The Process

The intact vascular endothelium is the primary barrier against hemorrhage. The endothelial cells that line the vessel wall normally inhibit coagulation and provide a smooth surface that permits rapid blood flow.

After vascular injury, vasoconstriction occurs and flowing blood comes in contact with the subendothelial matrix (Fig. 469-1). In flowing blood, when exposed to subendothelial matrix proteins, von Willebrand factor (VWF) changes conformation and provides the glue to which the platelet VWF receptor, the glycoprotein Ib complex, binds, tethering platelets to sites of injury. When the VWF receptor binds its ligand, complex signaling occurs from the outside membrane receptor to intracellular pathways, activating the platelets and triggering secretion of storage granules containing adenosine diphosphate (ADP), serotonin, and stored plasma and platelet membrane proteins. Upon activation, the platelet receptor for fibrinogen, α2bβ3, is switched on (“inside out” signaling) to bind fibrinogen and triggers the aggregation and recruitment of other platelets to form the platelet plug. Multiple physiologic agonists can trigger platelet activation and aggregation, including ADP, collagen, thrombin, and arachidonic acid. Aggregation involves the interaction of specific receptors on the platelet surface with plasma hemostatic proteins, primarily fibrinogen.

Figure 469-1 The clotting cascade, with sequential activation and amplification of clot formation. Many of the factors (F) are activated by the clotting factors shown above them in the cascade. The activated factors are designated by the addition of an a. On the right side, the major anticoagulants and the sites that they regulate are shown: Tissue factor pathway inhibitor (TFPI) regulates tissue factor (TF]); factor VIIa, protein C, and protein S (P-C/S) regulate factors VIII and V; and antithrombin III (AT-III) regulates factor Xa and thrombin (factor IIa). The dotted line shows that, in vivo, TF and factor VIIa activate both factors IX and X but that, in vitro, only the activation of factor X is measured. Unactivated factor VIII, when bound to its carrier protein, von Willebrand factor, is protected from protein C inactivation. When thrombin, or factor Xa activates factor VIII, it becomes unbound from von Willebrand factor, whereupon it can participate with factor IXa in the activation of factor X in the presence of phospholipid (PL) and Ca²⁺ (the “tenase” complex). Factor XIIIa cross links the fibrin clot and thereby makes it more stable. Prekallikrein, high molecular weight kininogen (HMWK), and factor XII are shown in blue because they do not have a physiologic role in coagulation, although they contribute to the clotting time in partial thromboplastin time (PTT).

One of the subendothelial matrix proteins that are exposed after vascular injury is tissue factor. Just as exposed subendothelial matrix proteins bind VWF, exposed tissue factor binds to factor VII and activates the clotting cascade, as shown in Figure 469-2. The activated clotting factor then initiates the activation of the next sequential clotting factor in a systematic manner. Our understanding of the sequence of steps in the cascade followed assignment of the numerals for the clotting factors for the participant proteins, and thus the sequence seems “out of numerical order.” During the process of platelet activation, internalized platelet phospholipids (primarily phosphatidylserine) become externalized and interact at 2 specific, rate-limiting steps in the clotting process—those involving the cofactors factor VIII (X-ase complex) and factor V (prothrombinase complex). Both of these reactions are localized to the platelet surface and bring together the active enzyme, an activated cofactor, and the zymogen that will form the next active enzyme in the cascade. This sequence results in amplification of the process, which supplies a burst of clotting where it is physiologically needed. In vivo, autocatalysis of factor VII generates small amounts of VIIa continuously, so the system is always poised to act. Near the bottom of the cascade, the multipotent enzyme thrombin is formed. Thrombin converts fibrinogen into fibrin, activates factors V, VIII, and XI, and aggregates platelets. Activation of factor XI by thrombin amplifies further thrombin generation and contributes to inhibition of fibrinolysis. Thrombin also activates factor XIII. The stable fibrin-platelet plug is ultimately formed through clot retraction and cross linking of the fibrin clot by factor XIIIa.

