H cause of morbidity after cardiac operations [l, 21. A

Reduction of Heparin Binding to and Inhibition of Platelets by Aprotinin Lindsay C. H. John, FRCS, Gareth M. Rees, FRCS, and Iren B. Kovacs, FRCPath Department of Cardiothoracic Surgery and the Thrombosis Unit, St. Bartholomew s Hospital, London, England The direct effect of aprotinin on in vitro platelet function was assessed by hemostatometry (n = 10). No significant enhancement was demonstrated. However, aprotinin reduced platelet inhibition secondary to heparin. Hemostatometry demonstrated a significant preservation of in vitro platelet function (n = 25) (p = 0.04), which was particularly marked (p = 0.003) in the subgroup (n = 7) demonstrating a severe inhibition of platelet function with heparin. Aprotinin significantly reduced the bind- ing of tritium-labeled heparin to both nonactivated (n = 25) (p = 0.004) and activated platelets (n = 25) (p < 0.0001). We conclude that interference with heparininduced inhibition of platelet function by aprotinin may be one of its hemostatic actions in cardiac surgery. This effect is probably secondary to aprotinin reducing binding of heparin to platelets. (Ann Thorac Surg 1993;55:1175-9) emorrhage after cardiopulmonary bypass is a major H cause of morbidity after cardiac operations [l, 21. A chief factor in this appears to be an acquired platelet dysfunction [l, 3,4]. One cause recently identified for this dysfunction is the use of heparin [5], which is an essential prerequisite for cardiopulmonary bypass. Aprotinin has been clearly demonstrated to reduce hemorrhage after bypass [6-81. A number of actions of aprotinin have been suggested to explain its hemostatic action, including preservation of platelet function during bypass [7]. Theoretically aprotinin may have such a beneficial effect either by a direct action enhancing platelet function or by an indirect action protecting the platelets from an inhibitory consequence of cardiopulmonary bypass. To date, however, in vitro tests of platelet function and coagulation have failed to show any hemostatic action with aprotinin. On the contrary, platelet aggregation was inhibited by esterase inhibitors including aprotinin [9, 101. This suggests that aprotinin does not directly enhance platelet function but instead probably has an indirect protective action. The aim of this study was to examine one such possible protective action, a prevention of heparin-mediated platelet inhibition. The method used to assess platelet function was hemostatometry, a technique that has the advantage of not requiring the use of anticoagulants and that has been used to demonstrate a possible association between inhibition of platelet function by heparin and postbypass hemorrhage [5]. Heparin has been demonstrated to bind directly to platelets [ll]. One way by which aprotinin may interfere with an inhibitory effect of heparin is by reducing this binding. This possibility was also explored in this study Accepted for publication Aug 21, 1992 Address reprint requests to Dr John, Department of Cardiothoracic Surgery, St. Bartholomew s Hospital, West Smithficld, London EClA 7BE, England. by examining the binding of tritium-labeled heparin to activated and nonactivated platelets with and without the presence of aprotinin. Material and Methods The following products were used in this study: Heparin (Monoparin-heparin sodium mucous [porcine]; CP Pharmaceuticals Ltd, Wrexham, UK) Aprotinin (Trasylol; Bayer, Leverkusen, Germany) Tritium-labeled heparin sodium salt {3H(G)}(NEN Products, Du Pont, Stevenage, UK) Adenosine diphosphate (Sigma, Watford, UK) Hemostatometry The hemostatometer is a recently developed method for assessing platelet function that has previously been described in detail [12, 131. Two and a half milliliters of non-anticoagulated blood is perfused through polyethylene tubing by paraffin oil displacement, a technique that has been shown to cause no measurable platelet damage. A hole is punched through the tubing, resulting in bleeding and a pressure drop. The pattern of pressure recovery shows the formation of hemostatic plugs in the holes. The initial recovery of the pressure represents platelet plug formation due to activation of platelets by shear stress and release of adenosine diphosphate from the shear-damaged platelets and red blood cells, and the later recovery is due to the stabilization of the platelet plug secondary to thrombin and fibrin formation such that it can resist the increasing pressure. The hemostasis curve is recorded and its area analyzed as H (mm Hg * s). The greater the value of H, the greater the relative inhibition of platelet function. As the blood is not anticoagulated, hemostasis is followed by coagulation manifested as a rapid pressure drop. The dynamic coagulation time is the time from the start of the measurement to the final pressure drop to zero. 0 1993 by The Society of Thoracic Surgeons 0003-4975/93/$6.00

