• View in gallery
    Figure 1—

    Mean ± SE percentage aggregation of feline platelets induced by ADP (A) and collagen (B). Each value represents results for blood samples from 6 cats. Saline (0.9% NaCl) solution was included in all assays as a negative control agent. *Value differs significantly (P < 0.05) from the value for the control agent.

  • View in gallery
    Figure 2—

    Mean ± SE percentage aggregation of feline platelets. Each value represents results for blood samples from 6 cats. A—Citrated feline plasma was incubated with various concentrations of adrenaline, noradrenaline, oxymetazoline, xylometazoline, or saline solution (negative control agent) for 1 minute before the addition of ADP (0.5 μmol/L). B—Citrated feline plasma was incubated with adrenaline (100 μmol/L), noradrenaline (100 μmol/L), oxymetazoline (10 μmol/L), xylometazoline (10 μmol/L), or saline solution for 1 minute before the addition of various concentrations of ADP (0.1 to 10 μmol/L). C— Citrated feline plasma was incubated with various concentrations of α-adrenoceptor agonists for 1 minute before the addition of ADP (0.5 μmol/L). D—Citrated feline plasma was incubated with various concentrations of adrenaline and saline solution for 1 minute before the addition of collagen (0.5 to 1.0 μg/mL). In panels A, C, and D, the mean ± SE value for the negative control solution is 6.2 ± 1.2% to 8.9 ± 1.7%, 5.5 ± 1.1% to 10.0 ± 3.0%, and 14.3 ± 1.1%, respectively. Notice that the scale on the y-axis of panel A differs from that of panels B, C, and D. See Figure 1 for remainder of key.

  • View in gallery
    Figure 3—

    Mean ± SE percentage aggregation of feline platelets induced by adrenaline (100 μmol/L) and ADP (1 μmol/L) for imidazoline and nonimidazoline α -adrenoceptor antagonists (A and B) and agonists (C and D). Each value represents results for blood samples from 6 cats. Each agent was added to citrated feline plasma. Adrenaline was added 0.5 minutes later, and ADP was added 0.5 minutes after that. Values are reported as a percentage of the value for the control agent (percentage aggregation of adrenaline-ADP with saline solution was assigned a value of 100%). See Figure 1 for remainder of key.

  • View in gallery
    Figure 4—

    Mean ± SE percentage aggregation of feline platelets induced by adrenaline (10 μmol/L) and collagen (1 to 3 μg/mL) for imidazoline and nonimidazoline α-adrenoceptor antagonists (A and B) and agonists (C and D). Each value represents results of blood samples from 4 or 5 cats. Each agent was added to citrated feline plasma. Adrenaline was added 0.5 minutes later, and collagen was added 0.5 minutes after that. Values are reported as a percentage of the value for the control agent (percentage aggregation of adrenaline-collagen with saline solution was assigned a value of 100%). See Figure 1 for remainder of key.

  • View in gallery
    Figure 5—

    Mean ± SE percentage aggregation of feline platelets induced by adrenaline (10 μmol/L) and collagen (1 to 3 μg/mL) for α2-adrenoceptor agonists and antagonists (A) and the combination of agonists and antagonists (B). Each value represents results for blood samples from 4 or 5 cats. Each agent was added to citrated feline plasma. Adrenaline was added 0.5 minutes later, and collagen was added 0.5 minutes after that. Values are reported as a percentage of the value for the control agent (percentage aggregation of adrenaline-collagen with saline solution was assigned a value of 100%). See Figure 1 for remainder of key.

  • View in gallery
    Figure 6—

    Mean ± SE percentage aggregation of feline platelets induced by ADP (10 μmol/L) alone for phentolamine, atipamezole, and yohimbine. Each value represents results for blood samples from 6 cats. Each agent was added 1 minute before the addition of ADP. Values are reported as a percentage of the value for the control agent (percentage aggregation of ADP with saline solution was assigned a value of 100%). See Figure 1 for remainder of key.

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Effects of imidazoline and nonimidazoline α-adrenoceptor agonists and antagonists, including xylazine, medetomidine, dexmedetomidine, yohimbine, and atipamezole, on aggregation of feline platelets

Takuya Matsukawa1United Graduate School of Veterinary Science, Yamaguchi University, Yamaguchi 753–8515, Japan.
2Joint Department of Veterinary Medicine, Faculty of Agriculture, Tottori University, Tottori 680–8553, Japan.

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Yoshiaki Hikasa2Joint Department of Veterinary Medicine, Faculty of Agriculture, Tottori University, Tottori 680–8553, Japan.

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Abstract

OBJECTIVE

To examine the effects of imidazoline and nonimidazoline α-adrenergic agents on aggregation of feline platelets.

SAMPLE

Blood samples from 12 healthy adult cats.

PROCEDURES

In 7 experiments, the effects of 23 imidazoline and nonimidazoline α-adrenoceptor agonists or antagonists on aggregation and antiaggregation of feline platelets were determined via a turbidimetric method. Collagen and ADP were used to initiate aggregation.

RESULTS

Platelet aggregation was not induced by α-adrenoceptor agonists alone. Adrenaline and noradrenaline induced a dose-dependent potentiation of ADP- or collagen-induced aggregation. Oxymetazoline and xylometazoline also induced a small potentiation of ADP-stimulated aggregation, but other α-adrenoceptor agonists did not induce potentiation. The α2-adrenoceptor antagonists and certain imidazoline α-adrenergic agents including phentolamine, yohimbine, atipamezole, clonidine, medetomidine, and dexmedetomidine inhibited adrenaline-potentiated aggregation induced by ADP or collagen in a dose-dependent manner. The imidazoline compound antazoline inhibited adrenaline-potentiated aggregation in a dose-dependent manner. Conversely, α1-adrenoceptor antagonists and nonimidazoline α-adrenergic agents including xylazine and prazosin were ineffective or less effective for inhibiting adrenaline-potentiated aggregation. Moxonidine also was ineffective for inhibiting adrenaline-potentiated aggregation induced by collagen. Medetomidine and xylazine did not reverse the inhibitory effect of atipamezole and yohimbine on adrenaline-potentiated aggregation.

CONCLUSIONS AND CLINICAL RELEVANCE

Adrenaline-potentiated aggregation of feline platelets may be mediated by α2-adrenoceptors, whereas imidazoline agents may inhibit in vitro platelet aggregation via imidazoline receptors. Imidazoline α-adrenergic agents may have clinical use for conditions in which there is platelet reactivity to adrenaline. Xylazine, medetomidine, and dexmedetomidine may be used clinically in cats with minimal concerns for adverse effects on platelet function.

