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Ann Thorac Surg 2001;72:2169-2175
© 2001 The Society of Thoracic Surgeons

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Review

The antithrombotic and antiinflammatory mechanisms of action of aprotinin

^a The British Heart Foundation Unit of Cardiovascular Medicine, Hammersmith Hospital, National Heart and Lung Institute, Imperial College School of Medicine, London, England, United Kingdom
^b The British Heart Foundation Unit of Cardiac Surgery, Hammersmith Hospital, National Heart and Lung Institute, Imperial College School of Medicine, London, England, United Kingdom

* Address reprint requests to Dr Landis, BHF Cardiovascular Medicine Unit, National Heart and Lung Institute, Imperial College School of Medicine, Hammersmith Hospital, Du Cane Rd, London, England W12 0NN, UK
e-mail: r.landis{at}ic.ac.uk

	Abstract

Top Footnotes Abstract Introduction The thrombosis debate: a... Antiinflammatory action of... Conclusion Acknowledgments References

 
Aprotinin (Trasylol) is generally regarded to be an effective hemostatic agent that prevents blood loss and preserves platelet function during cardiac surgery procedures requiring cardiopulmonary bypass (CBP). However, its clinical use has been limited by the concern that such a potent hemostatic agent might be prothrombotic, particularly in relation to coronary vein graft occlusion. In this review we present a mechanism of action that challenges such a viewpoint and explains how aprotinin can be simultaneously hemostatic and antithrombotic. Aprotinin achieves these two apparently disparate properties by selectively blocking the proteolytically activated thrombin receptor on platelets, the protease-activated receptor 1 (PAR1), while leaving other mechanisms of platelet aggregation unaffected. We also review recent research leading to the discovery of novel antiinflammatory targets for aprotinin. A better understanding of its mechanisms of action has led to the conclusion that aprotinin is a remarkable drug with the capacity to correct many of the imbalances that develop in the coagulation system and the inflammatory system after CPB. Nonetheless, it has been clinically underused for fear of causing thrombotic complications, a fear that in light of recent evidence may be unfounded.

	Introduction

Top Footnotes Abstract Introduction The thrombosis debate: a... Antiinflammatory action of... Conclusion Acknowledgments References

 
The discovery that aprotinin, a nonspecific protease inhibitor, was highly effective in reducing blood loss during and after cardiac surgery procedures was a serendipitous finding [1–4]. The researchers’ original hypothesis related not to hemostasis but to inflammation, specifically the potential for aprotinin, in a kallikrein-inhibitory dose, to attenuate the inflammatory response to CPB.

From the time of publication of the original studies in the late 1980s and continuing through the 1990s, there has been a perception that an agent so powerfully hemostatic might also be prothrombotic. Few, if any, cardiac surgeons doubt the hemostatic efficacy of aprotinin, though many remain concerned that its use in coronary artery surgery patients may compromise graft patency. In consequence, many cardiac surgeons have been reluctant to use aprotinin in their routine practice, particularly their coronary artery surgery practice.

	The thrombosis debate: a problem with rationale

Top Footnotes Abstract Introduction The thrombosis debate: a... Antiinflammatory action of... Conclusion Acknowledgments References

 
The clinical evidence
Attempts to resolve the controversy surrounding aprotinin by conducting clinical trials of coronary graft patency have not been notably successful. Each side of the argument may cite published evidence to support its position. Although the studies of Bidstrup and associates [5], Havel and associates [6], Lass and colleagues [7], Hayashida and colleagues [8], and Rich [9] found no evidence of reduced graft patency with aprotinin therapy, those of Cosgrove and associates [10], Van der Meer and colleagues [11], and Alderman and associates [12] reinforced the prothrombotic concerns. However, criticisms have been leveled at most, if not all, of these studies [13]. They vary considerably in relation to the aprotinin dosage used, in the imaging modes for graft patency, and in the postsurgery time interval at which graft patency was assessed. Confounding factors such as heparin dose during CPB, use of aspirin therapy pre- and postoperatively and, perhaps most important, the quality of the distal vessels—all have contributed to the heat of the debate, if not to the illumination of the underlying issue.

