Development of Nonthrombogenicity of Injured RabbitAortas Despite Inhibition of Platelet Adherence

Hallie M. Groves, Raelene L. Kinlough-Rathbone, and J. Fraser Mustard

After removal of the endothelium from normal rabbit aortas or after injury to theneointima, the injured surfaces rapidly become nonreactive to circulating platelets.Experiments were done to determine whether prevention of the initial interaction ofplatelets with the surfaces would influence the loss of vessel wall reactivity. Inhibitionof platelet accumulation on the subendothelium by the infusion of PGI2 (850 ng/kg/min) or the administration of dipyridamole (12.5 mg/kg initially followed by 5 mg/kg/hr)for periods of less than 8 hours inhibited platelet accumulation on the injured vesselsduring the infusion, but did not prevent the subsequent accumulation of platelets onthe surfaces when the infusions were stopped. If the animals were treated for 8 hours,platelets did not accumulate on the surface when the drugs were discontinued. Thus,an injured vessel wall can develop a nonthrombogenic surface even when plateletadherence is prevented, although approximately 8 hours are required before thesurface loses its ability to interact with platelets.(Arteriosclerosis: 6:189-195, March/April 1986)

Platelets rapidly accumulate on the surface of freshlyexposed subendothelium and release the contents of

their granules, among which is a growth factor that stimu-lates smooth muscle cells to form a thickened neoin-tima.1"5 After the initial accumulation of platelets on thesubendothelium that is exposed following deendotheliali-zation with a balloon catheter, the surface of the injuredvessel loses its reactivity to circulating platelets within 30minutes and few additional platelets accumulate.2'6 Within2 to 4 days of deendothelialization, many of the adherentplatelets are lost from the injured vessel, exposing a sur-face that appears morphologically similar to the suben-dothelium but which shows little reaction with circulatingplatelets.2 The factors responsible for injured vessels los-ing their ability to interact with circulating platelets are notunderstood, although there are several possibilities thatcould account for this.

The purpose of the present experiments was to deter-mine whether the initial interaction of platelets with dam-aged vessel wails contributes to the subsequent ioss ofvessel reactivity. Rabbits were treated with dipyridamoleor PGI2, both of which inhibit platelet interaction with sur-faces, including subendothelial structures,7"11 to deter-mine whether vessels upon which few platelets accumu-late retain their ability to attract circulating platelets.

From the Department of Pathology, McMaster University,Hamilton, Ontario, Canada.

Address for reprints: Dr. Raelene L. Kinlough-Rathbone, De-partment of Pathology, Room 3N26, McMaster University, 1200Main Street West, Hamilton, Ontario, L8N 3Z5 Canada.

Received July 8, 1985; revision accepted August 28, 1985.

Methods

Preparation of Suspensions of Washed51Cr-Labeled Platelets

Suspensions of washed rabbit platelets were preparedas previously described12'13 from blood collected into acid-citrate-dextrose anticoagulant14 through a polyethylenecannula placed in the carotid artery of rabbits anesthetizedwith sodium pentobarbital (40 mg/kg). The platelets werelabeled in the first washing solution for 30 to 45 minutes atroom temperature with Na2

51CrO4 (New England Nuclear,Canada Limited, Lachine, Quebec; 200-500 /JLC\ per /ng ofchromium; 150 /xC\ of 51Cr were used to label plateletsobtained from the blood of each rabbit). The platelets werewashed once with calcium-free Tyrode's solution contain-ing 0.35% bovine serum albumin (Pentex, Fraction V,Miles Laboratories, Kankakee, Illinois), were separated bycentrifugation, and were resuspended in platelet-poorplasma for injection into rabbits.

Animals

Male New Zealand White rabbits were used throughoutthe study. The weights of the animals varied from 2.2 to 3.2kg, and animals of approximately the same weight wereused within an experiment. All procedures conformed withinstitutional guidelines. Before surgery the animals wereanesthetized with sodium pentobarbital (40 mg/kg). Whenthe animals were to be killed by perfusion-fixation, theywere preanesthetized with ketamine (Ketaset; Rogar/STB,Division of BTA Products, London, Ontario; 100 mg intra-muscularly) and atropine (0.1 mg subcutaneously) 20 min-utes before they were anesthetized with an intravenousinjection of sodium pentobarbital (approximately 40mg/kg).