Abstract

OBJECTIVE

To examine the effects of imidazoline and nonimidazoline α-adrenergic agents on aggregation of feline platelets.

SAMPLE

Blood samples from 12 healthy adult cats.

PROCEDURES

In 7 experiments, the effects of 23 imidazoline and nonimidazoline α-adrenoceptor agonists or antagonists on aggregation and antiaggregation of feline platelets were determined via a turbidimetric method. Collagen and ADP were used to initiate aggregation.

RESULTS

Platelet aggregation was not induced by α-adrenoceptor agonists alone. Adrenaline and noradrenaline induced a dose-dependent potentiation of ADP- or collagen-induced aggregation. Oxymetazoline and xylometazoline also induced a small potentiation of ADP-stimulated aggregation, but other α-adrenoceptor agonists did not induce potentiation. The α2-adrenoceptor antagonists and certain imidazoline α-adrenergic agents including phentolamine, yohimbine, atipamezole, clonidine, medetomidine, and dexmedetomidine inhibited adrenaline-potentiated aggregation induced by ADP or collagen in a dose-dependent manner. The imidazoline compound antazoline inhibited adrenaline-potentiated aggregation in a dose-dependent manner. Conversely, α1-adrenoceptor antagonists and nonimidazoline α-adrenergic agents including xylazine and prazosin were ineffective or less effective for inhibiting adrenaline-potentiated aggregation. Moxonidine also was ineffective for inhibiting adrenaline-potentiated aggregation induced by collagen. Medetomidine and xylazine did not reverse the inhibitory effect of atipamezole and yohimbine on adrenaline-potentiated aggregation.

CONCLUSIONS AND CLINICAL RELEVANCE

Adrenaline-potentiated aggregation of feline platelets may be mediated by α2-adrenoceptors, whereas imidazoline agents may inhibit in vitro platelet aggregation via imidazoline receptors. Imidazoline α-adrenergic agents may have clinical use for conditions in which there is platelet reactivity to adrenaline. Xylazine, medetomidine, and dexmedetomidine may be used clinically in cats with minimal concerns for adverse effects on platelet function.

Platelet responses to catecholamines and other α-adrenergic agents differ widely among animal species.1–3 In humans, adrenaline- and noradrenaline-induced platelet aggregation is mediated by α2-adrenoceptors; this aggregation is blocked by α2-adrenoceptor antagonists but not by α1-adrenoceptor antagonists.4–7 Radioligand binding studies have revealed the existence of α2-adrenoceptors on platelet membranes of dogs, cats, rabbits,8 and humans9–12; however, there is a lack of α2-adrenoceptors on platelet membranes of rats, cattle, and horses.8 Adrenaline is considered a rather weak platelet agonist, the function of which is primarily to sensitize platelets to other activating agents in humans.13,14 Adrenaline alone does not induce platelet aggregation in dogs,15 cats,16,17 and rabbits,18 but it does potentiate platelet aggregation stimulated by other platelet agonists such as ADP, collagen, and thrombin. Adrenaline-potentiated platelet aggregation is also mediated by α2-adrenoceptors in dogs15 and rabbits.18 Conversely, in contrast to humans, dogs, and rabbits, adrenaline in cattle and horses does not potentiate platelet aggregation induced by other platelet agonists such as ADP, collagen, thrombin, or platelet-activating factor.19–22 Physiologic concentrations of adrenaline enhance shear-dependent platelet aggregation and platelet-to-platelet interactions on collagen.23,24

Overactivity of the sympathetic nervous system and high circulating catecholamine concentrations are induced in conditions such as pheochromocytoma,25 endotoxic shock,26,27 and acute stress28 in cats. Hypercatecholaminemia reportedly has an influence on hemostasis (eg, disseminated intravascular coagulation29 and thromboembolism30–32) through actions on platelets. Information about drugs that inhibit platelet aggregation stimulated by catecholamines may also be useful for the control and management of hemostasis in diseases or conditions associated with hypercatecholaminemia in small animals.

The imidazoline chemical structure is found in many pharmaceutical drugs with a variety of biological activities, including antifungal (eg, miconazole), antihypertensive (eg, losartan), antiulcer (eg, cimetidine), and antiplatelet agents that inhibit thromboxane A2 synthesis (eg, ozagrel). Some adrenoceptor agents have imidazoline-like chemical structures. Clonidine, an imidazole α2-adrenoceptor agonist, has a complex effect on platelets. For example, clonidine binds with a high affinity to α2-adrenoceptors on platelets but induces only limited platelet aggregation, in contrast to the effect of endogenous agonists such as adrenaline.4,5 Moreover, clonidine potentiates ADP-induced aggregation of human platelets but has inhibitory activity for adrenaline- and noradrenaline-induced platelet aggregation.10,33

Although the mechanism underlying these conflicting actions of clonidine is unclear, imidazoline agents may interact with non–α2-adrenoceptor binding sites on platelets.34–36 Two clonidine-related drugs reportedly inhibit platelet adenylate cyclase through non–α2-adrenoceptor mechanisms because their effects are not blocked by yohimbine.37 In addition, a clonidine-displacing substance extracted from bovine brain tissue38 is recognized as a noncatecholamine endogenous ligand and interacts with nonadrenoceptor sites in the brainstem, which was determined via the use of tritiated p-aminoclonidine.39 Nonadrenergic I1 and I2 receptors that are pharmacologically distinct from α2-adrenoceptors have been detected in human,40–43 canine, feline, bovine, and equine platelets.8 Furthermore, canine, feline, bovine, and equine platelets have I1 receptors that are defined by binding to tritiated clonidine, but murine and leporine platelets do not have I1 receptors. Conversely, platelets of all species have I2 receptors that are defined by binding to tritiated idazoxan.8 In addition, the density of I1 and I2 receptors and α2-adrenoceptors differs among animal species.8

These variations for receptors may reflect differences among animal species regarding the platelet aggregation response. However, there is no information available concerning the platelet aggregatory effects of α-adrenergic agents in cats. Comparative studies of the effects of imidazolines on aggregation of feline platelets may be important for the characterization of platelet receptors and may be useful to elucidate the function of imidazoline receptors.

In cats, α2-adrenoceptor agonists such as xylazine, medetomidine, and dexmedetomidine are widely used as sedative, analgesic, and muscle relaxant agents, whereas α2-adrenoceptor antagonists such as atipamezole and yohimbine are often used to reverse the effects of the aforementioned agonists. These drugs differ in that medetomidine, dexmedetomidine, and atipamezole have imidazoline-like chemical structures, whereas xylazine and yohimbine do not. It also may be important to determine whether imidazoline structures affect the response of platelets. Therefore, the objective of the study reported here was to investigate the effects of various imidazoline and nonimidazoline α-adrenergic agents on in vitro aggregation and antiaggregation of feline platelets.

