Biochfmi~ ¢1 ,~iqptlysica Acla. 1137(1992) 279-28b

279

1992 Elsevier Science Publishers B.V. All rights reserved 0167-4~89/92/$05.~

BBAMCR 13268

Inhibition of platelet adhesion and thrombus formation on a collagen-coated surface by novel c~bamoylpiperidine

antiplatelet agents B. Rita Alcvriadou

~, L a r : y V . M c l n t i r c

" and Andrew

Lassie b

"Cox Laburalor~ )'or Biomedical Engineeril~g Rice U~dLe~sily, Ifegslon, TX (USA) ~nd b Deparlmenl of Med,:c'/tmlChcmlsley, Health Science Center, Tl~e U.~irersity of Teanemee. Mere.vials, TN ~USAJ

(Reo~.ived 6 FebrlmW l~12) (Revised mawmseriptreceived 15 Ma~"1992)

Key words: Platele~ Collagen: Me, al t.h..~mbogenes[s:Ami~l~telet agent: C~tbam0yleipefidine The inhibito~ effect o[ two novel ant[platefe', agents, l,tO-bls[3~N,N-diethylcsrbamoyl)plperidino]decane dihydrobremide (G-il0) and 1,6-bis[3-(N,N-dietbylcathamoyl~ip~rldino]hexane dihydrobromide (G-II2), on platelet adhesion and subsequent aggregation on collagen-coated surface was ¢,~luamd under controlled flow. Glass coverslips coaled with bo~dne fibril|ar collagen type I were exposed 1o heparinized human whole blood that had been preincuhated with either aqueous solutions of one of the two cmrbamoylplperidine congeners or correspc~,,ding amounts (1.0-4.0 u.I/ml blood) of distilled water, at a wall shear rate of lO0O s -t, in a parallebplate pexfus[on chum.her. Epifloorcscence video microscopy with a micmphommctric measurement technique was used to visualize and quantify ~¢position of fluorescently-labeled platelets fix~m flowing whole blood onto the collagen-coated surface. At concentrations of 190 and 200 p.M, G-110 inhibited phtclet accumulation by 30 + 9% (4,S.E.) and 63 ± 3 .% ( :t: S.E.), respectively; while G-112 reduced platelet deposition by 19 :f. 3% ( 4-S.E.) and 31 + 2% ( ± S.E.) at concentrations of 200 and 400 I~M, reslmctivcly. Distal image preecssing techniques were used to analyze the dynamics of thmmbus growth on the collagen-coated surfaces. It was found that the compounds reduced the rate of thrombus growth by impeding both surface cOVerage and the nurpb,~r r~f-ptLiteIets per thrombus in a concentration-dependent manner. This study, together with other~ on related compotmds GT-I.2, BPAT-II7 and BPAT-I43. corroborates the nature of pivotal features in the molegadar structure of carbamoylpil~tidin¢ and nlp¢coto).lpip~la~2.1e derivatives which enhan0c desirable antithrombotk: effects, e.g. intramo]vrular distance between two tertiatv amines (ring ulbrogens) and levels of hydrophobicity.

latrnductima In ~eeking to dece[op novel potential antithrombotic agents, L a ~ l o and eoworkers employed carbamoylpiperidine, nipecotx3ylpiporazine and related congeners with gradual and systematic alterations in their molecular structure [1-3]. T h e tertiat3, an,~nes~ around the ring nitrogens, in those synthetic entities have been identified as aggregation-inhibitory specific functional groups [i,4-6]. T h e y are subject to broad variances ia protonation, that enables them to assume appropriate hydrophobia character for Penetrating the platelet membrane's lipid bilayer [7,8]. T h e penetrated amines could ga~,arate sufficient quantities o f cationic species to interact with and reduce the response-sensitivity of

Corccspondenc¢ to: I V . Mclnt~re. Cog Laeoralory [or BiOmedical Engineering, Rice Onivelsity, P.O. Box t892, Houston, TX 77251, USA.

anionic phospholipids, stabilizin 8 thereby membrane complexes of the dense tubular system and o f o t h e r membrane complexes that sequester calcium (Ca 2÷ ) the platelets [8-11]. in this manner, the compounds would impede o r block Ca z÷ release into the platelet's ¢ytosel by conventional agonists [12]. Since increase o f cytosolic free Ca z+ concentration is a key step among the intracollular mechanisms that mediate platelet activation i13], the compounds would elevate the threshold for triggering a.td sustaining viable platelet aggregation, and only stimuli of greater intensity could actuate the process [7]. By employing the compounds as molecular probes, it was possible tu interpret r:latelet ftmctlon response patterns in terms o f certain chemical parameters, and to introduce modifications in o r d e r to enhance desirable antis hrombotie properties [1,4,5,7,8]. T h e principal determinants from which the compounds derive their ~lggregatiominhibitory potency inclnd¢ their hydrophobia characteristics, the number of tertiary amino fang-

