Rat Platelet Aggregation by ATP Aggregonietrical and UCItrastructtiral Comparison Wiith Aggregations Induced by ADP and Collagen Chung-hsin Ts'ao, PhD
This paper describes the aggregation of rat platelets by adenosine triphosphate (ATP). The aggregometry of ATP-induced aggregation and the ultrastructure of ATP-aggregated platelets were compared and contrasted with those of adenosine diphosphate (ADP)-treated and collagen-treated samples. Human platelets were also studied alongside with rat specimens. Several lines of evidence indicate that the ATP-induced aggregation of rat platelet-rich plasma (PRP) is not a result of contaminating ADP in the ATP preparation. ATP did not cause aggregation of human platelets: it inhibited ADP- and collagen-induced human platelet aggregation. ATP pretreated with a creatine phosphate/creatine phosphokinase sy stem caused similar rat platelet aggregation as did ATP not treated with this system. The aggregometnr of ATP-induced aggregation of rat PRP was similar to that of collagen-induced aggregation but markedly different from that of ADP-induced aggregation. However, the nature of ATP-induced aggregation was similar to that induced by ADP. Both ATP- and ADP-induced rat platelet aggregations were not affected by adenosine, adenosine monophosphate, or acetylsalic-lic acid. The ultrastructure of ATP-aggregated platelets was similar to that of ADP-aggregated ones. It appears that either platelets of rats possess specific ATP receptors or the rat plasma contains a material, lacking or insufficiently present in human plasma, that converts ATP to ADP in a fashion similar to the release of ADP from platelet storage granules. (Am J Pathol 85:581-594, 1976)
THE FINDING THAT ADE\NOSINE DIPHOSPHATE (ADP)
causes
aggregation of human platelets 1 is of monumental importance. This observation probably has played an essential role in the stimulation and promotion of research activities in the areas of hemostasis and thrombosis. A great deal of our kno-ledge of the pathophvsiology of hemostasis thrombosis is due to this single discovery. Based on this discoverv, a tremendous amount of data has been accumulated in the past 15 y-ears. It is established that a high degree of structural specificity of adenine nucleotides is necessary for their ability- to aggregate blood platelets. Only ADP, and to a lesser extent adenosine tetraphosphate,23 are capable of inducing human platelet aggregation. Adenosine and its mono- and triphosphates (AMP and ATP) are not only incapable of clumping human platelets but also of having potent inhibitory effects on ADP-induced From the Departmenit of Pathologys. \orthw-estern L-nixersits Scho(l of Mledicine and North"estern \lemorial Hospital. Chicago. Illinois. Accepted for public-ation Jul% 16. 1976, xddress reprin't requests to Dr, C. Ts'ao. Department of Patholoy!-. Northsvestern U'niversits MIedical Center. \\iesle\ Pasilion. East Superior Street and Fairbanks Court. Chicago. IL 60611, 581
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aggregation.4'5 The adenine moiety of ADP is also essential. Replacing adenine with guanine or inosine abolishes the platelet-aggregating activity-.6 In addition, sizable variations of ADP-induced platelet aggregation are noted among animal species.7-9 For example, rat platelets are sensitive to ADP, but the aggregation is usually reversible. Adenosine or AMP exert no inhibition on ADP-induced rat platelet aggregation. Avian thrombocytes are not aggregated by ADP at all.10"'1 Recentlyr, we noted, after repeated trials, that rat platelet-rich plasma (PRP) was aggregable bv both ADP and ATP. This report describes our findings on ATP-induced rat platelet aggregation and compares the aggregometry, as well as the ultrastructure, of ATP-aggregated rat platelets with rat platelets aggregated bv ADP and collagen. Materials and Methods Bbod Samphs Albino rats (Sprague-Dawlev strain, Charles River, Wilmington, Mass.) of both sexes were used in these experiments. Ten male rats (weighing about 50 g each), 10 female rats (weighing about 230 g each), and 4 pregnant rats (weighing about 350 g each) were housed in separate cages and fed Purina Rat Chow and water ad libitum. Rat blood was obtained via cardiac puncture using a No. 19 gauge needle attached to a plastic disposable syringe while the animal was anesthetized with an intraperitoneal injection of sodium pentobarbital (Nembutal sodium, Abbott Laboratories, North Chicago, Ill., about 35 mg, kg body weight). The blood was anticoagulated with 3.2% trisodium citrate in a ratio of 9 parts of blood to 1 part of anticoagulant. Human blood was drawn from an antecubital vein of healthy volunteers of both sexes; one sample was acquired from a pregnant woman in her first trimester. Human subjects had not taken any medication known to affect platelet aggregation in the preceding week. Human blood was anticoagulated with buffered sodium citrate (0.1 NI). Platelet-rich and platelet-poor plasmas (PRP and PPP) were prepared by differential centrifugation."2 Most studies were performed on these "native" PRP samples. Some experiments were done on rat PRP after it was added with autologous PPP to achieve a platelet count comparable to that of human PRP. Platelets in PRP were counted by phase-contrast microscopy.
Reagent All reagents except creatine phosphate, creatine phosphokinase, and collagen w-ere purchased from Sigma Chemical Co., St. Louis. These reagents included adenosine, adenosine-5'-monophosphoric acid (AMP), disodium salts of adenosine-5'-diphosphate and adenosine-5'-triphosphate (ADP and ATP), purified apyrase, and acetylsalicylic acid (ASA). All Sigma reagents were stored at -20 C. Four ATP preparation from three laboratories in our institution, each having vaaning lengths of storage time, were used. One of the preparations was tested less than 24 hours after its receipt. The other three had been stored for 4, 6, and 14 months. According to the manufacturer, these ATP preparations are 99% pure but do contain 0.5 to 0.85 ADP. Creatine phosphate (CP) and creatine phosphokinase (CPK) were purchased from Calbiochemicals, La Jolla, Calif. These reagents were reconstituted in water in concentrations of 200 mM for CP and 2 mg/ml for CPK. This mixture was stored at -20 C. In the
Vol. 85, No. 3 December 1976
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beginning, of an experiment, a -(olume of 0 02 ml CP CPK X"-as mixed Xvith 0.48 ml ATP 0.2 m\l 1. and the mixture wsas incubated at 37 C for 3 minutes. The mixture "vas then chilled in an ice bath before it "as introduced to rat PRP (,ollagen "-as prepared from a strip of human skin according to the method of Green et a! 13 _SA -as first dissolved in acidified Tris-saline buffer (0.06-0.09 \t) at pH :3 The pH "as brought back to 6.8&sith NaOH. All other reagents 'vere dissolved in normal saline XAbbott Laboratories) to desired concentrations. usuallx- in the beginning of a set of experiments. Experimnents
Platelet aggregation studies were performed 'vith a Chronolog platelet aggregometer e(qllipped svith a recorder."2 An aliquot of 0:33 ml PRP wsas mixed mvith 0.02 ml saline or one of the reagents (adenosine. AMP. ASA. apyrase. or CP CPK) 1 to 11'- minutes prior to the addition of 0.05 ml aggregating agent eADP. ATP. or collagen The final concentration of each reagent "as stated in the text. Each aggregation reaction wvas recorded for 5 mintutes.
