Mechanisms of vascular graft thrombosis: Role of altered canine platelet sensitivity to thromboxane

THROMBOSIS RESEARCH 55; 695-707, 1989 0049-3848/89 $3.00 t .OO Printed in the USA. Copyright (c) 1989 Pergamon Press plc. All rights reserved.

HlKHAUISl4SOF VASCULAR GSAPT THROMBOSIS: ROLE OF ALTERED CAMIUE PLATELET SSUSITIVITY TO THROl'lBOXAD8

Hari A. HcGofft*, Brent T. Allen*, Terry Der*, Gregorio A. Sicard*, and Samuel A. Santorot+ *Vascular Surgery Section, Department of Surgery Washington University School of Medicine, St. Louis, UO

63110

*Division of Laboratory Medicine - Box 8118 Departments of Pathology and Uedicine Washington University School of Medicine St. Louis, HO 63110 USA accepted in revised form 21.6.1989 (Received 18.1.1989; by Editor N.U. Bang)

BSTSACT Using the standard turbidimetric method of platelet aggregation and quantitation of platelet secretion with 14C-Serotonin, we have examined the responsiveness of the platelets of mongrel dogs to arachidonic acid (AA), and the thromboxane agonist U46619 in the and presence absence of a subthreshold concentration of In response to stimulation with 750 PM AA, the epinephrine. platelets of 18 dogs produced irreversible aggregation (Group I), the platelets of 22 dogs showed, at most, reversible aggregation (Group II), while the platelets of 8 dogs demonstrated no In the presence of AA and a aggregatory response (Group III). subthreshold concentration of epinephrine (0.5 PM), the platelets of all three groups demonstrated enhanced aggregatory and secretory responses although the extent of 14C-Serotonin secretion differed significantly between all three groups. These in vitro differences in platelet aggregation correlate with the &D vivo deposition of platelets onto synthetic vascular grafts and the maintenance of graft patency. When stinnalated with 0.5 PM U46619 and a subthreshold concentration of epinephrine, the platelets of 97%

Key

words:

+To

whom all correspondence should be addressed

Platelet aggregation, secretion, thrombosis

695

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Group I dogs and 75% of Group II dogs exhibited irreversible while the platelets of all Group III dogs showed only aggregation, significant differences in addition, reversible aggregation. In the extent of 14C-Serotonin secretion to this combination of Further examination of the agonists were observed between groups. revealed that of U46619 on canine platelets effects specific although the aggregatory and secretory responses to U46619 vary between the different canine platelet populations, the threshold concentration of U46619 required to produce platelet shape change of the stable Quantitation groups. among all identical is pathway, the cyclooxygenase via AA produced metabolite of thromboxane B2 (TxB2), revealed no significant differences in the production of TxB2 by the platelets of these different suggest that the populations upon stimulation with AA. Our results mechanisms underlying the differences in responsiveness of canine due to differences in sensitivity of platelets to AA, are likely to the mechanism canine platelets to TxA2, and may be localized aggregation and secretion in responsible for mediating platelet response to TxA2.

INTRODUCTION Recent studies have indicated that platelets contribute significantly to the development of thromboembolic diseases such as coronary artery disease, strokes, TIAs, and peripheral vascular disease (1,2,3,4). Numerous attempts have been made to identify in vitro characteristics of human platelet function which might be predictive of the tendency to develop thromboembolic disorders, but none have yielded a consistently reliable test (5). In vitro of the mechanisms of platelet function should provide valuable study information regarding -in vivo reactivity of platelets and the development of thrombotic disease. Our laboratory has previously identified two populations of dogs which could be distinguished by both their -in vitro platelet aggregatory response to AA and their tendency to thrombose small diameter synthetic vascular grafts (6,7). Those dogs with platelets which aggregated irreversibly in response to AA tended to thrombose grafts in less than 1 month, while those dogs with platelets which did not aggregate irreversibly to AA tended to maintain patent grafts at one month. Other laboratories have also identified differences in canine platelet populations in response to AA and other platelet agonists, including ADP, collagen, epinephrine and serotonin. In addition, several groups have studied AA metabolite production in search of an explanation for these aggregatory differences but conflicting results have been obtained (8.9.10). In order to begin to elucidate the mechanisms underlying these differences in canine platelet aggregability. we have studied h -vitro aggregatory and secretory .responses via the AA/thromboxane A2 pathway by supplying (TxA2) exogenous AA, and by use of the TxA2 analogue, U46619. Furthermore, we have directly quantitated generation of the stable AA metabolite of the cyclooxygenase pathway, thromboxane B2 (TxB2).

