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From: Essential Cardiology: Principles and Practice, 2nd Ed.
Edited by: C. Rosendorff © Humana Press Inc., Totowa, NJ
5 Thrombosis
Yale Nemerson, MD and Mark B. Taubman, MD
INTRODUCTION
Thrombosis and hemostasis are similar processes, the former being pathologic and involving intravascular formation of aggregates of platelets and fibrin, and the latter resulting in the cessa- tion of bleeding after external injury to the vasculature. While it is not clear that these processes involve precisely the same biochemical and biophysical events, they appear to be sufficiently sim- ilar to be considered as a single process that results in quite different structures owing to the local environment, either within a vessel or at the site of bleeding.
The initial event in both instances likely is the exposure of tissue factor (TF) at the site of injury (1,2). In arterial thrombosis, the most frequent initiating event appears to be rupture or fissuring of an atheromatous plaque, which exposes TF; an event that enables the circulating blood to con- tact TF, thus activating the coagulation cascade (3).
BRIEF VIEW OF THE MECHANISM OF BLOOD COAGULATION Although for many years it was thought that coagulation was initiated via the so-called intrinsic system (so named because it was believed that all the components required for coagulation were
“intrinsic” to the blood), it is generally recognized that this system was an artifact of glass activa- tion (1). The prevailing view is that coagulation via the TF pathway is the principal means of throm- bin production. Some patients who are deficient in factor XI, however, have some hemorrhagic symptoms. Until recently, it was thought that factor XI was activated mainly when the blood con- tacted glass or a similar surface by a mechanism independent of TF. Two findings, however, offer alternative schemes, each consistent with TF being the only physiologic activator of the coagu- lation system. First, it was shown that factor XI could be activated on platelets via a mechanism independent of factor XII or glass. Interestingly, platelet factor XI is an alternatively spliced form of plasma factor XI (4), and its synthesis is independent (5). Alternatively, the major catalyst of factor XI activation may be thrombin, which activates this zymogen via limited proteolysis (6). For- mation of a thrombus involves many circulating proteins, blood platelets, and damage to the arterial wall with consequent exposure of TF. Because of this complexity, it is difficult to describe the entire process precisely. The clinical efficacy of anticoagulant and antiplatelet agents indicates that per- haps all these components are necessary but that none alone is sufficient for thrombus formation.
TF forms a complex with activated factor VII (VIIa), thereby forming a holoenzyme that initiates the coagulation cascade by activating factors IX and X (Fig. 1). The TF:VIIa complex has a regulatory subunit, TF, and a catalytic subunit, VIIa. The latter is a serine protease that has essen- tially no procoagulant activity unless it is in complex with TF. This theme—the assembly of holo- enzymes from regulatory and enzymatic species—is central to the understanding of coagulation, because is occurs three times in this process.
The vast majority of circulating factor VII is in the zymogen, or unactivated form, but small amounts of VIIa also circulate (7,8), and it is probably responsible for the initial activity of the
TF complex. When factor VII is bound to TF, it has little or no enzymatic activity; however, in the bound state (whose crystal structure has been described [9]), zymogen factor VII is in a conformation that renders it liable to limited proteolysis that results in its conversion to its active enzymatic form, VIIa (10,11). The complex of TF:VIIa has two substrates, factors IX and X. Their activated forms, IXa and Xa, respectively, form complexes with two circulating regulatory pro- teins; IXa with the antihemophilic factor, factor VIII, and Xa with factor V, forming the so-called prothrombinase complex. These complexes are similar to the TF:VIIa complex inasmuch as each contains a serine protease (factors IXa and Xa, respectively) and a regulatory protein (factors VIIIa and Va, respectively). Both factors VIII and V circulate as “pro-cofactors” and must be acti- vated via limited proteolysis to function in these complexes. Thrombin is likely the enzyme that is mainly responsible for activating these cofactors; thus, strong positive feedback results in explo- sive formation of thrombin. The last event in this cascade is the cross-linking of fibrin via the action of factor XIII, which, after being activated by thrombin, crosslinks the fibrin monomers. Once cross- linked, fibrin becomes resistant to lysis by plasmin, which is one explanation for lytic therapy losing efficacy over time.
