Healio
Healio
June 01, 2004
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Aprotinin: A Pharmacologic Overview

ABSTRACT

Aprotinin is a polypeptide with serine protease inhibitory activity of key enzymes associated with inflammatory, fibrinolytic, and hemostatic pathways. The drug binds directly to the fibrinolytic plasmin at the lower plasmin-inhibiting dose (plasma concentration, 137 KIU/mL), and the inflammatory mediator, kallikrein, using the higher kallikrein-inhibiting dose (plasma concentration, >250 KIU/mL). Aprotinin inhibits platelet glycoprotein loss (GpIb and GpIIb/IIIa receptors) associated with cardiopulmonary bypass and has been described as platelet sparing. Current literature supports direct anti-inflammatory effects through modulation of neutrophil activation, attachment, and transmigration, with resultant blunting in the rise of proinflammatory cytokine levels and deleterious tissue damaging enzymes. The pharmacologic properties of aprotinin may lead clinicians to consider this therapy for use as a hemostatic and anti-inflammatory agent in surgeries beyond its established use in coronary artery bypass graft surgery.

Figure 1 Figure 2
Figure 1. Aprotinin, a 58 amino acid polypeptide, contains three disulfide bridges (yellow circles). The reactive bond site of aprotinin is the lysine residue in position 15, within the Kunitz domain (blue circles) of the inhibitor.
Figure 2. Aprotinin inhibits a spectrum of human serine proteases over a range of concentrations.
Figures courtesy of Jerrold Levy, MD.

Aprotinin is a nonspecific serine protease inhibitor that was discovered in 1930 by a research group at the University of Munich. The group isolated an inhibitor of kallikrein from bovine lung tissue and pancreas. In 1936, Kunitz and Northrup isolated an inhibitor of trypsin, also from bovine pancreas. In 1959, the bovine trypsin inhibitor was launched in Germany as aprotinin for treatment of pancreatitis. In the late 1960s, it was determined that the kallikrein inhibitor and trypsin inhibitor (aprotinin) were the same. Acknowledging that kallikrein is a key inflammatory mediator, Royston et al1 developed a protocol to assess the ability of aprotinin to limit lung inflammation associated with cardiopulmonary bypass in the setting of coronary artery bypass graft surgery. The results of a study, published in 1987, unexpectedly showed significant reductions in bleeding and the administration of blood in a coronary artery bypass graft population.1 Further investigation revealed that aprotinin exhibited a significant inhibitory effect on plasmin and stabilized platelet glycoproteins. Aprotinin was subsequently studied and introduced as a pharmacologic agent for use in cardiopulmonary bypass associated coronary artery bypass graft in the United States in 1994. The initial indication for aprotinin use was for patients at highest risk of perioperative bleed (eg, redo coronary artery bypass graft, patients who could not receive blood transfusion, and Jehovah’s Witness). The current indication of aprotinin is prophylactic use to reduce perioperative blood loss and the need for blood transfusion in patients undergoing cardiopulmonary bypass in the course of coronary artery bypass graft surgery.2

Structure and Activity

Aprotinin is a small polypeptide molecule that consists of 58 amino acids with a molecular weight of 6512 daltons (Figure 1).2 Aprotinin belongs to the Kunitz family of serine protease inhibitors. The 15-lysine in the structure of aprotinin binds to a serine amino acid on the target enzyme in a tight, but reversible, manner. Aprotinin inhibits a spectrum of human serine proteases over a broad range of concentrations (Figure 2). Aprotinin binds to trypsin, plasmin, and kallikrein more readily and in much lower concentrations then to urokinase or thrombin. The inhibition constant (Ki) is approximately 500 million-fold higher for thrombin.3 As a result of aprotinin’s concentration-dependent binding to serine sites on specific compounds, the expected clinical efficacy is dependent on specific dosing to achieve targeted blood levels.

