The American Journal of PATHOLOGY March 1976 * Volume 82, Number 3
Dynamics of Thrombus Formation on an Artificial Surface In Vivo Effects of Antithrombotic Agents Reginald G. Mason, MD, PhD, Robert H. Wolf, MS, DVM, William H. Zucker, PhD, and Brian A. Shinoda, MS
A nonhuman primate model for in vivo evaluation of antithrombotic agents is described. In this model, the formation of a thrombus on a segment of Silastic tubing placed in the vena cava of a rhesus monkey is utilized to evaluate the effectiveness of antithrombotic agents. Thrombus formation in this model was found to occur rapidly, but this initial deposit quickly was followed by a reduction in thrombus weight. Eventually, after 2 hours of implantation of the test device, thrombus weight again increased and reached an apparent plateau. Three different antithrombotic agents were evaluated with this model. Warfarin therapy was found to decrease the thrombus weight in approximate proportion to its effect on the prothrombin time. Aspirin and dextran each produced a decrease in thrombus weight in 2 of 3 animals tested. Individual differences in response to thrombotic agents are apparent, but despite this, the model appears to offer advantages for in vlvo evaluation of antithrombotic agents. (Am J Pathol 82:445-456, 1976)
A NONHUMAN PRIMATE MODEL for the study of thrombus formation upon artificial surfaces-in vivo has been developed recently.' This model is a modification of the Gott vena cava ring test system 2 with the rhesus monkey used in place of the dog as the experimental animal. A previous study indicated that it was possible to compare semiquantitatively the reactivity of two different artificial surfaces with blood using this model.' From the Department of Pathology, University of North Carolina, Chapel Hill, North Carolina; The Department of Pathology, The Memorial Hospital, Pawtucket, Rhode Island; Brown University, Providence, Rhode Island; and The Delta Regional Primate Research Center, Covington, Louisiana. Supported in part by Contract PH 43-68-977 with the Artificial Kidney-Chronic Uremia Program of the National Institute of Arthritis, Metabolism, and Digestive Diseases and by Grants HL-14228, HL-46351, and HL-13296 from the National Heart and Lung Institute, National Institutes of Health. Accepted -for publication November 13, 1975. Address reprint requests to Dr. R. G. Mason, Department of Pathology, The Memorial Hospital, Pawtucket, RI 02860. 445
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There is a current need for development of models for study of the effects of antithrombotic agents. Numerous animal models have been designed for such studies, but none appears to be without some fault.3 The rhesus monkey model for study of thrombus formation on artificial surfaces was modified further to produce an in vivo system of reproducible thrombus generation for use in the study of effects of antithrombotic agents. The present investigation characterizes this thrombus generator model with determination of the optimal time for evaluation of the thrombotic deposit. Effects of three antithrombotic agents have been determined, and the ultrastructural characteristics of the thrombus have been studied in each case. Materials and Methods Test Devices
The rhesus monkey model has been described previously.' In the original model, the vena cava test rings were made of glass or silicone-coated glass. In the present modification, the vena cava rings are made of Silastic rubber tubing (Dow-Corning, Midland, Mich., Catalog No. 601-401, 3/16 inch internal diameter tubing). Previous studies have shown that the inner surface of this tubing is composed of poly(dimethyl siloxane).4 However, the cut ends of the tubing expose a quartz filler that is highly thrombogenic. Vena cava rings were 1.5 cm in length, and the cut ends of the tubing segments were smooth but without taper. Test rings were washed in dilute detergent (FL70, Fisher Scientific Co.), rinsed ten times in tap water and three times in distilled water, and dried in a 60 C electric oven. Vena cava rings were sterilized by ethylene oxide treatment prior to implantation. Time Sequential Study
In the time sequence study of thrombus formation on the surfaces of the Silastic vena cava rings, test rings were implanted for varying intervals of time from 0.5 to 4 hours and then removed and fixed in 4/3% glutaraldehyde prior to determination of the weight of adherent thrombus. The optimal time for quantitation of thrombus deposited upon vena cava rings thus was determined. This optimal time, 2 hours, was used in all subsequent studies of the effects of antithrombotic agents. Gravimetric Determination of Thrombus Weight For gravimetric determination of adherent thrombus weight, fixed vena cava rings were
dried at 60 C and weighed daily on an analytical balance until they maintained a stable weight, usually by 3 days. Following this weighing, thrombus was removed by digestion in a 40% urea-0.02 M KOH solution, and the cleaned vena cava ring was redried and reweighed as before. This second weight was subtracted from the initial weight to give the weight of the thrombus. Control studies indicated that the digestion process decreased the weight of Silastic vena cava rings by less than 1 mg; this weight change was deducted from final thrombus weights in each case.