280 tions, their basicity and their interatomie distance within the molecule. One of the must potent earbamoylpiperidine congarters, a , a "-bis[3-( N,N-diet hylearbamoyl)piperidiao]p-xylene dihydrubromide (GT-12), and two nipecotoylpiperazine derivatives A~,N'-bis(l-decyinipecotoyi)piperazin¢ dlhydriodlde (BPAT-117) and N,N'-bis(lhexTinipccotoyi)piperazinc dihydriodidc (BPAT-143) have been shown to inhibit adenosine diphosphate (~dDP)- and throm~:n-induced platelet aggregation in platelct-rich plasma (PRP) in an aggregom¢ter [1,4,7]. The two carhamoylpiporidinc entities, 1,10-bis[3-(N,Ndiethylcarbamoyl)piperidino]decane dihydrobromide (G-110) and 1,6-bis[3-(N,N-diethylcarbamoyi)piperidino]hexane dihydrobromide (G-I12L were found to be less potent than GT-12 and BPAT-117 [1,4,7]. Whole blood perfusion studies using a parallel-plate flow chamber, one side of which is coated with collagen, constitute a better model for studying 1he in-vivo procuss of thro~abns formation where platelets adhere and subsequently aggregate on an injured, exposed subendothelial surface, rather than aggregating in suspension [14,15[ GT-12, B P A T - i I 7 and BPAT-143 were tested and found effective antiplatelet agant~ in a whole blood perfosion model under controlled flow conditions [16,17]. In this study, the effect of the two less potent carbamoylpiperidine derivatives, G - I I 0 and G - I t 2 was e~amined. They were evaluated on platelet accumulation from flowing whole blood onto collagen-type-Icoated glass slides, using a parallel-plate flow chamber. .~m epifluorescent video microscope was employed t_o visualize real-time platelet deposition, with a microphotometric measurement technique to quantify end-point platelet accumulation as a function of axial positions along the collagen-coated surface. The video recording taken during the perfusinn period was subjected to digitat image analysis in order to obtain information on the kinetics of thrombus growth and on the morphologic characteristics of each thrombus. While both G-110 and G-112 were considerably less potent in the whole blood perfusion model, their comparative antithrombotic strengths clearly correlated with 1hose of the previnnsly examined GT-12, BPATo 117 and BPAT-143 in terms of their chemical features and the respective physicochemical characteristics. Our findings corroborated that hydrophobicity and other formerly identifed structural features [1,2,6,7] contributed to rendering the compounds more effective in reducing the potential of thrombos formation. Materials aad Mtthmls A suspension of 2.1 mg/mt type l acid-lusolublc collagen from bovine achilles tendon (Sigma, St. Louis, M e ) in 0.5 M acetic acid (pH 2.8) was prepared by the

