Therapeutics VI

Michael B. Bolger

Level III

Thrombus formation on a fissured or disrupted atherosclerotic plaque is the main pathogenetic mechanism for the acute coronary syndromes of myocardial infarction and unstable angina. Myocardial infarction results from an acute total occlusion of the artery, while unstable angina is secondary in most cases to mural thrombus formation. Thrombus formation has also been implicated in chronic atherosclerotic disease progression and in restenosis following coronary angioplasty. Therapeutic measures to treat thrombus rely on the ability of drugs to either prevent thrombus extension, dissolve its fibrin component, or prevent further platelet aggregation. Other measures rely on the ability of intracoronary techniques to open coronary arteries. The primary prevention of intracoronary thrombus formation is evolving. Measures to stabilize plaques or to reduce hypercoagulability are being tested or have been tested in recent trials.

Figure 1. Drawing of cross-sections of the identical, most proximal parts of 6 left anterior descending coronary arteries. The morphologic features of the intima range from adaptive intimal thickening always present in this lesion-prone location to a type VI lesion in advanced atherosclerotic disease. Adapted from Ambrose, John A. MD. Weinrauch, Michael MD. Thrombosis in Ischemic Heart Disease. [Article] Archives of Internal Medicine. 156(13):1382-1394, July 8, 1996.

Figure 2. A reconstruction in longitudinal section of an occluding thrombus due to plaque fissuring. The thrombus within the plaque labeled I is rich in platelets, stage II contains both platelets and fibrin, and the stage III thrombus, which is propagated in the lumen, is predominantly fibrin and red blood cells with a minimal platelet component. Adapted from Ambrose, John A. MD. Weinrauch, Michael MD. Thrombosis in Ischemic Heart Disease. [Article] Archives of Internal Medicine. 156(13):1382-1394, July 8, 1996.

Although the thrombolytic agents (SK, u-PA, t-PA, and their derivatives) are new clinical tools compared with the previous drugs, their history reaches as far back as the 1860s. All of the thrombolytics are large proteins, yet their current sources are diverse: SK and its congener anistreplase from bacterial cultures, u-PA from human kidney cell tissue cultures, and t-PA from recombinant DNA (derived from human melanoma cell lines) expressed in Chinese hamster ovary cell cultures.

The history of thrombolytic agents begins in 1861 with the report by von Brucke on the proteolytic activity of human urine. In 1886, Sahli noted urinary proteolytic activity with some specificity for fibrin. Following purification and isolation of the fibrinolytic enzyme in urine, Sobel et al coined the name "urokinase" in 1952. He and others demonstrated that u-PA is not a direct fibrin-digesting enzyme but rather an activator of endogenous plasminogen, thereby generating plasmin, which then consumes fibrin, fibrinogen, and other coagulation proteins. This mechanism was found to be shared by all current thrombolytic agents.

In the early 1930s, William Tillett, a bacteriologist discovered that the beta -hemolytic, group C streptococci, produced a fibrinolytic agent and called it "fibrinolysin." Ultimately, the more advanced techniques and basic hemostasis knowledge of Tillett's time enabled his colleague Milstone to show in 1941 that the streptococcal fibrinolysin did not directly dissolve fibrin in vitro; rather it required a plasma protein cofactor he called "plasma lysing factor." This factor later proved to be plasminogen. In 1944, fibrinolysin was renamed to "streptokinase (SK)." Sol Sherry became the first to administer crude SK to humans in early 1947.

The usefulness of both intravenous SK and u-PA in venous thromboembolism was shown in several studies in the 1960s and 1970s, leading to FDA approval of both agents for intravenous use in 1977. SK was approved for deep venous thrombosis, pulmonary embolism, thrombosed dialysis fistulas, and arterial thrombosis; u-PA was approved only for pulmonary embolism and thrombosed intravenous catheters. Further FDA approvals for other indications came in 1982 for intracoronary use of both SK and u-PA in treatment of acute MI and in 1987 for intravenous use of SK in acute MI (u-PA remains unapproved for intravenous use in acute MI, due to a relative lack of studies). Current commercial SK is still prepared by purification from bacterial cultures. u-PA, currently is produced only by Abbott, and is no longer prepared from human urine but instead by purification from human kidney cell cultures.

The thrombolytic era we now enjoy thus had to wait for final proof of the role of thrombosis and then the rediscovery of thrombolytic therapy, first with intracoronary use and finally coming full circle with intravenous use. This unfortunate delay of several decades in accepting the use of thrombolysis as the cornestone of treatment of acute MI carried a heavy toll in lives. Despite the demonstration by Herrick as early as 1912 of the central role of thrombosis in acute MI, doubt lingered about the issue until the landmark angiographic demonstration of DeWood et al in 1980 of acute thrombosis in most cases of acute MI.

