Introduction

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The synthetic AVP analog DDAVP is used in a variety of hemorrhagic disorders. It is used as an antihemorrhagic agent in hemophilia and in von Willebrand disease (Mannucci et al., 1977), and it has been used to reduce bleeding side effects caused by various compounds, including aspirin (Flordal and Sahlin, 1993), streptokinase (Johnstone et al., 1990), heparin (Shulman and Johnson, 1991) and hirudin (Bove et al., 1996). Despite the wide use of DDAVP in these clinical situations, the exact mechanism of DDAVP-induced release of hemostasis factors is not fully understood. DDAVP is a relatively V₂-specific AVP agonist with minimal smooth muscle activity and strong and prolonged antidiuretic action, but it also reveals AVP V_1a (Wun et al., 1995) and V₃ (also called V_1b) (Ammar et al., 1994) receptor agonist activity. DDAVP stimulates the release of FVIII, vWF and t-PA from endothelial cells (Abreg et al., 1979; Johnson et al., 1986; Lethagen, 1994). The DDAVP-induced clotting factor release has been postulated to involve extrarenal V₂-like receptors (Bichet et al., 1988). Reversal of the DDAVP clotting factor response by using selective V₂ receptor antagonists was employed as a method to test this hypothesis. However, only data with peptide V₂ receptor antagonists have been available, and reported effects of these substances on DDAVP-induced hemostasis factor release are controversial: SKF 105494 was found to be active in the monkey (Kinter et al., 1992), whereas d(CH₂)₅[D-lle²,lle⁴]AVP was inactive in the dog (Vilhardt and Barth, 1991). Peptide AVP V₂ receptor antagonists also showed interspecies differences in antagonizing the antidiuretic AVP action (Bichet et al., 1988). In addition, AVP analogs are not very selective V₂ antagonists, and chronically administered AVP analogs lose their antagonist properties and show an agonistic activity (Hofbauer et al., 1986). Furthermore, peptide AVP V₂ receptor antagonists are limited to parenteral use. SR 121463 and OPC 31260 are two novel, highly potent and selective nonpeptide antagonists for V₂ AVP receptors. They possess a high affinity for renal V₂ receptors, inhibit AVP-induced cAMP formation and reveal an aquaretic effect in rats, dogs and monkeys (Serradeil-Le Gal et al., 1996; Yamamura et al., 1992).

Animal models to investigate the mechanism of DDAVP-induced effects on the coagulation and fibrinolytic systems are lacking. Unlike humans, rats and pigs do not show a DDAVP-induced clotting factor response, but experiments with V₂ receptor agonists suggest that dogs and rhesus monkeys may be appropriate (Kinter et al., 1992; Vilhardt and Barth, 1991). The present experiments were designed to further characterize the mechanism of DDAVP-induced release of hemostasis factors in conscious dogs.

	Methods

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Drugs and dosage. DDAVP, oxytocin, d(CH₂)₅Tyr(Me)-AVP, bacitracine, d(CH₂)₅[D-lle²,lle⁴]AVP and D-Pal were from Sigma Chemical Co. (Lisle d'Abeau, France). BSA (type V) was obtained from IBF (Villeneuve La Garenne, France). EDTA, Tris and dimethylsulfoxide were from Merck-Clevenot (Nogent sur Marne, France). [³H]AVP (80 Ci/mmol) was purchased from New England Nuclear (Les Ulis, France). The V₂ receptor antagonist d(CH₂)₅[D-lle²,lle⁴]AVP was from Peninsula Lab Ltd. (Belmont, CA). The V_1a receptor antagonist SR 49059 (Serradeil-Le Gal et al., 1993), the V₂ receptor antagonists SR 121463 and OPC 31260 and the PAF receptor antagonist SR 27417 (Herbert et al., 1991) were from Sanofi Recherche (Toulouse, France). For the in vivo studies, DDAVP, d(CH₂)₅[D-lle²,lle⁴]AVP and SR 121463 were dissolved in saline. SR 49059 was dissolved in a solution containing ethanol, H₂O, glycerol and polyethylene glycol (60:30:5:5 v/v). SR 27417 and OPC 31260 were solubilized in 0.1 N HCl in saline (1:40 v/v). All substances were administered intravenously as solutions prepared daily before the administration. Control dogs were treated with saline. For in vitro binding experiments, SR 121463, SR 49059 and OPC-31260 were dissolved in dimethylsulfoxide (10² M) and then diluted in the test solvent.

