©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Calreticulin, an Antithrombotic Agent Which Binds to Vitamin K-dependent Coagulation Factors, Stimulates Endothelial Nitric Oxide Production, and Limits Thrombosis in Canine Coronary Arteries (*)

(Received for publication, August 3, 1994; and in revised form, November 18, 1994)

Keisuke Kuwabara (1) David J. Pinsky (1) Ann Marie Schmidt (1) Claude Benedict (2) Jerold Brett (1) Satoshi Ogawa (1) M. Johan Broekman (3) Aaron J. Marcus (3) (4) Robert R. Sciacca (1) Marek Michalak (5) Feng Wang (6) Yu Ching Pan (6) Saul Grunfeld (7) Stephen Patton (7) Tadeusz Malinski (7) David M. Stern (1)(§) Jane Ryan (1)

From the  (1)Departments of Physiology and Medicine, Columbia University, College of Physicians and Surgeons, New York, New York 10032, the (2)Department of Medicine, University of Texas Health Science Center, Houston Texas 77225, the Departments of (3)Medicine and (4)Pathology, Cornell University Medical College and Department of Veterans Affairs Medical Center, New York, New York 10010, the (5)Department of Biochemistry, University of Alberta, Edmonton T6G2E1, Canada, the (6)Roche Research Center, Hoffmann-LaRoche, Inc., Nutley, New Jersey 07110, and the (7)Oakland University Department of Chemistry, Rochester, Michigan 48309

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Coagulation Factor IX/IXa has been shown to bind to cellular surfaces, and Factor IXa expresses its procoagulant activity by assembling into the intrinsic Factor X activating complex (Factors IXa/VIIIa/X), which also forms on membrane surfaces. This led us to identify cellular proteins which bind Factor IX/IXa; an approx55-kDa polypeptide was purified to homogeneity from bovine lung extracts based on its capacity to bind I-Factor IX in a dose-dependent and saturable manner. From protein sequence data of the amino terminus and internal peptides, the approx55-kDa polypeptide was identified as calreticulin, a previously identified intracellular calcium-binding protein. Recombinant calreticulin bound vitamin K-dependent coagulation factors, I-Factor IX, I-Factor X, and I-prothrombin (K values of approx2.7, 3.2, and 8.3 nM, respectively), via interaction with its C-domain, although it did not affect the coagulant properties of these proteins. I-Calreticulin also bound to endothelial cells in vitro (K approx 7.4 nM), and mouse infusion studies showed an initial rapid phase of clearance in which calreticulin could be localized on the vascular endothelium. Exposure of endothelial cells to calreticulin led to dose-dependent, immediate, and sustained increase in the production of nitric oxide, as measured using a porphyrinic microsensor. In a canine electrically induced thrombosis model, intracoronary infusion of calreticulin (n = 7) prevented occlusion of the left circumflex coronary artery in a dose-dependent manner compared with vehicle-treated controls (n = 5). These results indicate that calreticulin interacts with the endothelium to stimulate release of nitric oxide and inhibit clot formation.


INTRODUCTION

The vitamin K-dependent coagulation Factor IX/IXa has an important role in hemostasis and can contribute to the pathogenesis of thrombosis (1, 2, 3, 4) . In view of the well-known rapid clearance of Factor IX from the intravascular space, the association of infused and endogenous Factor IX with the vessel wall(5, 6) , and in vitro studies demonstrating Factor IX binding to endothelium and platelets(7, 8, 9) , studies have been performed to characterize the molecular basis of this coagulation protein-cell surface interaction. At the level of the ligand, the amino-terminal -carboxyglutamic acid-containing domain of Factor IX has been shown to be essential for cell surface binding (10, 11, 12, 13, 14) . At the level of the cell surface site, previous studies have suggested that Factor IX binding involves a protease-sensitive polypeptide, which on endothelial cells had a predicted molecular mass of approx150 kDa(15) , and on platelets appeared to involve proteins with molecular masses of approx150 and approx250 kDa(16) .

To further characterize polypeptides which interact with Factor IX, we employed bovine lung extract as a starting material and isolated the major species which bind Factor IX. An approx55-kDa polypeptide was purified to homogeneity and proved to be identical to calreticulin, based on protein sequence analysis of the amino terminus and trypsin-cleaved internal peptides. Calreticulin demonstrated specific binding to Factor IX and also interacted similarly with Factors X and prothrombin, although calreticulin was distinct from the endothelial binding site. Interaction of calreticulin with these vitamin K-dependent coagulation factors did not change their coagulant properties. In contrast, calreticulin bound to endothelial cells in vitro, localized to the vessel wall after intravenous infusion, and stimulated production of nitric oxide. Intracoronary administration of calreticulin prevented coronary thrombosis in a canine model. These results suggest that calreticulin has a novel, potentially clinically applicable antithrombotic function.


EXPERIMENTAL PROCEDURES

Preparation of Reagents: Purification of Coagulation Factors, Preparation of Recombinant Calreticulin, and Radiolabeling

Bovine Factors IX, X (a pool of X(1) and X(2)), and prothrombin were purified to homogeneity by previously described methods (17, 18, 19) and were supplied by Enzyme Research Laboratories, Inc. (South Bend, IN). Factor IX was radiolabeled by the lactoperoxidase method (20) using Enzymobeads (Bio-Rad), and I-Factor IX was isolated as described(21) , to a specific radioactivity of approx2.9 times 10^4 cpm(^1)/ng. The radioactivity profile of I-Factor IX on SDS-PAGE (10%) showed a single peak of approx55 kDa. Factor X and prothrombin were radiolabeled by the same procedure to specific radioactivities of 3.4 times 10^4 and 2.7 times 10^4 cpm/ng, respectively. Rabbit serum albumin (Sigma) was also similarly radioiodinated to a final specific radioactivity of 8 times 10^4 cpm/ng. Other coagulation factors, human proteins C and S, bovine Factor Xa, human Factor Va, thrombin and antithrombin III were also obtained from Enzyme Research Laboratories.