1176 JOHN ET AL AM Thorac Surg 1993;55:1175-9 Platelet function was assessed by hemostatometry of the following blood samples from 35 cardiac surgical patients: Study 1 (n = 10): native blood alone; native blood with aprotinin (4 pmovl; and native blood with saline solution (equivalent volume to aprotinin). This study examined the direct in vitro effect of aprotinin on platelet function. Study 2 (n = 25): native blood alone; native blood with heparin (5 U/mL) and with aprotinin (4 pmovl); and native blood with heparin (5 U/mL) and with saline solution. This study examined the in vitro effect of aprotinin on the inhibition of platelet function by heparin. Radiolabeled Heparin Binding to Activated and Nonactivated Platelets Ten milliliters of citrated venous blood was withdrawn from each patient (1 ml of 3.8% trisodium citrate solution). To 5 ml of the citrated blood, aprotinin (4 pmol/l) was added, and to the other 5 ml, an equivalent volume of saline solution. Both samples were placed in a slowspeed rotator (26 rpm) for 10 minutes. Tritium-labeled heparin was added to each sample (2.4 pci), and the samples were again placed in the slow-speed rotator for 10 minutes and then centrifuged at 800 rpm for a further 10 minutes. One and a half milliliters of platelet-rich plasma was withdrawn from each sample. Two hundred microliters of the platelet-rich plasma was measured for radioactivity (after the addition of a tissue solubilizer [Solvease; BDH Chemicals, Poole, UK] and scintillation fluid [PCS; Amersham International, Amersham, UK]) and 100 pl was used for a platelet count estimation by spectrophotometry. One milliliter of platelet-rich plasma underwent high-speed centrifugation (12,000 rpm) for 5 minutes. Two hundred microliters of plasma was measured for radioactivity, and a relative platelet count was performed on 100 pl. The difference in the radiation count (Beckman LS 6000 IC p scintillation counter) between the platelet-rich plasma and the plasma samples was that due to plateletbound tritium-labeled heparin. To estimate relative platelet counts accurately in relatively small volumes, a spectrophotometer (UV-Visible Spectrophotometer, M330; Cam.Spec. Ltd, Cambridge, UK) was used at a wavelength of 540 nm. The difference in the spectrophotometric measurements between equivalent platelet-rich plasma and plasma specimens was a measure of the relative platelet count for which the radioactivity of the platelet-bound tritium-labeled heparin had been measured. To compare differences in heparin binding between different specimens and patients, the radioactivity counts were corrected to the equivalent platelet count for a spectrophotometric measurement of 1.0 at 540 nm. Binding studies were performed on 50 cardiac surgical patients as follows: Study 3 (n = 25): Measurements were made as described of platelet-bound tritium-labeled heparin in Table 1. Examination of Direct Effect of Aprotinin on Platelet Function In Vitro Variable Saline P (n = 10) Solution Aprotinin Value H (mm Hg * s) 2,298 f 480 3,008 k 609 NS CT (min) 16.9 f 0.9 24.9 f 2.5 0.02 a Data are shown as the mean * the standard error of the mean. CT = coagulation time; H = hemostatometry measurement; NS = not significant. the presence of aprotinin (4 -om) or an equivalent volume of saline solution. Study 4 (n = 25): Measurements were made as for study 3 but with the addition of adenosine diphosphate (30 pl/ml) to activate the platelets before high-speed centrifugation. Studies 3 and 4 examined in vitro the effect of aprotinin on the binding of tritium-labeled heparin to nonactivated and activated platelets, respectively. Study Population The study population was composed of cardiac surgcal patients from whom measurements were obtained either in the week before cardiac operation or within 1 year postoperatively. Eighty-five percent were male, and the mean age was 54.9 years. Of the patients, 71% were taking aspirin; 37%, calcium antagonists; and 8%, dipyridamole. Although they were on a regimen of antiplatelet medication, cardiac surgical patients were used, as this is a population in whom the hemostatic effects of aprotinin have been reported. All patients gave informed consent. Statistical Analysis The