~ t h o d described by Folio e t a l . [16], and the collagen concentration was determined by hydroxyprolinc analysis [18]. Before assembly in the flow chamber, a glass coverslip (No. 1, 24 × 50 ram; Coming, NY) was coated with 200/zl of the fibrillar collagen solution, which was spread over all but the first 1.5 em of the length of the surface of each slide. The slides were placed in a humid environment for a minimum of 45 min, and the excess collagen was rinsed off with 10 ml of isotonic saline. The collagen density on the glass surfaaas averaged about 20 ~ g / e m 2 (range --+2 fzg/craZ). G - I I 0 (C~oHeoN~O2Br2, M, 668.64) and G-I12 (C26Hs2N4OzB3"2, Mr 612-54) were designed, synthesized and stored as previously described [3,5]. Compound solutions were prepared fresh daily. Dissolving 66.86 mg of G - 1 1 0 in I.O m l of distilled water yielded a 100 mM stock solution; addition of 1.0 (2.0) /zl of the 100 mM solution for each ml of blood sample resulted in 100 (200)/LM final concentration of G-t 10 in blood. Similarly, dissolving 61.25 mg of G-112 in 1.0 ml of distilled water yielded a 100 mM stock solution; addition of 2.0 (4.0) g l of the 100 mM solution for each mi of blood resulted in 200 ( 4 0 0 ) / t M final concentration of G-112. Blood was collected into polypropylene test tubes from the antecubital vein of non-smoking and medication-free donors into the anticoagulant, porcine heparia (heparin sodium: EIIdus-Sinn, Cherry Hill, NJ') in 10 O / m l final concentration, and the fluorescent dye, mepacrine, (quinacrine dihydrochloride; Sigma) in :10 g M final concentration. Mepacrinc has no effect on normal platelet function at this concentration [19]. Before the porfusion, blood was incubated at 37°C for 15 min, and then an additional 5 rain with the appre~ priale amount of either distilled water, G-110 or G-112 solution. All blood samples were used within 5 h after veniponetm'e. Control runs at the beginning and end of flow chamber experimental sets demonstrated the same extent of platelet accumulation on collagen-coated slid¢~. Each glass coverslip w;th a collagen-coated surface of approx. 3 em 2 formed one side of a parallel-plate flow chamber, as described in detail elsewhere [14,15]. The height of the flow regime, 205 ~m, was dctermined by the thickness of a silicon gasket. The flow chamber was assembled and filled with isotonic saline. A syringe pump (Model 935; Harvard Apparatus, South Natick, IVlA) was ttsed to aspirate blood from the polypro~lene tube through the flow chamber, displacing the saline, at a constant flow rate for 2 rain. A flow rate of 5.3 ml/min yielded 1000 s - ~ wall shear rat¢ {40 dynes/am 2 wall shear stress) fltat corresponds to a high venous or low arteriolar shear rate. The test tube. tubing, and the flow chamber were maintained at 37~C by a thermostatic air bath (Model 279; Laboratory Proclucts, Boston, MA). T h e flow chamber was mounted

281 on an inverted-stage microscope (DIAPHOT-TMD; Nikon, Oardan City, NY) equipped with an epifluorescent illumination attachmem (TMD-EF; Nikon), a 60 × FLUOR objective, a 1 X projection lens (Nikon), and a silicon-intensified target (SIT) video camera (Model CI000; Hamamatsu, Walthafr. M:A) suitable for very low light levels. Epifluoresce2~e illuminazi0h was used to visualize platelet adhesion and subsequent platelet aggregation throughout the perfusion period. This was possible in whole blood because mepacrine is take~ up by the dense granules of platelets and the graanl~b of leukocytes, whereas any fluorescence from within the erythroeytes is quenched by hemoglobin. Leuko~ytes did not adhere to the collagen-coated surface coder the relafively high shear rate condition of the ex=erimeats. Macroscopic meesurement of platelet acec!mulation at the cud of the porft~iou period was do:=-.: by scanning the slide and continuously recording the locally averaged fluorescent intensity over the entire field of view, using a motorized microscope stage ar:l a computerized mierophotometrie measurement sys:em, This system consisted of a 40 x FLUOR objective (Nikon), a photodiode (Model PIN-10DP/SB; United Detector Technology, Hawthorne, CA), an amplifier with variable transimpedance gain (Model IOIC; United Detector Technology), a 12-bit analog to digital converter board (ACSE-12; Strawberry Tree Computers, Sunnyvale, CA) and a micro-computer (Macintosh SE; Apple Computer, Cupertino, CA). Menu-driven software (Analog Connection Workbench; Strawberry Tree Computers) was used to select sampling rate and create data files. The experiments were recorded in real time on a 0.f-inch video casette recorder (Model BR3100U, JVC; Industrial Audio/Video, Houston, TX). Digitization of videotape images taken at 0.38 mm distance downstream from the collagen interface, and further image processing and analysis performed by a digital image processor (IC-300 Modular Image Procossing Workstation; lnovision Corporation. Durham, NC) were nscd to obtain information on the morphology of each thromhus and the kinetics of thrombus growth, as well as for the recopstruction of three-dimensional models of individual thrombi. Determination of the height of thsombi and number of platalets that the:,' conRtined was pos~'ble by normalizing intensity and area measurements of a th~omhus to those of a single platelet. The perc=ntage of the total surface area covered by thr~mhi was computed dividing the area of the microscope field encompassing platclct aggregates and singlets by the area of the smallest rectangle enclosing all the thrombi with visible boundaries in the digitized frame. More detailed description of the equipment and its capabilities has been published elsewhere [14-17]. The collagen-coated slide used in each experiment was removed frum the flow chamber, dipped in a