Since the issue of the prothrombotic potential of aprotinin was first raised with the original Hammersmith research team in the late 1980s, we have consistently stated that this issue would be resolved only when the mechanisms of action of aprotinin became more completely understood. Despite the understandable concern that aprotinin might be prothrombotic, such a mechanism has never been supported by a convincing scientific rationale. In fact, with the cloning of the thrombin receptor on platelets, the prediction could be made that aprotinin, instead of potentiating the actions of thrombin, should, in fact, antagonize them.

The classic thrombin receptor on platelets is a protease-activated receptor
The cloning of the human thrombin receptor (the classic thrombin receptor or seven-transmembrane thrombin receptor) on platelets revealed a receptor belonging to the seven-transmembrane superfamily, but with a unique difference: Alone among seven-transmembrane receptors, it required proteolytic cleavage in order to transduce an intracellular activating signal [14]. Cleavage of the receptor is a result of the serine protease activity of thrombin, which is known to be competitively inhibited by aprotinin [15]. The immediate hypothesis could thus be posited that aprotinin should antagonize and not potentiate thrombin-induced platelet activation.

Recognition of the distinctive proteolytic activation mechanism of the thrombin receptor led to its being renamed the "protease-activated receptor 1" (PAR1) [14], now established as the prototypic member of a subfamily of four related thrombin receptors, PAR1 to PAR4 [16]. Signaling through PAR1 occurs via a sequential mechanism (Fig 1), involving initial binding of thrombin to a hirudin-like domain at amino acids 53 to 64, followed by proteolytic cleavage of the receptor at arginine 41 [17]. Cleavage is mediated by the serine protease activity of thrombin but can be mimicked by other unrelated serine proteases, such as trypsin, albeit at a lower affinity than thrombin itself [14]. Proteolysis unmasks a so-called tethered ligand sequence within the ectodomain, which then becomes available for docking in the ligand-binding pocket, causing intracellular signaling and platelet activation. The PAR series can therefore be thought of as receptors that carry their own ligands, but in a form that requires proteolysis to unmask each receptor’s ligand. If the tethered ligand is provided as a synthetic peptide in solution, however, it can bypass the requirement for proteolysis. The best known of these peptides is SFLLRN [18, 19], also known as "thrombin receptor– activating peptide 6" (TRAP6) or "PAR1–activating peptide" (PAR1AP). Conversely, the truncated peptide FLLRN acts as a competitive inhibitor of PAR1 activation and can be used to define the PAR1 component of thrombin’s action on platelets [19].

View larger version (25K): [in this window] [in a new window]   Fig 1. The proteolytic activation mechanism of the thrombin receptor PAR1. Thrombin initially binds to a hirudin-like motif on PAR1 before cleaving the receptor at arginine 41. Cleavage unmasks a "tethered ligand" sequence in the ectodomain, which is now available to dock within the ligand-binding pocket located between extracellular loops 2 and 3 of the transmembrane domain. Cleavage occurs via the serine protease activity of thrombin in conjunction with other "cofactors," some of which are serine proteases that may themselves be targets of aprotinin. Proteolytic activation of PAR1 is followed by translocation of heterotrimeric G protein signals, Ca2+ fluxing and downstream activation events, such as platelet aggregation and serotonin secretion. (For a good review on protease-activating receptors, see the article by Coughlin [16].)

Having confirmed PAR1 as the major thrombin receptor on platelets, we next studied the effect of aprotinin on proteolytic agonists (thrombin and trypsin) versus nonproteolytic agonists (collagen, adenosine diposphate (ADP), and epinephrine). These experiments showed that aprotinin specifically inhibited platelet aggregation induced by thrombin and trypsin, but not by collagen, ADP, and epinephrine (Fig 2). Thrombin-induced platelet aggregation was inhibited 42.6% ± 21.6% (mean ± SD) at 50 KIU/ml aprotinin (p = 0.0047), 61.0 (25.2% at 100 KIU/ml (p = 0.0001), and 86.6 (8.9% at 160 KIU/ml (p < 0.0001). The 50 KIU/ml level corresponds to the low dose of aprotinin; 160 KIU/ml provides a level below the high dose of aprotinin (200 to 250 KIU/mL) used in cardiac surgery requiring CPB. The lack of effect on collagen-, ADP-, and epinephrine-induced aggregation ruled out any possibility that aprotinin could have inhibited a common distal event in platelet aggregation, such as the binding of fibrinogen to GPIIbIIIa.

