Regulation of Hemostasis and Thrombosis

Vascular Bed.

It is very important that blood not clot within the vascular system. In the baseline state, vascular endothelial cells provide a nonthrombogenic interface with the circulating blood. Endothelial cells do not normally express molecules that support platelet adhesion or promote activation and activity of the coagulation proteins. In addition, the antithrombotic features of the endothelial surface go beyond simply being “inert” with respect to coagulation. The endothelium also expresses molecules that actively downregulate the coagulation reactions on its surface: principally thrombomodulin to localize activated protein C (APC) to the endothelial surface and heparan sulfates to localize antithrombin (AT) to the endothelial surface. A further discussion of these mechanisms is presented in the section on thrombosis. These properties are critical to preventing coagulation from being initiated at inappropriate sites within the vasculature and preventing appropriately initiated hemostatic reactions from spreading within the vascular tree.

Extravascular Tissues.

When an injury disrupts a blood vessel, it allows blood to contact extravascular cells and matrix. Extracellular matrix proteins—such as collagen, fibronectin, thrombospondin, and laminin—interact with adhesive receptors on blood platelets and support formation of the initial platelet plug at the site of injury, referred to as primary hemostasis . Perivascular tissues also express significant levels of tissue factor (TF). Exposure of TF to blood initiates the process of thrombin generation on the surfaces of adherent platelets and ultimately leads to stabilization of the initial platelet plug in a fibrin clot, referred to as secondary hemostasis . Different tissues express different complements of matrix components and procoagulants. Thus, the local tissue environment plays a role in determining the intensity of the procoagulant response to an injury.

Platelets.

Membrane receptors for collagen (glycoprotein [GP] VI) and other subendothelial and extravascular matrix proteins are present on the platelet membrane and mediate binding of unactivated platelets at sites of injury. Platelet binding is also mediated by von Willebrand factor (vWF) bridging between collagen and the platelet receptor GP Ib. These receptor-binding events also transmit an activation signal to the platelets. Full platelet activation also requires stimulation by thrombin that is produced as the coagulation reactions are initiated. The platelet surface receptor for fibrinogen, GPIIb/IIIa, rapidly changes conformation from an inactive to an active form on platelet activation. This conformational change allows platelet aggregates to be stabilized by binding to fibrinogen even before conversion to fibrin begins.

Platelet activation also initiates the synthesis of prostaglandins and thromboxanes—compounds that modulate platelet activation and promote vasoconstriction. Platelet adhesion and activation at a site of injury, in concert with local vasoconstriction, provides initial hemostasis for small-caliber vessels. Once hemostasis is achieved by these mechanisms, the subsequent stabilization of the platelet plug in a fibrin meshwork can proceed more effectively than if bleeding continues. Initial hemostasis may be established even if a deficiency of plasma coagulation proteins is present. The platelet plug is insufficient, however, to provide long-term hemostasis and delayed rebleeding occurs if it is not reinforced by a stable fibrin clot during secondary hemostasis. Even after overt bleeding (loss of red blood cells) is stopped by the stable fibrin clot, leakage of plasma proteins from the microvasculature continues. A hemostatic clot structure with a densely packed core of platelets is required to form a tight vascular seal that minimizes the leakage of plasma proteins at a site of injury.

It is becoming clear that, in addition to providing primary hemostasis following an overt injury, platelets also play more complex and subtle roles in maintaining vascular integrity. It has long been known that platelets maintain endothelial integrity in the microvasculature. A failure of this function is responsible for petechiae resulting from thrombocytopenia. However, platelets also directly prevent microvascular bleeding at sites of inflammation and angiogenesis by mechanisms that are independent of fibrin generation.

Coagulation Proteins.

Adequate levels and function of each of a series of procoagulant proteins are required for hemostasis. The coagulation proteins can be organized into several groups based on their structural features.

The vitamin K–dependent factors include factors II (prothrombin), VII, IX, and X. These each have a structural domain in which several glutamic acid residues are posttranslationally modified to gamma carboxy-glutamic acid (Gla) residues by a vitamin K–dependent carboxylase. The vitamin K cofactor is oxidized from a quinone to an epoxide in the process. A vitamin K epoxide reductase then cycles the vitamin K back to the quinone form to allow carboxylation of additional glutamic acid residues. The negatively charged Gla residues bind calcium ions. These binding interactions hold the Gla-containing proteins in their active conformation. The calcium-bound form of the Gla domain is responsible for mediating binding of the coagulation factors to phospholipid membranes. Lipids with negatively charged head groups, particularly phosphatidylserine, are required for binding and activity of the Gla-containing factors.

The carboxylation process is inhibited by the anticoagulant warfarin, which competes with vitamin K for binding to the reductase. This results in the production of undercarboxylated forms of the vitamin K–dependent proteins, which are nonfunctional. The vitamin K–dependent procoagulants are zymogens (inactive precursors) of serine proteases. Each is activated by cleavage of at least one peptide bond. The activated form is indicated by the letter “a.” Factors VIIa, IXa, and Xa each require calcium ions, a suitable cell (phospholipid) membrane surface, and a protein cofactor for their activity in hemostasis.

Factor IIa (thrombin) is a little different from the activated forms of the other vitamin K–dependent factors. Its Gla domain is released from the protease domain during activation. Thus, it no longer binds directly to phospholipid membranes. It also does not require a cofactor to cleave fibrinogen and initiate fibrin assembly or to activate platelet receptors. IIa that escapes the vicinity of a hemostatic plug can bind to a cofactor on endothelial cell surfaces, that is, thrombomodulin. After binding to thrombomodulin, IIa can no longer activate platelets or cleave fibrinogen. Instead, it triggers an antithrombotic pathway by activating protein C (PC) on the endothelial surface.

