Coagulation Pathways

The coagulation factors participate in a series of activating and feedback inhibition reactions, ending with the formation of an insoluble clot.⁵ A clot is the sum total of platelet-to-platelet interactions, leading to the formation of a platelet plug (initial stoppage of bleeding). The cross-linking of platelets to each other by way of the final insoluble fibrin leads to a stable clot. Clot is not simply the activation of proteins leading to more protein deposition. Clinicians have been shaped in their thinking about coagulation by the historic way that coagulation proteins were discovered and the resulting coagulation tests. It is that teaching of the coagulation cascade, with resultant monitoring technology, that has led to some transfusion behaviors. The way coagulation has been classically taught (protein cascades) is not the way coagulation proceeds biologically.

With few exceptions, the coagulation factors are glycoproteins (GPs) synthesized in the liver, which circulate as inactive molecules termed zymogens. Factor activation proceeds sequentially, each factor serving as substrate in an enzymatic reaction catalyzed by the previous factor in the sequence. Hence this classically has been termed a cascade or waterfall sequence. Cleavage of a polypeptide fragment changes an inactive zymogen to an active enzyme. The active form is termed a serine protease because the active site for its protein-splitting activity is a serine amino acid residue. Many reactions require the presence of calcium ion (Ca²⁺) and a phospholipid surface (platelet phosphatidylserine). The phospholipids occur most often either on the surface of an activated platelet or endothelial cell and occasionally on the surface of white cells. So anchored, their proximity to one another permits reaction rates profoundly accelerated (up to 300,000-fold) from those measured when the enzymes remain in solution. The factors form four interrelated arbitrary groups (Figure 31-2): the contact activation, intrinsic, extrinsic, and common pathways. They were so labeled historically by the human need for order. In biology, they are all highly interactive, occur simultaneously on the surface of cells, and have feedback loops with cross-reactions.

Figure 31-2 Depiction of coagulation protein activation sequence.

Asterisks denote participation of calcium ion. HMWK, high-molecular-weight kininogen; PK, prekallikrein.

Contact Activation

Factor XII, high-molecular-weight kininogen (HMWK), prekallikrein (PK), and factor XI form the contact or surface activation group. The in vivo events that activate factor XII remain unconfirmed. Clinicians do know that ex vivo contact with ionically charged surfaces will activate factor XII. Because factor XII autoactivates by undergoing a shape change in the presence of a negative charge, in vitro coagulation tests use glass, silica, kaolin, and other compounds with negative surface charge (see Chapter 17). The glycocalyx of endothelial cells has a repelling charge for coagulation proteins. One potential in vivo mechanism for factor XII activation is disruption of the endothelial cell layer, which exposes the underlying negatively charged collagen matrix. Activated platelets also provide negative charges on their membrane surfaces. HMWK anchors the other surface activation molecules, PK and factor XI, to damaged endothelium or activated platelets. Factor XIIa cleaves both factor XI, to form factor XIa, and PK, to form kallikrein. Figure 31-3 depicts the events of surface activation.

Figure 31-3 A–C, Activation of the contact factors XII, XI, and prekallikrein (PK). The cofactor, high-molecular-weight kininogen (HMWK), binds factor XI and PK to the endothelial surface. Kallikrein (K) amplifies factor XII activation.

(From Colman RW, Marder VJ, Salzman EW, Hirsh J: Overview of hemostasis. In Colman RW, Hirsh J, Marder VJ, Salzman EW [eds]: Hemostasis and Thrombosis, 3rd ed. Philadelphia: JB Lippincott, 1994, p 3.)

This system also interlinks to the complement cascade and fibrinolytic process as follows. Kallikrein converts HMWK to bradykinin, which activates the complement proteins. Kallikrein also may convert plasminogen to plasmin (see later). This latter function is quite weak, however, and of unknown significance in vivo.

It was rather tempting historically to attribute all coagulation abnormalities to activation of the contact system. This seemed an obvious explanation for the coagulopathy of CPB. The circuits are most often made of polyvinylchloride with a negatively charged surface. Today, it is known that the effects of contact activation in the entire scheme of CPB coagulation dysfunction are actually quite small. Of note, patients with completely absent factor XII do just fine and do not exhibit excess bleeding, nor are they particularly dry after CPB. Therefore, the presence or absence of factor XII in the evolution of humankind seems to not be critical for survival. It also should reveal that the surface activation mechanism is not the driving force behind the bleeding/consumptive coagulopathy of CPB.

Intrinsic System

Intrinsic activation forms factor XIa from the products of surface activation. Factor XIa splits factor IX to form factor IXa, with Ca²⁺ required for this process. Then factor IXa activates factor X with help from Ca²⁺, a phospholipid surface (platelet-phosphatidylserine), and a GP cofactor, factor VIIIa. Figure 31-4 displays a stylized version of factor X activation. The phospholipids and GP cofactors are on the surface of platelets.

Figure 31-4 Factor VIII facilitates the activation of factor X by factor IXa. Calcium tethers the molecules to the phospholipid surface.

(From Horrow JC: Desmopressin and antifibrinolytics. Int Anesthesiol Clin 28:230, 1990.)

Common Pathway

Using membrane phospholipids (phosphatidylserine) as a catalyst site, Ca²⁺ as a ligand, and factor Va as cofactor, factor Xa splits prothrombin (factor II) to thrombin (factor IIa). The combination of factors Xa, Va, and Ca²⁺ is termed the prothrombinase complex. Factor Xa anchors to the membrane surface (of platelets) via Ca²⁺. Factor Va, assembling next to it, initiates a rearrangement of the complex, vastly accelerating binding of the substrate, prothrombin. Most likely, the factor Xa formed from the previous reaction is channeled along the membrane to this next reaction step without detaching from the membrane.

Figure 31-5 depicts the steps involved in formation of thrombin from its precursor, prothrombin. The by-product, fragment F1.2, serves as a plasma marker of prothrombin activation. An alternative scheme generates a different species, meizothrombin, involved more specifically in activation of coagulation inhibitors.¹¹

Figure 31-5 Activation of prothrombin by factor Xa proceeds in a multistep fashion.

A, On the phospholipid surface, the prothrombinase complex consists of prothrombin (factor II), factor Xa, and factor Va. B, The first activation step in which the prothrombin fragment F1.2 is split from prothrombin to form prethrombin (II*). C, Molecular rearrangement of prethrombin yields thrombin.

