) and untransfected BHK cells (Delta) were incubated with either mixtures of 160 nM factor X, 10 nM factor VII, and 1 µM prothrombin (, Delta), 160 nM factor X and 1 µM prothrombin (), or 160 nM factor X, 10 nM S344A factor VII, and 1 µM prothrombin (). At selected times, thrombin formed in the supernatant was determined as described under ``Experimental Procedures.''




DISCUSSION

In the present study, we have transfected BHK cells with a plasmid containing the cDNA of human hepsin and investigated the ability of these Hepsin-1/BHK cells to initiate and support coagulation reactions. Evidence is presented herein that cell-surface-expressed hepsin converts single-chain zymogen factor VII to two-chain factor VIIa as a result of proteolytic cleavage of the Arg-Ile peptide bond in factor VII. In addition to the activation of factor VII, we provide evidence that factor VIIa generated on the Hepsin-1/BHK cell-surface activates factor X in what appears to be a tissue factor-independent reaction. Factor Xa formed subsequently converts prothrombin to thrombin in the absence of exogenous factor V/Va and phospholipids. Thus, in addition to its ability to regulate cell growth and maintain cell morphology (11) , cell-surface hepsin may be involved physiologically in initiating the extrinsic pathway of blood coagulation that ultimately leads to thrombin formation on these cells in the absence of demonstrable tissue factor.

The activation of factor VII by cell-surface hepsin was found to be calcium dependent with maximal activation occurring at 2-5 mM CaCl(2). Preliminary data strongly suggest that this calcium requirement, in all likelihood, involves the calcium-dependent binding of factor VII to anionic phospholipids on the cell-surface in close proximity to the extracellular serine protease domain of hepsin. In this regard, Hepsin-1/BHK cells failed to cleave and activate a gla-domainless derivative of factor VII under conditions of optimal factor VII activation (data not shown). In addition, human prothrombin fragment 1, as well as the isolated human factor VII gla-peptide, inhibited the activation of factor VII by Hepsin-1/BHK cells in a dose-dependent manner. In these latter studies, 10 µM factor VII gla-peptide and 10 µM prothrombin fragment 1 inhibited factor VII activation by Hepsin-1/BHK cells 40 and 70%, respectively. Accordingly, these results suggest that factor VII associates with the cell-surface in a calcium-dependent reaction to kinetically increase its rate of activation by cell-surface hepsin.

Our data indicating that factor VII activating activity was observed exclusively in the total membrane fraction of Hepsin-1/BHK cells confirms the previous finding of Tsuji et al.(10) who demonstrated that hepsin in an integral membrane protease in a molecular orientation of type II membrane-associated proteins. These investigators also showed that immunoreactive hepsin was virtually all removed from the HepG2 cell-surface after a brief incubation of the cells with 100 µg/ml trypsin at 0 °C. In contrast, our data indicate that hepsin factor VII activating activity on the Hepsin-1/BHK cell line was surprisingly resistant to trypsin digestion, as 20% of hepsin activity still remained after incubation of these cells with 500 µg/ml trypsin for 30 min at 37 °C. The reason(s) for this discrepancy is not readily apparent, but may relate to the characteristics of the peptide-specific antibody used by Tsuji et al.(10) for immunoblot analysis. In this regard, the antibody used by Tsuji et al.(10) for this experiment was raised against a synthetic peptide comprising the carboxyl-terminal region of human hepsin (Glu-Leu), and it is conceivable that trypsin readily cleaves the Lys-Th peptide bond in cell-surface hepsin resulting in loss of immunoreactivity without appreciably decreasing its proteolytic activity. Alternatively, our Hepsin-1/BHK cells may be expressing severalfold higher levels of hepsin in comparison to the HepG2 cells, and this difference may account for the relatively high levels of residual cell-surface hepsin activity following trypsinization observed in this study.

Preliminary immunoblot analyses of solubilized Hepsin-1/BHK cell total membrane fraction using rabbit anti-MBP/hepsin IgG indicated the presence of thee immunoreactive bands with apparent molecular weight values of 55,000, 48,000, and 33,000. These three immunoreactive bands were also observed in solubilized total membrane fractions derived from an equivalent number of Zem 229R-transfected BHK cells but at an intensity of 10% of that seen for the Hepsin-1/BHK cell membranes. Presumably, the latter bands represent cross-reactivity of anti-MBP/human hepsin IgG with hamster hepsin as was originally reported by Tsuji et al.(10) using peptide-specific antibodies based on the human hepsin sequence. The presence of endogenous hepsin on BHK cells naturally raises the question as to why these cells did not support factor VII activation in proportion to their hepsin content. While the answer to this question will require further experimentation, it is conceivable that the activation of factor VII by hepsin may be a species-specific reaction.

