Transfusion Medicine and Coagulation Disorders

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 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.

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 reaction sequence classically has been termed a cascade or waterfall . Cleavage of a polypeptide fragment changes an inactive zymogen to an active enzyme often by creating a conformational change of the protein, exposing an active site. 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 (Ca ²⁺ ) 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. 27.2 ): the contact activation, 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, creates chemotaxis of leukocytes and monocytes; releases t-PA, prostacyclin, and nitric oxide from endothelial cells; releases IL-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. Coagulation function truly centers on the effects of thrombin as far-reaching accelerant. The platelets, tissue factor, and contact activation all are interactive and 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 of reacting cells and proteins, which, in turn, leads to clot formation. 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—much larger than the sum of the parts. 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 it reduces platelet number, decreases platelet-to-platelet interactions, and decreases the concentration of protein substrates.

The many serine proteases that compose the coagulation pathways are balanced by serine protease inhibitors, termed serpins. Thus a 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 α ₁ -antitrypsin, α ₂ -macroglobulin, heparin cofactor II, α ₂ -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 partially 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 point is that activated AT III is active only against free thrombin (fibrin-bound thrombin cannot be seen by AT III). 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.

Another serpin, protein C, degrades factors Va and VIIIa. Like other vitamin K–dependent factors, it requires Ca ²⁺ 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 through a feedback loop to the endothelial cells to release t-PA.

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. 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. 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 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 GPI 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. Table 27.1 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.

The integrin GPs 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, then vWF binding and platelet arrest on the endothelium at these vWF ligand sites.

Platelet Activation and Aggregation

Platelet activation results after contact with collagen, when adenosine diphosphate (ADP), thrombin, or thromboxane A ₂ 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 Ca ²⁺ ; 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 A ₂ . Other platelet agonists besides ADP and collagen include serotonin, a weak agonist, and thrombin and thromboxane A ₂ , 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 Ca ²⁺ concentration, and stimulate platelet G protein. In addition, serotonin and thromboxane A ₂ are potent vasoconstrictors (particularly in the pulmonary vasculature). 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).

Prostaglandins and Aspirin

Endothelial cell cyclooxygenase synthesizes prostacyclin, which inhibits aggregation and dilates vessels. Platelet cyclooxygenase forms thromboxane A ₂ , 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.

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. Platelets have a life span of approximately 9 days, so the idea of taking somebody off aspirin for 5 to 7 days seems reasonable in that the majority of platelets circulating will not have cyclooxygenase poisoned by aspirin. Aspirin is a drug for which an increased risk for bleeding often has been demonstrated. Today, it probably is more likely that, in some patients, a mild-to-moderate increased risk for bleeding is possible.

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. 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 only for their vasorelaxing effect on large blood vessels. Nonsteroidal antiinflammatory drugs reversibly inhibit both endothelial cell and platelet cyclooxygenase.

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 27.2 ).