Recombinant rabbit calreticulin and discrete domains from calreticulin were expressed in Escherichia coli using the glutathione S-transferase (GST) fusion protein system with pGEX-3X plasmid(22) , as described previously(23) . Plasmids containing intact calreticulin or C-domain (amino acids 330-401) were expressed in BNN103 E. coli host(23) , and GST fusion proteins were purified to homogeneity by one-step glutathione-Sepharose 4B affinity chromatography (Pharmacia). Calreticulin or C-domain fusion protein was cleaved with Factor Xa, separated from Factor Xa using monoQ FPLC chromatography, reapplied to glutathione-Sepharose 4B, and the pass-through contained homogeneous recombinant calreticulin. Plasmids containing the N-domain (amino acids 1-182), the P-domain (amino acids 182-290), and C-domain were also expressed as fusion proteins. GSH and the GSH fusion proteins of the individual domains were purified as above, immobilized on polyvinylchloride wells (see polyvinylchloride plate binding assay, below), and used only in an experiment to compare the relative binding of I-Factor IX. Native rabbit calreticulin was isolated by a selective ammonium sulfate precipitation procedure in the presence of protease inhibitors followed by FPLC monoQ as described(24) . Calreticulin was radiolabeled using the same method for preparing I-Factor IX. Following radiolabeling, I-calreticulin was separated from free iodine by gel filtration, and the final tracer had a specific radioactivity of 3.0 times 10^4 cpm/ng, was >90% precipitable in trichloroacetic acid (20%), and migrated as a single band of approx55 kDa on SDS-PAGE (10%).

Assays to Detect I-Factor IX Binding Activity: Polyvinylchloride Plate Binding Assay (PVC Assay) and Endothelial Cell Binding Assay

PVC binding assays were performed with lung extracts, partially purified, and purified calreticulin. Samples, prepared as described below, were diluted in buffer containing 0.015 M NaC0(3), pH 9.2, 0.1 mM CaCl(2), and 0.05 ml of this material was incubated at 4 °C for 15 h in 96-well PVC plates. The mixture was then aspirated, the wells were washed once with washing buffer (10 mM HEPES, pH 7.55, 137 mM NaCl, 11 mM glucose, 4 mM KCl, 2.6 mM CaCl(2), 0.5 mg/ml bovine serum albumin) and 0.15 ml of blocking buffer (20 mM Tris, pH 7.4, 0.1 M NaCl, 1 mM CaCl(2), 50 mg/ml bovine serum albumin) was added to each well at 37 °C for 2 h. After this time, wells were washed again with washing buffer, and 0.05 ml of incubation buffer (minimal essential medium: 5 mg/ml bovine serum albumin, 7 mM HEPES pH 7.3) was added along with I-Factor IX, I-Factor X, or I-prothrombin alone or in the presence of a 100-fold molar excess of the respective, unlabeled protein for 2 h at 4 °C. The medium was then aspirated, and each well was washed six times (6 s total) with ice-cold washing buffer (0.15 ml/wash). Bound radioactivity was eluted during a 10-min incubation at 25 °C with 65 mM Tris, pH 7.9, 175 mM NaCl, 10 mM EDTA, and 0.5 mg/ml bovine serum albumin. Where indicated, crude lung extract prepared as above except without protease inhibitors was incubated with immobilized trypsin (10% (v/v); Sigma) for 1 h at 37 °C. Samples were diluted in buffer containing protease inhibitors as above, and the binding assay was performed. Similar results were obtained when lung extract proteins coated on PVC wells were exposed to trypsin, followed by washing, and inactivation of trypsin with protease inhibitors, and then a binding assay was performed as described above.

Equilibrium data were analyzed according to the equation of Klotz and Hunston (25) (B = nKA/[1 + K(a)], where B = specifically bound ligand (total binding, wells incubated with tracer alone, minus nonspecific binding, wells incubated with tracer in the presence of excess unlabeled material), n = sites/well, K = the dissociation constant, and A = free ligand concentration) using nonlinear least-squares analysis (Enzfitter). Data showing inhibition of I-Factor IX binding to calreticulin by unlabeled Factors IX, X, and prothrombin were fit to the equation(26) : b = B(m)[A]K(i)/(Ka;K(i) + K(a)[X] + K(i)[A]), where b = bound radioligand (I-Factor IX); [A] = concentration of radioligand; K(a) = K(m) in the absence of inhibitor; [X] = concentration of inhibitor (unlabeled Factor IX, X, or prothrombin); B(m) = maximal binding of radioligand in the absence of inhibitor.

Binding of I-calreticulin to endothelium was studied using confluent endothelial monolayers (0.32 cm^2/well). Bovine aortic endothelial cells were isolated and cultured as described previously(21) . Wells were incubated with binding buffer (0.05 ml/well) containing I-calreticulin alone (total binding) or in the presence of a 100-fold molar excess of unlabeled calreticulin (nonspecific binding). Following a 2-h incubation at 4 °C, wells were washed six times rapidly with ice-cold washing buffer as above. Where indicated, binding buffer was replaced by washing buffer, except that the amount of calcium chloride or EDTA was varied as stated. Dissociation was studied by the method of infinite dilution (27) . Following a binding assay, performed as described above, wells were washed and fresh binding buffer was added for the indicated time. Wells were then washed once and residual radioactivity determined.

