Extracorporeal Circulation

The Response of Humoral and Cellular Elements of Blood to Extracorporeal Circulation

THROMBOSIS AND BLEEDING

Initial Reactions in the Perfusion Circuit

When heparinized blood contacts any biomaterial, plasma proteins are instantly adsorbed (<1 s) onto the surface to form a monolayer of selected proteins.^370–372 For each protein the amount adsorbed depends on its bulk concentration in plasma and the intrinsic surface activity of the biomaterial. Different biomaterials have different intrinsic surface activities for each plasma protein. The physical and chemical composition of the biomaterial surface determine the intrinsic surface activity of the biomaterial, but intrinsic surface activity is not predictable from knowledge of chemical and physical characteristics. Thus intrinsic surface activity differs among biomaterial surfaces, among plasma proteins, and among different bulk concentrations of plasma proteins. Concentrations of plasma proteins on a given biomaterial differ from concentrations in bulk plasma. Similarly, concentrations of surface-adsorbed proteins from the same plasma differ on different biomaterials. The composition of the protein monolayer is specific for the biomaterial and for various concentrations of proteins in the plasma, but the topography of the adsorbed protein layer may not be uniform across the surface of the biomaterial.³⁷³ Thus it is not possible to predict the "thrombogenicity" of any biomaterial except by trial and error.

On most biomaterial surfaces fibrinogen is selectively adsorbed, but the adsorbed concentration of fibrinogen and other proteins may change over time.³⁷² Surface-adsorbed proteins "compete" for space on the biomaterial surface, but are tightly packed, irreversibly bound, and immobile. The density of surface-adsorbed proteins is 100 to 1000 times greater than the density of proteins in bulk plasma.³⁷³ The complexity of blood-biomaterial interactions is further compounded by the fact that adsorbed proteins often undergo limited conformational changes^374,375 that may expose "receptor" amino acid sequences that are recognized by specific blood cells or bulk plasma proteins. Conformational changes of adsorbed factor XII and fibrinogen initiate activation of the contact pathway and platelet surface adhesion, respectively; similar changes in complement protein 3 participate in activation of the complement system.³⁷⁵ For a given adsorbed protein these conformational changes may vary between biomaterial surfaces and in turn vary the reactivity of the adsorbed protein with cells and blood proteins in the bulk phase.

Thus heparinized blood does not directly contact biomaterial surfaces in extracorporeal perfusion circuits, but contacts monolayers of densely packed, immobile plasma proteins arranged in undefined mosaics that differ between locations and possibly across time. All biomaterial surfaces, including heparin-coated surfaces, are procoagulant;^367,376 only the endothelial cell is truly nonthrombogenic (Fig. 12-9).

View larger version (128K): [in this window] [in a new window]   Figure 12-9 Electron micrograph of a rabbit endothelial cell (E), the only known nonthrombogenic surface. Note the overlapping junctions with neighboring endothelial cells. Endothelial cells rest on the internal elastic lamina (I), which abut medial smooth muscle cells. The vessel lumen is at the top. (Reproduced with permission from Stemerman MB: Anatomy of the blood vessel wall, in Colman RW, Hirsh J, Marder VJ, Salzman E [eds]: Hemostasis and Thrombosis: Basic Principles and Clinical Practice, 2nd ed. Philadelphia, JB Lippincott, 1987; p 775.)

Extracorporeal perfusion and CPB are not possible without anticoagulation; the large procoagulant surface quickly overwhelms natural circulating anticoagulants—antithrombin, proteins C and S, tissue factor pathway inhibitor, and plasmin—to produce thrombin and thrombosis within the circuit. Thrombin is produced in extracorporeal perfusion systems with small surface areas and high-velocity flow,^368,377,378 but thrombosis may not be apparent if other procoagulants (e.g., addition of blood from wounds) are absent. Generation of thrombin varies widely between applications of extracorporeal technology (see below), but this powerful and potentially dangerous enzyme is produced whenever blood contacts a nonendothelial cell surface (Fig. 12-10).

View larger version (27K): [in this window] [in a new window]   Figure 12-10 Plasma thrombin-antithrombin (TAT) measurements of thrombin generation during CPB and clinical cardiac surgery of varying duration. (Data from Brister et al.366)

Heparin is also associated with some clinical idiosyncrasies. In some patients recent, prolonged parenteral heparin may reduce antithrombin concentrations and produce heparin resistance.^380,394,395 Insufficient antithrombin may also occur due to insufficient synthesis or increased consumption in some cyanotic infants, premature babies, cachectic patients, and patients with advanced liver or renal disease. The deficiency in antithrombin prevents heparin from prolonging activated clotting times to therapeutic levels. In these patients fresh frozen plasma is needed to increase plasma antithrombin concentrations to inhibit thrombin. Heparin rebound is a delayed anticoagulant effect after protamine neutralization due to the rapid metabolism of protamine and delayed seepage of heparin into the circulation from lymphatic tissues and other deposits. Heparin is also associated with an allergic response in some patients that produces heparin-induced thrombocytopenia (HIT) with or without thrombosis (see below). Lastly, heparin only partially suppresses thrombin formation during CPB and all applications of extracorporeal perfusion and mechanical circulatory and respiratory assistance despite doses two to three times those used for other indications (see Fig. 12-10).^365–368 Thus heparin is far from an ideal anticoagulant.

Potential alternatives for heparin during extracorporeal perfusion (ECP) include low-molecular-weight heparin, danaparoid (Organan), recombinant hirudin (Lepirudin), and the organic chemical argatroban (Texas Biotechnology Corp., Houston, Texas). All have important drawbacks and are approved for use in HIT and in patients with circulating IgG anti-heparin-PF4 complex antibodies (see below). Low-molecular-weight heparins have long half-lives in plasma (4 to 8 hours), require antithrombin as a cofactor, primarily inhibit factor Xa, and are not reversible by protamine.^396,397 Although less antigenic than standard heparin, low-molecular-weight heparins can stimulate production of IgG antiheparin-PF4 complex antibodies.³⁹⁷ Danaparoid is a mixture of heparin sulfate, dermatan sulfate, and chrondroitin sulfate that catalyzes antithrombin to inhibit thrombin and factor Xa. To a lesser extent, danaparoid also catalyzes inhibition of thrombin by heparin cofactor II. The anticoagulant effect is long lasting (plasma half-life 4.3 hours)³⁹⁸ and is not reversed by protamine.

Recombinant hirudin (Lepirudin) is a direct inhibitor of thrombin, is effective rapidly, does not have an effective antidote, is monitored by the partial thromboplastin time, is cleared by the kidney, and has a relatively short half-life in plasma (40 minutes).³⁹⁹ This drug has been successfully used during CPB and open heart surgery, but in many instances bleeding after bypass has been troublesome and substantial. A newer drug is a semisynthetic bivalent thrombin inhibitor composed of 12 amino acids from hirudin, which binds to exosite 1 of thrombin linked to an active site-directed moiety, D Phe Pro Arg Pro, by four glycines.⁴⁰⁰ This drug, bivalirudin (Angiomax), has a shorter half-life than hirudin and therefore may be safer. In addition, only a small amount is excreted by the kidney. In coronary angioplasty, bivalirudin was as effective as heparin but there was less bleeding. Argatroban is also a direct thrombin inhibitor⁴⁰¹ with rapid onset of action and short plasma half-life (40 to 50 minutes).⁴⁰² Argatroban is metabolized in the liver and is without an antidote, but can be monitored with partial thromboplastin times or activated clotting times. At present there is little clinical experience with argatroban or bivalirudin in cardiac surgical patients.

HEPARIN-ASSOCIATED THROMBOCYTOPENIA, HEPARIN-INDUCED THROMBOCYTOPENIA, AND HEPARIN-INDUCED THROMBOCYTOPENIA AND THROMBOSIS

Heparin-associated thrombocytopenia is a benign, nonimmune, 5 to 15% decrease in platelet count that occurs within a few hours to 3 days after heparin exposure. The etiology is due to mild platelet stimulation from multifactorial causes; bleeding does not occur; and the condition is clinically inconsequential.⁴⁰³

Heparin-induced thrombocytopenia (HIT) and heparin-induced thrombocytopenia and thrombosis (HITT) are different manifestations of the same immune disease. Heparin binds to platelets in the absence of an antibody and releases small amounts of platelet factor 4 (as occurs in heparin-associated thrombocytopenia). PF4 avidly binds heparin to form a heparin-PF4 (H-PF4) complex, which is antigenic in some people. In these individuals IgG antibodies to the H-PF4 complex are produced within 5 to 15 days after exposure to heparin and continue to circulate in the absence of more heparin for approximately 3 to 6 months.⁴⁰⁴ IgG-anti-H-PF4 antibodies plus H-PF4 complexes form HIT complexes, which unite IgG Fc terminals to platelet Fc receptors (Fig. 12-11). This binding strongly stimulates platelets to release more PF4.⁴⁰⁵ A self-perpetuating, accelerating cascade of platelet activation, release, and aggregation ensues. Since platelet granules contain several procoagulatory proteins (e.g., thrombin, fibronectin, factor V, fibrinogen, and von Willebrand factor), release also activates coagulation proteins to generate thrombin.