Electron microscopy "as done on PRP samples that had been exposed to ATP. ADP. \MIP. adenosine. or collagen for 212 minutes. Details of tissue preparation were described else"here 14 L.ltrathin sections "vere stained 'vith uranvl acetate and lead citrate and examined with a Philips-300 transmission electron microscope
Results
The platelet count in "native" rat PRP was over 1 million/cu mm; in human PRP it was 400.000 to 600,000 cu mm. Human and rat PRP sho-ed different aggregometrical patterns in response to aggregating agents. The sensitixity of indixidual PRP to a given aggregating agent varied in both species, but the variations seemed smaller among individual rat samples than among individual human samples. In the rat, undiluted PRP responded slightly better than diluted PRP. With the limited number of rats studied. we wvere not able to discern significant difference in the aggregability of platelets of male, female, and pregnant rats. The most interesting finding was that rat PRP was aggregable by ATP whereas human PRP was not. Details of ATP-induced rat platelet aggregation, as \vell as its aggregometrical and ultrastructural comparison with aggregations induced by ADP and collagen, are presented. These findings are also compared and contrasted wvith aggregation of human PRP. Aggregation of Rat Platelet-Rich Plasma Induced by ATP, ADP, and Collagen
Stirring of rat PRP in the aggregometer did not induce "spontaneous" aggregation. Addition of ATP, ADP, or collagen to PPP caused no change in light transmission. Exposure of rat PRP to any of the three agents resulted in platelet aggregation. Typical experiments depicting doseresponse relationships of ATP-. ADP-. and collagen-induced rat platelet aggregation are show-n in Text-figure 1. ATP treated wvith CP/ CPK caused similar platelet aggregation as did ATP treated wvith saline. The presence
584 - - I
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American Journal of Pathology 125.0 250
6.25 10.0
A
- AT-Fi-(.L RE 1-Light transmission recordings depictplatelet aggregation of rat PRP b%- XTP. ADP. and COllagen. The aggregometrv of ATP-induced aggregation is similar to that of collagen-induced aggregation but mark-edlv different from ADP-induced reaction. Numerals inidicate the final concentrations MA\1) for ATP and ADP in dilutions of original preparation for collagen. Rat platelets are more sensitise to XADP than to AtP. inv,
1/60 1/400
1/600
of CP CPK in rat PRP did not affect subsequent aggregation by ATP. The ATP-induced aggregation wvas not a result of high platelet count in rat PRP. Rat PRP diluted ith autologous PPP to give a platelet count of 400,000 to 600,000/cu mm did not affect the aggregation appreciably (Text-figure 2). There -as no noticeable difference in the pattern and the extent of aggregation induced by four ATP preparations. On a molar basis, rat platelets were much more sensitive to ADP than to ATP. The pattern of ATP-induced aggregation was also markedly different from that of ADP-induced aggregation. Aggregation induced by ADP mvas immediate and usually follow ed by rapid disaggregation. Increasing the concentration of ADP from to 10 jiM slowed down the rate of disaggregation ithout an equal increase in the extent of aggregation. ATP-induced aggregation had a lag period dunrng hich time the light transmission w-as slightly decreased. This photometrical change was very similar to platelet aggregation induced by collagen. Disaggregation was a
b
T \T-FI(.GL E 2-Light transmission changes of rat PRP exposed to ATP (final concentration. 2.3 \1M shomving the aggregation is independent from platelet count. In ( urce a. the PRP had a platelet count of 1.11.,.000 cu mm Curce b. the same PRP mixed swith autologous PPP resulted in a platelet ceunt of 41 3.000 coi mm In
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TEXT-F IGL HE :3-Absence of ATP aggregation of human PRP. In this experiment. a final concentration of 73 .lu\l ATP '----43 U.t-U. -IATP iin LLO tn LI LI IlL I" II "I %-.U .'W I --III JL f Up 2540 p.%1 concentration shomved similar results. ATP 2.5 ol consistently inhibited human platelet aggregation b- ADP (1.5 \M.- upper curve and collagen ,1 2400 dilution: louer curves
gradual in the ATP-induced reaction as compared to that in the ADPinduced aggregation. Increased concentrations of ATP resulted in shortening of the aggregation time with only moderate increase in the extent of aggregation. The shortening of the aggregation time with increased concentrations of ATP was again similar to collagen-induced aggregation in wvhich the collagen concentration wvas increased. It should be noted that the greatest extent of ATP-induced aggregation 'with anconcentration of ATP up to 250 IM wvas around 70%. Failure of ATP to Aggregate Human Platelet-Rich Plasma
ATP in concentrations up to 230 iMM failed to cause aggregation of human PRP. including a sample obtained from a pregnant wvoman. On the contrarx, ATP consistently inhibited ADP- and collagen-induced human platelet aggregation (Text-figure 3). Effects of ATP on ADP-lnduced Rat Platelet Aggregation
The effect of ATP on ADP-induced rat platelet aggregation w as related to the concentration of ADP that wvas used to aggregate rat platelets and to the time at which ATP was introduced to the reaction mixture. W'ith threshold concentrations of ADP (i.e., the minimum concentration that caused marked aggregation), addition of ATP (final concentration, 20 AL ) prior to disaggregation resulted in an apparent prevention of the disaggregation without promoting further increase in light transmission. When ATP was introduced during disaggregation or after the dis-
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I .- Lf
u
Control
TEXT-F- .t-RE 4-Effects of ATP on ADP-induced rat platelet aggregation. Upper Curres-The aggregation Nvas induced by 2 u\1 ADP. \-hich was followed by rapid disaggregation (control). Aliquots of ATP t20 A\l svere introduced to the reaction mixture at sarious times (arrows). When ATP %as delisvered prior to disaggregation. the decrease in light transmission was presented. When .TP %vas introduced during disaggregation. a second aggregation was noted. The extent of the second aggregation was greater "-hen ATP s,as added after platelets had completely disaggregated. Louer Curt-es-Exposure of rat platelets to a subthreshold concentration 0.42 NM of ADP did not affect subsequent aggregation by ATP.
aggregation had completed, further aggregation was observed (Textfigure 4). Addition of saline or adenosine to the mixture at any time during the ADP aggregation had no effect on the aggregation whatsoever. Effects of Adenosine and AMP on Platelet Aggregaton
Neither adenosine (final concentration, 25 jiM) nor ANMP (25 MNI) caused aggregation of rat or human PRP. Adenosine and ANIP exerted no appreciable effect on ATP- and ADP-induced rat platelet aggregation (Text-figure 5), but both inhibited human platelet aggregation. Although Con trol
Adenosine,
TEXT-F I(GUHE 5-Xbsence of effects of adenosine _25 g\l1 and X\IP _25 "M) on rat platelet aggregation induced by ATP l2 gM\l: upper c-urves) and ADP 2.5 4M1: louer curves). ASA also had no effect on these aggregations.
