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Monoclonal antibody—induced inhibition of platelet function: Effects on hemostasis and vascular graft thrombosis in baboons
Monoclonal antibody-induced inhibition of platclct function: Effects on hemostasis and vascular graft thrombosis in baboons Shlomo Torem, M.D., Peter A. Schneider, M.D., and Stephen R. Hanson, Ph.D.,
La Jolla, Calif. The usefulness of antiplatelet agents in vascular graft recipients remains controversial because of uncertainties regarding drug mechanisms of action and dose-duration effects. In our study, a well-characterized murine monoclonal antibody (LJ-CP8, 10 mg/kg) was infused into baboons to assess the hemostatic consequences and antithrombotlc effectiveness of blocking the platelet glycoprotein IIb-IIIa receptor for fibrinogen and other adhesive glycoproteins. Five treated animals and six control animals were evaluated with serial measurements of platelet count, bleeding time, and platelet aggregation ex vivo (in response to adenosine diphosphate and collagen). Indium Ill-labeled platelet deposition onto femoral vascaalar grafts (4 mm inner diameter Gore-Tex) implanted immediately after antibody infusion was measured by quantitative gamma camera imaging. Although the antibody did not alter circulating platelet counts, bleeding times were immediately prolonged to more than 30 minutes (vs. 4.8 -+- 0.4 minutes pretreatment) with only partial normalization by 48 hours (8.3 + 1.0 minutes, p < 0.05)0 Platelet aggregation in response to both collagen and adenosine diphosphate was abolished immediately and remained impaired for 48 hours. Despite the profound inhibition of platelet function, graft platelet deposition was equivalent postoperatively in both the treated and untreated groups (p > 0.5), averaging approximately 5 x 109 platelets per graft. Graft-associated indium 111-labeled platelet activity increased over 48 hours and was not reduced by the antibody treatment (p > 0.5 at all times). All grafts were removed at 8 days; only one graft from a treated animal was found patent. We conclude that (1) antibody-induced blockade of platelet glycoprotein IIb-IIIa is not antithrombotic in this setting and that (2) platelet hemostatic competence as assessed by bleeding time or aggregation measurements may not predict the capacity ofplatelets to contribute to graft thrombus formation. (J VAsc SURG 1988;7:172-80.)
The clinical usefulness of small-diameter vascular grafts remains limited because of the high frequency of early occlusion. The reactions of blood platelets contribute directly to graft failure because these blood elements may represent a primary component of forming arterial thrombP and also release factors that are mitogenic for smooth muscle cells? Subse-
From the Departments of Vascular and Thoracic Surgery (Drs. Torem and Schneider) mad Basic and Clinical Research (Dr. Hanson), Scripps Clinic and Research Foundation. Presented at the Thirty-fifth ScientificMeeting of the North American Chapter, The International Societyfor CardiovascularSurgery, Toronto, Ontario, Canada, June 8-9, 1987. Supported in part by research grants HL31469 and HL31950 from the National Institutes of Health, U.S. Public Health Service. Reprint requests: Dr. Stephen R. Hanson, Department of Basic and Clinical Research (BCR5), Scripps Clinic and Research Foundation, 10666 North Torrey Pines Rd., La Jolla, CA 92037.
172
quently the development of intimal proliferative lesions may mediate late-outcome events? Because the thrombogenici~ of both vascular grafts and injured arteries may diminish with time, 4-6 it has been suggested that early inhibition of platelet function may be important for maintaining graft patency. 7-I° Unfortunately, the efficacy of antiplatelet agents in graft recipients has not been well established, in part because of uncertainties regarding appropriate drug doses, duration of effects, and mechanisms of action. 8m In the current study, inhibition of platelet function was achieved by means of a well-characterized murine monoclonal antibody (LJ-CP8) against the platelet glycoprotein IIb-IIIa complex. This receptor binds fibrinogen and other adhesive glycoproteins, such as yon Willebrand factor and fibronectin, after stimulation by appropriate physiologic agonists such as thrombin or adenosine diphosphate (ADP). 1~ As
Volume 7 Number 1 lanuan, 1988
Antibody-induced pIatelet inhibition in vivo 173
Fig. 1. Indium ill-labeled platelet scintillation camera image of a femoral PTFE graft and contralateral limb. The image was taken 3 hours postoperatively in a control animal. Platelet 111InactivitT was clearlyvisualized and localized to the graft.
shown by studies in patients with Glanzmann thrombasthenia, who are deficient in membrane glycoproreins IIb and IIIa, and by studies in patients with severe afibrinogenemia, the inability of platelcts to bind fibrinogen normally may result in significant platelet dysfunction that is characterized by prolonged bleeding and abnormal aggregation.l~.12 The crucial role of the platelet glycoprotein IIb-IIIa receptor has been further documented by studies describing monoclonal antibodies that interact specifically with platelet glycoproteins IIb and/or IIIa, inhibit the binding of fibrinogen and other adhesive glycoproteins, and produce a thrombasthenic-like state in normal platelets in vitro. 13 On the basis of these observations, it has been suggested that such antibodies may be useful as antithrombotic therapeutic agents) 4 Therefore we have used a baboon animal model to assess the effects of injected antiglycoprotcin IIbIIIa antibody with respect to the following variables: platelet count, bleeding time, platelet aggregation, platelet deposition onto expanded polytetrafluoroethylene (PTFE) (Teflon) femoral artery grafts, and graft patency. A baboon model was chosen because this species is hemostatically similar to humans ~Sand has been shown to be useful for studying mechanisms of arterial injury repair after graft implantation. <~6
Therefore the purpose of this study was twofold: to asscss the antihemostatic and antithrombotic consequences of blocking the glycoprotein IIb-IIIa pathway for platclet activation and to detcrmine whether alterations in platelct hemostatic competence would predict graft outcome. MATERIAL A N D M E T H O D S
Animals studied. These studies used 11 normal male baboons (Papio anubis)weighing 12 to 17 kg and observed to be free of disease for at least 6 weeks before use. Six animals served as controls and five were given intravenous antibody. With standard surgical techniques and the use of 1% halothane for general anesthesia, expanded PTFE vascular grafts were inserted into the right superficial femoral artery (Gore-Tex, W. L. Gore & Associates, Inc., Flagstaff, Ariz.). All grafts had a fibril length of 30 Ixm and 4.0 mm internal diameter. The artery was dissected and the animals were given 100 U/kg of heparin. In the treated group, antibody was also injected at this time (discussed later). The artery was then clamped, divided, and replaced with a 4.0 cm segment of PTFE graft. End-to-end anastomoses were constructed with continuous 6-0 polypropylene sutures (Ethicon, Inc., Somerville, N.J.). Suture line bleeding was minimal in control animals. Surgical hemostasis was
)rournaI of VASCULAR SURGERY
174 Torero, Schneider, and Hanson
400
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/3.