Purification of approx55-kDa Polypeptide from Bovine Lung Extract

Bovine lung powder (30 g, Sigma) was added to 300 ml of 20 mM Tris, pH 7.4, 0.1 M NaCl, 1 mM phenylmethylsulfonyl fluoride, 0.1% trasylol, and 1% octyl-beta-glucoside for 16 h at 4 °C with constant mixing. Insoluble material was removed by centrifugation (11,000 times g) for 30 min at 4 °C, the supernatant (approx25 g) was filtered (0.45 µm), and applied to hydroxylapatite (300 ml, IBF, Savage, MD; column equilibration buffer: 20 mM Tris, pH 7.4, 0.1 M NaCl, 0.1% octyl-beta-glucoside, 1 ml/min flow rate). The column was washed until the absorbance at 280 nm was <0.01, and was then step-eluted with buffer containing 1 M NaCl. This wash was continued until the absorbance was <0.01, and the column was then eluted with 0.5 M NaPO(4), pH 7.4, containing 0.1% octyl-beta-glucoside. Fractions were collected (5 ml/fraction) and the protein content (A) and Factor IX binding activity was determined in the PVC assay (each fraction was tested at 1:100 dilution). Fractions with peak Factor IX binding activity were pooled, dialyzed overnight at 4 °C versus 20 mM Tris, pH 7.4 and 0.1% octyl-beta-glucoside, and then applied to FPLC monoQ (HR5/5; Pharmacia). The column was eluted with an ascending 0.05-1 M NaCl gradient, fractions were collected (1 ml/fraction; flow rate of 1 ml/min), and an aliquot of each was assayed as above. Samples with peak Factor IX binding activity were pooled (approx70 ml), concentrated by centrifugation on Centricon membranes (Amicon, Lexington, MA; molecular weight cut-off 10,000) (approx7 ml), and then applied to preparative nonreduced SDS-PAGE (10%; 28). Lanes of the gel were either stained with Coomassie Blue or were cut into 4-mm slices, incubated with 0.1 M sodium acetate, pH 8.3, and 0.1% octyl-beta-glucoside for 15 h at 4 °C. Tubes were then centrifuged (10,000 times g) for 5 min, and the supernatant was diluted 1:100 for testing in the PVC Factor IX binding assay. Where indicated, material eluted from the slices of the gel with peak Factor IX binding activity (corresponding to approx55 kDa) were again subjected to nonreduced SDS-PAGE (10%) and gel elution.

For sequence analysis, approx55-kDa polypeptide with peak Factor IX binding activity was subjected to SDS-PAGE (10%), transferred to polyvinylidene difluoride (PVDF) membranes, and the approx55 kDa was subjected to amino-terminal sequencing. Proteolytic digestion of protein adsorbed to PVDF pieces was performed by treating strips with 0.2% polyvinylpyrrolidine solution followed by trypsin in Tris-HCl, pH 8.5, as described(29) . The resultant peptide fragments were isolated by reversed-phase HPLC on a C(8) column (2.1 times 5 cm, YMC Inc., Morris Plain, NJ). Blotted protein samples on PVDF and peptide fragments recovered by HPLC peptide mapping were sequenced using an Applied Biosystems model 470A gas-phase sequencer with an ``on-line'' phenylthiohydantoin amino acid analyzer(30, 31) .

Coagulation Assays

Coagulation assays were carried out as described previously to assess Factor IXa-VIIIa-dependent activation of Factor X on endothelium and phospholipid(21, 32) , Factor Xa-Va-dependent activation of prothrombin on endothelium/phospholipid (33, 34, 35) , tissue factor/Factor VIIa-dependent activation of Factor X on endothelial cells stimulated with tumor necrosis factor(36) , thrombin/thrombomodulin (on bovine endothelial cells)-dependent activation of protein C(37) , and activated protein C/protein S-mediated inactivation of Factor Va on endothelial cells (38) (the latter study employed human endothelial cells and human activated protein C, protein S, and Factor Va, see below).

Infusion of Calreticulin into Mice

Mice (CD1) were infused via the tail vein with I-recombinant rabbit calreticulin or I-rabbit serum albumin (Sigma). Changes in plasma levels over time are expressed as the fraction of administered dose of radiolabeled albumin or calreticulin obtained in a 5-µl plasma aliquot obtained at the indicated times. Immunohistologic studies were performed on the lung tissue obtained from mice infused with unlabeled calreticulin. Five min following the infusion, animals were sacrificed, organs were removed and fixed by immersion in neutral-buffered formalin (10%), and embedded in paraffin by standard procedures. Sections were rehydrated, blocked with phosphate-buffered saline containing bovine serum albumin (1%) and normal goat serum (2%), and incubated with goat anti-rabbit calreticulin IgG (30 µg/ml; 40 min at 37 °C). Primary antibody was revealed using a rabbit anti-goat IgG avidin-biotin-conjugated system, as per the manufacturer's instructions (Sigma), with 3-amino-9-ethylcarbazole as chromogen.

To assess tissue deposition of I-calreticulin/I-albumin, the animals were sacrificed, organs removed, rinsed in phosphate-buffered saline, and dried overnight (80 °C). Subsequently, the weight and radioactivity (cpm) were measured(39) . The method of Spady and Dietschy (40) was used to calculate the tissue spaces employing the following formula: tissue space (microliters plasma/g dry tissue weight) = [cpm in tissue/(g dry weight times cpm/µl plasma)]. To correct for nonspecific tissue trapping of calreticulin, a tissue space for I-albumin was calculated. The specific tissue space of calreticulin was determined by subtracting the I-albumin tissue space from the I-calreticulin tissue space(39) .