hemolysing solution (ZAP-OGLOBIN II in Coulter lsoton 11; Coulter Electronics, Hialcah, FL) to lyse red blood cells (RBCs) sticking to the surface, and crushed into 1 ml of 1% Triton X-HKI colt iysing buffer(Sigma) to [yse the deposited platelets. The sample was soulcared for 5 s, and centrifuged at 2 4 0 × g for 10 min to remove the glass fragments. A lactate dehydrogenase (LDH) assay (LD-14 PL Kit; Gilford Systems, Oberlin, OH) of the test supernatant and controls made with the lysates of known platolot concentrations was used to determine the total number of platelets on the slide. By integrating the intensity distribution along the collagen-coated surface, as recorded b~, the mierophotometric measurement system, an intensity/platelet ratio was determined. This was used to convert fluorescent intensity to platelet density at any axial position down~tteaPa P e w the glass-collagen interface up to 23 ram, which is the total length of the collagen-coated surface exposed to blood flow [14-16]. By integrating the platelet density vs. distance carve up to any length of the slide, it was possible to estimate the total number of platelets accumulating on a portion of the collagencoated surface. The first 4 mm of the length of each slide downstream from the beginning of the collagen coating were used as the portion of the surface to compare platelet deposition from control and test blood samples. Platelet density profiles along the collagen-coated surfaces were statistically analyzed to determine differeaces between distances for each treatment and differences between treatments at each distance, using a two-way analysis of varlanco with repeated measures [20]. The between factor was the treatment greup with three levels, control and two different test concentrations. The within factor was the distance with 16 levels from 0.16 to 20.00 ram. Two-way analysis of vat/once, followed by Dunn's multiple comparison at the 0.05 alpha level [20], was used in order to determine differences in total platelet accumulation on the first 4 mm of the length of the collagen-coated surface I'c!ween controls and treated samples. Probability values of Jess than 0.05 were considered significant, and those of less than 0.01 highly significant. Results Platelet density profiles along the collagen-coated surfaces, up to 4 mm downstream from the glass*collagen interface for untreated blood and blood treated with either 100 or 200 p M G-110, are shown in Fig. !. Each carve represents an average of n experiments performed under identical conditions. The error bars represent standard errors of the mean of the data points at each axial position and blood treatment. Controls v,ere run on the same days with the same donors as the corresponding treated samples, Rapid

282

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accumu|ation of control platelets immediately after the c o l l a g e n i n t e r f a c e d e p l e t e d t h e supply o f p l a t e l e t s n e a r the surface, creating a diffuslon-limited region downs t r e a m . W h e n t h e n o r m a l p t a t e l e t r e s p o n s e w a s inhibRed, the s u r f a c e r e a c t i o n r a t e d e c r e a s e d a n d diffusion w a s n o t t h e r a t e - l l m i t i n g s t e p [2l|. T h i s e x p l a i n s why, in t h e p r e s e n c e o f 2 0 0 ~ . M G - I I 0 , approxim~!ely s a m e p l a t e l e t a c c u m u l a t i o n o c g u r e d a t all axial p o s i t i o n s o n the collagen-coated surface. 100 o r 2 0 0 / s M G - 1 1 0 i n h i b i t e d 3 0 ± 9 % (±S.E.. n=4) and 63_+3% (±S.E., n =5) of total platelet a c c u m u l a t i o n o n t h e first 4 m m d o w n s t r e a m from t h e g l a s s - c o l l a g e n i n t e r f a c e , respectively, c o m p a r e d t o control v a l u e s . A t w o - w a g analysis o f v a r i a n c e f o l l o w e d by D u n n ' s m u l t i p l e c o m p a r i s o n [20] w a s u s e d to d e t e r mine differences between total number of platelets accumulated f r o m control blood and blood trgated

Olsiance dawnstmam (ram) Fig. L Accaamlati~n of plat¢lcls (I~r 10~0 /~lllz ) on a glass surface (4 rca,'n> 12,~ ram) coated with lihdnar bovine collagen type t (coati~g d:n~iW 2~/= ~/¢m 2) ~s, disza~=~ pm t the gla~s-collagen interface using u~!reated normal whole blood, a~d that treated with either 1130 btM or 200 ttM G-lift. Employing lit U / m l porcine heparin as anticoagulant+ the b l ~ l was incubated with aqueous solutior~ of the test ~ p o u n d or the corresponding volumes of dislilled water for 5 rain, and per[used at 3"7~Cat a wall shear rate of loOO s -I for 2 rain. At each point on each ~Jl~C the mcan+S.E, of n eape~imc~t~ is pletted.