hemostatometric variable H was approximated to a normal distribution using log-transformed data. For radioactivity measurements, the raw data were used. Comparisons were made using Student s t test. Data are presented as the mean 2 the standard error of the mean. Results Study 1: Efect of Aprotinin on Platelet Function in Vitro No significant difference in platelet reactivity as measured by hemostatometry was shown between blood containing aprotinin or saline solution. Coagulation time was significantly prolonged in blood containing aprotinin (Table 1). Study 2: Effect of Aprotinin on Platelet Function in the Presence of Heparin A wide individual variation in platelet inhibition has been measured by hemostatometry [5]. A severe inhibition has been defined as R greater than 10, where R = H with heparidh without heparin. The presence of aprotinin significantly (p = 0.04) reduced this inhibition (Fig l), and this reduction was particularly significant (p = 0.003) in

Ann Thorac Surg 1993:jj: 1175-9 JOHN ET AL 1177 the subgroup with severe platelet inhibition by heparin (R > 10, or H >,000) (Fig 2). Figure 3 is an example of two hemostasis curves from identical blood containing heparin (5 U/mL) with aprotinin (4 pmoyl) or an equivalent volume of saline solution. Studies 3 and 4: Efect of Aprotinin on Heparin Binding to Nonactivated and Activated PZateIets There was a significant reduction in tritium-labeled heparin bound to both nonactivated ( p = 0.004) and activated platelets (p < 0.0001) in blood samples with aprotinin (4 pmol/l) compared with an equivalent volume of saline solution (Table 2). There was a significant difference in platelet-bound tritium-labeled heparin between activated and nonactivated platelets. This was substantially less significant (p = 0.04) with added aprotinin (4 pmoyl) than without it ( p = 0.0002). H (mmhg.s)~lo-~ 30 25 20 H (mrn Hg.4 x ~ O - ~ 30 / 10 25 5 20 A 6 10 5 A P =Om04 B (Paired t test) Fig I. Hemostatometric nieasirrements (H) from (A) blood containing heparin with saline solution and (B) blood containing heparin with aprotinin (n = 25). Shaded areas represent the mean t the standard error of the mean. P = 0-003 (Paired t test) Fig 2. Hemostatometric measurements (H) from (A) blood containing heparin with saline solution and (B) blood containing heparin with aprotinin in the subgroup demonstrating seuere platelet inhibition with heparin (n = 7). Shaded areas represent the mean? the standard error of the mean. Comment Aprotinin reduces blood loss after cardiopulmonary bypass. This has been reported for first-time operative procedures, redo procedures, and patients with endocarditis [6-8]. A number of suggestions have been made as to the mechanism for this hemostatic action. Aprotinin undoubtedly has an antifibrinolytic action, and this has been suggested as the relevant mechanism [8]. However, others [6, 14-16] have disputed the importance of hyperfibrinolysis as a cause of bleeding after bypass. Another suggestion has been that aprotinin prevents the activation and consumption of clotting factors by released proteases. Aprotinin has previously been shown [17] to inhibit rather than promote coagulation, a finding confirmed in the present study. This observation is interesting in view of the current controversy over thrombotic

1178 JOHN ET AL Ann Thorac Surg 1993;55:11759 Pressure (mm Hg) I Time (mind I Time (mind Fig 3. Hemostasis curves demonstrating a reduction of heparin-induced platelet inhibition in the presence of aprotinin: (A) blood containing heparin with saline solution and (B) blood containing heprlrin with aprotinin. events in patients receiving aprotinin. However, an anticoagulant effect is unlikely to account for a clinical hemostatic effect. The principal cause of the bleeding diathesis secondary to cardiopulmonary bypass has largely been attributed to a transient and severe defect in platelet function 11, 181. Modification of this platelet dysfunction may be a possible mode of action for the drug. Such a mechanism was supported by the observation of a significantly shorter prolongation of the skin bleeding time during cardiopulmonary bypass in the presence of aprotinin [7]. Prevention of platelet activation as assessed by reduction in thromboxane release has also been reported [19], as has the prevention of the loss of platelet membrane surface glycoprotein GpIb [IS]. Theoretically aprotinin may modify acquired platelet dysfunction either by a direct action enhancing platelet function or by an indirect action preventing inhibition of platelet function by damaging