Platelet aggregation in the asian elephant is not dependent on thromboxane B2 production

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Camp. Eiochem. Physiol.Vol. 94A, No. I,pp.47-51,1989 Printed in Great Britain

IC 1989 Pergamon Press plc

PLATELET AGGREGATION IN THE ASIAN ELEPHANT IS NOT DEPENDENT ON THROMBOXANE B, PRODUCTION* P. A. GENTRY, C. NIEMULLER, M. L. Ross and R. M. LIPTRAP Department of Biomedical Science, Ontario Veterinary College, University of Guelph, Guelph, Ontario NIG 2W1, Canada. Telephone: (519) 824-4120 (Receired 7 February 1989) Abstract-i.

The platelet aggregation response to several known platelet agonists was evaluated in four Asian elephants. The platelets were highly responsive to stimulation with platelet-activating factor (PAF) and collagen, less responsive to adenosine diphosphate (ADP) and non-responsive to arachidonic acid, serotonin and epinephrine. 2. Arachidonic acid (1 x 10m4M), while inducing no aggregation, caused the release of 1248 + 1147pg/pl (mean+ SD) of thromboxane B, (TXBJ, the stable metabolite of thromboxane A, from stimulated platelet. The addition of 1 x 10m4M ADP to platelets caused suboptimal aggregation and the release of only 25 f 10 pg TXB,/pl. 3. The calcium channel blocker, verapamil, produced a dose-dependent inhibition of PAF-induced but not collagen-induced aggregation. The cyclooxygenase inhibitor, acetylsalicylic acid, produced no inhibition of either collagen- or PAF-induced aggregation.

INTRODUCTION

platelet phospholipids (Packham and Mustard, 1984). The released AA can be further metabolized by the cyclooxygenase enzyme system to form products, such as thromboxane A, (TXA,), which, in some species, also serves as a potent aggregatory agent (Smith, 1986). When the phospholipase C enzyme system is activated in the stimulated platelet, diacylglycerol is formed. This can interact with the secondary messengers cyclic AMP, and possibly cyclic GMP, to induce calcium mobilization from intracellular platelet stores, resulting in the release of granular contents and the formation of irreversible platelet aggregates (Packham and Mustard, 1984; Majerus ef al., 1986; Brass and Laposata, 1987). It is well established that there are species differences in the responsiveness of blood platelets to different agonists and to different combinations of agonists. Excellent reviews have appeared on this subject (Belamarich, 1976; Dodds, 1978; Hwang, 1985; Meyers, 1986). From the cumulative information in the literature, a pattern appears to be emerging which suggests that most mammalian platelets fall into one of two categories depending on the relative importance of the biochemical pathways in the stimulated cell. In platelets from species such as man, horse, cat, rabbit, rat, mouse and guinea pig the AA pathway is well developed and the aggregation response of these platelets, following exposure to AA correlates with the amount of TXA, formed (Meyers, 1986). In contrast, in platelets from species such as the cow, sheep, pig and mink, it appears that the AA pathway is not well developed (Meyers et al., 1980; Meyers, 1986) and in the bovine it has been clearly established that platelet aggregation can occur normally even when the production of TXA, is effectively inhibited (Bondy and Gentry, 1988; Gentry et al., 1989). One of the purposes of this study

Since a report on the blood coagulation profile of the Asian elephant (Elephas maximus) was published by Lewis in 1974, in which it was shown that elephant platelets were relatively unresponsive to the agonists adenosine diphosphate (ADP) and collagen, there do not appear to have been further studies of plate-

let function in this species. Recently, as part of an unrelated study, we had the opportunity to obtain blood samples, at regular intervals, from four healthy Asian elephants and to further investigate platelet function. In mammals the blood platelet is a circulating, nonnucleated cell, derived from megakaryocytes, which participates in the initial stages of the blood coagulation process. Circulating platelets are sensitive to a variety of chemical and physical agents (Gentry and Downie, 1984; Stormorken, 1986). Once platelets are activated they lose their discoid shape and form pseudopods before adhering to each other and to damaged endothelium. During this process granular contents are released which can facilitate further aggregate formation (Packham and Mustar, i984; Lages, 1986). There appear to be at least two biochemical pathways activated during the initial phases of platelet aggregation and which can be briefly summarized as follows. Platelets agonists interact with receptors on the platelet membrane and induce alterations in the membrane phospholipids. If the phospholipase A, enzyme sytem is activated, arachidonic acid (AA) will be liberated from the

*Supported in part by the Natural Sciences and Engineering Council of Canada and the Ontario Ministry of Agriculture and Food. 47

P. A. GEWRY et al.

48

was to evaluate the biochemical response of elephant platelets with respect to the relative importance of the AA/TXA, pathway. To evaluate platelet aggregation, the response of elephant platelets to a number of known agonists, including ADP, collagen, platelet-activating factor (PAF), AA, serotonin and epinephrine, was assessed. The production of TXAz following treatment of the platelets with ADP and AA was monitored by measuring the stable metabolite of TXA,, TXB,, with a radioimmunoassay. The relative significance of the thromboxane pathway was further evaluated by examining the inhibitory capacity of the cyclooxygenase inhibitor acetylsalicylic acid (ASA) and the calcium channel blocker, verapamil.