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o Treated (5) • Controls (5)
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Time Post.Grafting (hours) Fig. 2. Measurements of circulating platelet count in untreated and antibody-treated animals over 48 hours after graft placement.
achieved after approximately 5 to 10 minutes in the antibody-treated group. The surgical site was closed in two layers, and the animals were allowed to recover for 3 hours before graft imaging. All procedures were performed in accordance with the "Principles of Laboratory Care" and the "Guide for the Care and Use of Laboratory Animals" (NIH Publication No. 80-23, revised 1978). Antibody preparation and characterization. LJ-CP8 was prepared with washed human platelets used as immunogen and was characterized by methods described previously. 17'~8 LJ-CP8 reacted with glycoproteins IIb and/or IIIa when the two were in complex, as shown by (1) its lack of reactivity with platelets from patients with Glanzmann thrombasthenia; (2) solid-phase immunoisolation studies demonstrating that the antibody bound to the complex of two platelet membrane proteins having the electrophoretic mobility of glycoproteins IIb and IIIb; and (3) lack of reactivity with normal platelets incubated with 5 mmol/L EDTA for 15 minutes at 37 ° C. 18LJ-CP8 was of the IgG~ subclass and purifed IgG was prepared from ascitic fluid as previously described} r LJ-CP8 labeled with iodine 125 bound to normal human platelets; an average of 47,000 molecules bound per cell with a dissociation constant (Ka) of 9.4 x 10 -8 mol/L} 8 The binding of LJ-CP8 to baboon platelets was not evaluated directly. However, LJ-CP8 completely inhibited the binding of baboon
fibrinogen, purified as described previously,19 to thrombin-stimulated baboon platelets. Similar results were obtained with human fibrinogen and platclets. The methods used to measure the binding of fibrinogen to platelets have been previously reported in detail} 7 When added at saturation concentrations, LJ-CP8 completely inhibited aggregation of human or baboon platelet-rich plasma (PRP) induced by ADP, collagen, or ~/-thrombin. In the present study, LI-CP8 (10 mg/kg) was injected intravenously into five baboons after the femoral artery was exposed, approximately 5 minutes before the vessel was clamped for grafting. Measurements of platelet function. Platelet counts were performed on whole blood collected in 2 mg/ml disodium EDTA with a whole blood analyzer (model 810, I. T. Baker, Allentown, Pa.). Before graft placement platelet counts averaged 282,000 _+ 90,000/~xl (+_ 1 SD), hematocrit values averaged 38% -_ 2% and were equivalent in both the treated and untreated groups. Bleeding time measurements were performed on the shaved volar surface of the forearm with the standard template method as previously described for studies in baboons. 2° Bleeding time measurements were performed in duplicate and were averaged. Platelet aggregation was measured with an aggregometer (Chrono-Log Corp., Havertown, Pa.) by recording the increase in light transmission through a stirred suspension of PRP maintained at 37 ° C. PRPs and
Volume 7 Number 1 January 1988
platelet-poor plasmas were prepared as previously described.2° In all cases, the platdets in PRP were adjusted to a count of 200,000 plate/ets/t,l. Aggregation was induced by the addition of 1 to 100 ~mol/L ADP (Sigma Chemical Co., St. Louis, Mo.) and 1 to 100 Ixg/ml collagen (Hormon, Munich, F.R.G.). The response to each agonist was compared with preoperative control studies. Since a doseresponse curve (agonist concentration vs. increase in light transmission) was generated at all sampling times after antibody administration, results were expressed as the EDs0, or effective dose of agonist required to induce half the maximal increase in light transmittance.i° Imaging studies. Autologous blood platelets were labeled with 1 mCi indium 111-oxine according to a method described previously. ~ Labeling efficiencies averaged 88%. The labeled platelets were injected approximately 2 hours before the surgical procedures. Graft imaging was performed at S, 24, and 48 hours postoperatively with scintillation camera (Picker Internationl, Inc., Northford, Conn. ~ interfaced with an image processing system (Medical Data Systems A a, Ann Arbor, Mich. ). The 172 keV rain peak was acquired with a high-sensitivity collhnator on the camera with a 20% energy window. At all time points, 5-minute static images were taken of the graft and contralateral limb and of a freshly drawn 5.0 ml whole blood standard. A typical image is shown in Fig. 1. After the graft procedures variable hematoma formation was noted at the surgical sites in the animals given intravenous antibodv. Therefore before imaging was done 3 hours postoperatively, the surgical sites in the treated animals were reopened and approximately 2 to 4 ml of blood was evacuated. Thereafter no further hematoma formation was noted, and the sites were closed and imaged. The surgical sites in control animals were also opened with no evidence of blood leakage. All grafts were patent at this time as demonstrated by direct Doppler scanning of the distal artery with a pencil probe. All sites subsequently remained dosed until the grafts were removed 8 days later. The rain emissions from regions of interest of identical size (approximately 4 x 8 cm) were measured for the grafted area and at a comparable location on the contralatcral control limb. The control region activity was subtracted from the grafted region activity to yield graft-deposited H~In activity only, that is, to subtract the circulating blood activity. To correct for differences in the amount of injected isotope, isotope decay, and dilution, the data were
Antibody-induced platelet inhibition in vivo 175
>30
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Fig. 3. Measurements of bleeding time in untreated and antibody-treated animals over 48 hours after graft placement. Antibody was iniected during the surgicalprocedure but before vessel reconstruction.
expressed as the graft-blood ratio of emissions, calculated by dividing the graft activity by the platclet activity contained in 1 ml of whole blood. I5 At the earliest imaging time (3 hours), it was also possible to calculate total graft platelet accumulation (labeled plus unlabelcd cells) by multiplying the graft-blood ratio by the circulating platelet count (platclets per milliliter). ~5 Statistical methods. Statistical comparisons were performed with the Student t test (two-tailed) for paired and unpaired data. Unless otherwise specified, values are given as the mean + 1 standard error of the mean. RESULTS Effect o f injected antibody on platelet count. It was initially important to determine whether the injection of monoclonal LJ-CP8 IgG caused an increase in platelet removal directly because changing platelet counts in treated vs. untreated animals could influence graft thrombus tbrmation and patency. Initial platelet counts in the treated and untreated animals averaged 290,000 + 24,000/~1 and 287,000 _+ 43,000/~,1, respectively. As shown in Fig. 2, no significant alterations in platelet count were observed in either treated or untreated animals during a 48-hour interval after the surgical procedure. Effect on bleeding time. Measurements of the
176
journal of" VASCULAR SURGERY
Torero, Schneider, and Hanson
COLLAGEN (19.2 k~glml)
~,OP (38.5 ~M)
~PRE
~
PRE 48Hrs. 24Hrs.
3Hrs. 1 Min.
l 1 Min.
Fig. 4. Effect of injected antibody on platelet aggregation in response to collagen and ADP. Platelet aggregation (increase in light transmission) in response to both agonists was initially absent (3 hours after injection) and partially normalized over 48 hours. Table I. Effect of antiglycoprotein IIb-IIIa antibody on platelet aggregation Effective dosefor half maximal aggregation (EDso) Study Control Baseline 3 hr LJ-CP8 (10 mg/kg) Baseline 3 hr 24 hr 48 hr
ADP (~mol/L)
Collagen Oxg/ ml)
2.8 -± 0.3 3.1 ± 0.7
2.4 ± 0.4 2.6 ± 0.8
3.7 ± 0.2 >100 12.8 ± 2.7 7.3 ± 2.1
4.9 + 1.2 >100 i9.4 _+ 1.3 9.2 ± 1.8
NOTE: All values are m e a n ± 1 standard error o f measurements made in five animals (treated) or six animals (untreated). Baseline measurements were made preoperatively, beforc antibody adminstration.
standard template bleeding time are given in Fig. 3. Bleeding times in control animals were equivalent before and after operation (4.8 _+ 0.2 minutes vs. 4.9 + 0.2 minutes). In the five animals given LJ-CP8, bleeding times were prolonged immediately to more than 30 minutes. Although the bleeding times progressively shortened on subsequent days, values were still abnormal at 24 hours (13.2 _ 1.1
minutes, p < 0.01) and 48 hours (8.3 _+ 1.0 minutes, p < 0.05). Effect on platelet aggregation. Typical aggregation tracings are shown in Fig. 4. Measurements of the agonist concentration (ADP and collagen) required to induce a half-maximal aggregation response (EDs0) are given in Table I. Control animals demonstrated a normal aggregation response before and after operation. Three hours after administration of LJ-CPS, platelets were totally unresponsive to ADP (up to i00 Ixmol/L) and collagen (up to 100 jxg/ml). Agonist EDs0 values at 24 hours were significantly greater than baseline values for both ADP (12.8 +_ 2.7 txmol/L, p < 0.05) and collagen (19.4 _+ 1.3 gg/ml, p < 0.001) but tended to normalize by 48 h o u r s after antibody administration (ADP EDs0:7.3 -+ 2.1 ~xM, p > 0.1; collagen EDs0: 9.2 - 1.8 ~g/ml, p > 0.05). Effect on graft platelet deposition. Serial measurements by scintillation camera imaging of the ratio of graft nlIn platelet radioactivity to circulating platelet radioactivity are shown in Fig. 5. For determinations made at 3, 24, and 48 hours postoperatively, no differences were seen between the treated and untreated groups (p > 0.5 at all times). Total platelet accumulation (labeled plus unlabeled cells) was also calculated from images taken 3 hours postopera-
Volume 7 Number 1 January 1988
Antibody-induced platelet inhibition in vivo 177
4° r E
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Time Post-Grafting (hours) Fig. 5. Ratio of graft-deposited Hlln_labeled platelet activity to circulating H'In-labeled platelet activity (per milliliter whole blood) over 48 hours after graft placement. No differences were observed between the untreated and treated groups.