Figure 469-2 The hemostatic mechanism. ADP, adenosine diphosphate; GAGs, glycosaminoglycans; NO, nitric oxide; PGI₂, prostacyclin; PAI, plasminogen activator inhibitor; TAFI, thrombin-activated fibrinolytic inhibitor; TXA₂, thromboxane A₂; VWF, von Willebrand factor.

Virtually all procoagulant proteins are balanced by an anticoagulant protein that regulates or inhibits procoagulant function. Four clinically important, naturally occurring anticoagulants regulate the extension of the clotting process. They are antithrombin III (AT-III), protein C, protein S, and tissue factor pathway inhibitor (TFPI). AT-III is a serine protease inhibitor that regulates factor Xa and thrombin primarily and factors IXa, XIa, and XIIa to a lesser extent. When thrombin in flowing blood encounters intact endothelium, thrombin binds to thrombomodulin, its endothelial receptor. The thrombin-thrombomodulin complex then converts protein C into activated protein C. In the presence of the cofactor protein S, activated protein C proteolyses and inactivates factor Va and factor VIIIa. Inactivated factor Va is, in fact, a functional anticoagulant that inhibits clotting. TFPI limits activation of factor X by factor VIIa and tissue factor and shifts the activation site of tissue factor and factor VIIa to that of factor IX (see Figs. 469-1 and 469-2).

Once a stable fibrin-platelet plug is formed, the fibrinolytic system limits its extension and also lyses the clot (fibrinolysis) to reestablish vascular integrity. Plasmin, generated from plasminogen by either urokinase-like or tissue-type plasminogen activator, degrades the fibrin clot. In the process of dissolving the fibrin clot, fibrin degradation products (FDPs) are produced. The fibrinolytic pathway is regulated by plasminogen activator inhibitors and α₂-antiplasmin as well as by the thrombin-activatable fibrinolysis inhibitor (TAFI). Finally, the flow of blood in and around the clot is crucial, because flowing blood returns to the liver, where activated clotting factor complexes are removed and new procoagulant and anticoagulant proteins are synthesized to maintain homeostasis of the hemostatic system.

Pathology

Congenital deficiency of an individual procoagulant protein leads to a bleeding disorder, whereas deficiency of an anticoagulant (clotting factor inhibitor) predisposes the patient to excessive thrombosis. In acquired hemostatic disorders, there are frequently multiple problems with homeostasis that perturb and dysregulate hemostasis. A primary illness (sepsis) and its secondary effects (shock and acidosis) activate coagulation and fibrinolysis and impair the host’s ability to restore normal hemostatic function. When sepsis triggers disseminated intravascular coagulation (DIC), platelets, procoagulant clotting factors, and anticoagulant proteins are consumed, leaving the hemostatic system unbalanced and prone to bleeding or clotting. Similarly, newborn infants and patients with severe liver disease have synthetic deficiencies of both procoagulant and anticoagulant proteins. Such dysregulation causes the patient to be predisposed to both hemorrhage and thrombosis with mild or moderate triggers that result in major alterations in the hemostatic process.

In the laboratory evaluation of hemostasis, parameters are manipulated to allow assessment of isolated aspects of hemostasis and limit the multifunctionality of some of its components. The coagulation process is studied in plasma anticoagulated with citrate to bind calcium, with added phospholipid to mimic the reaction surface normally provided by the platelet membrane and with a stimulus to trigger clotting. Calcium is added to restart the clotting process. This results in anomalies such that the in vivo physiologic pathway of clotting in which factor VIIa activates factor IX is bypassed; instead, in prothrombin time (PT), factor VIIa activates factor X. If this were truly the physiologic situation, then there would be an in vivo bypass mechanism that would ameliorate severe factor VIII and factor IX deficiencies, the 2 most common severe bleeding disorders.