Membrane preparations. Both kidneys from pentobarbital-anesthetized male mongrel dogs were chilled in ice-cold saline. The renomedullary regions, which constitutively express AVP V₂ receptors, were immediately dissected. Membranes were prepared according to the method of Stassen et al. (1982) as described recently (Serradeil-Le Gal et al., 1996) and stored as aliquots in liquid nitrogen at a final concentration of ~10 mg of protein/ml. Protein concentration was determined according to Bradford (1976) with BSA as a standard.

AVP V₂ binding assay. Renomedullary membranes (100-150 µg/assay) were incubated for 45 min at 25°C in a 50 mM Tris · HCl buffer, pH 8.1, containing 2 mM MgCl₂, 1 mM EDTA, 0.1% BSA, 0.1% bacitracin, 3 nM [³H]AVP and increasing amounts of the test compounds. The reaction was stopped by the addition of 4 ml of ice-cold buffer followed by filtration through GF/B Whatman glass microfiber filters. Filters were washed twice with 4 ml of ice-cold buffer and counted by liquid scintillation using a beta scintillation counter (Packard, Tricarb). Saturation experiments were performed with increasing concentrations of [³H]AVP (0.03-15 nM). Nonspecific binding was determined by incubation with 1 µM AVP. Data for equilibrium binding (K_d, apparent equilibrium dissociation constant, and B_max, maximum binding density) and competition experiments (IC₅₀, n_H) were analyzed by an iterative nonlinear regression program using the software RS.1 (Munson and Rodbard, 1980). The IC₅₀ value was defined as the concentration of inhibitor required to obtain 50% inhibition of the specific binding. Inhibition constant (K_i) values were calculated from the IC₅₀ values using the Cheng and Prusoff equation (1973).

Animals and procedures. Twelve-month-old male mongrel dogs weighing 18 to 26 kg were used. The animals were fed a standard laboratory chow (Doko, Fontaine-les-Vervins, France), and tap water was available ad libitum. Before the experiments, the dogs were fasted overnight. They were trained to stand quietly on a table. Blood (4 ml) was collected through venipuncture of cephalic veins immediately before and at indicated intervals after intravenous injections of DDAVP in tubes containing trisodium citrate (3.8%, 1/9 v/v) for measurements of vWF, FVIII and PAI-1 plasma concentrations. At <2 min after withdrawal, 300 µl of sodium acetate (0.2 M) was added to 300 µl of blood for determination of t-PA activity. Blood samples were immediately centrifuged at 4°C (1000 × g for 15 min). The plasma was kept at 80°C until use. Antagonists were injected intravenously 5 min before administration of DDAVP. Some dogs repeated the protocol with a minimum interval of 1 week between studies. In these dogs, no tolerance to DDAVP-induced release of hemostasis factors was observed.

Analytical methods. FVIII (VIII:C) levels in plasma were measured using an immunodepleted plasma from Diagnostica Stago (Asnières, France). vWF was measured by means of an enzyme immunosorbent assay procedure with the Asserachrom vWF kit (Diagnostica Stago, Asnières, France). Activity of t-PA was determined with Coaset t-PA by measuring the amidolytic activity of plasmin from the chromogenic substrate S-2251. The PAI plasma activity was determined with Coatest PAI (Chromogenix, Molndal, Sweden) by measuring the plasmin formed from plasminogen in the presence of t-PA from the chromogenic substrate S-2403.

Data analysis. Results are expressed as mean ± S.E.M. Statistical analyses were performed using the Mann-Whitney U test, and P < .05 was accepted as a significant difference. ID₅₀ values were calculated by fitting the logistic equations to the data by linear regression.

	Results

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Binding studies. AVP receptors in canine renomedullary membranes were characterized using [³H]AVP as a ligand. Saturation experiments, performed with increasing concentrations of [³H]AVP, showed that the specific binding was saturable. Scatchard analysis of the data (fig. 1) gave a linear plot consistent with the presence of a single class of high-affinity binding sites with a K_d and a B_max value of 0.32 ± 0.03 nM and 100 ± 4 fmol/mg of protein, respectively. A similar affinity has been reported with [³H]AVP in kidney preparations of rat, bovine and human origin (Manning et al., 1984; Serradeil-Le Gal et al., 1996; Yamamura et al., 1992).

