Metabolism of rabbit plasma-derived factor VII in relation to prothrombin in rabbits

Mark W. C. Hatton1, Morris A. Blajchman1,2, Sampath Sridhara1, Suzanne M. R. Southward1, Bonnie Ross1, Myron Kulzcycky1, and Bryan J. Clarke1

1 Department of Pathology and Molecular Medicine, McMaster University Health Sciences Centre, Hamilton, and 2 Canadian Blood Services, Hamilton, Ontario, Canada, L8N 3Z5


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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In the human circulation, factor VII is present in relatively low plasma concentration (0.01 µM) and has been reported to have a short half-life (t1/2; 6 h). In contrast, prothrombin is present in a relatively high plasma concentration (2 µM) and has a relatively long catabolic half-life (t1/2 = ~2-3 days). This report examines the metabolic characteristics of purified rabbit plasma factor VII and prothrombin, radiolabeled with 125I and 131I, respectively, in healthy young rabbits. From the plasma clearance curves of protein-bound radioactivities, fractional catabolic rates and compartmental distributions were calculated using a three-compartment model. Turnover of factor VII within the intravascular space (2.95 days) exceeded that of prothrombin (1.9 days). However, the whole body fractional catabolic rate of factor VII (0.34 days-1; catabolic t1/2 = 2.04 days) was significantly slower than that of prothrombin (0.53 days-1; t1/2 = 1.31 days). Furthermore, the fractional distributions of factor VII in the intravascular (0.14) and extravascular compartments (0.76) differed from those of prothrombin (0.29 and 0.53). Absolute quantities of factor VII and prothrombin catabolized by a 3-kg rabbit amounted to 0.18 and 24.0 mg/day, respectively (molar ratio of prothrombin to factor VII = 100). The molar ratio of catabolism was compared with the release rates of factor VII and prothrombin from rabbit livers perfused ex vivo. After correction for uptake of factor VII and prothrombin by the liver, the molar ratio of released prothrombin to factor VII in the perfusate was ~293:1 over a 0.25- to 3-h interval. These results indicate that, compared with prothrombin, factor VII in the healthy rabbit circulates as a relatively long-lived protein. This behavior does not reflect that reported for factor VII in the human circulation.

hemostasis; plasma proteins; plasma clearance; protein metabolism; liver perfusion


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

IN PREVIOUS STUDIES, we have compared the metabolisms of several rabbit hemostatic proteins in rabbits, e.g., prothrombin (13, 17), fibrinogen (14), antithrombin (10, 13), heparin cofactor II (10), and plasminogen (15, 16). Recently, we have used these data collectively in an attempt to understand the dynamics of these actively interdependent proteins, both during a hemostatic response to an acute injury in vivo, e.g., immediately after a deendothelializing injury to the rabbit aorta (9), and during a chronic hemostatic event in vivo, e.g., within the stroma of a procoagulant lung tumor in rabbits (12).

Little is known about the metabolism of factor VII in rabbits. In this report, we describe the clearance of plasma-derived factor VII, relative to rabbit prothrombin, from the circulation of the rabbit. Using a three-compartment model to measure their fractional catabolic rates and compartmental distributions, we have calculated the relative quantities of factor VII and prothrombin catabolized per day and, from these results, predicted the relative rates of release of these two proteins de novo into the circulation. To substantiate our findings, and assuming the liver to be the only source of factor VII and prothrombin in blood, we then measured the rates of release of these proteins by the rabbit liver perfused ex vivo.


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Materials. Dextran sulfate-Sepharose, DEAE-Sephacel, DEAE-Sepharose FF, and CNBr-activated Sepharose were purchased from Pharmacia (Baie Durfe, QC, Canada), Immobilon and 0.22-µm ultrafilters from Millipore (Mississauga, ON, Canada), benzamidine, epsilon -aminocaproic acid (EACA), diethanolamine, alkaline phosphatase-linked donkey anti-sheep IgG, penicillin-streptomycin and p-nitrophenyl phosphate from Sigma Chemical (St. Louis, MO), 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitroblue tetrazolium (NBT) and Affi-Gel 10 agarose from Bio-Rad (Mississauga, ON, Canada), and alkaline phosphatase-linked rabbit anti-chicken IgG from Dimension Labs (Mississauga, ON, Canada). Sulfosuccinimidyl-3-(4-hydroxyphenyl) propionate (sulfo-SHPP), Iodogen, and Tween-20 were purchased from Pierce (Rockford, IL), D-phenylalanyl-L-prolyl-L-arginyl-CHCl2 (PPACK), 1,5-dansyl-L-glutamyl-L-glycyl-L-arginyl-CHCl2 (DGGACK), bestatin, pepstatin A, and leupeptin from Calbiochem-Behring (San Diego, CA), bovine serum albumin (BSA) from Boehringer Mannheim (Laval, QC, Canada), and heparin (Hepalean) from Organon Teknika (Toronto, ON, Canada). Alkaline phosphate-conjugated streptavidin was purchased from Jackson Immuno Research Laboratories (BioCan, Mississauga, ON, Canada) and Immulon-II microtiter plates (Dynatech Laboratories) from VWR Scientific (Mississauga, ON, Canada).

