Platelet protection in coronary artery surgery: Benefits of heparin-coated circuits and high-dose aprotinin therapy

Platelet Protection in Coronary Artery Surgery: Benefits of Heparin-Coated Circuits and High-Dose Aprotinin Therapy Hans Peter Wendel, PhD, Hans-Joachim Schulze, MD, Wolfgang Heller, PhD, and Hans-Martin Hoffmeister, MD Objective: To examine the extent of platelet activation during extracorporeal circulation by using the combination of heparin-coated oxygenation systems and high-dose aprotinin therapy, and to examine the affinity and thereby the protective capacity of aprotinin to the glycoprotein (GP) receptors of the platelet membrane. Design: Experimental in vitro study. Setting: Research laboratory of a university hospital. Participants: Thirty-two volunteers (blood donors). Measurements and Main Results: Thirty-two oxygenation circuits of the same construction series (16 heparin-coated and 16 noncoated) were investigated in a closed system of a heart-lung machine model with fresh human whole blood. In each of these two groups, eight circuits with and eight without a high-dose aprotinin application (250 kallikrein inhibitory units [KIU]/mL) were investigated. In all four groups, the number of platelets declined continuously during the 90-minute recirculation period. Group I (no heparin coating, no aprotinin) showed the greatest reduction; group IV (heparin coating, aprotinin) had a significantly smaller decrease in platelet number (p < 0.01). Platelet

factor 4 (PF-4) levels, released from the s-granule, were in inverse proportion to the platelet loss. After 90 minutes of recirculation, the PF-4 values increased to 615.8% - 559.5% and 237.2% -+ 179.0% of the initial value for groups I and IV, respectively (p < 0.01). Affinity chromatography and immunoblotting techniques were used to evaluate the affinity of aprotinin for the GP receptors of the platelet membrane. The affinity appeared in the following order: GPIIb < GPIIla < GPIb. Conclusion: Heparin-coated oxygenation systems and additional aprotinin caused significantly less platelet damage in an in vitro cardiopulmonary bypass model. Chromatographic and immunologic methods could prove aprotinin's affinity for the platelet receptor proteins GPIb and GPIIb-Illa and therefore its probable role in diminishing the triggering of the platelet activation cascade. Copyright © 1999 by W.B. Saunders Company

INCE ITS FIRST use 40 years ago, 1 extracorporeal circulation (ECC) in cardiac surgery has developed into a routine procedure worldwide. Despite this, the propensity toward bleeding after surgery with ECC remains an unsolved phenomenon, a problem that can be traced to pathophysiologic changes in the hemostatic systems of fibrinolysis and coagulation. 2 The incomplete hemocompatibility of the perfusion systems used during ECC has a significant role in these changes. The thrombogenicity of the artificial devices mainly depends on the extent of their ability to mimic the activation and inhibition of platelet adhesion and aggregation, a process constantly occurring on the surface of the natural endothelium. 3 This equilibrium reaction lies behind plasmatic as well as cellular autoregulation and reverse-feedback mechanisms. The adsorption of plasma proteins such as thrombin, fibrinogen, and yon Willebrand's factor is considered a key mechanism for the further stimulation of platelets on artificial surfaces. 4 In light of this, the minimization of these activation mechanisms induced by the artificial surfaces during ECC assumes first priority. Two fundamental approaches to attaining this aim principally exist: improving the biocompatibility of the device's component materials or using pharmacologic means to inhibit the humoral activation cascades and protect the blood cells. The use of heparin-coated oxygenation devices promises a significantly

reduced activation of the plasma proteins and blood cells? and with a high-dose therapy of the antifibrinolytic proteinase inhibitor aprotinin, limited postoperative bleeding was shown. 6-9 Aprotinin's protective effect on the adhesion glycoproteins (GPIb and IIb-IIIa) of the platelet membrane is still controversialJ 0-13 The present in vitro study examined how the combination of heparin-coated oxygenation systems and high-dose aprotinin therapy affected platelet activation in a simulated ECC. In a second experiment, aprotinin's affinity for the GP receptors of the platelet membrane was investigated by affinity chromatography and immunoblotting techniques.