In 1979, Smith et al reported the rational design of anistreplase, a combination of SK and its target, plasminogen. This agent, which spontaneously deacylates slowly after injection and thus can be administered as a single bolus, was first used in humans with acute MI by Been et al and others in 1985, and it was approved for intravenous use in acute MI by the FDA in 1989. Anistreplase is manufactured by Beecham-Wulfing of Germany and distributed in the United States by Smithkline Beecham; it is prepared by combining human lys-plasminogen (isolated from pooled plasma) with chemically acylated SK harvested from bacterial cultures.

In 1947 t-PA was discovered in tissue slices that were shown by Astrup and Permin to lyse fibrin. Pennica et al reported the cloning of t-PA DNA derived from human melanoma cells and its expression by Escherichia coli in 1983. Clinical use evolved rapidly, leading to FDA approval of intravenous t-PA for acute MI in 1987 and for pulmonary embolism in 1990. Unlike the early t-PAs derived from a variety of human tissues or from melanoma cell culture, current commercial t-PA is prepared from recombinant DNA derived from human melanoma cells and expressed in the Chinese hamster ovary tissue culture system. Currently, only Genentech produces t-PA; Burroughs Wellcome has abandoned development of its t-PA, which differs from the native molecule by a single amino acid and by its double-chained structure. Genentech's product shares native t-PA's amino acid sequence, single-chained structure, and possibly its glycosylation.

Mueller, Richard L.. Scheidt, Stephen. History of Drugs for Thrombotic Disease: Discovery, Development, and Directions for the Future. [Article] Circulation. 89(1):432-449, January 1994.

Figure 3.. Plasminogen (plg) is activated to plasmin (plm) in the fluid phase by urokinase (u-PA), whereas fibrin-bound plg is activated by t-PA in the fibrin phase. The free-phase plm is inhibited by a₂-antiplasmin (AP) and macroglobulin (MG), whereas the bound-phase plm is protected from inhibition. The u-PA and t-PA are inhibited in the free phase by plasminogen activator inhibitor (PAI-1). Inhibition reactions were assumed to be irreversible. Soluble species can reversibly adsorb and desorb with fibrin under kinetically controlled conditions. The plg and plm compete for the same sites on the intact fibrin monomer, whereas t-PA binds a unique site.

108-119 CQCPEGFAGKCC

41-78 CRDEKTQMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQC

178-183 YCRNPD

266-271 YCRNPD

Human t-PA is a glycoprotein consisting of a single chain of 527 amino acids. Its molecular weight is about 70,000 daltons. Human t-PA contains 35 cysteines assigned to 17 disulfide bonds. A serine protease domain of about 260 residues is located at the carboxy-terminal end of this protein. A fibronectin "finger" domain (contributes to fibrin binding), two "kringle" domains (also bind to fibrin), and an epidermal growth factor domain are also present. The t-PA protease domain is approximately 35 to 40% homologous with typical serine proteases, such as bovine trypsin and chymotrypsin.

Overview of Three Commercially Available Thrombolytic Agents

Three thrombolytic drugs, streptokinase (SK), tissue plasminogen activator (t-PA), and anisoylated plasminogen streptokinase activated complex (APSAC), are commercially available and widely used in the treatment of AMI. These three drugs differ in their clearance, fibrin selectivity, plasminogen binding, and in the potential to induce allergic reactions Table 2. Unlike SK and APSAC, t-PA is a relatively fibrin-specific and clot-selective thrombolytic agent. By itself, t-PA is inactive, but when bound to fibrin, the fibrin/t-PA complex has a high affinity for clot-bound plasminogen and lyses fibrin on the surface of a thrombus. Despite its greater relative clot selectivity, t-PA does not cause less bleeding complications than any other non-clot-selective agent. Compared with SK and APSAC, t-PA causes no allergic reactions or hypotension and is now produced in large quantities using recombinant technology. It is still, however, about five to ten times as expensive as streptokinase.

Table 1. Comparison of Pharmacologic Characteristics of Thrombolytic Drugs*

Habib, Gabriel B. MD FCCP. Current Status of Thrombolysis in Acute Myocardial Infarction*: I. Optimal Selection and Delivery of a Thrombolytic Drug. [Review] Chest. 107(1):225-232, January 1995.