consequences of a cardiac operation. To date, there has been no reported evidence of aprotinin directly enhancing platelet function when tested in vitro. This study using the technique of hemostatometry also failed to demonstrate such a direct action. Therefore, if modification of platelet dysfunction by aprotinin is relevant to its hemostatic action, then it is most likely due to an indirect action protecting platelets from inhibition. Possible indirect actions include protection of platelets from plasmin, which has been observed to remove GpIb from platelets [ZO], and the reduction of factor XI1 and complement activation together with lysosomal enzyme release and the consequent destruction of von Willebrand factor and platelet membrane receptors [21]. However, the observation that a single dose of aprotinin at the commencement of cardiopulmonary bypass may be as effective as a continuous infusion [] is difficult to reconcile with the view that aprotinin acts as a hemostatic agent by preventing protease-mediated platelet damage or the consequences of complement activation, as protease release and complement activation occur throughout bypass. This suggests the possibility that there are other inhibitors of platelet function that aprotinin may protect against and that have not previously been appreciated. One such possible inhibitor is heparin, which is an essential prerequisite for cardiopulmonary bypass. It has recently been demonstrated using hemostatometry that an inhibition of platelet function by heparin is a probable etiological factor in hemorrhage after a cardiac operation [5]. In the present study, it has been demonstrated (again by hemostatometry) in vitro that aprotinin significantly reduced this inhibition. Therefore we suggest that interference with heparin-induced inhibition of platelet function may be one of the hemostatic actions of aprotinin in cardiac operations. How heparin modifies platelet function is unknown, but one possibility is that it is secondary to a direct binding of heparin to platelets. There is substantial evidence of such a direct binding both to nonactivated and especially to activated platelets [ll, 221. It has also been reported that aprotinin can bind to the platelet membrane [23]. In addition, aprotinin can bind directly to acidic glycoproteins, including heparin [24, 251, possibly as a result of its high basicity. These binding properties suggest that one way by which aprotinin could interfere with the interaction between heparin and platelets is by reducing the binding of the former to the latter. This possibility is strongly supported by the present study, which has clearly demonstrated a significant reduction in the bind- Table 2. Examination of Effect of Aprotinin on Binding of Tritium-Labeled Heparin to Activated and Nonactivated Platelets Corrected Corrected Radioactivity Radioactivity Count, Added Count, Added Variable Saline Solution (CPM) Aprotinin P M ) PI Value Nonactivated 19,378 t 3,766 8,020 2 3,466 0.004 platelets (n = 25) Activated 42,828 2 4,774,953 2 2,536 10.0001 platelets (n = 25) pz Value 0.0002 0.04 a Data are shown as the mean? the standard error of the mean y, value = significance of difference between samples containing saline solution and those with aprotinin (activated and nonactivated); p2 value = significance of difference between activated and nonactivated platelets (with saline solution or aprotinin).

Ann Thorac Surg 1993;55:1175-9 JOHN ET AL 1179 ing of tritium-labeled heparin to both nonactivated and activated platelets. In summary, the findings of this study were as follows: No significant direct enhancement of platelet function by aprotinin alone as assessed in vitro by hemostatometry. Significant reduction in heparin-associated platelet inhibition by aprotinin as assessed in vitro by hemosta tometry. Direct binding of tritium-labeled heparin to platelets, significantly more so to activated than nonactivated platelets. Significant reduction in binding of tritium-labeled heparin both to activated and nonactivated platelets by aprotinin. We conclude that a hemostatic action of aprotinin in cardiac operations may be to reduce the inhibition of platelets by heparin. It is probable that it does so by reducing the direct binding of heparin to platelets, especially to activated platelets, which may be a consequence of cardiopulmonary bypass [4]. A practical implication of these observations is the possibility of identifying those individuals who would most benefit from the use of aprotinin during a cardiac operation, ie, those who preoperatively demonstrate severe in vitro inhibition of platelet function that is due to heparin. This may also be relevant for patients who are heparinized for other than cardiac surgical reasons where hemorrhage as a major complication has long been recognized [26] and in whom the use of aprotinin may be potentially beneficial. This work was partly supported by the Joint Research Board of St. Bartholomew's Hospital. References 1. 