View larger version (25K): [in this window] [in a new window]   Figure 12-11 The generation of HIT complexes. Read each horizontal group of three left to right beginning at top left. See text for full explanation.

IgG anti-H-PF4 antibodies are detected in two ways. The serotonin release test detects the release of radioactive serotonin from normal platelets washed by the patient’s serum.⁴⁰⁷ An enzyme immunoassay measures IgG anti-H-PF4 antibodies directly. Both assays are equally sensitive in patients with clinical HIT, but the enzyme immunoassay is more sensitive in detecting IgG anti-H-PF4 antibodies in patients without other evidence of the disease.⁴⁰⁷

The clinical presentation of HIT may be insidious. If the platelet count was originally normal, the earliest sign is an abrupt decrease of at least 50% in platelet count (to less than 150,000/µL) in a patient who has had exposure to heparin within the past 5 to 15 days.⁴⁰⁴ This event is a preoperative stop sign for elective cardiac operations. After CPB, platelet counts below 80,000/µL should trigger an order to stop all heparin, including heparin flushes, and to obtain daily platelet counts. The patient should be thoroughly examined for deep vein thrombosis, extremity ischemia, stroke, myocardial infarction, or any evidence of intravascular thrombosis using ultrasound and appropriate radiographic technology. Any evidence of vascular thrombosis should prompt a plasma sample for IgG anti-H-PF4 antibodies. A positive antibody test confirms the diagnosis of HIT in patients with thrombocytopenia and HITT in those with either venous or arterial thrombosis or both. It is important to stress that HIT or HITT is a clinical diagnosis and that a positive antibody test is not required before stopping heparin.

Once the diagnosis of HIT or HITT is suspected, management must focus on prevention of further intravascular thrombosis. Bleeding is rarely the problem; intravascular thrombosis is. Neither heparin nor platelet transfusions should be given; platelet transfusions only add more PF4 if heparin and IgG anti-H-PF4 antibodies are still circulating. If heparin is proven absent from the circulation, platelet transfusions may be used very cautiously if the patient has significant nonsurgical bleeding. Surgical measures to reopen thrombosed large arteries are usually futile because the platelet-rich thrombus (white clot) often extends into small arteries and arterioles. An inferior vena cava filter is recommended if pulmonary embolism is likely or has occurred.

Modern management also includes full anticoagulation with recombinant hirudin (Lepirudin), argatroban, or possibly bivalirudin to prevent further extension of thrombosis or development of clinical intravascular thrombosis. This may occur in 40 to 50% of patients with HIT who are treated only with heparin cessation.⁴⁰⁸ At present there is little experience with argatroban in cardiac surgical patients with HITT, but the drug is a direct thrombin inhibitor, has attractive pharmacokinetics, and is approved for patients with HITT. Full anticoagulation with hirudin in fresh postoperative cardiac surgical patients is recommended, but the safety zone between bleeding and thrombosis is narrow. The patient must be carefully monitored for pericardial tamponade and signs of hidden bleeding. Hirudin is monitored by activated partial thromboplastin time and the range used is similar to that with intravenous heparin. The effective blood concentration of hirudin for thrombin inhibition is 0.5 to 1.5 µg/mL.⁴⁰⁹ To achieve this, 0.2-mg/kg/h infusions are recommended.⁴⁰⁹ Dose must be reduced in patients with renal failure because the kidney clears the drug. Argatroban is sometimes a better choice, but it should be remembered that it is difficult to manage in the presence of liver disease since it is metabolized in that organ. In most patients oral anticoagulation with warfarin is started at the same time as intravenous hirudin, but warfarin should not be started prior to hirudin.

Emergency or urgent open heart surgery with CPB using hirudin is possible in patients with circulating IgG anti-HPF4 antibodies. The therapeutic level of drug should be between 3.5 and 4.5 µg/mL during CPB.⁴⁰⁹ Greinacher recommends bolus doses of 0.25 mg/kg IV and 0.2 mg/kg in the priming volume followed by an infusion of 0.5 mg/min until 15 minutes before stopping CPB.⁴⁰⁹ At that time 5 mg of hirudin is added to the perfusate to prevent clotting within the heart-lung machine.

Patients who require elective cardiac surgery are best deferred until circulating IgG anti-H-PF4 antibodies are absent by enzyme immunoassay. Patients with a history of HIT who require elective cardiac surgery with CPB should have IgG anti-H-PF4 antibodies measured in their serum before surgery is scheduled. If antibodies are absent, elective surgery can be safely carried out using heparin anticoagulation, if the first re-exposure to heparin is the bolus dose given just before starting CPB. Since HIT requires the presence of the H-PF4 complex plus IgG anti-H-PF4 antibodies to form the HIT complex, and since it takes about 5 days to produce these antibodies, HIT or HITT will not occur if no further heparin is given after operation.

COAGULATION AND EXTRACORPOREAL PERFUSION THROMBIN GENERATION

Generation of thrombin during cardiopulmonary bypass and other applications of extracorporeal circulatory technology is the cause of the thrombotic and bleeding complications associated with ECP. Theoretically, if thrombin formation could be completely inhibited during ECP, the consumptive coagulopathy, which consumes coagulation proteins and platelets and causes bleeding complications, would not occur.

Thrombin generation and the fibrinolytic response primarily involve the extrinsic and intrinsic coagulation pathways, the contact and fibrinolytic plasma protein systems, and platelets, monocytes, and endothelial cells.

Contact System

The contact system includes four primary plasma proteins— factor XII, prekallikrein, high-molecular-weight kininogen, and C-1 inhibitor⁴¹⁰—and is activated during CPB and clinical cardiac surgery.⁴¹¹ This system is involved in complement and neutrophil activation and the inflammatory response to ECP, but is not involved in thrombin formation in vivo. However, when blood contacts a negatively charged surface (protein surfaces contain both positive and negative charges) in ECP, small amounts of factor XII are adsorbed and undergo a conformational change to factor XIIa.^373,412 Factor XIIa in the presence of high-molecular-weight kininogen activates factor XI and initiates the intrinsic coagulation pathway (Fig. 12-12). Thrombin also activates factor XI, and is the predominating agonist in vivo in pathologic states.⁴¹³

View larger version (65K): [in this window] [in a new window]   Figure 12-12 Steps in the generation of thrombin in the wound and in the perfusion circuit via the extrinsic, intrinsic, and common coagulation pathways. Ca++ = calcium ion; HMWK = high-molecular-weight kininogen; mono = monocyte; PK = prekallikrein; PL = cellular phospholipid surface; TF = tissue factor. Activated coagulation proteins are indicated by the suffix "a."

The intrinsic coagulation pathway probably does not generate thrombin in vivo, but does initiate thrombin formation when blood contacts nonendothelial cell surfaces such as perfusion circuits.^414,415 Factor XIa, produced by activation of the contact system and subsequently thrombin generation, activates factor IX, which forms part of the intrinsic tenase complex.^416,417 Factor XI is primarily activated by thrombin (see Fig. 12-12).

Extrinsic (Tissue Factor) Coagulation Pathway

The extrinsic coagulation pathway is the major coagulation pathway in vivo and is a major source of thrombin generation during CPB and clinical cardiac surgery.^418,419 Exposure of blood to tissue factor by direct contact in the wound or by wound blood aspirated into the ECP circuit initiates the extrinsic coagulation pathway.⁴¹⁷ Tissue factor (TF) is a cell-bound glycoprotein that is constitutively expressed on the cellular surfaces of fat, muscle, bone, epicardium, adventitia, injured endothelial cells, and many other cells except pericardium.^419–421 Plasma TF associated with wound monocytes is a second source of TF and may be an important source during CPB and clinical cardiac surgery.⁴²² Tissue factor is the cofactor for the activation of factor VII to factor VIIa, which is part of extrinsic tenase (see Fig. 12-12).

Tenase Complexes

Intrinsic and extrinsic tenase catalyze the activation of factor X to factor Xa (see Fig. 12-12). Extrinsic tenase is formed by the combination of tissue factor, factor VIIa, calcium, and a phospholipid surface to cleave a small peptide from factor X to form factor Xa.⁴¹⁷ Extrinsic tenase also generates small amounts of factor IXa,⁴²³ which greatly accelerates formation of intrinsic tenase and is the major pathway for the formation of factor Xa. Intrinsic tenase is produced by the combination of factor IXa, factor VIIIa, and calcium on the surface of an activated platelet,⁴²⁴ and catalyzes production of factor Xa 50 times faster than extrinsic tenase.⁴¹⁷ Factor Xa activates factors V and VII in feedback loops.