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Dtstlnmi dQwmImmm (m~) ':ix- 2, Platelet accumldation along oullagen-¢onted ~iurfa¢¢$ from nmmal Imparinized whule blood, and that in the presence of either 200 IzM or 400 p M G-112. Employing lo U/ml po~inc hcparln as anticoagulant, the blood wab incubated with aq ug,aas ~olutions of the test compeund or the corresponding volumes ot distilled water for 5 rain, and per[used at 3"P'C at a wall shear rate of 1000 s I for 2 min. At each point on each curve the mcan±S.E, of n experiments is plotted.

Time Isecondg} Fig, 3, Hittogran'~ or thromhus 5i~ at 15 s iznervals demon~oale the kinetics of thrombus growth for untres.ted blood (tOp flame) and for blood trezzt¢~ for 5 m;n ~'.[I ~00 ,u]*~ G-llO (hollO~T. frame) front same donor. Normal whole blood was treated with hoparin (I0 U/roll, incubated in the presence and aly-~enceof the test compound and peffused aL l ~ S I for 2 rain, Digital analysis was performed o.1 video ilxlages (60× } taken 0.38 mm downstream from the gla~collagen interface.

283 with G - I 1 0 : t h e control was found sigr=ificantly differe n t only f r o m b lo o d treated with the highest concentration o f G - l l 0 , 200 p-M. However, this was probably du¢ to t h e small n u m b e r of s a m p l e s available a n d the variations a m o n g different donors. A two-way analysis of variance with r e p e a t e d measures was u s e d to test the t hre e platelet density curves versus distauce. C o n t r o l platelet density d a t a points, in the first 0.5 ram, w e r e significantly greater t h a n the data points d o w n str eam; whi l e in t h e d r u g curves, data points in the region from 0.5 to 1.5 m m were significantly g r e a t e r t h a n th o se downs t re a m. Densities resulting from t r e a t m e n t w i t h 200 # M G - I 1 0 were significantly less than control densities t h r o u g h o u t the length o f the slide; while densities resulting f r o m t r e a t m e n t with I00 /LM G-1)O were less t h a n control densities only in the first 0.5 m m d o w n s t r e a m from t h e collagen interface.

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Time (seconds) FiB. S. Average plat¢lct composition of thrombi for tlrffreated blood and that treated with either 100 tim or 200/tM G-It0 (top frame), and for unlw..atad hhmd and that neared with either 200/~M or 4C~ ~zM G-112 (bnnon| t[atlle) from two different donors. Evaluation w ~ performed for the same t hrombi as in Fig, 3, for ¢~nnol and 2(10pM G-II0 in the top frame, and for the same throm~i as in Fig- 4, far ¢oeuol arid 41~ ~ M G-I 12 in the bottom frame.

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Fig. 4. Histograms of thrombus size at l$-s intervals far umreated blood (top frame) and for blood treated for 5 s i n -~ith 400 ,gM G-It2 (bnnom frame) fl~m same donor. Normal whole blood was treated with heparin (In U/ml). inellhaled ia the pre~nee and ab~ettce of the test e.oml~oundarid ~effused at 1000 s -t for 2 rain. Digital analysis ~vas performed on video imases (bOx) taken 0.38 ram downslream from Ihe glagsmollagen interface.

Fig, 2 shows p]atelgt density profiles from control a n d blood t r e a t e d with e i t h e r 200 or 4 0 0 / . t M ro-112 for t h e first 4 mrn o f collagen-coated surfaces. 200 ~ M O - I 12 reduced by 19 ± 3 % ( ~ S.E,, n = 5) total p l a t e l e t accumulation, c o m p a r e d to control values, i n the prese n c e of 400 p.M O - l l 2 , control total p l a t e l e t accomnlafinn was inhibited by 31 :k 2 % ( + S . E . , n ~ 7). T o t a l platelet deposition f r o m u n t r e a t e d blood, at the first 4 m m d o w n s t r e a m f r o m the b e g i n n i n g of the collagen coating, was f o u n d significantly different from total platelet deposition if b l o o d was treated with e i t h e r 200 o r 400 t t M G-[12; tested by two-way analy~;s o f variance followed by D u n n ' s multiple c o m p a r i s o n [20], A two-way analysis o f variance with r e p e a t e d m e a s u r e s s h o w e d that control platelet density d a t a points, in the first 1 m s , w e r e significantly g r e a t e r t h a n all others b e y o n d those. D e n s i t y d a t a points f r o m b l o o d incub a t e d with either 200 o r 400 ~ M G-II2~ io tho first 2 ram, were g r e a t e r than the d a t a points in the region