tively. Is Platelet deposition onto grafts placed in the treated animals (5.1 + 1.0 x 109 platelets per graft) was equivalent to values obtained in the control group (4.8 + 0.8 × 109 platelets per graft, p > 0.5). The extent of acute platelet deposition onto the grafts was also influenced by the circulating platelet count (Fig. 6). For all animals studied, a good correlation (r = 0.69) was obtained between platelet deposition and platelet count, indicating that platelet count alone was an important factor regulating acute thrombus formation. There were no apparent differences between treated and untreated animals in this regard. Effect on graft patency. All grafts were removed after 8 days. None of the grafts in untreated animals was patent. Only one graft from a treated animal was found to be patent. No technical problems were noted with any of the graft anastomoses. DISCUSSION This article documents that the infusion into baboons of a monoclonal antibody against the platelct glycoprotcin IIb-IIIa complex may markedly impair platelet hcmostatic function as determined by measurements of bleeding time and platelet aggregation ex vivo. In particular, the prolonged bleeding times
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Fig. 6. Effect of preoperative circulating platelet count on graft platelet deposition (labeled plus unlabeled cells) at 3 hours after grafting. The effect ofplatelet count appeared similar for both the treated and untreated groups.
observed in the present study are consistent with the bleeding tendency observed in patients with thrombasthcnia who lack platelct glycoprotcin IIb-IIIa, and whose platelets are unable to normally bind fibrin-
Jottmalof VASCULAR SURGERY
178 Torero, Schneider, and Hanson
ogen and other adhesive glycoproteins, such as von Willebrand factor and fibronectin, n Because all physiologic agonists are thought to mediate platelet aggregation by expression of this receptor, it is generally believed that platelet glycoprotein IIb-IIIa complex is of central importance for platelet thrombus formation in vivo. H However, we observed no significant benefit of the injected monoclonal antibody with respect to acute graft thrombus formation or graft patency. Several issues are relevant to these observations. First, the antibody was injected before vessel clamping and circulated for approximately one half hour before flow was restored through the grafted segment. Because the effects of this antibody are maximal within minutes after infusion (unpublished observations), the predosing interval was clearly adequate to ensure equilibrium binding to circulating platelets. Second, the dose of injected antibody (10 mg/kg) was chosen so that the initial plasma concentration in vivo would be about twice the concentration (> 100 lag/ml) required to saturate glycoprotein IIb-IIIa and abolish fibrinogen binding in vitro. The efficacy of this dose was also documented postoperatively, at which time bleeding times were essentially infinite, and platelet aggregation in response to ADP and collagen was absent. Third, the observation that all control grafts had failed by 8 days might suggest that technical or other factors unrelated to platelet accumulation may have contributed to early graft occlusion. However, graft platelet deposition was equivalent in treated and untreated animals at 3 hours postoperatively, when the effect of the antibody was maximal and all grafts were patent as documented by Doppler scanning. In addition, the n~In labeled-platelet imaging measurements demonstrated a thirty- to fort)gold enrichment in platelet numbers relative to graft luminal blood volume, whereas static or coagulated blood in the grafts would have reflected only normal blood pool radioactivity without an apparent accumulation of platelets. Finally, all graft anastomoses were examined at the time of removal and found to be technically acceptable. Thus we conclude that superficial femoral artery grafts in the baboon are intrinsically thrombogenic and consequently fail because of mechanisms involving the active accumulation of blood platelets. The most unexpected finding of this study was the observation that intravenous antibody did not modify graft platelet deposition despite the profound inhibition of platelet function shown by measurements of bleeding time and platelet aggregation ex vivo. In addition, previous studies with mono-
clonal antibodies against glycoprotein iIb-][IIa have shown that platelet deposition may be reduced, but not eliminated, on subendothehum perfused in vitro 21'= and on Dacron vascular grafts exposed in an arteriovenous shunt systemY These results may be reconciled in part by recent observations showing tha~ platelets exposed to strong agonists such as thrombin exhibit markedly increased numbers of surface-oriented binding sites associated with platelet glycoprotein IIb-IIIa) 8 With unstimulated platelets, these sites would be inaccessible to extracellular antibodies. On activation, platelets also release intracellular fibrinogen and von Willebrand factor, which may be bound to the glycoprotein IIb-IIIa receptor and subsequently support platelet aggregation. 24,25 Thus in stirred platelet suspensions, agonist-induced receptors may be readily blocked by antibodies, thereby preventing aggregation. Under the more solid-phase conditions of graft thrombus formation, platelet aggregation may have been favored by the close proximity of activated cells and local high concentrations of adhesive glycoproteins and physiologic agonists. In effect, the forming platelet thrombi may have been protected from the antithrombotic actions of the plasma antiglycoprotein IIb-IIIa antibody. Conversely, bleeding times may have been prolonged by the antibody as a consequence of particular modulating effects of vessel injury at the capillary level and microenvironmental factors influencing the intensity of platelet stimulation and local concentrations of relevant blood elements. Thus we conclude that the antithrombotic effectiveness of antibodies against platelet glycoprotein IIb-IIIa may not be predicted by tests of platelet hemostatic competence. However, since the results of the present report do not preclude the usefulness of this approach in other settings, further studies are warranted. The LJ-CP8 was given to us by Dr. Z. M. Ruggeri of Scripps Clinic and Research Foundation. We thank Jill Janik, Paul McFadden, Brandon Domitrowsky, and Stanley Robinson for their technical assistance and Drs. E. F. Bernstein, L. A. Harker, and Z. M. Ruggeri for helpful discussions. REFERENCES
1. StrattonJR,Thide BL, KitchieJL.Naturalhistoryofplatdet depositionon Dacronaorticbifurcationgraftsin the firstyear following implantation in man. Am J Cardiol 1983;52: 371-4. 2. DeuelTF, Huang JS. Platelet-derivedgrowth factor: structure, function, and roles in normal and transformedceils. J Clin Invest 1984;74:669-76. 3. ClowesAW. Current theories of arterial graft failure.Vase Diagn Ther 1982;3:41-52.