The cost-effectiveness of t-PA compared with SK can be assessed by dividing the difference in cost of t-PA and SK to the difference in life expectancy, assuming that the survival benefit at 30 days is maintained indefinitely. The GUSTO Economics and Quality-of-Life investigators estimated that the economic impact of t-PA compared with SK would amount to $21,820 per year of additional life saved. This is comparable or substantially more economical than that of other widely accepted therapies such as dialysis ($35,000), drug therapy for severe hypertension ($20,000), or cholestyramine for hypercholesterolemia ($180,000).

Streptokinase is a non-enzymatic fibrinolytic agent produced by beta-hemolytic streptococci. It was first isolated in 1933 and entered clinical use in the mid-1940s. Streptokinase by itself is not a plasminogen activator, but rather binds with free circulating plasminogen (or with plasmin) to form a complex which can then convert additional plasminogen to plasmin. Streptokinase activity is not enhanced in the presence of fibrin.

The principal plasma activity half-life is about 20 minutes, but an unbound fraction (about 15%) has a half-life of 80 minutes. Because it is produced from streptococcal bacteria, it often causes febrile reactions and other allergic problems. Its use results in high levels of antistreptococcal antibodies, thus it usually cannot be safely administered a second time within 6 months.

Urokinase is a physiologic thrombolytic agent that is produced in renal parenchymal cells. Urokinase directly cleaves plasminogen to produce plasmin. When purified from human urine, approximately 1500 L of urine are needed to yield enough urokinase to treat a single patient. Urokinase is also commercially available in a form produced by tissue culture, and recombinant DNA techniques have been developed for urokinase production in E. coli cultures as well.

In plasma, urokinase has a half-life of approximately 15 minutes. Allergic reactions are rare, and the agent can be administered repeatedly without antigenic problems.

Prourokinase is a relatively inactive precursor that must be converted to urokinase before it becomes active in vivo. It is produced from renal cell cultures as well as by recombinant DNA techniques. Its advantages over other plasminogen activators are that it is inactive in plasma and does not bind to or consume circulating inhibitors. Like tissue-type plasminogen activator, prourokinase is somewhat clot-specific, since the presence of fibrin enhances the conversion of prourokinase to active urokinase by an unknown mechanism.

Prourokinase itself is relatively inactive and a substantial amount is taken up and sequestered inside platelets; thus, the half-life is difficult to specify.

Tissue-type plasminogen activator (t-PA) is synthesized and made available by cells of the vascular endothelium. In vivo, it is the physiologic thrombolytic agent responsible for most of the body's natural efforts to prevent excessive thrombus propagation. Alteplase exerts is action on the endogenous fibrinolytic system to convert plasminogen to plasmin by directly hydrolyzing the arginine-valine bond in plasminogen. Unlike streptokinease or urokinase, most of the activity of alteplase is dependent on the presense of fibrin. Upon binding to to fibrin, the one-chain form of t-PA is converted to the two-chain form. Both forms have similar fibrinolytic and plasminogen-activating potential; however, the one-chain t-PA is considerably less active in the absence of fibrin. Alteplase that is bound to fibrin acquires a high affinity for plasminogen, which is responsible for an increased activity at the fibrin surface compared to the circulation.

Alteplase is administered by iv infusion. Hepatic clearance is responsible for the removal of 80% within 10 minutes. It is primarily excreted in the urine (80%).

In theory rt-PA should be effective only at the surface of fibrin clot, but a systemic lytic state is seen in practice, with moderate amounts of circulating fibrin degradation products. The agent may be readministered as necessary, as it is not antigenic and is almost never associated with any allergic manifestations.

Reteplase is a parenteral third-generation thrombolytic agent. It is a recombinant plasminogen activator (r-PA). Reteplase consists of only the kringle-2 and protease domains of the t-PA molecule. Compared to alteplase, reteplase is more potent and has a more rapid onset of action. Final FDA approval was granted on Oct. 30, 1996.

Reteplase is administered iv and is cleared from plasma at a rate of 250-450 mL/min by the liver and kidney. It is also cleared from the circulation by a₂-antiplasmin, and a₁-antitrypsin. Terminal half-life is approximately 170 min.

Anisoylated purified streptokinase activator complex (APSAC) is a complex of streptokinase and plasminogen that does not require free circulating plasminogen for its effectiveness. It has many theoretical benefits over streptokinase, but suffers antigenic problems similar to those of the parent compound.

The half-life of APSAC in plasma is somewhere between 40 minutes and 90 minutes.