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Antithrombotic Agents
The three antithrombotic agents tested were warfarin, acetylsalicvlic acid (aspirin), and dextran. Warfarin was given at a dosage of 1.0 mg/kg for 2 days prior to implantation of vena cava rings. Prothrombin time (PT) values were determined by a method described previously.5 Aspirin was given by nasogastric tube at a dosage of 30 mg/kg/day for 2 days prior to the surgery and on the day of surgery. Dextran was given intravenously as 10 ml of 6% Dextran 75 per kilogram immediately following implantation of the vena cava ring. In these studies of antithrombotic agents, vena cava rings were implanted for 2 hours only. Ultrastructural Studies
Procedures for glutaraldehyde fixation, dehydration, embedding, and sectioning of materials for examination by transmission electron microscopy (TEM) in an RCA EMU 3G instrument have been described.6 Similarly, procedures for preparation of specimens for examination by scanning electron microscopy (SEM) in a Coates and Welter 100-4 instrument have been described.4
Results Gravimetric Studies Time Sequential Studies of Thrombus Formation
A series of experiments was conducted order to determine the optimal interval for implantation of Silastic 'swna cava rings. Results of these studies are shown in Table 1. There was considerable thrombus formation upon Silastic rings implanted for as brief an interval as 30 minutes. The amount of variation in the thrombus deposited in two different rings was considerable in the 30-minute study. Silastic rings implanted for 1 hour contained less adherent thrombotic material than did those implanted for 30 minutes. Surprisingly, Silastic rings implanted for 1¼/4 hour had little thrombotic material adherent to them. On the other hand, Silastic rings implanted for 2 or 4 hours had approximately as much adherent Table 1-Time Sequence Studies of the Dynamics of Thrombus Formation on Silastic Vena Cava Rings In Vivo* Elapsed timet (hrs) Weight of thrombotic deposit (mg) 0.5 202 0.5 118 1.0 60 1.0 85 1.25 12 1.25 17 2.0 223 2.0 276 4.0 281 * Each horizontal line gives data from a test with a single vena cava ring. Each ring was implanted in a different monkey. t Time of exposure of Silastic vena cava ring to flowing blood in vivo.
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thrombotic material as did rings implanted for 30 minutes. These studies indicated that a 2-hour implantation interval appeared optimal for generation of maximum thrombus. Effects of Antithrombotic Agents
Three different antithrombotic agents were evaluated for their effects on the generation of thrombus upon Silastic rings. Data are presented in Table 2. The inhibitory effects of warfarin on thrombus deposition are evident and are correlated closely with prolongation of the PT value. In 1 animal with a PT of 20.5 seconds, the amount of thrombus deposited was equivalent to that formed in untreated animals. On the other hand, in animals with PT values of 27.6 or 39.5 seconds, the amount of thrombus deposited was markedly reduced. Both aspirin and dextran were shown to inhibit thrombus formation on Silastic rings in some, but not all, test animals. With each of these latter two antithrombotic agents, the amount of thrombus deposited was decreased in 2 test animals but was unaffected in a third animal. Ultrastructural Studies
Ultrastructural features of the thrombus generated on Silastic vena cava rings were studied both in the time sequential experiments and in the experiments evaluating effects of antithrombotic agents. The ultrastructural appearance of the thrombotic deposit was not appreciably different in the time sequential studies from that of the thrombus formed in the studies with antithrombotic drugs. Thrombus formed on Silastic vena cava rings was typical of that seen in Table 2-Effects of Antithrombotic Agents on Thrombus Formed on Silastic Vena Cava Rings Implanted for Two Hours*
PTt (test/control) (sec) 20.5/12.2 27.6/12.9 39.5/12.4
Weight of thrombotic deposit (mg) 280 Warfarin 43 Warfarin 27 Warfarin 273 Aspirin 100 Aspirin 53 _ Aspirin 245 Dextran 85 Dextran 44 Dextran * Each horizontal line gives data from a test with a single vena cava ring. Each ring was implanted in a different monkey. t PT = prothrombin time. Values are listed in order of decreasing thrombus weight for each of the three agents tested. Antithrombotic agent
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previous studies with glass and silicone-coated glass rings exposed to blood in vivo. The external appearance of an occluding thrombus removed intact from a Silastic vena cava ring is shown in Figure 1. This cylindrical structure has a fluted appearance at one end. The thrombus is shown to consist of a network of fibrin fibers (Figures 2 and 3) that has entrapped erythrocytes. Single and aggregated platelets are intimately associated with the fibrin network (Figures 2 and 3). Studies of mural thrombotic deposits were carried out in situ. Some mural thrombi were composed of a fibrin network with entrapped erythrocytes (Figure 4). In addition, in many areas leukocytes were adherent to the Silastic surface, as were single and aggregated platelets (Figure 5). The association of platelet aggregates with the fibrin network is illustrated clearly in Figure 6, where it can be seen that some platelet aggregates serve to anchor the fibrin to the Silastic surface. In some areas, adherent leukocytes were intimately associated with single and aggregated platelets (Figure 7), and some platelets appeared to stick to the surfaces of leukocytes. Transmission electron microscopy studies of the thrombus showed that adherent cells rested upon a layer of material adsorbed to the Silastic surface. Platelet aggregates were applied to this adsorbed layer and were associated with numerous fibrin fibers (Figure 8). In other areas, dense bundles of fibrin lay between adherent platelets and the adsorbed layer (Figure 9). That the cells identified as leukocytes by SEM were indeed leukocytes is demonstrated in Figure 10, where TEM study permits identification of nuclei and cytoplasmic granules. Leukocytes also were adherent to the adsorbed layer present upon the Silastic surface (Figure 11). Discussion
