Just as the formation of the platelet plug is a highly cooperative process, so is the concomitant formation of the fibrin clot. Amplification of a small signal (vascular injury) is transduced by a series of molecular interactions into a fibrin clot. The protagonists in this dramatic process are a set of plasma proteins that normally circulate as inactive zymogens. Figure 13-2 depicts a prototypical or consensus pathway. When the coagulation cascade is activated, either in vivo or in the test tube, a zymogen is converted into an active proteolytic enzyme capable of specifically cleaving the next zymogen in the set, and so on. This highly controlled limited proteolysis leads to the eventual formation of thrombin and generation of the fibrin clot.
Consensus pathway responsible for the rapid and controlled formation of the fibrin clot. Zymogen activation by limited proteolysis from inactive to active enzyme (left) and inhibition of the enzyme (E) by a specific inhibitor (I).
During the last century, biochemical studies of in vitro clot formation have gradually led to the identification of the specific proteins that participate in the coagulation cascade, along with in-depth understanding of how they interact in a precisely controlled and orchestrated fashion. A substantial portion of this cumulative knowledge has come from rigorous investigation of families with inherited defects of specific coagulation proteins (see Chapter 15).
Figure 13-3 depicts the coagulation proteins involved in clot formation prior to the formation of fibrin. The zymogens, shown in image A (prothrombin and factors VII, IX, X, and XI), are synthesized in the liver. As this figure demonstrates, they have some striking structural similarities. All have a signal peptide at the N-terminus, and except for factor XI, a propeptide is immediately adjacent. These N-terminal portions are cleaved within the hepatocyte prior to the release of the mature zymogen into the circulation. All five zymogens have a large catalytic domain at the C-terminus that, when activated, functions as a serine protease that specifically cleaves and activates the next protein in the coagulation cascade. Four of these zymogens, factors VII, IX, X, and prothrombin, undergo a crucial post-translational modification in the Golgi body prior to secretion into the plasma. Specific glutamic acid residues in the region adjacent to the propeptide undergo vitamin K–dependent carboxylation. This additional negative charge facilitates the binding of calcium ions, an essential cofactor for optimal function.
Proteins in the proximal coagulation cascade. A) Zymogens are converted to active enzymes by proteolytic cleavage. Blue rectangles at left = signal peptides; • • • • • = propeptide; Y = specific sites of glutamic acid carboxylation. The curved arrows to the right show sites of cleavage releasing the C-terminal catalytic domain (long blue rectangle on right), which now functions as an active serine protease. B) Cofactors serve as docking sites for zymogens. Tissue factor is a transmembrane protein. (The lipid bilayer is shown as a pair of vertical rectangles.) Factors VIII and V are activated by proteolytic cleavage at sites shown by the curved arrows. (Modified with permission from Furie B and Furie BC. The molecular basis of blood coagulation. Cell. 1988. 53: 505-518.)
Protein cofactors play an equally important role in the regulation of the coagulation cascade. They bind to platelet and endothelial cell membranes, serving as docking sites for the zymogens and contributing importantly to the amplification that is essential for the rapid formation of the fibrin clot. As shown in Figure 13-3B, tissue factor is a transmembrane protein on the surface of extravascular cells, cells within the vessel wall, and circulating microparticles. Like the zymogens mentioned earlier, factors VIII and V are actually pro-cofactors—soluble plasma proteins that require proteolytic cleavage for activation.
In the test tube, the formation of the fibrin clot proceeds by the intrinsic pathway depicted in Figure 13-4. Exposure of the plasma to the glass surface of the tube triggers the activation of factor XII to XIIa (the a stands for activated), which in turn, with the participation of high molecular weight kininogen, activates factor XI to factor XIa. Factor XIa, in the presence of calcium ions, catalyzes the activation of factor IX to factor IXa. Factor IXa forms a complex with activated factor VIII (factor VIIIa) on membrane surfaces in the presence of calcium ions to trigger the activation of factor X to factor Xa.