ATP-AGGREGATED RAT PLATELETS
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587
TEXT-FIGURE 6-Inhibition of collagen 1 160 dilutionwinduced aggregation of rat PRP by adenosine (23 g\M and A\IP k25 M\1\/. ASA also inhibited the aggregation.
adenosine and AMP did not affect ATP- and ADP-induced aggregation in the rat, they inhibited collagen-induced aggregation of rat PRP (Textfigure 6). The extent of inhibition was related to the concentration of collagen employed in the experiment. With suprathreshold concentrations of collagen, the inhibition was minimum and was manifested bv a slight prolongation of the aggregation time. With threshold concentrations of collagen, a decrease of the extent of aggregation w as also observed. Effects of ASA and Apyrase on Platelet Aggregation
ASA (final concentration, 0.83 m\1) had no effect on ATP- and ADPinduced aggregation of the rat sample; it abolished the secondary phase of human platelet aggregation induced by ADP. The effect of apyrase (0.1 mg ml) on ADP-induced human and rat platelet aggregation was similar. It did not affect the immediate response of these platelets to ADP but reduced the extent of aggregation and caused a precipitous disaggregation (Text-figure 7). In the presence of apyrase, the pattern of ATP-induced
TF\T-F-l(LURE 7-Similar effect of apyrase on XDP-induced aggregation of human kupper -tirrott
and
r:at 1"Livr
tirtac
PR P A TIP
LU1
centration used in the human sample w-as 1.3 ALM. in the rat. 23 w\l.
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American Journal of Pathology
T'.xi-vi(. ti S-LEffect of aps rase on rat platelet aiyreiation induced bx- 2.3 MNI ATP In the presence of apyrase. the pattern of XATP-induced avizrevation became identical to that induced b% ADP in the absence of apyrasse
aggregation of rat PRP became identical to aggregation induced by ADP in the absence of apyrase (Text-figure 8). Now the response w.as immediate, the extent of aggregation increased, and disaggregation precipitous. Both ASA and apyrase markedly inhibited collagen-induced aggregation of human and rat PRP. Ultrastructure of Rat Platekets Exposed to Adenosine, AMP, ADP, ATP, or Collagen
Rat platelets exposed to adenosine or AMP w-ere not clumped. These platelets retained their discoid shape without detectable alterations on their ultrastructure. Samples exposed to ADP, ATP. or collagen contained platelet aggregates of various sizes. The morphology of ADP- and ATPaggregated platelets wvas similar. These platelets were swollen with short, blunt pseudopods; granules of many platelets, especially those in ADPtreated samples. were centralized. Degranulated platelets were, however. not observed in the ADP- and ATP-treated specimens. Collagen-aggregated platelets wvere distinguished from those aggregated by ADP or ATP by the presence of numerous degranulated elements. Representative electron micrographs are show n in Figure 1 A-C. Discussion
We have repeatedly observed, with the light transmission method of Born.4 aggregation of rat platelets by ATP in citrated rat PRP. Stirring of rat PRP alone or addition of ATP to PPP caused no change in light transmission. The aggregation was confirmed by the demonstration of platelet aggregates in ATP-treated samples xvith electron microscopy. Although ATP is kno-n to cause aggregation of frog thrombocytes," to our know -ledge. it has not been shown to induce aggregation of platelets of any mammalian species. A previous report of human platelet aggregation by ATP 16 has been attributed to the ADP present in the ATP preparation.' In the present study. ve were not able to induce aggregation of human platelets by ATP with concentrations many times greater than
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were needed to clump rat platelets. On the contrary, ATP inhibited human platelet aggregation by ADP or collagen. The ATP-induced aggregation observed by us in the rat PRP does not seem to be a result of contaminating ADP in the ATP preparations. Although there was 0.3 to 0.8%c ADP in the ATP preparations, the amount of ADP [which would be less than 0.2M M in the ATP (10 to 23i5M) that caused marked rat platelet aggregation] was insufficient to aggregate rat platelets. In addition, the aggregation induced by 123 ILM ATP \vas not that much greater than the aggregation caused by 25 gNM ATP. The amount of contaminating ADP in the former would be five times greater than in the latter. Furthermore, ATP previously treated with CP CPK, which would be more than sufficient to convert trace amounts of contaminating ADP to ATP`17 caused similar aggregation as ATP not treated by this sv-stem. If ATP decomposes appreciably during storage to generate ADP. wve wvould have detected greater aggregation with a sample acquired 1 year ago than wvith one obtained 1 day ago. W\7e found no difference in the extent or the pattern of aggregation induced by four ATP preparations acquired by three laboratories at various times. The argument that ATP may somehowv "sensitize" rat platelets to make them extremely responsive to minute amounts of ADP is not supported by the ADP-ATP combination experiments. ATP did not potentiate ADPinduced aggregation -hen it was introduced to the reaction mixture before disaggregation had taken place. In these instances, only the decrease in light transmission, -hich was associated with the ADP-induced aggregation. was obliterated. The obliteration of the decrease in light transmission probably does not represent a true prevention of the disaggregation but rather an aggregation induced by ATP. The absence of further increments in light transmission under these circumstances is probably due to the fact that these platelets have reached their fullest aggregation. and additional ATP could not hav-e made the aggregation better. ATP was capable of causing further aggregation only after these platelets had disaggregated. Another piece of evidence favoring the notion that the ATP-induced aggregation of rat PRP is not due to contaminating ADP is that the patterns of aggregation induced by these tw o closely related nucleotides are completely different. In the case of ADP reactions, the onset of the aggregation was al-ay-s abrupt so long as the concentration of ADP wvas sufficient to elicit an aggregation. ATP-induced aggregation had a lag period. The similarity in the aggregometry of ATP- and collagen-induced rat