This concert of events, during which the platelets become activated, enables them to support coagulation and to form a nidus for thrombus formation via the action of the IIb/IIIa receptor that facilitates the formation of platelet masses by interacting with fibrin. The IIb/IIIa receptor is the target of the clinically effective antithrombotic monoclonal antibody Rheopro®. Leukocytes are
Fig. 1. (Upper panel) Schematic view of blood coagulation with tissue factor (TF) as the initiating species.
TF is shown as a complex with activated factor VII (VIIa) and that small amounts of this enzyme are present in normal blood. (Lower panel) Schematic view of a thrombus. The inset indicates the relationship between the time it takes a diffusing molecule to traverse a given distance. This relationship is such that as the distance doubles, the time to capture is squared.
also involved in thrombus formation. It is noteworthy that a blocking antibody to P selectin inhibited fibrin formation in an arteriovenous shunt model in vitro (12). P selectin is a protein stored in plate- let granules that translocates to the plasma membrane upon platelet activation. When on the plate- let surface, P selectin interacts withits cognate ligand, CD-15, on the surface of leukocytes. This observation raises the issue of the role of leukocytes in thrombus formation, which is addressed later.
NATURAL ANTICOAGULANT SYSTEMS
Natural anticoagulant systems can conveniently be divided into two classes: those that circu- late as inhibitory species and those that are activated during coagulation. Of those that require activation, that best studied is protein C, which, like factors VII, and X, is a vitamin K-dependent zymogen that must be activated by limited proteolysis (for a review, see ref. 13). The activation of protein C is accomplished by thrombin that is complexed with an endothelial surface protein, thrombomodulin. When thrombin is in this complex, the substrate specificity of thrombin is altered so that it activates protein C and thrombin-activatable fibrinolysis inhibitor (TAFI) (14) but does not clot fibrinogen. Activated protein C is a serine protease that attacks activated factors V and VIII, thus shutting down the coagulation cascade. Factor V Leiden is a genetic variant of factor V that is resistant to proteolysis by activated protein C. Those with this mutation exhibit increased throm- bosis, mostly venous, although serious arterial thrombosis is also increased (15–17). This is a reason- ably common mutation: some 5 to 6% of Caucasians possess it (18). Deficiencies of protein C are associated mainly with venous thromboembolic disease, although instances of arterial thrombosis have been reported (19).
TAFI is a recently described fibrinolysis inhibitor that is a form of procarboxidase B. When activated by thrombin or (>1000-fold faster) by thrombomodulin-thrombin complex, the resultant enzyme attacks the carboxy-terminal residues of proteins, resulting, in this case, in reduced plasmin/
plasminogen and tissue plasminogen activator (tPA) binding to fibrin (20). Thus, the formation of the thrombin-thrombomodulin complex results in the generation of an anticoagulant, activated protein C, and the antifibrinolytic (prothrombotic) species TAFI. Clearly, sorting out these phenom- ena with respect to thrombogenesis will be most difficult.
The blood also contains an inhibitory protein, tissue factor pathway inhibitor (TFPI), whose func- tioning is complex: TFPI has modest affinity for TF and thus it is not directly inhibitory. TFPI is in its most effective form when it is bound to factor Xa; this binary complex then attacks TF:VIIa, with which it forms an inactive quaternary complex, thus damping TF-initiated coagulation (21, 22). No clinical deficiency states of TFPI have yet been reported, so it is difficult to assess its role in preventing thrombosis. It is noteworthy, however, that mice whose gene for TFPI has been knocked out die in utero (23).