Thrombin cleaves the fibrinogen molecule to form soluble fibrin monomer and polypeptide fragments termed fibrinopeptides A and B. Fibrin monomers associate to form a soluble fibrin matrix. Factor XIII, activated by thrombin, cross-links these fibrin strands to form an insoluble clot. Patients with lower levels of factor XIII have been found to have more bleeding after cardiac surgery.^11,12

Modulators of the Coagulation Pathway

Thrombin, the most important coagulation modulator, exerts a pervasive influence throughout the coagulation factor pathways. It activates factors V, VIII, and XIII; cleaves fibrinogen to fibrin; stimulates platelet recruitment, chemotaxis of leukocytes and monocytes; releases t-PA, prostacyclin, and nitric oxide from endothelial cells; releases interleukin-1 from macrophages; and with thrombomodulin, activates protein C, a substance that then inactivates factors Va and VIIIa.¹² Note the negative feedback aspect of this last action (Figure 31-6). Coagulation function truly centers around the effects of thrombin. The platelets, tissue factor, and contact activation all are interactive and are activated by a rent in the surface of the endothelium or through the loss of endothelial coagulation control. Platelets adhere to a site of injury and, in turn, are activated, leading to sequestration of other platelets. It is the interaction of all of those factors together that eventually creates a critical mass. Once enough platelets are interacting together, with their attached surface concomitant serine protease reactions, then a thrombin burst is created. Only when enough thrombin activation has been encountered in a critical time point is a threshold exceeded, and the reactions become massive and much larger than the sum of the whole. It is thought that the concentration and ability of platelets to react fully affect the ability to have a critical thrombin burst. CPB may affect the ability to get that full thrombin burst because of its effects on platelet number, platelet-to-platelet interactions, and the decreased amounts of protein substrates.

Figure 31-6 Modulating effects of protein C on coagulation.

Thrombomodulin from endothelial cells accelerates thrombin activation of protein C (Pr C). In the presence of protein S (Pr S), activated protein C [(act Pr C)a] inactivates factors V and VIII. Protein C and protein S are vitamin K dependent.

The many serine proteases that compose the coagulation pathways are balanced by serine protease inhibitors, termed serpins.¹³ This biologic yin and yang leads to an excellent buffering capacity. It is only when the platelet-driven thrombin burst so overwhelms the body’s localized anticoagulation or inhibitors that clot proceeds forward. Serpins include α₁-antitrypsin, α₂-macroglobulin, heparin cofactor II, α₂-antiplasmin, antithrombin (AT; also termed antithrombin III [AT III]), and others.

AT III constitutes the most potent and widely distributed inhibitor of blood coagulation. It binds to the active site (serine) of thrombin, thus inhibiting action of thrombin. It also inhibits, to a much lesser extent, the activity of factors XIIa, XIa, IXa, and Xa; kallikrein; and the fibrinolytic molecule, plasmin. Thrombin bound to fibrin is protected from the action of AT, thus explaining the poor efficacy of heparin in treating established thrombosis. AT III is a relatively inactive zymogen. To be most effective, AT must bind to a unique pentasaccharide sequence contained on the wall of endothelial cells in the glycosaminoglycan surface known as heparan; the same active sequence is present in the drug heparin.

An important note is that activated AT III is active only against free thrombin (fibrin-bound thrombin cannot be seen by AT III).¹⁴ Prothrombin circulates in the plasma but is not affected by heparin-AT III complexes; it is only thrombin, and thrombin does not circulate freely. Most thrombin in its active form is either bound to GP binding sites of platelets or in fibrin matrices. When blood is put into a test tube and clot begins to form (such as in an activated coagulation time [ACT]), 96% of thrombin production is yet to come. Most thrombin generation is on the surface of platelets and on clot-held fibrinogen. Platelets, through their GP binding sites and phospholipid folds, protect activated thrombin from attack by AT III. Therefore, the biologic role of AT III is to create an anticoagulant surface on endothelial cells. It is not present biologically to sit and wait for a dose of heparin before CPB.

CPB dilutes AT III substantially, and the further consumption of AT III during CPB (thrombin generation) leads, in some patients, to profoundly low levels of this important inhibitor.¹⁵ Research work adding AT III back to the CPB circuit has shown promise in that by doing so there is better preservation of serine protease proteins and platelets. Unfortunately, no pharmaceutical company has decided to pursue this line of research to the point that it would be either commonplace or economically feasible to add large amounts of AT III to CPB and, therefore, avoid consumptive coagulopathies. Congenital AT III deficiency can lead to in utero fetal destruction if the fetus is homozygous for the abnormal AT III. However, patients who are heterozygous for AT III abnormalities have about 40% to 60% of normal AT III activity. They have a particularly high risk for deep vein thrombosis. Low AT III levels have been described during extracorporeal membrane oxygenation, and the addition of AT III to the extracorporeal membrane oxygenation circuit has been effective in improving outcome and decreasing bleeding in some circumstances.¹⁶ It is not known how useful it would be in all patients on CPB because only small trials have been performed, but these have been encouraging.^17,18 Both human AT III concentrate (harvested from multiple plasma donors and pasteurized) and a pharmaceutically engineered, goat milk–produced AT III (slightly different structure than human AT III) are commercially available.

Heparin cofactor II also inhibits thrombin, once it is activated. Although large doses of heparin activate heparin cofactor II, dermatans on endothelial cell surfaces activate it far more effectively, suggesting dermatans as alternative drugs to heparin.¹⁹ Dermatan sulfates are not available for use today in the United States.

Another serpin, protein C, degrades factors Va and VIIIa. Like other vitamin K–dependent factors, it requires Ca²⁺ to bind to phospholipid. Its cofactor, termed protein S, also exhibits vitamin K dependence. Genetic variants of protein C are less active and lead to increased risk for deep vein thrombosis and pulmonary embolism. When endothelial cells release thrombomodulin, thrombin then accelerates by 20,000-fold its activation of protein C²⁰ (see Figure 31-6). Activated protein C also promotes fibrinolysis through a feedback loop to the endothelial cells to release t-PA.²¹

Regulation of the extrinsic limb of the coagulation pathway occurs via tissue factor pathway inhibitor (TFPI), a glycosylated protein that associates with lipoproteins in plasma.²² TFPI is not a serpin. It impairs the catalytic properties of the factor VIIa-tissue factor complex on factor X activation. Both vascular endothelium and platelets appear to produce TFPI.^23,24 Heparin releases TFPI from endothelium, increasing TFPI plasma concentrations by as much as sixfold, which should be viewed as a biologic indicator of how poor heparin is as an anticoagulant. TFPI is not tested for in routine coagulation testing. It may be that some individuals with certain types of TFPI or who have very large amounts of circulating TFPI could be at risk for severe adverse bleeding after cardiac surgery.^25–30 This area is just beginning to be examined today both in terms of whether TFPI is responsible for abnormal bleeding and whether its genetic variants have abnormal bleeding or thrombosis.