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MATERIALS AUDMETIiODS Surgical Technique Placement of bilateral carotid and femoral polytetrafluroethylene (PTFE) grafts (W.G. Gore & Assoc., Flagstaff, AR) was performed as previously described by our laboratory (6,7). Briefly, carotid arteries and femoral arteries were exposed bilaterally under pentobarbital anesthesia was infused prior to cross clamping of (30 mg/kg). Heparin (100 &kg) vessels. PTFE grafts (4 mm x 6 cm) were implanted in each carotid and femoral artery in an end-to-end fashion with 7-O prolene (Ethicon, Inc., Somerville, RJ) suture by the triangulation technique. At the conclusion of the procedure, lllIn-labelled platelets were infused and flow was restored to these arteries. The incisions were closed in layers. Dogs received Cefamandol (500 mg/IV) (Eli Lilly & Co., Indianapolis, It?)immediately prior to and following surgery. Platelet labelling. scintigravhic imaging and analysis Platelets were labelled on the day of surgery and 24 hours prior to subsequent scanning with lllIndium as described by Heaton and as-sociates (11). Inunediatelyprior to scanning, autologous erythrocytes were labelled with ggTc and infused intravenously. The dogs were positioned under a large field of view scintillation camera fitted with a medium energy parallel-hole collimator interfaced to a digital computer. Separate 150,000 count images of carotid and femoral regions were collected, digitized and processed on the computer. Dogs were scanned at 24 hours, two weeks, and Platelet deposition was one month after implantation of the grafts. measured and expressed as Percent Indium Excess (%IE) as described by Powers et al (12). Patency was determined by the presence of palpable pulses and appearance of contiguous vessels on ggTc scanning. Aggregation Venipuncture was performed in either the cephalic, saphenous, or external jugular vein on mongrel dogs of both sexes weighing between 20-30 kg. Thirty-six milliliters of whole blood was collected in a plastic syringe containing 4 milliliters of 3.8% sodium citrate. Platelet rich plasma (PRP) was prepared in the standard fashion by centrifugation of the whole blood specimen at 180 x g for 15 min. Platelet poor plasma (PPP) was prepared by centrifugation of the remaining blood specimen at 2000 x g for The platelet count of each PRP specimen was adjusted to 15 min. 250,00O/rl with autologous PPP. Aggregation was performed by the standard turbidimetric method of Born (13) in a Payton (Payton Scientific Inc., Buffalo, NY) dual channel aggregometer coupled to a Linear (Irvine, CA) chart recorder. Briefly, the percent light transmittance was standardized by setting the recorder pen at 10% with PRP and at 90% with PPP. Adjusted PRP was incubated at 37°C for 5 min prior to aggregation. To 450 Pl of PRP, 50 ~1 of agonist was added and this mixture was stirred at 900 rpm for 5 min. The change in light transmittance was recorded for 5 min, and the character of the aggregation response was determined as either irreversible, reversible, or no aggregation as shown in Figure 1. The agonists used and the final concentrations were AA (750 pH), and U46619 (0.5 PM). Indomethacin at a concentration of 10 PM was used with U46619 to eliminate endogenous TxA2 production. In addition to testing alone, AA and U46619 were also examined in the presence of a subthreshold concentration of epinephrine (0.5 pH). AA was purchased from BioData Corporation (Hatboro, PA), epinephrine from Helena Laboratories (Beaumont, TX), and U46619 from Caymen Chemicals, Inc.