Common Coagulation Pathway

Factor Xa is the gateway protein of the common coagulation pathway. Factor Xa slowly cleaves prothrombin to alpha-thrombin, the active enzyme, and a fragment, F1.2, but the reaction is 300,000 times faster if catalyzed by the prothrombinase complex.⁴¹⁷ The prothrombinase complex is produced when factor Xa, in the presence of Ca²⁺, is anchored by factor Va onto a phospholipid surface provided by platelets, monocytes, or endothelial cells.⁴¹⁷ Either factor Xa or thrombin activates factor V to factor Va. The prothrombinase complex cleaves prothrombin to alpha-thrombin and a fragment, F1.2, and is the major pathway producing thrombin.⁴¹⁷ F1.2 is a useful marker of the reaction.

Thrombin

Thrombin is a powerful enzyme that accelerates its own formation by several feedback loops.⁴²⁵ Thrombin is the major activator of factor XI and the exclusive activator of factor VIII in the intrinsic pathway. Thrombin is a secondary activator of factor VII, but once formed may be the most important activator in the wound. Lastly, thrombin is the primary activator of factor V in the formation of the prothrombinase complex (see Fig. 12-12).

Thrombin has both procoagulant and anticoagulant properties.⁴²⁵ Thrombin is the enzyme that cleaves fibrinogen to fibrin and in the process creates two fragments, fibrinopeptides A and B. Thrombin activates platelets via the platelet thrombin receptor and thus may be the major agonist for platelets both in the wound and in the perfusion circuit. Thrombin also activates factor XIII to cross-link fibrin to an insoluble form and to attenuate fibrinolysis. Lastly, thrombin activates thrombin-activated fibrinolysis inhibitor, which alters fibrin to reduce lysis.⁴²⁵

Thrombin also stimulates the production of anticoagulants. Surface glycosaminoglycans, such as heparan sulfate, inhibit thrombin and coagulation via antithrombin. Thrombin stimulates endothelial cells to produce tissue plasminogen activator (t-PA), which is the major enzyme that cleaves plasminogen to plasmin. Thrombin also stimulates the production of nitric oxide and prostaglandin by endothelial cells. Thrombin in the presence of thrombomodulin activates protein C, which in the presence of protein S destroys activated factor V and VIII.

Thrombin Generation during Extracorporeal Perfusion

All applications of extracorporeal perfusion and exposure of blood to nonendothelial cell surfaces generate thrombin.^365–367 F1.2 is a protein fragment that is formed when prothrombin is cleaved to thrombin; thus F1.2 is a measure of thrombin generation but not of thrombin activity. F1.2 and thrombin-antithrombin complex increase progressively during clinical cardiac surgery with CPB, during applications of circulatory assist devices,^368,379 and during extracorporeal life support (see Fig. 12-10). The amount of thrombin produced seems to vary with the intensity of the stimuli for thrombin production and may vary with age, comorbid disease, and clinical health of the patient. The cytokines interleukin-1-beta (IL-1-beta) and tumor necrosis factor-alpha (TNF-alpha) are procoagulant and inhibit the thrombomodulin/protein C anticoagulant pathway and stimulate production of type I plasminogen activator inhibitor.⁴²⁶ Complex cardiac surgery that requires several hours of CPB produces more F1.2³⁶⁶ than short procedures with minimal exposure of circulating blood to the wound.⁴²⁷ Thrombin generation varies with the amount and type of anticoagulant used; surface area of the blood-biomaterial interface; duration of exposure to the surface; turbulence, stagnation, and cavitation within perfusion circuits; and to a lesser degree temperature and the "thromboresistant" characteristics of biomaterial surfaces.⁴²⁸ Very high concentrations of heparin, sufficient to increase spontaneous bleeding, reduce F1.2 production, probably by interfering with thrombin-activated feedback loops,⁴²⁹ since heparin does not directly inhibit thrombin formation.

For many years blood contact with the biomaterials of the perfusion circuit was thought to be the major stimulus to thrombin formation during CPB and open heart surgery. Increasing evidence indicates that the wound is the major source of thrombin generation during CPB and clinical cardiac surgery.^430–431 This understanding has encouraged development of strategies to reduce the amounts of circulating thrombin during clinical cardiac surgery by either discarding wound blood⁴³² or by exclusively salvaging red cells by centrifugation and washing in a cell saver. The reduced thrombin formation in the perfusion circuit has also supported misguided strategies for reducing the systemic heparin dose during first-time coronary revascularization procedures using heparin-bonded circuits.⁴²⁷ While there is no good evidence that heparin-bonded circuits reduce thrombin generation,³⁶⁷ there is strong evidence that discarding wound plasma or limiting exposure of circulating blood to the wound (e.g., less bleeding in the wound) does reduce the circulating thrombin burden.^376,432

CELLULAR PROCOAGULANTS AND ANTICOAGULANTS

Platelets

Platelets are activated by thrombin, contact with the surface of nonendothelial cells, heparin, and platelet-activating factor produced by a variety of cells during all applications of extracorporeal perfusion and/or recirculation of anticoagulated blood that has been exposed to a wound. Circulating thrombin and platelet contact with surface-adsorbed fibrinogen in the perfusion circuit are probably the earliest and strongest agonists. Circulating thrombin, although rapidly inhibited by antithrombin, is a powerful agonist and binds avidly to two specific thrombin receptors on platelets: PAR-1 and GPIb-alpha.⁴³³ As CPB continues, C5a, C5b-9,^434,435 plasmin,⁴³⁶ hypothermia,⁴³⁷ platelet-activating factor (PAF), interleukin-6,⁴³⁸ cathepsin G, serotonin, epinephrine, eicosanoids, and other agonists also activate platelets and contribute to their loss and dysfunction.

The initial platelet reaction to agonists is shape change. Circulating discoid platelets extend pseudopods, centralize granules, express glycoprotein Ib (GPIb) and GPIIb/IIIa receptors,⁴³⁹ and secrete soluble and bound P selectin receptors from alpha granules.⁴⁴⁰ GPIIb/IIIa (alpha_IIbbeta3) receptors almost instantaneously bind platelets to exposed binding sites on the alpha- and gamma-chains on surface-adsorbed fibrinogen (Fig. 12-13).⁴⁴¹ The number of adherent platelets is proportional to the amount of surface-adsorbed fibrinogen recognized by fibrinogen antibody,⁴⁴² but the density of adherent platelets also varies with the chemical and physical composition of the surface biomaterial.^443,444 Rough surfaces accumulate more platelets than smooth surfaces.⁴⁴⁵ Fewer platelets adhere to polyurethane, cuprophane, and PMEA (poly-2-methoxyethylacrylate) than to silicone rubber.^374,428 Platelet adhesion and aggregate formation reduce the circulating platelet count, which is already reduced by dilution with pump priming solutions.

View larger version (22K): [in this window] [in a new window]   Figure 12-13 Adhesion of activated platelets binding to surface-adsorbed fibrinogen via GPIIb/IIIa (IIbβ3) receptors. The same receptors bind plasma fibrinogen molecules to form platelet aggregates.

A small percentage of activated platelets synthesize and release a variety of chemicals and proteins from granules that include thromboxane A₂,⁴⁵⁰ platelet factor 4, beta-thromboglobulin,⁴⁵¹ P-selectin, and serotonin. Platelet lysosomes release neutral proteases and acid hydrolases.⁴⁵²

During ECP the circulating platelet pool is reduced by dilution, adhesion, aggregation, destruction, and consumption. The platelet mass consists of a reduced number of morphologically normal platelets, platelets with pseudopod formation, new and larger platelets released from megakaryocytes,⁴⁵³ partially and completely degranulated platelets, platelet membrane fragments, platelet microparticles, and resealed platelets that have lost some of their membrane receptors.^{446,447,452,453} Most of the circulating platelets appear structurally normal,⁴⁵³ but bleeding times increase and remain prolonged for several hours after protamine.⁴⁵⁴ The functional state of the circulating intact platelet during and early after CPB is reduced, but it is not clear whether this functional defect is intrinsic or extrinsic to the platelet. Flow cytometry studies of circulating intact platelets show little change in platelet membrane receptors.⁴⁵⁵ In prolonged applications of ECP, platelets are consumed and may or may not be adequately replaced by new platelets from the bone marrow.⁴⁵⁶

Monocytes

During CPB and clinical cardiac surgery the concentration of plasma tissue factor, which normally is 0.26 to 1.1 pM,^422,457 doubles to 2.0 pM.⁴³² During cardiac surgery wound plasma contains 6 to 11 pM.⁴³² In the wound with calcium present, monocytes associate with plasma tissue factor to rapidly accelerate the conversion of factor VII to factor VIIa.⁴⁵⁸ This association is specific for monocytes—the reaction is essentially nil for platelets, neutrophils, and lymphocytes—and does not occur if monocytes, plasma tissue factor, or factor VII is not present. Monocytes also synthesize and express tissue factor, but this process, which peaks 3 to 4 hours after monocytes are activated,⁴⁵⁸ is not a major source of tissue factor during CPB and clinical heart surgery but does occur during prolonged perfusions.⁴⁵⁹ Plasma microparticles, also present in wound plasma, are procoagulant⁴⁶⁰ and monocytes may express the procoagulant CD 11b receptor,⁴⁶¹ but the clinical importance of these pathways in thrombin generation is not clear and probably minor. The major sources of tissue factor in the wound are the combination of monocytes, plasma tissue factor, and cell-bound tissue factor.