von Willebrand factor (vWF), a massive molecule composed of disulfide-linked glycosylated peptides, associates with factor VIII in plasma, protecting it from proteolytic enzymes. It circulates in the plasma in its coiled inactive form.³¹ Disruption of the endothelium either allows for binding of vWF from the plasma or allows for expression of vWF from tissue and from endothelial cells. Once bound, vWF uncoils to its full length and exposes a hitherto cryptic domain in the molecule. This A-1 domain has a very high affinity for platelet GPs. Initially, vWF attaches to the glycoprotein Iα (GPIα) platelet receptor, which slows the platelet forward movements against the shear forces of blood flow. Shear forces are activators of platelets. As the platelet’s forward movement along the endothelial brush border is slowed (because of vWF attachment), shear forces actually increase; thus, the binding of vWF to GP1 acts to provide a feedback loop for individual platelets, further activating them. The activation of vWF and its attachment to the platelet are not enough to bind the platelet to the endothelium, but it creates a membrane signal that allows for early shape change and expression of other GPs, GPIb and GPIIb/IIIa. Then, secondary GPIb binding connects to other vWF nearby, binding the platelet and beginning the activation sequence. It bridges normal platelets to damaged subendothelium by attaching to the GPIb platelet receptor. An ensuing platelet shape change then releases thromboxane, β-thromboglobulin, and serotonin, and exposes GPIIb/IIIa, which binds fibrinogen.

Deficiency States

Decreased amounts of coagulation proteins may be inherited or acquired. Deficiencies of each part of the coagulation pathway are considered in turn. Table 31-1 summarizes the coagulation factors, their activation sequences, and vehicles for factor replacement when deficient.

Platelet Adhesion

Capillary blood exhibits laminar flow, which maximizes the likelihood of interaction of platelets with the vessel wall. Red cells and white cells stream near the center of the vessels and marginate platelets. However, turbulence causes reactions in endothelium that lead to the secretion of vWF, adhesive molecules, and tissue factor. Shear stress is high as fast-moving platelets interact with the endothelium. When the vascular endothelium becomes denuded or injured, the platelet has the opportunity to contact vWF, which is bound to the exposed collagen of the subendothelium. A platelet membrane component, GPIb, attaches to vWF, thus anchoring the platelet to the vessel wall. Independently, platelet membrane GPIa and GPIIa and IX may attach directly to exposed collagen, furthering the adhesion stage.^44–46

After activation (see later), additional adhesive mechanisms come into play. Release of selectin GPs from α-granules allows their membrane expression, thus promoting platelet-leukocyte adhesion. This interaction ultimately may allow expression of tissue factor on monocytes, thus amplifying coagulation.⁴⁷

The integrin GPs form diverse types of membrane receptors from combinations of 20 α and 8 β subunits.⁴⁸ One such combination is GPIIb/IIIa, a platelet membrane component that initially participates in platelet adhesion. Platelet activation causes a conformational change in GPIIb/IIIa, which results in its aggregator activity.

Platelet adhesion begins rapidly—within 1 minute of endothelial injury—and completely covers exposed subendothelium within 20 minutes.⁴⁵ It begins with decreased platelet velocity when GPIb/IX and vWF mediate adhesion, followed by platelet activation, GPIIb/IIIa conformational change, then vWF binding and platelet arrest on the endothelium at these vWF ligand sites.^44,46

Adhesion requires margination of platelets; high hematocrits concentrate RBCs in the central regions of a vessel, promoting marginal placement of platelets. Dilute hematocrit (e.g., post-CPB) impairs this effect, thus adversely affecting platelet adhesion. Low hematocrit also may affect platelet prostaglandin levels as RBCs are required for preprocessing of arachidonic acid before platelets make thromboxane. Because of rheology, RBC transfusions have been thought by some to improve hemostasis. However, transfusion should not be used primarily to achieve this goal because RBC concentrates carry a high concentration of cytokines and platelet-activating factor, which may contribute to platelet dysfunction or consumptive coagulopathy. Some of the most recent data-based studies looking at only several units of blood transfusion have noted that when transfusion is used, the postoperative chest tube output is greater. That observation is, however, fundamentally different from one in which if a patient is bleeding and has a particularly low hematocrit, the use of RBCs may well increase margination of platelets.

Platelet Activation and Aggregation

Platelet activation results after contact with collagen, when adenosine diphosphate (ADP), thrombin, or thromboxane A₂ binds to membrane receptors, or from certain platelet-to-platelet interactions. Platelets then release the contents of their dense (δ) granules and α granules. Dense granules contain serotonin, ADP, and Ca²⁺; α granules contain platelet factor V (previously termed platelet factor 1), β-thromboglobulin, platelet factor 4 (PF4), P-selectin, and various integrin proteins (vWF, fibrinogen, vitronectin, and fibronectin). Simultaneously, platelets use their microskeletal system to change shape from a disk to a sphere, which changes platelet membrane GPIIb/IIIa exposure. Released ADP recruits additional platelets to the site of injury and stimulates platelet G protein, which, in turn, activates membrane phospholipase. This results in the formation of arachidonate, which platelet cyclooxygenase converts to thromboxane A₂. Other platelet agonists besides ADP and collagen include serotonin, a weak agonist, and thrombin and thromboxane A₂, both potent agonists. Thrombin is by far the most potent platelet agonist, and it can overcome all other platelet antagonists, as well as inhibitors. In total, more than 70 agonists can produce platelet activation and aggregation.

Agonists induce a graded platelet shape change (the amount based on the relative amount of stimulation), increase platelet intracellular Ca²⁺ concentration, and stimulate platelet G protein. In addition, serotonin and thromboxane A₂ are potent vasoconstrictors (particularly in the pulmonary vasculature).⁴⁹ The presence of sufficient agonist material results in platelet aggregation. Aggregation occurs when the integrin proteins (mostly fibrinogen) released from α granules form molecular bridges between the GPIIb/IIIa receptors of adjacent platelets (the final common platelet pathway).