View larger version (28K): [in this window] [in a new window]   Fig 2. Selective blocking of PAR1-mediated activation of platelets by aprotinin. This schematic summarizes the effect of aprotinin on platelet activation in response to proteolysis-dependent agonists (ie. thrombin and trypsin) versus proteolysis-independent agonists (ie, collagen, ADP or epinephrine). Aprotinin selectively blocks proteolysis-dependent modes of activation mediated via the PAR1 receptor, but not proteolysis-independent modes, which are mediated via the collagen, ADP, or epinephrine receptors (after Poullis and associates [20]).

Aprotinin is simultaneously hemostatic and antithrombotic
An interesting feature of platelets in which the thrombin response has been blocked by aprotinin is their continued capacity to aggregate with respect to collagen, ADP, or epinephrine [20]. The clinical implication for surgery requiring CPB is that aprotinin would be expected to prevent the participation of thrombin-activated platelets in the coagulation cascade, thereby exerting a net antithrombotic effect, while maintaining the hemostatic capacity of platelets in surgical wounds (sites at which collagen and ADP are likely to be generated).

Aprotinin can act on a wide range of potential targets in vivo, and its net clinical effect is determined by its relative affinity to its various substrates. The dominant hemostatic effect observed clinically is likely a result of the inhibition of plasmin, which aprotinin targets with a relatively high inhibition constant (K_i = 9 x 10 to 11 mol/L) compared to kallikrein (K_i = 3x10 to 8 mol/L) or thrombin (K_i = 6 x 10 to 6 mol/L). The weak inhibition constant for thrombin contrasts with the ability of aprotinin, even at "half-dose" levels, to inhibit PAR1 activation. The inhibition constant quoted in the literature refers to the amidolytic activity of thrombin for peptide bonds. Because PAR1 activation by thrombin is blocked at a dose almost 60 times lower than that predicted to block amidolytic activity, it is possible that aprotinin will target other interactions distinct from the serine protease activity of thrombin during PAR1 activation. Possible explanations include inhibition of membrane-associated proteases other than thrombin that play a necessary role in PAR1 signaling or in preventing the proper association between thrombin and the hirudin-like binding motif in PAR1. Recent evidence supports the idea that aprotinin may target cofactors that form a signaling complex with PAR1. Known cofactors to date include the membrane-type serine protease-1 [21], the coagulation factors VIIa and Xa (themselves presented in association with their own cofactors) [23], and other protease-activated receptors [22]. The blockade of PAR1-mediated responses in platelets, at concentrations equivalent to "half-dose" aprotinin, is therefore likely to be mediated by the inhibition of other serine proteases involved in PAR1 signaling.

"Platelet preservation" explained
The antithrombotic action of aprotinin described above may also explain its "platelet preservation" properties reported in the clinic [24–26]. Platelet dysfunction is a well-recognized problem associated with CPB [27, 28], which is caused by degranulation and consequent "exhaustion" of platelets secondary to the generation of thrombin during operation [29, 30]. Exhausted platelets contribute to excessive postoperative bleeding, which may necessitate platelet transfusion [31, 32]. A likely mechanism of platelet preservation, therefore, is that aprotinin protects platelets from desensitization by thrombin generated during operation, resulting in a net increase in the number of platelets available to participate in hemostasis at wound and suture sites.

	Antiinflammatory action of aprotinin: a multi-tiered mechanism

Top Footnotes Abstract Introduction The thrombosis debate: a... Antiinflammatory action of... Conclusion Acknowledgments References

 
Inflammatory response to CPB
It has been extensively demonstrated and well reviewed elsewhere that surgery requiring CPB elicits a systemic inflammatory response [33–35]. Consequences of the inflammatory response range from prolonged hospital stay, to neurocognitive disorders, to strokes, acute lung injury, multiple organ failure, and even death. Although aprotinin is used in heart surgery mainly for its hemostatic effect, it has also been recognized to provide a significant antiinflammatory benefit. In the following section we will review recent research that has led to the discovery of both soluble and cell-associated targets within the inflammatory system, and that has revealed a multi-tiered mechanism of antiinflammatory action for aprotinin.