189

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190 ARTERIOSCLEROSIS VOL 6, No 2, MARCH/APRIL 1986

Removal of Aortic Endothellum or Injury ofNeolntlma with a Balloon Catheter

The aortic endothelium was removed with a ballooncatheter as previously described2 by using a 4F embolec-tomy catheter (Edwards Laboratories, Santa Ana, Califor-nia). The neointima that had formed 7 days after deendo-thelialization was also damaged by the passage of aballoon catheter.6

Platelet Accumulation on Damaged Aortas InVivo

Platelets (8 ml of 3 x 109 platelets per ml) labeled with51Cr were injected into rabbits 18 hours before a ballooncatheter was used to remove the aortic endothelium ordamage the aortic neointima. The animals received anintravenous injection of heparin (1000 U) immediately be-fore they were perfused through a carotid cannula withLocke's-Ringer solution containing heparin (1 U/ml) fol-lowed by fixation with 4% paraformaldehyde in phosphatebuffer. The radioactivity associated with the vessels wasmeasured, and platelet accumulation was calculated aspreviously described.2 Calculations were based on thespecific radioactivity of platelets in the circulation immedi-ately before the insertion of the balloon catheter.

Administration of Dlpyrldamole

Dipyridamole (a gift of Boehringer Ingleheim, Quebec)was dissolved in 0.1 N HCI, and the pH was adjusted to3.0. An intravenous injection of dipyridamole (12.5 mg/kg)was given 10 minutes before the aorta was injured. Tomaintain the drug concentration in the plasma for longerperiods, hourly injections of dipyridamole (5 mg/kg) weregiven for 3 or 7 hours through a cannula introduced into thesuperior vena cava. The accumulation of platelets on thesurface of the injured aorta was measured at the end of thetreatment period or 9 hours after the last injection of dipyri-damole.

Infusions of PGI2The PGI2 used in these experiments was a generous gift

of the Upjohn Company, Kalamazoo, Michigan, and Salva-dor Moncada, Wellcome Research Laboratories, Becken-ham, England. A polyethylene cannula (PE 190) was intro-duced through the left common carotid artery of ananesthetized rabbit and passed into the ascending aorta towithin approximately 0.5 cm of the aortic valve. A solutionof PGI2 in 0.1 M Tris buffer (pH 9.0) or a solution of Trisbuffer (placebo) was infused with a Harvard pump (Model927) calibrated to deliver 0.1 ml/min/kg body weight. Insome experiments the infusion of PGI2 (850 ng/kg/min)was begun 30 seconds before the aorta was injured with aballoon catheter, and continued for 10 minutes after theinjury. In other experiments in which PGI2 was infused for 8hours, the infusion pump was calibrated so that the animalreceived the drug (850 ng/kg/min) or placebo solution atthe rate of 1 ml/hr/kg. For these longer infusion periods, thecannula through which the drug was administered waspassed subcutaneously from a scalp incision and insertedinto the common carotid artery to within approximately 0.5cm of the aortic valve. The infusion of PGI2 was begun 10minutes before the aorta was injured with a balloon cath-eter. During the infusion, the syringes containing PGI2

were packed in ice to minimize degradation. After injury ofthe aorta with a balloon catheter, each animal was placedin an individual plastic basket at the same level as the

pump and allowed to recover in a quiet, darkened room.The animals were monitored continuously during the re-mainder of the infusion period to ensure that their activityduring and following recovery did not interfere with thecontinuous infusion of PGI2. Blood pressure was recordedduring the infusion of PGI2 by a pressure-sensitive trans-ducer placed in the right carotid artery and connected to aMingograf 34 (Elma Schonander, Stockholm, Sweden).Shortly before the end of the infusion, the animals wereanesthetized and prepared for perfusion-fixation.

Scanning Electron Microscopy

Segments of aorta approximately 2.5 cm long were dis-sected free of extraneous tissue and cut open to exposethe luminal surface. The adventitial surface was attachedto a coverglass with cyano-acrylic glue (Eastman 910).15

The tissue was kept moist with a buffer solution.15 Thespecimen was post-fixed in osmium tetroxide, dehydratedthrough graded ethanol, critical-point dried from CO2,mounted on a scanning electron microscope (SEM) stubwith double-sided tape, and coated with gold. Specimenswere examined in a Philips 501 SEM.