A polyspecific antibody against purified rabbit prothrombin was raised in a laying hen and extracted and partially purified from the egg yolks (22). The IgG preparation was then affinity purified by being passed through a column consisting of rabbit prothrombin coupled to CNBr-activated Sepharose. An antibody preparation against purified human prothrombin was raised in a sheep. Sheep IgG was prepared and affinity purified using a column of human prothrombin coupled to Sepharose as above. Antibody against rabbit factor VII was raised in a sheep immunized with purified recombinant rabbit factor VII (prepared in our laboratory; see Ref. 26) and purified as described previously (26).

Preparation of rabbit factor VII and prothrombin. Blood was collected by carotid cannulation from anesthetized healthy New Zealand White (NZW) rabbits into acid-citrate-dextrose (ACD) anticoagulant (1). After centrifugation (3,000 g for 10 min), platelet-poor plasma was recovered, and various protease inhibitors were immediately added, i.e., DGGACK and PPACK, to a final concentration of 5 × 10-6 M for prothrombin preparation and benzamidine (10-2 M) for factor VII preparation.

Starting with 1 liter of rabbit ACD plasma containing DGGACK and PPACK inhibitors, prothrombin was isolated by BaCl2 precipitation and purified by serial chromatographic steps (DEAE-Sephacel, dextran sulfate-Sepharose), as described previously (17). After activation in the presence of factor Xa, thromboplastin, and prothrombin-deficient human plasma (as a source of factor V), 1 mg of purified rabbit prothrombin yielded 1,300 (±190) IU of thrombin (17).

Factor VII was isolated from the BaCl2 precipitate from 1 liter of rabbit ACD plasma to which had been added the following: 10-2 M benzamidine, 1.5 × 10-3 M EDTA, 1.5 × 10-3 M EACA, and a protease inhibitor cocktail (3 × 10-6 M bestatin; 3 × 10-6 M pepstatin, and 2 × 10-6 M leupeptin). Precipitation by BaCl2 was carried out as described previously for recombinant rabbit factor VII (26). Semipurified factor VII in BaCl2 extract (0.6 l) was then directly purified by antibody-affinity chromatography [using polyclonal sheep anti-rabbit factor VII coupled to Affi-Gel 10 agarose (5-10 mg IgG/ml of gel)], as described (26). The eluted factor VII was concentrated by pressure filtration (Amicon) using a YM-10 ultrafiltration membrane (molecular mass cut-off, 10 kDa) and the buffer was changed to 25 mM sodium citrate, pH 6.0, containing 2.5 mM benzamidine and 1.5 mM EDTA.

Factor VII was further purified on a DEAE-fast flow column (2 ml) which separated gamma -carboxy-glutamic acid (Gla)-domainless factor VII from Gla-containing factor VII. The DEAE column was initially equilibrated with 25 mM sodium citrate containing 2.5 mM benzamidine and 1.5 mM EDTA. Factor VII from the previous step was applied to the column, and the column was washed with 10 ml of equilibration buffer followed by 2.5 ml of equilibration buffer containing 100 mM NaCl. The flow-through contained essentially Gla-domainless factor VII. Gla-containing factor VII was eluted with 10 ml of 250 mM NaCl in equilibration buffer and was concentrated using Millipore Ultrafree-4 centrifugal filter device (mol mass cut-off, 5 kDa). For long-term storage (at -70°C), 2.5 mM benzamidine and 1.5 mM EDTA were included in the buffer. Immediately before radiolabeling, the buffer was changed to 50 mM HEPES, pH 7.4, containing 100 mM NaCl. The final yield from 1 liter of plasma was 250 µg of Gla-containing rabbit factor VII in a volume of 500 µl. After activation, 1 mg of purified factor VII yielded 1,736 (±186) U of factor VIIa (26).

Protein purity was determined by polyacrylamide gel electrophoresis (PAGE) in the presence of 0.1% SDS (19). Gels were either fixed and stained with Coomassie brilliant blue in methanol-acetic acid-water (4:1:5, vol/vol) and dried or were subjected to electroblotting, during which the protein content of the gel was transferred to Immobilon (27). Blots were developed to detect either factor VII-related components by use of sheep anti-rabbit factor VII followed by alkaline phosphatase-linked anti-sheep IgG and then by the BCIP/NBT staining procedure (26) or prothrombin-related components by use of chicken anti-rabbit prothrombin followed by alkaline-phosphatase-linked anti-chicken IgG and then stained using BCIP/NBT (17).

Measurement of plasma concentrations of factor VII and prothrombin. Approximately 1 ml of blood was taken from an ear artery into 0.25 ml of ACD and weighed before centrifugation to prepare plasma. Knowing the volume of ACD and the total weight of blood/ACD and assuming a hematocrit of 0.42 for rabbit blood allowed us to correct for the ACD content of plasma during the measurement of factor VII and prothrombin concentrations in blood.