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platelet aggregation is intriguing. The prolonged aggregation time is expected in the collagen-induced aggregation. For platelet aggregation induced by collagen is a result of platelet release; the lag period is presumablv needed for sufficient materials to be released from some platelets to initiate the aggregation of other platelets. Indeed, collageninduced aggregation was inhibited by ASA, a well-known inhibitor. The ATP-induced aggregation apparently does not involve the platelet release, and the aggregation is not inhibited by ASA. The meaning of lengthened aggregation time in the ATP reactions will not be known until the mechanism of ATP-induced rat platelet aggregation is better understood. At the present time, only speculations can be offered. An obvious speculation is that the rat platelet surface contains specific ATP receptors, and the interaction between ATP and these receptors is slow in nature. Secondly, rat plasma contains a component (adenosine triphosphatase ATPase or ATPase-like material) that converts exogenous ATP to ADP in a fashion similar to the release of ADP (by collagen stimulation) from storage granules. The second speculation could explain the identical aggregometrv of ATP- and collagen-induced aggregations and the many similarities between ATP- and ADP-induced aggregations. However, this postulation requires additional assumptions: a) this material is lacking or insufficiently present in human plasma and b) the affinitv between rat platelets and ADP is so great that ADP binds to platelet receptors as soon as it is generated from ATP and before it is converted to ATP by the CP/CPK svstem. There is no evidence for any of these assumptions at the present time. Rat platelets behave verv differently from human platelets in many respects. In addition to the literature cited earlier concerning their responses to ADP, and the observations described in this report, rat platelets are not aggregable by epinephrine,7 ristocetin,'8"9 or bovine fibrinogen."' Human platelets are aggregable bv all these agents. The lack of aggregation of rat platelets by these agents is apparently not due to the absence of factors or presence of inhibitors in the rat plasma, for addition of human plasma to rat PRP did not result in aggregation by epinephrine or ristocetin and rat plasma did not inhibit human platelet aggregation bv these two substances."8 In the present study, we showed that ADP- and ATP-induced rat platelet aggregation was not inhibited bv a concentration of ASA which abolished the secondarv wave of human platelet aggregation by ADP. An earlier observation on the reduction of the severity of thrombocvtopenia induced bv an intravenous injection of ADP by ASA in the rat 2 is not confirmed by another report.21 A very recent article ' has shown that in order to have ADP-induced aggregation in-
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December 1976
hiibited. a much higher le-el of cy-clic AMP must be reached in the rat platelets than that is necessary in human platelets. There is little doubt that qualitative as w-ell as quantitative differences do exist betwveen human and rat platelets with respect to their responses to a certain stimulus. On the other hand, there are many similarities betwveen rat and human platelets. For example. the basic ultrastructure of human and rat platelets is the same: platelets of both species are aggregable by collagen and thrombin: prostaglandin E1 and pyrimidine derivatives (RA 233, VK 744) are capable of inhibiting both human and rat platelet aggregation by ADP. Despite these similarities, the numerous dissimilarities betw een human and rat platelets are sufficient to warrant further investigative efforts for the purpose of elucidation of the behavior of rat platelets. These investigations are particularly important in view of the wvide use of this animal species in hemostasis thrombosis research. WN"e believe that before a better understanding of the properties of rat platelets and that of rat plasma is achieved, caution must be exercised in interpreting research data obtained from the rat system mvith respect to human situations. References 1.
2.
*3. 4.
.3. 6.
S. 9. 10. 11. 12.
13.
aarder A. Janisen J. Leland S. Hellem A. Owven PA: Adenosine diphosphate in red eells as the factor in the adhesix-eness of human blood platelets. Nature 192 :331-532. 1961. Clav ton S. Born GVR. Cross MJ: Inhibition of the aggregation of blood platelets by nlcleotides. Nature 200:1:3-1:39. 1963 SUIhegg BA. Hellem AJ. Odegaard AE: Investigations on adenosine diphosphate ADP) induced platelet adhesiveness in vitro. II. Studies on the mechanism. Thromb Diath Haemorrh 11 :305-316. 1964 Born GVR. Cross MJ: The aggregation of blood platelets. J Physiol 168:178-193. 1963 Born GVR. Cross NIJ: Inhibitioni of the aggregation of blood platelets by substances related to adenosine diphosphate. J Phvsiol 166:29P-30P. 1963 Marcus AJ. Zucker IB: The Physiology of Blood Platelets. Newv York. Grune & Stratton. 1965. Siinakos Z. Caen JP: Platelet aggregation in mammalians (human. rat. rabbit. guinea-pig, horse. dog): A comparative study. Thromb Diath Haemorrh 17:99-111. 1967 Philp RB: Species-dependent inhibition of platelet aggregation by adenosine and dipyridamole: Paradoxical potentiation in the rat. Thromb Diat Haemorrh 23:129-1:39. 1970 Macmillan DC. Sim AK: A comparative study of platelet aggregation in man and laboratorv animals. Thromb Diath Haemorrh 24::385-394. 1970 Belamarich FA. Fusari MH. Shepro D. Kien M: In vitro studies of aggregation of non-mammalian thrombocvtes. Nature 212:1579-1380. 1966 Stiller RA. Belamarich FA. Shepro D: Aggregation and release in thrombocvtes of the duck. Am J Physiol 229:206-210. 1973 Ts'ao C-H. Wirman JA. Ruder EA: Altered in vitro functions of platelets prepared by the Haemonetics blood processor. J Lab Clin Med 86:315-325. 1973 Green D. Dunne B. Schmid FR. Rossi EC. Louis G: A study of the variable
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14. 15.
16. 17.
1I8. 19. 20.
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response of human platelets to collagen: Relation to aspirin-induced inhibition of aggregation. Am J Clin Pathol 60:920-926. 1973 Ts'ao C. Green D. Rossi EC: Ultrastructure of ristocetin aggregated normal human platelets: Tortuous boundaries and swollen granules. Scand J Haematol 11:287-297. 197:3 Stiller RA. Belamarich FA. Shepro D: Frog thrombocytes: Aggregation and release reaction. Throm Diath Haemorrh :32:685-694. 1974 Mitchell JAR. Sharp AA: Platelet clumping in -itro. Br J Haematol 10:78-93. 1964 Packham MA. Guccione MA. Chang PL. Mustard JF: Platelet aggregation and release: effects of low)- concentrations of thrombin and collagen. Am J Physiol 225:38-47. 197:3 Tsaao C. Perry I. Glagov S: Effect of human plasma on monkey and rat platelet aggregation. Fed Proc 33611. 1974 (Abstr) DeGaetano G. Donati MB. Revers-Degli Innocenti I. Roncaglioni MC: Defective ristocetin and bovine factor V1III-induced platelet aggregation in normal rats. Experimentia :31:500-502. 1975 Kobavashi I. Didisham P: Systemic effects of ADP-induced aggregation and their modification by aspirin and by pyridinolcarbamate. Thromb Diath Haemorrh
:30:178-190. 197:3
21. Rev ers-Degli Innocenti I. deGaetano G: Platelet aggregation and haemolysis induced in rats by intravenous infusion of ADP: Effect of potentially antithrombotic drugs. Scand J Haematol 1:3::331-3337. 1974 22. \tichel H. Caen JP. Born GCVR. Mliller R. d'Auriac GA. Meyer P: Relation between the inhibition of aggregation and the concentration of cAMP in human and rat platelets. Br j Haematol :33:27.-38. 1976
Acknowledgments The atuthor zwould like to thank Dr. Ennio Rossi for his helpful discussions. Cassandra Smith pro'ided excellent technical assistance, and Ellen Rohde typed the manuscript.