MATERIALS

AND METHODS

Animals

Four healthy Asian elephants (Hephas maximus), three males (17, 18 and 21 years of age) and one female (25 years of age), owned bv the African Lion Safari, Ontario. Canada. were ‘used for -the study. The elephants were bled by venepuncture of a vein on the underside of an ear with an 18 gauge butterfly needle connected to a plastic syringe. Blood samples were collected between 0800 and 0900 hr at regular intervals. Preparation of blood samples

After collection, blood was immediately transferred, in aliquots of 9 ml, to siliconized glass centrifuge tubes containing 1 ml of 3.8% trisodium citrate. Platelet-rich plasma (PRP) was prepared by centrifuging the whole blood at 150g for 10 min at 20°C and removing the upper two thirds of the PRP with a siliconized Pasteur pipette. Platelet-poor plasma (PPP) was obtained by recentrifuging the remaining blood at 25OOg for 15 min at 4°C. The unopette method (Becton-Dickinson Co., Rutherford, NJ) was used to determine platelet counts, and PRP was adjusted to 200,000 platelets/p 1 with homologous PPP. Platelet aggregation studies

Platelet aggregation was monitored in a Payton Dual Channel aggregometer (Payton Associated Ltd., Scarborough, Ont.). For each experiment, the aggregometer was calibrated with PPP to establish the 100% aggregation limit, corresponding to 100% transmittance through the plasma. The corresponding PRP sample was used to establish the 0% aggregation limit (0% light transmittance through the plasma). The extent of aggregation in a sample was determined from the difference in light transmission through the plasma before and after addition of an aggregation agent. All plasma samples were assayed in duplicate and each

animal was tested in triplicate for each agonist and inhibitor studied. For platelet aggregation studies, PRP, in aliquots of 215~11,was incubated with 25~11of Tris-buffered saline (0.05 M Tris, pH 7.4, TBS) at 37°C for 2 min. Platelet aggregation was stimulated with 25 ~1 of aggregating agent. To study the response of elephant platelets to combinations of aggregating agents, PRP was incubated with 25 ~1 of the first agent, and the aggregation response monitored for 2min when 25 ~1 of the second aggregating agent was added, and the response recorded for a further 10min. To evaluate the effect of inhibitory agents on the aggregation response, PRP, in aliquots of 215 ~1, was incubated with 10 ~1 of inhibitor or 10 ~1 of inhibitor solvent at 37°C for 5min. Platelet aggregation was initiated with 25~1 of aggregating agent, and the percent maximum aggregation response was measured from the aggregometer tracing.

Radioimmunoassax of platelet thromboxane Bz production

Elephant blood was collected, and PRP (200,000 platelets/PI) was prepared as described. Platelets were treated as for aggregation, except that the aggregated sample was removed from the aggregometer 5 min after addition of aggregating agent, and immediately centrifuged in a Beckman 150 microfuge for 5 min at 4°C. Thromboxane B, (TXB,) levels were determined from 100~1 aliquots of supernatant using a thromboxane B2 [jH] radioimmunoassay kit (New England Nuclear, Lachine, Que.), All samples were assayed at a minimum of two dilutions, in duplicate and TXB, values determined from a standard curve prepared simultaneously with the sample analysis. Preparation of aggregating agents and inhibitors

ADP was dissolved in TBS to a concentration of 10 mM. Collagen (collagen reagent. calf skin) was reconstituted and diluted with deionized water as recommended by the manufacturer. PAF was dissolved in 0.5 ml ethanol, and 5.0 ~1 of this solution was added to IOOml Modified Tyrodes (with 0.1% bovine serum albumin) to yield a stock solution of 0.5 pg/ml. AA was dissolved in I : I methanol: saline (v/v) to prepare a 40 mM solution. Epinephrine reagent was reconstituted and diluted with deionized water as recommended by the manufacturer. Serotonin (5-hydroxytryptamine) was prepared to a concentration of IOmM in SOml TBS @H 7.4) containing 0.1% L-ascorbic acid. Acetylsalicyhc acid (ASA) was dissolved in 0.3 M sodium acetate to prepare a stock solution of 0.1 M concentration. Verapamil was prepared in methanol to a concentration of lOmM, and dilutions for aggregation were made with I:9 methanol: modified Tyrodes solution (v/v). All aggregating agents and inhibitors, with the exception of PAF, were purchased from Sigma Chemical Co. (St. Louis, MO). PAF was purchased from Calbiochem (San Diego. CA). Statistical anal.vsis

Results were analysed with Student’s t-test on a Monroe programmable calculator (Monroe Calculation Corp., Kitchener, Ont.).