An enzyme-linked immunosorbent assay (ELISA) was developed to measure rabbit plasma concentrations of factor VII. Briefly, a polyclonal sheep anti-rabbit factor VII antibody (250 ng/well) was coated onto an Immulon-II microtiter plate in carbonate antigen coating buffer overnight at 4°C. After the plate was washed with phosphate-buffered saline, pH 7.4, containing 0.025% Tween-20 (PBS-T), the nonspecific binding sites were saturated with a blocking buffer, 3.5 mg BSA/ml of PBS-T (PBS-T/BSA) containing 28 mM benzamidine, for 2 h at room temperature. The plate was washed three times with PBS-T, and 100 µl of serially diluted standards containing affinity purified plasma-derived rabbit factor VII (1-25 ng/ml) and suitably diluted rabbit plasma samples were placed, each in triplicate, into the wells; all standards and plasma samples were made up in PBS-T/BSA buffer. The plate was incubated for 2 h at room temperature. After washing with PBS-T, factor VII bound to the capture antibody was detected by adding 100 µl of biotinylated sheep anti-rabbit factor VII to each well (1.5 µg/ml for 1 h at room temperature). The plate was then washed with PBS-T containing 1 M NaCl before 100 µl of streptavidin conjugated to alkaline phosphatase were added to each well (0.66 µg/ml for 1 h at room temperature). After the plate was washed with PBS-T, 100 µl of the substrate p-nitrophenyl phosphate (1 mg/ml in diethanolamine buffer) were placed into each well in a timed fashion. Color development was allowed to proceed for 20-30 min, after which the reaction was stopped by adding 25 µl of 0.5 M EDTA to each well in a timed fashion. The plate was read at 405 nm in a Bio-Tek EL-808 automatic microplate reader. After correction for the ACD content of plasma samples, and with an assumed hematocrit of 0.42 for rabbit blood, samples from 16 NZW rabbits were calculated to contain a mean concentration of 7.9 ± 0.2 nmol/l of blood.

The concentration of prothrombin in normal rabbit plasma has been measured previously using a biological activity assay (17) to be 90% of that of human prothrombin in normal human plasma [which is generally considered to be 2 µmol/l (21)]. Consequently, prothrombin in rabbit plasma was calculated to be 1.8 µmol/l (17). In addition, an ELISA for rabbit prothrombin has been used previously (13) with affinity-purified chicken anti-rabbit prothrombin followed by alkaline phosphatase-linked rabbit anti-chicken IgG and then applying a standard substrate, p-nitrophenyl phosphate. By use of this ELISA, prothrombin concentration of normal rabbit plasma amounted to 1.93 ± 0.07 µmol/l (13). From these measurements, and with a hematocrit of 0.42, mean rabbit blood concentrations of prothrombin were calculated to be 1.05 ± 0.05 µmol/l.

Radiolabeling factor VII and prothrombin. For the catabolism study, only samples of rabbit Gla-containing factor VII were compared with rabbit prothrombin. Both factor VII and prothrombin were radiolabeled using the Iodogen-coated glass vial method described by Fraker and Speck (7). The interior surface of flat-bottomed glass vials was coated with 10 µg of Iodogen to a height of 3 mm, and the vials were stored at 4°C over a drying agent. A mixture, consisting of factor VII (20-40 µg) in 0.4 ml 0.2 M Na phosphate, pH 7.4, was placed in the vial. Finally, 1 mCi of 125I was added, and the reaction was stirred for 60 s at 23°C. The radiolabeled protein was removed and placed in a dialysis sac (diameter: 0.6 cm) and dialyzed overnight at 4°C against 250 ml (changed 4 times) of 0.15 M NaCl buffered by 0.01 M Na phosphate, pH 7.4. Prothrombin was radiolabeled with 131I by means of a similar reaction procedure but in the presence of 50 mM EACA (to prevent aggregation of 131I-labeled prothrombin) as explained previously (17). Preparations of 125I-labeled factor VII (of specific radioactivity: 5.7 ± 1.6 × 106 dpm/µg) and 131I-prothrombin (1.3 ± 0.3 × 107 dpm/µg) were stored at 4°C and used in experimental work within 24 h of labeling.

In a few initial experiments, a method that radiolabels proteins through -NH2 groups (usually epsilon -NH2 of lysyl residues) was compared with the Iodogen method in labeling factor VII and prothrombin. In this method, a modified Bolton-Hunter-type reagent, sulfo-SHPP, was used as described previously (14). All reactions were performed at room temperature. Briefly, sulfo-SHPP was first radiolabeled in an Iodogen-coated glass vial. Then, radiolabeled sulfo-SHPP (in the absence of free I2 and Iodogen) was allowed to react with factor VII (or prothrombin). Reaction products were then dialyzed four times against 250 ml 0.01 M PBS (pH 7.45) for 18 h at 4°C; 125I-labeled factor VII or 131I-prothrombin was used for a plasma clearance experiment within 24 h of labeling. With the use of the sulfo-SHPP method, specific radioactivities of the recovered proteins were relatively low, amounting to 6-8 × 105 and 6-7 × 105 dpm/µg for 125I-factor VII and 131I-prothrombin, respectively. Furthermore, losses of both radiolabeled proteins during the dialysis step were significant with the sulfo-SHPP method (20-25% losses) compared with losses observed with the Iodogen method (9-10%) above.