Canine Coronary Thrombosis Model

In vivo coronary thrombosis was induced with electric current in dogs as described previously(4, 41) . This model involved instrumentation of the left circumflex coronary artery with a Doppler flow probe to measure coronary blood flow velocity and placement of a needle electrode intraluminally to initiate thrombus formation. Sampling catheters were present in the left atrium and coronary sinus, and length-segment crystals were placed in the anterior and posterior myocardial walls. Following a 30-min period to allow stabilization of hemodynamic parameters, current was applied (150 µA) to the needle electrode until a 50% increase in blood flow velocity occurred. This has been shown to correlate with a 40-50% decrease in cross-sectional area of the lumen due to thrombus formation at the site of placement of the electrode(41) . At this point, the current was turned off, and the animals received an intracoronary infusion of either saline (0.5 ml) or the indicated amount of calreticulin in 0.5 ml of saline. Left circumflex coronary blood flow, myocardial wall motion, and other hemodynamic parameters were monitored continuously(4, 41) . Blood samples were collected prior to calreticulin infusion and after the infusion at 1, 5, 10, 15, 30, 60, 90, 120, and 200 min. Clotting times (activated partial thromboplastin time and prothrombin time) were not affected by intracoronary infusion of calreticulin.

Measurement of Nitric Oxide Levels

Nitric oxide (NO) was measured using a porphyrinic microsensor prepared using cyclic voltammetric scanning to deposit a film of polymeric porphyrin (Ni(II) tetrakis(3-methoxy-4-hydroxyphenyl)porphyrin) on a thermally sharpened carbon fiber electrode, which was then coated with nafion as described (42) . This microsensor was placed on the surface of a confluent monolayer of bovine aortic endothelial cells (third passage) and served as the working electrode, with a platinum wire counterelectrode and a saturated calomel reference electrode. A PAR model 264A voltammetric analyzer with a PAR model 181 current-sensitive preamplifier were used for amperometric measurements at a potential of 0.68 V. NO measurements were standardized using aqueous NO standard prepared as described(43) . Experiments were performed using recombinant rabbit calreticulin or rabbit serum albumin (for controls), diluted to the indicated concentrations using phosphate-buffered saline (pH 7.4).


RESULTS

Characterization and Purification of Factor IX Binding Activity in Bovine Lung Extracts

In order to characterize cellular proteins that might interact with Factor IX, we began with extracts of bovine lung, a rich source of endothelium used previously for the purification of endothelial cell-associated receptors(44, 45) . A solid state binding assay was developed in which lung extracts were immobilized on the surface of PVC wells, and subsequent binding of I-Factor IX was examined. Specific I-Factor IX binding (the difference between binding in wells incubated with I-Factor IX alone compared with wells incubated with a 100-fold excess of unlabeled protein) was observed in this assay system; binding was proportional to the concentration of protein in the lung extract incubated with the PVC well, was not observed in control wells coated with albumin, and was blocked by pretreatment of the extract with trypsin (Fig. 1A). As the concentration of I-Factor IX was varied over a wide range, binding to immobilized lung extract was observed to be dose-dependent, with K(d) approx 1.6 nM (Fig. 1B), close to the known affinity of Factor IX for its binding sites on intact endothelial cells and platelets(8, 9) . However, in contrast to the specific interaction of Factor IX with these cellular surfaces, the binding of Factor IX to lung extract was not selective for this vitamin K-dependent coagulation factor, as competition was also observed with unlabeled Factor X and prothrombin (Fig. 1C).


Figure 1: Binding of I-Factor IX to lung extract immobilized in PVC wells. A, dependence on extract concentration. PVC wells were incubated with dilutions of lung extract (protein concentration as indicated) for 15 h at 4 °C, additional sites on wells were blocked by exposure to blocking buffer, and then a binding assay was performed by addition of I-Factor IX alone (6.3 nM; total binding) or in the presence of a 100-fold molar excess of unlabeled Factor IX (nonspecific binding) for 2 h at 4 °C. Wells were then washed, bound I-Factor IX was eluted by exposure to EDTA-containing buffer, and specific binding (total minus nonspecific binding) was determined (mean ± S.E. of triplicate determinations). B, dependence on I-Factor IX concentration. The experiment was performed as in A above, except that the concentration of lung extract was fixed (4 µg/ml), and the concentration of I-Factor IX was varied as indicated. Specific binding is plotted versus the concentration of I-Factor IX added to the wells, and the inset shows Scatchard analysis of the same data. Parameters of binding were: K = 1.4 ± 0.13 nM and n = 4.32 ± 0.17 fmol/well. C, competition study with unlabeled Factors IX, X, and prothrombin. Lung extract (4 µg/ml) was adsorbed to PVC wells as above, excess sites on the wells were blocked with albumin, and then a binding assay was performed by adding I-Factor IX alone (6.3 nM) or in the presence of a 100-fold molar excess of unlabeled Factors IX, X, or prothrombin (II) for 2 h at 4 °C. Unbound tracer was removed by washing, bound I-Factor IX was eluted by exposure to EDTA-containing buffer, and binding (mean ± S.E. of triplicate determinations) is shown.