Fgure 1-Electron micrographs of rat platelets aggregated by 25 ,M ATP (A), 2.5 ADP (B), and a 1:160 dilution of collagen (C). The specimens were fixed at 2.5 minutes afterJM exposure of one of these agents. At this time, disaggregation in the ADP-treated sample had not commenced. The ultrastrucure of ATP-aggregated platelets is similar to that of ADP-aggregated platelets. There were no degranulated platelets in these samples. Many collagen-aggregated platelets were degranulated. (A, x 5200; B, x 4750; C, x 4500)
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[End of Article]
This paper describes the aggregation of rat platelets by adenosine triphosphate (ATP). The aggregometry of ATP-induced aggregation and the ultrastructure of ATP-aggregated platelets were compared and contrasted with those of adenosine diphosphate (ADP)-treated and collagen-treated samples. Human platelets were also studied alongside with rat specimens. Several lines of evidence indicate that the ATP-induced aggregation of rat platelet-rich plasma (PRP) is not a result of contaminating ADP in the ATP preparation. ATP did not cause aggregation of human platelets: it inhibited ADP- and collagen-induced human platelet aggregation. ATP pretreated with a creatine phosphate/creatine phosphokinase sy stem caused similar rat platelet aggregation as did ATP not treated with this system. The aggregometnr of ATP-induced aggregation of rat PRP was similar to that of collagen-induced aggregation but markedly different from that of ADP-induced aggregation. However, the nature of ATP-induced aggregation was similar to that induced by ADP. Both ATP- and ADP-induced rat platelet aggregations were not affected by adenosine, adenosine monophosphate, or acetylsalic-lic acid. The ultrastructure of ATP-aggregated platelets was similar to that of ADP-aggregated ones. It appears that either platelets of rats possess specific ATP receptors or the rat plasma contains a material, lacking or insufficiently present in human plasma, that converts ATP to ADP in a fashion similar to the release of ADP from platelet storage granules. (Am J Pathol 85:581-594, 1976)
THE FINDING THAT ADE\NOSINE DIPHOSPHATE (ADP)
causes
aggregation of human platelets 1 is of monumental importance. This observation probably has played an essential role in the stimulation and promotion of research activities in the areas of hemostasis and thrombosis. A great deal of our kno-ledge of the pathophvsiology of hemostasis thrombosis is due to this single discovery. Based on this discoverv, a tremendous amount of data has been accumulated in the past 15 y-ears. It is established that a high degree of structural specificity of adenine nucleotides is necessary for their ability- to aggregate blood platelets. Only ADP, and to a lesser extent adenosine tetraphosphate,23 are capable of inducing human platelet aggregation. Adenosine and its mono- and triphosphates (AMP and ATP) are not only incapable of clumping human platelets but also of having potent inhibitory effects on ADP-induced From the Departmenit of Pathologys. \orthw-estern L-nixersits Scho(l of Mledicine and North"estern \lemorial Hospital. Chicago. Illinois. Accepted for public-ation Jul% 16. 1976, xddress reprin't requests to Dr, C. Ts'ao. Department of Patholoy!-. Northsvestern U'niversits MIedical Center. \\iesle\ Pasilion. East Superior Street and Fairbanks Court. Chicago. IL 60611, 581
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aggregation.4'5 The adenine moiety of ADP is also essential. Replacing adenine with guanine or inosine abolishes the platelet-aggregating activity-.6 In addition, sizable variations of ADP-induced platelet aggregation are noted among animal species.7-9 For example, rat platelets are sensitive to ADP, but the aggregation is usually reversible. Adenosine or AMP exert no inhibition on ADP-induced rat platelet aggregation. Avian thrombocytes are not aggregated by ADP at all.10"'1 Recentlyr, we noted, after repeated trials, that rat platelet-rich plasma (PRP) was aggregable bv both ADP and ATP. This report describes our findings on ATP-induced rat platelet aggregation and compares the aggregometry, as well as the ultrastructure, of ATP-aggregated rat platelets with rat platelets aggregated bv ADP and collagen. Materials and Methods Bbod Samphs Albino rats (Sprague-Dawlev strain, Charles River, Wilmington, Mass.) of both sexes were used in these experiments. Ten male rats (weighing about 50 g each), 10 female rats (weighing about 230 g each), and 4 pregnant rats (weighing about 350 g each) were housed in separate cages and fed Purina Rat Chow and water ad libitum. Rat blood was obtained via cardiac puncture using a No. 19 gauge needle attached to a plastic disposable syringe while the animal was anesthetized with an intraperitoneal injection of sodium pentobarbital (Nembutal sodium, Abbott Laboratories, North Chicago, Ill., about 35 mg, kg body weight). The blood was anticoagulated with 3.2% trisodium citrate in a ratio of 9 parts of blood to 1 part of anticoagulant. Human blood was drawn from an antecubital vein of healthy volunteers of both sexes; one sample was acquired from a pregnant woman in her first trimester. Human subjects had not taken any medication known to affect platelet aggregation in the preceding week. Human blood was anticoagulated with buffered sodium citrate (0.1 NI). Platelet-rich and platelet-poor plasmas (PRP and PPP) were prepared by differential centrifugation."2 Most studies were performed on these "native" PRP samples. Some experiments were done on rat PRP after it was added with autologous PPP to achieve a platelet count comparable to that of human PRP. Platelets in PRP were counted by phase-contrast microscopy.
Reagent All reagents except creatine phosphate, creatine phosphokinase, and collagen w-ere purchased from Sigma Chemical Co., St. Louis. These reagents included adenosine, adenosine-5'-monophosphoric acid (AMP), disodium salts of adenosine-5'-diphosphate and adenosine-5'-triphosphate (ADP and ATP), purified apyrase, and acetylsalicylic acid (ASA). All Sigma reagents were stored at -20 C. Four ATP preparation from three laboratories in our institution, each having vaaning lengths of storage time, were used. One of the preparations was tested less than 24 hours after its receipt. The other three had been stored for 4, 6, and 14 months. According to the manufacturer, these ATP preparations are 99% pure but do contain 0.5 to 0.85 ADP. Creatine phosphate (CP) and creatine phosphokinase (CPK) were purchased from Calbiochemicals, La Jolla, Calif. These reagents were reconstituted in water in concentrations of 200 mM for CP and 2 mg/ml for CPK. This mixture was stored at -20 C. In the
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beginning, of an experiment, a -(olume of 0 02 ml CP CPK X"-as mixed Xvith 0.48 ml ATP 0.2 m\l 1. and the mixture wsas incubated at 37 C for 3 minutes. The mixture "vas then chilled in an ice bath before it "as introduced to rat PRP (,ollagen "-as prepared from a strip of human skin according to the method of Green et a! 13 _SA -as first dissolved in acidified Tris-saline buffer (0.06-0.09 \t) at pH :3 The pH "as brought back to 6.8&sith NaOH. All other reagents 'vere dissolved in normal saline XAbbott Laboratories) to desired concentrations. usuallx- in the beginning of a set of experiments. Experimnents
Platelet aggregation studies were performed 'vith a Chronolog platelet aggregometer e(qllipped svith a recorder."2 An aliquot of 0:33 ml PRP wsas mixed mvith 0.02 ml saline or one of the reagents (adenosine. AMP. ASA. apyrase. or CP CPK) 1 to 11'- minutes prior to the addition of 0.05 ml aggregating agent eADP. ATP. or collagen The final concentration of each reagent "as stated in the text. Each aggregation reaction wvas recorded for 5 mintutes.