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Antibody-induced platelet inhibition in vivo 179
4. McCollum CN, Kester RC, Rajah SM, Learoyd P, Pepper M. Arterial graft maturation: the duration of thrombotic activity in Dacron aortobifemoral grafts measured by platelet and fibrinogen kinetics. Br J Surg 1981;68:61-4. 5. Groves HM, Kinlough-Rathbone RL, Richardson M, Moore S, Mustard JF. Platelet interaction with damaged rabbit aorta. Lab Invest 1979;40:194-200. 6. Clowes AW, Kirkman TR, Clowes MM. Mechanisms of artcrial graft failure. II. Chronic endothelial and smooth muscle cell proliferation in healing polytetrafluoroethylene prostheses. J VAse SURG 1986;3:877-84. 7. Weiss HJ. Platdets: pathophysiology and antiplatelet drug therapy. New York: Alan R. Liss, 1982:95-9. 8. Kohler TR, Kaufinan JL, Kacoyanis G, et al. Effect of aspirin and dipyridamole on the patency of lower extremity bypass grafts. Surgery 1984;96:463-6. 9. Stratton JR, Ritchie JL. Reduction of indium-ill platelet deposition on Dacron vascular grafts in humans by aspirin plus dipyridamole. Circulation 1986;73:328-30. 10. Harker LA. Antiplatelet drugs in the management of patients with thrombotic disorders. Semin Thromb Hemost 1986; 12:134-55. 11. Coller BS, Scudder LE, Peerschke EI, Kalomiris EL, Steinberg M. Platelet physiology and platelet membrane glycoproteins. In: Jolles G, Legrand YJ, Nurden A, eds. Biology and pathology ofplatelet-vessel wall interactions. New York: Academic Press, 1986:181-200. 12. DeMarco L, Girolami A, Zimmerman TS, Ruggeri ZM. yon Willebrand factor interaction with the glycoprotein IIb/IIIa complex. Its role in platelet function as demonstrated in patients with congenital afibrinogenemia. J Clin Invest 1986; 77:1272-7. 13. Coller BS, Peerschke EI, Scudder LE, Sullivan CA. A routine monoclonal antibody that completely blocks the binding of fibrinogen to platelets produces a thrombasthenic-like state in normal ptatelets and binds to glycoproteins IIb and/or Ilia. J Clin Invest 1983;72:325-38. 14. Coller BS, Scudder LE. Inhibition of dog platelet function by in vivo infusion of F(ab')2 fragments of a monoclonal antibody to the platelet glycoprotein IIb/IIIa receptor. Blood 1985;66:1456-9.
15. Hanson SR, Kotze HF, Savage B, Harker LA. Platdet interactions with Dacron vascular grafts: a model of acute thrombosis in baboons. Arteriosclerosis 1985;5:595-603. 16. Clowes AW, Gown AM, Hanson SR, Reidy MA. Mechanisms of arterial graft failure. 1. Role of cellular proliferation in early healing of PTFE prostheses. Am J Pathol 1985; 118:43-54. 17. Lombardo VT, Hodson E, Roberts JR, Kunicki TJ, Zimmerman TS, Ruggeri ZM. Independent modulation of yon Willebrand factor and fibrinogen binding to the platelet membrane glycoprotein IIb/IIIa complex as demonstrated by monoclonal antibody. J Clin Invest 1985;76:1950-8. 18. Niiya K, Hodson E, Bader R, et al. Increased surface expression of the membrane glycoprotein IIb/IIIa complex induced by platelet activation. Relationship to the binding of fibrinogen and platelet aggregation. Blood 1987;70:475-83. 19. Weathersby PK, Horbett TA, Hoffman AS. Solution stability of bovine fibrinogen. Thromb Res 1977;10:245-52. 20. Malpass TW, Hanson SR, Savage B, Hessel EA II, Harker LA. Prevention of acquired transient defect in platelet plug formation by infused prostacyclin. Blood 1981;57:736-40. 21. Sakariassen KS, Nievelstein PFEM, Coller BS, Sixma JJ. The role of platelet membrane glycoproteins Ib and IIb-IIIa in platelet adherence to human artery subendorhelium. Br J Haematol 1986;63:681-91. 22. Weiss HJ, Turitto VT, Baumgartuer HR. Platelet adhesion and thrombus formation on subendothdium in platelets deficient in glycoproteins IIb-IIIa, Ib, and storage granules. Blood 1986;67:322-30. 23. Hanson SR, Pareti FI, Ruggeri Z, et aL Antibody-induced platelet inhibition reduces thrombus formation in vivo. Clin Res 1986;34:658A. 24. Courtois G, Ryekewaert J-J, Woods VL, Ginsberg MH, Plow Et;, Marguerie GA. Expression of intracellular fibrinogen on the surface of stimulated platelets. Eur J Biochem 1986; 159:61-7. 25. Parker RI, Gralnick HR. Identification of platelet glycoprotein IIb/IIIa as the major binding site for released ptareletyon Willebrand factor. Blood 1986;68:732-6.
DISCUSSION
surfaces, but here its role is shared with at least one other protein, yon Willebrand's factor, which is especially important in adhesion ofplatelets to subendothelial structures in the vessel wall and probably to artificial surfaces as well. yon Willebrand's factor will interact with glycoprotein IIb-IIIa, but its principal receptor is another glycoprotein, GPIb. The significance of this is illustrated by the fact that platelet adhesion to surfaces is essentially normal in Glanzmann's disease, a deficiency of glycoprotein IIb-IIIa in which fibrinogen binding to platelets and platelct aggregation are markedly reduced. In this study the monoclonal antibodies created a form of acquired thrombasthcnia. The platelet receptors for fibrinogen were blocked, but the platelet receptors for von Willebrand's factor were left intact. This may account for the puzzling finding that, despite
Dr. Edwin W. Salzman (Boston, Mass.). This provocative article raises several interesting issues, of which the most far-reaching is the use of a monoclonal antibody against fibrinogen as an antithrombotic drug. Fibrinogen is a necessary cofactor for platelet aggregation, in which it is thought to serve as a bridge between contiguous platelets. I f aggregation is induced by so-called weak agonists like A D P or epinephrine, the fibrinogen must be furnished exogenously, but with strong agonists such as thrombin or collagen the platelet can secrete sufficient fibrinogen from internal storage granules. Platelets have specific membrane receptors for fibrinogen, glycoproteins IIb-IIIa, whose affinity for the protein increases when the platelets are activated. Fibrinogen is also involved in adhesion o f platelets to
180
Torero, Schneider, and Hanson
the long bleeding time and impairment of fibrinogen binding and platelet aggregation in the baboons that received the monoclonal antibodies, there was no reduction in labeled platelets accumulated in the grafts and there was no improvemcnt in graft patency. It is possible that the number of platelets detected in the grafts after 3 hours could have been retained there by adhesion to the graft surface alone without aggregation. I calculate that a smooth cylinder of the same dimensions as the graft could accommodate 7 x 10 r platelets in a monolayer, mad the fibrillar nature of the graft could easily account for the difference without invoking platelet aggregation. This question could be readily answered by microscopy; therefore my first question to Dr. Torero is: has your group performed microscopy under these circumstances? We do not know when the grafts occluded--it was sometime between 3 hours and 8 days--but the effects of the monoclonal antibody were largely gone by 48 hours, at a time when at least 20% of the affected platelets would have been replaced from the bone marrow. Continuing interaction with the .recovering platelet population was more than enough to explain the eventual graft closure,
Journal of VASCULAR SURGERY
which might have been avoidable by a longer period of suppression of platelet function. Perhaps Dr. Torem could tell us whether repeated administration of these antibodies continues to be effective. Exploitation of monoclonal antibodies in therapy might be thwarted by a loss of efficiency of this murine protein after repeated administration. Dr. Torem. Thank you for your comments, Dr. Salzman. Microscopy was not done because no grafts were harvested immediately after the injection of antibody. However, even with the antibody present an average of 5 x 10 9 platelets accumulated, which would consist of about I0% of the total graft luminal volume. We believe that platelet adhesion alone could not account for such deposits, even considering the fibrillar nature of the graft material. We conclude that platelet aggregates did form, perhaps as a consequence of the increased expression of fibrinogen receptors after platelet activation at sites of forming thrombus. Referring to your second question, we do intend to perform a second study with serial injections of the antibody to prolong its antithrombotic effects.
