Key Points
- 1.
It is easiest to think of coagulation as a wave of biologic activity occurring at the site of tissue injury, consisting of initiation, acceleration, control, and lysis.
- 2.
Hemostasis is part of a larger body system: inflammation. The protein reactions in coagulation have important roles in signaling inflammation.
- 3.
Thrombin is the most important coagulation modulator, interacting with multiple coagulation factors, platelets, tissue plasminogen activator, prostacyclin, nitric oxide, and various white blood cells.
- 4.
The serine proteases that compose the coagulation pathway are balanced by serine protease inhibitors, termed serpins. Antithrombin is the most important inhibitor of blood coagulation, but others include heparin cofactor II and alpha 1 antitrypsin.
- 5.
Platelets are the most complex part of the coagulation process, and antiplatelet drugs are important therapeutic agents.
- 6.
Heparin requires antithrombin to anticoagulate blood and is not an ideal anticoagulant for cardiopulmonary bypass. Newer anticoagulants are actively being sought to replace heparin.
- 7.
Protamine can have many adverse effects. Ideally, a new anticoagulant will not require reversal with a toxic substance such as protamine.
- 8.
Antifibrinolytic drugs are often given during cardiac surgery; these drugs include ε-aminocaproic acid and tranexamic acid.
- 9.
Recombinant factor VIIa is an off-label “rescue agent” to stop bleeding during cardiac surgery, but it can also be prothrombotic, which is an off-label use of the drug.
- 10.
Every effort should be made to avoid transfusion of banked blood products during routine cardiac surgery. In fact, bloodless surgery is a reality in many cases. Patient blood management, including techniques to reduce coagulation precursors, has been shown to be cost effective and to have better outcomes than routine surgery.
- 11.
The evolving risks of transfusion have shifted from viral transmission to transfusion-related acute lung injury and immunosuppression. Those patients who receive allogeneic blood have a measurable increased rate of perioperative serious infection (approximately 16% increase per unit transfused).
- 12.
Cardiac centers that have adopted multidisciplinary blood management strategies have improved patient outcomes and decreased costs. The careful application of these strategies in use of coagulation drugs and products is very beneficial.
- 13.
New purified human protein adjuncts are replacing fresh-frozen plasma and cryoprecipitate with four-agent prothrombin complex concentrate and human lyophilized fibrinogen.
Coagulation and bleeding assume particular importance when surgery is performed on the heart and great vessels using extracorporeal circulation. This chapter provides an understanding of the depth and breadth of hemostasis relating to cardiac procedures, beginning with coagulation pathophysiology. The pharmacology of heparin and protamine follows. This background is then applied to treatment of the bleeding patient.
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Overview of Hemostasis
Proper hemostasis requires the participation of innumerable biologic elements ( Box 27.1 ). This section groups them 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 (perhaps >800) and control mechanisms, all interacting simultaneously. The interaction of the platelets, endothelial cells, and proteins either to activate or to deactivate coagulation is a highly buffered and controlled process. It is perhaps easiest to think of coagulation as a wave of biologic activity occurring at the site of tissue injury ( Fig. 27.1 ). 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 really is begun with endothelial cell destruction or dysfunction. This initiation phase leads to binding of platelets, as well as protein activations; both happen nearly simultaneously, and each has feedbacks into the other. Platelets adhere, creating an activation or acceleration phase that gathers many cells to the site of injury. From that adhesion a large number of events of cellular/protein messaging cascade. As the activation phase ramps up into an explosive set of reactions, counter-reactions are spun off, leading to control proteins damping the reactions. It is easiest, conceptually, to think of these control mechanisms as analogous to a nuclear reactor. The activation phase would continue to grow and overcome the whole organism unless control rods were inserted (eg, thrombomodulin, proteins C and S, and tissue plasminogen activator [t-PA]) to stop the spread of the reaction. The surrounding normal endothelium acts quite differently from the disturbed (ischemic) endothelium. Eventually, the control reactions overpower the acceleration reactions and lysis comes into play. A key concept is that hemostasis is part of a larger body system: inflammation. Most, if not all, of the protein reactions of coagulation control have importance in signaling inflammation leading to 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.