RESULTS

The platelets obtained from each of the four elephants available for this study responded in a similar manner to each agonist and each inhibitor examined. Of the platelet agonists examined, the elephant platelets appeared most responsive to collagen and PAF, only partially responsive to ADP, and unresponsive to either AA, epinephrine or serotonin (Tables 1 and 2). Although both collagen and PAF could induce maximal, irreversible aggregating, the initial rate of aggregation was greater when PAF was the agonist and less PAF (0.2 ng/ml) than collagen was required to initiate a near-maximal aggregation response (Table 1). Unlike the aggregation response to either PAF or collagen, the ADP response was independent of the concentration of agonist and the aggregates which formed appeared unstable since deaggregation was frequently observed. While the platelets from a number of species are relatively insensitive to agonist such as epinephrine and serotonin, frequently, these agents will exert a synergistic aggregation response when added in combinations with ADP (Kinlough-Rathbone and Mustard, 1986). To determine the response of the elephant, platelets were exposed to either epinephrine, or serotonin singly and in combination with ADP (Table 2). Epinephrine, at concentrations of

49

Platelet aggregation in the Asian elephant Table 1. Platelet

aggregationresponseto stimulationwith various aggregating agents Initial velocity change OD/min (mean + SD)

Agonist

ReversibiliW

ADP

1 x IO-‘M I x IO-‘M I x IO-‘M

15.4f 1.0 15.6rt: 1.0 1I .O + 0.6

C0llagan

40 @g/ml) 20 &g/m0 10 &g/mu 5 (umlt

11.0* 1.2 7.0 + 2.0 3.4 F 0.6 1.4*0.4

33.4 + 33.0 + 21.2 f 88.6 k 73.6 * 60.8 f 25.3 i

3.6 x W’M 9.0 x lo‘-” M

29.2 + 2.8 30.6 I 3.2

85.6 + 3.9 69.3 ; 5.5

-

28.1 + 4.1

_+

PAF

2.3 x lo-‘” M * + . reversible aggregation; -,

15.2 _t 2.2 irreversible aggregation.

I x lob4 M and 1 x 10e5 M, had no effect on the platelets since the aggregometer tracings indicated that not even shape change had occurred. Furthermore, the aggregation response was similar whether ADP alone or a combination of ADP and epinephrine was used to initiate aggregation. While serotonin (1 x lo-’ M) by itself also failed to induce an aggregation response, it did produce evidence of shape change in the treated samples. The extent of the aggregation response in platelets exposed to the combination of serotonin and ADP was significantly reduced (P < 0.05) by 25 and 47.3%, respectively, in response to 1 x 1O-3 M and 1 x low4 M serotonin, respectively, compared to the response to ADP alone (Table 2). A slight, but not statistically significant (P > 0.05) reduction in the aggregation response was also observed when the platelets were stimulated with a combination of AA (1 x 10-j M) and ADP (1 x 10m4M) (Table 2). In contrast, when the platelets were exposed to a lower concentration of ADP (1 x 10m4M) before the addition of ADP, the aggregation response was increased 2-fold compared to the response to ADP alone. This increase was statistically significant (P < 0.01). Since it has been reported, for most species, that the extent of aggregation to AA correlates with the amount of TXB, formed in the stimulated platelets (Meyers, 1986), the amount of TXB, formed in the elephant platelets exposed to AA alone and in combination with ADP was examined. The amount of

Table 2. Platelet response to combinations of aggregating agents

Agonist

Maximum aggregation response percent (mean of:SD)

ADP Epinephrine Epinephrine Epinephrine Epincphrine

1x 10-4M 1 x 10-*M + ADP

36.4 & 5.6 0 42.4 & 2.9

+ ADP

30.2 & 10.1

ADP Serotonin Serotonin Serotonin Serotonin

1 x 10-4M 1 x lo-‘M + ADP 1 x lo-‘M + ADP

43.5 + 2.7

1 x 10-‘M

ADP I x 10-4M AA 1 x IO-“M ADPiAA AA I x IO-‘M ADP+AA *P < 0.05, tP < 0.01.