S

From the Division of Thoracic, Cardiac, and Vascular Surgery, Departments of Surgery and Internal Medicine III, University of Tuebingen, Tuebingen; and the Clinic and Policlinic for Thoracic and Cardiovascular Surgery, University of Wuerzburg, Wuerzburg, Germany. Address reprint requests to Hans Peter Wendel, PhD, Clinical Research Laboratory, Division of Thoracic, Cardiac, and Vascular Surgery, Department of Surgery, Eberhard-Karls-University, Calwerstr 7/1, 72076 Tuebingen, Germany. Copyright © 1999 by W.B. Saunders Company 1053-0770/99/1304-0003510.00/0 388

KEY WORDS: aprotinin, platelets, platelet membrane receptors, biocompatibility, heparin coating, extracorporeal circulation, cardiopulmonary bypass surgery

MATERIALS AND METHODS

The experiments were performed within a well-established in vitro heart-lung machine (HLM) model to evaluate the practicality of new components (artificial devices) or drugs used in ECC. TM This model served to investigate the potential of artificial surfaces for activating various mechanisms within the hemostatic systems, without the body's self-regulatory compensation mechanisms. Thirty-two membrane oxygenators (Minimax; Medtronic, Anaheim, CA) comprised the test series. Sixteen of these oxygenation systems had heparin coating (Carmeda, Oslo, Norway; bioactive surface). In each of these two groups, eight machines with and eight without aprotinin (250 kallikrein inhibitory units (K1U)/mL; Trasylol; Bayer, Leverkusen, Germany) were studied. Hence, the test series consisted of four different groups with eight oxygenators each (group I, no heparin coating, no aprotinin; group II, heparin coating, no aprotinin; group HI, no heparin coating, aprotinin; group IV, heparin coating, aprotinin). Fully recalcifled fresh acid citrate dextrose blood (250 mL) was circulated in this closed system by using a roller pump (Sarns, Inc, Ann Arbor, MI) for 90 minutes. Blood from one volunteer was used for one oxygenator run. A constant blood flow of 1,850 mL/min and a mean arterial pressure of 60 mmHg were maintained, while a hypothermia apparatus (type Q 102; Haake, Berlin, Germany) held the temperature of the arterial oxygenator exit at 28°C. The priming volume, in accord with operation conditions, consisted of 25 mL of glucose solution (5%; Delta Pharma, Pfullingen, Germany), 58 mL of Ringer's lactate (Schiwa, Glandorf,

Journal of Cardiothoracic and Vascular Anesthesia, Vo113, No 4 (August), 1999: pp 388-392

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Fig 1. Plateiet counts (mean _+ SD) during 90-minute recirculation in an HLM model. Group I, no heparin coating, no aprotinin; group II, heparin coating, no aprotinin; group III, no heparin coating, aprotinin; group IV: heparin coating, aprotinin. II, group 1; [], group 2; [], group 3; [], group 4.

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_~latelets(%)

100 90 80 70 60 50 40 30 20 10 0

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Germany), 10 mL of sodium hydrogencarbonate (NaHCO3 8.4%; Braun Melsungen AG, Melsungen, Germany), and 12 mL of calcium (10% Ca; Braun Melsungen AG) for complete recalcification of the acid citrate dextrose blood. The oxygenator and tubing were prerinsed with 350 mL of Ringer's lactate for 30 minutes, and the solution was discarded. A gaseous mix of 77% N2, 20% O2, and 3% CO2 provided the oxygenation. For every trial run, a first blood sample was taken from the blood bag (control) and seven samples from the arterial exit of the oxygenator, the latter after 1, 5, 10, 20, 30, 60, and 90 minutes of recirculation. An exactly calculated amount of heparin (Liquemin; Hoffman-LaRoche, Basel, Switzerland) was added to the blood, thus maintaining a concentration of 3 IU/mL in the entire filling volume. Determination of the platelet count was performed manually under a light microscope with the help of a Neubauer chamber. According to the methods of Pelzer et all5 a sandwich enzyme immunoassay (BehringWerke, Marburg, Germany) was used to determine the amount of human platelet factor 4 (PF-4) in the plasma. In brief, platelet-poor plasma (anticoagulant, 10% [vol] ethylenediaminetetraacetic acidheparin [50 g/L]) was incubated in tubes precoated with a polyclonal antibody (rabbit antihuman PF-4). Incubation was renewed after three washing steps with a conjugated antibody (rabbit antihuman PF-4peroxidase); after the addition of substrate, the absorbance at 492 nm was read, and the concentration calculated from a double-logarithmic calibration curve. Gradient centrifugation (Nycoprep; Nycomed, Oslo, Norway) enabled the isolation of platelets from the blood, which were then mechanically lysed in a dismembrator (LKB, Freiburg, Germany). To test the affinity between the GPs of the platelet membrane and aprotinin, a 2.5-mL aprotinin-agarose column (Sigma, Deisenhofen, Germany) was used. A peristaltic pump was fitted to the front of the colmnn. At the end of the column, a flux photometer set at 280 nm (dual-path monitor, UV-2; Pharmacia, Freiburg, Germany) with attached writer (Servogor 311; Zeiss, Oberkochen, Germany) was used to measure the protein content of the eluates. The eluates were finally collected by a fraction assembler (Frac-100; Pharmacia). The aprotinin-agarose column was equilibrated with 10 column volumes of a 15-mmol/L sodiumphosphate buffer, pH 8.0. The previously attained platelet lysate was next buffer-changed with the equilibration buffer before being moved onto the column. After the lysate had been loaded, it was once again washed with the equilibration buffer to remove any unbound proteins. The elution of the bound proteins occurred by using a NaC1 gradient (0 to 1.5 mol/L of NaC1 in equilibration buffer); the eluates were then evenly distributed in 1-mL fractions at 4°C. The immunologic detection of the fractionated eluates on the basis of the content of receptor proteins from the platelet membrane relied on the dot blot procedure (96-well dot blot apparatus; Bio-Rad, Munich, Germany). A nitrocellulose membrane with 0.45%tm diameter pores (Sigma) was soaked in tris buffered saline (TBS) (20 mmol/L of

