Thrombolytic Agents used in Trials and Clinical Practice

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Thrombolytic drugs lyze preexisting thrombus in both arteries and veins and re-establish tissue perfusion.4 They exert their action through conversion of plasminogen to plasmin, which then degrades fibrin, a major structural component of the thrombus.37 The action of thrombolytic drugs is achieved by either potentiating endogenous fibrinolytic pathways or mimicking natural thrombolytic molecules.1 Currently available thrombolytic agents are derived from bacterial products or manufactured using recombinant DNA technology. They differ in their efficiency, fibrin selectivity, and side-effect profile. Even for the same thrombolytic agent, different doses, different administration regimens, and concomitant use of adjunctive agents can modify its patency rates. However, these differences are only marginal.1 Many limitations exist among the available generations of thrombolytic agents, raising the need for continued research for better agents. The indications of fibrinolytic therapy are summarized in Table 1 and contraindications in Table 2. First-generation thrombolytic agents

There are three agents belonging to this group: streptokinase, urokinase, and anisoylated purified streptokinase activator complex (APSAC). They are not fibrin specific or site specific, and they act anywhere in the blood, converting circulating plasminogen to plasmin. Eventually, this causes depletion of body plasminogen, disturbing the equilibrium between circulating plasminogen and plasminogen in the thrombus (plasminogen steal) and reducing clot lysis.

Table 1 Indications of fibrinolytic therapy (ACC guidelines) Class I

1. In the absence of contraindications, fibrinolytic therapy should be administered to STEMI patients with symptom onset within the prior 12h and STelevation greater than 0.1 mVin at least 2 contiguous precordial leads or at least 2 adjacent limb leads (Level of Evidence: A)

2. In the absence of contraindications, fibrinolytic therapy should be administered to STEMI patients with symptom onset within the prior 12 h and new or presumably new LBBB (Level of Evidence: A)

Class IIa

1. In the absence of contraindications, it is reasonable to administer fibrinolytic therapy to STEMI patients with symptom onset within the prior 12 h and 12-lead ECG findings consistent with a true posterior MI (Level of Evidence: C)

2. In the absence of contraindications, it is reasonable to administer fibrinolytic therapy to patients with symptoms of STEMI beginning within the prior 12-24h who have continuing ischemic symptoms and STelevation greater than 0.1 mVin at least 2 contiguous precordial leads or at least 2 adjacent limb leads (Level of Evidence: B)

Class III

1. Fibrinolytic therapy should not be administered to asymptomatic patients whose initial symptoms of STEMI began more than 24 h earlier (Level of Evidence: C)

2. Fibrinolytic therapy should not be administered to patients whose 12-lead ECG shows only ST-segment depression except if a true posterior MI is suspected (Level of Evidence: A)

Table 2 Contraindications to fibrinolytic therapy (ACC guidelines) Absolute contraindications

1. Any prior ICH

2. Known structural cerebral vascular lesion (e.g., AVM/aneurysm)

3. Known malignant intracranial neoplasm

4. Ischemic stroke within 3 months EXCEPT acute ischemic stroke within 3 h

5. Suspected aortic dissection

6. Active bleeding or bleeding diathesis (excluding menses)

7. Significant closed-headi or facial trauma within 3 months Relative contraindications

1. History of chronic, severe, poorly controlled hypertension

2. Severe uncontrolled hypertension on presentation (SBP greater than 180mmHg or DBP greater that 110mmHg)

3. History of prior ischemic stroke greater than 3 months, dementia, or known intracranial pathology not covered in contraindications

4. Traumatic or prolonged (greater than 10min) CPR or major surgery (within the last 3 weeks)

5. Recent (within 2-4 weeks) internal bleeding

6. Noncompressible vascular punctures

7. For streptokinase/anistreplase: prior exposure (more than 5 days ago) or prior allergic reaction to these agents

8. Pregnancy

9. Active peptic ulcer disease

10. Current use of anticoagulants: the higher the INR, the higher the risk of bleeding

CPR, cardiopulmonary resuscitation; AVM, arteriovenous malformation; SBP, systolic blood pressure; DBP, diastolic blood pressure; INR, international normalized ratio. Streptokinase