Effect of aprotinin on leukocyte activation and cytotoxic mediator release
Within the circulation, aprotinin has been shown to significantly reduce neutrophil activation at 15 to 60 minutes after reversal of CPB, as assessed by diminished expression of Mac-1 (CD11b/CD18) [36–38]. There is no clinical evidence for a similar effect on circulating monocyte/macrophage cells [38], although, at very high concentrations (1200 KIU/ml), aprotinin can inhibit Mac-1 and tissue factor expression by monocytes in vitro [39]. The prevailing evidence from the blood compartment, therefore, is that the neutrophil is the main antiinflammatory target for aprotinin during CPB.

It is possible, however, that the alveolar macrophage compartment may be more efficiently targeted than the circulating compartment during CPB. In endotoxemia-induced lung injury, alveolar macrophages play an important role in the early phase of disease through the secretion of chemoattractants that initiate or amplify the main wave of neutrophil infiltration to the lungs [40, 41]. The effect of aprotinin on alveolar macrophages therefore deserves consideration in future studies.

Once they have populated an organ, neutrophils are the main protagonists of acute inflammatory injury. Their activity is mediated in large part through the release of cytotoxic mediators, such as oxygen-free radicals and chaotropic enzymes, that degrade or remodel the extracellular matrix [42, 43]. Important enzymes in this process are the azurophilic granule components elastase and myeloperoxidase (MPO), as well as proteases of the matrix metalloproteinase family. Aprotinin has been shown to target azurophilic granule release and to block secretion of MPO and neutrophil elastase induced by neutrophil chemoattractants [44, 45]. Aprotinin may therefore exert a potent combined protective effect on neutrophils, first by preventing their activation within the circulation and second by preventing secretion of histotoxic mediators within the tissues.

Effect of aprotinin on the leukocyte–endothelial cell adhesion cascade
The development of organ injury in systemic inflammatory response syndrome (SIRS) is characterized by an initial wave of leukocyte infiltration into the affected organ, followed by activation of the sequestered leukocytes, elaboration of chemoattractants, and amplification of the inflammatory response. Once started, this process can lead to the rapid accumulation of large numbers of harmful inflammatory cells within major organs. The prevention of the first wave of leukocyte infiltration should therefore be a priority of antiinflammatory therapy, and this is another area where aprotinin has been recently shown to be effective.

The sequestration of leukocytes into tissues proceeds in a process known as the "leukocyte-endothelial cell adhesion cascade"—an orderly series of contact interactions between circulating leukocytes and vascular endothelial lining cells. The adhesion cascade is broken into three distinct phases (summarized in Figure 3): leukocyte rolling, firm adhesion, and extravasation, each controlled by adhesion molecules specialized to their task (reviewed by Frenette and Wagner [46]). Broadly speaking, the initial rolling phase under hydrodynamic shear flow is mediated by the selectin family of adhesion molecules; the firm adhesion step, by the so-called integrins; and the extravasation step, by endothelial adhesion molecules belonging to the immunoglobulin supergene family. Proteases secreted by the migrating leukocyte may also play a role in extravasation and subendothelial migration, by digesting the cellular and matrix barriers ahead of the migrating cell. The molecular details of this process as they are currently understood are described in Figure 3.

View larger version (21K): [in this window] [in a new window]   Fig 3. The leukocyte–endothelial cell adhesion cascade. The three main phases of the adhesion cascade are shown: 1. leukocyte rolling, 2. firm adhesion, and 3. extravasation. Also indicated are the points at which the major categories of adhesion molecules and proteases play a role. The initial tethering and rolling phase under hydrodynamic shear flow is mediated by the selectin family of adhesion molecules, E-, P- and L-selectin, which are characterized by rapid on:off rates for their lectin-type ligands. The firm adhesion step is mediated by the integrins, such as Mac-1, LFA-1 and VLA-4, which have evolved as conditional adhesion molecules on leukocytes that require prior activation, usually via a chemokine, before they can mediate adherence to endothelium. Endothelial counterligands for the integrins are members of the immunoglobulin supergene family and include ICAM-1, ICAM-2, and CD31. Chemokines are generated from within the tissues and are transported to the apical side of the blood vessel where they are "presented" on the glycocalyx to rolling leukocytes looking for signs of underlying tissue injury or infection. The extravasation and tissue migration steps involve further contributions from proteases of the elastase and metalloproteinase families, which are secreted at the leading edge of leukocytes, where they digest cell:cell junctions or subendothelial matrix components ahead of the migrating cell. (For a good review of the leukocyte–endothelial cell adhesion cascade, see Frenette and Wagner [46].)