Coagulation factor activation
Platelet function
Vascular endothelium
Fibrinolysis and modulators of coagulation
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 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.
Contact Activation
Factor XII, high-molecular-weight kininogen (HMWK), prekallikrein (PK), and factor XI form the contact or surface activation group. 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. 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.
Intrinsic System
Intrinsic activation forms factor XIa from the products of surface activation. Factor XIa splits factor IX to form factor IXa, with Ca 2+ required for this process. Then factor IXa activates factor X with help from Ca 2+ , a phospholipid surface (platelet-phosphatidylserine), and a GP cofactor, factor VIIIa.
Extrinsic System
Activation of factor X can proceed independently of factor XII by substances classically thought to be extrinsic to the vasculature. Any number of endothelial cell insults can lead to the production of tissue factor by the endothelial cell. At rest, the endothelial cell is quite antithrombotic. However, with ischemia, reperfusion, sepsis, or cytokines (particularly interleukin [IL]-6), the endothelial cell will stimulate its production of intracellular nuclear factor-κB and send messages for the production of messenger RNA for tissue factor production. This can happen quickly and the resting endothelial cell can turn out large amounts of tissue factor. It is widely held today that the activation of tissue factor is what drives many of the abnormalities of coagulation after cardiac surgery, rather than contact activation. Thromboplastin, also known as tissue factor, released from tissues into the vasculature, acts as a cofactor for initial activation of factor X by factor VII. Factors VII and X then activate one another with the help of platelet phospholipid and Ca 2+ , thus rapidly generating factor Xa. (Factor VIIa also activates factor IX, thus linking the extrinsic and intrinsic paths.)
Common Pathway
Factor Xa splits prothrombin (factor II) to thrombin (factor IIa). The combination of factors Xa, Va, and Ca 2+ is termed the prothrombinase complex —a critical step. Factor Xa anchors to the membrane surface (of platelets) via Ca 2+ . 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.
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.
Vitamin K
Those factors that require calcium (II, VII, IX, X) depend on vitamin K to add between 9 and 12 γ-carboxyl groups to glutamic acid residues near their amino terminal. Calcium tethers the negatively charged carboxyl groups to the phospholipid surface (platelets), thus facilitating molecular interactions. Some inhibitory proteins also depend on vitamin K (proteins C and S) for their functional completion.
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.
| Factor | Activated By | Acts On | Minimal Amount Needed | Replacement Source | Alternate Name and Comments |
|---|---|---|---|---|---|
| XIII | IIa | Fibrin | <5% | FFP, CRYO | Fibrin-stabilizing factor; not a serine protease, but an enzyme |
| XII | Endothelium | XI | None | Not needed | Hageman factor; activation enhanced by XIIa |
| XI | XIIa | IX | 15%–25% | FFP | Plasma thromboplastin antecedent |
| X | VIIa or IXa | II | 10%–20% | FFP, 9C | Stuart-Prower factor; vitamin K–dependent |
| IX | VIIa or XIa | X | 25%–30% | FFP, 9C, PCC | Christmas factor; vitamin K–dependent |
| VIII | IIa | X | >30% | CRYO, 8C, FFP | Antihemophilic factor; a cofactor; RES synthesis |
| VII | Xa | X | 10%–20% | FFP, PCC | Serum prothrombin conversion accelerator; vitamin K–dependent |
| V | IIa | II | <25% | FFP | Proaccelerin; a cofactor; RES and liver synthesis |
| IV | — | — | — | — | Calcium ion; binds II, VII, IX, X to phospholipid |
| III | — | X | — | — | Thromboplastin/tissue factor; a cofactor |
| II | Xa | I | 20%–40% | FFP, PCC | Prothrombin; vitamin K–dependent |
| I | IIa | — | 1 g/L | CRYO, FFP, FC | Fibrinogen; activated product is soluble fibrin |
| vWF | — | VIII | See VIII | CRYO, FFP | von Willebrand factor; endothelial cell synthesis |
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 about 10 to 15 minutes because 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 release coagulation factors; secrete active substances affecting themselves, other platelets, the endothelium, and other coagulation factors; and alter shape (through active actin-myosin contraction) to expose membrane GPs essential to hemostasis. 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.
