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.

Figure 24-2 Depiction of coagulation protein activation sequence. Asterisks denote participation of calcium ion. PK = prekallikrein; HMWK = high-molecular-weight kininogen.

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 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).

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 while thromboxane synthesis ceases, decreasing platelet activation and aggregation. High doses of aspirin inhibit the enzyme at both cyclooxygenase sites.

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.

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).

Extrinsic Fibrinolysis

Endothelial cells synthesize and release t-PA. Both t-PA and a related substance, urokinase plasminogen activator (u-PA), 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.

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.

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 (ASPAC) 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 (rt-PA; Alteplase) is a second-generation agent that is made by recombinant DNA technology and is relatively fibrin specific.

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.

Figure 24-4 The fibrinolytic pathway. Antifibrinolytic drugs inhibit fibrinolysis by binding to both plasminogen and plasmin. Intrinsic blood activators (factor XIIa), extrinsic tissue activators (t-PA, u-PA), and exogenous activators (streptokinase, ASPAC) 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.

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).

Figure 24-5 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.)

Source and Biological Role

Heparin is found mostly in the lungs, intestines, and liver of mammals, with skin, lymph nodes, and thymus providing less plentiful sources. 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 biological role relating to immune function. Heparin may assist white blood cell movements in the interstitium after an immunologic response has been triggered.

Most commercial preparations of heparin now use pig intestine, 40,000 pounds of which yield 5 kg of heparin. Prevention of postoperative thrombosis constituted the initial clinical use of heparin in 1935.

Potency

Heparin potency is determined by comparing the test specimen against a known standard’s ability to prolong coagulation. Current United States Pharmacopeia (USP) and British Pharmacopoeia (BP) assays use a PT-like method on pooled sheep’s plasma obtained from slaughterhouses.

UFH dose should not be specified by weight (milligrams) because of the diversity of anticoagulant activity expected from so heterogeneous a compound. 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. 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 biological activity. None of these measures has anything to do with the effect of a unit on anticoagulation effect for human cardiac surgery.