Streptokinase is approved for use in myocardial infarction, pulmonary embolism, deep venous thrombosis, arteriovenous-cannula occlusions, and peripheral arterial occlusions.30 Streptokinase is an enzyme with a molecular weight of 47 000 Da35 derived from the culture filtrate of Lancefield group C5 b-hemolytic streptococci.1'35 Streptokinase lyses thrombus by potentiating the body's own fibrinolytic pathways.1 It acts indirectly2,37 through binding with free circulating plasminogen to form a 1:1 complex, resulting in a conformational change and exposure of an active site that can convert additional plasminogen into plasmin, the main thrombolytic enzyme in the body.4 The resultant plasmin is of two types: fibrin-bound and unbound. Fibrin-bound plasmin causes direct fibrinolysis of the thrombus,2 while unbound circulating plasmin leads to systemic fibrinolysis and a hypocoagulable state due to the depletion of fibrinogen, plasminogen, and factors V and VIII.1'35'37 In addition, streptokinase increases levels of activated protein C, which enhances clot lysis.35 The greatest benefit of streptokinase appears to be achieved by early i.v. administration.4

Streptokinase has no metabolites. The complex is inactivated, in part, by antistreptococcal antibodies, resulting from prior infection,38 and eliminated through the liver.35 It has two half-lives: a fast one (11-13 min) due to inhibition by the circulating antibodies; and a slow one (23-29 min) due to loss of the enzyme activity.4 The unbound fraction, which constitutes about 15%, has a serum half-life of 80 min.30 On the other hand, fibrin degradation products can be detected in the serum for up to 24 h, which means that the patient remains somewhat 'anticoagulated' even without the use of heparin.35,37 Urokinase

Urokinase is the most familiar thrombolytic agent among interventional radiologists and it is often used for peripheral vascular thrombosis.37 It is approved for pulmonary embolism and for lysis of coronary thrombi, but not for mortality reduction in acute myocardial infarction.4,35

Urokinase is a trypsin-like1 enzyme that is produced endogenously by renal parenchymal cells30 and found in urine. Approximately 1500 L of urine are needed to yield enough urokinase to treat a single patient.30 There are two forms of urokinase, which differ in molecular weight but have similar clinical effect.39 Commercially available urokinase, the low-molecular-weight form (32 400 Da), is produced from cultured human neonatal kidney cells. It is a two-polypeptide chain serine protease, containing 411 amino acid residues4,35: an A chain of 2000 Da is linked by a sulfhydryl bond to a B chain of 30 400 Da. Recombinant techniques are used to produce urokinase in Escherichia coli.30 Unlike streptokinase, urokinase directly cleaves plasminogen to produce plasmin.1 Although this process is slightly increased in the presence of fibrin, urokinase produces circulating unbound plasmin, which means that it not only degrades fibrin clots but also fibrinogen and other plasma proteins leading to systemic fibrinolysis.40

In plasma, urokinase has a half-life of approximately 15 min (7-20 min). It is rapidly metabolized and cleared by the liver38 with small fractions being excreted in bile and urine. Plasma levels can be elevated two- to fourfold1 and clearance is reduced in patients with hepatic impairment. Moreover, due to its short half-life, re-thrombosis may occur within 15-30 min of therapy cessation. Heparin is commonly used during and after urokinase adminstration to minimize this risk.40 Anisoylated purified streptokinase activator complex (APSAC, anistreplase)