Agonists for activating monocytes during CPB and clinical cardiac surgery include C5a,⁴⁶² endotoxin, IL-6, IL-1-beta, TNF-alpha, and monocyte chemotactic protein-1 (MCP-1). Monocytes and macrophages produce MCP-1, IL-1-beta, IL-6, and TNF-alpha;^463,464 express tissue factor,⁴⁵⁸ Mac-1,⁴⁶¹ L-selectin, and MCP-1;⁴⁶⁵ and form aggregates with platelets.⁴⁴⁰ For the most part, monocyte reactions are slow and peak concentrations of cytokines occur several hours after CPB ends.^466–468

Endothelial Cells

Endothelial cells, charged with maintaining the fluidity of circulating blood and the integrity of the vascular system, are activated during CPB and clinical cardiac surgery by thrombin, C5a,⁴⁶⁹ IL-1, and TNF-alpha.⁴⁷⁰ Endothelial cells produce both procoagulants and anticoagulants. Procoagulant activities of endothelial cells include expression of tissue factor and production of a host of procoagulant proteins, including collagen, elastin, microfibillar protein, laminin, fibronectin, thrombospondin, von Willebrand factor, factor V, platelet-activating factor, and plasminogen activator inhibitor-1, and the vasoconstrictors endothelin-1 and renin. Endothelial cells also bind von Willebrand factor, vibronectin, and factors IXa and Xa. Anticoagulant activities of endothelial cells include the production of t-PA, heparin sulfate, dermatan sulfate, protein S (which accelerates the activation of protein C), tissue factor inhibitor protein, thrombomodulin and protease nexin 1 (which both bind thrombin), prostacyclin,⁴⁷¹ nitric oxide, and adenosine. Prostacyclin concentrations increase rapidly at the beginning of CPB and then begin to decrease.⁴⁷² During clinical cardiac surgery endothelin-1 peaks several hours after CPB ends.⁴⁷³

Except for expression of tissue factor and expression of CD11b/CD18 (Mac-1), which is weakly procoagulant, endothelial cell receptors do not participate heavily in thrombin generation during ECP.

Neutrophils

During ECP neutrophils express Mac-1 receptors,⁴⁷⁴ which bind factor X and fibrinogen and weakly facilitate thrombin formation. Neutrophils secrete elastase, which can destroy protease inhibitors such as antithrombin and coagulation factors such as factor V and may contribute significantly to the equilibrium between the fluid and gel forms of blood.

FIBRINOLYSIS

Circulating thrombin activates endothelial cells to produce t-PA, which binds avidly to fibrin.^475–477 Endothelial cells are the principal source of t-PA.⁴⁷⁶ The combination of t-PA, fibrin, and plasminogen cleaves plasminogen to plasmin; plasmin cleaves fibrin.⁴⁷⁶ This reaction produces the protein fragment D-dimer, which is a useful marker of fibrinolysis, and a marker of thrombin activity because fibrin is cleaved from fibrinogen by thrombin. Kallikrein produced by the contact system cleaves pro-urokinase to urokinase; however, this enzyme is less important in fibrinolysis than t-PA because urokinase binds poorly to fibrin.⁴⁷⁷ F1.2, D-dimer, and fibrinopeptide A (produced by the conversion of fibrinogen to fibrin) increase during extracorporeal perfusion, indicating ongoing thrombin production, fibrin formation, and fibrinolysis.^{366,367,478,479} D-dimer and other fibrin degradation products are themselves anticoagulants inhibiting fibrin polymerization.⁴⁷⁶

Fibrinolysis is controlled by native protease inhibitors, alpha₂-antiplasmin, alpha₂-macroglobulin, and plasminogen activator inhibitor-1.⁴⁷⁷ Plasminogen activator inhibitor-1, produced by endothelial cells, directly inhibits t-PA and urokinase, but little is produced during CPB and open cardiac surgery.⁴⁸⁰ Alpha₂-antiplasmin rapidly inhibits unbound plasmin, preventing the enzyme from circulating, but poorly inhibits plasmin bound to fibrin. Alpha₂-Macroglobulin is a slow inhibitor of plasmin.

Plasmin is both a stimulator and inhibitor of platelets, depending on concentration and temperature.⁴⁸¹ High concentrations of plasmin at normothermia and low concentrations during hypothermia cause conformational changes in platelets, centralization of platelet granules, and internalization of platelet GPIb receptors but not GPIIb/IIIa receptors.⁴⁸²

CONSUMPTIVE COAGULOPATHY

Simultaneous and ongoing thrombin formation and fibrinolysis is by definition a consumptive coagulopathy⁴⁸³ and is present in all applications of ECP. In the normal state the fluidity of blood and the integrity of the vascular system are established and maintained by an equilibrium between procoagulants favoring clot and anticoagulants favoring liquidity (Fig. 12-14A). Blood contact with ECP systems and the wound disrupts this equilibrium to produce a massive procoagulant stimulus that overwhelms natural anticoagulants; therefore an exogenous anticoagulant, heparin, is required for nearly all applications of ECP (Fig. 12-14B). Exceptions are only possible in applications that produce a relatively weak procoagulant stimulus and a minimal thrombin burden that can be contained by natural anticoagulants. Surgeons must realize that any blood exposure to nonendothelial cell surfaces, including prosthetic heart valves, produces a procoagulant stimulus whether or not clot is produced. Except for the healthy endothelial cell, no nonthrombogenic surface exists.

View larger version (60K): [in this window] [in a new window]   Figure 12-14 (A) The balance between procoagulant and anticoagulant forces that produces an equilibrium that allows blood to circulate. (B). During CPB and OHS (open heart surgery) the normal equilibrium is disturbed by changes in both procoagulants and anticoagulants. Imbalance of procoagulants risks thrombosis; an imbalance of anticoagulants risks bleeding.

MANAGEMENT OF BLEEDING

The cornerstone of bleeding management is meticulous surgical hemostasis during all phases of an operation. The surgical techniques, topical agents, and customary drugs used do not need reiteration for trained surgeons. Most cardiac surgical operations involving CPB are accompanied by net blood losses between 200 and 600 mL. Reoperations, complex procedures, prolonged (>3 hours) cardiopulmonary bypass, and patient factors listed above may be associated with excessive and ongoing blood losses. Most surgeons use an antifibrinolytic, such as aprotinin or epsilon-aminocaproic acid, to reduce fibrinolysis in prolonged or complex operations. Problem patients who bleed excessively after heparin neutralization require an attempt to rebalance pro- and anticoagulants to near normal pre-CPB concentrations.

The most useful tests in the operating room are an activated clotting time or a protamine titration test to assess the presence of heparin, prothrombin time to uncover deficiency in the extrinsic coagulation pathway, and platelet count. If heparin is neutralized, the partial thromboplastin time may be measured to assess possible deficiency of coagulation proteins. Other tests such as measurements of fibrinogen, template bleeding time, and the thromboelastography are controversial and/or difficult to obtain. Platelet counts below 80,000 to 100,000/µL require platelet transfusions in bleeding patients, except those with IgG anti-H-PF4 antibodies, to add functioning platelets to the mass of partially dysfunctional platelets.

Measurements of F1.2 and D-dimer are two tests that can be very helpful and probably should be made available on an emergency basis in hospitals that perform complex procedures and offer mechanical circulatory and respiratory assistance. F1.2 measures thrombin formation by factor Xa, and if absent or low, there may be a deficiency in the concentrations of coagulation proteins; fresh frozen plasma is needed. If F1.2 and D-dimer (a measurement of fibrinolytic activity) are both elevated, thrombin is being formed and an antifibrinolytic (aprotinin or epsilon-aminocaproic acid) is needed to neutralize plasmin. If both markers or F1.2 remain elevated after the antifibrinolytic drug, this indicates continuing thrombin generation and the cause (e.g., usually infection) should be aggressively treated with antibiotics. Some thrombin is needed to stop bleeding, but excessive thrombin production feeds the consumptive coagulopathy. As with disseminated intravascular coagulopathy,⁴⁸³ no guaranteed therapeutic recipe is known; success requires patience, persistence, and judicious use of platelets, antifibrinolytics, specific clotting factors, and replacement transfusions to rebalance the coagulation equilibrium at near normal concentrations of the constituents.

THE INFLAMMATORY RESPONSE

The inflammatory response to CPB is initiated by contact between heparinized blood and nonendothelial cell surfaces.^484–486 Blood contact with nonendothelial cell surfaces in the wound and in the perfusion circuit activates plasma zymogens and cellular blood elements that constitute part of the body’s defense reaction to all noxious substances including infectious agents, toxins, foreign antigens, allergens, and also injuries. All surgery, like accidental trauma, triggers an acute inflammatory response, but the continuous exposure of heparinized blood to nonendothelial cell surfaces followed by reinfusion of wound blood and recirculation within the body greatly magnifies this response in operations in which CPB is used. Although far from fully described and understood, this primary "blood injury" produces a unique response, which is different in detail from that caused by other threats to homeostasis.