The present studies have illustrated the potential usefulness of a nonhuman primate model for thrombus generation in testing the effects of antithrombotic agents in vivo. This nonhuman primate model is obviously not suitable for screening large numbers of antithrombotic agents, since only a single datum is obtained from each animal. In addition, the rhesus monkeys are expensive and difficult to obtain. Nevertheless, the model appears suitable for evaluating the effects of highly promising candidate antithrombotic agents prior to their testing in humans. The value of the time sequential study for selection of the optimal interval for vena cava ring implantation is obvious. Had an implantation time of 1 hour or 1 ¼/4 hours been chosen blindly, the amount of thrombus generated would have been small indeed. The decrease in the amount of
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thrombus from the 30-minute specimen to the 1.-hour or 1¼/4-hour specimens is likely due to activation of the fibrinolytic system with digestion of some of the thrombus previously deposited. This interpretation is speculative, since we did not document activation of the fibrinolytic system in these studies. Nevertheless, the increase in the amount of thrombus deposit from the 1-hour and I1/4¼-hour specimens to the 2- and 4-hour specimens suggests that the activated fibrinolytic system subsequently had been inactivated permitting additional thrombus to accumulate. The agreement between the weights of thrombus in similar specimens exposed to blood in different animals was good, with the exception of the 30-minute specimens. This degree of agreement of data is encouraging, since small differences in activation and inactivation of the intrinsic blood coagulation and fibrinolytic systems could influence thrombus deposit weight considerably. The observed effects of the three antithrombotic agents 7,8 on thrombus deposition upon Silastic vena cava rings indicate that the model test system can indeed be used to evaluate such agents. The effect of warfarin clearly was correlated with the inhibitory action of this agent on the blood coagulation system as is indicated by the PT values. A prolongation of the PT time indicates impairment of the intrinsic blood coagulation system, and this was associated with a decrease in the amount of thrombus deposited upon Silastic vena cava rings. With both aspirin and dextran, the antithrombotic effect was demonstrated in 2 of 3 animals. Despite the presence of the antithrombotic agent, the vena cava ring from the third animal in each case showed a weight of thrombus equivalent to that found in controls. These findings suggest that there are individual variations in the response of test animals to Warfarin, aspirin, and dextran. It is likely that by increasing the dosage of the three agents, an antithrombotic effect could have been demonstrated even in these "resistant" animals. The present findings illustrate further the apparently unavoidable variations encountered in work with laboratory animals and show the need for multiple observations with each test agent. Ultrastructural studies of the different thrombotic deposits demonstrate that they are typical thrombi composed of fibrin, platelets, leukocytes, and entrapped erythrocytes. The presence of leukocytes within such thrombi, especially adherent to the artificial surface, was found in a previous study.' Similar findings of leukocytes adherent to artificial surfaces have been reported in studies of hemodialysis units 9,10 and blood oxygenators."1 The role of leukocytes in thrombus formation in unclear, although it is known that leukocytes may contribute to thrombus formation by several different pathways. 12,13 Similarly, leukocytes contribute also to the phenomenon of
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fibrinolysis.14 The lack of effect of the three different antithrombotic agents on the composition of the thrombus indicates that none of these agents functions by inhibiting completely the fibrin-forming or platelet adhesion-aggregation sequences. Indeed, while the amount of thrombus was decreased in most studies in the presence of each of the antithrombotic agents, the ultrastructural composition of the thrombus that did form was not appreciably different from that of controls. Of interest was the fact that platelets appeared no different in thrombi formed in aspirin-treated animals than in control thrombi or in thrombi from dextran-treated animals, even though these agents influence platelet function.7"8 The present studies report on one step of an ongoing program of development of animal models for the study of thrombus formation and for evaluation of the effects of antithrombotic agents. The present model permits study of thrombus formation in vivo in a nonhuman primate. Although some differences do exist in the blood coagulation, platelet adhesion-aggregation, and fibrinolytic systems of nonhuman primates and humans,15", these differences are generally less marked than those that exist between humans and lower mammals. References 1. 2.
3. 4.
5. 6. 7. 8.
9.
10.