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(Ann Arbor, HI). Louis, MO).

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Indomethacin was obtained from Sigma Diagnostics (St.

Secretion 14C-Serotonin secretion was performed in conjunction with aggregation studies. Adjusted PRP was incubated with 14C-Serotonin (0.01 @i/ml) for 30 min prior to testing. 14C-Serotonin (54 mCi/mole) was purchased from Amersham, Inc. (Arlington Heights, IL). After incubation, aggregation was performed as described above. Five minutes after the addition of agonist to the aggregometer cuvette, the cuvette contents were centrifuged One hundred microliters at 12,000 x g in a microcentrifuge for 2 min. (ul) of supernatant were then removed for counting in a scintillation The extent of secretion was determined as a percent of platelet vial. associated 14C-Serotonin released upon agonist stimulation by the formula: X release

=

specimen cpm - background cum total cpm - background cpm

x

100

The total counts per minute (cpm) was obtained by counting a 100 ~1 specimen of unactivated, uncentrifuged labelled PRP. The background cpm (or the label not taken up by the platelets) was determined by counting 100 l.11 of supernatant following centrifugation of unactivated PRP. Preliminary studies in our laboratory demonstrated no significant difference in the extent of 14C-Serotonin secretion when performed with and without the addition of cold 4% formalin - 0.1 H EDTA solution prior to centrifugation of the samples (9). Thromboxane B2 (TxB2) generation A separate set of 13 dogs was studied for platelet TxB2 generation. Venipuncture was performed and PRP was prepared as described above. Aggregation was performed using 750 ph AA as the agonist and the aggregatory response was characterized. After 5 minutes of aggregation, the cuvette contents were centrifuged at 12,000 x g for 2 minutes, and a 300 )rl aliquot of supernatant was removed and stored at -80°C for future quantitation. Previous determination of TxB2 from unactivated, centrifuged PRP samples levels approximately 1% of levels from 750 @I AA revealed TxB2 activated, centrifuged samples. All specimens were treated in the same manner with the same time intervals for aggregation, centrifugation, and aspiration of supernatant in order to eliminate variability of TxB2 production between samples due to centrifugation alone. The production of the arachidonic acid metabolite thromboxane A2 (measured as determined was using a competitive binding TxB2) radioimmunoassay as previously described Briefly, TxD2 was (14). extracted from plasma with octadecyl (Cl81 columns (J.T. Baker Chemical co., Phillipsburg, NJ). Efficiency of extraction was calculated by recovery of 3H-TxB2 (New England measuring the tracer Nuclear, Boston, MA) added to samples prior to extraction. Samples were then combined with specific rabbit antisera (Advanced Hagnetics Inc., Cambridge, MA) to TxD2. After a minimum of 12 hours incubation at 4"C, a dextran-coated charcoal solution was added to remove the excess tritium from the samples. Triplicate antibody-bound aliquots were then counted in a liquid scintillation counter. A standard curve prepared from a stock concentrate of 100 ng/ml diluted with bovine gamma globulin-phosphate buffer with a range of 200 pg to 0.18 pg/O.l ml was included with each assay. The cross-reactivity of TxB2 antisera with prostaglandins and other metabolites was minimal as previously reported (14).