The principal blood elements involved in this acute defense reaction are contact and complement plasma protein systems, neutrophils, monocytes, endothelial cells, and to a lesser extent platelets. Lymphocytes are also altered by CPB,^487,488 but are more involved in the immune response to foreign proteins and acute rejection and do not materially contribute to the acute response to CPB. Likewise, eosinophils and basophil/mast cells are primarily activated by IL-5 and IgE antibodies, respectively, and have prominent roles in allergy, parasitic diseases, and histamine production. When activated during CPB, the principal blood elements release vasoactive and cytotoxic substances, produce cell signaling inflammatory and inhibitory cytokines, express complementary cellular receptors that interact with specific cell signaling substances and other cells, and generate a host of vasoactive and cytotoxic substances that circulate.⁴⁸⁹ Normally these reactive blood elements mediate and regulate the defense reaction,^490–492 but during CPB an orderly, targeted response is overwhelmed by the massive activation and circulation of these reactive blood elements.

Admittedly there is considerable overlap between the plasma and blood cellular responses involved in bleeding and thrombosis, ischemia/reperfusion,⁴⁹³ acute rejection, and acute and chronic inflammation, but these responses are separated in this book in the interest of simplification. This section offers a simplified overview of the acute inflammatory response to cardiopulmonary bypass; the detailed interactions of the body’s defense system against hurtful stimuli are under active and intense investigation and are far beyond the author’s expertise.

PRIMARY BLOOD CONSTITUENTS

Complement

The complement system constitutes a group of more than 30 plasma proteins that interact to produce powerful vasoactive anaphylatoxins, C3a, C4a, and C5a, and the terminal complement cytotoxic complex, C5b-9.⁴⁹⁴ Complement is activated by three pathways, but only the classical and alternative pathways are involved in cardiopulmonary bypass,^495,496 although a role for the mannose-lectin pathway has not been excluded. Direct contact between heparinized blood and the synthetic surfaces of the extracorporeal perfusion circuit activates the contact plasma proteins and the classical complement pathway.⁴⁹⁵ Activation of C1, possibly by activated factor XIIa, sequentially activates C2 and C4 to form C4b2a (classical C3 convertase) that cleaves C3 to form C3a and C3b (Fig. 12-15).⁴⁹⁴

View larger version (61K): [in this window] [in a new window]   Figure 12-15 Steps in activation of the classical and alternative complement pathways and formation of the membrane attack complex, C5b-9. (Adapted with permission from Walport494 and Plumb and Sadetz.684)

The complement system is activated at three different times during CPB and cardiac surgery: during blood contact with nonendothelial cell surfaces^495,499 and wound exudate containing tissue factor;⁴⁸⁵ after protamine administration and formation of the protamine-heparin complex;^495,500 and after reperfusion of the ischemic, arrested heart.⁴⁹³ CPB and myocardial reperfusion activate complement by both the classical and alternative pathways; the heparin-protamine complex activates complement by the classical pathway.⁴⁹⁵ Other agonists that activate the classical pathway during CPB include endotoxin,⁴⁹⁶ apoptotic cells, and C-reactive protein.⁴⁹⁴

The two C3 convertases effectively merge the two complement pathways by producing C3b, which activates C5 to C5a and C5b (see Fig. 12-15). C3a and C5a are potent vasoactive anaphylatoxins. C5a, which avidly binds to neutrophils and therefore is difficult to detect in plasma, is the major agonist. C3b acts as an opsonin, which binds target cell hydroxyl groups and renders them susceptible to phagocytic cells expressing specific receptors for C3b.^494,497 C5b is the first component of the terminal pathway that ultimately leads to formation of the membrane attack complex, C5b-9. In prokaryotic cells like erythrocytes, C5b-9 creates transmembrane pores, which cause death by intracellular swelling following loss of the intracellular/interstitial osmotic gradient. In eukaryotic cells, deposits of C5b-9 may not be immediately lethal but may eventually cause injury mediated by release of arachidonic acid metabolites (thromboxane A₂ and leukotrienes) and oxygen free radicals by macrophages and neutrophils, respectively.⁴⁹⁷

Together, C5a and C5b-9 play major roles in promoting neutrophil–endothelial cell interactions through upregulation of specific adhesion molecules (see below). Importantly, C5b-9 may also activate platelets and promote platelet-monocyte aggregates.⁵⁰¹ As such, these complement proteins contribute to neutrophil loss from the circulation by adhesion to surface-bound platelets,⁵⁰¹ but more importantly to endothelial cells. The interaction between complement proteins and neutrophils contributes to postoperative organ damage in both adults⁵⁰² and in children.⁵⁰³

Normally, several regulatory proteins modulate the inflammatory actions of C5a and C5b-9 by inactivating convertases, which cleave C3 and C5,⁵⁰⁴ but these inhibitors are usually overwhelmed during CPB. Two proteins, factors H and I, are soluble; three others, complement receptor 1 (CD35), decay accelerating factor, and membrane cofactor protein (CD46), are membrane bound.⁴⁹⁴ Factor I cleaves C3 into inactive iC3b, which cannot form C3 convertase, but can be an opsonin.⁵⁰⁵ Factor H is the dominant complement regulatory protein and competes with factor B in binding to C3.⁴⁹⁴ CD59 and homologous restriction factor are direct inhibitors of the membrane attack complex.^497,506

Neutrophils

Leukocyte counts decrease in response to hemodilution during CPB and increase moderately after operation.^486,507 Only a few neutrophils attach to synthetic surfaces, to each other, or to platelets and monocytes.^507,508 Nevertheless, neutrophils are strongly activated during CPB (Fig. 12-16).^486,509 The principal agonists are kallikrein⁵¹⁰ and C5a^511,512 produced by the contact and complement systems, respectively.^511,513,514 C5a, generated early during CPB and clinical cardiac surgery, is a particularly potent chemotactic protein that induces neutrophil chemotaxis, degranulation, and superoxide generation.⁵¹⁵ Other agonists involved during CPB include IL-1-beta,⁵¹⁶ TNF-alpha,^492,517 IL-8,⁵¹⁸ C5b-9,⁵¹² factor XIIa,⁵¹⁹ heparin, histamine, hypochlorous acids, and products of arachidonate metabolism (leukotriene B₄),⁴⁹² platelet activating factor (PAF), and thromboxane A₂.⁵¹⁵ Lastly, CPB, perhaps mediated by IL-6 and IL-8,⁵²⁰ partially inhibits neutrophil apoptosis and prolongs the period of neutrophil activity.⁵²¹

View larger version (122K): [in this window] [in a new window]   Figure 12-16 Scanning electron micrographs of resting neutrophils (left) and 5 seconds after exposure to a chemoattractant (right). (Reproduced with permission from Baggiolini M: Chemokines and leukocyte traffic. Nature 1998; 392:565.)

View larger version (42K): [in this window] [in a new window]   Figure 12-17 Mechanism of arrest and transmigration of neutrophils into the interstitial space. Neutrophils constitutively express L-selectin, which binds to endothelial cell glycoprotein ligands. Simultaneously, early response cytokines stimulate endothelial cells to rapidly express P-selectin and later E-selectin receptors, which weakly bind neutrophil P-selectin glycoprotein-1 (PSGL-1) ligands. Marginated neutrophils, which are slowed by local vasoconstriction and reduced blood flow, lightly adhere to endothelial cells via selectin expression and begin to roll. Neutrophils activated by C5a, kallikrein, and early response cytokines express β-2 CD11b and c receptors, which bind firmly to cytokine-activated endothelial cell integrins, intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). Once arrested, L-selectins are shed and platelet-endothelial cell adhesion molecule (PECAM) receptors on endothelial cell surfaces mediate neutrophil transmigration through endothelial cell junctions, led by chemoattractants into the interstitial space. PMN = polymorphonuclear leukocyte; PSGL-1 = P-selectin glycoprotein ligand-1.