Materials and Methods

Sample

Twelve healthy adult mixed-breed cats (8 males and 4 females) provided blood samples for platelet aggregation experiments. Cats were 2 to 7 years of age and comprised 8 males and 4 females; body weight ranged from 3.2 to 5.0 kg. They were housed in a laboratory with appropriate animal management facilities and fed a standard commercial dry food; water was available ad libitum.

Prior to each sample collection, cats were examined (physical examination and hematologic analysis) to ensure that they were healthy. The study protocol was approved by the Animal Research Committee of Tottori University.

Sample collection and preparation of citrated platelet plasma

Food was withheld from cats for at least 6 hours before blood sample collection. Jugular blood samples (9 mL) were collected with a 21-gauge needle into a 10-mL plastic syringe containing 3.8% sodium citrate solution (ratio, 1 part anticoagulant to 9 parts blood). Samples were repeatedly collected from the cats at intervals of ≥ 2 weeks until all experiments were completed.

Citrated platelet plasma was prepared in accordance with a modification of methods described elsewhere.15,17 Blood samples were centrifuged at 90 to 110 × g for 10 to 15 minutes to obtain PRP. The PPP was then obtained by centrifuging the citrated blood at 1,500 × g for 15 minutes. The final platelet count was adjusted to 25 to 30 × 104 platelets/μL via dilution with autologous PPP.

Aggregation experiments

The study consisted of 7 platelet aggregation experiments, which were performed as previously described.5,15,18,22 Briefly, a turbidimetric method was used. An aliquot (200 μL) of PRP was placed in an aggregometera at 37°C, and an aliquot (22 μL) of test agent was added to the PRP 1 minute later. The percentage aggregation was standardized via the assumption that PPP and PRP represented 100% and 0% light transmission, respectively.

Drugs used in the study included L-adrenaline,b L-noradrenaline,b phenoxybenzamine HCl,b p-aminoclonidine HCl,c antazoline HCl,c clonidine HCl,c idazoxan HCl,c methoxamine HCl,c moxonidine HCl,c naphazoline HCl,c oxymetazoline HCl,c phentolamine HCl,c L-phenylephrine HCl,c prazosin HCl,c tolazoline HCl,c xylazine HCl,c xylometazoline HCl,c yohimbine HCl,c tramazoline HCl,d atipamezole HCl,e detomidine HCl,e medetomidine HCl,e dexmedetomidine HCl,f ADP,g and collagen.g Adrenaline and noradrenaline were dissolved in 0.04M HCl solution and then diluted with sterile saline (0.9% NaCl) solution. Prazosin and phenoxybenzamine were dissolved in sterile distilled water and then diluted with sterile saline solution. All other drugs were dissolved in sterile saline solution. In addition, sterile saline solution was used as a negative control agent throughout the experiments.

Both prazosin and phenoxybenzamine could be dissolved in sterile distilled water at concentrations up to 100 μmol/L, but both agents at higher concentrations (1 mmol/L) became cloudy and could not be completely dissolved in distilled water. Because cloudy solutions influence the percentage aggregation on the basis of light transmission, we did not determine percentage aggregation of both agents at 1 mmol/L. In addition, we did not determine the percentage aggregation of higher concentrations of medetomidine (1 mmol/L) and dexmedetomidine (0.1 to 1 mmol/L) because we used the drug solution products rather than drug powders for both those agents.

The drugs were categorized as α-adrenoceptor agonists (adrenaline, noradrenaline, clonidine, p-aminoclonidine, xylazine, medetomidine, detomidine, dexmedetomidine, oxymetazoline, xylometazoline, moxonidine, tramazoline, naphazoline, phenylephrine, and methoxamine), α-adrenoceptor antagonists (yohimbine, phentolamine, atipamezole, idazoxan, tolazoline, phenoxybenzamine, and prazosin), imidazoline α-adrenoceptor agonists (clonidine, p-aminoclonidine, medetomidine, detomidine, dexmedetomidine, oxymetazoline, xylometazoline, moxonidine, tramazoline, and naphazoline), imidazoline α-adrenoceptor antagonists (phentolamine, atipamezole, idazoxan, and tolazoline), nonimidazoline α-adrenoceptor agonists (adrenaline, noradrenaline, xylazine, phenylephrine, and methoxamine), nonimidazoline α-adrenoceptor antagonists (yohimbine, phenoxybenzamine, and prazosin), and imidazoline non–α-adrenoceptor agonists (antazoline).

In experiment 1, the platelet aggregation effects of α-adrenergic agents alone were evaluated. An aliquot of PRP was placed in the aggregometer; 1 minute later, 21 α-adrenoceptor agonists or antagonists (adrenaline, noradrenaline, clonidine, xylazine, medetomidine, detomidine, dexmedetomidine, oxymetazoline, xylometazoline, moxonidine, tramazoline, naphazoline, phenylephrine, methoxamine, antazoline, yohimbine, phentolamine, atipamezole, idazoxan, tolazoline, and prazosin) at final concentrations of 0.1 nmol/L to 1 mmol/L (except for medetomidine at 1 mmol/L, dexmedetomidine at 0.1 to 1 mmol/L, and prazosin at 1 mmol/L) were added to the PRP (time 0), and the maximum percentage aggregation was recorded during the subsequent 10-minute interval.

In experiment 2, the aggregation effects of ADP or collagen were examined. An aliquot of PRP was placed in the aggregometer; 1 minute later, ADP (0 to 10 μmol/L) or collagen (0 to 10 μg/mL) was added to the PRP (time 0), and the maximum percentage aggregation was recorded during the subsequent 10-minute interval.

In experiment 3, the stimulatory or inhibitory effects of α-adrenergic agents on ADP- or collagen-induced aggregation were examined. An aliquot of PRP was placed in the aggregometer; 1 minute later, 12 α-adrenergic agents (adrenaline, noradrenaline, oxymetazoline, xylometazoline, clonidine, medetomidine, detomidine, xylazine, naphazoline, tramazoline, phenylephrine, and methoxamine) at final concentrations of 1 nmol/L to 1 mmol/L, except for medetomidine at 1 mmol/L, were added and 1 minute after that, ADP (0.5 μmol/L) or collagen (0.5 or 1 μg/mL) was added (time 0). In addition, dose-dependent effects of ADP on platelet aggregation were evaluated. Adrenaline (100 μmol/L), noradrenaline (100 μmol/L), oxymetazoline (10 μmol/L), or xylometazoline (10 μmol/L) were added to PRP; 1 minute later, ADP (0.1 to 10 μmol/L) was added (time 0). The maximum percentage aggregation during the 10-minute interval after the addition of ADP or collagen was recorded.