Electron microscopy "as done on PRP samples that had been exposed to ATP. ADP. \MIP. adenosine. or collagen for 212 minutes. Details of tissue preparation were described else"here 14 L.ltrathin sections "vere stained 'vith uranvl acetate and lead citrate and examined with a Philips-300 transmission electron microscope
Results
The platelet count in "native" rat PRP was over 1 million/cu mm; in human PRP it was 400.000 to 600,000 cu mm. Human and rat PRP sho-ed different aggregometrical patterns in response to aggregating agents. The sensitixity of indixidual PRP to a given aggregating agent varied in both species, but the variations seemed smaller among individual rat samples than among individual human samples. In the rat, undiluted PRP responded slightly better than diluted PRP. With the limited number of rats studied. we wvere not able to discern significant difference in the aggregability of platelets of male, female, and pregnant rats. The most interesting finding was that rat PRP was aggregable by ATP whereas human PRP was not. Details of ATP-induced rat platelet aggregation, as \vell as its aggregometrical and ultrastructural comparison with aggregations induced by ADP and collagen, are presented. These findings are also compared and contrasted wvith aggregation of human PRP. Aggregation of Rat Platelet-Rich Plasma Induced by ATP, ADP, and Collagen
Stirring of rat PRP in the aggregometer did not induce "spontaneous" aggregation. Addition of ATP, ADP, or collagen to PPP caused no change in light transmission. Exposure of rat PRP to any of the three agents resulted in platelet aggregation. Typical experiments depicting doseresponse relationships of ATP-. ADP-. and collagen-induced rat platelet aggregation are show-n in Text-figure 1. ATP treated wvith CP/ CPK caused similar platelet aggregation as did ATP treated wvith saline. The presence
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A
- AT-Fi-(.L RE 1-Light transmission recordings depictplatelet aggregation of rat PRP b%- XTP. ADP. and COllagen. The aggregometrv of ATP-induced aggregation is similar to that of collagen-induced aggregation but mark-edlv different from ADP-induced reaction. Numerals inidicate the final concentrations MA\1) for ATP and ADP in dilutions of original preparation for collagen. Rat platelets are more sensitise to XADP than to AtP. inv,
1/60 1/400
1/600
of CP CPK in rat PRP did not affect subsequent aggregation by ATP. The ATP-induced aggregation wvas not a result of high platelet count in rat PRP. Rat PRP diluted ith autologous PPP to give a platelet count of 400,000 to 600,000/cu mm did not affect the aggregation appreciably (Text-figure 2). There -as no noticeable difference in the pattern and the extent of aggregation induced by four ATP preparations. On a molar basis, rat platelets were much more sensitive to ADP than to ATP. The pattern of ATP-induced aggregation was also markedly different from that of ADP-induced aggregation. Aggregation induced by ADP mvas immediate and usually follow ed by rapid disaggregation. Increasing the concentration of ADP from to 10 jiM slowed down the rate of disaggregation ithout an equal increase in the extent of aggregation. ATP-induced aggregation had a lag period dunrng hich time the light transmission w-as slightly decreased. This photometrical change was very similar to platelet aggregation induced by collagen. Disaggregation was a
b
T \T-FI(.GL E 2-Light transmission changes of rat PRP exposed to ATP (final concentration. 2.3 \1M shomving the aggregation is independent from platelet count. In ( urce a. the PRP had a platelet count of 1.11.,.000 cu mm Curce b. the same PRP mixed swith autologous PPP resulted in a platelet ceunt of 41 3.000 coi mm In
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TEXT-F IGL HE :3-Absence of ATP aggregation of human PRP. In this experiment. a final concentration of 73 .lu\l ATP '----43 U.t-U. -IATP iin LLO tn LI LI IlL I" II "I %-.U .'W I --III JL f Up 2540 p.%1 concentration shomved similar results. ATP 2.5 ol consistently inhibited human platelet aggregation b- ADP (1.5 \M.- upper curve and collagen ,1 2400 dilution: louer curves
gradual in the ATP-induced reaction as compared to that in the ADPinduced aggregation. Increased concentrations of ATP resulted in shortening of the aggregation time with only moderate increase in the extent of aggregation. The shortening of the aggregation time with increased concentrations of ATP was again similar to collagen-induced aggregation in wvhich the collagen concentration wvas increased. It should be noted that the greatest extent of ATP-induced aggregation 'with anconcentration of ATP up to 250 IM wvas around 70%. Failure of ATP to Aggregate Human Platelet-Rich Plasma
ATP in concentrations up to 230 iMM failed to cause aggregation of human PRP. including a sample obtained from a pregnant wvoman. On the contrarx, ATP consistently inhibited ADP- and collagen-induced human platelet aggregation (Text-figure 3). Effects of ATP on ADP-lnduced Rat Platelet Aggregation
The effect of ATP on ADP-induced rat platelet aggregation w as related to the concentration of ADP that wvas used to aggregate rat platelets and to the time at which ATP was introduced to the reaction mixture. W'ith threshold concentrations of ADP (i.e., the minimum concentration that caused marked aggregation), addition of ATP (final concentration, 20 AL ) prior to disaggregation resulted in an apparent prevention of the disaggregation without promoting further increase in light transmission. When ATP was introduced during disaggregation or after the dis-
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I .- Lf
u
Control
TEXT-F- .t-RE 4-Effects of ATP on ADP-induced rat platelet aggregation. Upper Curres-The aggregation Nvas induced by 2 u\1 ADP. \-hich was followed by rapid disaggregation (control). Aliquots of ATP t20 A\l svere introduced to the reaction mixture at sarious times (arrows). When ATP %as delisvered prior to disaggregation. the decrease in light transmission was presented. When .TP %vas introduced during disaggregation. a second aggregation was noted. The extent of the second aggregation was greater "-hen ATP s,as added after platelets had completely disaggregated. Louer Curt-es-Exposure of rat platelets to a subthreshold concentration 0.42 NM of ADP did not affect subsequent aggregation by ATP.
aggregation had completed, further aggregation was observed (Textfigure 4). Addition of saline or adenosine to the mixture at any time during the ADP aggregation had no effect on the aggregation whatsoever. Effects of Adenosine and AMP on Platelet Aggregaton
Neither adenosine (final concentration, 25 jiM) nor ANMP (25 MNI) caused aggregation of rat or human PRP. Adenosine and ANIP exerted no appreciable effect on ATP- and ADP-induced rat platelet aggregation (Text-figure 5), but both inhibited human platelet aggregation. Although Con trol
Adenosine,
TEXT-F I(GUHE 5-Xbsence of effects of adenosine _25 g\l1 and X\IP _25 "M) on rat platelet aggregation induced by ATP l2 gM\l: upper c-urves) and ADP 2.5 4M1: louer curves). ASA also had no effect on these aggregations.