2. 3. 4. 5. 6. Harker LA, Malpass TW, Branson HE, Hessel EA, Slichter SJ. Mechanism of abnormal bleeding in patients undergoing cardiopulmonary bypass: acquired transient platelet dysfunction associated with selective alpha-granule release. Blood 1980;56:82&34. Talamonti MS, LoCicero J, Hoyne WP, Sanders JH, Michaelis LL. Early reexploration for excessive postoperative bleeding lowers wound complication rates in open heart surgery. Am Surg 1987;53:1024. Mammen EF, Koets MH, Washington BC, et al. Hemostasis changes during cardiopulmonary bypass. Semin Thromb Hemost 1985;11:281-92. Colman RW. Platelet and neutrophil activation in cardiopulmonary bypass. Ann Thorac Surg 1990;49:32-4. John LCH, Rees GM, Kovacs IB. Inhibition of platelet function by heparin: an etiological factor in post bypass hemorrhage. J Thorac Cardiovasc Surg (in press). Royston D, Bidstrup BP, Taylor KM, Sapsford RN. Effect of aprotinin on need for blood transfusion after repeat openheart surgery. Lancet 1987;2:1289-91. 7. Bidstrup BP, Royston D, Sapsford RN, Taylor KM. Reduction in blood loss and blood use after cardiopulmonary bypass with high dose aprotinin (Trasylol). J Thorac Cardiovasc Surg 1989;97:364-72. 8. Have1 M, Teufelsbauer H, Knob1 P, et al. Effect of intraoperative aprotinin administration on postoperative bleeding in patients undergoing cardiopulmonary bypass operation. J Thorac Cardiovasc Surg 1991;101:968-72. 9. Mc Nicol GP, Prentice CRM, Douglas AS. Trasylol and failure in hemostasis. In: Donatelli R, ed. Proteases and antiproteases in cardioangiology. Stuttgart/New York: FK Schattauer Verlag, 1970:89-97. 10 Aoki N, Naito K, Yoshida N. Inhibition of platelet aggregation by protease inhibitors. Possible involvement of proteases in platelet aggregation. Blood 1978;52:1-12. 11 Horne MK, Chao SC. Heparin binding to resting and activated platelets. Blood 1989;74:238-43. 12. Kovacs IB, Hutton RA, Kernoff PBA. Hemostatic evaluation in bleeding disorders from native blood: clinical experience with the Hemostatometer. Am J Clin Pathol 1989;91:271-9. 13 Ratnatunga CP, Rees GM, Kovacs IB. Preoperative hemostatic activity and excessive bleeding after cardiopulmonary bypass. Ann Thorac Surg 1991;52:250-7. 14 Woodman RC, Harker LA. Bleeding complications associated with cardiopulmonary bypass. Blood 1990;76:1680-97. Van Oeveren W, Harder MR, Roozendaal KJ, Eijsman L, Wildevuur CRH. Aprotinin protects platelets against the initial effects of cardiopulmonary bypass. J Thorac Cardiovasc Surg 1990;99:788-97. 16 Harker LA. Bleeding after cardiopulmonary bypass. N Engl J Med 1986;314:1446-8. 17 De Smet AE, Joen MCN, van Oeveren W, et al. Increased anticoagulation during cardiopulmonary bypass by aprotinin. J Thorac Cardiovasc Surg 1990;100:520-7. 18 Zilla P, Fasol R, Groscurth P, Klepetko W, Reichenspurner H, Wolner E. Blood platelets in cardiopulmonary bypass operations. Recovery occurs after initial stimulation rather than continual activation. J Thorac Cardiovasc Surg 1989;97 379-88. 19 Van Oeveren W, Jansen NJG, Bidstrup BP, et al. Effects of aprotinin on hemostatic mechanisms during cardiopulmonary bypass. Ann Thorac Surg 1987;44:640-5. 20 Adelman B, Michelson AD, Loscalzo J, Greenberg J, Handin RI. Plasmin effect on platelet glycoprotein Ib-von Willebrand factor interactions. Blood 1985;65:3240. 21 Fraedrich G, Weber C, Bernard C, Hettwer A, Schlosser V. Reduction of blood transfusion requirement in open heart surgery by administration of high doses of aprotininpreliminary results. Thorac Cardiovasc Surg 1989;3789-91. 22 Sobel M, Adelman B. Characterization of platelet binding of heparin and other glycosaminoglycans. Thromb Res 1988;50: 8-26. 23 Kiernan JA, Stoddart RW. Fluorescent-labelled aprotinin: a new reagent for the histochemical detection of acid muco substances. Histochemie 1973;34:77-84. 24 Stoddart RW, Kiernan JA. Aprotinin, a carbohydrate-binding protein. Histochemie 1973;34:275-80. 25 Heine H, Forster FJ, Neufahrt A. Zur Wechselwirkung zwischen dem basischen Polvpeptid Trasvlol und Proteoglvkanen. IV. lntrazellulare binding. Med Welt 1976;27177L7. 26. Levine MN, Hirsh J. 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