Pharmacodynamic Effects

Hemostatic derangements associated with cardiopulmonary bypass include platelet function defects, coagulation defects (dilutional, consumptive, hypothermia), hyperfibrinolysis, and alterations in vascular (endothelial) function. Many of the compounds and enzymes involved in these processes are serine proteases, namely, clotting factors, plasminogen and plasmin, and kallikrein.

TD CLASS="cap1">Reprinted with permission from Peters DC, Noble S. Aprotinin: an update of its pharmacology and therapeutic use in open heart surgery and coronary artery bypass surgery. Drugs. 1999; 57:233-260.
Figure 3
Figure 3. A schematic diagram showing the roles of kallikrein and plasmin as mediators in the coagulation, fibrinolytic, kinin, angiotensin, and complement systems after activation at a foreign surface.
Abbreviations: C1 = complement 1, HMWK = high molecular weight kininogen.

To discuss fully the pharmacodynamic effects of aprotinin as they relate to patient management, an overview of cardiopulmonary bypass induced alterations in hemostasis and the propagation of the systemic inflammatory response should be reviewed (Figure 3).4 Blood contact with the negatively charged foreign surface of the bypass circuit (tubing) initiates the activation of the intrinsic clotting cascade via Hageman factor (factor XII) conversion to active factor XII (XIIa). Through a series of like enzyme activations, key mediators related to coagulation (thrombin, factor IIa), fibrinolysis (plasmin), and inflammation (kallikrein) are produced. The net results are activation of clot formation and clot lysis to maintain hemostatic balance. Additionally, an undesirable inflammatory response from kinin generation (bradykinin) and activation of the complement system via kallikrein are observed. Adsorption of fibrinogen and other plasma proteins to the surface of the cardiopulmonary bypass circuit promotes platelet aggregation and consumption, therefore necessitating anticoagulation with heparin while a patient is on cardiopulmonary bypass. Although patients undergoing orthopedic procedures do not use cardiopulmonary bypass, skin incision activates the extrinsic clotting cascade and resultant fibrinolytic pathway leading to hemostatic imbalance. This imbalance, as well as surgical manipulation and associated trauma, produces a similar inflammatory response in a patient undergoing orthopedic surgery.

Figure 4
Figure 4. After transmigration, neutrophil degranulates and releases proinflammatory intracellular cytokines and tissue damaging enzymes.
Reprinted with permission from Elliott MJ, Finn AH. Interaction between neutrophils and endothelium. Ann Thorac Surg. 1993; 56:1503-1508.

Unlike the lysine analogs (amino-caproic acid, tranexamic acid) that inhibit the formation of plasmin, the effect of aprotinin on reestablishing a balance between clot formation and clot lysis is multifactorial and consists of a direct binding to plasmin and kallikrein in a concentration-dependent manner.3 The drug binds to kallikrein early in the coagulation cascade thereby attenuating the domino effect that follows (Figure 3). This modulates the coagulation and fibrinolytic derangements as well as the systemic inflammatory response syndrome associated with cardiopulmonary bypass.4,5 Other effects on balancing hemostasis include reduction in the formation of fibrin degradation products (D-dimers), increases in the activity of plasmin activator inhibitor and alpha-2 antiplasmin, and an attenuation of the release of tissue plasminogen activator from the endothelial cells, all of which limit the degree of clot lysis and patient blood loss.6-9

Aprotinin preserves platelet glycoprotein receptor function, which is associated with platelet adhesion (GPIa) and aggregation (GPIIb/IIIa).10-12 This key effect on platelets may provide additional hemostatic support, particularly in a patient undergoing cardiopulmonary bypass in whom platelets undergo significant physical destruction by the pump and elevated plasmin levels. Aprotinin blunts the effect of thrombin stimulation of the platelet via the protease activated receptor that prevents undesired “overaggregatation” and thus platelet consumption, while allowing for effective platelet stimulation and appropriate adhesion and aggregation from other in vivo sources such as circulating adenosine diphosphate and adrenalin.13 The outcome to a surgical patient is suppression of bleeding and a reduction in blood and blood product administration.