CBPfA) 941-D

Maximum aggregation percent (mean + SD)

0

22.2:

2.F

29.2:

2.2’

40.4 i. 2.2 2.5 + I .8 27.3 + 6.0 *7.7”, s.it

1.7 2.3 1.2 6.0 9.0 4.2 5.5

L+z If * -

TXB, released from the platelets, following exposure to 1 x 10e3 M AA, was greater than could be measured within the sensitivity of the assay procedure despite extensive dilution of the samples (Table 3). With the lower concentration of AA (1 x 10e4 M) the amount of TXB, released was 1248 + 1147 pgjlO0 ~1 (mean _t SD). The large SD reflects the variable response found for the four animats. The addition of 1 x IO-* M ADP to the PRP produced only minimal TXB, formation with only 25 & 1Opg TXB, per 100 ~1 being detected (Table 3). There was no significant difference (P > 0.05) in the amount of TXB, produced when both AA and ADP were added to the PRP compared to the amount formed when these agents were added singly. The amount of TXB, formed in the presence of both AA and ADP was 1801 f 773 pg per 100 ~1, which was similar to the expected value of 1273 Ifi 1156 pg per 100 ~1. In the bovine platelet it appears that calcium mobilization is important for aggregation (Bondy and Gentry, 1988). In order to evaluate whether calcium mobili~tion is equally impo~ant in the response of elephant platelets, the effect of verapamil, a calcium channel blocking agent, on the aggregation induced by PAF and collagen was examined (Table 4). The exposure of platelets to verapamil for 2 min before the addition of PAF produced a dosedependent inhibition of the aggregation response. At a concentration of verapamil of 5 x 10v4 M, the aggregation response to 0.9 x lo-’ M PAF was completely inhibited. A significant (P < 0.01) inhibition of 66.7% in the aggregation response was observed when the verapamil concentration was reduced to 5 x lo-‘M. Verapamil, at all the concentrations tested, failed to inhibit the aggregation response induced by collagen. The preincubation of the elephant platelets with ASA at concentrations as high as 1 x 10T2 M failed to induce an inhibitory response in both PAF- and collagen-stimulated platelets (Table 4).

Table 3. Comparison of thromboxane B2 release from platelets stimulated to aggregate with ADP and archidonic acid Agonist

pg mB, per 100~1 (mean + SD)

AA 1 x IO-‘M AA 1 x IO-‘M ADP 1 x IO-‘M ADP I x IO-‘M+AA 1 x IO-‘M

> 5000 1248 * 1147 25& 10 1801 f 773

50

P.

A. GENTRY et al.

DISCUSSION

The aggregation response of the platelets from the four Asian elephants to the agonists, ADP and collagen, was similar to that previously reported (Lewis, 1974). Consequently it may be assumed that, even though only a few elephants were available to the study, the results may be representative of the type of platelet responses common to Asian elephants. Similar responses were found for the three males and the single female suggesting that there are no significant sex-linked differences in the platelet response. Since ADP is generally referred to as the “universal agonist”. the relative unresponsiveness of the elephant platelets to this aggregating agent indicates that this platelet is potentially biochemically different from those of other mammals so far investigated (Calkins et al., 1974; Addonizio et al.. 1978; Dodds, 1978; Meyers et al., 1979; Feingold et al., 1986; Meyers, 1986). The elephant platelets are less responsive to ADP that even the bovine platelet, which has been previously shown to be less sensitive to this agonist than for, example, human platelets (Bondy and Gentry, 1988). In nonmammalian species the thrombocyte is the circulating cell which is considered equivalent to the mammalian platelet (Ratnoff, 1987). Unlike the platelet, the thromb~yte is a nucleated cell, possibly of blast cell or hemocytoblast origin, which is generally non-responsive to ADP (Belamarich ef al., 1976; Ratnoff, 1987). The elephant platelet may represent a class of mammalian platelets which have shared biochemical characteristics with the nonmammalian thrombocyte. The reversible nature of the aggregates which formed in response to ADP was similar to that previously reported by Lewis (1974). and also supports the concept that ADP is not a critical agonist for the formation of hemostatically functional platelet aggregates in the elephant. A variable response to collagen was observed in this study. A commercial calf skin collagen preparation was effective as an agonist capable of inducing maximal, irreversible aggregation (Table 1). This particular collagen preparation was the only one of several tested which was found to induce aggregation (data not shown). The concentrations of PAF which induced aggregation in the elephant platelet suspensions are similar to those which are effective in bovine, equine and human platelet suspensions (Suquet and Leid, 1983; Cargill et al., 1983; Liggitt et al., 1984; Bondy and Gentry, 1988). PAF is a biologically active phosphoglyceride which initiates aggregation through a calcium-calmodulin system (Kuster et al., 1986). In the bovine platelet this PAF-induced biochemi~a1 pathway appears to be more important for aggregation than the thromboxane-dependent pathway (Bondy and Gentry, 1988; Gentry et al., 1989) and the elephant platelet appears to be similar in this regard. Not only is the elephant platelet unresponsive when AA is the sole agonist added to the platelets suspensions, but also there appears to be no correlation between the extent of aggregation and the amount of TXB, formed. Further, ASA, the cyclooxygenase inhibitor which prevents the formation of TXA, from AA, does not inhibit platelet aggregation. The elephant platelet is not unique in these features.