In experiment 4, the inhibitory effects of imidazoline and nonimidazoline α-adrenergic agents on platelet aggregation induced by adrenaline and ADP were examined. An aliquot of PRP was placed in the aggregometer; 1 minute later, 19 agents (yohimbine, atipamezole, idazoxan, phentolamine, tolazoline, phenoxybenzamine, prazosin, naphazoline, tramazoline, xylometazoline, antazoline, clonidine, medetomidine, oxymetazoline, detomidine, p-aminoclonidine, phenylephrine, xylazine, and methoxamine) at final concentrations of 1 nmol/L to 1 mmol/L (except for medetomidine, phenoxybenzamine, and prazosin at 1 mmol/L) were added. Then 0.5 minutes later, adrenaline (100 μmol/L) was added, and 0.5 minutes after that, ADP (1 μmol/L) was added (time 0). The maximum percentage aggregation was determined during the 10-minute interval after the addition of ADP.

In experiment 5, the inhibitory effects of imidazoline and nonimidazoline α-adrenergic agents on platelet aggregation induced by adrenaline and collagen were examined. An aliquot of PRP was placed in the aggregometer; 1 minute later, 14 agents (yohimbine, atipamezole, idazoxan, phentolamine, tolazoline, prazosin, naphazoline, antazoline, clonidine, medetomidine, oxymetazoline, dexmedetomidine, moxonidine, and xylazine) at final concentrations of 1 nmol/L to 100 μmol/L, except for dexmedetomidine at 100 μmol/L, were added. Adrenaline (10 μmol/L) was added 0.5 minutes later, and collagen (1, 2, or 3 μg/mL) was added 0.5 minutes after that (time 0). The maximum percentage aggregation was determined during the 10-minute interval after the addition of collagen.

In experiment 6, the effects of a combination of α2-adrenoceptor agonists and antagonists on platelet aggregation induced by adrenaline and collagen were examined. An aliquot of PRP was placed in the aggregometer; 1 minute later, 2 agonists (yohimbine and atipamezole), 2 antagonists (xylazine and medetomidine), and their combinations were added (final concentrations, 1 nmol/L to 100 μmol/L). Adrenaline (10 μmol/L) was added 0.5 minutes later, and collagen (1, 2, or 3 μg/mL) was added 0.5 minutes after that (time 0). The maximum percentage aggregation was determined during the 10-minute interval after the addition of collagen.

In experiment 7, the effects of α-adrenoceptor antagonists on platelet aggregation induced by ADP were examined. An aliquot of PRP was placed in the aggregometer; 1 minute later, 3 antagonists (phentolamine, atipamezole, and yohimbine) at final concentrations of 1 nmol/L to 100 μmol/L were added. Then, 1 minute later, ADP (10 μmol/L) was added (time 0). The maximum percentage aggregation was determined during the 10-minute interval after the addition of ADP.

Statistical analysis

Statistical analysis was performed with commercially available software.h Data are reported as the mean ± SE.

To determine the inhibitory effect of the agents on platelet aggregation, the IC50 was obtained from the concentration-response curve. The IC50, ED50, and percentage aggregation data were assessed for normality of distribution with the Shapiro-Wilk test. When the data were normally distributed, the Student t test was used for comparisons between agents. When the data were not normally distributed, the Wilcoxon-Mann-Whitney test was used to determine significant differences. The paired t test was used to determine significant differences for change in percentage aggregation that were expressed as a percentage of the value for the control agent, which was assigned a value of 100%. For all tests, differences were considered significant at values of P < 0.05.

Results

Effects of α-adrenergic agents on aggregation of feline platelets (experiment 1)

None of the 21 agents tested at concentrations ranging from 0.1 nmol/L to 1 mmol/L elicited aggregation in feline platelets (data not shown).

Effects of ADP or collagen on aggregation of feline platelets (experiment 2)

Both ADP and collagen induced aggregation of feline platelets in a dose-dependent manner (Figure 1). The aggregatory effects of ADP at concentrations exceeding 0.5 μmol/L or of collagen at concentrations exceeding 0.5 μg/mL were significantly different from those of the control agent (saline solution). On the basis of these results, the concentration close to the submaximal amount of aggregation (< 25%) of ADP (0.5 μmol/L) or collagen (0.5 to 1 μg/mL) was chosen to examine the stimulatory effects of α-adrenergic agents on ADP- or collagen-induced platelet aggregation. By contrast, an ADP concentration (10 μmol/L) that induced almost complete platelet aggregation (> 70% aggregation) was chosen to examine the inhibitory effects of α-adrenergic agents on ADP-induced platelet aggregation.

Figure 1—
Figure 1—

Mean ± SE percentage aggregation of feline platelets induced by ADP (A) and collagen (B). Each value represents results for blood samples from 6 cats. Saline (0.9% NaCl) solution was included in all assays as a negative control agent. *Value differs significantly (P < 0.05) from the value for the control agent.

Citation: American Journal of Veterinary Research 81, 2; 10.2460/ajvr.81.2.159

Effects of α-adrenoceptor agonists on aggregation of feline platelets stimulated by ADP or collagen (experiment 3)

Adrenaline and noradrenaline at concentrations of 1 μmol/L to 1 mmol/L potentiated in a dose-dependent manner the platelet aggregation stimulated by a low dose (0.5 μmol/L) of ADP (Figure 2). The maximum and full aggregatory effect for the mean percentage aggregation was observed with adrenaline and noradrenaline at a concentration of 1 mmol/L. However, there was no significant difference in mean aggregation values between adrenaline concentrations of 100 μmol/L and 1 mmol/L or between noradrenaline concentrations of 100 μmol/L and 1 mmol/L. The mean percentage aggregation for adrenaline concentrations of 100 μmol and 1 mmol/L was significantly greater than for adrenaline at a concentration of 10 μmol/L. A small but significantly different potentiation of the ADP-stimulated platelet aggregation was also observed in response to oxymetazoline and xylometazoline at concentrations of 1 to 100 μmol/L, and the maximum aggregatory effect was observed at an oxymetazoline concentration of 1 μmol/L and a xylometazoline concentration of 10 μmol/L. There were no significant differences between oxymetazoline and adrenaline and between xylometazoline and adrenaline at concentrations of 1 or 10 μmol/L. Oxymetazoline and xylometazoline at higher concentrations (100 μmol/L to 1 mmol/L) had an inhibitory effect.