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Platelet shave change PRP was prepared as described above and placed into a Payton aggregometer cuvette. The sensitivity of the aggregometer was increased in order to augment changes in light transmittance through the cuvette indica-tive of platelet shape change. To 450 ~1 of PRP, 50 PL of U46619 was added, and the change in light transmittance was recorded for 2 minutes. The final concentrations of U46619 tested were 0.005, 0.01, 0.05, 0.1, 0.5, 1.0, and 5.0 uM. Indomethacin at a concentration of 10 uM was present The minimum in all specimens to inhibit endogenous TxA2 formation. concentration of U46619 which produced platelet shape change, designated as the threshold concentration, was recorded for each dog's platelets. Statistical analysis Analyses of secretion, The data were expressed as mean f SEH. threshold concentrations for aggregation, and TxB2 generation were done by the paired Student's t-test. Platelet deposition (XIE) was analyzed by the Mann-Whitney rank test. Patency was evaluated by Chi-Square test. Animal Care Animal care complied with the "Principles of Laboratory Animal Care" and the "Guide for the Care and Use of Laboratory Animals" (National Institutes of Health Publication No. 86-23, Revised 1985).

Arachidonic Acid (AA) - Induced Platelet Aggregation At least two separate and independent studies of aggregation in response to 750 uU AA were performed on PRP from each dog. The platelets of 18 dogs produced irreversible aggregation in response to 750 uH AA, and were classified as Group I. Of these 18 dogs, 5 had platelets which produced irreversible aggregation in one study and reversible aggregation in a subsequent study. The platelets of 22 dogs demonstrated, at most, reversible aggregation to 750 uFl AA. Five of these 22 dogs had platelets which showed reversible aggregation on one study but no aggregation on a subsequent study. These 22 dogs were classified as Group II.

FIG. 1 Aggregatory responses to 750 @I AA of platelets from 48 dogs.

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The platelets of 8 dogs produced no aggregatory response to any concentration of AA upon repeated testing. These 8 dogs comprised Group III (Figure 1). The extent of 14C-Serotonin secretion from the platelets of Group I dogs was significantly greater in response to 750 uH AA than from platelets of Group II dogs (p <.OOl) or Group III dogs (p < .OOl). There was no significant difference in the extent of 14C-Serotonin secretion between platelets from Group II and III dogs (Figure 2).

FIG. 2 Extent of 14C-Serotonin secretion from platelets of three groups of dogs in response to 750 uM AA in the absence and presence of 0.5 ph epinephrine.

When the responsiveness to 750 )rHAA was examined in the presence of a subthreshold concentration of 0.5 MM epinephrine, (a concentration insufficient to elicit any aggregatory or secretory response alone), aggregatory responses were augmented in all three groups as judged by increases in the magnitude and rate of aggregation. This enhancement was most marked in the platelets of Group II dogs in which 18 of the 22 dogs increased their response from reversible to irreversible aggregation. The platelets of Group I dogs continued to manifest irreversible aggregation, while the platelets of 7 of 8 Group III dogs changed from no aggregation to reversible aggregation. One dog of Group III had platelets which produced irreversible aggregation in res onse to potentiation of AA with subthreshold epinephrine. The extent of P4C-Serotonin secretion in response to 750 PM AA and 0.5 $I epinephrine in combination was increased from platelets of Group I and II dogs but not significantly in Group III dogs. Significant differences were observed in the extent of 14C-Serotonin secretion between all three groups (Figure 2). Furthermore, those Group II dogs with platelets which aggregated irreversibly to the combination of 750 ~EI AA and 0.5 ut! epinephrine demonstrated platelet secretion of 25.0 ? 2.6 X 14C-Serotonin which was significantly lower than the 38.7 f 2.3% 14C-Serotonin secreted from the platelets of Group I dogs (p < .005).

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Platelet Imaging and Analysis of Patency A group of 10 dogs was selected to have grafts implanted. Preooerative b vitro evaluation of the platelet aggregacory response to AA revealed 5 dogs with platelets which aggregated irreversibly to AA, and 5 dogs with platelets which did not aggregate irreversibly, including one dog with platelets which produced no aggregation to AA. The dual isotope method for determination of plate:;: d;~~~~lt..o~ll~ grafts was employed using lllIn-labelled platelets RBC. Scanning performed at 24 hours revealed significantly greater platelet deposition, calculated as XIE, among dogs with platelets which aggregated irreversibly to AA than dogs with platelets which did not aggregate irreversibly to AA (Table 1). Comparison of the %IE between the two groups at two weeks and one month was impaired due to the occlusion of most of the grafts in the first group.