Platelet Disorders

Dysfunctional vWF produces von Willebrand disease (vWD), an autosomal dominant disorder of variable expressivity.³⁴ With an incidence of 1.4 to 5 cases per 1000 population, vWD is the most common inherited coagulopathy. Patients present with mucocutaneous hemorrhages rather than hemarthroses. Common symptoms include epistaxis, ecchymoses, and excessive bleeding after trauma, with surgery, or during menses. Because vWF concentrations vary greatly with time, symptoms have variable expressivity. Desmopressin reverses the prolonged bleeding time in patients with mild vWD.⁵⁰ As with hemophilia A, severe cases of vWD do not benefit from desmopressin therapy. In one rare class of vWD (type IIB, 3% to 5% of vWD), desmopressin aggregates platelets, inducing thrombocytopenia and worsening rather than helping hemostasis. Table 31-2 summarizes features of the more common types of vWD. When blood products are needed, cryoprecipitate constitutes the replacement vehicle of choice in vWD, although recent factor VIII concentrates retain vWF activity and have been used successfully during cardiac surgery.⁵¹

The addition of agonist (ADP or collagen) to platelets allows measurement of platelet aggregation in vitro. In Glanzmann thrombasthenia, the GPIIb/IIIa receptor is absent, preventing aggregation. However, ristocetin, a cationic antibiotic similar to vancomycin, can agglutinate platelets directly via GPIb receptors and vWF. Absence of the GPIb receptor, Bernard–Soulier syndrome, prevents adhesion and agglutination with ristocetin, but aggregation to ADP is normal, because the GPIIb/IIIa receptor is intact. Patients with vWD also exhibit impaired platelet adhesion and normal aggregation. Decreased amounts of vWF antigen distinguish it from the Bernard–Soulier syndrome. In platelet storage pool deficiency, impairment of dense granule secretion yields no ADP on adhesion. In vitro addition of collagen will not aggregate platelets because of absence of ADP release, whereas added ADP will initiate some aggregation. Table 31-3 summarizes these diagnostic findings. Uremia impairs the secretory and aggregating functions of platelets, resulting in an increased bleeding time. However, the most common effect of renal dysfunction is hypercoagulability. It is only with severe uremia that the platelets are poisoned. It is, therefore, a common misconception in the operating room that a patient with mild-to-moderate renal failure will be at increased risk for bleeding. The utilization of thromboelastography (TEG) can help in deciding whether the extent of renal failure is causing hypocoagulability. The cause and clinical significance remain poorly defined (see Chapter 17).

Prostaglandins and Aspirin

Endothelial cell cyclooxygenase synthesizes prostacyclin, which inhibits aggregation and dilates vessels. Platelet cyclooxygenase forms thromboxane A₂, a potent aggregating agent and vasoconstrictor. Aspirin irreversibly acetylates cyclooxygenase, rendering it inactive. Low doses of aspirin, 80 to 100 mg, easily overcome the finite amount of cyclooxygenase available in the nucleus-free platelets. However, endothelial cells can synthesize new cyclooxygenase. Thus, with low doses of aspirin, prostacyclin synthesis continues whereas thromboxane synthesis ceases, decreasing platelet activation and aggregation. High doses of aspirin inhibit the enzyme at both cyclooxygenase sites.⁴⁹

Reversible platelet aggregation is blocked by aspirin, as the platelet cyclooxygenase is inhibited. However, the more powerful agonists that yield the calcium release response can still aggregate and activate platelets, because cyclooxygenase is not required for those pathways (Figure 31-8).

Figure 31-8 Pathways for reversible (left column) and irreversible (right column) platelet aggregation. Note the different agents that activate distinct receptors. Aspirin inhibits reversible platelet aggregation via the phospholipase A₂ pathway by affecting the arachidonic acid enzyme cyclooxygenase, but it does not prevent more powerful agonists from aggregating platelets by directly stimulating phospholipase C pathway receptors.

(Adapted from Kroll MH, Schafer A: Biochemical mechanisms of platelet activation. Blood 74:1181, 1989.)

In many centers, a majority of the patients presenting for coronary artery bypass grafting (CABG) will have received aspirin within 7 days of surgery in hopes of preventing coronary thrombosis.⁵² Aspirin is a drug for which an increased risk for bleeding often has been demonstrated.⁵³ Although most early research studies stated that aspirin leads to increased bleeding, since the mid-1990s, that early impression has not been confirmed. Today, it probably is more likely that, in some patients, a mild-to-moderate increased risk for bleeding is possible.

Unfortunately, most of the early studies of aspirin were not blinded, and it may be that the pervading belief that aspirin caused increased bleeding was actually self-fulfilling. Since the early 1990s, there have been a number of therapeutic changes including use of lower doses and greater use of antifibrinolytics. These changes alone may have decreased the overall risk for bleeding from aspirin. Follow-up prospective studies have, thus, yielded varied results. In the largest cohort of 772 men, aspirin increased bleeding after CABG by 33%,⁵⁴ and a group of 101 aspirin-taking patients bled 25% to 56% more than a control group.⁵⁵ In another study, the need to explore the mediastinum for excessive bleeding nearly doubled (factor of 1.82) in patients taking aspirin.⁵⁶ However, many other studies have not shown increased bleeding with aspirin.^57–62 Although a single aspirin can irreversibly inhibit platelet cyclooxygenase for the life of the platelet, aspirin-related bleeding after surgery usually requires more extensive exposure to the drug.

Drug-Induced Platelet Abnormalities

Many other agents inhibit platelet function. β-Lactam antibiotics coat the platelet membrane, whereas the cephalosporins are rather profound but short-term platelet inhibitors.⁶³ Many cardiac surgeons may not realize that their standard drug regimen for antibiotics may be far more of a bleeding risk than aspirin. Hundreds of drugs can inhibit platelet function. Calcium channel blockers, nitrates, and β-blockers are ones commonly used in cardiac surgery. Nitrates are effective antiplatelet agents, and that may be part of why they are of such benefit in angina, not just for their vasorelaxing effect on large blood vessels. Nonsteroidal anti-inflammatory drugs reversibly inhibit both endothelial cell and platelet cyclooxygenase. In addition, anecdotal reports of platelet inhibition, without clear confirmatory studies, exist for many pharmaceuticals including dextran, and for innumerable foods (e.g., onion, garlic, alcohol) and spices (e.g., ginger, tumeric, cloves).²⁴

Rofecoxib (Vioxx), a cyclooxygenase 2 (COX-2) inhibitor, was withdrawn from the U.S. market because of its cardiovascular risk profile (a small increase in associated acute myocardial infarctions).⁶⁴ This COX-2 inhibitor has the highest selectivity for COX-2 versus COX-1 and thus leads to an imbalance between thromboxane A₂ and prostacyclin production. The other COX-2 inhibitors are currently also undergoing cardiovascular investigation.

In addition to the partial inhibitory effects of aspirin and the other drugs mentioned earlier, new therapies that inhibit platelet function in a more specific manner have been developed. These drugs include platelet adhesion inhibitor agents, platelet-ADP-receptor antagonists, and GPIIb/IIIa receptor inhibitors (Table 31-4).⁶⁵

Extrinsic Fibrinolysis

Endothelial cells synthesize and release t-PA. Both t-PA and a related substance, urokinase plasminogen activator, are serine proteases that split plasminogen to form plasmin. The activity of t-PA magnifies on binding to fibrin. In this manner, also, plasmin formation remains localized to sites of clot formation. Epinephrine, bradykinin, thrombin, and factor Xa cause endothelium to release t-PA, as do venous occlusion and CPB.⁷⁴ Fibrinolysis during and after CPB is discussed later.