Figure 5
Figure 5. Dose-response of aprotinin and blood loss in surgeries.
Reprinted with permission from Kovesi T, Royston D. Dose-response of aprotinin and blood loss in surgeries. Vox Sang. 2003; 84:2-10.

Assessments of the anti-inflammatory effects, beyond the modulation of kallikrein, have also been studied. In animal models, aprotinin has been shown to protect against kallikrein-induced rises in serum bradykinin and associated deleterious effects on brain edema.2 Aprotinin also directly blunts the inflammatory response at the leukocyte activation level through inhibition of the up-regulation of the pro-inflammatory CD11b integrin. This integrin plays a key role in the attachment of the neutrophil to the endothelial surface during an inflammatory response and also facilitates subsequent transmigration of the neutrophil into the extravascular space. After transmigration, the neutrophil degranulates and releases proinflammatory intracellular cytokines (eg, IL-6) and tissue damaging enzymes (eg, elastase) (Figure 4). This direct effect of aprotinin on modulating neutrophil transmigration has been quantified and shown to be statistically significant in animal models and humans.14 Animal and human data have shown that aprotinin attenuates neutrophil infiltration into tissue, blunts the rise of pro-inflammatory cytokines (IL-6, IL-8, TNF alpha) and enzymes (elastase), as well as elevates levels of the anti-inflammatory cytokine IL-10.15-19 A blunting effect on complement activation and the respective levels of C3a and C5a has also been observed.20

Preliminary evidence based on animal models indicate that aprotinin may have a cardioprotective effect, particularly pertaining to myocardial ischemia reperfusion injury. Effects on surrogate markers (troponin T, CK-MB) in humans undergoing cardiopulmonary bypass coronary artery bypass graft have supported these initial findings.21 Finally, aprotinin has been shown to attenuate increases in nitric oxide and inducible nitric oxide synthase in in vitro experimental models, which may assist in maintaining vascular tone during the surgical procedure.22

Pharmacokinetic Properties

Aprotinin is inactive via the oral route and must be given intravenously. After administration, this water-soluble drug rapidly distributes into the extravascular space. The post-distribution half-life is about 2.5 hours, with a terminal elimination half-life of 4 to 5 hours. Aprotinin is filtered by the glomeruli of the kidneys and reabsorbed by the proximal tubules. It is stored in phagolysosomes and then undergoes slow degradation by lysosomal enzymes. Less than 10% of the drug is excreted unchanged.2

>Dosing

Aprotinin is measured using kallikrein inhibitor units (KIU), which is defined as the amount of aprotinin that decreases the activity of 2 biologic units of kallikrein by 50%. Dosing is based primarily on KIU that equates to the following relationship: 100,000 KIU of aprotinin equals 14 mg of aprotinin. Currently, two approved doses exist for use in cardiopulmonary bypass associated coronary artery bypass graft. The full “kallikrein inhibitory” dose, also known as the full Hammersmith, consists of a 2 million KIU loading dose given intravenously over 20 to 30 minutes, followed by a 500,000 KIU/hr continuous infusion. This is accompanied by a 2 million KIU aliquot administered into the pump prime solution of the cardiopulmonary bypass machine before initiation of bypass.1,2 The half plasmin inhibitory dose, or half-Hammersmith, is one-half of the full Hammersmith loading dose, continuous infusion and pump prime dose. In either instance, a 10,000 KIU (1 mL) intravenous test dose is administered in the operating suite 10 minutes before the loading dose as a test dose to assess for potential hypersensitivity reaction in the patient.2

Figure 6
Figure 6. Dose-response of aprotinin and blood loss in spine surgeries.
Data summarized from Khoshhal K, Mukhtar I, Clark P, et al. Efficacy of aprotinin in reducing blood loss in spinal fusion for idiopathic scoliosis. J Pediatr Orthop. 2003; 23:661-664; Cole JW, Murray DJ, Snider RJ, et al. Aprotinin reduces blood loss during spinal surgery in children. Spine. 2003; 28:2482-2485; Urban MK, Beckman J, Gordon M, et al. The efficacy of antifibrinolytics in the reduction of blood loss during complex adult reconstructive surgery. Spine. 2001; 26:1152-1156; Lentschener C, Cottin P, Bouaziz H, et al. Reduction of blood loss and transfusion requirement by aprotinin in posterior lumbar spine fusion. Anesth Analag. 1999; 89:590-597.