TABLE 1 Platelet Deposition and Patency of Small Diameter Vascular Grafts Response to 750 )lNAA

l"In

Deposition (%IE)* 24 Hour (N)t

96 1 Month Graft Patency

Irreversible aggregation

209 f 39 (16)

15% (20)

Reversible or no aggregation

113 t 12 (20)

50% (20)

<.02*

<.025§

P Values *Wean f SEH

)Number of grafts studied

%ann

- Whitney Test

(N)t

SChi-Square Test

Graft patency was determined at 24 hours, two weeks, and one month by palpation of the femoral vessels and by the presence of contiguous arterial vessels in the carotid region as revealed by ggTc scanning. In dogs with platelets which aggregated irreversibly, I7 of the 20 grafts were occluded by one month, while in dogs with platelets which did not aggregate irreversibly, 10 of 20 grafts were occluded at one month. Analysis of patency at one month by the Chi-Square test revealed significant differences between these two canine populations. U46619 U46619 is an analogue of thromboxane A2 (TxA2) and functions through the PGH2/TxA2 endoperoxide receptor (15,16,17). By supplying this compound exogenously in the presence of indomethacin to inhibit endogenous production, we bypassed AA metabolism eliminating any potential variability among canine platelets to produce TxA2. Canine platelets were exposed to U46619 in the presence or absence of 0.5 PM epinephrine. U46619 alone did not produce irreversible aggregation in the platelets of any dog. Differences between the platelet responses of the three groups of dogs were most marked when stimulated with the combination of 0.5 PM U46619 and Aggregatory response to this combination of agonists 0.5 #I epinephrine . was studied at least twice on the platelets from 35 of 48 dogs. Within Group I, the platelets of only one of 14 dogs produced inconsistent results

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of irreversible aggregation in one study and reversible aggregation in a separate study. The platelets of 3 of 18 dogs of Group II demonstrated similar inconsistent results in the 2 independent studies, while the platelets of the 3 dogs studied twice in Group III consistently demonstrated only reversible aggregation. The combination of 0.5 PM U46619 and 0.5 pl4 epinephrine produced irreversible aggregation in the platelets of 97% of Group I dogs and 7% of Group II dogs and only reversible aggregation in the platelets of 25% of Group II dogs and all Group III dogs (Table 2). TABLE 2 Platelet Responsiveness to U46619 (0.5 ullw) + Epinephrine (0.5 uR)

Aggregation (X response) R IR

14C-Serotonin Secretion (Percent)

Group

n

I

18

97

II

22

75

25

9.1 +-1.8

III

8

0

100

2.1 f 0.9

3.

15.1 +-1.8**

*mean t SRI4 *paired Student's t-test

U46619 alone failed to elicit significant secretion of 14C-Serotonin from platelets of any dogs; however, the combination of 0.5 pliU46619 and 0.5 uH subthreshold epinephrine produced significant amounts of secretion from the platelets of Group I and II dogs but not from Group III dogs. Application of the paired Student's t-test demonstrated the extent of 14C-Serotonin secretion between the platelets of the three groups to be significantly different at p co.05 (Table 2). Platelet shape change Recent studies relying solely on receptor binding information support the presence of only one binding site for TxA2 (17,181. Conversely, several other studies which have examined receptor binding and functional effects of TxA2 analogues on platelets have indicated the presence of two distinct classes of receptors for TxA2 on platelets (16,19,20,21). According to Rorinelli et al (16). one receptor appears to mediate platelet shape change and myosin light chain phosphorylation at low agonist concentrations with high affinity binding while the second receptor elicits aggregation and secretion at higher agonist concentrations with lower affinity binding. To determine whether differences in one or both of these proposed receptors for TxA2 could be responsible for differences in canine platelet reactivity to AA, we studied platelet shape change along with aggregation and secretion in response to U46619. We classified 36 dogs into three groups according to their platelet aggregatory response to AA as previously described. Differences in aggregation in response to U46619 and subthreshold epinephrine similar to to those shown in Table 2 were observed. Significant differences (p c.001) were also observed in the extents of 14C-Serotonin secretion between Groups I and III and Groups II and XII but not between Groups I and II (p >.l) for this sub population of dogs. In contrast to the striking differences observed in aggregation and secretion, the threshold