APSAC was the third thrombolytic agent to be developed. It has a molecular weight of 131 000 Da35 and consists of streptokinase in a noncovalent 1:1 complex with plasminogen.4 It does not require free circulating plasminogen to be effective. APSAC is catalytically inert because of the acylation of the catalytic site of plasminogen (having the catalytic site temporarily blocked by a ^-anisoyl group), which protects the catalytic center of the complex from premature neutralization.35 However, the affinity of plasminogen binding to fibrin is maintained.4 APSAC acts as an indirect plasminogen activator35 and is nonfibrin selective, and so activates both circulating and clot-bound plasminogen, but is most active within the thrombus.2

Inside the circulation, anistreplase undergoes spontaneous deacylation to form the active complex of plasminogen-streptokinase. This conversion occurs with a half-life of 90-100 min,2,37 which lengthens its thrombolytic effect after i.v. injection.4 The active complex is metabolized in the liver35 and has the longest half-life among all thrombolytic agents, ranging from 90 min4 to 100 min.35 Second-generation thrombolytic agents

These agents include t-PA and scu-PA (prourokinase). The second-generation agents are more fibrin selective and although they were developed to avoid systemic thrombolytic state, they can still cause a mild to moderate decrease in the levels of circulating fibrinogen and plasminogen.35 Tissue-type plasminogen activator (alteplase)

Alteplase was the first recombinant t-PA to be produced. It is the most familiar fibrinolytic agent in emergency departments and the most often used agent for treatment of coronary artery thrombosis, pulmonary embolism, and acute stroke.30 Alteplase is expensive, costing approximately 8-10 times more than streptokinase per dose.35'37 In vivo, t-PA is synthesized by the vascular endothelial cells and is considered the physiologic thrombolytic agent that is responsible for most of the body's natural efforts to prevent thrombus propagation. Based on the results of Stroke Trials sponsored by the National Institute of Neurological Disorders and Stroke (NINDS), intravenous thrombolysis with rt-PA was approved as the 'first-ever' effective treatment for ischemic stroke during the first 3 h of symptom onset.36 rt-PA is a sterile purified glycoprotein molecule consisting of 527 amino acids35 that is structurally identical to endogenous t-PA.1,41 It has a molecular weight of 70 000 Da35 and is produced by recombinant technology1,4,35 from a human melanoma cell line.35 The molecule contains five domains: finger, epidermal growth factor, kringle 1, kringle 2, and serum protease.2 There are two different forms of t-PA based on the number of chains: t-PA (the two-chain form duteplase and alteplase by recombinant technology) and rt-PA (one chain form).2,35 It consists predominantly of the single-chain form (rt-PA), but upon exposure to fibrin, rt-PA is converted to the two-chain dimer.4

t-PA is a naturally occurring enzyme (serine protease).42 It is the principal physiological activator of plasminogen in the blood and has a high binding affinity for fibrin4 at the site of a thrombus, directly2,35,37 activating clot-bound plasminogen only.42 While this might seem an advantage, this selectivity is not absolute; circulating plasminogen may also be activated by large thrombolytic doses or lengthy treatment.4 Moreover, its action is fibrin-enhanced; that is, in the absence of fibrin, t-PA is a weak plasminogen activator.42 It is rapidly cleared from plasma with an initial half-life of approximately 5 min (4-10 min)5,35; however, its effect at the clot persists for over an hour35 ( 72min), but the concentration of circulating t-PA would be expected to return to endogenous circulating levels of 510ngmL_ 1 within 30 min.42 Having a short plasma half-life necessitates its administration as a bolus injection, followed by a short continuous infusion.2 Heparin is usually coadministered to avoid reocclusion.2,35 The plasma clearance is 380-570 mL min _ 1,35 and is primarily mediated by the liver.2,35,43

t-PA is not associated with hypotension35 and is not antigenic; it can be readministered as necessary1 and may be considered for use in patients who have high antibody titer against streptokinase.35 The activity of t-PA is enhanced in the presence of fibrin, resulting in thrombus-specific fibrinolysis.1 In practice, however, t-PA causes a milder form of systemic fibrinogenolysis4,35 than streptokinase at equi-effective doses, but the incidence of bleeding is similar with both agents.4 Moreover, t-PA is considered more efficacious than streptokinase in establishing coronary reperfusion4 and has a slight mortality advantage over streptokinase due to its accelerated administration because of the shorter halflife. Unfortunately, this occurs at the cost of a marginal increase in stroke rate.1