Figure 2—
Figure 2—

Mean ± SE percentage aggregation of feline platelets. Each value represents results for blood samples from 6 cats. A—Citrated feline plasma was incubated with various concentrations of adrenaline, noradrenaline, oxymetazoline, xylometazoline, or saline solution (negative control agent) for 1 minute before the addition of ADP (0.5 μmol/L). B—Citrated feline plasma was incubated with adrenaline (100 μmol/L), noradrenaline (100 μmol/L), oxymetazoline (10 μmol/L), xylometazoline (10 μmol/L), or saline solution for 1 minute before the addition of various concentrations of ADP (0.1 to 10 μmol/L). C— Citrated feline plasma was incubated with various concentrations of α-adrenoceptor agonists for 1 minute before the addition of ADP (0.5 μmol/L). D—Citrated feline plasma was incubated with various concentrations of adrenaline and saline solution for 1 minute before the addition of collagen (0.5 to 1.0 μg/mL). In panels A, C, and D, the mean ± SE value for the negative control solution is 6.2 ± 1.2% to 8.9 ± 1.7%, 5.5 ± 1.1% to 10.0 ± 3.0%, and 14.3 ± 1.1%, respectively. Notice that the scale on the y-axis of panel A differs from that of panels B, C, and D. See Figure 1 for remainder of key.

Citation: American Journal of Veterinary Research 81, 2; 10.2460/ajvr.81.2.159

Incubation of platelets with adrenaline (100 μmol/L), noradrenaline (100 μmol/L), oxymetazoline (10 μmol/L), or xylometazoline (10 μmol/L) before the addition of ADP resulted in dose-dependent leftward shifts of the concentration-effect curve for ADP, compared with result for incubation with saline solution before the addition of ADP (Figure 2). The mean ± SE ED50 of ADP that caused 50% aggregation was 0.66 ± 0.16 μmol/L after incubation with adrenaline, 0.94 ± 0.17 μmol/L after incubation with noradrenaline, 0.55 ± 0.1 μmol/L after incubation with in oxymetazoline, 0.43 ± 0.1 μmol/L after incubation with xylometazoline, and 1.89 ± 0.18 μmol/L after incubation with the control agent. The ED50 values of ADP for these 4 agents were significantly lower than for the control agent. Conversely, clonidine, medetomidine, detomidine, xylazine, naphazoline, tramazoline, phenylephrine, and methoxamine did not potentiate the ADP-stimulated platelet aggregation at concentrations of 1 nmol/L to 1 mmol/L.

Adrenaline potentiated in a dose-dependent manner the platelet aggregation stimulated by collagen (0.5 to 1 mg/mL; Figure 2). The maximum and full aggregatory effect was observed with adrenaline at a concentration of 100 μmol/L.

Effects of imidazoline and nonimidazoline α-adrenergic agents on aggregation of feline platelets induced by the combination of adrenaline and ADP (experiment 4)

The α2-adrenoceptor antagonist yohimbine and 4 imidazoline α2-adrenoceptor antagonists (phentolamine, atipamezole, idazoxan, and tolazoline) at concentrations of 1 μmol/L to 1 mmol/L inhibited in a dose-dependent manner the full platelet aggregation induced by the combination of adrenaline at a concentration of 100 μmol/L and ADP at a concentration of 1 μmol/L (Figure 3). By contrast, the nonimidazoline α1-adrenoceptor antagonist phenoxybenzamine significantly inhibited platelet aggregation induced by adrenaline and ADP only at a high phenoxybenzamine concentration of 100 μmol/L, but the inhibition was less (by approx 65%). Another nonimidazoline α-adrenoceptor antagonist (prazosin) at concentrations up to 100 μmol/L was not effective at inhibiting the adrenaline-ADP-induced aggregation. Conversely, 8 imidazoline α-adrenoceptor agonists (oxymetazoline, naphazoline, tramazoline, clonidine, p-aminoclonidine, xylometazoline, medetomidine, and detomidine) at concentrations of 1 μmol/L to 1 mmol/L also inhibited in a dose-dependent manner the adrenaline-ADP-induced platelet aggregation. Antazoline, an imidazoline devoid of α2-adrenergic activity, at concentrations of 1 μmol/L to 1 mmol/L also inhibited in a dose-dependent manner the adrenaline-ADP-induced platelet aggregation. By contrast, 2 non-imidazoline α-adrenoceptor agonists (xylazine and phenylephrine) significantly inhibited the adrenaline-ADP-induced aggregation at a high concentration of xylazine or phenylephrine of 1 mmol/L, but the inhibition was less (by approx 40%). Another nonimidazoline α-adrenoceptor agonist (methoxamine) at all concentrations was not effective at inhibiting the adrenaline-ADP-induced aggregation.

Figure 3—
Figure 3—

Mean ± SE percentage aggregation of feline platelets induced by adrenaline (100 μmol/L) and ADP (1 μmol/L) for imidazoline and nonimidazoline α -adrenoceptor antagonists (A and B) and agonists (C and D). Each value represents results for blood samples from 6 cats. Each agent was added to citrated feline plasma. Adrenaline was added 0.5 minutes later, and ADP was added 0.5 minutes after that. Values are reported as a percentage of the value for the control agent (percentage aggregation of adrenaline-ADP with saline solution was assigned a value of 100%). See Figure 1 for remainder of key.

Citation: American Journal of Veterinary Research 81, 2; 10.2460/ajvr.81.2.159

The IC50 values obtained for the inhibition of adrenaline-ADP-induced platelet aggregation were summarized (Table 1). The order of potencies determined on the basis of the IC50 values was as follows: phentolamine > atipamezole > idazoxan > naphazoline > antazoline > xylometazoline, yohimbine, tramazoline, oxymetazoline, and clonidine > medetomidine > tolazoline > detomidine > p-aminoclonicine > phenoxybenzamine. The potencies of atipamezole, idazoxan, and naphazoline for inhibiting aggregation were not significantly different from that of phentolamine. The potencies of yohimbine, oxymetazoline, clonidine, and medetomidine were significantly less (9- to 17-fold difference) than that of phentolamine. The potencies of detomidine, p-aminoclonidine, and phenoxybenzamine were significantly less (36- to 91-fold difference) than that of phentolamine. The IC50 value was not obtained for xylazine, phenylephrine, methoxamine, and prazosin.

Table 1—

Mean ± SE potency and IC50 ratio* of imidazoline and nonimidazoline α-adrenergic agents for the inhibition of aggregation of feline platelets induced by adrenaline (100 μmol/L) and ADP (1 μmol/L).