Intrinsic Fibrinolysis

Factor XIIa, formed during the contact phase of coagulation, cleaves plasminogen to plasmin. The plasmin so formed then facilitates additional cleavage of plasminogen by factor XIIa, forming a positive feedback loop. Kallikrein also can activate plasminogen; the physiologic importance of this pathway for fibrin breakdown has not been established.

Exogenous Activators

Streptokinase (made by bacteria) and urokinase (found in human urine) both cleave plasminogen to plasmin, but do so with low fibrin affinity. Thus, systemic plasminemia and fibrinogenolysis, as well as fibrinolysis, ensue. Acetylated streptokinase plasminogen activator complex provides an active site, which is not available until deacetylation occurs in blood. Its systemic lytic activity lies intermediate to those of t-PA and streptokinase. Recombinant t-PA (Alteplase) is a second-generation agent that is made by recombinant DNA technology and is relatively fibrin specific.

Clinical Applications

Figure 31-9 illustrates the fibrinolytic pathway, with activators and inhibitors. Streptokinase, acetylated streptokinase plasminogen activator complex, and t-PA find application in the lysis of thrombi associated with myocardial infarction. These intravenous agents “dissolve” clots that form on atheromatous plaque. Clinically significant bleeding may result from administration of any of these exogenous activators or streptokinase.⁷⁵

Figure 31-9 The fibrinolytic pathway.

Antifibrinolytic drugs inhibit fibrinolysis by binding to both plasminogen and plasmin. Intrinsic blood activators (factor XIIa), extrinsic tissue activators (tissue plasminogen activator, urokinase plasminogen activator), and exogenous activators (streptokinase, acetylated streptokinase plasminogen activator complex) split plasminogen to form plasmin.

(From Horrow JC, Hlavacek J, Strong MD, et al: Prophylactic tranexamic acid decreases bleeding after cardiac operations. J Thorac Cardiovasc Surg 99:70, 1990.)

Fibrinolysis also accompanies CPB. This undesirable breakdown of clot after surgery may contribute to postoperative hemorrhage and the need to administer allogeneic blood products. Regardless of how they are formed, the breakdown products of fibrin intercalate into sheets of normally forming fibrin monomers, thus preventing cross-linking. In this way, extensive fibrinolysis exerts an antihemostatic action. Factor XIII is an underappreciated coagulation protein. It circulates and, when activated, cross-links fibrin strands and protects fibrin from the lytic actions of plasmin. It has been known for some time that low levels of factor XIII are associated with increased hemorrhage after CPB. Factor XIII levels are reduced by hemodilution, but it also appears that there is active destruction in some patients with CPB. Several new studies have begun testing adding factor XIII to patients and assessing bleeding.^76–78 The problem, however, is that currently there is no good way to assess factor XIII levels. Clearly, factor XIII will be quite expensive, and using it for most patients may well be ill advised.

Chemical Structure

In the 1920s, Howell’s group isolated heparin and identified it as a carbohydrate containing glucuronic acid residues. In the 1930s, Jorpes demonstrated a hexosamine component to heparin (glucosamine, in particular) that is present in a ratio of 1:1 with glucuronic acid. Of greater importance, he discovered that heparin contains many sulfate groups—two per uronic acid residue—making it one of the strongest acids found in living things. In the 1950s, Jorpes’ group identified the sulfate groups at the N-position on glucosamine, where solely acetyl groups previously were thought to reside. In the 1960s, they corrected identification of the uronic acid as l-iduronic acid, an epimer of d-glucuronic acid, and refined its structural detail.⁸⁰

The N-sulfated-d-glucosamine and l-iduronic acid residues of heparin alternate in copolymer fashion to form chains of varying length (Figure 31-10). As a linear anionic polyelectrolyte, the negative charges being supplied by sulfate groups, heparin demonstrates a wide spectrum of activity with enzymes, hormones, biogenic amines, and plasma proteins. A pentasaccharide segment binds to AT.⁸¹ Heparin is a heterogenous compound; the carbohydrates vary in both length and side-chain composition, yielding a range of molecular weights from 5000 to 30,000, with most chains between 12,000 and 19,000.⁸² Today, the standard heparin is called unfractionated heparin (UFH).

Figure 31-10 An octasaccharide fragment of heparin, a substituted alternating copolymer of iduronic acid and glucosamine. The leftmost sugar is iduronic acid. Note the numerous sulfate groups and the acetyl substitution on the second sugar. Variations in sugar substitutions and in chain length produce molecular heterogeneity. Brackets indicate the pentasaccharide sequence that binds to antithrombin.

(From Rodén L: Highlights in the history of heparin. In Lane DA, Lindahl U [eds]: Heparin. Boca Raton, FL: CRC Press, 1989, p 1.)

Source and Biologic Role

Heparin is found mostly in the lungs, intestines, and liver of mammals, with skin, lymph nodes, and thymus providing less plentiful sources.⁸⁴ Abundance of heparin in tissues rich in mast cells suggests these as the source of the compound. Its presence in tissues with environmental contact suggests a biologic role relating to immune function. Heparin may assist white blood cell movements in the interstitium after an immunologic response has been triggered. Mollusks have no coagulation system yet possess heparin, arguing against a biologic role in hemostasis. It is clear that heparin, per se, was never intended biologically to be circulating in large dosages throughout the vascular tree.

Most commercial preparations of heparin now use pig intestine, 40,000 pounds of which yield 5 kg heparin.⁸⁰ Prevention of postoperative thrombosis constituted the initial clinical use of heparin in 1935 by Best, Jaques, and colleagues in Toronto, and by Crafoord in Stockholm.⁸⁰

Potency

Heparin potency is determined by comparing the test specimen against a known standard’s ability to prolong coagulation.⁸⁵ Current United States Pharmacopeia (USP) and British Pharmacopoeia (BP) assays use a PT-like method on pooled sheep’s plasma obtained from slaughterhouses. The plasma commonly is contaminated with tissue extracts or other hemostatically active substances. The European Pharmacopoeia’s method, an aPTT on fresh sheep’s plasma, is superior to that of the USP.⁸⁶ Modern research assays use human FFP, an aPTT-like method, and require linear log versus log plots of standard and test samples.⁸⁵

UFH dose should not be specified by weight (milligrams) because of the diversity of anticoagulant activity expected from so heterogenous a compound. Unfortunately, because of the flawed USP assay, even units of activity often do not reflect clinical effects. As originally defined, 1 unit heparin prolongs the clotting of cat’s blood for only 24 hours at 0°C.⁸⁷ Milligram usage, introduced in 1937 as a Swedish standard, was superseded by the first international standard, in which 130 units of activity corresponded to 1 mg. The fourth international standard uses a porcine mucosal preparation.⁸⁵

One USP unit of heparin activity is the quantity that prevents 1.0 mL of citrated sheep’s plasma from clotting for 1 hour after addition of calcium.⁸⁸ Units cannot be cross-compared among heparins of different sources, such as mucosal versus lung, or low-molecular-weight heparin (LMWH) versus UFH, or even lot to lot, because the assay used may or may not reflect actual differences in biologic activity. None of these measures has anything to do with the effect of a unit on anticoagulation effect for human cardiac surgery.