Rodman NF, Wolf RH, Mason RG: Venous thrombosis on prosthetic surfaces: Evolution and blood coagulation studies in a nonhuman primate model. Am J Pathol 7:229-242, 1974 Gott VL, Furuse A: Antithrombogenic surfaces, classification and in vivo evaluation. Fed Proc 30:1679-1685, 1971 Herrman RG: Drugs and experimental hemostasis. Platelets, Drugs and Thrombosis. Edited by J Hirsh, JF Cade, AS Gallus, E Schonbaum. Basel, S. Karger, 1975, pp. 145-157 Mason RG, Zucker WH, Shinoda BA, Chuang HY, Kingdon HS, Clark HG: Study of the reactions of blood with artificial surfaces: Use of the thrombogenerator. Lab Invest 31:143-155, 1974 Langdell RD, Wagner RH, Brinkhous KM: Effect of antihemophilic factor on onestage clotting tests: A presumptive test for hemophilia and a simple one-stage antihemophilic factor assay procedure. J Lab Clin Med 41:637-647, 1953 Zucker WH, Shermer RW, Mason RG: Ultrastructural comparison of human plattelets separated from blood by various means. Am J Pathol 77:255-268, 1974 Mustard JF, Packham MA: Factors influencing platelet function: adhesion, release and aggregation. Pharmacol Rev 22:97-187, 1970 Mason RG, Sarji K, Brinkhous KM: Antithrombotic agents: Their effects on platelets and methods for their evaluation. Principles and Techniques of Human Research and Therapeutics. Edited by FG McMahon. Mount Kisco, N.Y., Futura Publications, 1974, pp. 183-199 Ahearn DJ, Marshall JW, Nothum RJ, Esterly JA, Nolph KD, Maher JF: Morphologic studies of dialysis membranes: Adherence of blood components to air rinsed coils. Trans Am Soc Artif Intern Org 19:435-439, 1973 Zucker WH, Morton BD, Erlandson SE: Ultrastructural aspects of blood: Artificial
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11. 12.
13. 14. 15.
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surface reactions in experimental and clinical hemodialysis. Fed Proc 32:836 Abs 1973 Lautier A, Bonnet A, Volter F, Hung BM, Laurent D: Studies on oxygenator membrane surface in long term extracorporeal assistance. Trans Am Soc Artif Intern Org 20:307-312, 1974 Niemetz J, Fani K: Role of leukocytes in blood coagulation and the generalized Shwartzman reaction. Nature [New Biol] 232:247-248, 1971 Niemetz J, Fani K: Thrombogenic activity of leukocytes. Blood 42:47-59, 1973 Wflnschmann-Henderson B, Horwitz DL, Astrup T: Release of plasminogen activator from viable leukocytes of man, baboon, dog and rabbit. Proc Soc Exp Biol Med 141:634-638, 1972 Todd ME, McDevitt E, Goldsmith El: Blood-clotting mechanisms of nonhuman primates: Choice of the baboon model to simulate man. J Med Primatol 1:132-141, 1972
16. Mason RG, Read MS: Some species differences in fibrinolysis and blood coagulation. J Biomed Mater Res 5:121-128, 1971 Dr. Zucker's present address is School of Natural Resources, The University of the South Pacific, PO Box 1168. Suva, Fiji.
I
3a
4
4
Figure 1-A low magnification SEM view of a thrombus that completely occluded a Silastic vena cava ring. The uppermost portion of the thrombus protruded from the ring and formed a fluted flange. (x 25) Figure 2-A SEM view of the surface of the thrombus illustrated in Figure 1. It can be seen that a fibrin network entraps numerous erythrocytes. Single and aggregated platelets are associated with the fibrin network. (x 7100) Figure 3-A SEM view of the inner aspects of the thrombus illustrated in Figure 1. The fibrin network is closely applied to erythrocytes and platelets. (x 9600) Figure 4-A SEM view of a mural thrombus formed upon a Silastic vena cava ring. In some areas, a dense fibrin network entraps erythrocytes. In other areas, erythrocytes and a few fibrin fibers are adherent to the surface of the ring. (x 600)
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Figure 5-A mural thrombus as seen by SEM. A single leukocyte had adhered to the surface and spread upon it. A number of loose platelet aggregates are present, and two of these are associated with the tangled mass of fibrin fibers. (x 5500)
Figure 6-A SEM view to illustrate the association of small and large platelet aggregates with the fibrin net. In addition, two smaller platelet aggregates associated with fibrin fibers have spread upon the surface of the ring. (x 2500) Figure 7-A mural thrombotic deposit that consists largely of single and aggregated platelets with numerous short and long pseudopods. Several platelets are adherent to the surface of a leukocyte. Fibrin is not demonstrated in this area. (x 4750)
Figure 8-A TEM view of an adherent platelet aggregate. The platelets within this aggregate still retain some of their cytoplasmic organelles. Platelets are adherent to a layer of absorbed material (arrow) present on the surface of the Silastic vena cava ring. Numerous fibrin fibrils are present. (Uranyl acetate and lead citrate, x 10,050)
5
6
42
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1,
:;
. :.,.