We therefore carried out intravital microscopy in rats to study the effect of aprotinin on the three main stages of the leukocyte–endothelial cell adhesion cascade [44]. Intravital microscopy permits direct visualization of leukocyte trafficking in the mesenteric microcirculation through the transparent mesentery, and it therefore has the power to discern each of the three steps in the adhesion cascade. The representative video clips located at the URL: http://www.med.ic.ac.uk/divisions/35/aprotinin.mpg illustrates the effect of aprotinin on leukocyte recruitment to a topically applied chemoattractant, N-Formyl L-Methyl L-Leucyl L-Phenylalanine (fMLP).

The first video clip (before application of fMLP) shows the leukocyte rolling step and demonstrates no significant difference in rolling behavior between aprotinin and control groups. The second video clip depicts firm adhesion of leukocytes to the vessel wall at 20 minutes following fMLP. Again, there is no observable difference in the number of leukocytes firmly adhered between aprotinin and control groups, but there is a discernible drop in the number of extravasated leukocytes in the aprotinin group. The final video clip reveals substantial extravasation of leukocytes at 40 minutes into the surrounding tissue in the control group but few if any in the aprotinin group. Inhibition of leukocyte extravasation at 40 minutes was 73% (p = 0.032). The total dose of aprotinin infused into rats during this procedure, which lasted approximately 75 minutes from the point of incision to exteriorize the mesentery to the final 40 minute time point on the video, was approximately 60,000 KIU/kg, which compares to the total dose given to patients throughout CPB surgery (60,000 to 120, 000 KIU/kg body weight).

It is clear that aprotinin, infused at concentrations relevant to cardiac surgery, exerts no effect on either rolling or adhesion of leukocytes, but it significantly inhibits the passage of leukocytes through the endothelial wall. This observation was supported by parallel in vitro experiments that showed that aprotinin blocked the transmigration of neutrophils through cultured human endothelial cells in response to three different chemoattractants: fMLP, interleukin-8, and platelet-activating factor. By pretreating either neutrophils or endothelial cells separately, aprotinin was found to target each cell type individually, with maximum effects achieved when both were targeted [44]. Our demonstration that aprotinin can block leukocyte extravasation may help explain a previous report that showed that aprotinin can prevent neutrophil accumulation in the bronchial alveolar fluid of CPB patients [50].

Effect of aprotinin on endothelial and bronchial epithelial cells
Systemic activation of endothelial cells by proinflammatory cytokines plays a major role in the margination of leukocytes and pathogenesis of SIRS, yet endothelium-specific actions of aprotinin have been difficult to pinpoint, particularly in vivo, because of the relative inaccessibility of the vascular endothelial compartment. Indirect evidence has suggested, however, that aprotinin can target vascular endothelium: In vivo studies showed that aprotinin can inhibit trypsin-induced vascular permeability in the lung and bronchoalveolar neutrophil accumulation after CPB [50, 51]. More recent in vitro evidence from our group has suggested that aprotinin can act directly on cultured endothelial cells to inhibit adhesion molecule expression and neutrophil transmigration in endothelial monolayers stimulated with tumor necrosis factor alpha [52].

Another cell-type targeted by aprotinin is the bronchial epithelial cell, which has been shown both in culture and in the airways of CPB patients to synthesize significantly lower levels of inducible nitric oxide (iNOS) in the presence of aprotinin [53, 54]. This would be expected to translate into protection against reperfusion injury by inhibiting the synthesis of endogenous nitric oxide secondary to iNOS generation. Animal models have confirmed that reperfusion injury is prevented by aprotinin and that vascular permeability in the lung is protected by aprotinin [51, 55].