View larger version (21K): [in this window] [in a new window]   Fig. 1.   Scatchard plot of [3H]AVP binding to canine renomedullary membranes. Saturation experiments were conducted in the presence of increasing concentrations of [3H]AVP (0.03-15 nM) and 140 µg/assay membranes as described in the text. Results shown are the mean of three experiments performed in triplicate.

DDAVP-induced release of hemostasis factors. In conscious dogs, initial plasma concentrations of FVIII and vWF immediately before DDAVP treatment amounted to 100.1 ± 7.2% and 97.7 ± 7.2% of the calibration curves of the test kits (n = 16). Initial plasma activities for t-PA and PAI were 6.9 ± 0.4 IU/ml and 32.0 ± 4.4 arbitrary units/ml, respectively (n = 16). In vehicle-treated controls, plasma levels of these four parameters were stable for the duration of the study (4 hr, n = 3). As shown in figure 2, DDAVP (0.1-1 µg/kg i.v.) induced a dose-related increase in plasma levels of FVIII, vWF and t-PA as measured 30 min after DDAVP administration. The time courses of the variations of FVIII, vWF and t-PA plasma levels after a single intravenous administration of 1 µg/kg DDAVP are illustrated in figure 3 (controls). Maximum increases in FVIII (43%), vWF (52%) and t-PA (50%) plasma levels were observed at 30 min after DDAVP administration. Plasma concentrations of FVIII returned to base-line values in a linear fashion within 4 hr. The decrease in vWF plasma concentrations was delayed. As a consequence, elevated vWF concentrations were still observed 4 hr after DDAVP administration (22%, P < .05). Plasma activity of t-PA returned to pretreatment values within 1 hr and tended to be lower than pretreatment values at 2 and 4 hr (14% and 21%, P > .05). No significant changes in PAI-1 plasma activity were recorded during the 4-hr observation period (data not shown).

View larger version (13K): [in this window] [in a new window]   Fig. 2.   Effect of DDAVP on plasma concentrations of FVIII, vWF and t-PA in conscious dogs. The indicated doses of DDAVP were administered i.v. After 30 min, blood samples were withdrawn, and FVIII, vWF and t-PA levels were measured as described in the text. The results are expressed as percent changes of plasma concentrations at 30 min compared with pretreatment values. * P < .05 compared with pretreatment values: n = 3-5 in the low- and medium-dose groups, n = 16-17 in the high-dose group.

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Effects of the V2 receptor antagonist SR 121463 on DDAVP-induced increases in plasma concentrations of FVIII, vWF and t-PA in conscious dogs. SR 121463 at doses of 10 (), 30 () or 100 µg/kg () or saline () were administered iv 5 min before DDAVP (1 µg/kg i.v.). Plasma concentrations of FVIII, vWF and t-PA levels were measured as described in the text (n = 3 in the SR 121463-treated groups, n = 16 in the control group). 100% refers to the values measured before DDAVP administration. For clarity, S.E.M. bars are presented for the saline-treated group only. * P < .05 compared with the groups treated with saline.

Influence of V_1a and V₂ receptor antagonism on DDAVP effects. Pretreatment with the V₂ receptor antagonist SR 121463 inhibited DDAVP-induced (1 µg/kg i.v.) increases in FVIII, vWF and t-PA plasma levels in a dose-dependent manner (fig. 3). ID₅₀ values for inhibition of DDAVP-induced increases in FVIII, vWF and t-PA at 30 min were 14.0 ± 4.0, 12.4 ± 3.0 and 16.7 ± 1.0 µg/kg i.v., respectively. Increases in plasma concentrations of these three proteins after DDAVP injection were completely eliminated at 100 µg/kg SR 121463 i.v.

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Comparison of the effects of three V2 receptor antagonists on DDAVP-induced increases in plasma concentrations of FVIII. Saline (), SR 121463 (100 µg/kg i.v., ), OPC 31260 (300 µg/kg i.v., ) or d(CH2)5[D-lle2,lle4]AVP (30 µg/kg i.v., ) were administered 5 min before DDAVP (1 µg/kg i.v.). Plasma concentrations of FVIII were measured as described in the text (n = 3 in the groups treated with the V2 receptor antagonists, n = 16 in the control group). 100% refers to the values measured before DDAVP administration. * P < .05 compared with the groups treated with saline.