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 ).
| Drug Type | Composition | Mechanism | Indications | Route | Half-Life | Metabolism |
|---|---|---|---|---|---|---|
| Aspirin | Acetylsalicylic acid | Irreversible COX inhibition | CAD, AMI, PVD, PCI, ACS | Oral | 10 days | Liver, kidney |
| NSAIDs | Multiple | Reversible COX inhibition | Pain | Oral | 2 days | Liver, kidney |
| Adhesion inhibitors (eg, dipyridamole) | Multiple | Block adhesion to vessels | VHD, PVD | Oral | 12 hours | Liver |
| ADP Receptor Antagonists | ||||||
| —Clopidogrel (Plavix), prasugrel (Effient) | Thienopyridines | Irreversible | AMI, CVA, PVD, ACS, PCI | Oral | 5 days | Liver |
| —Ticagrelor (Brilinta) | Nonthienopyridine | Reversible | AMI, CVA, PVD, ACS, PCI | Oral | 3–5 days | Liver |
| —Cangrelor (Kengreal) | Nonthienopyridine | Reversible | AMI, CVA, PVD, ACS, PCI | IV | 3–10 min | Blood |
| PAR-1 Inhibitors | ||||||
| —Vorapaxar (Zontivity) | PAR-1 antagonist | Irreversible—inhibits thrombin-induced platelet activation | AMI, PVD | Oral | 20 hr–4 wk | Liver |
| GPIIb/IIIa Receptor Inhibitors | ||||||
| —Abciximab (ReoPro) | Monoclonal antibody | Nonspecific—binds to other receptors | PCI, ACS | IV | 12–18 hours | Plasma proteinase |
| —Eptifibatide (Integrilin) | Peptide | Reversible—specific to GPIIb/IIIa | PCI, ACS | IV | 2–4 hours | Kidney |
| —Tirofiban (Aggrastat) | Nonpeptide-tyrosine derivative | Reversible—specific to GPIIb/IIIa | PCI, ACS, AMI, PVD | IV | 2–4 hours | Kidney |
Adenosine Diphosphate Receptor Antagonists
Clopidogrel (Plavix), and prasugrel (Effient), are thienopyridine derivatives that inhibit the ADP receptor pathway to platelet activation. They have a slow onset of action because they must be converted to active drugs, and their potent effects last the lifetime of the platelets affected (5–10 days). Clopidogrel and prasugrel are the preferred drugs. They are administered orally once daily to inhibit platelet function and are quite effective in decreasing myocardial infarctions (MIs) after percutaneous coronary interventions (PCIs). The combination of aspirin and clopidogrel has led to increased bleeding, but is used in an effort to keep vessels and stents open. Recently, two new nonthienopyridine ADP P 2 Y 12 inhibitors have become available. Ticagrelor is a direct-acting oral drug, and cangrelor is a short-acting intravenous agent. The latter drug may be a very valuable bridging drug for use in the PCI laboratory and perioperative period.
Glycoprotein Iib/Iiia Receptor Inhibitors
These are the most potent (>90% platelet inhibition) because they act at the final common pathway of platelet aggregation with fibrinogen, no matter which agonist began the process. All of the drugs mentioned earlier work at earlier phases of activation of platelet function. These drugs are all administered by intravenous infusion, and they do not work orally. The GPIIb/IIIa inhibitors often are used in patients taking aspirin because they do not block thromboxane A 2 production. The dose of heparin usually is reduced when used with these drugs (ie, PCI to avoid bleeding at the vascular puncture sites). Platelet activity can be monitored to determine the extent of blockade. Excessive bleeding requires allowing the short-acting drugs to wear off, while possibly administering platelets to patients receiving the long-acting drug abciximab. Most studies have found increased bleeding in patients receiving these drugs who required emergency CABG.