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concentration of U46619 necessary to produce platelet shape change (0.05 wBn) was found to be the same for the platelets of all 36 dogs examined (Table 3). TABLE 3 Shape Change Induced by U46619 U46619 0.5 plf+ Epinephrine 0.5 vM

Aggregation (X response) R IR

Group

n

I

12

96

4

II

18

7s

25

III

6

0

100

*mean f SEM

U46619

I%-Serotonin Secretion (Percent)

Threshold concentration for Shape change

11.9 t 2.1*+

0.05

8.8 f 1.7

0.05

1.1 * 0.5

0.05

*paired Student's t-test

Thromboxane B.J (TxB,) generation Using 750 pU &AA as a stimulus, we quantitated TxB9 generation by radioimmunoaasay from the platelets of a separate group of !l3 dogs. Within this group of 13 dogs, we identified 4 dogs with platelets which exhibited irreversible aggregation to AA and 9 dogs which produced only reversible aggregation (8 dogs) or no aggregation (1 dog) in response to AA. We found no significant difference (p >.l) in the amount of TxA2 produced by these two populations of dogs (Table 4). It is noteworthy that the platelets from the one dog exhibiting no response to AA produced a quantity of TxA2 (8.0 x 102 ng/ml) which exceeded the mean value of that produced by the dogs exhibiting irreversible aggregation in response to TxA2. I

-

_

TABLE 4 TxB2 Generation from 750 PM AA (X102 ng/ml)

Irreversible Aggregation

TxB2 n *

mean f SEW

6.0 +-0.4*t 4

tpaired Students t-test

Reversible or no aggregation

5.3 t 0.4 9

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DISCUSSIOW Canine models have been used extensively in cardiac and vascular surgery Although normally functioning human platelets aggregate research. a variable percentage of canine platelets aggregate irreversibly to AA, only Several investigators have identified canine irreversibly to AA (8.9,22). populations which differ in their aggregatory responses to AA as well as other such as ADP, epinephrine and serotonin. Clemmons and Meyers (9) agonists, studied extensively the effects of platelet agonists on different breeds of Their dogs and found platelet responsiveness to AA to be dependent on breed. results suggested that variability of canine platelet reactivity was in part In response to serotonin stimulation, they dependent upon AA metabolism. found a significantly greater amount of platelet TxB2 produced from dogs with platelets which aggregated irreversibly in response to AA than from dogs with platelets which did not aggregate irreversibly (9). Johnson et al., however, found no difference in generation of malondialdehyde, a by-product of of these two populations of dogs reduction. between the platelets TZ pIn a subsequent that differences in platelet study [23 I, they postulated levels of cAHP might be responsible for the differing platelet responsiveness to AA, but such differences were not demonstrated. In our own laboratory, we noted that those dogs with platelets aggregating irreversibly in vitro to AA were likely to thrombose small diameter synthetic grafts by one month whereas those dogs with platelets which failed to aggregate irreversibly to AA tended to maintain patent synthetic grafts at 1 month (6). Since platelet adhesion, activation, and aggregation are thought to be responsible for acute occlusion of small diameter synthetic grafts, the existence of canine populations which differ in both in vitro platelet reactivity and h m thrombotic tendency provides an opportunity to study the mechanisms responsible for differences in canine platelet function and thrombotic tendency and the relationship between the two. While the results of several previous studies have suggested that the differences in aggregation observed with canine platelets are dependent upon the AA/cycle-oxygenase pathway, the nature of the underlying difference(s) has not been elucidated. For example, the differences in aggregation could be due to a quantitative difference in the metabolism of AA and generation of TxA2 or to a difference in the sensitivity of canine platelets to the active AA/cyclooxygenase pathway end product, TxA2. We explored the role of the AA/cyclooxygenase pathway in the aggregatory differences of canine platelets by studying in vitro platelet responses to AA, and the TxA2 mimetic, U46619, under conditions where endogenous generation of TxA2 was inhibited. In addition, we directly quantitated the generation of the stable metabolite, TxB2 following stimulation with AA. Although Burke et al (24) have examined platelet aggregation produced by U46619 in a small group of dogs exhibiting a homogeneous aggregation response to AA, there have been no previous studies of platelet responsiveness to U46619 in dog platelets which demonstrate differential aggregatory patterns to AA. We also correlated the tendency of dogs to thrombose synthetic vascular grafts in vivo -with the in vitro determinants of platelet aggregability. We were able to classify dogs into three groups by their & vitro platelet aggregatory response to AA. Group I dogs possessed platelets which aggregated irreversibly to AA; dogs with platelets which demonstrated reversible aggregation to AA were classified as group II; and dogs with platelets which produced no aggregatory response to AA comprised group III.