Measurement of Plasma Dipyridamole

Plasma dipyridamole was measured by the method ofZak et al.16

Platelet Aggregation

The aggregation of platelets in citrated plasma wasmeasured as previously described.17

Analysis of Data

The individual values in Table 1 were logarithmicallytransformed before an analysis of variance was used totest the variation among the treatment groups. Student's ftest was used to compare the values among the groups.For Tables 2-6, the significance of the difference amongvalues was calculated by using untransformed data andStudent's / test.

Table 1. Effect of a Single Injection of Dipyridamoleon Platelet Accumulation on Subendothellum In Vivo

Treatment

Timeafter No. ofinjury rabbits

Plateletaccumulation

(noVmrn2)

PlaceboDipyridamole

10 min10 min30 min

1 hr2hr4 hr

995666

52,500 ±8,40016,900±1,80013,400±1,50017,900 + 2,70032,100 ±5,20043,900±8,100

ABCDH

I

F

Values are means ± SEM. Rabbits were given an intravenousinjection of dipyridamole (12.5 mg/kg) 10 minutes before trie en-dothelium was removed from the aorta with a balloon catheter.The animals were killed by perfusion-fixation at the times indicat-ed following injury and the accumulation of platelets on the sur-face was measured. The significance of the differences in plateletaccumulation among the groups was calculated as described inthe Methods section and are as follows: A vs B = p < 0.0005; B vsD = p < 0.45; A vs F = p < 0.3; D vs E = p < 0.005.

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LOSS OF THROMBOGENICITY IN INJURED VESSELS Groves et al. 191

Table 2. Effect of Administration of DipyrldamoleHourly for 4 Hours on the Accumulation of Platelets onthe Subendothellum In Vivo

Platelet accumulation(no./mm2) after injury

Treatment 4 hr 12 hr

PlaceboDipyridamoleSignificance of difference

between means

38,100±3,20015,500 ±5,400

p < 0.005

35,100± 10,10026,200 + 3,000

p < 0.25

Values are means ± SEM for six rabbits. Dipyridamole (12.5mg/kg) was administered intravenously 10 minutes before remov-al of the endothelium and was then given hourly at a concentrationof 5 mg/kg for 3 hours following injury. The animals were killed byperfusion-fixation 4 or 12 hours following deendothelializatbn,and the accumulation of platelets on the surface was measured.

Table 3. Effect of Administration of DipyridamoleHourly for 8 Hours on the Accumulation of Platelets onthe Subendothelium In Vivo

Platelet accumulation(noVmm2) after injury

Treatment 8 hr 16 hr

PlaceboDipyridamoleSignificance of difference

between means

32,700 ±6,60012,700 ±2,500

p < 0.01

23,800 ±3,40013,400 ±1,500

p < 0.01

Values are means ± SEM for nine animals. Dipyridamole (12.5mg/kg) was administered intravenously 10 minutes before remov-al of the endothelium, and thereafter at a concentration of 5 mg/kghourly for 7 hours. The animals were killed by perfusion-fixation 8or 16 hours following deendothelializatbn, and the accumulationof platelets on the surface was measured.

Results

Experiments with Dipyridamole

A single injection of dipyridamole (12.5 mg/kg) 10 min-utes before deendothelialization significantly reducedplatelet accumulation on the subendothelium compared tothe accumulation in placebo-treated animals; this effectwas maximal during the first hour (Table 1). Four hoursafter this single injection of dipyridamole, platelet accumu-lation on the deendothelialized aortas was not significantlydifferent from that in the placebo-treated group.

The administration of dipyridamole immediately beforeinjury and at hourly intervals for 3 hours significantly re-duced the number of platelets that accumulated on thedeendothelialized aorta measured 4 hours after the injury.Nine hours after the last injection of dipyridamole (12 hoursfollowing injury) platelet accumulation was similar in dipyri-damole- and placebo-treated animals (Table 2).