.:;";;..X;k. VW
9
11
10
9
10
Figure 9-A TEM view of an adherent platelet aggregate with dense deposits of fibrin present in the space between the platelet aggregate and the Silastic surface. In one area the layer of absorbed material (arrow) is illustrated. (Uranyl acetate and lead citrate, x 19,500) Figure 10-A TEM view to illustrate two adherent leukocytes. These leukocytes have spread only partially upon the Silastic surface. Fibrin fibrils are present, as are erythrocytes. (Uranyl acetate and lead citrate, 3000) Figure 11-A TEM view of a leukocyte adherent to the layer of absorbed material present on Silastic. This leukocyte contains numerous cytoplasmic granules of several types and is identified as a neutrophil. (Uranyl acetate and lead citrate, x 6700) X
Dynamics of Thrombus Formation on an Artificial Surface In Vivo Effects of Antithrombotic Agents Reginald G. Mason, MD, PhD, Robert H. Wolf, MS, DVM, William H. Zucker, PhD, and Brian A. Shinoda, MS
A nonhuman primate model for in vivo evaluation of antithrombotic agents is described. In this model, the formation of a thrombus on a segment of Silastic tubing placed in the vena cava of a rhesus monkey is utilized to evaluate the effectiveness of antithrombotic agents. Thrombus formation in this model was found to occur rapidly, but this initial deposit quickly was followed by a reduction in thrombus weight. Eventually, after 2 hours of implantation of the test device, thrombus weight again increased and reached an apparent plateau. Three different antithrombotic agents were evaluated with this model. Warfarin therapy was found to decrease the thrombus weight in approximate proportion to its effect on the prothrombin time. Aspirin and dextran each produced a decrease in thrombus weight in 2 of 3 animals tested. Individual differences in response to thrombotic agents are apparent, but despite this, the model appears to offer advantages for in vlvo evaluation of antithrombotic agents. (Am J Pathol 82:445-456, 1976)
A NONHUMAN PRIMATE MODEL for the study of thrombus formation upon artificial surfaces-in vivo has been developed recently.' This model is a modification of the Gott vena cava ring test system 2 with the rhesus monkey used in place of the dog as the experimental animal. A previous study indicated that it was possible to compare semiquantitatively the reactivity of two different artificial surfaces with blood using this model.' From the Department of Pathology, University of North Carolina, Chapel Hill, North Carolina; The Department of Pathology, The Memorial Hospital, Pawtucket, Rhode Island; Brown University, Providence, Rhode Island; and The Delta Regional Primate Research Center, Covington, Louisiana. Supported in part by Contract PH 43-68-977 with the Artificial Kidney-Chronic Uremia Program of the National Institute of Arthritis, Metabolism, and Digestive Diseases and by Grants HL-14228, HL-46351, and HL-13296 from the National Heart and Lung Institute, National Institutes of Health. Accepted -for publication November 13, 1975. Address reprint requests to Dr. R. G. Mason, Department of Pathology, The Memorial Hospital, Pawtucket, RI 02860. 445
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There is a current need for development of models for study of the effects of antithrombotic agents. Numerous animal models have been designed for such studies, but none appears to be without some fault.3 The rhesus monkey model for study of thrombus formation on artificial surfaces was modified further to produce an in vivo system of reproducible thrombus generation for use in the study of effects of antithrombotic agents. The present investigation characterizes this thrombus generator model with determination of the optimal time for evaluation of the thrombotic deposit. Effects of three antithrombotic agents have been determined, and the ultrastructural characteristics of the thrombus have been studied in each case. Materials and Methods Test Devices
The rhesus monkey model has been described previously.' In the original model, the vena cava test rings were made of glass or silicone-coated glass. In the present modification, the vena cava rings are made of Silastic rubber tubing (Dow-Corning, Midland, Mich., Catalog No. 601-401, 3/16 inch internal diameter tubing). Previous studies have shown that the inner surface of this tubing is composed of poly(dimethyl siloxane).4 However, the cut ends of the tubing expose a quartz filler that is highly thrombogenic. Vena cava rings were 1.5 cm in length, and the cut ends of the tubing segments were smooth but without taper. Test rings were washed in dilute detergent (FL70, Fisher Scientific Co.), rinsed ten times in tap water and three times in distilled water, and dried in a 60 C electric oven. Vena cava rings were sterilized by ethylene oxide treatment prior to implantation. Time Sequential Study
In the time sequence study of thrombus formation on the surfaces of the Silastic vena cava rings, test rings were implanted for varying intervals of time from 0.5 to 4 hours and then removed and fixed in 4/3% glutaraldehyde prior to determination of the weight of adherent thrombus. The optimal time for quantitation of thrombus deposited upon vena cava rings thus was determined. This optimal time, 2 hours, was used in all subsequent studies of the effects of antithrombotic agents. Gravimetric Determination of Thrombus Weight For gravimetric determination of adherent thrombus weight, fixed vena cava rings were
dried at 60 C and weighed daily on an analytical balance until they maintained a stable weight, usually by 3 days. Following this weighing, thrombus was removed by digestion in a 40% urea-0.02 M KOH solution, and the cleaned vena cava ring was redried and reweighed as before. This second weight was subtracted from the initial weight to give the weight of the thrombus. Control studies indicated that the digestion process decreased the weight of Silastic vena cava rings by less than 1 mg; this weight change was deducted from final thrombus weights in each case.