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, α 2 -antiplasmin, quickly consumes any plasmin formed from localized fibrinolysis. Thus localized fibrinolysis, not systemic fibrinogenolysis, accompanies normal hemostasis.
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.
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 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
Fig. 27.3 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 MI. 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. 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.
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Overview of Hemostasis
Proper hemostasis requires the participation of innumerable biologic elements ( Box 27.1 ). This section groups them 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 (perhaps >800) and control mechanisms, all interacting simultaneously. The interaction of the platelets, endothelial cells, and proteins either to activate or to deactivate coagulation is a highly buffered and controlled process. It is perhaps easiest to think of coagulation as a wave of biologic activity occurring at the site of tissue injury ( Fig. 27.1 ). 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 really is begun with endothelial cell destruction or dysfunction. This initiation phase leads to binding of platelets, as well as protein activations; both happen nearly simultaneously, and each has feedbacks into the other. Platelets adhere, creating an activation or acceleration phase that gathers many cells to the site of injury. From that adhesion a large number of events of cellular/protein messaging cascade. As the activation phase ramps up into an explosive set of reactions, counter-reactions are spun off, leading to control proteins damping the reactions. It is easiest, conceptually, to think of these control mechanisms as analogous to a nuclear reactor. The activation phase would continue to grow and overcome the whole organism unless control rods were inserted (eg, thrombomodulin, proteins C and S, and tissue plasminogen activator [t-PA]) to stop the spread of the reaction. The surrounding normal endothelium acts quite differently from the disturbed (ischemic) endothelium. Eventually, the control reactions overpower the acceleration reactions and lysis comes into play. A key concept is that hemostasis is part of a larger body system: inflammation. Most, if not all, of the protein reactions of coagulation control have importance in signaling inflammation leading to 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.
Coagulation factor activation
Platelet function
Vascular endothelium
Fibrinolysis and modulators of coagulation
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 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.
Contact Activation
Factor XII, high-molecular-weight kininogen (HMWK), prekallikrein (PK), and factor XI form the contact or surface activation group. 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. 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.
Intrinsic System
Intrinsic activation forms factor XIa from the products of surface activation. Factor XIa splits factor IX to form factor IXa, with Ca 2+ required for this process. Then factor IXa activates factor X with help from Ca 2+ , a phospholipid surface (platelet-phosphatidylserine), and a GP cofactor, factor VIIIa.
Extrinsic System
Activation of factor X can proceed independently of factor XII by substances classically thought to be extrinsic to the vasculature. Any number of endothelial cell insults can lead to the production of tissue factor by the endothelial cell. At rest, the endothelial cell is quite antithrombotic. However, with ischemia, reperfusion, sepsis, or cytokines (particularly interleukin [IL]-6), the endothelial cell will stimulate its production of intracellular nuclear factor-κB and send messages for the production of messenger RNA for tissue factor production. This can happen quickly and the resting endothelial cell can turn out large amounts of tissue factor. It is widely held today that the activation of tissue factor is what drives many of the abnormalities of coagulation after cardiac surgery, rather than contact activation. Thromboplastin, also known as tissue factor, released from tissues into the vasculature, acts as a cofactor for initial activation of factor X by factor VII. Factors VII and X then activate one another with the help of platelet phospholipid and Ca 2+ , thus rapidly generating factor Xa. (Factor VIIa also activates factor IX, thus linking the extrinsic and intrinsic paths.)
Common Pathway
Factor Xa splits prothrombin (factor II) to thrombin (factor IIa). The combination of factors Xa, Va, and Ca 2+ is termed the prothrombinase complex —a critical step. Factor Xa anchors to the membrane surface (of platelets) via Ca 2+ . 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.
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.
Vitamin K
Those factors that require calcium (II, VII, IX, X) depend on vitamin K to add between 9 and 12 γ-carboxyl groups to glutamic acid residues near their amino terminal. Calcium tethers the negatively charged carboxyl groups to the phospholipid surface (platelets), thus facilitating molecular interactions. Some inhibitory proteins also depend on vitamin K (proteins C and S) for their functional completion.
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.
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