Transfusion Medicine and Coagulation Disorders

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Chapter 24 Transfusion Medicine and Coagulation Disorders

Coagulation and bleeding assume particular importance when operations are performed on the heart using extracorporeal circulation. This chapter begins with a discussion of the depth and breadth of hemostasis relating to cardiac procedures, beginning with coagulation pathophysiology. The pharmacology of heparin and protamine is described next. This background is then applied to treatment of the bleeding patient.

OVERVIEW OF HEMOSTASIS

Proper hemostasis requires the participation of innumerable biological elements (Box 24-1). They can be divided into four topics to facilitate understanding: coagulation factors, platelet function, the endothelium, and fibrinolysis. The reader must realize this is for simplicity of learning and that in biology the activation creates many reactions and control mechanisms, all interacting simultaneously. The interaction of the platelets, endothelial cells, and proteins to either activate or deactivate coagulation is a highly buffered and controlled process. It is perhaps easiest to think of coagulation as a wave of biological activity occurring at the site of tissue injury (Fig. 24-1). Although there are subcomponents to coagulation itself the injury/control leading to hemostasis is a four-part event: initiation, acceleration, control, and lysis (recanalization/fibrinolysis). The initiation phase begins with tissue damage, which is really begun with endothelial cell destruction or dysfunction. This initiationphase leads to binding of platelets, as well as protein activations; both happen nearly simultaneously and each has feedbacks into the other. Platelets adhere and create an activation or acceleration phase that gathers many cells to the site of injury and creates a large number of biochemical protein cascade events. As the activation phase ramps up into an explosive set of reactions, counter-reactions are spun off, leading to control proteins damping the reactions. The surrounding normal endothelium exerts control over the reactions. Eventually the control reactions overpower the acceleration reactions and lysis comes into play.

image

Figure 24-1 Coagulation is a sine wave of activity at the site of tissue injury. It goes through four stages: initiation, acceleration, control, and lysis/recanalization.

(Redrawn with permission from Spiess BD: Coagulation function and monitoring. In Lichtor JL [ed]: Atlas of Clinical Anesthesia. Philadelphia, Current Medicine, 1996.)

The other key concept is that hemostasis is part of a larger body system—inflammation. Most of the protein reactions of coagulation control have importance in signaling inflammation and other healing mechanisms. It is no wonder that cardiopulmonary bypass (CPB) has such profound inflammatory effects when it is considered that each of the activated coagulation proteins and cell lines then feeds into upregulation of inflammation.

Protein Coagulation Activations

Coagulation Pathways

The coagulation factors participate in a series of activating and feedback inhibition reactions, ending with the formation of an insoluble clot. A clot is the total of platelet-to-platelet interactions leading to the formation of a platelet plug and then the cross-linking of platelets to each other by way of the final insoluble fibrin that leads to a stable clot. Clotting is not simply the activation of proteins leading to more protein deposition.

With few exceptions, the coagulation factors are glycoproteins synthesized in the liver, which circulate as inactive molecules termed zymogens. Factor activation proceeds sequentially, with each factor serving as substrate in an enzymatic reaction catalyzed by the previous factor in the sequence. Hence, this has classically been called 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 (Fig. 24-2): the contact activation and the intrinsic, extrinsic, and common pathways.

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 and chemotaxis of leukocytes and monocytes; releases tissue plasminogen activator (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. Note the negative feedback aspect of this last action. The latest thinking on coagulation function centers around the effects of thrombin. The platelets, tissuefactor, 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, then a threshold is 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 due to its effects on platelet number, platelet-to-platelet interactions, and the decreased amounts of protein substrates.

The many serine proteases that compose the coagulation pathways are balanced by serine protease inhibitors, termed serpins. This biological 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 (also termed antithrombin III), and others.