The other major circulating anticoagulant is antithrombin III, which forms a stable complex with several of the coagulation enzymes, most prominently thrombin, and activated factor X. This reac- tion is markedly accelerated by heparin and similar compounds and is the mechanism by which hep- arin exerts its anticoagulant activity (24). Like deficiencies of protein C, antithrombin III deficiencies are associated mainly with venous thrombosis, although recent data indicate that low levels of this protein are predictive of future cardiac events (25).
FIBRINOLYSIS
Just as coagulation involves multiple enzymatic and regulatory proteins, fibrinolysis, the pro- cess by which fibrin is lysed to reestablish blood flow, involves multiple proteins and reactions, the details of which are beyond the scope of this chapter. Plasminogen is the circulating zymogen of plasmin, a serine protease that has high specificity for fibrin. It is activated in vivo by plasmi- nogen activators that are released from tissue stores by ischemia. The activators generate plasmin, the active fibrinolytic enzyme, from the zymogen plasminogen. Plasminogen activation inhibitors 1 and 2 oppose the activation of plasmin and thus are prothrombotic (26–28). These inhibitors appear to be the major components of the fibrinolytic system that are associated with thrombotic risk.
Interestingly, while hemophilia A patients are protected against myocardial infarction (29), heredi- tary absence of factor XI affords no such protection (30).
TISSUE FACTOR AND ATHEROSCLEROTIC PLAQUE
As noted above, occlusive thrombi of the coronary arteries are thought to be a consequence of plaque rupture and are the leading cause of death in the Western world; because of this and because TF in plaques is felt to be necessary for thrombosis, many studies have focused on the presence of this protein in plaques. As early as 1972, TF was detected in plaque by immunostaining, although the antibody was undoubtedly polyspecific (31). Subsequent experiments with antibodies raised against pure TF confirmed these findings (32), as did results obtained with monoclonal antibodies and the use of haptene-labeled factor VIIa, a specific probe for TF (33). Because immunostaining reflects only localization of antigen, and binding of VIIa to TF the localization of TF-binding sites, it follows that neither of these techniques demonstrates TF activity in plaque. Direct enzymatic assay of TF harvested from plaque has been reported, and the majority of samples demonstrated activity (34,35). The activity, however, was low, and it is not certain that the specimens contained only plaque; thus, the meaning of these findings is somewhat questionable. What clearly is required to demonstrate unambiguously active TF in plaque is an enzyme histochemical assay for TF, which, however, has not been reported.
Circulating Tissue Factor: A Thrombogenic Species
Recent experiments have demonstrated that native, normal human blood forms TF-dependent thrombi on collagen-coated glass slides in a laminar flow chamber. The fact that these thrombi contain fibrin indicates that the deposited TF is biochemically active; furthermore, inclusion of active site-inhibited VIIa (a potent TF inhibitor) essentially abolished both fibrin and thrombus formation on the collagen surface (36). This finding contradicts many statements in the literature, including our own, that circulating TF is of no consequence. Further, these experiments suggest that exposure of collagen on blood vessels may be sufficient to initiate thrombus formation, although it seems likely that vessel wall TF initiates thrombus formation, whereas circulating TF may be responsible for its propagation. The apparent mechanism by which blood-borne TF can initiate thrombosis ex vivo works as follows: The first event appears to be binding of platelets to collagen;
thereafter, neutrophils and monocytes bind to the platelets (probably via P selectin and other mole- cules as yet to be identified). The leukocytes, which contain TF, apparently deposit TF-containing membranous structures on the platelets, thus rendering them highly thrombogenic. These experi- ments were designed to mimic thrombosis in vivo in the sense that they involved laminar flow at arteriolar shear rates (1000 to 2000/s). We imagine that the shear field, which is also encountered in mildly stenosed coronary arteries, favors (1) delivery of leukocytes to the nascent thrombus and (2) their fragmentation in situ. Thus, as the thrombus grows the platelets become surrounded with TF-containing vesicles and membranous structures that are competent to initiate coagulation and support thrombus propagation.