Neutrophils vary considerably among individuals in expression of adhesive receptors⁵⁴³ and responsiveness to chemoattractants during CPB. There also is substantial variation in measurements of soluble and cellular adhesion receptors.⁵³⁴ The presence of diabetes,⁵⁴⁴ oxidative stress,⁵⁴⁵ and perhaps genetic factors (see below) influences expression of cellular and soluble adhesive receptors and cytokines, which affect neutrophil adhesion and release of granule contents. It is difficult to show a correlation between markers of neutrophil activation and measurements of organ dysfunction.⁵⁴⁶

Neutrophils contain a potent arsenal of proteolytic and cytotoxic substances. Azurophilic granules contain lysozyme, myeloperoxidase, cationic proteins, elastase, collagenases, proteinase 3, acid hydrolases, defensins, and phospholipase.⁵⁴⁷ Specific granules contain beta-2 integrins, lactoferrin, lysozyme, type IV collagenase, histaminase, heparanase, complement activator, alkaline phosphatase, and membrane-associated NADPH (nicotinamide adenine dinucleotide phosphate, reduced form) oxidase.⁵¹⁵ Activated neutrophils, in a "respiratory burst," also produce cytotoxic reactive oxygen and nitrogen intermediates including superoxide anion, hydrogen peroxide, hydroxyl radicals, singlet oxygen molecules, N-chloramines, hypochlorous acids, and peroxynitrite.^490,548 Finally, neutrophils produce arachidonate metabolites, prostaglandins, leukotrienes, and platelet-activating factor. During CPB these vasoactive and cytotoxic substances are produced and released into the extracellular environment and circulation.^486,489 Circulation of these substances mediates many of the manifestations of the "whole body inflammatory response" or "systemic inflammatory response syndrome" associated with CPB and clinical cardiac surgery.⁵⁴⁹

Monocytes

Monocytes and macrophages (tissue monocytes) are relatively large, long-lived cells that are involved in both acute and chronic inflammation. Monocytes respond to chemical signals, are mobile, phagocytize microorganisms and cell fragments, produce and secrete chemical mediators, participate in the immune response, and generate cytotoxins.⁵⁵⁰ Monocytes are activated during CPB⁵⁵¹ and have a major role in thrombin formation.⁵⁵² Monocytes also produce and release many inflammatory mediators during acute inflammation including proinflammatory cytokines (principally TNF-alpha, IL-1-beta, IL-6, IL-8, and MCP-1), reactive oxygen and nitrogen intermediates, and prostaglandins.⁵²⁴

The mechanism by which monocytes are initially activated during CPB is not known, but the most likely candidates are C5a,⁵⁵⁰ thrombin,⁵⁵³ platelet factor 4, and bradykinin,⁵⁵⁴ which are four potent agonists rapidly generated from blood contact with nonendothelial cell surfaces. Monocytes possess a huge list of surface receptors,⁵⁵⁰ but those apt to be involved in the inflammatory response to CPB are C5a and three other complement proteins (IL-1, CD11b/CD18, and CD 11c/CD18), leukotriene B₄, and the C-C family of chemokine receptors.⁵⁵⁰ Monocytes also possess C-reactive protein receptors, which when activated, strongly upgrade proinflammatory cytokine production.⁴⁹¹

Monocytes are the major source of the early response cytokines IL-1-beta and TNF-alpha,^491,554 which play an important role in directing both neutrophils and monocytes to local sites of inflammation. Monocytes are also the major producer of IL-8,⁴⁹¹ which also is produced by neutrophils⁴⁹² and induces neutrophil chemotaxis.⁴⁹⁰ Other cytokines produced by monocytes include IL-1-alpha, IL-6, and IL-10.⁴⁹¹ Monocytes also produce important growth factors, matrix proteins, interferons, and a variety of enzymes, including elastase, collagenases, acid hydrolases, prostaglandins, and lipooxygenase products,⁵²⁴ and contain myeloperoxidase, which converts hydrogen peroxide into more powerful oxidants.

Endothelial Cells

Endothelial cells are activated during CPB and open heart surgery by a variety of agonists. The principal agonists for endothelial cell activation during CPB are thrombin, C5a,⁵⁵⁵ and the cytokines IL-1-beta and TNF-alpha.^537,556 Other agonists, such as endotoxin, histamine, and interferon-gamma (from lymphocytes), are less important during CPB, and endothelial cells are largely unresponsive to chemokines.⁵²⁵

IL-1-beta and TNF-alpha induce the early expression of P-selectin and the later synthesis and expression of E-selectin, which are involved in the initial stages of neutrophil and monocyte adhesion.⁴⁹⁰ The two cytokines also induce expression of ICAM-1 and vascular cell adhesion molecule-1, which firmly bind neutrophils and monocytes to the endothelium and initiate leukocyte trafficking to the extravascular space (see Fig. 12-17).^492,525,537 Experimentally ICAM-1 is upregulated during CPB in pulmonary vessels⁵⁵⁷ and there is evidence that P- and E-selectins are upregulated during CPB and in myocardial ischemia-reperfusion sequences. IL-1-beta and TNF-alpha induce endothelial cell production of the chemotactic proteins IL-8 and MCP-1, and induce production of prostaglandin I₂ (prostacyclin) by the cyclooxygenase pathway^532,558 and nitric oxide by nitric oxide synthase.^532,559 These two vasodilators reduce shear stress and increase vascular permeability and therefore enhance leukocyte adhesion and transmigration. Lastly, IL-1-beta and TNF-alpha stimulate endothelial cell production of proinflammatory cytokines, IL-1, IL-6, IL-8, MCP-1, and PAF.⁵²⁵

In addition to nitric oxide and prostacyclin, endothelial cells produce the vasoconstrictor endothelin-1^489,560 and inactivate other vasoactive mediators, including histamine, norepinephrine, and bradykinin.⁵⁶¹ Prostacyclin concentrations increase rapidly at the beginning of CPB and then begin to decrease.⁵⁶² Endothelin-1 peaks several hours after CPB ends.⁵⁶³

Platelets

Platelets are probably initially activated during CPB by thrombin, which is the most potent platelet agonist, but plasma epinephrine, PAF, vasopressin,⁵⁶⁴ cathepsin G⁵⁶⁵ from other cells, serotonin, and adenosine diphosphate (ADP) secreted by platelets, and internally generated thromboxane A₂⁵⁶⁶ contribute to activation as CPB continues.⁴⁸⁹ Platelets possess several protease-activated receptors⁵⁶⁴ to most of these agonists and to collagen, which has an important role in adhesion and thrombus formation. Collagen binding causes release of thromboxane A₂ and ADP, which help recruit platelets.⁵⁶⁴ Platelets contribute to the inflammatory response by synthesis and release of eicosanoids;⁵⁶⁶ serotonin from dense granules; IL-1-beta;⁵⁶⁷ CXC chemokines, PF4, neutrophil-activating protein-2, IL-8, and endothelial cell neutrophil attractant-78; and C-C chemokines, macrophage inflammatory protein-1a, MCP-3, and RANTES⁵⁶⁸ from alpha granules. Platelets also produce and release acid hydrolases from membrane-bound lysozymes. Platelet-secreted cytokines, neutrophil-activating protein-2, RANTES, PF4, IL-1-beta, IL-8, and endothelial cell neutrophil attractant-78 may be particularly involved in the inflammatory response to CPB because of strong activation of platelets in both the wound and perfusion circuit.

Circulating monocytes and neutrophils constitutively express P-selectin glycoprotein-1, which interacts with aggregated platelets via P-selectin expressed on activated platelets.⁵²⁶ Platelets aggregrate using platelet GPIIb/IIIa (alpha2beta3) receptors attached to symmetric fibrinogen molecules to form bridges between platelets. During CPB platelets aggregate with each other and also to monocytes and neutrophils.^507,561

OTHER MEDIATORS OF INFLAMMATION

Anaphylatoxins

The anaphylatoxins C3a, C4a, and C5a are bioactive protein fragments released by cleavage of complement proteins C3, C4, and C5. These fragments have potent proinflammatory and immunoregulatory functions and contract smooth muscle cells, increase vascular permeability, serve as chemoattractants, and in the case of C5a, activate neutrophils and monocytes.⁵¹⁴ Anaphylatoxins contribute to increased pulmonary vascular resistance, edema, and neutrophil sequestration and an increase in extravascular water during CPB. The duration of postoperative ventilation directly correlates with plasma C3a concentrations.^569–570 C3a and C5a are important mediators in ischemia/reperfusion injuries.

Cytokines

Cytokines are small, cell-signaling peptides produced and released into blood or the extravascular environment by both blood and tissue cells. Cytokines stimulate specific receptors on other cells to initiate a response in that cell. All blood leukocytes and endothelium produce cytokines, but many tissue cells including fibroblasts, smooth muscle cells, cardiac myocytes, keratinocytes, chrondrocytes, hepatocytes, microglial cells, astrocytes, endometrial cells, and epithelial cells also produce cytokines.^537,554,571 IL-1-beta and TNF-alpha are early response cytokines that are promptly produced at the site of injury by resident macrophages.⁵³⁷ These cytokines stimulate surrounding stromal and parenchymal cells to produce more IL-1-beta and TNF-alpha and chemokines, particularly IL-8 and MCP-1, which are powerful chemoattractants for neutrophils and macrophages, respectively. Together with IL-6, the cytokine that regulates production of acute-phase proteins (e.g., C-reactive protein and alpha₂-macroglobulin) by the liver,⁵⁷² these five cytokines are the major proinflammatory cytokines involved in the acute inflammatory response to CPB.

The major anti-inflammatory cytokine involved during CPB is IL-10.⁵⁷³ IL-10 inhibits synthesis of proinflammatory cytokines by monocytes and macrophages⁵⁷⁴ and induces production of IL-1 receptor antagonist (IL-1ra), which downgrades the response to IL-1.^554,575 IL-13 down-regulates production of IL-1, IL-8, and IL-10 and reduces monocyte production of reactive oxidants;⁵⁷⁶ its role during CPB is undetermined.