La Jolla, Calif. The usefulness of antiplatelet agents in vascular graft recipients remains controversial because of uncertainties regarding drug mechanisms of action and dose-duration effects. In our study, a well-characterized murine monoclonal antibody (LJ-CP8, 10 mg/kg) was infused into baboons to assess the hemostatic consequences and antithrombotlc effectiveness of blocking the platelet glycoprotein IIb-IIIa receptor for fibrinogen and other adhesive glycoproteins. Five treated animals and six control animals were evaluated with serial measurements of platelet count, bleeding time, and platelet aggregation ex vivo (in response to adenosine diphosphate and collagen). Indium Ill-labeled platelet deposition onto femoral vascaalar grafts (4 mm inner diameter Gore-Tex) implanted immediately after antibody infusion was measured by quantitative gamma camera imaging. Although the antibody did not alter circulating platelet counts, bleeding times were immediately prolonged to more than 30 minutes (vs. 4.8 -+- 0.4 minutes pretreatment) with only partial normalization by 48 hours (8.3 + 1.0 minutes, p < 0.05)0 Platelet aggregation in response to both collagen and adenosine diphosphate was abolished immediately and remained impaired for 48 hours. Despite the profound inhibition of platelet function, graft platelet deposition was equivalent postoperatively in both the treated and untreated groups (p > 0.5), averaging approximately 5 x 109 platelets per graft. Graft-associated indium 111-labeled platelet activity increased over 48 hours and was not reduced by the antibody treatment (p > 0.5 at all times). All grafts were removed at 8 days; only one graft from a treated animal was found patent. We conclude that (1) antibody-induced blockade of platelet glycoprotein IIb-IIIa is not antithrombotic in this setting and that (2) platelet hemostatic competence as assessed by bleeding time or aggregation measurements may not predict the capacity ofplatelets to contribute to graft thrombus formation. (J VAsc SURG 1988;7:172-80.)
The clinical usefulness of small-diameter vascular grafts remains limited because of the high frequency of early occlusion. The reactions of blood platelets contribute directly to graft failure because these blood elements may represent a primary component of forming arterial thrombP and also release factors that are mitogenic for smooth muscle cells? Subse-
From the Departments of Vascular and Thoracic Surgery (Drs. Torem and Schneider) mad Basic and Clinical Research (Dr. Hanson), Scripps Clinic and Research Foundation. Presented at the Thirty-fifth ScientificMeeting of the North American Chapter, The International Societyfor CardiovascularSurgery, Toronto, Ontario, Canada, June 8-9, 1987. Supported in part by research grants HL31469 and HL31950 from the National Institutes of Health, U.S. Public Health Service. Reprint requests: Dr. Stephen R. Hanson, Department of Basic and Clinical Research (BCR5), Scripps Clinic and Research Foundation, 10666 North Torrey Pines Rd., La Jolla, CA 92037.
172
quently the development of intimal proliferative lesions may mediate late-outcome events? Because the thrombogenici~ of both vascular grafts and injured arteries may diminish with time, 4-6 it has been suggested that early inhibition of platelet function may be important for maintaining graft patency. 7-I° Unfortunately, the efficacy of antiplatelet agents in graft recipients has not been well established, in part because of uncertainties regarding appropriate drug doses, duration of effects, and mechanisms of action. 8m In the current study, inhibition of platelet function was achieved by means of a well-characterized murine monoclonal antibody (LJ-CP8) against the platelet glycoprotein IIb-IIIa complex. This receptor binds fibrinogen and other adhesive glycoproteins, such as yon Willebrand factor and fibronectin, after stimulation by appropriate physiologic agonists such as thrombin or adenosine diphosphate (ADP). 1~ As
Volume 7 Number 1 lanuan, 1988
Antibody-induced pIatelet inhibition in vivo 173
Fig. 1. Indium ill-labeled platelet scintillation camera image of a femoral PTFE graft and contralateral limb. The image was taken 3 hours postoperatively in a control animal. Platelet 111InactivitT was clearlyvisualized and localized to the graft.
shown by studies in patients with Glanzmann thrombasthenia, who are deficient in membrane glycoproreins IIb and IIIa, and by studies in patients with severe afibrinogenemia, the inability of platelcts to bind fibrinogen normally may result in significant platelet dysfunction that is characterized by prolonged bleeding and abnormal aggregation.l~.12 The crucial role of the platelet glycoprotein IIb-IIIa receptor has been further documented by studies describing monoclonal antibodies that interact specifically with platelet glycoproteins IIb and/or IIIa, inhibit the binding of fibrinogen and other adhesive glycoproteins, and produce a thrombasthenic-like state in normal platelets in vitro. 13 On the basis of these observations, it has been suggested that such antibodies may be useful as antithrombotic therapeutic agents) 4 Therefore we have used a baboon animal model to assess the effects of injected antiglycoprotcin IIbIIIa antibody with respect to the following variables: platelet count, bleeding time, platelet aggregation, platelet deposition onto expanded polytetrafluoroethylene (PTFE) (Teflon) femoral artery grafts, and graft patency. A baboon model was chosen because this species is hemostatically similar to humans ~Sand has been shown to be useful for studying mechanisms of arterial injury repair after graft implantation. <~6
Therefore the purpose of this study was twofold: to asscss the antihemostatic and antithrombotic consequences of blocking the glycoprotein IIb-IIIa pathway for platclet activation and to detcrmine whether alterations in platelct hemostatic competence would predict graft outcome. MATERIAL A N D M E T H O D S
Animals studied. These studies used 11 normal male baboons (Papio anubis)weighing 12 to 17 kg and observed to be free of disease for at least 6 weeks before use. Six animals served as controls and five were given intravenous antibody. With standard surgical techniques and the use of 1% halothane for general anesthesia, expanded PTFE vascular grafts were inserted into the right superficial femoral artery (Gore-Tex, W. L. Gore & Associates, Inc., Flagstaff, Ariz.). All grafts had a fibril length of 30 Ixm and 4.0 mm internal diameter. The artery was dissected and the animals were given 100 U/kg of heparin. In the treated group, antibody was also injected at this time (discussed later). The artery was then clamped, divided, and replaced with a 4.0 cm segment of PTFE graft. End-to-end anastomoses were constructed with continuous 6-0 polypropylene sutures (Ethicon, Inc., Somerville, N.J.). Suture line bleeding was minimal in control animals. Surgical hemostasis was
)rournaI of VASCULAR SURGERY
174 Torero, Schneider, and Hanson
400
A
X
:3.
/3.
300
2OO
e-
¢..3
100
] LJ-CP8 (10 mg/kg)
o Treated (5) • Controls (5)
oo IL I
I
I
0 3
24
.._.1
48
Time Post.Grafting (hours) Fig. 2. Measurements of circulating platelet count in untreated and antibody-treated animals over 48 hours after graft placement.