Ii111 5'

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tris-HC1, 500 mmol/L of NaC1, pH 7.5) before being built into the dot blot chamber. After its insertion, it was rehydrated with 100 gL of TBS per well (vacuum). Every 100-pL sample was filtered passively for 30 to 40 minutes, and thereafter, 300 gL of blocking reagent (TBS with 0.05% Tween, 1.5% bovine serum albumin, 1% ethanolamine) blocked the nonspecific binding sites for 60 minutes (passive filtration). After three washings with TTBS (TBS + 0.05% [Tween; Boehringer Mannheim, Mannheim, Germany]), the respective primary antibodies (monoclonal antibodies: mouse antihuman CD 42b, CD61, CD41b; all from Dianova, Hamburg, Germany) were incubated for 30 minutes. After another triple washing, 30 more minutes of passive filtration proceeded with AP-conjugated secondary antibody (rabbit antimouse immunoglobulin G-alkaline phosphatase; Sigma). Two washings each with TTBS and TBS followed the completion of this filtration step, and after that, 100 gL of precipitating AP substrate (Sigma) was incubated in the dark for approximately 30 minutes. A vacuum removed the excess substrate, whereas water rinsed the wells and thus stopped the reaction. To distinguish changes in the plasma protein levels caused by dilution from those attributable to consumption, the values of plasma factors, determined from diluted blood, underwent a hematocrit correction. Because of the dispersion of plasma protein concentrations found in the blood donors, the initial value of each individual experiment was defined as 100%, with the individual values after each extraction calculated as percentages thereof. The statistical software SPSS processed the data (SPSS Software, Inc, Chicago, IL), and the results were expressed as mean + SD. Differences between the groups were calculated by univariate analysis of variance; p less than 0.05 was considered significant. RESULTS

For all test groups, the number of platelets declined continuously during the 90-minute recirculation period (Fig 1). Group I (no heparin coating, no aprotinin) s h o w e d the greatest reduction; the last sample collected had a mere 29.5% -+ 10.7% o f the original platelets. In contrast, group IV (heparin coating, aprotinin) had the least decrease o f platelets. After 90 minutes o f recirculation, 69.2% _+ 16.1% o f the original number were still detectable. Furthermore, the total number o f platelets after just 60 minutes o f recirculation was significantly greater in the heparin-coated groups II and IV than in the uncoated groups I and III ( p < 0.01). The aprotinin treatment led to an additional positive effect, albeit one that showed no statistically significant differences in comparison with the other groups within the limits o f the random sampling ( p > 0.1). The c~-granule released PF-4 in inverse proportion to the platelet loss (Fig 2). After 90 minutes o f recirculation, the amount o f PF-4 increased to 615.8% + 559.5% and 452.6% _+

