An Overview of Thrombolytic Agents

An Overview of Thrombolytic

Agents*

Joseph U>scalzo, M.D., Ph.D.t

The use of thrombolytic therapy has increased considerably in the past five years, particularly in patients with acute myocardial infarction. The agents that have been used in humans thus far include streptokinase and urokinase, as well as tissue-type plasminogen activator and, most recently, single-chain urokinase-type plasminogen activator or pro-urokinase. Each of these agents works by very different mechanisms to activate plasminogen and, as a result, to lyse fibrin clots. This article reviews the mechanisms by which pathophysiologic thrombi develop, the pharmacologic agents available to lyse thrombi, and the mechanisms of action of these agents.

hrombotic disorders of the vasculature produce T considerable mortality and morbidity in Western society. In particular, acute myocardial infarction, stroke, peripheral arterial occlusion, and venous thromboembolism are extremely prevalent among hospitalized patients and have thrombus as their underlying pathophysiologic substrate, either within an apparently normal vessel or at a site of atheroma (atherothrombosis). This article considers the mechanisms by which pathophysiologic thrombi develop and reviews the pharmacologic agents available to lyse thrombi, their mechanisms of action, and their proper use. NORMAL MECHANISMS OF HEMOSTASIS

Under normal circumstances, a finely regulated and very complex group of interrelated biochemical and cellular interactions serve to staunch the flow of blood at sites of acute vascular injury (Fig 1). Platelet *From the Center for Research in Thrombolysis, Brigham and Women's Hospital and Harvard Medical School, Boston. tChief, Cardiology Division, Brockton/West Roxbury Veterans Administration Medical Center. Reprint requests: Dr. Loscalzo, Department of Medicine, 75 Francis Street, Boston 02115

adhesion to the subendothelium is responsible for primary hemostasis and is mediated by the polymeric plasma glycoprotein, von Willebrand factor. 1 Additional platelets are recruited as the platelet-rich thrombus evolves, with fibrinogen serving as a cohesive bridge between platelets. 2 With platelet recruitment and activation, and on exposure of tissue factor in the perivasculature at sites of injury, the prothrombinase complex forms, which converts prothrombin to thrombin and thence fibrinogen into fibrin as the complete hemostatic plug evolves. Thrombin has a variety of effects on hemostasis that depend on both its topologic site of action and the substrate involved (Fig 2). The best-known action of thrombin is the conversion of soluble plasma fibrinogen into insoluble polymeric fibrin. In addition, thrombin activates factors V and VIII, and also activates platelets and induces their aggregation. These prothrombotic actions are critical at sites of vessel injury for arresting hemorrhage acutely. In contrast, thrombin also acts to limit the extent of thrombosis by activating the endogenous anticoagulant, protein C, when bound to thrombomodulin on the endothelial cell surface. Factor XIII is also activated by thrombin• and, as a result, chemically cross-links fibrin polymers to stabilize the clot. Finally, thrombin stimulates the endothelial cell to release tissue-type plasminogen activator (t-PA) and, with a different dose-response relationship, the inhibitor of t-PA, plasminogen activator inhibitor type 1 (PAI-1). 5 PATHOPHYSIOLOGY OF THROMBOSIS

Thrombosis has been defined by MacFarlane as "hemostasis at the wrong place."6 In thrombotic disorders of the arterial vasculature, that place is generally an atheroma. Atheromas are typically not very

Matrix PlateletFibrin Plug FIGURE 1. Formation of a platelet-fibrin plug at a site of endovascular injury. SMC =smooth muscle cell; EC =endothelial cell. CHEST I 97 I 4 I APRIL, 1990 I Supplement

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Crosslinked

Fibrin

V,1ZIII

Fibrin__)