The administration of dipyridamole immediately beforeinjury and hourly for 7 hours afterward reduced plateletaccumulation measured 8 hours after injury, and duringthe following 8 hours (9 hours after the last injection ofdipyridamole) additional platelets did not accumulate (Ta-ble 3). At the end of the administration of dipyridamole, theplasma concentration of the drug was 0.352 ± 0.18 /ig/mland 9 hours later was 0.002 ± 0.001 /ig/ml, a concentra-tion considerably below that required to inhibit plateletfunction. At 9 hours after the last injection of dipyridamole,there was no difference in the extent of platelet ag-gregation induced by collagen, ADP (5 ^iM) or sodiumarachidonate (500 /xM) compared with platelets from con-trol animals.

Experiments with PGI2Because dipyridamole inhibited platelet accumulation

on deendothelializated rabbit aortas by only about 60%, itis possible that platelets that interacted with the injuredvessel might have been responsible for altering its surfaceproperties. Therefore, PGI2, a more potent inhibitor ofplatelet function, was used to inhibit platelet accumulationon the damaged vessels. When the infusion of PGI2 wasbegun before the exposure of the subendothelium andmaintained for 10 minutes after injury, platelet accumula-tion at the end of the infusion was reduced by more than85%, as estimated by the accumulation of 51Cr (Table 4).However, 10 minutes after the end of the PGI2 infusion,

Table 4. Effect of Infusing PGI2 for 10 Minutes on theAccumulation of Platelets on the Subendothelium InVivo

Platelet accumulation(nc/mm2) after Injury

Treatment 10 min 20 min

Control (no drug)PGI2

Significance of differencebetween means

55,300 ±5,5007,300 ±1,300

p < 0.005

38,400 ±3,200

Values are means ± SEM for seven animals. The infusions ofPGI2 (850 ng/kg/min) were begun 1 minute before the aortas weredeendothelialized and continued for 10 minutes thereafter. Theanimals were killed by perfusion-fixation at the end of the PGI2

infusion or 10 minutes after the end of the infusion, and the accu-mulation of platelets on the surface was measured.

Table 5. Effect of Infusing PGI2 for 8 Hours on theAccumulation of Platelets on the Subendothelium InVivo

Platelet accumulation(no./mm2) after injury

Treatment 8hr 8.5 hr

Control (no drug)PlaceboPGI2

Significance of differencebetween means

36,600±4,10030,700 ±4,200

8,600 + 4,400

p < 0.005*

5,800+1,800

p < 0.3f

Values are means ± SEM for four animals. The infusions of PGI2

(850 ng/kg/min) or Tris buffer were started 10 minutes before theaortas were deendothelialized and were continued for 8 hoursfollowing injury. The animals were killed by perfusion-fixation atthe end of the PGI2 infusion, or 30 minutes after the end of theinfusion and the accumulation of platelets on the surface wasmeasured.

The significance of the difference in platelet accumulation be-tween the control group (no drug) and the PGI2-treated groupperfused at 8 hours following injury.

tThe significance of the difference in platelet accumulation forthe PGI2-treated group perfused at 8 hours and the group per-fused at 8.5 hours after injury.

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192 ARTERIOSCLEROSIS VOL 6, No 2, MARCH/APRIL 1986

Figure 1. A. Scanning electron micrograph of the surface ofa normal rabbit aorta subjected to injury with a balloon catheter10 minutes previously. The surface Is covered with a layer ofplatelets (PLT). B. Scanning electron micrograph of a normalrabbit aorta subjected to injury with a balloon catheter. Theaorta was perfusion-fixed at the end of a 10-minute infusion ofPGI2. Platelets are not present on the subendothelium (SE)(SE). C. Scanning electron micrograph of a normal rabbitaorta subjected to injury with a balloon catheter. The aorta wasperfusion-fixed 10 minutes after completion of a 10-minuteinfusion of PGI2. The surface is covered by a layer of platelets(PLT). Bar = 10 fim.

Figure 2. A. Scanning electron micrograph of the surface ofa normal rabbit aorta subjected to injury with a balloon catheter8 hours previously. The surface Is covered with a layer ofplatelets (PLT). B. Scanning electron micrograph of a normalrabbit aorta subjected to injury with a balloon catheter. Theaorta was perfusion-fixed at the end of an 8-hour infusion ofPGI2. Very few platelets are present on the subendothelium(SE). C. Scanning electron micrograph of a normal rabbitaorta subjected to injury with a balloon catheter. The aorta wasperfusion-fixed 30 minutes after completion of an 8-hour Infu-sion of PGI2. Only a few platelets are associated with thesubendothelium. Bar = 10 fim.