Actions and Interactions

Heparin exerts its anticoagulant activity via AT III, one of the many circulating serine protein inhibitors (serpins), which counter the effects of circulating proteases.¹⁰¹ The major inhibitor of thrombin and factors IXa and Xa is AT III; that of the contact activation factors XIIa and XIa is α₁-proteinase inhibitor; kallikrein inhibition arises mostly from C1 inhibitor. AT activity is greatly decreased at a site of vascular damage, underscoring its primary role as a scavenger for clotting enzymes that escape into the general circulation.

AT inhibits serine proteases even without heparin. The extent to which heparin accelerates AT inhibition depends on the substrate enzyme; UFH accelerates the formation of the thrombin-AT complex by 2000-fold, but accelerates formation of the factor Xa-AT complex by only 1200-fold¹⁰² (Table 31-5). In contrast, LMWH fragments preferentially inhibit factor Xa. Enzyme inhibition proceeds by formation of a ternary complex consisting of heparin, AT, and the proteinase to be inhibited (e.g., thrombin, factor Xa). For UFH, inhibition of thrombin occurs only on simultaneous binding to both AT and thrombin. This condition requires a heparin fragment of at least 18 residues.^101,103 A pentasaccharide sequence binds to AT (see Figure 31-10). LMWHs, consisting of chains 8 to 16 units long, preferentially inhibit factor Xa. In this case, the heparin fragment activates AT, which then sequentially inactivates factor Xa; heparin and factor Xa do not directly interact (Figure 31-12).^104,105

TABLE 31-5 Some Coagulation Factor Inhibitors and the Effects of Heparin

Figure 31-12 Antithrombin interaction with factor Xa may occur with either low-molecular-weight heparin (A) or standard unfractionated heparin (B). Inhibition of thrombin (factor IIa), however, requires simultaneous binding of the heparin molecule to both antithrombin and thrombin (C).

(From Holmer E, Soderberg K, Bergqvist D, Lindahl U: Heparin and its low-molecular-weight derivatives. Anticoagulant and antithrombotic properties. Haemostasis 16[Suppl 2]:1, 1986.)

Several investigators have demonstrated continued formation of fibrinopeptides A^106,107 and B¹⁰⁸ (Figure 31-13), as well as prothrombin fragment F1.2 and thrombin-AT complexes,¹⁰⁹ despite clearly acceptable anticoagulation for CPB by many criteria. These substances indicate thrombin activity. The clinical significance of this ongoing thrombin activity has had limited study. The ACT must be more prolonged to prevent fibrin formation during cardiac surgery compared with during extracorporeal circulation without surgery because surgery itself incites coagulation. UFH in conjunction with AT appears to work in plasma only on free thrombin. When considering what is known today about thrombin burst and thrombin activity, heparin appears to be relatively inefficient because there is not much free thrombin. Thrombin is held on the surface of activated platelets at various GP binding sites including the GPIIb/IIIa site. Most thrombin is fibrin bound, and heparin-AT complexes do not bind at all to this thrombin unless the level of heparin is pushed far above what is used routinely for CPB. The idea behind using heparin for CPB is that by creating a large circulating concentration of activated AT, whenever a thrombin molecule is produced, an available AT molecule will be there to immediately bind to it before it can have any further activating effect. Clearly, that is unrealistic with the knowledge that thrombin exerts its main activity by binding to the surface of platelets.

Figure 31-13 Serial measurements of fibrinopeptides A (top) and B (bottom) during cardiopulmonary bypass (CPB) in 20 patients. Asterisks denote statistically significant changes compared with the preoperative measurement. Note continued presence of these markers of thrombin activity despite heparin administration.

(From Tanaka K, Takao M, Yada I, et al: Alterations in coagulation and fibrinolysis associated with cardiopulmonary bypass during open heart surgery. J Cardiothorac Anesth 3:181, 1989.)

Bovine versus Porcine Preparations

Bovine lung heparin contains greater amounts of iduronic acid and sulfoamino groups than pork mucosal heparin. Because endothelial endoglycosidases degrade heparin at sulfoamino groups, elimination of beef lung heparin proceeds more quickly than that of pork mucosal heparin. Its AT III affinity and anticoagulant activity are less than those of porcine heparin. Either preparation can establish suitable anticoagulation for CPB. Beef lung heparin may be more amenable to protamine neutralization than the pork mucosal preparation because it exerts less anti–factor Xa activity.¹¹⁰ HIT is less common with pork heparin (see later). This information may be of historic value only because bovine heparin is no longer available in the United States.

Mechanism

Although several observations suggest that decreased levels of AT III mediate heparin resistance, these observations lack sufficient evidence to establish this relation.¹¹⁶ In one study of 500 CABG patients, 21% demonstrated heparin resistance, and 65% of these responded to added AT III; this translates into 35% being nonresponders.¹¹² First, a patient with congenital AT III deficiency displayed heparin resistance¹¹⁷; this hardly proves that all heparin resistance stems from AT III deficiency. Second, of six patients with venous thrombosis, three receiving heparin infusions displayed a 25% shorter half-life for AT III compared with the three untreated patients.¹¹⁸ Accelerated AT III consumption could have resulted from the thrombotic process rather than the heparin infusion. Third, plasma levels of AT III decreased by 17% to 33% during heparin administration by intravenous or subcutaneous routes.^119–124 It is possible that this measurement resulted merely from formation of heparin-AT complexes. Perhaps accelerated elimination of AT III arises from some modification of the protein during or after its interaction with heparin.¹²³ Also, it has been postulated that excesses of platelet activity, releases of PF4, could neutralize heparin in these patients. That has yet to be studied, as has a great deal to do with hypercoagulable states in cardiac surgery.¹²⁵

Hemodilution accompanying CPB decreases AT levels to about half of normal levels.⁹⁷ There are, however, outlier patients who have profoundly low AT levels. It is possible to see AT III levels as low as 20% of normal, and these levels correspond to levels seen in septic shock and diffuse intravascular coagulation.¹⁵ However, supplemental AT may not prolong the ACT, which means that the heparin available has been bound to sufficient or available AT. The only way that the ACT would be prolonged is if there is excess heparin beyond available AT. Reports of heparin resistance for CPB ascribe its occurrence variously to the use of autotransfusion,¹²⁴ previous heparin therapy,^126–129 infection,^130,131 and ventricular aneurysm with thrombus.^128,130 The differential diagnosis also includes hypereosinophilia, oral contraceptive therapy, consumptive coagulopathy, thrombocytosis, and congenital AT deficiency.¹²⁸ Heparin resistance also might occur in patients with subclinical thrombotic processes releasing PF4.