achieved after approximately 5 to 10 minutes in the antibody-treated group. The surgical site was closed in two layers, and the animals were allowed to recover for 3 hours before graft imaging. All procedures were performed in accordance with the "Principles of Laboratory Care" and the "Guide for the Care and Use of Laboratory Animals" (NIH Publication No. 80-23, revised 1978). Antibody preparation and characterization. LJ-CP8 was prepared with washed human platelets used as immunogen and was characterized by methods described previously. 17'~8 LJ-CP8 reacted with glycoproteins IIb and/or IIIa when the two were in complex, as shown by (1) its lack of reactivity with platelets from patients with Glanzmann thrombasthenia; (2) solid-phase immunoisolation studies demonstrating that the antibody bound to the complex of two platelet membrane proteins having the electrophoretic mobility of glycoproteins IIb and IIIb; and (3) lack of reactivity with normal platelets incubated with 5 mmol/L EDTA for 15 minutes at 37 ° C. 18LJ-CP8 was of the IgG~ subclass and purifed IgG was prepared from ascitic fluid as previously described} r LJ-CP8 labeled with iodine 125 bound to normal human platelets; an average of 47,000 molecules bound per cell with a dissociation constant (Ka) of 9.4 x 10 -8 mol/L} 8 The binding of LJ-CP8 to baboon platelets was not evaluated directly. However, LJ-CP8 completely inhibited the binding of baboon
fibrinogen, purified as described previously,19 to thrombin-stimulated baboon platelets. Similar results were obtained with human fibrinogen and platclets. The methods used to measure the binding of fibrinogen to platelets have been previously reported in detail} 7 When added at saturation concentrations, LJ-CP8 completely inhibited aggregation of human or baboon platelet-rich plasma (PRP) induced by ADP, collagen, or ~/-thrombin. In the present study, LI-CP8 (10 mg/kg) was injected intravenously into five baboons after the femoral artery was exposed, approximately 5 minutes before the vessel was clamped for grafting. Measurements of platelet function. Platelet counts were performed on whole blood collected in 2 mg/ml disodium EDTA with a whole blood analyzer (model 810, I. T. Baker, Allentown, Pa.). Before graft placement platelet counts averaged 282,000 _+ 90,000/~xl (+_ 1 SD), hematocrit values averaged 38% -_ 2% and were equivalent in both the treated and untreated groups. Bleeding time measurements were performed on the shaved volar surface of the forearm with the standard template method as previously described for studies in baboons. 2° Bleeding time measurements were performed in duplicate and were averaged. Platelet aggregation was measured with an aggregometer (Chrono-Log Corp., Havertown, Pa.) by recording the increase in light transmission through a stirred suspension of PRP maintained at 37 ° C. PRPs and
Volume 7 Number 1 January 1988
platelet-poor plasmas were prepared as previously described.2° In all cases, the platdets in PRP were adjusted to a count of 200,000 plate/ets/t,l. Aggregation was induced by the addition of 1 to 100 ~mol/L ADP (Sigma Chemical Co., St. Louis, Mo.) and 1 to 100 Ixg/ml collagen (Hormon, Munich, F.R.G.). The response to each agonist was compared with preoperative control studies. Since a doseresponse curve (agonist concentration vs. increase in light transmission) was generated at all sampling times after antibody administration, results were expressed as the EDs0, or effective dose of agonist required to induce half the maximal increase in light transmittance.i° Imaging studies. Autologous blood platelets were labeled with 1 mCi indium 111-oxine according to a method described previously. ~ Labeling efficiencies averaged 88%. The labeled platelets were injected approximately 2 hours before the surgical procedures. Graft imaging was performed at S, 24, and 48 hours postoperatively with scintillation camera (Picker Internationl, Inc., Northford, Conn. ~ interfaced with an image processing system (Medical Data Systems A a, Ann Arbor, Mich. ). The 172 keV rain peak was acquired with a high-sensitivity collhnator on the camera with a 20% energy window. At all time points, 5-minute static images were taken of the graft and contralateral limb and of a freshly drawn 5.0 ml whole blood standard. A typical image is shown in Fig. 1. After the graft procedures variable hematoma formation was noted at the surgical sites in the animals given intravenous antibodv. Therefore before imaging was done 3 hours postoperatively, the surgical sites in the treated animals were reopened and approximately 2 to 4 ml of blood was evacuated. Thereafter no further hematoma formation was noted, and the sites were closed and imaged. The surgical sites in control animals were also opened with no evidence of blood leakage. All grafts were patent at this time as demonstrated by direct Doppler scanning of the distal artery with a pencil probe. All sites subsequently remained dosed until the grafts were removed 8 days later. The rain emissions from regions of interest of identical size (approximately 4 x 8 cm) were measured for the grafted area and at a comparable location on the contralatcral control limb. The control region activity was subtracted from the grafted region activity to yield graft-deposited H~In activity only, that is, to subtract the circulating blood activity. To correct for differences in the amount of injected isotope, isotope decay, and dilution, the data were
Antibody-induced platelet inhibition in vivo 175
>30
25
~ ~J-CP8 (5)
A
.,,-,
.=_=
._~
20
15
== . m
CO
.~ Controls (6) 0
= = 0 3
= 24
= 48
Time Post-Grafting (hours)
Fig. 3. Measurements of bleeding time in untreated and antibody-treated animals over 48 hours after graft placement. Antibody was iniected during the surgicalprocedure but before vessel reconstruction.
expressed as the graft-blood ratio of emissions, calculated by dividing the graft activity by the platclet activity contained in 1 ml of whole blood. I5 At the earliest imaging time (3 hours), it was also possible to calculate total graft platelet accumulation (labeled plus unlabelcd cells) by multiplying the graft-blood ratio by the circulating platelet count (platclets per milliliter). ~5 Statistical methods. Statistical comparisons were performed with the Student t test (two-tailed) for paired and unpaired data. Unless otherwise specified, values are given as the mean + 1 standard error of the mean. RESULTS Effect o f injected antibody on platelet count. It was initially important to determine whether the injection of monoclonal LJ-CP8 IgG caused an increase in platelet removal directly because changing platelet counts in treated vs. untreated animals could influence graft thrombus tbrmation and patency. Initial platelet counts in the treated and untreated animals averaged 290,000 + 24,000/~1 and 287,000 _+ 43,000/~,1, respectively. As shown in Fig. 2, no significant alterations in platelet count were observed in either treated or untreated animals during a 48-hour interval after the surgical procedure. Effect on bleeding time. Measurements of the
176
journal of" VASCULAR SURGERY
Torero, Schneider, and Hanson
COLLAGEN (19.2 k~glml)
~,OP (38.5 ~M)
~PRE
~
PRE 48Hrs. 24Hrs.
3Hrs. 1 Min.
l 1 Min.
Fig. 4. Effect of injected antibody on platelet aggregation in response to collagen and ADP. Platelet aggregation (increase in light transmission) in response to both agonists was initially absent (3 hours after injection) and partially normalized over 48 hours. Table I. Effect of antiglycoprotein IIb-IIIa antibody on platelet aggregation Effective dosefor half maximal aggregation (EDso) Study Control Baseline 3 hr LJ-CP8 (10 mg/kg) Baseline 3 hr 24 hr 48 hr
ADP (~mol/L)
Collagen Oxg/ ml)
2.8 -± 0.3 3.1 ± 0.7
2.4 ± 0.4 2.6 ± 0.8
3.7 ± 0.2 >100 12.8 ± 2.7 7.3 ± 2.1
4.9 + 1.2 >100 i9.4 _+ 1.3 9.2 ± 1.8
NOTE: All values are m e a n ± 1 standard error o f measurements made in five animals (treated) or six animals (untreated). Baseline measurements were made preoperatively, beforc antibody adminstration.
standard template bleeding time are given in Fig. 3. Bleeding times in control animals were equivalent before and after operation (4.8 _+ 0.2 minutes vs. 4.9 + 0.2 minutes). In the five animals given LJ-CP8, bleeding times were prolonged immediately to more than 30 minutes. Although the bleeding times progressively shortened on subsequent days, values were still abnormal at 24 hours (13.2 _ 1.1
minutes, p < 0.01) and 48 hours (8.3 _+ 1.0 minutes, p < 0.05). Effect on platelet aggregation. Typical aggregation tracings are shown in Fig. 4. Measurements of the agonist concentration (ADP and collagen) required to induce a half-maximal aggregation response (EDs0) are given in Table I. Control animals demonstrated a normal aggregation response before and after operation. Three hours after administration of LJ-CPS, platelets were totally unresponsive to ADP (up to i00 Ixmol/L) and collagen (up to 100 jxg/ml). Agonist EDs0 values at 24 hours were significantly greater than baseline values for both ADP (12.8 +_ 2.7 txmol/L, p < 0.05) and collagen (19.4 _+ 1.3 gg/ml, p < 0.001) but tended to normalize by 48 h o u r s after antibody administration (ADP EDs0:7.3 -+ 2.1 ~xM, p > 0.1; collagen EDs0: 9.2 - 1.8 ~g/ml, p > 0.05). Effect on graft platelet deposition. Serial measurements by scintillation camera imaging of the ratio of graft nlIn platelet radioactivity to circulating platelet radioactivity are shown in Fig. 5. For determinations made at 3, 24, and 48 hours postoperatively, no differences were seen between the treated and untreated groups (p > 0.5 at all times). Total platelet accumulation (labeled plus unlabeled cells) was also calculated from images taken 3 hours postopera-
Volume 7 Number 1 January 1988
Antibody-induced platelet inhibition in vivo 177
4° r E
Controls (5)
I,==, v
=a
30
E o
LJ-CP8 (5)
20
O
E 10
0
~ 3
, . 24
.