xmo

___ __....,.. Prate in C Platelets

....:.-~1---~r-----...Y::::Endotheltol

Protein C Platelet Aggregates

0

cell, Thrombomodulin

t-PA

Endothelial Cell PAI -1

thrombogenic when quiescent, but on activation these vascular lesions can promote thrombosis intensely. 7 Activation of an atherosclerotic plaque can occur through a variety of mechanisms (Fig 3). A plaque can fissure as a result of an abrupt increase in shear force accompanying a transient increase in blood pressure. Turbulent flow created by the presence of a protruding atheroma within the vessel lumen induces acceleration of blood flow that can alter endothelial integrity, promoting exposure of the procoagulant subendothelium. The platelets of patients with hypercholesterolemia and hypertension, two important atherogenic risk factors, are hyperaggregable8 •9 ; as a result, thrombus formation may be more active than in normal individuals. A transient increase in the transplaque pressure gradient within the enriched network of vasa vasorum feeding the plaque may promote intraplaque hemorrhage and plaque expansion, 10 thereby further compromising the vessel lumen and promoting thrombus formation. Finally, thrombus can develop as a result of an increase in the prothrombotic properties of the blood or plasma, or of the endothelium in the vicinity of the plaque. 11 Whatever the mechanism operative in a given patient, the net result is the deposition of platelets and fibrin at a vascular site that does not warrant such hemostatic "protection." Consequently, the vessel ultimately becomes occluded, and depending on the vascular bed and territory it serves, acute clinical ischemic syndromes evolve. MECHANISMS OF FIBRINOLYSIS

Plasminogen and a 2 -Antiplasmin

Once formed, the protective hemostatic plug or the pathophysiologic thrombus must, as healing occurs, be dissolved in order to restore blood flow to tissue subtended by the injured vessel. The fibrinolytic 1188

FIGURE 2. The multiple effects of thrombin on hemostatic detenninants.

system serves this purpose and does so by elaborating the serine protease, plasmin, from its precursor plasma zymogen, plasminogen (Fig 4). Plasminogen is a oo,ooo-dalton protein synthesized primarily in the liver that is comprised of a carboxyterminal serine protease domain and five aminoterminal "kringle" domains that mediate binding to fibrin and to cell surface receptors. Proteolytic cleavage of the Arg 560Val 561 bond by certain plasminogen activators con-

vasa. vasorum

l

-t

BP -Turbulent Flow - Prothrombotic Diathesis

~ FIGURE

=plaque hemorrhage

3. Potential mechanisms of activation of an atherosclerotic

plaque. Overview of Thrombolytic Agents (Joseph l..o8celzo)

COOH

Activation Domain (Arg 560-Val561) 4. Stntclure of plasminogen. The five triple-looped domains represent the fibrin-binding kringle domains. The serine protease active site is located nearest to the carhoxyterminus. FIGURE

verts this inactive precursor into proteolytically active plasmin. Levels of plasminogen in the circulation average approximately 2.4 !J.M, and the zymogen has a plasma half-life of approximately 0.8 day. a 2 -Antiplasmin is a serine protease inhibitor (serpin) that is also synthesized in the liver and is a very rapid inhibitor of plasmin. Sufficient a 2-antiplasmin exists in the circulation to inhibit approximately one-half of all potential plasmin activity. By binding to both the serine protease active site and the kringle domains, a 2 -antiplasmin inhibits plasmin activity. In contrast, as a result of the binding of plasmin to fibrin through the kringle domains, the enzyme is in part protected from a 2-antiplasmins inhibition. Other plasma serpins can also inhibit plasmin, hut not as effectively as ~antiplasmin. Plasminogen Activators

Plasminogen activators can be categorized in several ways (Table 1). Classification schemes can be devised on the basis of the source of the agent (human or bacterial, endogenous or exogenous), the propensity Table I-Classifications ofPlaaminogen Activation* Exogenous Streptokinase APSAC

Endogenous t-PA u-PAs

Surface activated t-PA scu-PA

Not surface activated Streptokinase LMW-tcUK

Enzymes t-PA u-PAs APSAC

Not enzymes Streptokinase

*APSAC=anisoylated plasminogen-SK activator complex, t-PAtissue-type plasminogen activator, scu-PA = single-chain urokinase-type plasminogen activator (pro-urokinase), LMW-tc-UK = low molecular weight two-chain urokinase, u-PA =urokinase-type plasminogen activator.