Proinflammatory cytokines increase during and after clinical cardiac surgery using CPB, but peak concentrations usually occur 12 to 24 hours after CPB ends (Fig. 12-18).^{570,577–581} Measured amounts differ greatly in timing and within and between studies, probably because of differences in the duration of CPB, perfusion temperatures,⁵⁸² perfusion equipment, and aortic cross-clamp times; differences in methods of myocardial protection; possibly variable concentrations of inhibitory cytokines;^583–585 and perhaps exogenous factors such as priming solutions, anesthesia, and intravascular drugs.^{570,577–581} Some of the variation in measurements between studies also may be due to patient factors such as age, left ventricular function, and genetic factors.⁵⁸⁶ The presence of the APOE4 allele (one of the common human polymorphisms of the gene encoding apolipoprotein E) is associated with increased TNF-alpha and IL-8.⁵⁸⁶ Patients who are homozygous for TNF-beta-2 have elevated levels of TNF-alpha and IL-8 during both on- and off-pump cardiac surgery.⁵⁸⁷ Carriers of APOE4 have reduced concentrations of IL-1ra, the inhibitory peptide of IL-1.⁵⁸⁸ Additional hints of a genetic role in the acute inflammatory response are the association between postoperative serum creatinine and different APO-epsilon alleles⁵⁸⁹ and the association between length of stay after coronary artery surgery and 174GG polymorphism of the IL-6 gene.⁵⁹⁰

View larger version (11K): [in this window] [in a new window]   Figure 12-18 Changes in IL-1-β (A) and IL-6 (B) in 30 patients who had elective first-time myocardial revascularization. Letters on the x axis represent the following events: A, induction of anesthesia; B, 5 minutes after heparin; C, 10 minutes after starting CPB; D, end of CPB; E, 20 minutes after protamine; F, 3 hours after CPB; G, 24 hours after CPB. (Redrawn from Steinberg et al.466)

Neutrophils, monocytes, and macrophages produce reactive oxidants, which are cytotoxic inside the phagosome, but act as cytotoxic mediators of acute inflammation outside. Four enzymes generate a large menu of reactive oxidants: NADPH (nicotinamide adenine dinucleotide phosphate, reduced form) oxidase, superoxide dismutase, nitric oxide synthase, and myeloperoxidase.⁵⁴⁸ The enzyme NADPH oxidase adds a free electron to molecular oxygen to create superoxide (O₂^–) and two hydrogen ions, H⁺. Superoxide dismutase catalyzes the conversion of superoxide to hydrogen peroxide, H₂O₂, and molecular oxygen. Nitric oxide synthase produces nitric oxide (NO) from NADPH, arginine, and oxygen, and myeloperoxidase uses H₂O₂ to oxidize halide ions to hypochlorous acids.^591–592 The four products produced by these enzymes, O₂^–, H₂O₂, NO, and hypochlorous acids, generate all reactive oxidants from nonenzymatic reactions with other molecules or ions.⁵⁴⁸

Free radicals have one or more unpaired electrons and are highly reactive in scavenging hydrogen ions from other molecules. OH^• is produced from H₂O₂ by low-valence iron or copper ions, which are reduced to the original low valence after the reaction by various reducing agents, such as ascorbic acid. Secondary free radicals, containing carbon, oxygen, nitrogen, or sulfur, are formed when a free radical reacts with molecules that lack unpaired electrons;⁵⁴⁸ this self-perpetuating sequence produces a chain reaction of highly cytotoxic substances.

Endotoxins

Endotoxins, including lipopolysaccharides, are fragments of bacteria that are powerful agonists for complement,⁵⁹³ neutrophils, monocytes, and other leukocytes. Endotoxins have been detected during CPB^593–596 and after aortic cross-clamping using a very sensitive bioassay.^597,598 Sources include contaminants in sterilized infusion solutions, the bypass circuit, and possibly the gastrointestinal tract due to changes in microvascular intestinal perfusion, which may translocate bacteria.⁵⁹⁹ Intestinal microvascular blood flow is sensitive to both flow rate and duration of CPB. In some instances leakage of endotoxin into the systemic circulation occurs if clearance by the hepatic Kupffer cells fails. The quantitative significance of the role of endotoxins in the acute inflammatory response to CPB is unknown.

Metalloproteinases

CPB induces the synthesis and release of matrix metalloproteinases,⁶⁰⁰ which are one of the four major classes of mammalian proteinases. These proteolytic enzymes have a major role in degradation of collagens and proteins in the extracellular matrix and vascular basement membrane and in the pathogenesis of atherosclerosis and postinfarction left ventricular remodeling. The significance and possible injury produced by activation of these interstitial degradation enzymes over the long term remain to be determined.

Angry Blood

Blood circulating during clinical cardiac surgery with cardiopulmonary bypass can be a stew of vasoactive and cytotoxic substances, activated blood cells, and microemboli. Shear stress, turbulence, cavitation, and other rheologic forces and C5b-9 cause hemolysis of some red cells. Complement anaphylatoxins,⁵¹⁴ bradykinin formed by activation of the contact proteins,^490,513 and proinflammatory cytokines stimulate endothelial cells to contract, allowing extravasation of intravascular fluid into the extravascular space.⁶⁰¹ Numerous circulating vasoactive substances cause vasoconstriction or vasodilatation of heterogeneous regional vascular networks.⁴⁸⁹ As neutrophils and monocytes migrate across the endothelial cell barrier, stromal and parenchymal cells are exposed to a cytotoxic environment mediated by neutral proteases, collagenases and gelatinases, reactive oxidants, lipid peroxides, C5b-9, and other cytotoxins.^{486,545,602,603} This injury is magnified by microemboli produced from platelet-leukocyte aggregates, lipids, and other blood elements and emboli from other sources (see section 12C). The manifestations of the inflammatory response include systemic symptoms such as malaise, fever, increased heart rate, mild hypotension,⁵⁸² interstitial fluid accumulation,⁶⁰⁴ and temporary organ dysfunction, particularly of the brain, heart, lungs, and kidneys.

The magnitude of this defense reaction during and after CPB is influenced by many exogenous factors that include the surface area of the perfusion circuit, the duration of blood contact with extravascular surfaces, the amount of unwashed cardiotomy suction blood returned to the patient, general health and preoperative organ function of the patient, blood loss and replacement, organ ischemia and reperfusion injury, sepsis, different degrees of hypothermia, periods of circulatory arrest, genetic profiles, corticosteroids, and other pharmacologic agents. When well managed, these factors result in a postoperative patient with few, if any, overt manifestations of inflammation.

CONTROL OF THE ACUTE INFLAMMATORY RESPONSE TO CARDIOPULMONARY BYPASS

Off-Pump Cardiac Surgery

Myocardial revascularization without either CPB or cardioplegia reduces the acute inflammatory response but does not prevent it.^605–607 The response to surgical trauma, manipulation of the heart, pericardial suction, heparin, protamine, other drugs, and anesthesia activates the extrinsic clotting system and produces an increase in the markers of acute inflammation, C3a, C5b-9, proinflammatory cytokines (TNF-alpha, IL-6, and IL-8), neutrophil elastase, and reactive oxidants,⁵⁴⁵ but the magnitude of the response is significantly less than that observed with CPB.^606–608 Although it has not been shown that the attenuated acute inflammatory response directly reduces organ dysfunction,^605,608 elderly patients and those with reduced renal and pulmonary function often tolerate off-pump surgery with less morbidity and mortality than patients treated with CPB.^608–611

Perfusion Temperature

Release of mediators of inflammation is temperature sensitive. Normothermic CPB increases the release of cytokines and other cellular and soluble mediators of inflammation,⁵⁸² whereas hypothermia reduces production and release of these mediators until rewarming begins.⁶¹² Perfusion at tepid temperatures between 32 and 34°C is a reasonable compromise for many operations requiring 1 to 2 hours of CPB.^580,602

Perfusion Circuit Coatings

Ionic- or covalent-bonded heparin perfusion circuits are the most widely used surface coatings and are often combined with reduced doses of systemic heparin in first-time myocardial revascularization patients.⁶¹³ It is well established that heparin is an agonist for platelets, complement, factor XII, and leukocytes, but there is no reproducible evidence that heparin coating either produces a nonthrombogenic surface or reduces activation of the clotting cascade.^614–615 A review of a large portion of this literature concluded that heparin-bonded circuits reduced concentrations of the terminal complement complex, C5b-9 (Fig. 12-19),⁶¹⁶ but for nearly every study showing a beneficial anti-inflammatory or antithrombotic effect, another study shows no effect.⁶¹⁷ Clinical trials that have combined heparin-coated circuits with reduced systemic heparin and exclusion of field-aspirated blood from the perfusion circuit have demonstrated modest clinical benefits.⁶¹⁸ However, most trials, including a large European trial of 805 patients, have not observed clinical benefits except in certain subsets of patients that are not the same between studies^{617,619–624} and which report sporadic differences that barely reach statistical significance.^624,625 Excluding unwashed field blood from the perfusion circuit reduces admixture of high concentrations of thrombin,⁵⁵² fibrinolysins,⁶²⁶ cytokines, activated complement,⁴²² and leukocytes to the perfusate. Exclusion of these inflammatory mediators may be more important in reducing the amounts of vasoactive and cytotoxic substances circulating within the body than the heparin surface coating.