Lung extract was subjected to chromatography on hydroxylapatite, and Factor IX binding activity was recovered with the major protein peak step-eluted with 0.5 M NaPO(4). Fractions with Factor IX binding activity were pooled and applied to FPLC MonoQ, and Factor IX binding activity was detected in fractions corresponding to a small protein peak eluted as the column was developed with an ascending salt gradient at approx0.4-0.5 M NaCl. Fractions with peak Factor IX binding activity were pooled and subjected to nonreduced SDS-PAGE (Fig. 2, lane 1), and a complex pattern of bands was observed by Coomassie Blue staining. When material in slices of the gel was eluted and tested in the PVC assay, Factor IX binding activity was observed only in fractions which comigrated with a protein band corresponding to approx55 kDa (Fig. 2, lane 2). The latter eluted material was subjected to reduced or non-reduced SDS-PAGE (Fig. 2, lanes 3 and 4, respectively), and in each case a single band, approx55 kDa, was observed. Elution of this protein from the non-reduced gel (Fig. 2, lane 4) demonstrated the presence of Factor IX binding activity in the PVC assay (Fig. 2, lane 5).


Figure 2: SDS-PAGE and gel elution of approx55-kDa polypeptide derived from lung extract which binds Factor IX. Lane 1, nonreduced SDS-PAGE (10%) of the pool from MonoQ with Factor IX binding activity visualized by Coomassie Blue staining. Lane 2, activity profile of material eluted from the indicated slice in lane 1. A nonstained lane on the gel otherwise identical to lane 1 was sliced (approx4-mm pieces), subjected to elution as described in the text, and used to coat PVC wells for a binding assay with I-Factor IX alone (6.3 nM) or in the presence of an 100-fold excess of unlabeled Factor IX. Factor IX binding activity is seen to be maximal in the slices (nos. 5 and 6) corresponding to approx55 kDa. Data are expressed as counts/min bound/sample of extract. Lanes 3 and 4, the material eluted from slices nos. 5-6 in lane 2 was subjected to reduced (lane 3) or nonreduced (lane 4) SDS-PAGE (10%), and material on the gel was visualized by silver staining. Lane 5, activity profile of the material in lane 4; the material eluted from slices no. 5 and 6 in lane 4 was subjected to nonreduced SDS-PAGE (10%) and again the gel was sliced and proteins eluted. The latter eluted material was tested in the PVC assay for its ability to bind I-Factor IX. Details of methods are described in the text, and for activity assays (lanes 2 and 5), the mean of duplicate determinations is shown. Apparent molecular weights, shown by the arrows, were interpolated from semilogarithmic plots based on the migration of standard proteins run simultaneously (phosphorylase B, 97.4 kDa; bovine serum albumin, 69 kDa; ovalbumin, 46 kDa; carbonic anhydrase, 30 kDa; trypsin inhibitor, 21.5 kDa; lysozyme, 14.3 kDa).



The polypeptide in the approx55-kDa polypeptide band was then subjected to protein sequencing. A portion of the amino-terminal sequence aligned closely with that previously reported for rabbit (46) calreticulin, as well as the human, dog, rat, and pig counterparts(24, 47, 48, 49) . In addition, the band was cleaved with trypsin, fragments were separated on reversed phase HPLC and subjected to sequence analysis, and found to match those in rabbit calreticulin: no. 1 residues 262-265; no. 2 residues 32-36 and 39-43, and no. 3, residues 306-322(46) .

Recombinant Calreticulin and Its COOH-terminal Domain Interact with Vitamin K-dependent Coagulation Factors

These data indicated that the approx55-kDa polypeptide purified from bovine lung extract was calreticulin. Consistent with this, on nonreduced and reduced SDS-PAGE purified recombinant rabbit calreticulin (Fig. 3, lanes 2 and 4, respectively; bovine calreticulin has not been cloned) and the approx55-kDa protein from bovine lung extract (Fig. 3, lanes 1 and 3, respectively) were shown to comigrate. Further, recombinant rabbit calreticulin and the approx55-kDa polypeptide interacted with the vitamin K-dependent coagulation proteins similarly in the PVC assay. Specific I-Factor IX binding was observed in wells with adsorbed calreticulin, and the binding was proportional to the amount of calreticulin incubated with the well (Fig. 4A). Comparable experiments performed with different concentrations of calreticulin to coat wells in the PVC assay using either I-Factor X or I-prothrombin as the tracer demonstrated similar specific binding to that for I-Factor IX (data not shown). In each case, interaction of the vitamin K-dependent coagulation factor with calreticulin was dependent on its concentration, demonstrating K(d) values of approx2.7, 3.2, and 8.3 nM, for studies with I-Factors IX, X, and prothrombin, respectively (Fig. 4, B-D). Control wells coated with albumin in place of calreticulin showed no specific binding.


Figure 3: SDS-PAGE of approx55 kDa polypeptide and purified, recombinant rabbit calreticulin. The approx55-kDa polypeptide purified from bovine lung extract (1 µg/lane; lanes 1 and 3) or purified recombinant rabbit calreticulin prepared in E. coli (1 µg/lane; lanes 2 and 4) was subjected to nonreduced (lanes 1 and 2) or reduced (lanes 3 and 4) SDS-PAGE (10%). Gels were stained with Coomassie Blue. Migration of molecular mass markers is shown (numbers indicate molecular masses in kDa).