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LOSS OF THROMBOGENICITY IN INJURED VESSELS Groves et al. 193

Table 6. Effect of Infusing PGI2 for 8 Hours on theAccumulation of Platelets on the Injured Neolntlma InVivo

Treatment

Experiment 1Control (no drug)PGI2

Experiment 2Control (no drug)PGI2

Platelet accumulation(noVmm2) after injury

8hr

23,8006,700

13,7001,200

8.5 hr

4,800

3,900

The infusions of PGI2 (850 mg/kg/min) were begun 10 minutesbefore injury with a balloon catheter of the neointima that hadformed by 7 days following removal of the endothelium. The PGI2

infusion was continued for 8 hours following Injury. The animalswere killed by perfusion-flxation at the end of the PGI2 infusion (8hours) or 30 minutes after the end of the infusion (8.5 hours), andthe accumulation of platelets on the surface was measured.

platelet accumulation had Increased over fivefold. Duringthe infusion of PGI2, the mean arterial blood pressure ofthe anesthetized animals decreased by approximately25%. Figure 1 shows that very few platelets were detect-able by SEM on the deendothelialized surface of the aortaduring a 10-minute infusion of PGI2 compared with thelayer of platelets on the surface of the vessels of the ani-mals that received no drug. This figure also shows thatplatelets accumulated on the injured surface when the in-fusion of PGI2 was stopped.

Platelet accumulation at the end of the 8-hour infusion ofPGI2 was approximately 25% of the control value. Again,few platelets were detectable on the surface by SEM (Fig-ure 2). However, in contrast to the results following short-term PGI2 infusion, additional platelets did not accumulateduring the 30-minute period after the infusion was stopped(Table 5). Infusion of PGI2 for 6 hours did not preventsubsequent platelet accumulation (data not shown).

Injury to the neointima leads to platelet accumulationthrough a process involving connective tissue and throm-bin.3'5 In two experiments, we examined the effect of PGI2

infusion on platelet accumulation on the injured neointima.When PGI2 was infused for 8 hours after injury to theneointima, few platelets accumulated on the injured aorta,and during the 30 minutes after the end of the infusion, noadditional platelets accumulated (Table 6, Figure 3).

Discussion

The results from these studies confirm those of earlierexperiments in which it was shown that dipyridamole inhib-its platelet adhesion to the subendothelium.7 As previouslyshown by Adelman and his co-workers,18 these resultsalso demonstrate that the administration in vivo of highconcentrations of PGI2 strongly inhibits platelet adhesionto the subendothelium; the concentrations of PGI2 requiredto inhibit platelet adhesion to the subendothelium caused afall in blood pressure.

A single infusion of dipyridamole significantly reducedplatelet accumulation on the subendothelium over a period

Figure 3. A. Scanning electron micrograph of the surface ofa rabbit aorta that had been deendothelialized with a ballooncatheter 7 days previously, 8 hours after a second injury with aballoon catheter. Platelets (PLT) adhere to the damagedneointima. CT = connective tissue. B. Scanning electronmicrograph of the damaged neointima that had formed 7 daysafter the aorta was deendothelialized with a balloon catheter.The aorta was perfusion-fixed at the end of an 8-hour infusionof PGI2. There are few platelets on the surface. SMC =smooth muscle cells. C. Scanning electron micrograph of thedamaged neointima that had formed 7 days after the aorta wasdeendothelialized with a balloon catheter. The aorta was per-fusion-fixed 30 minutes after the end of an 8-hour infusion ofPGI2. Few platelets are present on the surface. Bar = 10 ̂ m.

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194 ARTERIOSCLEROSIS VOL 6, No 2, MARCH/APRIL 1986

of 3 hours, but when the blood levels of dipyridamole de-creased, platelets accumulated on the subendothelium.However, when dipyridamole was given for at least 8hours, platelets did not reaccumulate on the deendothelia-lized surface.