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Antithrombotic Agents
The three antithrombotic agents tested were warfarin, acetylsalicvlic acid (aspirin), and dextran. Warfarin was given at a dosage of 1.0 mg/kg for 2 days prior to implantation of vena cava rings. Prothrombin time (PT) values were determined by a method described previously.5 Aspirin was given by nasogastric tube at a dosage of 30 mg/kg/day for 2 days prior to the surgery and on the day of surgery. Dextran was given intravenously as 10 ml of 6% Dextran 75 per kilogram immediately following implantation of the vena cava ring. In these studies of antithrombotic agents, vena cava rings were implanted for 2 hours only. Ultrastructural Studies
Procedures for glutaraldehyde fixation, dehydration, embedding, and sectioning of materials for examination by transmission electron microscopy (TEM) in an RCA EMU 3G instrument have been described.6 Similarly, procedures for preparation of specimens for examination by scanning electron microscopy (SEM) in a Coates and Welter 100-4 instrument have been described.4
Results Gravimetric Studies Time Sequential Studies of Thrombus Formation
A series of experiments was conducted order to determine the optimal interval for implantation of Silastic 'swna cava rings. Results of these studies are shown in Table 1. There was considerable thrombus formation upon Silastic rings implanted for as brief an interval as 30 minutes. The amount of variation in the thrombus deposited in two different rings was considerable in the 30-minute study. Silastic rings implanted for 1 hour contained less adherent thrombotic material than did those implanted for 30 minutes. Surprisingly, Silastic rings implanted for 1¼/4 hour had little thrombotic material adherent to them. On the other hand, Silastic rings implanted for 2 or 4 hours had approximately as much adherent Table 1-Time Sequence Studies of the Dynamics of Thrombus Formation on Silastic Vena Cava Rings In Vivo* Elapsed timet (hrs) Weight of thrombotic deposit (mg) 0.5 202 0.5 118 1.0 60 1.0 85 1.25 12 1.25 17 2.0 223 2.0 276 4.0 281 * Each horizontal line gives data from a test with a single vena cava ring. Each ring was implanted in a different monkey. t Time of exposure of Silastic vena cava ring to flowing blood in vivo.
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thrombotic material as did rings implanted for 30 minutes. These studies indicated that a 2-hour implantation interval appeared optimal for generation of maximum thrombus. Effects of Antithrombotic Agents
Three different antithrombotic agents were evaluated for their effects on the generation of thrombus upon Silastic rings. Data are presented in Table 2. The inhibitory effects of warfarin on thrombus deposition are evident and are correlated closely with prolongation of the PT value. In 1 animal with a PT of 20.5 seconds, the amount of thrombus deposited was equivalent to that formed in untreated animals. On the other hand, in animals with PT values of 27.6 or 39.5 seconds, the amount of thrombus deposited was markedly reduced. Both aspirin and dextran were shown to inhibit thrombus formation on Silastic rings in some, but not all, test animals. With each of these latter two antithrombotic agents, the amount of thrombus deposited was decreased in 2 test animals but was unaffected in a third animal. Ultrastructural Studies
Ultrastructural features of the thrombus generated on Silastic vena cava rings were studied both in the time sequential experiments and in the experiments evaluating effects of antithrombotic agents. The ultrastructural appearance of the thrombotic deposit was not appreciably different in the time sequential studies from that of the thrombus formed in the studies with antithrombotic drugs. Thrombus formed on Silastic vena cava rings was typical of that seen in Table 2-Effects of Antithrombotic Agents on Thrombus Formed on Silastic Vena Cava Rings Implanted for Two Hours*
PTt (test/control) (sec) 20.5/12.2 27.6/12.9 39.5/12.4
Weight of thrombotic deposit (mg) 280 Warfarin 43 Warfarin 27 Warfarin 273 Aspirin 100 Aspirin 53 _ Aspirin 245 Dextran 85 Dextran 44 Dextran * Each horizontal line gives data from a test with a single vena cava ring. Each ring was implanted in a different monkey. t PT = prothrombin time. Values are listed in order of decreasing thrombus weight for each of the three agents tested. Antithrombotic agent
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previous studies with glass and silicone-coated glass rings exposed to blood in vivo. The external appearance of an occluding thrombus removed intact from a Silastic vena cava ring is shown in Figure 1. This cylindrical structure has a fluted appearance at one end. The thrombus is shown to consist of a network of fibrin fibers (Figures 2 and 3) that has entrapped erythrocytes. Single and aggregated platelets are intimately associated with the fibrin network (Figures 2 and 3). Studies of mural thrombotic deposits were carried out in situ. Some mural thrombi were composed of a fibrin network with entrapped erythrocytes (Figure 4). In addition, in many areas leukocytes were adherent to the Silastic surface, as were single and aggregated platelets (Figure 5). The association of platelet aggregates with the fibrin network is illustrated clearly in Figure 6, where it can be seen that some platelet aggregates serve to anchor the fibrin to the Silastic surface. In some areas, adherent leukocytes were intimately associated with single and aggregated platelets (Figure 7), and some platelets appeared to stick to the surfaces of leukocytes. Transmission electron microscopy studies of the thrombus showed that adherent cells rested upon a layer of material adsorbed to the Silastic surface. Platelet aggregates were applied to this adsorbed layer and were associated with numerous fibrin fibers (Figure 8). In other areas, dense bundles of fibrin lay between adherent platelets and the adsorbed layer (Figure 9). That the cells identified as leukocytes by SEM were indeed leukocytes is demonstrated in Figure 10, where TEM study permits identification of nuclei and cytoplasmic granules. Leukocytes also were adherent to the adsorbed layer present upon the Silastic surface (Figure 11). Discussion