The half-dose regimen achieves mean concentrations of about 137 KIU/mL, which is adequate to suppress rises in plasmin levels. Strategically, the full dose regimen is given to achieve mean target plasma KIU levels (250 KIU/mL) necessary to inhibit kallikrein (and plasmin). Dose response relating total amount of aprotinin and blood loss has been reviewed by Royston1 (Figure 5) and in pivotal trials used to gain Food and Drug Administration approval. Similar observations can be made when total dose is plotted with respect to blood loss from published research in the area of spinal orthopedic surgery (Figure 6).

Safety

Hypersensitivity reactions are rare, occurring no more frequently than placebo (<0.1%) with no prior exposure.2 However, the incidence of hypersensitivity increases to approximately 5% within a few weeks of initial aprotinin exposure and then dissipates over the next 6 months to an incidence of 0.9% thereafter.23 Because aprotinin is excreted and metabolized by the renal route, studies have assessed renal safety. Modest transient elevations in serum creatinine have been observed in some patients undergoing cardiac surgery with a return to baseline at discharge or first office visit. To date, however, studies have shown no propensity for increased incidence of renal toxicity in terms of acute renal failure and need for dialysis.24

Summary

Aprotinin is a unique agent with multiple pharmacologic properties that modulates a variety of key enzymes associated with cardiac surgeries. Most notable is the ability to bind directly to the fibrinolytic, plasmin, and the inflammatory mediator, kallikrein. In addition, aprotinin has platelet glycoprotein stabilizing effects as well as platelet-sparing effects, thus providing another mechanism by which homeostasis can be maintained during a surgical procedure in which activation of the procoagulant and fibrinolytic pathways are underway. Current literature supports direct anti-inflammatory effects through modulation of neutrophil activation, attachment and transmigration, with resultant blunting in the rise of proinflammatory cytokine levels and deleterious tissue-damaging enzymes. These pharmacologic properties may lead one to consider this agent as a hemostatic and anti-inflammatory advance in other surgeries beyond the established use in coronary artery bypass graft.