.
.
m 48
Time Post-Grafting (hours) Fig. 5. Ratio of graft-deposited Hlln_labeled platelet activity to circulating H'In-labeled platelet activity (per milliliter whole blood) over 48 hours after graft placement. No differences were observed between the untreated and treated groups.
tively. Is Platelet deposition onto grafts placed in the treated animals (5.1 + 1.0 x 109 platelets per graft) was equivalent to values obtained in the control group (4.8 + 0.8 × 109 platelets per graft, p > 0.5). The extent of acute platelet deposition onto the grafts was also influenced by the circulating platelet count (Fig. 6). For all animals studied, a good correlation (r = 0.69) was obtained between platelet deposition and platelet count, indicating that platelet count alone was an important factor regulating acute thrombus formation. There were no apparent differences between treated and untreated animals in this regard. Effect on graft patency. All grafts were removed after 8 days. None of the grafts in untreated animals was patent. Only one graft from a treated animal was found to be patent. No technical problems were noted with any of the graft anastomoses. DISCUSSION This article documents that the infusion into baboons of a monoclonal antibody against the platelct glycoprotcin IIb-IIIa complex may markedly impair platelet hcmostatic function as determined by measurements of bleeding time and platelet aggregation ex vivo. In particular, the prolonged bleeding times
10
¢n
""
8
×
6 m
.e//,~ A .~
r=0.69
4
o
2 I---
0
I
I
I
i
I
100
200
300
400
500
Circulating Platelet Count (per FI × 10 3)
Fig. 6. Effect of preoperative circulating platelet count on graft platelet deposition (labeled plus unlabeled cells) at 3 hours after grafting. The effect ofplatelet count appeared similar for both the treated and untreated groups.
observed in the present study are consistent with the bleeding tendency observed in patients with thrombasthcnia who lack platelct glycoprotcin IIb-IIIa, and whose platelets are unable to normally bind fibrin-
Jottmalof VASCULAR SURGERY
178 Torero, Schneider, and Hanson
ogen and other adhesive glycoproteins, such as von Willebrand factor and fibronectin, n Because all physiologic agonists are thought to mediate platelet aggregation by expression of this receptor, it is generally believed that platelet glycoprotein IIb-IIIa complex is of central importance for platelet thrombus formation in vivo. H However, we observed no significant benefit of the injected monoclonal antibody with respect to acute graft thrombus formation or graft patency. Several issues are relevant to these observations. First, the antibody was injected before vessel clamping and circulated for approximately one half hour before flow was restored through the grafted segment. Because the effects of this antibody are maximal within minutes after infusion (unpublished observations), the predosing interval was clearly adequate to ensure equilibrium binding to circulating platelets. Second, the dose of injected antibody (10 mg/kg) was chosen so that the initial plasma concentration in vivo would be about twice the concentration (> 100 lag/ml) required to saturate glycoprotein IIb-IIIa and abolish fibrinogen binding in vitro. The efficacy of this dose was also documented postoperatively, at which time bleeding times were essentially infinite, and platelet aggregation in response to ADP and collagen was absent. Third, the observation that all control grafts had failed by 8 days might suggest that technical or other factors unrelated to platelet accumulation may have contributed to early graft occlusion. However, graft platelet deposition was equivalent in treated and untreated animals at 3 hours postoperatively, when the effect of the antibody was maximal and all grafts were patent as documented by Doppler scanning. In addition, the n~In labeled-platelet imaging measurements demonstrated a thirty- to fort)gold enrichment in platelet numbers relative to graft luminal blood volume, whereas static or coagulated blood in the grafts would have reflected only normal blood pool radioactivity without an apparent accumulation of platelets. Finally, all graft anastomoses were examined at the time of removal and found to be technically acceptable. Thus we conclude that superficial femoral artery grafts in the baboon are intrinsically thrombogenic and consequently fail because of mechanisms involving the active accumulation of blood platelets. The most unexpected finding of this study was the observation that intravenous antibody did not modify graft platelet deposition despite the profound inhibition of platelet function shown by measurements of bleeding time and platelet aggregation ex vivo. In addition, previous studies with mono-
clonal antibodies against glycoprotein iIb-][IIa have shown that platelet deposition may be reduced, but not eliminated, on subendothehum perfused in vitro 21'= and on Dacron vascular grafts exposed in an arteriovenous shunt systemY These results may be reconciled in part by recent observations showing tha~ platelets exposed to strong agonists such as thrombin exhibit markedly increased numbers of surface-oriented binding sites associated with platelet glycoprotein IIb-IIIa) 8 With unstimulated platelets, these sites would be inaccessible to extracellular antibodies. On activation, platelets also release intracellular fibrinogen and von Willebrand factor, which may be bound to the glycoprotein IIb-IIIa receptor and subsequently support platelet aggregation. 24,25 Thus in stirred platelet suspensions, agonist-induced receptors may be readily blocked by antibodies, thereby preventing aggregation. Under the more solid-phase conditions of graft thrombus formation, platelet aggregation may have been favored by the close proximity of activated cells and local high concentrations of adhesive glycoproteins and physiologic agonists. In effect, the forming platelet thrombi may have been protected from the antithrombotic actions of the plasma antiglycoprotein IIb-IIIa antibody. Conversely, bleeding times may have been prolonged by the antibody as a consequence of particular modulating effects of vessel injury at the capillary level and microenvironmental factors influencing the intensity of platelet stimulation and local concentrations of relevant blood elements. Thus we conclude that the antithrombotic effectiveness of antibodies against platelet glycoprotein IIb-IIIa may not be predicted by tests of platelet hemostatic competence. However, since the results of the present report do not preclude the usefulness of this approach in other settings, further studies are warranted. The LJ-CP8 was given to us by Dr. Z. M. Ruggeri of Scripps Clinic and Research Foundation. We thank Jill Janik, Paul McFadden, Brandon Domitrowsky, and Stanley Robinson for their technical assistance and Drs. E. F. Bernstein, L. A. Harker, and Z. M. Ruggeri for helpful discussions. REFERENCES
1. StrattonJR,Thide BL, KitchieJL.Naturalhistoryofplatdet depositionon Dacronaorticbifurcationgraftsin the firstyear following implantation in man. Am J Cardiol 1983;52: 371-4. 2. DeuelTF, Huang JS. Platelet-derivedgrowth factor: structure, function, and roles in normal and transformedceils. J Clin Invest 1984;74:669-76. 3. ClowesAW. Current theories of arterial graft failure.Vase Diagn Ther 1982;3:41-52.