The present studies have illustrated the potential usefulness of a nonhuman primate model for thrombus generation in testing the effects of antithrombotic agents in vivo. This nonhuman primate model is obviously not suitable for screening large numbers of antithrombotic agents, since only a single datum is obtained from each animal. In addition, the rhesus monkeys are expensive and difficult to obtain. Nevertheless, the model appears suitable for evaluating the effects of highly promising candidate antithrombotic agents prior to their testing in humans. The value of the time sequential study for selection of the optimal interval for vena cava ring implantation is obvious. Had an implantation time of 1 hour or 1 ¼/4 hours been chosen blindly, the amount of thrombus generated would have been small indeed. The decrease in the amount of
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thrombus from the 30-minute specimen to the 1.-hour or 1¼/4-hour specimens is likely due to activation of the fibrinolytic system with digestion of some of the thrombus previously deposited. This interpretation is speculative, since we did not document activation of the fibrinolytic system in these studies. Nevertheless, the increase in the amount of thrombus deposit from the 1-hour and I1/4¼-hour specimens to the 2- and 4-hour specimens suggests that the activated fibrinolytic system subsequently had been inactivated permitting additional thrombus to accumulate. The agreement between the weights of thrombus in similar specimens exposed to blood in different animals was good, with the exception of the 30-minute specimens. This degree of agreement of data is encouraging, since small differences in activation and inactivation of the intrinsic blood coagulation and fibrinolytic systems could influence thrombus deposit weight considerably. The observed effects of the three antithrombotic agents 7,8 on thrombus deposition upon Silastic vena cava rings indicate that the model test system can indeed be used to evaluate such agents. The effect of warfarin clearly was correlated with the inhibitory action of this agent on the blood coagulation system as is indicated by the PT values. A prolongation of the PT time indicates impairment of the intrinsic blood coagulation system, and this was associated with a decrease in the amount of thrombus deposited upon Silastic vena cava rings. With both aspirin and dextran, the antithrombotic effect was demonstrated in 2 of 3 animals. Despite the presence of the antithrombotic agent, the vena cava ring from the third animal in each case showed a weight of thrombus equivalent to that found in controls. These findings suggest that there are individual variations in the response of test animals to Warfarin, aspirin, and dextran. It is likely that by increasing the dosage of the three agents, an antithrombotic effect could have been demonstrated even in these "resistant" animals. The present findings illustrate further the apparently unavoidable variations encountered in work with laboratory animals and show the need for multiple observations with each test agent. Ultrastructural studies of the different thrombotic deposits demonstrate that they are typical thrombi composed of fibrin, platelets, leukocytes, and entrapped erythrocytes. The presence of leukocytes within such thrombi, especially adherent to the artificial surface, was found in a previous study.' Similar findings of leukocytes adherent to artificial surfaces have been reported in studies of hemodialysis units 9,10 and blood oxygenators."1 The role of leukocytes in thrombus formation in unclear, although it is known that leukocytes may contribute to thrombus formation by several different pathways. 12,13 Similarly, leukocytes contribute also to the phenomenon of
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fibrinolysis.14 The lack of effect of the three different antithrombotic agents on the composition of the thrombus indicates that none of these agents functions by inhibiting completely the fibrin-forming or platelet adhesion-aggregation sequences. Indeed, while the amount of thrombus was decreased in most studies in the presence of each of the antithrombotic agents, the ultrastructural composition of the thrombus that did form was not appreciably different from that of controls. Of interest was the fact that platelets appeared no different in thrombi formed in aspirin-treated animals than in control thrombi or in thrombi from dextran-treated animals, even though these agents influence platelet function.7"8 The present studies report on one step of an ongoing program of development of animal models for the study of thrombus formation and for evaluation of the effects of antithrombotic agents. The present model permits study of thrombus formation in vivo in a nonhuman primate. Although some differences do exist in the blood coagulation, platelet adhesion-aggregation, and fibrinolytic systems of nonhuman primates and humans,15", these differences are generally less marked than those that exist between humans and lower mammals. References 1. 2.
3. 4.
5. 6. 7. 8.
9.
10.