AgentIC50 (× 10−6 mol/L)IC50 ratio
Phentolamine2.1 ± 0.41.0
Atipamezole4.6 ± 0.72.2
Idazoxan6.8 ± 2.03.3
Naphazoline10.4 ± 2.15.0
Antazoline15.9 ± 2.07.6
Xylometazoline18.5 ± 2.88.9
Yohimbine18.6 ± 4.68.9
Tramazoline18.7 ± 3.39.0
Oxymetazoline22.4 ± 6.110.7
Clonidine26.1 ± 4.212.5
Medetomidine36.4 ± 4.017.4
Tolazoline59.7 ± 17.128.6
Detomidine76.0 ± 12.236.3
p-Aminoclonidine90.4 ± 13.143.2
Phenoxybenzamine189.0 ± 24.890.6
XylazineNDND
PhenylephrineNDND
MethoxamineNDND
PrazosinNDND

Each value represents results for blood samples from 6 cats.

Ratios are expressed in relation to the value for phentolamine, which was assigned a value of 1.0.

ND = Not determined.

Effects of imidazoline and nonimidazoline α-adrenergic agents on aggregation of feline platelets induced by the combination of adrenaline and collagen (experiment 5)

The α2-adrenoceptor antagonist yohimbine and 4 imidazoline α2-adrenoceptor antagonists (phentolamine, idazoxan, atipamezole, and tolazoline) at concentrations of 0.1 or 10 μmol/L to 100 μmol/L inhibited in a dose-dependent manner the full platelet aggregation induced by the combination of adrenaline at a concentration of 10 μmol/L and collagen at a concentration of 1 to 3 μg/mL (Figure 4). By contrast, the nonimidazoline α1-adrenoceptor antagonist prazosin at all concentrations was not effective at inhibiting platelet aggregation induced by adrenaline and collagen. Conversely, 5 imidazoline α-adrenoceptor agonists (oxymetazoline, naphazoline, clonidine, medetomidine, and dexmedetomidine) at concentrations of 1 μmol/L to 100 μmol/L also inhibited in a dose-dependent manner the adrenaline-collagen–induced aggregation. Antazoline, an imidazoline devoid of α2-adrenergic activity, at concentrations of 1 μmol/L to 100 μmol/L also inhibited in a dose-dependent manner the adrenaline-collagen–induced platelet aggregation. By contrast, the nonimidazoline α-adrenoceptor agonist xylazine was not effective at inhibiting the platelet aggregation induced by adrenaline and collagen. Similarly, the imidazoline α2-adrenoceptor agonist moxonidine was not effective at inhibiting the adrenaline-collagen–induced platelet aggregation.

Figure 4—
Figure 4—

Mean ± SE percentage aggregation of feline platelets induced by adrenaline (10 μmol/L) and collagen (1 to 3 μg/mL) for imidazoline and nonimidazoline α-adrenoceptor antagonists (A and B) and agonists (C and D). Each value represents results of blood samples from 4 or 5 cats. Each agent was added to citrated feline plasma. Adrenaline was added 0.5 minutes later, and collagen was added 0.5 minutes after that. Values are reported as a percentage of the value for the control agent (percentage aggregation of adrenaline-collagen with saline solution was assigned a value of 100%). See Figure 1 for remainder of key.

Citation: American Journal of Veterinary Research 81, 2; 10.2460/ajvr.81.2.159

The IC50 values obtained for the inhibition of adrenaline-collagen–induced platelet aggregation were summarized (Table 2). The order of potencies determined on the basis of the IC50 values was as follows: phentolamine > idazoxan, oxymetazoline, and yohimbine > naphazoline and clonidine > atipamezole and tolazoline > antazoline > medetomidine and dexmedetomidine. The potencies of idazoxan and oxymetazoline at inhibiting aggregation were not significantly different from that of phentolamine. The potencies of yohimbine, naphazoline, and clonidine were significantly less (5-, 40- and 49-fold difference, respectively) than that of phentolamine. The potencies of atipamezole, tolazoline, and antazoline were significantly less (126- to 458-fold difference) than that of phentolamine. The potencies of medetomidine and dexmedetomidine were significantly less (1,286- and 1,300-fold difference) than that of phentolamine. The IC50 value was not obtained for xylazine, moxonidine, and prazosin.

Table 2—

Mean ± SE potency and IC50 ratio* of imidazoline and nonimidazoline α-adrenergic agents for the inhibition of aggregation of feline platelets induced by adrenaline (10 μmol/L) and collagen (1 to 3 μg/mL).

AgentIC50 (× 10−6 mol/L)IC50 ratio
Phentolamine0.7 ± 0.51.0
Idazoxan1.9 ± 1.12.7
Oxymetazoline2.1 ± 1.93.1
Yohimbine3.2 ± 0.24.7
Naphazoline27.6 ± 16.740.0
Clonidine33.6 ± 21.648.7
Atipamezole87.2 ± 35.0126.0
Tolazoline87.7 ± 36.2127.0
Antazoline316.0 ± 243.0458.0
Medetomidine887.0 ± 565.01,286.0
Dexmedetomidine897.0 ± 570.01,300.0
PrazosinNDND
XylazineNDND
MoxonidineNDND

Each value represents results for blood samples from 4 or 5 cats.

See Table 1 for remainder of key.

Effects of the combination of α2-adrenoceptor agonists and antagonists on aggregation of feline platelets induced by adrenaline and collagen (experiment 6)

Concentration-effect curves of α2-adrenoceptor antagonists and agonists alone and in combination on adrenaline-collagen–induced aggregation of feline platelets were plotted (Figure 5). Mean ± SE IC50 values for yohimbine, atipamezole, medetomidine, yohimbine plus xylazine, atipamezole plus xylazine, yohimbine plus medetomidine, and atipamezole plus medetomidine were 3.11 ± 0.24 × 10−6 mol/L, 73.4 ± 477 × 10−6 mol/L, 420 ± 409 × 10−6 mol/L, 6.41 ± 2.82 × 10−6 mol/L, 71.7 ±61.3 × 10−6 mol/L, 3.60 ± 0.25 × 10−6 mol/L, and 257 ± 160 × 10−6 mol/L, respectively; the IC50 value was not obtained for xylazine. There were no significant differences in IC50 values among yohimbine, yohimbine plus xylazine, or yohimbine plus medetomidine or among atipamezole, atipamezole plus xylazine, and atipamezole plus medetomidine. Therefore, the α2-adrenoceptor agonists xylazine and medetomidine did not reverse the inhibitory effects of the α2-adrenoceptor antagonists yohimbine and atipamezole for adrenaline-collagen–induced platelet aggregation.