Volume 7 Number 1 January 1988
Antibody-induced platelet inhibition in vivo 179
4. McCollum CN, Kester RC, Rajah SM, Learoyd P, Pepper M. Arterial graft maturation: the duration of thrombotic activity in Dacron aortobifemoral grafts measured by platelet and fibrinogen kinetics. Br J Surg 1981;68:61-4. 5. Groves HM, Kinlough-Rathbone RL, Richardson M, Moore S, Mustard JF. Platelet interaction with damaged rabbit aorta. Lab Invest 1979;40:194-200. 6. Clowes AW, Kirkman TR, Clowes MM. Mechanisms of artcrial graft failure. II. Chronic endothelial and smooth muscle cell proliferation in healing polytetrafluoroethylene prostheses. J VAse SURG 1986;3:877-84. 7. Weiss HJ. Platdets: pathophysiology and antiplatelet drug therapy. New York: Alan R. Liss, 1982:95-9. 8. Kohler TR, Kaufinan JL, Kacoyanis G, et al. Effect of aspirin and dipyridamole on the patency of lower extremity bypass grafts. Surgery 1984;96:463-6. 9. Stratton JR, Ritchie JL. Reduction of indium-ill platelet deposition on Dacron vascular grafts in humans by aspirin plus dipyridamole. Circulation 1986;73:328-30. 10. Harker LA. Antiplatelet drugs in the management of patients with thrombotic disorders. Semin Thromb Hemost 1986; 12:134-55. 11. Coller BS, Scudder LE, Peerschke EI, Kalomiris EL, Steinberg M. Platelet physiology and platelet membrane glycoproteins. In: Jolles G, Legrand YJ, Nurden A, eds. Biology and pathology ofplatelet-vessel wall interactions. New York: Academic Press, 1986:181-200. 12. DeMarco L, Girolami A, Zimmerman TS, Ruggeri ZM. yon Willebrand factor interaction with the glycoprotein IIb/IIIa complex. Its role in platelet function as demonstrated in patients with congenital afibrinogenemia. J Clin Invest 1986; 77:1272-7. 13. Coller BS, Peerschke EI, Scudder LE, Sullivan CA. A routine monoclonal antibody that completely blocks the binding of fibrinogen to platelets produces a thrombasthenic-like state in normal ptatelets and binds to glycoproteins IIb and/or Ilia. J Clin Invest 1983;72:325-38. 14. Coller BS, Scudder LE. Inhibition of dog platelet function by in vivo infusion of F(ab')2 fragments of a monoclonal antibody to the platelet glycoprotein IIb/IIIa receptor. Blood 1985;66:1456-9.
15. Hanson SR, Kotze HF, Savage B, Harker LA. Platdet interactions with Dacron vascular grafts: a model of acute thrombosis in baboons. Arteriosclerosis 1985;5:595-603. 16. Clowes AW, Gown AM, Hanson SR, Reidy MA. Mechanisms of arterial graft failure. 1. Role of cellular proliferation in early healing of PTFE prostheses. Am J Pathol 1985; 118:43-54. 17. Lombardo VT, Hodson E, Roberts JR, Kunicki TJ, Zimmerman TS, Ruggeri ZM. Independent modulation of yon Willebrand factor and fibrinogen binding to the platelet membrane glycoprotein IIb/IIIa complex as demonstrated by monoclonal antibody. J Clin Invest 1985;76:1950-8. 18. Niiya K, Hodson E, Bader R, et al. Increased surface expression of the membrane glycoprotein IIb/IIIa complex induced by platelet activation. Relationship to the binding of fibrinogen and platelet aggregation. Blood 1987;70:475-83. 19. Weathersby PK, Horbett TA, Hoffman AS. Solution stability of bovine fibrinogen. Thromb Res 1977;10:245-52. 20. Malpass TW, Hanson SR, Savage B, Hessel EA II, Harker LA. Prevention of acquired transient defect in platelet plug formation by infused prostacyclin. Blood 1981;57:736-40. 21. Sakariassen KS, Nievelstein PFEM, Coller BS, Sixma JJ. The role of platelet membrane glycoproteins Ib and IIb-IIIa in platelet adherence to human artery subendorhelium. Br J Haematol 1986;63:681-91. 22. Weiss HJ, Turitto VT, Baumgartuer HR. Platelet adhesion and thrombus formation on subendothdium in platelets deficient in glycoproteins IIb-IIIa, Ib, and storage granules. Blood 1986;67:322-30. 23. Hanson SR, Pareti FI, Ruggeri Z, et aL Antibody-induced platelet inhibition reduces thrombus formation in vivo. Clin Res 1986;34:658A. 24. Courtois G, Ryekewaert J-J, Woods VL, Ginsberg MH, Plow Et;, Marguerie GA. Expression of intracellular fibrinogen on the surface of stimulated platelets. Eur J Biochem 1986; 159:61-7. 25. Parker RI, Gralnick HR. Identification of platelet glycoprotein IIb/IIIa as the major binding site for released ptareletyon Willebrand factor. Blood 1986;68:732-6.
DISCUSSION
surfaces, but here its role is shared with at least one other protein, yon Willebrand's factor, which is especially important in adhesion ofplatelets to subendothelial structures in the vessel wall and probably to artificial surfaces as well. yon Willebrand's factor will interact with glycoprotein IIb-IIIa, but its principal receptor is another glycoprotein, GPIb. The significance of this is illustrated by the fact that platelet adhesion to surfaces is essentially normal in Glanzmann's disease, a deficiency of glycoprotein IIb-IIIa in which fibrinogen binding to platelets and platelct aggregation are markedly reduced. In this study the monoclonal antibodies created a form of acquired thrombasthcnia. The platelet receptors for fibrinogen were blocked, but the platelet receptors for von Willebrand's factor were left intact. This may account for the puzzling finding that, despite
Dr. Edwin W. Salzman (Boston, Mass.). This provocative article raises several interesting issues, of which the most far-reaching is the use of a monoclonal antibody against fibrinogen as an antithrombotic drug. Fibrinogen is a necessary cofactor for platelet aggregation, in which it is thought to serve as a bridge between contiguous platelets. I f aggregation is induced by so-called weak agonists like A D P or epinephrine, the fibrinogen must be furnished exogenously, but with strong agonists such as thrombin or collagen the platelet can secrete sufficient fibrinogen from internal storage granules. Platelets have specific membrane receptors for fibrinogen, glycoproteins IIb-IIIa, whose affinity for the protein increases when the platelets are activated. Fibrinogen is also involved in adhesion o f platelets to
180
Torero, Schneider, and Hanson
the long bleeding time and impairment of fibrinogen binding and platelet aggregation in the baboons that received the monoclonal antibodies, there was no reduction in labeled platelets accumulated in the grafts and there was no improvemcnt in graft patency. It is possible that the number of platelets detected in the grafts after 3 hours could have been retained there by adhesion to the graft surface alone without aggregation. I calculate that a smooth cylinder of the same dimensions as the graft could accommodate 7 x 10 r platelets in a monolayer, mad the fibrillar nature of the graft could easily account for the difference without invoking platelet aggregation. This question could be readily answered by microscopy; therefore my first question to Dr. Torero is: has your group performed microscopy under these circumstances? We do not know when the grafts occluded--it was sometime between 3 hours and 8 days--but the effects of the monoclonal antibody were largely gone by 48 hours, at a time when at least 20% of the affected platelets would have been replaced from the bone marrow. Continuing interaction with the .recovering platelet population was more than enough to explain the eventual graft closure,
Journal of VASCULAR SURGERY
which might have been avoidable by a longer period of suppression of platelet function. Perhaps Dr. Torem could tell us whether repeated administration of these antibodies continues to be effective. Exploitation of monoclonal antibodies in therapy might be thwarted by a loss of efficiency of this murine protein after repeated administration. Dr. Torem. Thank you for your comments, Dr. Salzman. Microscopy was not done because no grafts were harvested immediately after the injection of antibody. However, even with the antibody present an average of 5 x 10 9 platelets accumulated, which would consist of about I0% of the total graft luminal volume. We believe that platelet adhesion alone could not account for such deposits, even considering the fibrillar nature of the graft material. We conclude that platelet aggregates did form, perhaps as a consequence of the increased expression of fibrinogen receptors after platelet activation at sites of forming thrombus. Referring to your second question, we do intend to perform a second study with serial injections of the antibody to prolong its antithrombotic effects.