The individual anticoagulant response to heparin varies tremendously.¹³² Some presumed cases of heparin resistance may represent nothing more than this normal variation. Regardless of cause, measurement of each individual’s anticoagulant response to heparin therapy for CPB is warranted.¹³³ Heparin resistance helps focus the debate regarding whether anticoagulation monitoring should measure heparin concentrations or heparin effect; the goal of anticoagulation is not to achieve heparin presence in plasma but to inhibit the action of thrombin on fibrinogen, platelets, and endothelial cells (see Chapter 17). Therefore, the effect of heparin usually is measured.

Treatment

Most commonly, additional heparin prolongs the ACT sufficiently for the conduct of CPB. Amounts up to 800 U/kg may be necessary to obtain an ACT of 400 to 480 seconds or longer. Although administration of FFP, which contains AT,¹³⁴ should correct AT depletion (Figure 31-14) and suitably prolong the ACT,¹³⁵ such exposure to transfusion-borne infectious diseases should be avoided whenever possible. Today, the risks of blood transfusion have shifted away from blood-borne viral transmission, with transfusion-related acute lung injury being noted as the greatest mortality-associated event with transfusion. FFP and platelet transfusions carry the greatest risk for transfusion-related acute lung injury.^136,137 Although transfusion-related acute lung injury has not been studied in relation to use of FFP for heparin resistance, it makes great sense to use one of the AT products. This modality is reserved for the rare refractory case. Rather than administer FFP, centers normally accepting only ACTs of 480 seconds or longer for CPB might consider accepting 400 seconds or less, or administering AT III concentrate.^135,138

Figure 31-14 Activated coagulation time response in seconds (vertical axis) to heparin administered in units per kilogram (horizontal axis) to a single patient. A, Baseline measurement; (B) after 300 U/kg; (C) after an additional 300 U/kg; (D) soon after C; (E) after 2 units of fresh frozen plasma.

(From Sabbagh AH, Chung GKT, Shuttleworth P, et al: Fresh frozen plasma: A solution to heparin resistance during cardiopulmonary bypass. Ann Thorac Surg 37:466, 1984.)

AT concentrate specifically addresses AT deficiency.15–18,¹³⁴ Two products are available for utilization. One is a recombinant DNA engineered product made from goat’s milk and the other is a purified human plasma harvest derivative. There are currently no head-to-head studies to recommend one over the other at this time. The literature supports success in treating heparin resistance during cardiac surgery.^116,139 A multicenter study on the efficacy of using a recombinant human antithrombin in heparin-resistant patients undergoing CPB was published.¹⁴⁰ The patients received 75 U/kg recombinant human AT, which was effective in restoring heparin responsiveness in most patients. However, some patients still required FFP, and the patients bled more than did a control group after surgery.

Incidence and Timing

Although initial reports placed the incidence of heparin rebound after cardiac surgery at about 50%, modifications in the timing and amount of protamine administration decreased the incidence.^143,144 Heparin rebound can occur as soon as 1 hour after protamine neutralization.¹²⁷ When present, prolonged coagulation times or more direct evidence of circulating heparin may persist for 6 hours or longer.^97,144–147

Treatment and Prevention

Although still debated by a few, most clinicians accept heparin rebound as a real phenomenon. However, clinical bleeding does not always accompany heparin rebound. When it does, administration of supplemental protamine will neutralize the remaining heparin (Box 31-3). Can the initial protamine dose be adjusted to prevent heparin rebound? Available studies yield conflicting results. All six patients who received protamine based on the estimated amount of remaining heparin developed heparin rebound, compared with none of six who received protamine based on the total administered dose of heparin.¹⁴⁴ However, 42% of patients receiving large, fixed doses of both drugs experienced heparin rebound and increased bleeding compared with patients whose doses were titrated to clotting assays¹⁴⁵ (Figure 31-15). Likewise, patients receiving smaller doses of protamine bled less than those receiving protamine doses based on the total amount of heparin administered.¹⁴⁷ In contrast, a fourth study recommended that the ratio of protamine given to heparin remaining be as much as 1.6 mg/100 units.⁹⁷

Figure 31-15 Heparin rebound measured by protamine titration in 10 of 24 patients given heparin by a fixed-time protocol. Parentheses denote number of patients.

(From Kaul TK, Crow MJ, Rajah SM, et al: Heparin administration during extracorporeal circulation. J Thorac Cardiovasc Surg 78:95, 1979.)

It should be noted that in vitro work shows that as little as one-third the heparin dose is all the protamine that is actually required to reverse the heparin in a test tube. It is still difficult to know exactly what the best heparin reversal dose is by protamine. Some effort at titration with a heparin dose–response curve seems worthwhile.

Although larger initial doses of protamine may decrease the likelihood of heparin rebound, two potential complications of protamine overdosage must be considered: adverse cardiovascular sequelae of protamine administration and the anticoagulant effects of protamine itself. Although protamine is an in vitro anticoagulant, doses up to 6 mg/kg in volunteers, in the absence of heparin, do not prolong the clotting time.¹⁴⁸ However, doses four times in excess of a neutralizing dose doubled the ACT in dogs.¹⁴⁹ The dose after CPB that causes anticoagulation in patients remains unknown. Clinical studies comparing fixed-ratio protamine doses with protocols that gauge protamine dose to remaining heparin activity and protamine drug lot potency demonstrated decreased doses of protamine, decreased chest tube drainage after surgery, and fewer transfusions.^150,151

Mechanism

These patients with HITT demonstrate a heparin-dependent antibody, usually IgG, although others are described, which aggregates platelets in the presence of heparin.^161,162 During heparin therapy, measured antibody titers remain low because of antibody binding to platelets. Titers rise after heparin therapy ceases; but paradoxically, antibody may be undetectable a few months later.¹⁶⁵ Two other features are unexpected: First, the antibody does not aggregate platelets in the presence of excess heparin; and second, not all re-exposed patients experience development of thrombocytopenia.^166–168

The platelet surface contains complexes of heparin and PF4. Affected patients have an antibody to this complex. Antibody binding activates platelets via their FcγII receptors and activates endothelium^169–171 (Figure 31-16). The activation of the platelet surface triggers a secondary thrombin release. Platelets can attach to each other creating what is known as a white-clot syndrome; but if secondary thrombin generation is created through antibody activation of the platelets, then a fibrin clot can be the result. In the absence of heparin, the heparin-PF4 antigen cannot form. However, there seems to be some sort of continuum between idiopathic thrombocytopenia and HIT.