Figure 4: Binding of Factors IX, X, and prothrombin to recombinant calreticulin. A, dependence on calreticulin concentration. PVC wells were incubated with the indicated concentration of recombinant calreticulin, excess sites were blocked with albumin-containing buffer, and then a binding assay was performed by adding I-Factor IX (6.3 nM; A) alone or in the presence of a 100-fold excess of the respective unlabeled protein. Specific binding is shown (mean ± S.E. of triplicate determinations). B-D, dependence on concentration of Factors IX, X, and prothrombin. PVC wells were incubated with recombinant calreticulin (9.1 pmol/well), excess sites were blocked with blocking buffer, and then a binding assay was performed by adding the indicated concentration of either I-Factor IX (B), I-Factor X (C), or I-prothrombin (D) alone or in the presence of a 100-fold excess of the respective unlabeled protein. Specific binding is plotted versus free/added I-labeled clotting factor (either Factor IX, X, or prothrombin). Data were analyzed by the nonlinear least-squares program, and the curve indicates the best fit line. Parameters of binding were for panel B, K = 2.73 ± 0.36 nM and n = 21.8 ± 1.13 fmol/well; for panel C, K = 3.24 ± 0.69 nM and n = 32.9 ± 2.44 fmol/well; and for panel D, K = 8.26 ± 0.52 nM and n = 27.6 ± 0.91 fmol/well. The inset shows Scatchard analysis of the same data.



In competition experiments performed to determine if each of these vitamin K-dependent coagulation factors interacted with identical/overlapping sites on calreticulin, dose-dependent inhibition of I-Factor IX binding to calreticulin was observed on addition of unlabeled Factors IX, X, and prothrombin (Fig. 5, A-C, respectively). Although the relationship fits closely to a model of competitive inhibition (26) showing K(i) values of approx6, 5, and 12 nM for studies with unlabeled Factors IX, X, and prothrombin, respectively (Fig. 5, A-C, insets), these values are viewed as approximate. These data suggest that the K-dependent coagulation factors interacted with similar/overlapping sites on calreticulin.


Figure 5: Competitive binding study: effect of unlabeled Factors IX, X, and prothrombin on the binding of I-Factor IX to recombinant calreticulin. Wells were incubated with calreticulin (9.1 pmol/well) for 15 h at 4 °C, excess sites in the wells were blocked with albumin-containing buffer, and then a binding assay was performed by adding three different concentrations of I-Factor IX (circle = 6 nM; square = 3 nM; triangle = 1.5 nM) in the presence of the indicated concentrations of either unlabeled Factor IX (A), unlabeled Factor X (B), or unlabeled prothrombin (C). Inset, Dixon plot showing 1/bound (1/B, fmol) versus protein added (nM).



The interaction of calreticulin with I-Factor IX was calcium dependent, with maximal effect by an added calcium concentration between 2-5 mM (data not shown). Since calreticulin has two calcium-binding domains, a high affinity, low capacity site (P-domain) and a low affinity, high capacity site (C-domain), experiments were performed with each of these domains, as well as amino-terminal N-domain (50; for this experiment only, calreticulin domains were expressed and studied as glutathione S-transferase fusion proteins). The C-domain bound I-Factor IX, whereas the P-domain, N-domain, and glutathione S-transferase control protein displayed only background binding (data not shown). Studies with I-Factor IX and C-domain indicated that binding was concentration dependent, with an apparent K(d) approx 3.3 nM (Fig. 6). Similar results were obtained with respect to the interaction of I-Factor X and I-prothrombin with calreticulin C-domain (apparent K(d) values were 4.8 and 6.0 nM, respectively; data not shown).


Figure 6: Interaction of I-Factor IX with the C-domain of calreticulin. Wells were incubated with C-domain (0.5 µg/well) for 15 h at 4 °C, excess sites were blocked with blocking buffer, and then a binding assay was performed with the indicated concentrations of I-Factor IX (6.3 nM) alone or in the presence of a 30-fold excess of the unlabeled respective protein. Specific binding is plotted versus the concentration of free/added tracer. Data were analyzed by the nonlinear least-squares program, and the curve indicates the best fit line. The inset shows Scatchard analysis of the same data.



In view of the interaction of calreticulin with multiple vitamin K-dependent coagulation factors, we investigated the effect of calreticulin on coagulant reactions involving these proteins. The following reactions were studied: tissue factor/Factor VIIa-mediated activation of Factor X (using matrices prepared from endothelial cells exposed to tumor necrosis factor as the source of tissue factor; 36), Factor IXa-VIIIa-mediated activation of Factor X (using crude cephalin as the phospholipid surface or endothelium), Factor Xa-Va-mediated activation of prothrombin (using cephalin as the phospholipid surface or endothelium), and thrombomodulin-dependent, thrombin-mediated activation of protein C (using intact bovine endothelial cells as the source of thrombomodulin; 37). No inhibition of these reactions was observed over a range of calreticulin concentrations. Furthermore, addition of calreticulin to recalcified human or bovine plasma did not delay clotting initiated with Factor IXa or Factor Xa (employing cephalin as the phospholipid source; data not shown).

Infusion of Calreticulin into Mice

To assess cellular interactions of calreticulin, I-calreticulin was infused intravenously into mice. Infusion of I-calreticulin demonstrated an initial rapid phase of clearance, followed by a slower phase similar to that observed with albumin infusion (Fig. 7A). To be certain that the initial rapid phase of I-calreticulin was representative of the entire preparation of radioiodinated material, and not a subpopulation of tracer possibly damaged during the radiolabeling procedure, experiments were performed in which plasma from animals infused with I-calreticulin was infused into other mice. A comparable early and rapid phase of calreticulin removal from the plasma was observed. The tissue deposition of I-calreticulin 2 min after its infusion was studied by comparing accumulation of I-calreticulin with I-albumin(39, 40) . Radiolabel was present in the most vascular organs, with the greatest amount in the lung. The trichloroacetic acid precipitability of I-calreticulin present in the plasma or lung was not significantly changed 10 and 60 min after its infusion, suggesting that rapid degradation was not occurring (data not shown). Immunohistochemical studies showed that infused calreticulin was associated with the vessel wall (Fig. 7B, panel I, lung), whereas no staining was observed in rabbit serum albumin-treated controls (Fig. 7B, panel II) or when anti-rabbit calreticulin IgG was replaced with nonimmune IgG (data not shown).