Figure 5—
Figure 5—

Mean ± SE percentage aggregation of feline platelets induced by adrenaline (10 μmol/L) and collagen (1 to 3 μg/mL) for α2-adrenoceptor agonists and antagonists (A) and the combination of agonists and antagonists (B). Each value represents results for blood samples from 4 or 5 cats. Each agent was added to citrated feline plasma. Adrenaline was added 0.5 minutes later, and collagen was added 0.5 minutes after that. Values are reported as a percentage of the value for the control agent (percentage aggregation of adrenaline-collagen with saline solution was assigned a value of 100%). See Figure 1 for remainder of key.

Citation: American Journal of Veterinary Research 81, 2; 10.2460/ajvr.81.2.159

Effect of phentolamine, atipamezole, and yohimbine on full aggregation of feline platelets induced by ADP alone (experiment 7)

Phentolamine, atipamezole, and yohimbine, which effectively inhibited the full platelet aggregation induced by adrenaline-ADP, were not effective or were less effective at inhibiting the full platelet aggregation induced by ADP (10 μmol/L) alone (Figure 6). The IC50 value was not obtained for these agents.

Figure 6—
Figure 6—

Mean ± SE percentage aggregation of feline platelets induced by ADP (10 μmol/L) alone for phentolamine, atipamezole, and yohimbine. Each value represents results for blood samples from 6 cats. Each agent was added 1 minute before the addition of ADP. Values are reported as a percentage of the value for the control agent (percentage aggregation of ADP with saline solution was assigned a value of 100%). See Figure 1 for remainder of key.

Citation: American Journal of Veterinary Research 81, 2; 10.2460/ajvr.81.2.159

Discussion

Results of the study reported here confirmed those of previous investigations16,17 that indicated that adrenaline alone did not induce a change in aggregation of feline platelets and instead potentiated platelet aggregation stimulated by other platelet agonists including ADP and collagen. In addition, results of the present study indicated that noradrenaline potentiated ADP-stimulated platelet aggregation in a dose-dependent manner, and both oxymetazoline and xylometazoline (within limited concentrations, 1 to 100 μmol/L) induced a small potentiation of the ADP-stimulated platelet aggregation in feline platelets. However, other α-adrenoceptor agonists (clonidine, medetomidine, detomidine, xylazine, naphazoline, tramazoline, phenylephrine, and methoxamine) did not induce this potentiating effect. These findings were similar to those for canine platelets.15 Clonidine reportedly can induce aggregation in human platelets to a limited degree4,5,33,44–46 and can potentiate ADP-induced platelet aggregation in humans10 and rabbits.18 However, the present study showed that clonidine did not potentiate ADP-induced aggregation in feline platelets, whereas oxymetazoline and xylometazoline caused a small potentiation of the ADP-induced platelet aggregation, which is in agreement with results reported for dogs15 and cattle.22 The present results, in combination with results of the aforementioned studies,4,5,10,15,18,22,33,44–46 highlight species-specific variations in the potentiation of platelet aggregation.

Platelet α-adrenoceptors in humans have been characterized pharmacologically as Gi-coupled α2-adrenoceptors of the α2A-subtype, although a decrease in cAMP alone may not be the only cause of aggregation.47–53 In domestic animals, binding experiments with radiolabeled adrenoceptor agonists and antagonists have revealed the expression of α2-adrenoceptors on canine, feline, leporine, and murine platelets but not on bovine or equine platelets.8 The density of platelet α2-adrenoceptors evaluated by the use of specific tritiated yohimbine binding is reportedly lower in cats than in dogs8 and humans.48 In the present study, adrenaline-potentiated aggregation of feline platelets stimulated by low concentrations of ADP or collagen was inhibited in a dose-dependent manner by the α2-adrenoceptor antagonists atipamezole, yohimbine, phentolamine, idazoxan, and tolazoline, whereas the α1-adrenoceptor antagonists phenoxybenzamine and prazosin were not effective or were less effective at inhibiting the adrenaline-potentiated aggregation.

In previous reports,54–56 the order of affinity of antagonists for α2-adrenoceptors is atipamezole > yohimbine > idazoxan > phentolamine > tolazoline > prazosin. In the study reported here, the order of potency of α2-adrenoceptor agents for the inhibition of adrenaline-potentiated platelet aggregation was not in agreement with the order of affinity values for α2-adrenoceptors. However, although the mechanism for adrenaline-induced intraplatelet signaling is unclear, adrenaline is known to increase the release of arachidonic acid from platelet membranes via the phosphorylation of p38 mitogen-activated protein kinase and cytosolic phospholipase A2 via the α2A-adrenoceptors and its Na+-effector sites.51 In addition, adrenaline-potentiated platelet aggregation is not mediated by β-adrenoceptors because the β-adrenoceptor antagonist propranolol is less effective at inhibiting adrenaline-ADP–induced aggregation.2 Therefore, results of the present study suggested a partial involvement of the α2-adrenoceptor-mediated cascade in aggregation of feline platelets and the simultaneous involvement of other pathways in addition to the involvement of α2-adrenoceptors.

Furthermore, in the present study, certain imidazoline or α2-adrenoceptor antagonists (or both) were able to completely inhibit the full platelet aggregation induced by adrenaline-ADP or adrenaline-collagen but were not effective or were less effective at inhibiting full platelet aggregation induced by ADP alone. These findings suggested that the inhibitory effect of imidazoline agents on feline platelet aggregation is more specific for the action of adrenaline via α2-adrenoceptors rather than the action of ADP as an inducer of platelet aggregation.

Imidazoline and α2-adrenoceptor agonists including naphazoline, antazoline, xylometazoline, tramazoline, oxymetazoline, clonidine, medetomidine, dexmedetomidine, detomidine, and p-aminoclonidine, but not moxonidine, caused dose-dependent inhibition of adrenaline-ADP– or adrenaline-collagen–induced aggregation of feline platelets in the study reported here. These results for feline platelets were similar to results for canine platelets, except for a different order for α2-adrenoceptor activity,15 and to results for human platelets.7 The imidazoline agent antazoline, which lacks α2-adrenoceptor activity, inhibited adrenaline-potentiated platelet aggregation, suggesting that it interacts with non–α2-adrenoceptor sites on feline platelets. Furthermore, in the present study, neither xylazine nor medetomidine reversed the inhibitory effects of the α2-adrenoceptor antagonists yohimbine and atipamezole for adrenaline-collagen–induced platelet aggregation, suggesting that both yohimbine and atipamezole also interacted with non–α2-adrenoceptor sites on feline platelets.