Antithrombin (ATIII) constitutes the most important inhibitor of blood coagulation. It binds to the active site (serine) of thrombin, thus inhibiting thrombin’s action. 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 antithrombin, thus explaining the poor efficacy of heparin in treating established thrombosis. ATIII is a relatively inactive zymogen. To be most effective antithrombin 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 ATIII is active only against free thrombin. Most thrombin in its active form is either bound to glycoprotein-binding sites of platelets or in fibrin matrices. When blood is put into a test tube and clot begins to form (e.g., in an activated coagulation time [ACT]), 96% of thrombin production is yet to come. The vast majority of thrombin generation is on the surface of platelets and on clot-held fibrinogen. Platelets through their glycoprotein-binding sites and phospholipid folds protect activated thrombin from attack by ATIII. Therefore, the biological role of ATIII 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.

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 C. Activated protein C also promotes fibrinolysis.

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.2 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. Heparin releases TFPI from endothelium, increasing TFPI plasma concentrations by as much as sixfold.2

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. 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 glycoproteins. Initially, vWF attaches to the GPIα platelet receptor, which slows platelet shear forces. This is not enough to bind the platelet, but it creates a membrane signal that allows for early shape change and expression of other glycoproteins, 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.

Platelet Function

Most clinicians think first of the coagulation proteins when considering hemostasis. Although no one element of the many that participate in hemostasis assumes dominance, platelets may be the most complex. Without platelets, there is no coagulation and no hemostasis. Without the proteins, there is hemostasis, but it lasts only 10 to 15 minutes as the platelet plug is inherently unstable and breaks apart under the shear stress of the vasculature. Platelets provide phospholipid for coagulation factor reactions; contain their own microskeletal system and coagulation factors; secrete active substances affecting themselves, other platelets, the endothelium, and other coagulation factors; and alter shape to expose membrane glycoproteins essential to hemostasis. Platelets have perhaps as many as 30 to 50 different types of cell receptors. The initial response to vascular injury is formation of a platelet plug. Good hemostatic response depends on proper functioning of platelet adhesion, activation, and aggregation (Fig. 24-3).

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 leads 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, glycoprotein (GP)Ib, 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.

The integrin glycoproteins form diverse types of membrane receptors from combinations of 20 α and 8 β subunits. 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. It begins with decreased platelet velocity when GPIb/IX and vWF mediate adhesion, followed by platelet activation, GPIIb/IIIa conformational change, and then vWF binding and platelet arrest on the endothelium at these vWF ligand sites.3

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, P-selectin, and various integrin proteins (vWF, fibrinogen, vitronectin, and fibronectin). Simultaneously, platelets employ 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, there are more than 70 agonists that can produce platelet activation and aggregation.

Agonists induce a shape change, increase platelet intracellular Ca2+ concentration, and stimulate platelet G protein. In addition, serotonin and thromboxane A2 are potent vasoconstrictors. 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).

Drug-Induced Platelet Abnormalities

Many other agents inhibit platelet function.4 β-Lactam antibiotics coat the platelet membrane, whereas the cephalosporins are rather profound but short-term platelet inhibitors. 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 utilized 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 vaso-relaxing effect on large blood vessels. Nonsteroidal anti-inflammatory drugs (NSAIDs) reversibly inhibit both endothelial cell and platelet cyclooxygenase.

In addition to the partial inhibitory effects of aspirin and the other drugs just mentioned, new therapies have been developed that inhibit platelet function in a more specific manner. These drugs include platelet adhesion inhibitor agents, platelet-ADP-receptor antagonists, and GPIIb/IIIa receptor inhibitors (Table 24-1).

Vascular Endothelium

The cells that form the intima of vessels provide an excellent nonthrombogenic surface. Characteristics of this surface, which may account for its nonthrombogenicity, include negative charge; incorporation of heparan sulfate in the grid substance; the release of prostacyclin, nitric oxide, adenosine, and protease inhibitors by endothelial cells; binding and clearance of activated coagulation factors both directly, as occurs with thrombin, and indirectly, as evidenced by the action of thrombomodulin to inactivate factors Va and VIIIa via protein C; and stimulation of fibrinolysis.