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The extent of platelet granule secretion, as measured with 14C-Serotonin, paralleled the aggregation results in that only those platelets aggregating irreversibly demonstrated significant amounts of secretion. When stimulation with AA was potentiated with a subthreshold concentration of epinephrine, both aggregation and secretion were augmented in all three groups although differences in the extent of response persisted between the three groups. Of interest, we noted that while potentiation with epinephrine enabled 82% of the platelets of Group II dogs to mount an irreversible aggregation to AA, platelets from only one of the eight Group III dogs revealed this change. In agreement with our earlier findings (6,7), dogs with platelets which aggregated irreversibly in response to AA exhibited greater platelet deposition onto synthetic vascular grafts and reduced graft patency when compared to dogs with platelets not exhibiting irreversible aggregation in response to AA. These results suggested that differences in canine platelet reactivity might be due either to the amount of bioactive AA/cyclooxygenase end products, such as TxA2. generated or the sensitivity of canine platelets to the We addressed this question by two AA/cyclooxygenase end products. approaches. First, we circumvented the need for AA metabolism by providing U46619, a TxA2 analogue, exogenously under conditions where endogenous TxA2 production was inhibited. Second, we directly measured TxB2, the stable metabolite of TxA2, by RIA. We found that U46619 alone did not produce irreversible aggregation or significant amounts of secretion from canine platelets as previously described by others (25). When potentiated with subthreshold concentrations of epinephrine, irreversible aggregation and secretion did occur in platelets of 97% Group I dogs and 75% of Group II dogs. The platelets of twenty-five percent of group II dogs and all Group III dogs exhibited reversible aggregation to this combination of agents. Because the platelets of all groups were able to mount at least a reversible aggregatory response when stimulated with U46619 and subthreshold epinephrine, it is likely that all canine platelets possess receptors for TxA2. Differences in receptor number or function, however, are not excluded by this observation. This finding, that when exposed to identical concentrations of U46619 and subthreshold epinephrine, differences in aggregation and secretion persisted be-tween the three groups, suggests that differing extents of TxA2 production are unlikely to account completely for the differences in platelet reactivity and that altered sensitivity to TxA2 likely contributes significantly. Our observation that no significant differences in TxB2 production exist between the three groups following platelet stimulation with AA provides additional support for the above suggestion. It is likely that this difference in sensitivity underlies the observed differences in platelet aggregability and thrombotic tendency. Furthermore, we observed a consistent threshold concentration for platelet shape change in the presence of marked differences in the extents of platelet aggregation and secretion of granule contents which suggests that variability in sensitivity to TxA2 may be localized to the mechanism which mediates aggregation and secretion in response to TxA2.