The purity and integrity of the radiolabeled proteins were examined by PAGE in the presence of 0.1% SDS (19). A mixture of prestained protein standards (SeeBlue; Helixx Technologies, Scarborough, ON, Canada) were loaded in one lane of all gels. After SDS-PAGE, gels were fixed and stained with Coomassie brilliant blue, dried, and then exposed to photographic film (X-OMAT AR film; Eastman Kodak, Rochester, NY).

Clearance of factor VII and prothrombin from the rabbit circulation. Proposed animal experiments were approved by the Animal Research Ethics Board of McMaster University, and all experiments were conducted within the guidelines recommended by the Canadian Council of Animal Care (2).

NZW male rabbits (2.5-3.3 kg) were obtained from a local supplier. On arrival at the Animal Facility (McMaster University), the rabbits were housed normally and supplied with food and water ad libitum for >= 7 days before use in experiments.

The procedure for measuring the clearance of 125I-factor VII and 131I-prothrombin was similar to that used previously for measuring the clearance of other radiolabeled plasma proteins (10, 13, 17). Only the results of experiments in which proteins radiolabeled by use of the Iodogen method are reported below. Briefly, a mixed dose of 125I-factor VII (1.62 ± 0.37 µg/kg) and 131I-prothrombin (3.07 ± 0.51 µg/kg) contained in a total volume of 1.0 ml sterile saline was sampled (i.e., a known weight taken and added to 1.0 ml saline for radioactivity measurement) and then injected intravenously (ear vein) into each NZW rabbit (weight range: 2.8-3.1 kg). As each rabbit was injected, the time was noted. Each syringe was weighed immediately before and after injection to calculate the volume of each injected sample.

After injection, blood samples (1-2 ml blood anticoagulated with 0.25 ml ACD) were taken from the medial ear artery at 5 and 30 min, 1, 2, 4, 6, 8, 24, and 32 h, and then daily for 6 days. Each blood sample was centrifuged (3,000 g, 3 min), and plasma was aspirated and sampled for total radioactivity measurement. In addition, plasma samples were subjected to precipitation using 20% (wt/vol) trichloroacetic acid (TCA) to determine the proportion of protein-bound radioactivity (as cpm/ml of plasma). Further corrections were made for ACD content of plasma and for 131I "crossover" into the 125I channel. For each isotope, the 5-min value was assumed to represent 100% of the dose, and all samples obtained subsequently were related to the 5-min value. These percentages constituted the plasma clearance curves for factor VII and prothrombin.

Calculation of fractional catabolic rates, catabolic half-lives and compartmental distributions of factor VII and prothrombin. Calculation of the plasma curve components was undertaken by a "curve-peeling" technique using linear regression analysis to define the components of each exponential (6). As the curves of factor VII and prothrombin were best described by three exponentials rather than two, a three-compartment model, proposed earlier (3) to explain the clearance of 125I-labeled antithrombin III from the rabbit circulation, was used to calculate the dynamics of the behaviors of factor VII and prothrombin. With the use of the terminology employed by Carlson et al. (3), the three compartments are defined as: compartment 1, intravascular space; compartment 2, noncirculating vascular wall; compartment 3, extravascular space. Each curve is described by
*A<SUB>p</SUB><IT>=</IT>C<SUB>1</SUB><IT>e</IT><SUP><IT>−</IT>a<SUB>1</SUB><IT>t</IT></SUP> + C<SUB>2</SUB><IT>e</IT><SUP>−a<SUB>2</SUB><IT>t</IT></SUP> + C<SUB>3</SUB><IT>e</IT><SUP>−a<SUB>3</SUB><IT>t</IT></SUP>
where *Ap is that fraction of the radiolabeled protein remaining in the circulation at t days after injection, C1, C2, and C3 are the fractional constants for compartments 1, 2, and 3; and terms a1, a2, and a3 are the respective rate constants of exchange between those compartments. From the C and a values of each plasma curve, fractional catabolic rates were calculated for the plasma compartment (j3), the plasma plus noncirculating vascular wall compartments (j3.5), and the total body (jT). The j values were used to calculate the distribution of each protein among the plasma (Ap), noncirculating vascular wall (Aw), and extravascular (Ae) compartments (3).

Measurement of comparative release rates of factor VII and prothrombin from the perfused rabbit liver ex vivo. Our technique for perfusion of a rabbit liver (15, 16) was modeled on that for a rat liver (18). A temperature-controlled cabinet, complete with organ bath, perfusate reservoir, peristaltic pump, and circuitry for oxygenation (95% O2-5% CO2) and debubbling of the perfusion fluid was used as described by Miller (23). Ten healthy NZW rabbits (male; mean weight: 3.09 ± 0.25 kg) were housed for 1 wk before use in these experiments.