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227.8% of the initial value for groups I and III, respectively (no heparin coating). Both groups with a heparin coating underwent a lesser increase in PF-4 levels: group II reached 293.1% + 156.3% of the original value, whereas group IV (with aprotinin) only increased to 237.2% -+ 179.0%. These numbers indicate that the aprofinin treatment decreased the release of PF-4 in heparin-coated as well as uncoated groups (groups III and IV), although the reduction was not significantlyless than that in the groups without aprotinin (p > 0.15). The heparin-coated systems (groups II and IV), however, showed a significantly lower PF-4 content than the uncoated systems after recirculating for 90 minutes (p < 0.001). The proteins bound to the aprotinin-agarose column were eluted in relation to their aprotinin affinity by using a NaC1 gradient (0 to 1.5 tool/L). In the 280-nm spectrum, a main peak in the 8th fraction and a temporally later and fainter peak in the 12th fraction could be discerned (Fig 3). The individual fractions from the affinity chromatography were then examined with respect to the content of membrane receptor proteins. This semiquantitative analysis used the dot blot procedure and determined that a quantitatively high binding to the aprotinin affinity column existed for the GP receptor CD61 (GPIIIa [VNR [3-chain]), especially concentrated in fractions 3 to 8. The platelet receptor GPIIb was also detected, albeit more weakly, with a maximum concentration in fractions 3 to 6. Finally, GP CD42b (GPIb) showed the strongest aprotinin affinity of all the

40

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Fig 2. Platelet factor 4 concentrations (mean ± SD) during 90minute recirculation in an HLM model. II, group 1; [], group 2; [], group 3; [], group 4.

proteins. It possessed the highest concentration in fractions 7 to 13 and could only be eluted from the column with a relatively high salt concentration.

DISCUSSION

During the several hours of a cardiopulmonary bypass (CPB) operation performed with the assistance of an HLM, the patient's blood comes into contact with roughly 3 m2 of nonphysiologic artificial surfaces. This exposure contributes to the so-called postperfusion syndrome, a nonspecific, wholebody inflammatory reaction. This syndrome varies in extent between individuals but can escalate in severe cases into sepsis, adult respiratory distress syndrome, and multiorgan failure. 16-18 Therefore, it was hypothesized relatively early that improving the biocompatibility of the ECC materials coming into contact with the patients' blood would contribute considerably to alleviating postperfusion syndrome. Several research groups, 1921 in particular those of Vroman21 and Mulzer and Brash, 2° proved that the adhesion of plasma proteins to an artificial surface represents a significant criterion for their thrombogenicity. Fibrinogen adsorbed on the surface leads to a strong activation of platelets, which in turn causes adhesion, aggregation, and, in advanced stages, thromboembolic processes. 22,23 This present study examined the extent to which the advanta-

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Fig 3. Affinity chromatography of aprotinin-agarose. Elution of the platelet proteins (A = 280 nm; left ordinate) with a NaCI gradient (0 to 1.5 mol/L, right ordinate). X axis = number of collected fractions (1 mL each).

HEPARIN COATING AND APROTININ

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geous hemocompatibility of heparin-coated oxygenators could he increased through the addition of the protease inhibitor aprotinin. To that end, it relied on an HLM model with fresh human blood. The additional application of aprotinin was of particular interest because it inhibits both kallikrein and plasminogen. It also probably exercises a protective influence on the adhesion GPs (GPIb and GPIIb-IIIa) of the platelet membrane.1012 The contact between blood and the ECC device's nonphysiologic surface results in several cascade reactions, among them a pronounced platelet activation and the subsequent release of intracellular platelet proteins, a4-26 The number of platelets decreased continually during the recirculation phase of all four test groups of this study. The most precipitous decrease occurred in group I (no heparin coating, no aprotinin); the platelet count was 29% of the initial value after the 90-minute recirculation. Nonetheless, it appears highly unlikely that more than two thirds of the platelets were destroyed. Stanford et al22 showed that when fibrinogen adheres to the nonphysiologic surface, it triggers an increased platelet deposition. Presumably, under the control of fibrinogen receptors, the platelets acquire a high affinity to the surface-bound fibrinogen. This explanation agrees with the results of Wenger et al, 27 who showed that the fibrinogen receptors of the platelet membrane decreased steadily during CPB. In group IV (heparin coated, aprotinin), the platelet count decreased by only one third and the PF-4 levels increased only 2.3-fold, whereas it (PF-4 levels) increased to 6.1 times the initial value in group I (p < 0.01). A number of other studies confirmed that heparin-coated oxygenators, together with reduced heparin doses, lead to platelet protection with decreased blood loss. %-3° In this study, groups II and IV (heparin coating) performed significantly better than groups I and III (no coating) in regard to platelet loss and the release of PF-4 (p < 0.01). By means of affinity chromatography and immunoblotting, the direct affinity of aprotinin for the GPs of the platelet's membrane could be shown. Semiquantitative data processing showed GPIb to have the highest aprotinin affinity, followed by