One interesting finding of our studies is the fact that newly formed factor VIIa converts appreciable amounts of factor X to factor Xa on the Hepsin-1/BHK cell-surface in the absence of tissue factor. While several studies have demonstrated the slow activation of factor X by factor VIIa in the presence of calcium and phospholipids(4, 5, 41, 42) , our data corroborate a recent report that certain cell types can support the tissue factor-independent activation of factor X by factor VIIa(43) . In the latter study, human monocytes, but not an endothelial cell line, accelerated the activation of factor X by factor VIIa in a reaction that was not inhibited by either anti-tissue factor antibody or a 100-fold molar excess of prothrombin fragment 1(43) . The inability of prothrombin fragment 1 to inhibit this reaction suggests that monocytes express a specific factor VIIa-binding protein that augments factor X activation by factor VIIa, rather than binding of factor VIIa to anionic cell-surface phospholipid through its -carboxyglutamic acid domain. With respect to our results, it is conceivable that Hepsin-1/BHK cells synthesize and express cell-surface hamster tissue factor that weakly interacts with nascent human factor VIIa and augments the activation of factor X by factor VIIa. Our evidence, although indirect, suggests that this is not the case as neither Zem-transfected BHK cells nor factor VII-transfected BHK cell total membranes supported factor VII activation, assuming that cell-surface hamster tissue factor would support autoactivation of factor VII as well as it would support factor X activation by factor VIIa. In addition, pretreatment of Hepsin-1/BHK cells with high levels of goat anti-rabbit tissue factor apoprotein IgG, an antibody shown to strongly inhibit the prothrombin time of hamster plasma and hamster brain thromboplastin, had no measureable effect on the ability of these cells to generate factor Xa in the presence of factors VIIa and X (data not shown).

Our studies do not shed light on either the mechanism of hepsin activation or the number of functional, activated hepsin molecules on the surface of the Hepsin-1/BHK cells. The findings of Leytus et al.(9) predict that hepsin is synthesized as an inactive zymogen that is converted to an active serine protease by cleavage of the Arg-Ile peptide bond in the extracellular domain of hepsin. The identity or the origin of the protease responsible for this cleavage on our Hepsin-1/BHK cells is not known. One possibility includes an exogenous source such as proteases present in the fetal calf serum used in culturing these cells. Alternatively, hepsin may be synthesized as a single-chain zymogen but undergoes intracellular cleavage and activation prior to insertion into the membrane. In light of our results demonstrating inhibition of factor VII activating activity on Hepsin-1/BHK cells by antithrombin III, it is also uncertain as to what extent the cell-surface-activated hepsin is inactivated by antithrombin III (or other serpins) present in the 10% fetal calf serum. It is perhaps worthwhile to mention that our Hepsin-1/BHK cells were initially cultured in T-75 flasks and dislodged from this flask for subculturing by gentle bumping in EDTA-containing buffer rather than trypsinization. Subsequent treatment of these cells with trypsin, however, failed to result in a measurable transient increase in factor VII activating activity that preceded the time-dependent decline in the ability of the cell to activate factor VII. This result suggests that either all cell-surface hepsin was fully activated or that simultaneous degradation and activation of hepsin occurred precluding any measurable increase in activated hepsin activity under these conditions. Accordingly, the efficacy of factor VII activation by cell-surface hepsin is unknown at this point and will presumably require the isolation of hepsin to homogeneity in order to address this important question. In preliminary studies, however, the ability of Hepsin-1/BHK cells to activate factor VII was equivalent to that seen for factor VII activation on untransfected BHK cells in the presence of either 20 pM factor Xa or 10 nM factor IXa (data not shown).

Our finding that cell-surface-expressed hepsin activates factor VII leading to significant thrombin formation on these cells may be relevant to coagulation activation and fibrin deposition in situ on certain tumor tissues and thereby contribute to the progression of malignancy. While tissue factor synthesis and expression by several tumor types has been implicated as the major contributor to fibrin deposition on these tumor cells, there are specific instances of tumor tissues that support fibrin formation yet lack immunoreactive tissue factor(7) . In some of these tumor types, such as malignant melanoma, functional factor Xa has been observed without detectable tissue factor and factor VII antigen(44) , suggesting that these cells synthesize cancer procoagulant, a cysteinyl protease(45) , that activates factor X directly. In other tumor types, such as renal cell carcinoma, significant fibrin was observed in tumor stroma despite the absence of demonstrable tissue factor antigen on these tumor cells (46) . It is tempting to speculate that hepsin synthesis may be dramatically up-regulated in these, and other, tissue factor-deficient tumor cells and contribute to coagulation activation on these cells. In this regard, efforts to address this possibility using hepsin-specific antibodies and immunohistochemical techniques are currently ongoing in our laboratory.


FOOTNOTES

*
This work was supported in part by research grants from the National Institutes of Health (HL35246) and Blood Systems, Inc. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Pathology, University of New Mexico, School of Medicine, Albuquerque, NM 87131-5301. Tel.: 505-277-0094; Fax: 505-277-0593.

(^1)
The abbreviations used are: BHK, baby hamster kidney; PAGE, polyacrylamide gel electrophoresis; TF, tissue factor; TF, recombinant COOH-terminal truncated TF-containing residues 1-219; gla, -carboxyglutamic acid; EGRck, glutamyl-glycyl-arginyl chloromethyl ketone; TPCK, N-tosyl-L-phenylalanine chloromethyl ketone; CAPS, 3-cyclohexylamino-1-propanesulfonic acid; DMEM, Dulbecco's modified Eagle's medium; MBP, maltose binding protein; IPTG, isopropyl-thiogalactopyranoside; MTX, methotrexate.


ACKNOWLEDGEMENTS

We are grateful to Drs. Gordon Vehar (Genentech, South San Francisco) and Ole Nordfang (Novo Nordisk, Copenhagen) for providing us with preparations of the truncated tissue factor apoprotein and full-length tissue factor pathway inhibitor, respectively, used in this study. We also thank Drs. Earl Davie and Steven Leytus for providing us with the full-length cDNA for human hepsin, and Dr. L. V. M. Rao for the goat anti-rabbit tissue factor IgG. Finally, the excellent technical assistance of Nancy Basore and Joseph Kuijper is gratefully acknowledged.


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