Details of anesthesia and the procedure for excision of the liver without previous exsanguination and placement of the afferent and efferent cannulas have been described previously (15, 16). The following procedures were performed inside the temperature-controlled cabinet (37°C). A freshly excised liver was placed in a Petri dish containing ultrafiltered (0.22-µm filter; Millipore) Krebs-Henseleit (KH) physiological solution [containing 3% (wt/vol) BSA, 5 mM glucose, heparin (10 USP U/ml) and penicillin-streptomycin (final concentrations: 10,000 U/l and 10 mg/l, respectively), pH 7.4], and immediately perfused by a hand-operated syringe (connected to the hepatic portal vein cannula) with 120 ml of KH solution to clear much of the blood content of the organ. The partially perfused liver was then connected to a circuit containing 500 ml of oxygenated KH solution (as described earlier) driven by a rotary pump (Cole-Palmer, Chicago, IL). This perfusion solution, after passing through the liver, was not recirculated.

For 6 of the 10 livers, when the liver was essentially free of blood (at ~20 min after surgery was completed), recirculation-perfusion with a fresh batch (300 ml) of ultrafiltered KH solution was commenced at a mean flow rate of 50 ml/min. Perfusate pH was maintained at 7.42-7.45 by the addition of a few drops of acid (1 M HCl) or base (1 M NaOH) to the reservoir (which always contained ~180 ml of perfusate) by use of a Pasteur pipette. Samples (1-2 ml) of perfusate were taken at 0, 0.25, 0.5, 0.75, and 1 h and then at 0.5-h intervals for up to 3 h and were measured for their glucose content (undertaken by Laboratory Medicine Services, Chedoke-McMaster Hospitals, Hamilton, ON, Canada) and for factor VII and prothrombin concentration by means of the ELISAs described earlier.

For 4 of the 10 livers, after the liver was free of blood, samples of freshly labeled 125I-factor VII (6.8 ± 0.3 µg) and 131I-prothrombin (9.5 ± 1.9 µg) were added to 300 ml of freshly ultrafiltered KH solution for recirculation-perfusion. Samples (1 ml) of perfusate were taken at intervals (as above) over 3 h to measure 125I and 131I (as TCA-precipitable, protein-bound radioactivity) remaining in the perfusate. We assumed that these values reflected the relative rates of uptake of factor VII and prothrombin (or their related complexes or fragments) by the liver ex vivo. These relative rates of uptake were used to correct the observed rates of release to more finely determine the rates of release of factor VII and prothrombin by the liver ex vivo.

Statistical calculations. Data are presented as means ± SE. Tests of significance were made using the Student's t-test (two tailed). Where not stated, P > 0.05 and is considered not significant.


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ABSTRACT
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MATERIALS AND METHODS
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Purity of plasma-derived 125I-factor VII and 131I-prothrombin. Purified rabbit factor VII appeared, after radiolabeling with 125I, as a single band on SDS-PAGE of Mr 55 kDa (Fig. 1, lane 4). In comparison, unlabeled recombinant rabbit factor VII, stained with Coomassie brilliant blue, migrated as a slightly smaller protein (lane 2; Mr, 53 kDa). This observation indicated that factor VII was not autoactivated to factor VIIa during the radiolabeling procedure. After reduction by beta -mercaptoethanol, 125I-factor VII migrated as a single band of Mr 57 kDa. Rabbit prothrombin, before and after radiolabeling, migrated as a single band of Mr 72 kDa, as described previously (13, 15) (Fig. 1, lanes 1 and 3, respectively).


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Fig. 1.   Electrophoresis of radiolabeled plasma-derived samples of carboxy-glutamic acid (Gla)-containing rabbit factor VII and rabbit prothrombin in 10% polyacrylamide gel in the presence of 0.1% SDS. Note that unlabeled rabbit recombinant factor VII (Mr, 53 kDa) and plasma-derived prothrombin were used as "carrier" proteins for 125I-labeled factor VII and 131I-labeled prothrombin, respectively. Lanes 1 and 2 were stained with Coomassie brilliant blue and then exposed to autoradiographic film to provide lanes 3 and 4, respectively. Lanes 1 and 3: prothrombin + 131I-prothrombin; lanes 2 and 4: rabbit recombinant factor VII + 125I-factor VII; lane 5: prestained standard proteins (Mr in kDa).

After reaction with human factor Xa in the presence of thromboplastin and Ca2+, 125I-factor VII was proteolytically cleaved to yield 125I-factor VIIa, as shown by the presence of the heavy and light chains of factor VIIa on SDS-PAGE/autoradiography when reducing conditions were used (data not shown). By the same means, 131I-prothrombin was lysed to liberate characteristic intermediate forms during the formation of thrombin, as described previously (13, 15).

Clearance of 125I-factor VII and 131I-prothrombin from the circulation of normal rabbits. Three different preparations of purified factor VII were studied. Doses of these 125I-factor VII preparations differed to a small extent (range 0.8-2.4 µg/kg). Each dose represented only a minor contribution (3-8%) to the estimated plasma pool of factor VII in the rabbit circulation.