The blood coagulation cascade in vitro. A fibrin clot can be formed by activation of either the intrinsic or extrinsic pathway. In the test tube, surface contacts trigger the intrinsic pathway. Key: diamonds = zymogens; circles = active enzymes and cofactors; pink rectangles = pro-cofactors; green rectangles = bimolecular complexes; HMWK = high molecular weight kininogen; TF = tissue factor; PT = prothrombin; T in small circle = thrombin; FG = fibrinogen; F = fibrin. (Modified with permission from Furie B and Furie BC. The molecular basis of blood coagulation. In Hoffman R, Benz EJ, Shattil SJ, et al, eds. Hematology, Basic Principles and Practice, 3rd Edition, New York USA, Churchill Livingstone, 2001:1784.)
According to conventional wisdom, the extrinsic pathway depicted in Figure 13-4 is responsible for the initiation of the coagulation cascade in vivo. Tissue factor is constitutively expressed on cells within blood vessels. Upon vessel injury, tissue factor is exposed and binds to minute amounts of activated factor VII (factor VIIa) that are normally present in plasma. The procoagulant complex factor VIIa/tissue factor then initiates the coagulation cascade by converting factor X to factor Xa. As Figure 13-4 shows, this is the point at which the extrinsic and intrinsic pathways converge.
However, in vivo coagulation is more complicated. Low concentrations of tissue factor may be concentrated in the thrombus early in its development. The model that best reflects in vivo coagulation in humans is depicted in Figure 13-5. As mentioned earlier, the primary initiating trigger is the exposure of tissue factor on the injured blood vessel. Tissue factor, along with phosphatidylserine on the plasma membrane of platelets and endothelial cells, forms a complex with activated factor VII (factor VIIa), which not only catalyzes the activation of factor X but also the activation of factor IX. The latter event is of little importance in the test tube but is critical in vivo, because individuals with deficiencies of factor IX or its cofactor, factor VIII, suffer from hemophilia (Chapter 15), a severe bleeding disorder.
The blood coagulation cascade in vivo. The primary initiating event is the exposure of tissue factor (TF) on the injured blood vessel. Tissue factor forms a complex with activated factor VII (FVIIa), which not only catalyzes the activation of factor X but also the activation of factor IX. This dual role of the FVIIa/tissue factor complex contributes to the dramatic amplification of blood coagulation in vivo. Activated coagulation factors are shown as circles. (Modified with permission from Furie B and Furie BC. The molecular basis of blood coagulation. In Hoffman R, Benz EJ, Shattil SJ, et al, eds. Hematology, Basic Principles and Practice, 3rd Edition, New York USA, Churchill Livingstone, 2001:1785.)
At each successive step in the bottom of the cascade, the concentration of the respective zymogen increases, thus favoring amplification of clot formation. It follows then that by far the most abundant coagulation protein in the plasma is the structural protein fibrinogen, which is polymerized to form fibrin in the final step in the cascade. As shown in Figure 13-6A, fibrinogen is a heterodimer composed of a pair of three subunits, Aα, Bβ, and γ, linked by disulfide bonds. Upon cleavage by thrombin (factor IIa), small fibrinopeptides, designated fibrinopeptide A and fibrinopeptide B, are released from the Aα and Bβ subunits, respectively. Measurement of fibrinopeptide A reflects the conversion of fibrinogen to fibrin and can be useful in monitoring patients with disseminated intravascular coagulation (Chapter 16). Upon cleavage, fibrinogen undergoes a conformational change that enables it to form noncovalent polymers via end-to-end interactions as well as side-to-side interactions between the linear strands. The strength and durability of the fibrin polymer is then greatly enhanced by the action of factor XIIIa, which catalyzes the formation of covalent crosslinks between the fibrin strands.
Fibrinogen and fibrin. A) Diagram of the dimeric structure of fibrinogen showing its three subunits connected by disulfide bonds. Fibrinopeptides A and B (FPA and FPB) are cleaved by thrombin, enabling fibrinogen to polymerize into fibrin by forming end-to-end as well as side-to-side contacts, as shown in panel B. (Modified with permission from Furie B and Furie BC. The molecular basis of blood coagulation. In Hoffman R, Benz EJ, Shattil SJ, et al, eds. Hematology, Basic Principles and Practice, 3rd Edition, New York USA, Churchill Livingstone: 2001: 1797, 1798.)