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TEXT-FIGURE 6-Inhibition of collagen 1 160 dilutionwinduced aggregation of rat PRP by adenosine (23 g\M and A\IP k25 M\1\/. ASA also inhibited the aggregation.
adenosine and AMP did not affect ATP- and ADP-induced aggregation in the rat, they inhibited collagen-induced aggregation of rat PRP (Textfigure 6). The extent of inhibition was related to the concentration of collagen employed in the experiment. With suprathreshold concentrations of collagen, the inhibition was minimum and was manifested bv a slight prolongation of the aggregation time. With threshold concentrations of collagen, a decrease of the extent of aggregation w as also observed. Effects of ASA and Apyrase on Platelet Aggregation
ASA (final concentration, 0.83 m\1) had no effect on ATP- and ADPinduced aggregation of the rat sample; it abolished the secondary phase of human platelet aggregation induced by ADP. The effect of apyrase (0.1 mg ml) on ADP-induced human and rat platelet aggregation was similar. It did not affect the immediate response of these platelets to ADP but reduced the extent of aggregation and caused a precipitous disaggregation (Text-figure 7). In the presence of apyrase, the pattern of ATP-induced
TF\T-F-l(LURE 7-Similar effect of apyrase on XDP-induced aggregation of human kupper -tirrott
and
r:at 1"Livr
tirtac
PR P A TIP
LU1
centration used in the human sample w-as 1.3 ALM. in the rat. 23 w\l.
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T'.xi-vi(. ti S-LEffect of aps rase on rat platelet aiyreiation induced bx- 2.3 MNI ATP In the presence of apyrase. the pattern of XATP-induced avizrevation became identical to that induced b% ADP in the absence of apyrasse
aggregation of rat PRP became identical to aggregation induced by ADP in the absence of apyrase (Text-figure 8). Now the response w.as immediate, the extent of aggregation increased, and disaggregation precipitous. Both ASA and apyrase markedly inhibited collagen-induced aggregation of human and rat PRP. Ultrastructure of Rat Platekets Exposed to Adenosine, AMP, ADP, ATP, or Collagen
Rat platelets exposed to adenosine or AMP w-ere not clumped. These platelets retained their discoid shape without detectable alterations on their ultrastructure. Samples exposed to ADP, ATP. or collagen contained platelet aggregates of various sizes. The morphology of ADP- and ATPaggregated platelets wvas similar. These platelets were swollen with short, blunt pseudopods; granules of many platelets, especially those in ADPtreated samples. were centralized. Degranulated platelets were, however. not observed in the ADP- and ATP-treated specimens. Collagen-aggregated platelets wvere distinguished from those aggregated by ADP or ATP by the presence of numerous degranulated elements. Representative electron micrographs are show n in Figure 1 A-C. Discussion
We have repeatedly observed, with the light transmission method of Born.4 aggregation of rat platelets by ATP in citrated rat PRP. Stirring of rat PRP alone or addition of ATP to PPP caused no change in light transmission. The aggregation was confirmed by the demonstration of platelet aggregates in ATP-treated samples xvith electron microscopy. Although ATP is kno-n to cause aggregation of frog thrombocytes," to our know -ledge. it has not been shown to induce aggregation of platelets of any mammalian species. A previous report of human platelet aggregation by ATP 16 has been attributed to the ADP present in the ATP preparation.' In the present study. ve were not able to induce aggregation of human platelets by ATP with concentrations many times greater than
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were needed to clump rat platelets. On the contrary, ATP inhibited human platelet aggregation by ADP or collagen. The ATP-induced aggregation observed by us in the rat PRP does not seem to be a result of contaminating ADP in the ATP preparations. Although there was 0.3 to 0.8%c ADP in the ATP preparations, the amount of ADP [which would be less than 0.2M M in the ATP (10 to 23i5M) that caused marked rat platelet aggregation] was insufficient to aggregate rat platelets. In addition, the aggregation induced by 123 ILM ATP \vas not that much greater than the aggregation caused by 25 gNM ATP. The amount of contaminating ADP in the former would be five times greater than in the latter. Furthermore, ATP previously treated with CP CPK, which would be more than sufficient to convert trace amounts of contaminating ADP to ATP`17 caused similar aggregation as ATP not treated by this sv-stem. If ATP decomposes appreciably during storage to generate ADP. wve wvould have detected greater aggregation with a sample acquired 1 year ago than wvith one obtained 1 day ago. W\7e found no difference in the extent or the pattern of aggregation induced by four ATP preparations acquired by three laboratories at various times. The argument that ATP may somehowv "sensitize" rat platelets to make them extremely responsive to minute amounts of ADP is not supported by the ADP-ATP combination experiments. ATP did not potentiate ADPinduced aggregation -hen it was introduced to the reaction mixture before disaggregation had taken place. In these instances, only the decrease in light transmission, -hich was associated with the ADP-induced aggregation. was obliterated. The obliteration of the decrease in light transmission probably does not represent a true prevention of the disaggregation but rather an aggregation induced by ATP. The absence of further increments in light transmission under these circumstances is probably due to the fact that these platelets have reached their fullest aggregation. and additional ATP could not hav-e made the aggregation better. ATP was capable of causing further aggregation only after these platelets had disaggregated. Another piece of evidence favoring the notion that the ATP-induced aggregation of rat PRP is not due to contaminating ADP is that the patterns of aggregation induced by these tw o closely related nucleotides are completely different. In the case of ADP reactions, the onset of the aggregation was al-ay-s abrupt so long as the concentration of ADP wvas sufficient to elicit an aggregation. ATP-induced aggregation had a lag period. The similarity in the aggregometry of ATP- and collagen-induced rat