GPIIb and IIIa, which appear in a complex under physiologic conditions (fibrinogen receptor). Whether the measured affinity of aprotinin for the platelet receptor is also important in vivo still remains unclear because many contradictory opinions cloud the current discussion over the platelet-protecting effects of high-dose aprotinin therapy. Wachtfogel et a131 showed that aprotinin could reduce platelet activation in a perfusion model. Lavee et a112 and Mohr et a132 proved through electron microscope analyses that aprotinin treatment significantly limited pseudopodia formation (the first step in platelet activation). Other research groups hypothesized that aprotinin inhibits the plasmin-mediated platelet activation.33,34Again, clinical studies could only partially confirm these in vitro results. 1°,35-37Wahba et a113 found that aprotinin had only a minimal, insignificant influence on the expression of GPIlb-IIIa during CPB surgery. Recent in vitro research showed that the combined use of aprotinin and heparin-bonded circuits reduced contact-phase activation.38 A case report also showed improved patient outcome after aortic aneurysm repair. 39 Preliminary clinical studies, investigating this combination, could show reduced blood-cell activation4° and a decrease in intensive care unit stay. 41 This study used an in vitro HLM model with fresh human blood and showed that heparin-coated oxygenation systems and additional aprotinin application caused significantly less platelet damage. Moreover, chromatographic and immunologic methods could prove aprotinin's affinity for the platelet receptor proteins GPlb and GPIIb-IIIa. This affinity apparently restricts the activity of the thrombin, von Willebrand's and fibrinogen receptors and therefore probably diminishes the triggering of the platelet activation cascade and promotes aprotinin's direct inhibition of the plasmin-mediated platelet activation during ECC. Therefore, it can be postulated that more biocompatible materials and the supplementary use of aprotinin may positively influence the platelet-related postperfusion syndromes arising from ECC procedures, particularly hyperfibrinolysis and blood loss.

REFERENCES

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30. Thelin S, Bagge L, Hultman J, et al: Heparin-coated cardiopulmonary bypass circuits reduce blood cell trauma. Eur J Cardiothorac Surg 5:486-491, 1991 31. Wachtfogel YT, Kucich U, Hack CE, et al: Aprotinin inhibits the contact; Neutrophil and platelet activation systems during simulated extracorporeal perfusion. J Thorac Cardiovasc Surg 106:1-10, 1993 32. Mohr R, Goor DA, Lusky A, Lavee J: Aprotinin prevents cardiopulmonary bypass-induced platelet dysfunction. A scanning electron microscope study. Circulation 86:405-409, 1992 33. Cramer EM, Lu H, Caen JR et al: Different redistribution of platelet glycoproteins lb and IIb-IIIa after plasmin stimulation. Blood 77:694-699, 1991 34. Lu H, Sofia C, Cramer EM, et al: Temperature dependence of plasmin-induced platelet activation or inhibition of human platelets. Blood 77:996-1105, 1991 35. Tabuchi N, De Haan J, Boonstra PW, et al: Aprotinin effect on platelet function and clotting during cardiopulmonary bypass. Eur J Cardiothorac Surg 8:87-90, 1994 36. Orchard MA, Goodchild CS, Prentice CRM, et al: Aprotinin reduces cardiopulmonary bypass-induced blood loss and inhibits fibrinolysis without influencing platelets. Br J Haematol 85:533-541, 1993 37. Blanhut B, Gross C, Necek S, et al: Effects of high-dose aprotinin on blood loss, platelet function, fibrinolysis, complement and renal function after cardiopulmonary bypass. J Thorac Cardiovasc Surg 101:958-967, 1991 38. Wendel HP, Heller W, Gallimore MJ: Heparin-coated devices and high-dose aprotinin optimally inhibit contact system activation in an in vitro cardiopulmonary bypass model. Immunopharmacology 32:128-131, 1996 39. Gallagher JM, Brown ME, Gasior TA: Combined use of aprotinin and a heparin-bonded cardiopulmonary bypass system for aortic aneurysm repair. J Cardiothorac Vasc Anesth 9:728-730, 1995 40. Banfreton C, Jansen PG, Le Besnerais P, et al: Hepafin coating with aprotinin reduces blood activation during coronary artery operations. Ann Thorac Surg 63:50-56, 1997 41. Jansen PG, Banffeton C, Le Besnerais P, et al: Heparin-coated circuits and aprotinin prime for coronary artery bypass grafting. Ann Thorac Surg 61:1363-1366, 1996