Figure 7: Infusion of recombinant rabbit calreticulin into mice. A, clearance of infused I-calreticulin from the plasma. Mice were infused via the tail vein with I-calreticulin or I-albumin, and at the indicated times, blood was withdrawn for determination of plasma radioactivity, with data expressed as the percent recovery (100 times measured cpm in 5 µl of plasma/administered cpm). B, immunohistologic study of rabbit calreticulin infused into mice. Mice were infused with rabbit calreticulin or rabbit serum albumin (120 µg/animal); 5 min later the animals were sacrificed, tissues were washed, and prepared for immunostaining with anti-rabbit calreticulin IgG. Panel I shows lung tissue from an animal infused with calreticulin, compared with an albumin-infused control (panel II). Magnification, times 480. bullet, albumin; circle, calreticulin.



Effect of Calreticulin on Coronary Thrombosis in a Canine Model

In view of the association of infused calreticulin with the vessel wall, experiments were performed in 12 dogs using a thrombosis model to determine if it could modulate the procoagulant response. Formation of a thrombus was induced by placement of a needle electrode into the left circumflex coronary artery and application of electric current, as described previously(4, 41) . When the cross-section of the artery was 40-50% occluded by thrombus, the current was discontinued and animals received either saline or calreticulin administered into the left circumflex artery as a bolus (Fig. 8). All animals treated with saline (n = 5) developed occlusive thrombosis with cessation of blood flow within 20-45 min. In contrast, animals receiving calreticulin (1.2 mg; n = 3) did not develop thrombosis, and the vessel stayed open for the duration of the experiment (180 min). The antithrombotic properties of calreticulin in this model were roughly proportional to the infused dose, with maximal effect at 1.2 mg/animal and steadily diminishing to finally no effect at 200 µg/animal (Fig. 8).


Figure 8: Effect of calreticulin on coronary thrombosis in a canine model. Following instrumentation of the left circumflex artery, current was applied to the needle electrode until a 50% increase in mean blood flow velocity occurred (this corresponds to a approx50% decrease in cross-sectional area). The current was then turned off, a bolus of the indicated amount of calreticulin or saline was given into the left circumflex coronary artery (in each case, volume was 0.5 ml), and vessel patency was monitored as described in the text.



Interaction of Calreticulin with Endothelial Cells

These data suggested that calreticulin interacted with endothelium, and thereby could potentially augment anticoagulant mechanisms on the endothelial cell surface. Therefore, binding studies were performed with I-calreticulin and confluent monolayers of bovine aortic endothelial cells. I-Calreticulin bound to endothelial cells in a time-dependent and reversible manner (data not shown), which was proportional to the dose of added radioligand (K(d) approx 7.4 nM) (Fig. 9). It should be noted that calreticulin was unlikely to be related to the endothelial cell-binding site for I-Factor IX, as addition of excess unlabeled calreticulin did not alter I-Factor IX-EC interaction, and anti-human calreticulin IgG did not inhibit binding of I-Factor IX to endothelial cells (data not shown).


Figure 9: Binding of calreticulin to endothelial cells. Dose dependence. confluent monolayers of bovine aortic endothelial cells (passage 6; 0.32 cm^2/well) (endothelial cells) were incubated with the indicated concentration of I-calreticulin alone or in the presence of 100-fold excess of unlabeled protein. Monolayers were washed, bound radioactivity was eluted, and specific binding is plotted versus the free/added concentration of I-calreticulin. Data were analyzed by nonlinear least-squares analysis, and the curve indicates the best fit line. Parameters of binding were K = 7.37 ± 0.93 nM and n = 4.28 ± 0.23 fmol/well.



Effect of Calreticulin on Endothelial Nitric Oxide Production

Since calreticulin bound to the endothelium, we considered whether it might augment natural endothelial cell antithrombotic mechanisms, such as release of tissue plasminogen activator, elaboration of prostacyclin, and/or production of NO. Although calreticulin had no effect on endothelial cell plasminogen activator or prostacyclin elaboration (data not shown), it did stimulate NO production. Nitric oxide production by confluent monolayers of bovine aortic endothelial cells was measured using a porphyrinic microsensor capable of detecting nitric oxide release at extremely low levels (nanomolar range) at the endothelial surface(42) . In these experiments, calreticulin caused a dose-dependent release of nitric oxide that was not observed when control albumin was applied, with sustained production observed at the endothelial surface following application of 40 nM calreticulin (Fig. 10). These experiments demonstrate that an average of 14 ± 2 fmol of NO/s/1 nM calreticulin are detected by the porphyrinic microsensor (Fig. 10). This amount represents NO electrolyzed on the surface area of the microsensor and is directly proportional to the total amount of NO released. These concentrations of calreticulin which induce endothelial cell nitric oxide release are comparable to those which result in occupancy of endothelial binding sites (Fig. 9).