Table 4. Comparison of the inhibitors verapamil and acetylsalicyciic acid on platekt aggregation Maximum aggregation response (%) PAF Collagen 0.9 x 10-9M 20 P s/l Inhibitor (mean k SD) (mean i SD) -.Control 78.3 i 6.1 98.0 t I I .4 Verapamil 5 x IO 4M I x IO--‘M 5 x IO-‘M 1x IO-*M ASA 4 x 10.“M 1 x 10”M

0 17.7 + 4.0 26.1 + 6.0 69.2 + 10.3

91.9 * 6.2 90.4 f 3.9 96.0 + S.7 -

78.9 2 6. I 71.1 i 5.6

103.3 It 11.4

Rat platelets and the platelets from some breeds of dogs are also insensitive to AA stimulation (Nishizawa et al., 1983; Chignard and Vargaftig, 1976). Bovine platelets are not inhibited by ASA (Bondy and Gentry, 1988; Gentry et al., 1989) and aggregation of the bovine platelet, like that of the sheep and mink is not dependent on TXB, formation (Meyers et al., 1980; Bondy and Gentry, 1988). With the limited information available it is difficult to interpret the aggregation response to the combined action of AA and ADP. When 1 x 1Oa4M AA was added to the platelet suspensions aiong with ADP, a significant enhancement of aggregation was observed, but when the concentration of AA was increased to 1 x low3 M, no enhancement of the aggregation response compared to ADP alone was recorded despite the extensive formation of TXB,. One possible interpretation of these observations is that a metabolite of AA, other than TXB,, is important in the biochemical pathway of aggregation in the elephant platelet. If this is the case, the failure of the higher concentration of AA to enhance the aggregation response may be due to the fact that TXB, is being formed at the expense of other metabolite(s). Alternatively, TXB, itself may be an inhibitor of aggregation in the elephant platelet. The existence of a biochemical pathway, other than the thromboxane pathway, is also suggested from the aggregation studies in which verapamil was included in the platelets suspensions. Verapamil caused a dose-dependent inhibition of PAF-induced aggregation (Table 4) in an analogous manner to that reported for the bovine platelet (Bondy and Gentry, 1988). Since it has been shown that PAF stimulates calcium mobilization in the bovine platelet by inducing phosphoinositide and polyphosphoinositide turnover in the platelet membranes (Bondy and Gentry, 1989), this pathway may also be functional in the elephant platelet. However, collagen-induced aggregation must occur through another pathway since, both verapamil and ASA failed to impair aggregation initiated by the addition of collagen to the samples. The nonresponsiveness of the elephant platelets to stimulation with epinephrine is similar to the finding reported for most mammalian species (Dodds, 1978; Meyers, 1986); however, the lack of any potentiating effect between epinephrine and ADP was an unexpected finding (Kinlough-Rathbone and Mustard, 1986). Even more surprising was the inhibitory nature of the combination of serotonin and ADP compared to the response of the platelets to ADP alone. Since shape change but no aggregation was observed in the

51

Platelet aggregation in the Asian elephant piatelet suspensions exposed only to serotonin, it is possible that serotonin is able to interact with pIatelet membrane receptors in the elephant cell. This interaction then either blocks subsequent interaction of ADP with the appropriate receptors or the interaction of serotonin induces biochemical changes in the cell which inhibit the aggregation response to ADP. Since no evidence of either shape change or aggregation was observed in the platelets exposed only to epinephrine, the difference in the epineph~ne and serotonin responses in the presence of ADP may be attributed to the possibility that epinephrine does not interact with any membrane receptors on the elephant platelet. REFERENCES Addonizio V. P., Edmunds L. H. and Colman R. W. (1978) The function of monkey (M. mulafta) platelets compared to platelets of pig, sheep, and man. J. Lab. cfin. Med. 91, 989997.

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