Figure 31-16 Presumed mechanism of the interaction among heparin, platelets, and antibody in heparin-induced thrombocytopenia.

Top, Platelet factor 4 (PF4) released from platelet granules is bound to the platelet surface. Middle, Heparin and PF4 complexes form. Bottom, The antibody binds to the PF4-heparin complex and activates platelet FcγII receptors.

In the absence of an endothelial defect, the only responses to the antibody-antigen interaction are platelet consumption and thrombocytopenia. Atheroma rupture, endovascular interventions such as balloon angioplasty, vascular surgery, and other procedures that disrupt endothelium can provide a nidus for platelet adhesion and subsequent activation. PF4, released with platelet activation, binds to heparin locally, thus not only removing the inhibition of coagulation but also generating additional antigenic material (Figure 31-17). Clumps of aggregated platelets thrombose vessels, resulting in organ and limb infarction. Amputation, death, or both often occur with established HIT with thrombosis (HITT). The presence of heparin-PF4 antibodies recently has been associated with other adverse effects. It appears that if a patient undergoes cardiac surgery with positive antibodies, the risk for mortality or myocardial infarction, or both, may at least double.

Figure 31-17 Mechanism of thrombosis accompanying heparin-induced thrombocytopenia.

Normally, heparin and antithrombin (AT) form a complex that inhibits coagulation. Platelet factor 4 (PF4), released from platelets on activation, binds heparin and drives the dissociation reaction of the AT-heparin complex to the right, restoring coagulation locally. Restored coagulation mechanisms and activated platelets form thrombus in the presence of vascular injury.

(Adapted from Parmet JL, Horrow JC: Hematologic diseases. In Benumof J [ed]: Anesthesia and Uncommon Diseases, 3rd ed. Philadelphia: WB Saunders Company, 1997.)

Incidence and Diagnosis

Estimates of the true incidence of HIT are confounded by different diagnostic thresholds for platelet count, varying efforts to detect other causes, and incomplete reports.^172,173 After 7 days of therapy with UFH, probably 1% of patients experience development of HIT; after 14 days of therapy, the prevalence rate is 3%.¹⁷¹ Using a platelet count of 100,000/mm³, multiple reports comprising more than 1200 patients revealed an overall incidence rate of HIT of 5.5% with bovine heparin and 1.0% with porcine heparin.¹⁶¹ Other recent research has found the preoperative incidence rate of enzyme-linked immunosorbent assay (ELISA)–positive patients to be between 6.5% to 10%. This means that antibodies are present, and that may not mean that thrombocytopenia is occurring. Of great interest is that many more patients develop positive tests for ELISA antibodies by days 7 to 30 after cardiac surgery. Somewhere between 25% and 50% of patients develop these antibodies.¹⁶² In a study of patients after cardiac surgery wherein all patients were screened for HIT antibodies, the group also looked for platelet counts well less than the patient’s baseline¹⁷⁴; 21 of 153 patients (14%) tested positive for antibodies by heparin induced platelet activation (HIPA) testing. Those patients with a low platelet count and a high HIT antibody titer after surgery were at very high risk for mortality (59%). Therefore, a decline in platelet count or persistence of a low platelet count after cardiac surgery should be considered HIT until proved otherwise and taken very seriously. Anticoagulation with alternative nontriggering anticoagulants is imperative.¹⁷⁴ Warfarin should be avoided until such time as the platelet count is recovered and vitamin K therapy can be instituted; this is because the use of warfarin compounds will decrease protein C, which has been known to trigger an HITT crisis.

In the bivalirudin trials, wherein patients thought to have HIT were given an alternative anticoagulant, the presence of antibodies after surgery was associated with adverse events. Particularly worrisome were the presence of a low platelet count or a blunted return toward a normal platelet count after surgery.^175–177

Some particular lots of heparin may be more likely to cause HIT than others.¹⁷⁸ HIT can occur not only during therapeutic heparin administration but with low prophylactic doses, although the incidence is dose related. Even heparin flush solution or heparin-bonded intravascular catheters can incite HIT.^179–182 Cases of platelet-to-platelet adhesion creating a “white clot” in otherwise normal patients have been observed in the oxygenator and the reservoir of CPB machines. The fact that such events have been reported even when all other tests appeared normal signals the unpredictable nature of the heparin-PF4 antibody, as well as the biologic activity of UFH.

Although HIT usually begins 3 to 15 days (median, 10 days) after heparin infusions commence, it can occur within hours in a patient previously exposed to heparin. Platelet count steadily decreases to a nadir between 20,000 and 150,000/mm³. Absolute thrombocytopenia is not necessary; only a significant decrease in platelet count matters, as witnessed by patients with thrombocytosis who experience development of thrombosis with normal platelet counts after prolonged exposure to heparin. Occasionally, thrombocytopenia resolves spontaneously despite continuation of heparin infusion.¹⁸³

Clinical diagnosis of HIT requires a new decrease in platelet count during heparin infusion. Laboratory confirmation is obtained from several available tests. In the serotonin release assay, patient plasma, donor platelets, and heparin are combined. The donor platelets contain radiolabeled serotonin, which is released when donor platelets are activated by the antigen-antibody complex. Measurement of serotonin release during platelet aggregation at both low and high heparin concentrations provides excellent sensitivity and specificity.¹⁶⁶

A second assay measures more traditional markers of platelet degranulation in a mixture of heparin, patient plasma, and donor platelets.¹⁸⁴ The most specific test is an ELISA for antibodies to the heparin-PF4 complex.^{160,161,185,186}

The type, source, and lot of heparin used affect the outcome of these tests. The heterogenous nature of heparin demands that a lot-specific drug be used in serotonin release assays, especially when used to determine suitability of future administration. For example, if LMWH administration is planned for a patient who experienced development of HIT II with UFH, testing of patient plasma with the lot of LMWH to be given should precede its administration.

Measurement of platelet-associated IgG is poorly specific for HIT because of numerous other causes of antiplatelet IgG. This test should not be used in the diagnosis of HIT.