for enhanced enzymatic activity on a fibrin or cell surface (surface/fibrin selectivity), or the mechanism of action (enzymatic vs nonenzymatic). Each of these methods of classification is useful in helping to characterize the diverse nature of plasminogen activators, but regardless of how one defines these agents, they all serve one primary purpose-the conversion of plasminogen to plasmin. Each of the currently available agents is briefly reviewed below. Streptokinase (SK), the oldest and best-known plasminogen activator, is a 47 ,000-dalton protein produced by the Lancefield group C strains of ~-hemolytic streptococci. It is unique not only with regard to its source, hut also in terms of its mechanism of action. In contrast to all of the other plasminogen activators, SK is not an enzyme and therefore does not elaborate plasmin activity by proteolytic cleavage of plasminogen. Instead, SK binds nonrovalently to plasminogen and thereby confers plasmin activity on the complex. 12 The SK-plasminogen complex then acts on other plasminogen molecules to cleave the Arg 560-Val 561 bond and, as a result, generate plasmin (Fig 5). SK has a plasma half-life of 30 min, and because it is a bacterial product, its use may be associated with allergic reactions and antibody-mediated inhibition of plasminogen activation. Another form of SK that has recently been developed is the anisoylated plasminogen-SK activator complex (APSAC). This hybrid molecule is an inactive derivative produced by acylation of the active center of plasminogen (see Fig 5). The complex cannot act on plasminogen until deacylation has occurred, which happens spontaneously in plasma. 13 The plasma halflife of APSAC is 70 min. This complex does not appear to be inhibited by endogenous serpins. Urokinase-type plasminogen activators comprise a family of endogenous molecules synthesized by endothelial and mononuclear cells. The parent molecule is single-chain urokinase-type plasminogen activator (scu-PA), also known as pro-urokinase (PUK). 14 This protein is 54,000 daltons in molecular weight and may have some intrinsic enzymatic activity, but far less than its proteolytic derivative, high molecular weight two-chain urokinase (HMW-tc-UK). As a result of proteolytic cleavage of the Lys 158-IIe 159 bond of scu-PA, HMW-tc-UK is produced, which is itself further cleaved to the 33,000-dalton conventional product, low molecular weight two-chain urokinase (LMW-tc-UK). Urokinase-type plasminogen activators also contain a carboxyterminal serine protease domain as well as an aminoterminal kringle domain. The structural relationships among these molecules are shown in Figure 6. HMW-tc-UK and LMW-tc-UK have plasma half-lives of 10 min, while scu-PA has a plasma half-life of 5 min. Circulating plasma concentrations of scu-PA are approximately 5 to 10 nglml; CHEST I 97 I 4 I APRIL. 1990 I Supplement

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+~ SK

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SK- Plasminogen

APSAC neither HMW-tc-UK nor LMW-tc-UK circulates in any appreciably measurable concentration in plasma under normal circumstances. Tissue-type plasminogen activator (t-PA) is also synthesized by endothelial cells as a single-chain polypeptide of 72,()()()-dalton molecular weight. 15 Proteolytic cleavage of the Arg 275-Ile 276 bond by plasmin, kallikrein, or factor Xa converts this singlechain form (sct-PA) into a two-chain species (tct-PA); both have reasonable enzymatic activities. By analogy with the other fibrinolytic serine proteases, t-PA has a carboxyterminal serine protease domain and two aminoterminal kringle domains. In addition to these kringle regions, the aminoterminal portion of the molecule contains a fibronectin-like "finger" domain which, in conjunction with the second kringle, confers fibrin binding properties on the molecule (Fig 7). t-PA circulates at a plasma concentration of approximately 5 to 10 ng/ml with a half-life of approximately 4 min. 17 Moduwtors of Plasminogen Activation

Factors that modify the plasminogen activation mechanisms are complex in their interrelationships. Plasmin itself is as varied in its effects as is thrombin (Fig 8). Not only does plasmin proteolyze fibrin, but it can also digest fibrinogen, promoting the formation of a systemic lytic state. In the process of fibrin(ogen)olysis, a variety of proteolytic degradation products (FOPs) are released that can infiuence the extent and tempo of the fibrinolytic response. FOPs inhibit fibrin polymerization and platelet aggregation, thereby acting as natural anticoagulants. Certain of these fragments, such as fragment 0, enhance plasminogen activation by t-PA much as fibrin itself does, 18 1208

FIGURE 5. Mechanism of action of streptokinase and APSAC (anisoylated plasminogen-streptokinase activator complex).