References

  1. Royston D, Bidstrup BP, Taylor KM, Sapsford RN. Effect of aprotinin on need for blood transfusion after repeat open-heart surgery. Lancet. 1987; 2:1289-1291.
  2. Aprotinin (Trasylol, aprotinin injection) package insert. Bayer Corporation, West Haven, Conn. March 2001.
  3. Fritz H, Wunderer G. Biochemistry and applications of aprotinin, the kallikrein inhibitor from bovine organs. Arzneimittelforschung. 1983; 33:479-494.
  4. Peters DC, Noble S. Aprotinin: an update of its pharmacology and therapeutic use in open heart surgery and coronary artery bypass surgery. Drugs. 1999; 57:233-260.
  5. Royston D. Preventing the inflammatory response to open-heart surgery: the role of aprotinin and other protease inhibitors. Int J Cardiol. 1996; 53(Suppl):S11-S37.
  6. Lu H, Du Buit C, Soria J, et al. Postoperative hemostasis and fibrinolysis in patients undergoing cardiopulmonary bypass with or without aprotinin therapy. Thromb Haemost. 1994; 72:438-443.
  7. Mastroroberto P, Chello M, Zofrea S, Marchese AR. Suppressed fibrinolysis after administration of low-dose aprotinin: reduced level of plasmin-alpha2-plasmin inhibitor complexes and postoperative blood loss. Eur J Cardiothorac Surg. 1995; 9:143-145.
  8. Van Oeveren W, Jansen NJ, Bidstrup BP, et al. Effects of aprotinin on hemostatic mechanisms during cardiopulmonary bypass. Ann Thorac Surg. 1987; 44:640-645.
  9. Blauhut B, Gross C, Necek S, Doran JE, Spath P, Lundsgaard-Hansen P. Effects of high-dose aprotinin on blood loss, platelet function, fibrinolysis, complement, and renal function after cardiopulmonary bypass. J Thorac Cardiovasc Surg. 1991; 101:958-967.
  10. Van Oeveren W, Harder MP, Roozendaal KJ, Eijsman L, Wildevuur CR. Aprotinin protects platelets against the initial effect of cardiopulmonary bypass. J Thorac Cardiovasc Surg. 1990; 99:788-796; discussion 796-787.
  11. Primack C, Walenga JM, Koza MJ, Shankey TV, Pifarre R. Aprotinin modulation of platelet activation in patients undergoing cardiopulmonary bypass operations. Ann Thorac Surg. 1996; 61:1188-1193.
  12. Lavee J, Savion N, Smolinsky A, Goor DA, Mohr R. Platelet protection by aprotinin in cardiopulmonary bypass: electron microscopic study. Ann Thorac Surg. 1992; 53:477-481.
  13. Poullis M, Manning R, Laffan M, Haskard DO, Taylor KM, Landis RC. The antithrombotic effect of aprotinin: actions mediated via the proteaseactivated receptor 1. J Thorac Cardiovasc Surg. 2000; 120:370-378.
  14. Kamiya T, Katayama Y, Kashiwagi F, Terashi A. The role of bradykinin in mediating ischemic brain edema in rats. Stroke. 1993; 24:571-575; discussion 575-576.
  15. Asimakopoulos G, Thompson R, Nourshargh S, et al. An anti-inflammatory property of aprotinin detected at the level of leukocyte extravasation. J Thorac Cardiovasc Surg. 2000; 120:361-369.
  16. Hill GE, Pohorecki R, Alonso A, Rennard SI, Robbins RA. Aprotinin reduces interleukin-8 production and lung neutrophil accumulation after cardiopulmonary bypass. Anesth Analg. 1996; 83:696-700.
  17. Whitten CW, Latson TW, Allison PM, Levy JH, Salmenpera MT, Oflaherty DN. Does aprotinin inhibit cardiopulmonary bypass-induced inflammation? Anesthesiology. 1992; 77:A266.
  18. Stammers AH, Huffman S, Alonso A, et al. The antiinflammatory effects of aprotinin in patients undergoing cardiac surgery with cardiopulmonary bypass. J Extra Corpor Technol. 1997; 29:114-122.
  19. Rahman A, Ustunda B, Burma O, Ozercan IH, Cekirdekci A, Bayar MK. Does aprotinin reduce lung reperfusion damage after cardiopulmonary bypass? Eur J Cardiothorac Surg. 2000; 18:583-588.
  20. Himmelfarb J, Holbrook D, McMonagle E. Effects of aprotinin on complement and granulocyte activation during ex vivo hemodialysis. Am J Kidney Dis. 1994; 24:901-906.
  21. Wendel HP, Heller W, Michel J, et al. Lower cardiac troponin T levels in patients undergoing cardiopulmonary bypass and receiving high-dose aprotinin therapy indicate reduction of perioperative myocardial damage. J Thorac Cardiovasc Surg. 1995; 109:1164-1172.
  22. Hill GE, Robbins RA. Aprotinin but not tranexamic acid inhibits cytokine-induced inducible nitric oxide synthase expression. Anesth Analg. 1997; 84:1198-1202.
  23. Dietrich W. Incidence of hypersensitivity reactions. Ann Thorac Surg. 1998; 65:S60-S64; discussion S74-S66.
  24. Lemmer JH Jr, Stanford W, Bonney SL, et al. Aprotinin for coronary artery bypass grafting: effect on postoperative renal function. Ann Thorac Surg. 1995; 59:132-136.