PC and protein S (PS) are also vitamin K–dependent factors. They do not act as procoagulants but rather as antithrombotics on endothelial surfaces. PC is the zymogen of a protease, while PS has no enzymatic activity but serves as a cofactor for APC. The APC/PS complex cleaves and inactivates FVa and FVIIIa, thus preventing propagation of thrombin generation on normal healthy endothelium.

Factors V and VIII are large structurally related glycoproteins that act as cofactors. They have no enzymatic activity of their own, but when activated by proteolytic cleavage dramatically enhance the proteolytic activity of factors Xa and IXa, respectively.

Factor VIII circulates in a noncovalent complex with vWF, which prolongs its half life in the circulation. The vWF-FVIII complex binds to the platelet surface primarily via GPIb as vWF mediates adhesion of platelets to collagen under high shear conditions. Cleavage and activation of FVIII releases it from vWF so that it can assemble into a complex with FIXa on the platelet surface, where it activates FX.

FV circulates in the plasma and is packaged in the alpha granules of platelets during their development from megakaryocytes. It is released upon platelet activation in a partially activated form. Both plasma and platelet-derived FV can be fully activated by cleavage by FXa or IIa. The FVa then assembles into a complex with FXa on the platelet surface, where it activates prothrombin to IIa.

TF is also a cofactor but is structurally unrelated to any of the other coagulation factors. Instead, it is related to one class of cytokine receptors. This lineage emphasizes the close evolutionary and physiologic links between the coagulation system and the other components of the host response to injury. Rather than circulating in the plasma, as do the other coagulation factors, TF is a transmembrane protein. TF serves as the cellular receptor and cofactor for FVIIa. It is primarily expressed on cells outside the vascular space under normal conditions, though monocytes and endothelial cells can express TF in response to inflammatory cytokines. The FVIIa/TF complex can activate both FIX and FX and is the major initiator of hemostatic coagulation.

Another group of related proteins are the contact factors : factors XI and XII and prekallekrein (PK) and high molecular weight kininogen (HMK). These proteins share the feature of binding to charged surfaces. The only one of this group that is needed for normal hemostasis is factor XI. However, the other contact factors may play a role in thrombosis in some settings. FXI is a zymogen that can be activated to a protease by FXIIa but is likely activated primarily by thrombin during the hemostatic process. FXIa, in turn, activates FIX.

Fibrinogen provides the key structural component of the hemostatic clot. Two small peptides, fibrinopeptides A and B, are cleaved from fibrinogen by thrombin; the resulting fibrin monomer polymerizes into a network of fibers. The fibrin polymer is then stabilized further when it is crosslinked by activated factor XIII. FXIIIa is a transglutaminase present in plasma and platelets that is activated by thrombin coincident with fibrin formation.

Thrombin plays a key role in activating procoagulant and anticoagulant factors; it also has a key role in triggering formation of fibrin. In addition, thrombin has cytokine-like activities that bridge the transition between hemostasis, inflammatory/immune responses, and wound healing. Thrombin is truly a multifunctional molecule that impacts the host response to injury at many levels.

Even before the structure and function of the various factors were defined, their interactions had been studied during plasma clotting. In the 1960s, two groups proposed a “waterfall” or “cascade” model of the interactions of the coagulation factors leading to thrombin generation. These schemes were composed of a sequential series of steps in which activation of one clotting factor led to the activation of another, finally leading to a burst of thrombin generation. At that time, each clotting factor was thought to exist as a proenzyme that was activated by proteolysis. The existence of cofactors without enzymatic activity was not recognized until later. The original models were subsequently modified as information about the coagulation factors accumulated and eventually evolved into the “Y-shaped” scheme shown in Fig. 8.1 .

The cascade model shows distinct “intrinsic” and “extrinsic” pathways that are initiated by FXIIa and FVIIa/TF, respectively. The pathways converge on a common pathway at the level of the FXa/FVa (prothrombinase) complex.

This scheme was not proposed as a literal model of the hemostatic process in vivo; rather, it was derived from studies of plasma clotting in a test tube and was intended to represent the biochemical interactions of the procoagulant factors. In fact, the coagulation cascade reflects very well the process of plasma clotting, as in the PT and aPTT tests. However, the lack of any other clear and predictive concept of hemostasis has meant that, until recently, most physicians have also viewed the cascade as a model of physiology and the PT and aPTT as reflecting the risk of clinical bleeding.

The limitations of the coagulation cascade as a model of the hemostatic process in vivo are highlighted by certain clinical observations. Patients deficient in the initial components of the intrinsic pathway—FXII, high molecular weight kininogen, or PK—have a greatly prolonged aPTT but no bleeding tendency. Patients deficient in FXI also have a prolonged aPTT but usually have a mild to moderate bleeding tendency. Other components of the intrinsic pathway clearly have a critical role in hemostasis since patients deficient in factor VIII or IX have a serious bleeding tendency even though the extrinsic pathway is intact. Similarly, patients deficient in FVII also have a serious bleeding tendency even though the intrinsic pathway is intact. Thus, although the cascade model accurately reflects the protein interactions that lead to plasma clotting and is an essential guide to interpretation of PT and aPTT results, it is not an adequate model of hemostasis in vivo.