Rodman NF, Wolf RH, Mason RG: Venous thrombosis on prosthetic surfaces: Evolution and blood coagulation studies in a nonhuman primate model. Am J Pathol 7:229-242, 1974 Gott VL, Furuse A: Antithrombogenic surfaces, classification and in vivo evaluation. Fed Proc 30:1679-1685, 1971 Herrman RG: Drugs and experimental hemostasis. Platelets, Drugs and Thrombosis. Edited by J Hirsh, JF Cade, AS Gallus, E Schonbaum. Basel, S. Karger, 1975, pp. 145-157 Mason RG, Zucker WH, Shinoda BA, Chuang HY, Kingdon HS, Clark HG: Study of the reactions of blood with artificial surfaces: Use of the thrombogenerator. Lab Invest 31:143-155, 1974 Langdell RD, Wagner RH, Brinkhous KM: Effect of antihemophilic factor on onestage clotting tests: A presumptive test for hemophilia and a simple one-stage antihemophilic factor assay procedure. J Lab Clin Med 41:637-647, 1953 Zucker WH, Shermer RW, Mason RG: Ultrastructural comparison of human plattelets separated from blood by various means. Am J Pathol 77:255-268, 1974 Mustard JF, Packham MA: Factors influencing platelet function: adhesion, release and aggregation. Pharmacol Rev 22:97-187, 1970 Mason RG, Sarji K, Brinkhous KM: Antithrombotic agents: Their effects on platelets and methods for their evaluation. Principles and Techniques of Human Research and Therapeutics. Edited by FG McMahon. Mount Kisco, N.Y., Futura Publications, 1974, pp. 183-199 Ahearn DJ, Marshall JW, Nothum RJ, Esterly JA, Nolph KD, Maher JF: Morphologic studies of dialysis membranes: Adherence of blood components to air rinsed coils. Trans Am Soc Artif Intern Org 19:435-439, 1973 Zucker WH, Morton BD, Erlandson SE: Ultrastructural aspects of blood: Artificial
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11. 12.
13. 14. 15.
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surface reactions in experimental and clinical hemodialysis. Fed Proc 32:836 Abs 1973 Lautier A, Bonnet A, Volter F, Hung BM, Laurent D: Studies on oxygenator membrane surface in long term extracorporeal assistance. Trans Am Soc Artif Intern Org 20:307-312, 1974 Niemetz J, Fani K: Role of leukocytes in blood coagulation and the generalized Shwartzman reaction. Nature [New Biol] 232:247-248, 1971 Niemetz J, Fani K: Thrombogenic activity of leukocytes. Blood 42:47-59, 1973 Wflnschmann-Henderson B, Horwitz DL, Astrup T: Release of plasminogen activator from viable leukocytes of man, baboon, dog and rabbit. Proc Soc Exp Biol Med 141:634-638, 1972 Todd ME, McDevitt E, Goldsmith El: Blood-clotting mechanisms of nonhuman primates: Choice of the baboon model to simulate man. J Med Primatol 1:132-141, 1972
16. Mason RG, Read MS: Some species differences in fibrinolysis and blood coagulation. J Biomed Mater Res 5:121-128, 1971 Dr. Zucker's present address is School of Natural Resources, The University of the South Pacific, PO Box 1168. Suva, Fiji.
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Figure 1-A low magnification SEM view of a thrombus that completely occluded a Silastic vena cava ring. The uppermost portion of the thrombus protruded from the ring and formed a fluted flange. (x 25) Figure 2-A SEM view of the surface of the thrombus illustrated in Figure 1. It can be seen that a fibrin network entraps numerous erythrocytes. Single and aggregated platelets are associated with the fibrin network. (x 7100) Figure 3-A SEM view of the inner aspects of the thrombus illustrated in Figure 1. The fibrin network is closely applied to erythrocytes and platelets. (x 9600) Figure 4-A SEM view of a mural thrombus formed upon a Silastic vena cava ring. In some areas, a dense fibrin network entraps erythrocytes. In other areas, erythrocytes and a few fibrin fibers are adherent to the surface of the ring. (x 600)
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Figure 5-A mural thrombus as seen by SEM. A single leukocyte had adhered to the surface and spread upon it. A number of loose platelet aggregates are present, and two of these are associated with the tangled mass of fibrin fibers. (x 5500)
Figure 6-A SEM view to illustrate the association of small and large platelet aggregates with the fibrin net. In addition, two smaller platelet aggregates associated with fibrin fibers have spread upon the surface of the ring. (x 2500) Figure 7-A mural thrombotic deposit that consists largely of single and aggregated platelets with numerous short and long pseudopods. Several platelets are adherent to the surface of a leukocyte. Fibrin is not demonstrated in this area. (x 4750)
Figure 8-A TEM view of an adherent platelet aggregate. The platelets within this aggregate still retain some of their cytoplasmic organelles. Platelets are adherent to a layer of absorbed material (arrow) present on the surface of the Silastic vena cava ring. Numerous fibrin fibrils are present. (Uranyl acetate and lead citrate, x 10,050)
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Figure 9-A TEM view of an adherent platelet aggregate with dense deposits of fibrin present in the space between the platelet aggregate and the Silastic surface. In one area the layer of absorbed material (arrow) is illustrated. (Uranyl acetate and lead citrate, x 19,500) Figure 10-A TEM view to illustrate two adherent leukocytes. These leukocytes have spread only partially upon the Silastic surface. Fibrin fibrils are present, as are erythrocytes. (Uranyl acetate and lead citrate, 3000) Figure 11-A TEM view of a leukocyte adherent to the layer of absorbed material present on Silastic. This leukocyte contains numerous cytoplasmic granules of several types and is identified as a neutrophil. (Uranyl acetate and lead citrate, x 6700) X