PGI2 was a more effective inhibitor of platelet accumula-tion on the subendothelium than dipyridamole. The resultsof these experiments with PGI2 indicate that when plateletinteraction with the subendothelium is extensively inhibit-ed, about 8 hours are required before the surface becomesnonreactive to platelet accumulation. Infusions of PGI2 forshorter periods of time did not inhibit subsequent plateletaccumulation when the infusion was stopped. Adelmanand co-workers18 also found that, with short-term PGI2infusions, platelet accumulation on the subendotheliumwas restored when the infusion was stopped. In the experi-ments with dipyridamole and PGI2, platelet adherenceafter these treatments was measured when the circulatinglevels of these drugs were very low; for dipyridamole thiswas 9 hours after the last injection and for PGI2 it was 30minutes after the infusion was stopped.

Thus, it seems reasonable to conclude that the loss ofreactivity of the subendothelium to platelet accumulationafter 8 hours is not a result of platelet interaction with theinjury site. It is also unlikely that loss of vessel wall reactiv-ity can be attributed to plasma proteins or other plasmafactors masking the sites on the subendothelium withwhich platelets interact, since deposition of plasma pro-teins should take place almost immediately. It is probablethat there is a change in the vessel wall that accounts forthis effect. PGI2 formation by the vessel wall is one factorthat could inhibit platelet adhesion to the subendothelium.In earlier studies, we found that inhibition of PGI2 produc-tion by damaged vessels did not cause a further increasein platelet accumulation, indicating that PGI2 productionwas not likely to be responsible for the loss of vessel wallreactivity.19 Eldor and his colleagues20 demonstrated thatPGI2 production by deendothelialized vessels is consider-ably diminished; this observation provides additional evi-dence that increased PGI2 production by injured vesselsprobably does not account for the loss of reactivity.

The two experiments involving the injured neointimashow that the PG^-sensitive platelet accumulation followsa similar pattern to that observed for the subendothelium.However, with the injured neointima both thrombin genera-tion and platelet-connective tissue interaction are involvedin the initial platelet accumulation.36 The combination ofheparin and PGI2 has been shown to be required to pro-duce maximum inhibition of platelet accumulation resultingfrom damage of the neointima.21 Although our experimentsdo not provide any information about the coagulation com-ponent, Piepgras and his colleagues22 found that afterendarterectomy in cats, an infusion of heparin for a periodof 6 hours prevented thrombus formation and resulted in asurface that was nonreactive to the formation of platelet-fibrin thrombi when heparin was discontinued.

In contrast to the results of the present experiments inwhich platelet adherence was inhibited and approximately8 hours were required before the injured surface becamenonreactive to platelets, we have previously demonstratedthat if platelet adherence is not inhibited, the monolayerof platelets that immediately covers the injured vesselwall provides a surface to which other platelets do notadhere.2-6

The results from our experiments indicate that within 6 to8 hours after injury with a balloon catheter, the aortas ofrabbits become nonreactive to circulating platelets. If

drugs that inhibit platelet adherence are administered for asufficient period of time, the injured vessel wall loses itsreactivity to circulating platelets. Inhibition of platelet ad-herence to the subendothelium by the infusion of PGI2 hasbeen shown to prevent the accumulation of released plate-let proteins in the vessel wall.18 Thus, it is possible toprevent the initial delivery to the injured vessel of the plate-let factor that is mitogenic for smooth muscle cells, and iftrie therapy is continued until the vessel wall becomesnonreactive, it may be possible to stop the therapy withlittle subsequent platelet interaction with the damaged ves-sel surface.

References1. Baumgartner HR. The role of blood flow in platelet adhesion,

fibrin deposition, and formation of mural thrombi. MterovascRes 1973:5:167-179

2. Groves HM, Klnlough-Rathbone RL, Richardson M,Moore S, Mustard JF. Platelet interaction with damaged rab-bit aorta. Lab Invest 1979;40:194-200

3. Stemerman MB, Ross R. Experimental atherosclerosis. I.Fibrous plaque formation in primates, an electron microscopestudy. J Exp Med 1972;136:769-789

4. Ross R, Glomset JA. Atherosclerosis and the arterialsmooth muscle cell. Science 1973:180:1332-1339

5. Ross R, Glomset JA, Kariya B, Harfcer LA. A platelet-de-pendent serum factor that stimulates the proliferation of arte-rial smooth muscle cells in vitro. Proc Natl Acad Sci USA1974:71:1207-1210

6. Groves HM, Klnlough-Rathbone RL, Richardson M,Moore S, Mustard JF. Thrombin generation and fibrin forma-tion following injury to rabbit neointima. Studies of vessel wallreactivity and platelet survival. Lab Invest 1982;46:605-612