Radiolabeled factor VII passed more rapidly from the intra- to the extravascular space than 131I-prothrombin during the 1st day after coinjection of 125I-factor VII and 131I-prothrombin (Fig. 2). This comparatively rapid migration of factor VII was confirmed by the values obtained for j3, which measures the rate of turnover of factor VII within the intravascular space. In three experiments, j3 values ranged from 2.3 to 3.1 days-1 (Table 1). In comparison, rabbit prothrombin, measured in 17 rabbits, gave a mean j3 of 1.87 ± 0.14 days-1, a value significantly less than the mean value (i.e., 2.65 ± 0.10 days) obtained for factor VII (P < 0.001). However, the terminal slopes of the plasma clearance curves (Fig. 2) and the measurements of fractional catabolic rate for the whole animal, jT (Table 1), indicated that factor VII was catabolized more slowly than prothrombin (mean jT for factor VII, 0.34 days-1; for prothrombin, 0.53 days-1; P < 0.05). These values of jT correspond to mean catabolic half-lives (t1/2) of 2.04 days for factor VII and 1.31 days for prothrombin in rabbits.


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Fig. 2.   Mean plasma clearance curves of 125I-factor VII (open circle ; n = 11 rabbits) and 131I-prothrombin (; n = 17 rabbits) from the circulation of rabbits. Data were derived from protein-bound radioactivities, as described in the text. Details of protein dynamics are given in Table 1.


                              
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Table 1.   Fractional catabolic rates and compartmental distributions of rabbit factor VII and prothrombin in healthy rabbits in relation to dose

The distribution profiles of factor VII and prothrombin, measured using the respective fractional catabolic rates obtained from the 125I-factor VII and 131I-prothrombin plasma curves, differed substantially. Thus the proportions of factor VII resident in the circulation (Ap) and associated reversibly with the vascular wall (Aw) and within the extravascular space (Ae) amounted to 0.14, 0.10, and 0.76, respectively. Similar calculations for the fractional distribution of prothrombin amounted to 0.29, 0.18, and 0.53 (Table 1).

In one experiment, the integrity of circulating 125I-factor VII was investigated by SDS-PAGE of whole plasma taken from six rabbits at 30 min after injection, followed by exposure to autoradiographic film for 20 days. In all cases, 125I-factor VII appeared intact both before and after reduction by beta -mercaptoethanol. There was no evidence of small molecular mass 125I-labeled products, although in three plasmas, some of the radioactivity in the load did not enter the gel.

Comparative release of factor VII and prothrombin from perfused rabbit liver ex vivo. ELISAs were used to measure the factor VII and prothrombin concentrations of liver perfusates. Livers from six healthy rabbits, perfused ex vivo over a 3-h period, liberated prothrombin significantly faster than factor VII (Fig. 3). By regression analysis, the mean slopes of the factor VII and prothrombin curves indicated that 0.03 nmol · l-1 · h-1 and 12.80 nmol · l-1 · h-1, respectively, were released over the 0.25- to 3-h interval, a molar ratio of 428:1. Analysis of the perfusate at 3 h by SDS-PAGE followed by immunoblotting revealed that prothrombin-related protein was circulating largely in a high molecular mass form (Mr, ~160-180 kDa), which possibly consists of vitronectin-thrombin-antithrombin ternary complexes (6), a trace of prothrombin (72 kDa), and smaller molecular mass prothrombin-related fragments (ranging from Mr 32 to 50 kDa; Fig. 4). In comparison, factor VII-related protein appeared to a small extent as two high molecular mass components (~90 and 120 kDa) but primarily as an ~50-kDa band (which appeared distorted, presumably by the high concentration of albumin) together with smaller molecular mass fragments (31-38 kDa).


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Fig. 3.   Mean data of factor VII (open circle )- and prothrombin ()-related proteins released from 6 rabbit livers during recirculation-perfusion over 3 h ex vivo.



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Fig. 4.   SDS-PAGE/Western blots of prothrombin- and factor VII-related proteins released from rabbit livers over 3 h ex vivo relative to prestained protein and purified factor VII and prothrombin standards. Blots were developed as described in the text against anti-rabbit prothrombin (A) and against anti-rabbit factor VII (B). A: lane 1, rabbit plasma; lane 2, rabbit prothrombin; lane 3, liver perfusate sampled at 0 min; lane 4, liver perfusate sampled after 3 h of perfusion. B: lane 1, rabbit plasma; lane 2, rabbit recombinant factor VII (Mr, 53 kDa); lane 3, liver perfusate sampled at 0 min; lane 4, liver perfusate sampled after 3 h of perfusion. The high molecular mass band in lanes 3 and 4 of B is an unknown contaminant of the perfusion solution. Molecular masses of prestained standards are in kDa.