On the basis of these results, it would be difficult to envisage how the α2-adrenoceptors could be the mediator of the observed responses. Both feline platelets and canine platelets have nonadrenergic I1 receptor sites (as determined by the use of labeled tritiated clonidine) and I2 receptor sites (as determined by the use of labeled tritiated idazoxan).8 The order of potency of imidazoline agents for the inhibition of adrenaline-potentiated platelet aggregation in the present study appeared to be in agreement with the order of affinity values for platelet I1 receptors or I2 receptors.8 In a report of a comparative study57 of the effects of imidazoline α-adrenergic agents on intraplatelet cAMP and thromboxane B2 that involved the use of canine platelets with both I1 and I2 receptors and leporine platelets that lacked I1 receptors, it was suggested that imidazoline α2-adrenergic agents suppress cAMP production via the α2-adrenoceptor while exerting a negative effect on generation of thromboxane B2 via the arachidonic acid–thromboxane A2 pathway. Therefore, it would seem logical to conclude that imidazoline agents inhibit platelet aggregation via nonadrenoceptor binding sites, including I1 and I2 receptors, on feline platelets.

Yohimbine and idazoxan exhibit only modest selectivity for rat α2-receptors, compared with selectivity for 5HT1A receptors.58,59 Oxymetazoline also stimulates 5HT receptors, including 5HT1A, and can mobilize a second signaling system.60,61 Furthermore, noradrenaline induces heterologous desensitization of the 5HT1 receptors in human platelets through activation of protein kinase C.62 Therefore, it is also possible that the effect of imidazoline or α-adrenergic agents on platelet aggregation may be partially mediated by serotonin receptors, including 5HT1A. In the present study, both oxymetazoline and xylometazoline induced a small potentiation of the ADP-induced platelet aggregation, but both agents at higher concentrations had an inhibitory effect on potentiation of the ADP-induced platelet aggregation. Although the precise mechanism for this effect was unknown, it may have been attributable to the complicated actions via 5HT receptors, α2-adrenoceptors, and I1 and I2 receptors on feline platelets.8,63

Several drugs with α2-adrenoceptor activity are clinically available. For felids, the α2-adrenoceptor agonists xylazine, medetomidine, and dexmedetomidine are used for sedation and analgesia and as a premedication for general anesthesia, whereas the antagonists atipamezole and yohimbine are used to reverse the effects of the aforementioned α2-adrenoceptor agonists. On the basis of the pharmacokinetic data for xylazine, medetomidine, and dexmedetomidine, which have typically been administered systemically at clinically recommended doses to cats and dogs,64–66 results of the present study suggested that the α2-adrenoceptor agonists xylazine, medetomidine, and dexmedetomidine may be used in cats with minimal concern for adverse effects on platelet function and hemostasis because xylazine did not inhibit platelet aggregation and both medetomidine and dexmedetomidine did not inhibit in vitro platelet aggregation at the estimated blood concentrations of both agents in clinical use. However, the α2-adrenoceptor antagonists phentolamine, yohimbine, and atipamezole may also have inhibitory effects on feline hemostasis during certain events (eg, blood vessel damage and collagen exposure). Because the findings reported here represented results of in vitro experiments, it will be necessary to investigate the effects of various agents on aggregation of feline platelets in vivo or ex vivo.

Overactivity of the sympathetic nervous system and increased systemic catecholamine concentrations have been suggested to possibly influence hemostasis via actions on platelets, coagulation and fibrinolytic factors, and endogenous anticoagulants, thereby leading to activation of both the coagulation and fibrinolytic systems.31 Hypercatecholaminemia occurs in conditions such as pheochromocytoma,25 endotoxic shock,26,27 and acute stress28 in cats. Risk factors for poor short-term survival in dogs with pheochromocytoma involve disseminated intravascular coagulation.29 Fatal thromboembolism has been also reported in a cat with pheochromocytoma.30 In addition, low-dose endotoxic infusion induces platelet aggregation,32 and intravascular coagulation is manifested during endotoxin shock in cats.67 Therefore, the results of the study reported here suggested that imidazoline α-adrenergic agents may have clinical benefits for the hypercoagulatory state that accompanies hypercatecholaminemia or for the conditions in which there is platelet reactivity to adrenaline because catecholamines have a stimulatory effect on platelet aggregation. However, additional research will be required to examine the effects of α-adrenergic agents on in vivo or ex vivo platelet aggregation under various pathological conditions in cats.

In the present study, both adrenaline and noradrenaline potentiated in a dose-dependent manner aggregation of feline platelets induced by ADP or collagen, but other α-adrenoceptor agonists, except for oxymetazoline and xylometazoline, did not potentiate platelet aggregation induced by ADP. Furthermore, results indicated that the α2-adrenoceptor antagonists and certain imidazoline α-adrenergic agents (or both) inhibited, in a dose-dependent manner, adrenaline-potentiated aggregation induced by ADP or collagen, whereas α1-adrenoceptor antagonists and nonimidazoline α-adrenergic agents were ineffective or less effective at inhibiting adrenaline-potentiated aggregation and that the α2-adrenoceptor agonists medetomidine and xylazine did not reverse the inhibitory effects of the α2-adrenoceptor antagonists atipamezole and yohimbine on adrenaline-potentiated aggregation. The results also suggested that adrenaline-potentiated aggregation was mediated by α2-adrenoceptors, whereas imidazoline agents inhibited platelet aggregation via imidazoline receptors in cats. Although findings supported the notion that clinically recommended doses of xylazine, medetomidine, and dexmedetomidine may be used in feline practice with minimal concern for adverse effects on platelet function, additional in vivo or ex vivo studies will be required to examine the effects of these agents on platelet aggregation.

Acknowledgments

Dr. Hikasa was supported in part by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (No. 18580316).

ABBREVIATIONS

5HT

5-Hydroxytryptamine

ED50

Concentration of an agent that causes a half-maximal response

I1

Imidazoline-preferring binding site 1

I2

Imidazoline-preferring binding site 2

IC50

Concentration of an agent at which the response is inhibited by half

PPP

Platelet-poor plasma

PRP

Platelet-rich plasma

Footnotes

a.

MCM Hema tracer 804, LMS Co Ltd, Tokyo, Japan.

b.

Tokyo Kasei Industries Co, Tokyo, Japan.

c.

Sigma Chemical Co, St Louis, Mo.

d.

Boehringer-Ingelheim Corp, Hyogo, Japan.

e.

Farmos Group Ltd, Turku, Finland.

f.

Maruishi Pharmaceutical Co Ltd, Osaka, Japan.

g.

LMS Co Ltd, Tokyo, Japan.

h.

Prism, version 7.0, GraphPad Software Inc, San Diego, Calif.

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Contributor Notes

Address correspondence to Dr. Hikasa (hikasa@tottori-u.ac.jp).