Nitric oxide vasodilates blood vessels and inhibits platelets. Its mechanism involves activation of guanylate cyclase with eventual uptake of calcium into intracellular storage sites. Prostacyclin (PGI2) possesses powerful vasodilator and antiplatelet properties. Endothelium-derived prostacyclin opposes the vasoconstrictor effects of platelet-produced thromboxane A2. Prostacyclin also inhibits platelet aggregation, disaggregates clumped platelets, and, at high concentrations, inhibits platelet adhesion. Prostacyclin increases intracellular concentrations of cAMP, which inhibits aggregation. Thromboxane acts in an opposite manner. The mechanism of prostacyclin action is stimulation of adenylyl cyclase, leading to reduced intracellular calcium concentrations. Some vascular beds (e.g., lung) and atherosclerotic vessels secrete thromboxane, endothelins, and angiotensin, all vasoconstrictors, as well as prostacyclin. Activation of platelets releases endoperoxides and arachidonate. These substances, utilized by nearby damaged endothelial cells, provide substrate for prostacyclin production.

Fibrinolysis

Fibrin breakdown, a normal hematologic activity, is localized to the vicinity of a clot. It remodels formed clot and removes thrombus when endothelium heals. Like clot formation, clot breakdown may occur by intrinsic and extrinsic pathways. As with clot formation, the extrinsic pathway plays the dominant role in clot breakdown. Each pathway activates plasminogen, a serine protease synthesized by the liver, which circulates in zymogen form. Cleavage of plasminogen by the proper serine protease forms plasmin. Plasmin splits fibrinogen or fibrin at specific sites. Plasmin is the principal enzyme of fibrinolysis, just as thrombin is principal to clot formation. Plasma normally contains no circulating plasmin, because a scavenging protein, α2-antiplasmin, quickly consumes any plasmin formed from localized fibrinolysis. Thus, localized fibrinolysis, not systemic fibrinogenolysis, accompanies normal hemostasis.

Clinical Applications

Figure 24-4 illustrates the fibrinolytic pathway, with activators and inhibitors. Streptokinase, ASPAC, 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.

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.

HEPARIN

Pharmacology

Chemical Structure

The N-sulfated-D-glucosamine and L-iduronic acid residues of heparin alternate in copolymer fashion to form chains of varying length (Fig. 24-5). As a linear anionic polyelectrolyte, with 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 antithrombin. Heparin is a heterogeneous compound: the carbohydrates vary in both length and side chain composition, yielding a range of molecular weights from 5 000 to 30,000, with most chains between 12,000 and 19,000. Today, the standard heparin is called unfractionated heparin (UFH).

Pharmacokinetics and Pharmacodynamics

The heterogeneity of UFH molecules produces variability in the relationship of dose administered to plasma level of drug. In addition, the relationship of plasma level to biological effect varies with the test system. A three-compartment model describes heparin kinetics in healthy humans: rapid initial disappearance, saturable clearance observed in the lower dose range, and exponential first-order decay at higher doses. The rapid initial disappearance may arise from endothelial cell uptake. The reticuloendothelial system, with its endoglycosidases and endosulfatases, and uptake into monocytes, may represent the saturable phase of heparin kinetics. Finally, renal clearance via active tubular secretion of heparin, much of it desulfated, explains heparin’s exponential clearance.

Male gender and cigarette smoking are associated with more rapid heparin clearance. The resistance of patients with deep vein thrombosis or pulmonary embolism to heparin therapy may be due to the release from thrombi of platelet factor 4 (PF4), a known heparin antagonist. Chronic renal failure prolongs elimination of high, but not low, heparin doses. Chronic liver disease does not change elimination.

Loading doses for CPB (200 to 400 units/kg) are substantially higher than those used to treat venous thrombosis (70 to 150 units/kg). Plasma heparin levels, determined fluorometrically, vary widely (2 to 4 units/mL) after doses of heparin administered to patients about to undergo CPB. The ACT response to these doses of heparin displays even greater dispersion. However, the clinical response to heparin administered to various patients is more consistent than suggested by in vitro measurements.

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