Encryption of Cell Surface Tissue Factor:
What Is the Biologically Active Species?
The fact that blood-borne TF is active in experimental thrombogenesis suggests that there is a mechanism for controlling its activity in blood cells, One possibility is that cell surface TF in vivo is entirely encrypted, by which we mean that while it is capable of binding VIIa and specific antibodies, cell surface TF is catalytically inactive. The phenomenon of encryption or dormancy on the cell surface was suggested many years ago (37) and was subsequently explored and docu- mented using contemporary techniques(38–40).It has been suggested that on the cell surface TF exists as inactive dimers and that it must be monomerized to exhibit procoagulant activity (41).
Quantitative studies using cultured cells have shown that the majority of surface TF is encrypted
(40). One possibility that is difficult to explore is that, in vivo, cell-surface TF is inactive; if so, that fact raises the question of the state of the active species. Extracellular TF has been noted in arterial adventitia and in the plaque, which raises the possibility that it is the active pool (42). It is well documented that, for optimal activity, TF requires acidic phosphatides to be exposed. It is presumed that normally these molecules are on the inner leaflets of plasma membranes and render these mem- branes more or less inactive. Extracellular TF, however, is present on membrane fragments and vesi- cles, which lack the energy necessary to maintain phospholipid asymmetry; therefore, one expects acidic phosphatides to be randomly distributed, the net result being that extracellular TF quite likely is procoagulant.
Tissue Factor in Arterial Injury
In addition to its association with acute coronary syndromes such as myocardial infarction and unstable angina, thrombosis is also a concomitant of acute arterial injury, such as that produced by coronary angioplasty, directional atherectomy, and coronary artery stenting (43,44). TF anti- gen is induced in the smooth muscle cells near the intimal border in rat (45), rabbit (46–48), and porcine (49) models of arterial injury; the significance of this induction is subject to the same concerns raised earlier in the discussion of the atherosclerotic plaque. It has been demonstrated that TF activity in the injured rat aortic media increased coordinately with TF mRNA and antigen (45); however, activity was measured in homogenized aortic sections and therefore could have come from encrypted or intracellular stores not capable of initiating coagulation in vivo.
The relevance of TF induction after balloon injury can be questioned on the grounds that injury to normal rat and rabbit arteries does not result in deposition of macroscopic fibrin, the end product of TF activation, even when medial smooth muscle is injured. However, fibrin deposition occurs rapidly when previously injured rabbit arteries are subjected to a second injury 1 to 2 wk later. Fibrin deposition and microthrombi were not seen at any time after single injuries to normal rat aortas but were present on the luminal surface within 30 min of a second injury. TF antigen was not detectable in the endothelium or media during the first 4 h after injury; TF antigen was abundant in the media by 24 h and then declined to baseline levels over the next 2 d. TF antigen subsequently accumu- lated in the developing intima and was abundant throughout the intima after 2 wk, at the time of the second injury. Whole-mount preparations showed minimal TF antigen on the surface of unin- jured or once-injured vessels, but the second injury rapidly exposed surface TF antigen. Rapid expo- sure of intimal TF to the circulation may be necessary to generate fibrin and produce thrombosis.
Other studies have suggested that the induction of TF by arterial injury is functionally impor- tant. Antibodies to TF inhibited the variations in cyclic flow in rabbits subjected to arterial injury and mechanical stenosis and inhibited thrombus formation in a rabbit femoral artery eversion graft preparations. TFPI has also been shown to inhibit angiographic restenosis and intimal hyperplasia in balloon-injured atherosclerotic rabbits and to attenuate stenosis in balloon-injured hyperlipide- mic pigs. Once again, the precise location of the functionally important TF remains to be determined and awaits the development of an in situ activity assay.
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RECOMMENDED READING
Belting M, Dorrell MI, Sandgren S, et al. Regulation of angiogenesis by tissue factor cytoplasmic domain signaling. Nat Med 2004;10:502–509.
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