The risk of intracranial hemorrhage is higher with t-PA than streptokinase (0.7% versus 0.5%). Having a shorter plasma half-life, the rate of re-thrombosis after t-PA is greater than streptokinase,4 which raises the need for continuous infusion in order to achieve its greatest efficacy,1 and since t-PA is more expensive and toxic than streptokinase, the latter is the agent of choice for coronary thrombolysis.35

Prourokinase is a new fibrinolytic agent that is currently undergoing clinical trials for a variety of indications. It is a single chain urokinase35 and has been produced both in glycosylated (ABT-74187) and nonglycosylated (saruplase) forms.2,35 It is a relatively inactive precursor that must be converted to urokinase before it becomes active in vivo. However, it displays selectivity for clots by binding to fibrin before activation.37 The mechanism of action of the nonglycosylated form is unclear, but it is known to be nonfibrin specific.35 Its advantage over other plasminogen activators is that it is inactive in plasma and so does not consume circulating inhibitors. Also, it is somehow clot specific, where it needs fibrin to be converted by an unknown mechanism into active urokinase.30 It is usually administered as a bolus followed by intravenous infusion, but single-bolus regimens are now being developed.35 Third-generation thrombolytic agents

These groups of agents have been developed through modifications of the basic t-PA structure. They are either: conjugates of plasminogen activators with monoclonal antibodies against fibrin, platelets, or thrombomodulin; mutants, variants, and hybrids of t-PA and prourokinase (amediplase); or new molecules of animal (vampire bat) or bacterial (Staphylococcus aureus) origin.35 These molecular variations have yielded agents with better pharmacological properties than t-PA, a longer half-life, resistance to plasma protease inhibitors, and more selective fibrin binding.4,35 Several of these agents are being developed including reteplase (r-PA, retevase), lanoteplase (nPA), tenecteplase (TNKase), pamiteplase (YM866; Solinase), staphylokinase, and novel modified tissue plasminogen activator (E6010).

Reteplase is the first third-generation thrombolytic agent to be approved for use in acute MI to improve postinfarct ventricular function, lessen the incidence of congestive heart failure, and reduce mortality.35 It is a synthetic nonglycosylated deletion mutein of t-PA containing 355 of the 527 amino acids of the native tissue plasminogen activator; it lacks the finger, epidermal growth factor, and kringle 1 domains2'44 as well as carbohydrate side chains.2 This results in a prolonged half-life and less fibrin specificity than t-PA.37'44 It has a molecular weight of 39 500 Da and is produced in E. coli by recombinant DNA technology.1 The gene for a fragment of t-PA is inserted into E. coli, and the protein is then extracted from the bacteria and processed to convert it into an active thrombolytic.30'37

Patients receiving reteplase have faster clot resolution than those receiving t-PA, owing to the fact that reteplase binds less tightly to fibrin, allowing for more free diffusion through the clot rather than only binding to the surface as t-PA does. In a controlled trial, 64% of patients who received a double bolus of reteplase showed a decrease in fibrinogen levels to below 100mgdL— 1 within 2h. However, the mean fibrinogen levels returned to baseline within 48 h.45 Moreover, at high concentrations, it does not compete with plasminogen for fibrin-binding sites, allowing plasminogen at the site of the clot to be transformed into clot-dissolving plasmin.30

The above-mentioned structural modifications result in a fivefold decrease in fibrin binding and an extended halflife (11-19 min).35,37,44 The longer half-life allows for administration of reteplase by double-bolus infusion rather than a prolonged infusion.35,37,44 It undergoes renal (and some hepatic) clearance30 at a rate of 250-450 mLmin— 1. Lanoteplase (nPA, novel plasminogen activator)