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platelet aggregation is intriguing. The prolonged aggregation time is expected in the collagen-induced aggregation. For platelet aggregation induced by collagen is a result of platelet release; the lag period is presumablv needed for sufficient materials to be released from some platelets to initiate the aggregation of other platelets. Indeed, collageninduced aggregation was inhibited by ASA, a well-known inhibitor. The ATP-induced aggregation apparently does not involve the platelet release, and the aggregation is not inhibited by ASA. The meaning of lengthened aggregation time in the ATP reactions will not be known until the mechanism of ATP-induced rat platelet aggregation is better understood. At the present time, only speculations can be offered. An obvious speculation is that the rat platelet surface contains specific ATP receptors, and the interaction between ATP and these receptors is slow in nature. Secondly, rat plasma contains a component (adenosine triphosphatase ATPase or ATPase-like material) that converts exogenous ATP to ADP in a fashion similar to the release of ADP (by collagen stimulation) from storage granules. The second speculation could explain the identical aggregometrv of ATP- and collagen-induced aggregations and the many similarities between ATP- and ADP-induced aggregations. However, this postulation requires additional assumptions: a) this material is lacking or insufficiently present in human plasma and b) the affinitv between rat platelets and ADP is so great that ADP binds to platelet receptors as soon as it is generated from ATP and before it is converted to ATP by the CP/CPK svstem. There is no evidence for any of these assumptions at the present time. Rat platelets behave verv differently from human platelets in many respects. In addition to the literature cited earlier concerning their responses to ADP, and the observations described in this report, rat platelets are not aggregable by epinephrine,7 ristocetin,'8"9 or bovine fibrinogen."' Human platelets are aggregable bv all these agents. The lack of aggregation of rat platelets by these agents is apparently not due to the absence of factors or presence of inhibitors in the rat plasma, for addition of human plasma to rat PRP did not result in aggregation by epinephrine or ristocetin and rat plasma did not inhibit human platelet aggregation bv these two substances."8 In the present study, we showed that ADP- and ATP-induced rat platelet aggregation was not inhibited bv a concentration of ASA which abolished the secondarv wave of human platelet aggregation by ADP. An earlier observation on the reduction of the severity of thrombocvtopenia induced bv an intravenous injection of ADP by ASA in the rat 2 is not confirmed by another report.21 A very recent article ' has shown that in order to have ADP-induced aggregation in-
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hiibited. a much higher le-el of cy-clic AMP must be reached in the rat platelets than that is necessary in human platelets. There is little doubt that qualitative as w-ell as quantitative differences do exist betwveen human and rat platelets with respect to their responses to a certain stimulus. On the other hand, there are many similarities betwveen rat and human platelets. For example. the basic ultrastructure of human and rat platelets is the same: platelets of both species are aggregable by collagen and thrombin: prostaglandin E1 and pyrimidine derivatives (RA 233, VK 744) are capable of inhibiting both human and rat platelet aggregation by ADP. Despite these similarities, the numerous dissimilarities betw een human and rat platelets are sufficient to warrant further investigative efforts for the purpose of elucidation of the behavior of rat platelets. These investigations are particularly important in view of the wvide use of this animal species in hemostasis thrombosis research. WN"e believe that before a better understanding of the properties of rat platelets and that of rat plasma is achieved, caution must be exercised in interpreting research data obtained from the rat system mvith respect to human situations. References 1.
2.
*3. 4.
.3. 6.
S. 9. 10. 11. 12.
13.
aarder A. Janisen J. Leland S. Hellem A. Owven PA: Adenosine diphosphate in red eells as the factor in the adhesix-eness of human blood platelets. Nature 192 :331-532. 1961. Clav ton S. Born GVR. Cross MJ: Inhibition of the aggregation of blood platelets by nlcleotides. Nature 200:1:3-1:39. 1963 SUIhegg BA. Hellem AJ. Odegaard AE: Investigations on adenosine diphosphate ADP) induced platelet adhesiveness in vitro. II. Studies on the mechanism. Thromb Diath Haemorrh 11 :305-316. 1964 Born GVR. Cross MJ: The aggregation of blood platelets. J Physiol 168:178-193. 1963 Born GVR. Cross NIJ: Inhibitioni of the aggregation of blood platelets by substances related to adenosine diphosphate. J Phvsiol 166:29P-30P. 1963 Marcus AJ. Zucker IB: The Physiology of Blood Platelets. Newv York. Grune & Stratton. 1965. Siinakos Z. Caen JP: Platelet aggregation in mammalians (human. rat. rabbit. guinea-pig, horse. dog): A comparative study. Thromb Diath Haemorrh 17:99-111. 1967 Philp RB: Species-dependent inhibition of platelet aggregation by adenosine and dipyridamole: Paradoxical potentiation in the rat. Thromb Diat Haemorrh 23:129-1:39. 1970 Macmillan DC. Sim AK: A comparative study of platelet aggregation in man and laboratorv animals. Thromb Diath Haemorrh 24::385-394. 1970 Belamarich FA. Fusari MH. Shepro D. Kien M: In vitro studies of aggregation of non-mammalian thrombocvtes. Nature 212:1579-1380. 1966 Stiller RA. Belamarich FA. Shepro D: Aggregation and release in thrombocvtes of the duck. Am J Physiol 229:206-210. 1973 Ts'ao C-H. Wirman JA. Ruder EA: Altered in vitro functions of platelets prepared by the Haemonetics blood processor. J Lab Clin Med 86:315-325. 1973 Green D. Dunne B. Schmid FR. Rossi EC. Louis G: A study of the variable
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16. 17.
1I8. 19. 20.
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response of human platelets to collagen: Relation to aspirin-induced inhibition of aggregation. Am J Clin Pathol 60:920-926. 1973 Ts'ao C. Green D. Rossi EC: Ultrastructure of ristocetin aggregated normal human platelets: Tortuous boundaries and swollen granules. Scand J Haematol 11:287-297. 197:3 Stiller RA. Belamarich FA. Shepro D: Frog thrombocytes: Aggregation and release reaction. Throm Diath Haemorrh :32:685-694. 1974 Mitchell JAR. Sharp AA: Platelet clumping in -itro. Br J Haematol 10:78-93. 1964 Packham MA. Guccione MA. Chang PL. Mustard JF: Platelet aggregation and release: effects of low)- concentrations of thrombin and collagen. Am J Physiol 225:38-47. 197:3 Tsaao C. Perry I. Glagov S: Effect of human plasma on monkey and rat platelet aggregation. Fed Proc 33611. 1974 (Abstr) DeGaetano G. Donati MB. Revers-Degli Innocenti I. Roncaglioni MC: Defective ristocetin and bovine factor V1III-induced platelet aggregation in normal rats. Experimentia :31:500-502. 1975 Kobavashi I. Didisham P: Systemic effects of ADP-induced aggregation and their modification by aspirin and by pyridinolcarbamate. Thromb Diath Haemorrh
:30:178-190. 197:3
21. Rev ers-Degli Innocenti I. deGaetano G: Platelet aggregation and haemolysis induced in rats by intravenous infusion of ADP: Effect of potentially antithrombotic drugs. Scand J Haematol 1:3::331-3337. 1974 22. \tichel H. Caen JP. Born GCVR. Mliller R. d'Auriac GA. Meyer P: Relation between the inhibition of aggregation and the concentration of cAMP in human and rat platelets. Br j Haematol :33:27.-38. 1976
Acknowledgments The atuthor zwould like to thank Dr. Ennio Rossi for his helpful discussions. Cassandra Smith pro'ided excellent technical assistance, and Ellen Rohde typed the manuscript.
Fgure 1-Electron micrographs of rat platelets aggregated by 25 ,M ATP (A), 2.5 ADP (B), and a 1:160 dilution of collagen (C). The specimens were fixed at 2.5 minutes afterJM exposure of one of these agents. At this time, disaggregation in the ADP-treated sample had not commenced. The ultrastrucure of ATP-aggregated platelets is similar to that of ADP-aggregated platelets. There were no degranulated platelets in these samples. Many collagen-aggregated platelets were degranulated. (A, x 5200; B, x 4750; C, x 4500)
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