Molecular targets of aprotinin within the inflammatory system
There are two basic types of aprotinin target in the inflammatory system: (1) soluble proteases and (2) cell-associated proteases. Most research to date has focused on soluble substrates for aprotinin, such as kallikrein and plasmin. Kallikrein inhibition has been shown to diminish contact activation of leukocytes and platelets during CPB, but whether the necessary dosage to inhibit kallikrein can be achieved clinically remains in doubt [45]. The observed block in leukocyte extravasation [44] may be due to the targeting of soluble proteases, either directly, as in the case of elastase secreted by the migrating cell [56], or indirectly, as in the case of matrix metalloproteinase activation by plasmin, a process that appears to play an essential role in macrophage migration through the vessel wall [57, 58].

Cell-associated target molecules are comparatively difficult to identify, especially in vivo, due to the confounding effects of soluble targets. The use of defined model systems in vitro has been helpful in this regard and has suggested that aprotinin can directly target cell-associated proteases, for example during neutrophil L-selectin shedding [59]. Likely receptor targets are the protease-activating receptors, which, although poorly studied on leukocytes, are known to be expressed throughout the vasculature, on leukocytes, platelets, and endothelium [60].

On platelets we have demonstrated that aprotinin can inhibit PAR1-dependent activation by thrombin and trypsin [20] and it is therefore highly probable that aprotinin can target protease-activating receptors on other cell types as well. For example, endothelial PAR2 is a strong candidate molecule to explain the protective effect of aprotinin on vascular permeability induced by trypsin, because trypsin is a potent proteolytic agonist of PAR2 [51]. The recent demonstration that the protease-activating receptors can be cofactored through interaction with other cell surface proteases, such as membrane-type serine protease-1 [21], coagulation factors upstream of thrombin in the coagulation cascade [22], and other protease-activated receptors [23], has added a further layer of complexity to PAR signaling. At the same time, recognition of the cofactoring of aprotinin has increased the likelihood that these receptors will play a central role in the antiinflammatory mechanism of action of aprotinin during CPB. Much work remains to be done to elucidate the molecular targets for aprotinin within the inflammatory system.

Summary of the antiinflammatory actions of aprotinin
The distribution of serine proteases throughout the vasculature and the key role they play in inflammation may explain how aprotinin, as a nonspecific protease inhibitor, can exert such a multi-tiered antiinflammatory mechanism of action. Its activity ranges from reduced contact activation of platelets and leukocytes, to reduced extravasation and degranulation of leukocytes, reduced systemic cytokine production, reduced endothelial cell activation, reduced vascular permeability, and reduced bronchial epithelial cell activation.

	Conclusion

Top Footnotes Abstract Introduction The thrombosis debate: a... Antiinflammatory action of... Conclusion Acknowledgments References

 
The multi-tiered antiinflammatory mechanism of aprotinin suggests that it should provide significant clinical benefit in the prevention of SIRS after cardiopulmonary bypass surgery. This expectation has been borne out in clinical practice, particularly in the case of high-risk patients, in whom the length of hospital stay after CPB is significantly reduced with aprotinin compared to other antiinflammatory strategies. This benefit more than offsets the initial cost of the drug used during surgery [61]. We hope that by uncovering the antithrombotic mechanism of action in platelets we may have eased the understandable concern regarding the safety of aprotinin and that this drug will find more widespread acceptance in routine cardiac and coronary artery bypass surgery.

	Acknowledgments

Top Footnotes Abstract Introduction The thrombosis debate: a... Antiinflammatory action of... Conclusion Acknowledgments References

 
This work was supported by the British Heart Foundation.

	Footnotes

Top Footnotes Abstract Introduction The thrombosis debate: a... Antiinflammatory action of... Conclusion Acknowledgments References

 
A videoclip of this procedure can be viewed on the Internet at http://www.sts.org/section/atsvideo.

	References

Top Footnotes Abstract Introduction The thrombosis debate: a... Antiinflammatory action of... Conclusion Acknowledgments References

 

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