Lanoteplase is a deletion mutant of t-PA lacking the finger, epidermal growth factor, and one amino acid substitution in the kringle 1 domain, leading to deletion of a glycosylation site.2,35 Tenecteplase (TNK-t-PA, TNKase)

Tenecteplase is a tissue plasminogen activator with a molecular weight of 70 000 Da,35 produced by genetic engineering.37 To create tenecteplase, a 527 amino acid glycoprotein molecule, the human gene for t-PA was modified using 3 amino acid substitutions:37 a substitution of threonine 103 with asparagine and asparagine 117 with glutamine within the kringle 1 domain, and a tetra-alanine substitution at amino acids 296-299 in the protease domain.2,35 These mutations resulted in a decrease of plasma clearance, prolonged half-life, higher degree of fibrin specificity, and increased resistance to PAI-1 as compared to t-PA. The FDA has approved the use of tenecteplase in acute MI.

Tenecteplase is a modified form of human t-PA that binds more avidly to fibrin,4 and directly converts plasminogen to plasmin.35 This process relatively increases in the presence of fibrin, giving tenecteplase the advantage of being more specific with minimal systemic effect. However, this specificity is not absolute; a 4-15% decrease in circulating fibrinogen and 11-24% decrease in plasminogen has been reported following administration. Moreover, its clinical significance with regard to safety or efficacy has not been established.

A single bolus of tenecteplase administered to patients with acute myocardial infarction exhibits biphasic disposition from the plasma. The initial half-life is 20min (15-24 min),35 about four times that of t-PA,4 and is considered to be the longest elimination half-life among t-PA derivatives.4,35,37 The terminal phase half-life of tenecteplase is 90-130 min. The initial volume of distribution is weight related and approximates plasma volume. The main route of elimination is liver, at a clearance rate of 99-119 mLmin— 1.37 Staphylokinase

Staphylokinase was known to possess profibrinolytic properties more than four decades ago.35 It is produced by certain strains of S. aureus. It acts on the surface of the clot to form a plasmin-staphylokinase complex,2 which has high fibrin specificity, only activating plasminogen trapped in the thrombus.35 After administration, staphylokinase-related antigen disappears from plasma in a biphasic manner, with an initial half-life of 6.3 min and a terminal half-life of 37 min.35

As compared to t-PA, studies suggest that staphylokinase may have less procoagulant effects. Furthermore, it is highly antigenic; patients develop neutralizing antibodies in about 1-2 weeks and the titer remains elevated for several months after therapy cessation.35 This limits the use of a second dose until safer, more effective new variants that have less immunogenicity are developed.2 Fourth-generation thrombolytic agents Recombinant desmodus salivary plasminogen activator-1 (rDSPA-1, desmoteplase) A naturally occurring enzyme in the saliva of the blood-feeding vampire bat (Desmodus rotundus) is genetically related to t-PA.47 It consists of four different proteases — D. rotundus salivary plasminogen activators (DSPAs). DSPA-1 is the full-length variant with a greater than 72% sequence homology to human t-PA. Unlike t-PA, it exists as single-chain molecules48 and it is critically dependent on fibrin.47,49,50 DSPA-1 targets and destroys fibrin;47 it is more fibrin dependent and fibrin specific than t-PA.47 Its catalytic efficiency is enhanced 13 000-fold47 in the presence of fibrin, while that of t-PA increases only by 72-fold.49

It has high fibrin specificity and selectivity, and a longer half-life compared to other thrombolytic agents.48'51 Compared to t-PA, it is non-neurotoxic, causes less fibrinogenolysis,50 less antiplasmin consumption, and results in faster and more sustained reperfusion as demonstrated by animal studies.52 Furthermore, it can be given to acute ischemic stroke patients within 3-9 h of onset of symptoms.51 DSPA is safe and results in improved perfusion and low mortality rates without associated symptomatic intracerebral hemorrhage.51

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