Figure 10: Effect of calreticulin on endothelial production of nitric oxide. Bovine endothelial cells (third passage) were plated on coverslips and placed on a warmed microscope stage (37 °C). Nitric oxide was measured using a porphyrinic microsensor as the working electrode, placed with the aid of a micromanipulator. A saturated calomel electrode served as the reference electrode and a platinum wire electrode as the counterelectrode. A, amperometric recordings were taken at a potential of 0.68 V, with an aqueous nitric oxide standard used for microsensor calibration. Recombinant rabbit calreticulin (or albumin as control) in phosphate-buffered saline was applied, and nitric oxide measurements obtained. B, nitric oxide levels at the endothelial surface as a function of applied recombinant rabbit calreticulin concentration. Measurements were obtained as described above using a porphyrinic microsensor, with total amount of NO quantified by integration of the area under the amperometric curves. Data are expressed as the mean ± S.E.




DISCUSSION

To characterize cell-associated polypeptides which interact with Factor IX, approx55-kDa polypeptide was isolated from lung extracts based on its capacity to bind Factor IX. The approx55 kDa polypeptide co-migrated on SDS-PAGE with calreticulin and displayed virtual sequence identity with calreticulin of five different species. Furthermore, recombinant calreticulin bound Factor IX in a manner analogous to the approx55-kDa lung-derived polypeptide. These data indicated that the polypeptide isolated from lung was calreticulin (previously identified as an intracellular calcium-binding protein present in a wide range of cell types)(46, 50) , but it proved to be unrelated to the endothelial cell Factor IX-binding site, as indicated by several lines of evidence: (i) the binding of Factor IX to either the approx55-kDa polypeptide or calreticulin was not selective since the interaction with other vitamin K-dependent factors, including Factor X, prothrombin, and protein S (for the latter, data not shown), was very similar and the coagulation proteins cross-competed, whereas binding to the endothelial cell surface is selective for Factor IX(7, 8) ; (ii) addition of excess unlabeled calreticulin to binding mixtures of I-Factor IX-endothelial cell incubation mixtures did not prevent specific binding; and (iii) antibody to human calreticulin did not block the binding of I-Factor IX to human endothelial cells (data not shown). Thus, the interaction of Factor IX with the endothelium does not appear to involve calreticulin.

Although the binding of calreticulin to vitamin K-dependent coagulation factors seems to be relatively non-selective with respective to the target clotting factor, it specifically involves the C-domain of calreticulin. The first evidence supporting the possibility of an interaction between calreticulin and vitamin K-dependent coagulation proteins derives from the observation that calreticulin was a persistent contaminant co-purifying with protein Z, requiring preparative electrophoresis to effect a complete separation(51) . However, the same authors found that calreticulin was present in apparently normal human plasma at low levels(51) , despite the presence of an endoplasmic retention sequence and two lysosome targeting signals (50) . These data suggest that calreticulin is likely to bind to plasma vitamin K-dependent coagulation proteins under physiologic conditions, although the functional significance of this interaction is unclear, especially in view of our inability to find any effect of calreticulin on the participation of Factor VIIa, Factors IX/IXa, X/Xa, prothrombin, protein C/activated protein C or protein S in coagulation-related reactions.

The results of our in vivo clearance studies indicated that infused calreticulin became rapidly associated with endothelium of the vessel wall. To explore a possible mechanism whereby calreticulin could limit thrombus formation at localized endothelial cell sites without the development of a systemic anticoagulant effect, modulation of endothelial cell production of NO was studied. An important role for NO produced by the endothelium is to inhibit platelet aggregation and serotonin release, even in the presence of powerful proaggregants and secretogogues, such as thrombin. Measurement of endothelial cell-derived NO using a sensitive and specific porphyrinic microsensor showed that calreticulin increased NO production and that this was dose dependent over a range of calreticulin concentrations which paralleled those resulting in occupancy of endothelial cell calreticulin-binding sites. In addition, recombinant rabbit calreticulin appears to inhibit thrombin-mediated platelet aggregation and serotonin release (data not shown), suggesting the potential clinical relevance of these results. By stimulating a potent anticoagulant mechanism localized to the vascular wall, the nonsystemic nature of calreticulin's anticoagulant effects may be understood. Although local stimulation of nitric oxide production could account for the localized antithrombotic effect of calreticulin in vivo, the relationship between occupancy of endothelial cell calreticulin-binding sites and calreticulin-stimulated NO production by endothelial cells is at present unclear.

Taken together, these data suggest the hypothesis that calreticulin, an intracellular calcium-binding protein, when infused into the intravascular space binds to endothelial cells, stimulates NO production, blocks platelet adhesion and activation, and thereby prevents thrombus formation. Although further studies will be required to define the mechanisms through which calreticulin exerts its effects in vivo and in vitro, if these findings can be extrapolated to a range of thrombosis models, calreticulin may prove to have novel antithrombotic properties of potential therapeutic value. In support of the possible utility of calreticulin in this context, our recent pilot studies have shown that calreticulin infused intravenously (i.e. not at the site of thrombosis) also has an antithrombotic effect.


FOOTNOTES

*
This work was supported by Public Health Service Grants AG00602, HL34625, HL42833, HL42507, and HL21006, the Council for Tobacco Research(1971), American Heart Association (to D. J. P. and C. B.), and the New York Heart Association. 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.

§
This work was completed during the tenure of a GenentechEstablished Investigator Award from the American Heart Association. To whom correspondence should be addressed: Dept. of Physiology, P & S 11-518, Columbia University, College of Physicians and Surgeons, 630 W. 168th St., New York, NY 10032. Tel.: 212-305-1615; Fax: 212-305-5337.

(^1)
The abbreviations used are: cpm, counts/min; PVDF, polyvinylidene difluoride; PVC, polyvinylchloride plate binding assay; NO, nitric oxide; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase; FPLC, fast protein liquid chromatography; HPLC, high performance liquid chromatography.


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