View larger version (16K): [in this window] [in a new window]   Fig. 5.   Effect of the V1a receptor antagonist SR 49059 and of the PAF receptor antagonist SR 27417 on DDAVP-induced increases in plasma concentrations of FVIII, vWF and t-PA. Saline (), SR 49059 (1 mg/kg i.v., ) or SR 27417 (1 mg/kg i.v., ) were administered 5 min before DDAVP (1 µg/kg i.v.). Plasma concentrations of FVIII, vWF and t-PA were measured as described in the text (n = 3 in the groups treated with SR 49059 or SR 27417, n = 16 in the control group). 100% refers to the values measured before DDAVP administration. Differences between the SR 49059- or SR 27417-treated groups and the control group were not significant (P > .05).

Influence of PAF receptor antagonism on DDAVP effects. Another series of in vivo experiments was performed to examine whether PAF was involved in the DDAVP-induced release of hemostasis factors as suggested by Hashemi et al. (1993). At 5 min before DDAVP (1 µg/kg i.v.) administration, dogs were pretreated i.v. with 1 mg/kg of the PAF receptor antagonist SR 27417. As can be seen in figure 5, SR 27417 pretreatment did not modify DDAVP-induced increases in FVIII, vWF and t-PA levels in plasma. At this high dose, SR 27417 alone did not influence the measured parameters during the 4-hr observation period.

	Discussion

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The present results confirm that the intravenous administration of DDAVP induces a release of FVIII, vWF and t-PA in conscious dogs. Comparable increases in plasma levels of these proteins with similar kinetics have been previously described in conscious dogs (Vilhardt and Barth, 1991) and anesthetized monkeys (Kinter et al., 1992), whereas humans are more sensitive to DDAVP-induced release of vWF and t-PA (Mannucci et al., 1975). The possible influence of DDAVP on PAI-1 plasma activity has not previously been reported. Present results in conscious dogs do not demonstrate evidence of a involvement of PAI-1 in the effect of DDAVP to release hemostasis factors.

SR 121463 and OPC 31260, two novel, selective nonpeptide antagonists for V₂ receptors, possess a high affinity for renal V₂ receptors and inhibit AVP-induced cAMP formation (Manning et al., 1984; Serradeil-Le Gal et al., 1996; Yamamura et al., 1992). Both compounds also revealed potent in vivo activities: OPC 31260 exerted an aquaretic effect in rats, dogs, monkeys and humans, and SR 121463 showed aquaretic activity in rats (Fujisawa et al., 1993; Serradeil-Le Gal et al., 1996; Yamamura et al., 1992). In the present study, SR 121463 inhibited DDAVP-induced releases of FVIII and vWF in a dose-dependent manner with ID₅₀ values of 14.0 ± 4.0 and 12.4 ± 3.0 µg/kg i.v., respectively. OPC 31260 revealed a lower activity than SR 121463 in inhibiting DDAVP-induced clotting factor release. In addition, the present study is the first demonstration of an inhibition of DDAVP-induced t-PA release via V₂ receptor antagonism.

The inhibitory actions of SR 121463 and OPC 31260 on the DDAVP-induced clotting factor response are in agreement with observations of Kinter et al. (1992), who reported total prevention of DDAVP-induced clotting factor release by the peptide V₂ receptor antagonist SK&F 105494 in rhesus monkeys. Contrary to this, Vilhardt and Barth (1991) reported failure to block DDAVP-induced release of FVIII and t-PA by the V₂ receptor antagonist d(CH₂)₅[D-lle²,lle⁴]AVP in dogs and concluded that the ability of DDAVP to release FVIII and t-PA does not involve V₂ receptors. This conclusion, however, must be considered premature. Despite close structural similarities between AVP V₂ receptors cloned in several species such as rat, pig, cow and humans (Lolait et al., 1994; Birnbaumer et al., 1992; Gorbulev et al., 1993), marked interspecies differences exist for AVP V₂ receptors on the basis of affinity and efficacy of certain AVP analogs (Ufer et al., 1995; Guillon et al., 1982). Peptide V₂ receptor antagonists with branched side-chain amino acid substitutions at positions 2 and 4, such as d(CH₂)₅[D-lle²,lle⁴]AVP, show substantial interspecies variability in affinity for renal V₂ receptors; they are inactive as antidiuretic antagonists in dogs (Kinter et al., 1992). In line with these findings are the present in vivo observations that d(CH₂)₅[D-lle²,lle⁴]AVP did not reveal an inhibitory effect on DDAVP-induced release of hemostasis factors. These data suggest a low affinity of d(CH₂)₅[D-lle²,lle⁴]AVP to canine V₂ receptors.