suggesting that fibrinolysis can be considered autocatalytic through the inHuence of the proteolytic products produced by the action of plasmin on the fibrin clot. sct-PA can also be converted to tct-PA by plasmin, and this conversion has kinetic implications in that tct-PA has a greater catalytic efficiency than sct-PA in the absence of fibrin than does its precursor. 19•20 Plasmin can also act on the platelet to modulate the aggregation response and thereby alter platelet recruitment to the dissolving fibrin surface. 21 -23 The native form of plasminogen secreted by the liver has a glutamic acid residue at its carboxyterminus (Glu-plasminogen). Limited proteolysis by plasmin converts this molecule into a modified, slightly smaller protein with a lysine, valine, or methionine at the aminoterminus (so-called Lys-plasminogen).2-1 Lysplasminogen has a greater affinity for fibrin than Gluplasminogen, but Glu-plasminogen is a better substrate for scu-PA and HMW-tc-UK than is Lys-plasminogen. Thus, plasmin modification of plasminogen can alter its localization as well as its suitability as a substrate for plasminogen activators. In addition to the serpin inhibitor(s) of plasmin mentioned above, at least two classes of inhibitors of endogenous plasminogen activators have been identified. Plasminogen activator inhibitor type I (PAI-l) is synthesized by endothelial cells26 and hepatocytes as well as by platelets. It is responsible for most of the plasminogen activator inhibitor found in plasma. Plasminogen activator inhibitor type 2 (PAI-2) is synthesized in placenta27 and mononuclear cells. 28 Both serpins inhibit both t-PA and u-PAs, but PAI-l is a more potent inhibitor of t-PA than of u-PAs. Other plasma serpins that inhibit or attenuate t-PAs activity include « 2-antiplasmin, a 1-antitrypsin, 29 and Cl ester-

ase inhibitor. 30

c 0 0

H

scu-PA

l

HMW-tc-UK

l LMW-tc-UK 6. Structural relationships among the urokinase-type plasminogen activators. scu-PA =single-chain urokinase-type plasminogen activator, HMW-tc-UK=high molecular weight two-chain urokinase. FIGURE

Fibrin/CeU Surface Specificity The current trend in the use of plasminogen activators was predicated primarily on the so-called fibrin selectivity of t-PA. Neither SK nor LMW-tc-UK contains fibrin-binding domains; as a result, these agents are as effective at activating circulating plasminogen as fibrin-bound plasminogen. The resulting generation of a systemic lytic state has been thought by many investigators to be responsible for the hemorrhagic complications associated with the use of these agents, and it was for this reason that the clinical use of t-PA and scu-PA gained such favor early on. t-PA, HMWtc-UK, and scu-PA have been defined as fibrinselective compared with SK and LMW-tc-UK because in the presence of fibrin their catalytic efficiencies are significantly increased. t-PA's fibrin selectivity is the result of fibrin binding of t-PA and a concomitant conformational change that dramatically reduces the Michaelis constant of enzyme for substrate. 19 scu-PA's fibrin selectivity is dependent more on the avidity of enzyme for fibrin-bound substrate than on a direct effect of fibrin binding of scu-PA. 215 In practice, however, these so-called fibrin-selective agents have not been as selective as originally hoped. Therapeutically efficacious doses of both t-PA31 and scu-PA32•33 have been associated with reductions in fibrinogen, plasminogen, and az-antiplasmin levels, indicative of the development of a systemic lytic state, although less so than with SK or LMW-tc-UK. The incidence of bleeding complications has also not been shown to be significantly different for t-PA compared with SK.31 Hence, the importance of fibrin selectivity in therapeutic decisions appears moot at this time. Interestingly, the reason for the hemorrhagic equivalence of fibrin-selective and nonselective agents may be the result of another binding property of fibrinselective agents, namely cell-surface binding. Both t-PA and scu-PA bind to endothelial cells,34 platelets,35 and mononuclear cells36 and, as a result, have enhanced catalytic efficiencies. Local elaboration of plasmin in the microvasculature where the endothelial

FIGURE 7. The structure of tissue-type plasminogen activator. The F domain represents the fibronectin-like "finger" domain, the E domain represents the EGFlike domain, and K, and K. refer to the first and second kringle domains. The serine protease active site is located closest to the carboxyterminus.

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FOPs 10

Fibrin

11

Aggregation Inhibition

12 13

Disaggregation glu- plasminogen

14

lys -plasminogen FIGURE 8. The multiple effects of plasmin on hemostatic determinants.

surface area-to-blood volume ratio is great may be responsible for the lysis of local hemostatic plugs that contributes to the hemorrhage diathesis of plasminogen activators.

15

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FUTURE DIRECTIONS

The development of mutant and chimeric plasminogen activators using the techniques of molecular biology promises to be a potentially useful program that will enhance the safety and efficacy of these very useful agents. 37 •38 Defining the specific domains of these molecules that are responsible for fibrin and cell-surface binding, linking these molecules to antibodies specific for fibrin, 39 and altering their halflives40 all may prove useful approaches in the development of an ideal plasminogen activator enzyme that activates the fibrinolytic system with topologic selectivity.

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