In view of the range of perfusate products related to factor VII and to prothrombin, we considered it necessary to apply a correction for the relative rate of uptake of factor VII (as 125I-factor VII) and prothrombin (as 131I-prothrombin). With the use of four livers, the starting recirculation perfusates were dosed with similar molar quantities of 125I-factor VII and 131I-prothrombin, and perfusion was performed for 3 h. In all livers, 125I-factor VII (or its related products of metabolism) was cleared more rapidly than 131I-prothrombin (or its related products). The mean molar ratio of uptake of factor VII compared with prothrombin was 1.46:1, i.e., 1.46 molecules of factor VII were taken up by the liver for each molecule of prothrombin. With this value applied to correct the mean slopes of factor VII and prothrombin in Fig. 3, the rabbit liver ex vivo released 293 molecules of prothrombin for each molecule of factor VII.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Using radiolabeled plasma-derived proteins, we have described the relative turnovers of rabbit factor VII and prothrombin in NZW rabbits. To our knowledge, this is the first reported study of the dynamics of factor VII catabolism in rabbits. We have also measured the relative rates of release of factor VII and prothrombin by the perfused rabbit liver ex vivo to complement the catabolic study.

From data obtained in this study, all plasma curves for both proteins contained only three exponentials with the curve-stripping procedure. This observation suggested that the behaviors of both factor VII and prothrombin were best represented by a three-compartment model (5). Of two possible three-compartment models tested by Carlson et al. (3), the series (or catenate) model best described the behavior of a liver-synthesized hemostatic plasma protein, rabbit antithrombin-III (hereinafter termed "antithrombin"). In that study, the authors deduced that a substantial proportion of antithrombin was contained in association with the vascular wall, a conclusion that others have since verified using other approaches (11, 20, 31). We have also studied the in vivo behaviors of the alpha - and beta -glycoforms of rabbit antithrombin (30) and found good agreement with the results previously reported for the rabbit antithrombin glycoforms (4). In addition, we have used the same three-compartment method to compare the in vivo behaviors of antithrombin and prothrombin in the rabbit circulation (13). We consider that, in addition to antithrombin, other hemostatic factors including factor VII are also concentrated, relatively, at or within the vascular wall to support or control hemostasis.

Using a three-compartment catenate model (3), we have calculated the fractional catabolic rates and compartment distributions of factor VII and prothrombin in the rabbit. The distribution of 125I-factor VII at equilibrium revealed that only a relatively small proportion of factor VII (14%) is contained within the intravascular space compared with prothrombin (29%), the remaining factor VII being either bound to the endothelium (11%) or located within the extravascular space (75%). This profile of distribution of factor VII may be partly explained by the distribution of the principle cofactor for factor VII, tissue factor (TF). TF is not expressed constitutively by endothelial cells or by blood cells but is expressed constitutively as a cell membrane-binding site by many extravascular cells, particularly those surrounding organs and cells of the epidermis and in the adventitia of blood vessels (22). Although, under physiological conditions, factor VII may not be bound directly to TF at these extravascular locations, we speculate that the hemostatic system in the rabbit (and other mammals) has evolved to ensure that, should an injury occur, a supply of factor VII is located in close proximity to TF to maximize blood coagulation at the site of injury.

We have calculated the relative turnovers of factor VII and prothrombin as follows. With the assumption of a blood volume of 58 ml/kg, a healthy 3-kg rabbit has a calculated blood volume of 174 ml. For factor VII, blood concentration is 7.9 nmol/l (using a mol wt of 55,000, equivalent to 434.5 µg/l); then 174 ml of blood contains 75.6 µg of factor VII. By use of the value for Ap in Table 1, total factor VII in the 3-kg rabbit is 75.6/0.14, i.e., 540.0 µg. When jT for factor VII is applied, the rabbit catabolizes 540.0 × 0.34 µg/day, i.e., 183.6 µg/day. To sustain this loss of factor VII, the liver must release 183.6 µg/day (0.00335 µmol/day). A similar calculation for prothrombin, with a normal blood concentration of 1.05 µmol/l, indicates that the rabbit catabolizes 24.04 mg/day. Therefore, to maintain a constant concentration of prothrombin in rabbit blood, the liver must release 24.04 mg/day (equivalent to 0.335 µmol/day).From these measurements, we conclude that the molar ratio of prothrombin to factor VII released by the rabbit liver would be 0.335:0.00335, i.e., 100:1, or 100 prothrombin molecules released for each molecule of factor VII. For comparison, the molar ratio prothrombin/factor VII in rabbit blood is 1.05:0.0079, i.e., 133:1.