7. Groves HM, Klnlough-Rathbone RL, Cazenave J-P, De-Jana E, Richardson M, Mustard JF. Effect of dipyridamoleand prostacyclin on rabbit platelet adherence in vitro and invtvo. J Lab Clin Med 1982:99:548-558

8. Oblath RW, Buckley FO, Green RM, Schwartz SI,DeWeese JA. Prevention of platelet aggregation and adher-ence to prosthetic vascular grafts by aspirin and dipyrida-mole. Surgery 1978:84:37-44

9. Mustard JF, Packham MA. Platelets, thrombosis and drugs.In: Avery GS, ed. Cardiovascular drugs, vol 3. Antithromboticdrugs. New York: Adis Press, 1978;1-83

10. Cazenave J-P, Dejana E, Klnlough-Rathbone RL, Rich-ardson M, Packham MA, Mustard JF. Prostaglandins l2 andE, reduce rabbit and human platelet adherence without inhib-iting serotonin release from adherent platelets. Thromb Res1979:15:273-279

11. Hlggs EA, Moncada S, Vane JR, Caen JP, Michel H, Tobe-lem G. Effects of prostacyclin (PGI2) on platelet adhesion torabbit arterial subendothelium. Prostaglandins 1978;16:17-22

12. Ardlle NG, Packham MA, Mustard JF. Adenosine diphos-phate-induced platelet aggregation in suspensions ofwashed rabbit platelets. Br J Haematol 1970;19:7-17

13. Ardlle NG, Perry DW, Packham MA, Mustard JF. Influenceof apyrase on stability of suspensions of washed rabbit plate-lets. Proc Soc Exp Biol Med 1971;136:1021-1023

14. Aster RH, Jandl JH. Platelet sequestration In man. I. Meth-ods. J Clin Invest 1964;43:843-855

15. Richardson M, Moore S. Preparation of large curved biologi-cal surfaces for scanning electron microscopy. Artery 1980;6:409-417

16. Zak S, Tallan HH, Qulnn GP, Fratta I, Greengard P. Thedetermination and physiological distribution of dipyridamoleand its glucuronides in biological material. J Pharmacol ExpTher 1963:141:392-398

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LOSS OF THROMBOGENICITY IN INJURED VESSELS Groves et al. 195

17. Kinlough-Rathbone RL, Packham MA, Mustard JF. Plate-let aggregation; In: Harker LA, Zimmerman TS, eds. Mea-surements of platelet function. New York: Churchill Living-stone, 1983;64-91

18. Adelman B, Stemerman BM, Mennell D, Handin Rl. Theinteraction of platelets with aortic subendothelium. Inhibitionof adhesion and secretion by prostaglandin l2. Blood 1981;58:198-205

19. Dejana E, Cazenave J-P, Groves HM, et al. The effect ofaspirin inhibition of PGI2 production on platelet adherence tonormal and damaged rabbit aortae. Thromb Res 1980; 17:453-464

20. Eldor A, Falcone DJ, Hajjar DP, Minlck CR, Weksler BB.Recovery of prostacyclin production by de-endothelializedrabbit aorta. Critical role of neointimal smooth muscle cells. JClin Invest 1981;67:735-741

21. Adelman B, Stemerman MB, Handin Rl. Interaction ofplatelets and fibrin with injured rabbit aortic neointima. Effectof prostaglandin l2 and heparin. Arteriosclerosis 1983;3:141-148

22. Piepgras DG, Sundt TM, Didisheim P. Effect of anticoagu-lants and inhibition of platelet aggregation on thrombotic oc-clusion of endarterectomized cat carotid arteries. Stroke1976;7:248-254

Index Terms: thrombogenicity of vesselsplatelet adhesion

platelet adherence • PGI2 • vessel injury • dipyridamole

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H M Groves, R L Kinlough-Rathbone and J F Mustardadherence.

Development of nonthrombogenicity of injured rabbit aortas despite inhibition of platelet

Print ISSN: 1079-5642. Online ISSN: 1524-4636 Copyright © 1986 American Heart Association, Inc. All rights reserved.

Avenue, Dallas, TX 75231is published by the American Heart Association, 7272 GreenvilleArteriosclerosis, Thrombosis, and Vascular Biology

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