View larger version (12K): [in this window] [in a new window]   Figure 12-19 Changes in C5b-9 (TCC) terminal complement complex in heparin-coated (n = 15) and uncoated (n = 14) perfusion circuits during myocardial revascularization. The two curves are significantly different by ANOVA (p = .004).(Reproduced with permission from Videm et al.173)

Modified Ultrafiltration

Although effective in pediatric cardiac surgery,^634–635 ultrafiltration to remove intravascular (and extravascular) water and inflammatory substances has produced mixed results in adults.⁶³⁶ Dialysis during CPB in adults may be beneficial in removing water, potassium, and protein wastes in patients with renal insufficiency.

Leukocyte Filtration

The role of neutrophils in the acute inflammatory response has led to development of leukocyte-depleting filters for the CPB circuit. Multiple groups have investigated these filters in clinical trials, but consistent efficacy in reducing markers of neutrophil activation and improvement in respiratory or renal function are lacking. Most clinical studies fail to document significant leukocyte depletion or clinical benefits.^637–640 Washing cardiotomy suction blood and using leukodepleted allogeneic red cell and platelet transfusions has reduced the interest in leukocyte filtration. Active sequestration of leukocytes and platelets using a separate cell separator during CPB may have beneficial clinical effects,^644,645 but requires a separate inflow cannula and separator system.

Complement Inhibitors

The central role of complement in the acute inflammatory response to CPB provides ample rationale for inhibition. The anaphylatoxins and C5b-9 are direct mediators of the inflammatory response, and C5a is the principal agonist for activating neutrophils and is a potent chemoattractant for neutrophils, monocytes, macrophages, eosinophils, basophils, and microglial cells.⁵¹⁴ C1-inhibitor is a natural inhibitor of complement C1 components C1s and C1r, factor XIIa, kallikrein, and factor XIa.⁵⁰⁴ Factor H and C4BP inhibit C3 and C5 convertase subunits, but are poor inhibitors of induced activation of the complement system.⁵⁰⁴ None of these inhibitors are attractive candidates for inhibiting complement activation during CPB.

The sequential activation cascade with convergence of the classical and alternative pathways at C3 offers many opportunities for inhibition by recombinant proteins.⁶⁴¹ Using a humanized, recombinant antibody to C5 (h5G1.1-scFv), Fitch and associates demonstrated that generation of C5b-9 was completely blocked in a dose-response manner (Fig. 12-20) and that neutrophil and monocyte CD11b/CD18 expression was attenuated in patients during and for several hours after clinical cardiac surgery using CPB.⁶⁴² Large scale clinical trials that have followed but are not yet published have shown modest improvements in morbidity and mortality.

View larger version (16K): [in this window] [in a new window]   Figure 12-20 Inhibition of C5b-9, complement terminal attack complex, with placebo (solid circles) and 2 µg/kg of h5G1.1-scFv (open circles) during clinical cardiac surgery with CPB. Letters on the x axis represent the following events: A, before heparin; B, 5 minutes after drug; C, 5 minutes after cooling to 28°C; D, after beginning rewarming; E, 5 minutes after reaching 32°C; F, 5 minutes after reaching 37°C; G, 5 minutes after CPB; H, 2 hours after CPB; I, 12 hours after CPB; J, 24 hours after CPB. h5G1.1-scFv completely inhibited formation of the C5b-9 terminal attack complex. (Data redrawn from Fitch et al.643)

Other complement recombinant protein inhibitors have been developed and are under active investigation and in clinical trials because of the importance of this plasma protein system in CPB, ischemia/reperfusion, and injuries that summon the acute inflammatory response.^{504,646–648} Although any effective and safe inhibitor is welcome, C3 may be a better target for inhibition because both activation pathways are blocked at the point of convergence and because C3 concentrations in plasma are 15 times greater than those of C5.^{504,649–651}

Glucocorticoids

Many investigators have used glucocorticoids to suppress the acute inflammatory response to CPB and clinical cardiac surgery, but beneficial effects in adult patients have been inconsistent.^652–654 Steroids reduce release of rapid-response cytokines, TNF-alpha, and IL-1-beta from macrophages,⁶⁵⁵ enhance release of IL-10,^656–657 and suppress expression of endothelial cell selectins and neutrophil integrins.⁶⁵⁸ Clinically, glucocorticoids decrease endotoxin release,⁶⁵⁹ shift the cytokine balance towards the anti-inflammatory side,^659–661 and decrease expression of neutrophil integrins.⁶⁵² Clinical results from a few randomized trials are conflicting: one study observed earlier extubation and reduced shivering,⁶⁶² but another found increased blood glucose levels and delayed extubation.^663,664 Differences in specific steroids, dosing, and timing may explain some of these discrepancies.

Recent observations regarding the inhibitory effect of glucocorticoids on transcription factor nuclear factor-B (NF-B) may provide a rationale for using glucocorticoids to suppress the acute inflammatory response to CPB.⁶⁶⁵ This inducible transcription factor controls the expression of genes encoding a wide array of proinflammatory mediators, including cytokines, inducible NO synthase, and adhesion molecules, and is activated by IL-1-beta, TNF-alpha, reactive oxidants, and other noxious stimuli.^{654,666–668} Given the multiplicity and redundancy of pathways involved in the inflammatory response to bypass, inhibition of a common "upstream" control point in transcriptional regulation of inflammatory genes is an attractive strategy.

Protease Inhibitors

Aprotinin is a natural serine protease inhibitor in the kinin superfamily that strongly inhibits plasmin and weakly inhibits kallikrein.⁶⁶⁹ Plasma concentrations of 4 to 10 KIU (kallikrein inhibitory units) of aprotinin completely inhibit plasmin, but 250 to 400 KIU are required to fully inhibit kallikrein.⁶⁶⁹ Clinical doses of aprotinin totally inhibit plasmin, but are not sufficient to completely inhibit kallikrein.⁶⁷⁰ The antifibrinolytic and platelet-sparing effects of the drug are well known and significantly reduce blood losses during and after complex cardiac surgery.^671–672 The anti-inflammatory effects of aprotinin are more difficult to quantitate and may reflect multiple mechanisms including partial kallikrein inhibition, direct effects, and inhibition of NF-B.⁶⁷³

In vitro aprotinin inhibits kallikrein formation, and attenuates complement activation and release of platelet beta thromboglobulin and neutrophil elastase.⁶⁷⁴ Aprotinin also reduces neutrophil transmigration and expression of ICAM-1 and vascular cell adhesion molecule-1 by endothelial cells.^675–676 Clinically, aprotinin reduces circulating TNF-alpha, IL-6, IL-8, and neutrophil CD11b expression,^{653,677–678} and synergistically increases IL-10 synthesis.^657,678 The drug may also attenuate neutrophil activation and myocardial damage during aortic cross-clamping⁶⁷⁹ and reduce overall mortality.⁶⁷² Nevertheless, low- or high-dose aprotinin used in large, randomized controlled clinical trials fails to show a reduction in proinflammatory cytokines, activated complement, neutrophil elastase, and myeloperoxidase.⁶⁸⁰ Thus the efficacy of aprotinin as an anti-inflammatory agent remains unresolved.

Nafamostat mesilate is a trypsin-like protease inhibitor that inhibits platelet aggregation and release, formation of kallikrein, and factor XIIa and neutrophil elastase release during in vitro extracorporeal recirculation.⁶⁸¹ Early clinical trials show that nafamostat mesilate inhibits fibrinolysis, preserves platelet numbers and function, reduces blood loss, and attenuates the acute inflammatory response by suppressing IL-6, IL-8, and malondialdehyde formation and neutrophil integrin expression.^682–683

Comment

As described above, CPB and clinical cardiac surgery can produce a broad and acute inflammatory response that varies in degree among patients. The cause is the continuous recirculation of blood that is sequentially in contact with the wound, perfusion circuit, and intravascular compartment, to which is added the washout of reperfused ischemic organs and tissues. The acute inflammatory response together with microembolization is responsible for most of the morbidity of CPB and clinical cardiac surgery. Given the magnitude and diversity of the acute inflammatory response, it appears unlikely that drug cocktails or indirect measures directed against specific mediators of this response will prove more than mildly effective. Efforts to temporarily inhibit the more important mediators, specifically complement⁶⁸⁴ and neutrophils, during the perioperative period are more attractive and achievable targets that could produce more immediate clinical benefits. Because our patients are vulnerable to infection and other forms of injury during and immediately after operation, and because the acute inflammatory response is an important first step in healing, the clinician must remember that temporary, reversible inhibitors are probably safer than permanent inhibitors.