From Fig. 3, the mean net release rates of factor VII and prothrombin by the perfused liver ex vivo were 0.03 and 12.80 nmol · l-1 · h-1, respectively, i.e., a molar ratio of prothrombin to factor VII of 428:1. As factor VII (or its products) was taken up by the liver 1.46 times faster than prothrombin, the corrected molar ratio of prothrombin to factor VII released by the liver amounts to 293:1. There are several possible reasons for the apparent discrepancy between the calculated molar ratio, 100:1, from the catabolism study and the observed molar ratio, 293:1, obtained by liver perfusion. We assume that the liver is the only source of these proteins but that, ex vivo, the perfused freshly excised liver may not perform with the same efficiency as the liver in vivo. It is possible that iodination could alter properties of either protein and distort the observed clearance profile from the rabbit. As a response to this possible criticism, we radiolabeled prothrombin and factor VII by means of the 125I-labeled sulfo-SHPP method. However, the plasma clearance rates and distributions obtained for sulfo-SHPP-labeled 125I-factor VII and 125I-prothrombin were similar to those of factor VII and prothrombin radiolabeled using the Iodogen method (data not shown). Furthermore, the ELISAs used to quantify factor VII or prothrombin in liver perfusates could be variably sensitive to smaller molecular mass fragments of, or higher molecular mass complexes containing, these proteins. As shown in Fig. 4, 3-h perfusates contained both high molecular mass complexes and smaller molecular mass fragments of both factor VII and prothrombin compared with the native proteins. We assume that factor VII and prothrombin released into the perfusate ex vivo are either activated to VIIa and thrombin, respectively, to yield stable enzyme-inhibitor complexes or are cleaved proteolytically into fragments during perfusion. Although the capture antibodies used in both ELISAs were affinity-purified preparations, nothing is known of the efficiency of reaction between these polyspecific antibodies and either the complexes or fragments of factor VII and prothrombin. For this reason, we consider that the calculations based on the ELISAs are a potential weakness of our experimental approach. It should also be noted that the SE values of the data points in Fig. 3 are relatively large.

We conclude that the catabolic t1/2 of factor VII, 2.04 days, exceeds that of prothrombin (1.31 days) in rabbits. To our knowledge, there are no other direct animal or human studies of homologous factor VII metabolism for comparison with this study. Hasselback and Hjort (8) and van Oosterom and colleagues (28, 29) calculated a plasma half-life for factor VII in humans and rats as 5.7-5.9 and 3.3 h, respectively. These values were calculated from the rates of consumption of plasma factor VII as the human or rat was receiving treatment with warfarin, an inhibitor of posttranslational gamma -carboxylation of all vitamin K-dependent proteins. By comparison, Rivard et al. (25) measured the clearance of human plasma factor VII immunologically after a single infusion of the preparation (dose: 2.6 IU/kg; estimated infusion time: 12 min) into the circulation of congenitally factor VII-deficient patients. A "biologic half-life" of 6.5 h was reported for factor VII in this context. However, not one of these investigations has evaluated factor VII behavior in a blood circulation with a normal intra- and extravascular factor VII concentration and distribution. From our observations (see Clearance of 125I-factor VII and 131I-prothrombin from the circulation of normal rabbits), a relatively small proportion of factor VII is resident in the intravascular space, whereas a relatively large proportion occupies the extravascular space. In a patient being treated with warfarin, a small drop in the extravascular factor VII concentration may make a relatively high demand on, and result in a rapid decrease of, the depleting intravascular supply of factor VII. Furthermore, any possible pharmacological effects of warfarin on factor VII catabolism cannot be excluded.

Conceivably, factor VII metabolism in rabbits may differ significantly from that in rats and humans, although a comparison of the catabolic profiles and blood concentrations of other hemostatic proteins of the rabbit (10, 13-17) reveals that they are similar to those of a healthy human. But let us assume that the plasma clearance results obtained in the rabbit using 125I-factor VII do not mimic the behavior of native factor VII, and that native factor VII in the rabbit is catabolized with a half-life of, say, 6 h. Knowing that the blood concentrations of factor VII and prothrombin in rabbits are similar to those of humans, then, by making a calculation as above, we would estimate that prothrombin would need to be released from the rabbit liver at a 16:1 molar ratio compared with factor VII. This possibility was not supported by the liver perfusion results.

In summary, we have determined, from the plasma clearance of rabbit 125I-factor VII and rabbit 131I-prothrombin, new information about the dynamics of the behavior of factor VII in relation to prothrombin in rabbits. From the rates of catabolism and the fractional distributions of these two proteins, we calculated the rates of release of factor VII and prothrombin per day to maintain plasma levels. This was equivalent to 100 molecules of prothrombin released from the liver for each molecule of released factor VII. Our attempt to confirm this conclusion by analyzing rabbit liver perfusates was considered only partially successful, due, possibly, to technical problems in accurately quantifying the assortment of circulating factor VII-related and prothrombin-related products present in liver perfusates.


    ACKNOWLEDGEMENTS

We acknowledge the awards of operating grants from the Heart and Stroke Foundation of Canada (to M. W. C. Hatton, M. A. Blajchman, and B. J. Clarke), and from the Canadian Blood Services (to M. A. Blajchman and B. J. Clarke).


    FOOTNOTES

Address for reprint requests and other correspondence: M. W. C. Hatton, Dept. of Pathology (Room HSC-4N67), McMaster Univ. Health Sciences Centre, 1200 Main St. West, Hamilton ON, Canada, L8N 3Z5 (E-mail: hattonm{at}mcmaster.ca).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 2 November 2000; accepted in final form 19 April 2001.


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ABSTRACT
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DISCUSSION
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Am J Physiol Endocrinol Metab 281(3):E507-E515
0193-1849/01 $5.00 Copyright © 2001 the American Physiological Society




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