We gratefully acknowledge the technical assistance of Albert0 Rojales and the assistance of Jane Huth in the preparation of the manuscript. This investigation was supported by NIH grant HL 40506, a grant-in-aid from the American Heart Association with funds contributed in part by the UAU is the Uissouri Affiliate and by a grant from the Monsanto Co. recipient of National Research Service Award HL 0774. SAS is an Established Investigator of the American Heart Association.

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1. Steering Committee of the Physicians Health Study Research Group. "Preliminary Report: Findings from the aspirin component of the ongoing physicians health study." 13.Engl. J. Wed. 318, 262-264, 1988. 2. HARKER, L.A. Clinical trials evaluating platelet-modifying drugs in patients with atherosclerotic cardiovascular disease and thrombosis. Circulation 73, 206-223, 1986. 3. EICHNER, E.R. Platelets, carotids, antithrombotic role of antiplatelet diets. Am. J. Bed. 77, 513-523, 1984.

and coronaries: Critique on agents, exercise, and certain

4. WARD, A.S., PORTER, N., PRESTON, F.E. and MORRIS-JONES, W. with peripheral vascular aggregation in patients Atherosclerosis 29. 63+8, 1978. 5. PACKHAM, R.A. Methods for detection Thromb. Haem. 40. 175-195, 1978.

of

Platelet disease.

hypersensitive platelets.

6. ALLEN, B.T., SICARD, G.A., UATHIAS, C.J., WELCH, H.J. and CLARK, R.E. Platelet deposition on vascular grafts. Ann. Surg. 203, 318-328, 1986. 7. FREEUAN, B.B., SICARD, G.A., ALLEN, B.T., EEUBERGgR, S.R., HATHIAS, C.J. Endogenous canine thrombogenecity is equally and ANDERSON, C.B. important in vascular graft patency as platelet inhibition. Surg. For. VKKKVII, 453-456, 1986. 8. JOHNSON, G.J., LEIS, L.A., RAO, G.H.R. and WHITE, J.G. Arachidonate induced aggregation in the dog. Thromb. Res. I4, 147-154, 1979. 9. CLEUMONS, R.M. and HEYLRS, K.H. Acquisition and aggregation of canine blood platelets. Basic mechanisms of function and differences because of breed. Am. J. Vet. Res. 45, 137-144, 1984. 10. KAPLAN, S., HARCOE, K.F., SAUVAGE. L.R., ZURt4IT,H., WU, H., EATHISLN, S.R. and WALKER, H.W. The effect of predetermined thrombotic potential of the recipient on small caliber graft performance. J. Vast. Sura. 2, 311-321. 1986. 11. HEATON, W.A., DAVLS, H.H., and WELCH, H.J. Indium-111; a new radionuclide label for studying human platelet kinetics. Br.J, Hematol. 46, 613-622, 1979. 12. POWERS, W.J., HOPKINS, K.T., and WELCH, H.J. radiotracer method for quantitative lllIn Thromb. Res. 34, 135-145, 1984. 13. BORN, G.V.R. and CROSS, B.J. J. Physiol. E. 178-195, 1963.

The

Validation of the dual platelet scintigraphy.

aggregation of blood platelets.

14. SICARD, G.A., ALLEN, B.T., LONG, J.A., WELCH, B.J., GRIFFIN, A., CLARK, R.E. and ANDERSON, C.B. Prostaglandin production and platelet reactivity of small diameter grafts. J. Vast. Surg. 1, 776-781, 1984.

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15. BUUDY, G. The synthesis of prostaglandin endoperoxide analogs. Lett. 24. 1957-1960, 1975. 16. BOBINELLI, T.A., NIEWLAROWSKI , S. , DANIEL, J.L. and WITH, Receptor-mediated effects of a PGH2 analogue (U46619) on platelets. Am. J. Physiol. 253, H1035-H1043, 1987.

707

Tetra &

J.B. human

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