The present in vitro results with canine renomedullary membrane preparations confirm this conclusion. The affinity of d(CH₂)₅[D-lle²,lle⁴]AVP was more than 2 orders of magnitude lower than that of SR 121463. d(CH₂)₅[D-lle²,lle⁴]AVP strongly discriminates between dog and rat V₂ receptors because this compound, which is well known for its potent anti-V₂ diuretic properties in rats (pA₂ = 8.04), was found in this study to be ~100-fold less potent at dog (K_i = 122 nM) than at rat (K_i = 1.1 nM) V₂ receptors (Manning et al., 1984). The present in vitro findings also give a good explanation for the differences in in vivo activity between SR 121463 and OPC 31260 to inhibit the release of hemostasis factors. The observed affinity of OPC 31260 in this study is in agreement with previous results obtained in rat, bovine and human preparations (Serradeil-Le Gal et al., 1996).

Although the agonist activity of DDAVP is generally considered to be V₂ mediated, DDAVP also possesses V_1a and V₃ receptor agonist activity (Wun et al., 1995; Ammar et al., 1994). A V_1a receptor-mediated mechanism has been described for DDAVP-induced platelet activation (Wun et al., 1995). In the current model, however, the V_1a receptor antagonist SR 49059 had no effect on the DDAVP-induced increases in FVIII, vWF and t-PA plasma levels. Taken together, these data strongly suggest that the stimulation of extrarenal V₂-like receptors is the main mechanism involved in the DDAVP-induced release of FVIII, vWF and t-PA. This conclusion is also in agreement with the clinical findings that male patients with congenital X chromosome-linked nephrogenic diabetes insipidus do not exhibit a rise in FVIII plasma concentrations when treated with DDAVP (Kobrinsky et al., 1985). Diuresis in these patients is due to mutation of renal V₂ receptors; perhaps the missing DDAVP-clotting factor response is due to the same mutation of extrarenal V₂(-like) receptors. There is no evidence as yet to the location of the V₂ AVP receptors that are involved in the DDAVP response and intracellular signaling mechanisms. Because bilateral nephrectomy did not prevent the effect of DDAVP in the present experiments, it appears that the receptors responsible for the increase in hemostasis factors are extrarenal. Further evidence for the existence of extrarenal V₂ receptors has been previously provided. The DDAVP-induced releases of cAMP in anephric dogs and of hemostasis factors in surgically anephric patients were not different from those in the control groups with intact kidneys (Liard, 1992; Mannucci et al., 1975). It is tempting to speculate that the extrarenal V₂ receptors accounting for the DDAVP-induced increase in hemostasis factors might be on endothelial cells; however, there is no proof of the existence of V₂ receptors on endothelial cells.

The involvement of PAF in the DDAVP-clotting factor response has been postulated by Hashemi et al. (1993). Their in vitro findings indicated that the stimulation of endothelial cells by DDAVP may be an indirect effect mediated through stimulation of a monocyte V₂ receptor. In turn, PAF would be secreted by DDAVP-treated monocytes and enhance the release of vWF from endothelial cells. Present in vivo experiments with the PAF receptor antagonist SR 27417, however, do not indicate an involvement of such a mechanism in the DDAVP-induced release vWF or in the release of FVIII and t-PA.

In summary, the present in vivo results in conscious dogs, together with the in vitro findings in canine renomedullary membranes, strongly support the conclusion that the effect of DDAVP on hemostasis factors occurs via a specific interaction with extrarenal V₂ or V₂-like receptors, whose localization remains to be further explored. The present data argue that the effect of DDAVP does not involve PAF or V_1a receptors.