284 downstream. Densities resulting from blood treated with 200 # M G-112 were significantly less than control densities in the first 1 mm of collagen-coated surface; while densities resulting from blood treated with 400 /zM G-112 were les.s than control densities in the first 2 mm of collagen-coated surface. Microscopic analysis of the dynamics of thrombus growth was obtained b~, digital an alysis of video images taken every 15 s during the perfusion. All images were from microscope fields at 0.38 mm downstream from the collagen interface. Fig. 3 shows histograms of thrombus size for •ntreated blood in the top frame and for blood treated with 200 tiM G - l l 0 in the bottom frame. Each bar in the histogram shows the number of thrombL in the field of view (60 × ), that were composed of the indicated number of platelets. The distribution of sizes at each time was ~kewed tc~/~ards the lower platelet numbers in the case of the drug compared to the control. Fewer ot the big aggregates

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( > 500 platelets/thrombus) were formed in the presence of O-110, at the end of 2 rain perfusion. Histograms of thrombus size when blood was treated with the lower concentration of G-110 demonstrated a milder effect on the kinetics of thromhos growth (data not shown). Similarly, histograms of thrombus size for untreated brood in the top frame and for that treated with the highest concentration G-112 tested, 400 # M , in the bottom frame, are plotted in Fig. 4. Fig. 5 shows the average platelet composition of all the thrombi within the frame analyzed for untreated blood and that treated with either 100 v.M or 200 p_M G-IIO (top frame), and for untreated blood and that treated with either 200 tiM or 400 tiM G-I12 (bottom frame). The number of plateiets per thrombus were lower for the treated cases, but not signifieantly, due to the broad distribution of tbrombus sizes (Figs. 3 and 4). Quantitative comparison of the surface area covered by thrombi, in the region 0,38 mm downstream from the collagen interface, is shown in Fig. 6 for the same video images previously examined. With the exception of 200 I*M G - l l 0 (top frame), the rest of the compound incubations decreased modestly the percentage of surface coverage, comgared to respective controls. The effect of toe compounds G-I10 and G-I12 on tbrombus morphology can also be observed qualitatively by comparing the three-dimensional structures of thrombi covering the collagen-coated surface in the field of view from untreated blood and that treated with 200 /zM G - i 10, and from untreated blood anti that treated with 400 /*M (3-112 (Fig. 7). All frames were taken at 0.38 mm downsh'eam from the collagen interface at the end of 2-rain perfusiogs. T h e figure demonstrates that, when blood is incubated with the highest of the tested concentrations of either G - I I 0 or G-112, both the size of the thrombi decreases and the surface coverage becomes lower. Discussion

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Fig. 8. Surface coverage by tltrombi after peffugion of untreated and that treated with either tO0 pM or 200 ~M G-[~0 (lop Frame). and of untreated blood and that treated with either 200/zM or 400 btM O-112{'0on~.mfrafll¢),from two different donors. Digital anal~is was aerfo~med for the same [hrombi as in Fi~. 5, top and bottom frames.

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The rcsu!~.s obtained in our experiments showed float bis(dialk'ylcarbamoylpipeddigo)alkanes O - l l 0 and (3-112 are effective in inhibiting platelet adhesion and subsequent aggregation from flowing blood onto a collagen-coated surface, in vitro. G - t i e in concentrations of 100 and 200 t i m reduced platelet accumulation, on the first 4 mm of the reactive surface, by 30 ± 9% (_+ S.E.) and 63 _+3% (±S.E.), respectively, relative to controls. G-112 was less potent: it impeded 19 +_ 3% ( _+S.E.) and 31 +_ 2% (+_ S.E.) of control plate)et accumulation, at concentrations of 200 and 400 t~M, respectively. The compounds exerted their inhibitory potent3, in the microscopic scale by reducing the number of platelets adhering to the surface (measured as snrface area covered by thtombi; Fig. 6) and the number of platelets attached to the previously adherent ones

285

Fig. 7. Three-dimensional r¢oonstructions o[ platelets dep0siledonlo collagen-cratedsurfaces (3.5-10-4 cm2) froal snnealcd normal whole blood and that treated with 200 ~M G-110,and from untreated normal whote blood and that treated with 400 ,~M G-112,[tom two ditf,~rcnt donors. Digitalanalysiswas performed [or the samu thg~bi sttLdiudin Fi~s.3 and 4, respectively.Flowwas from )en to rightand ma~uification used 60~