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(Circulation. 1996;93:857-865.)
© 1996 American Heart Association, Inc.

Articles

Fibrin-Selective Thrombolytic Therapy for Acute Myocardial Infarction

D. Collen, MD, PhD

From the Center for Molecular and Vascular Biology, University of Leuven, and Center for Transgene Technology and Gene Therapy, Flanders Interuniversity Institute of Biotechnology, Campus Gasthuisberg KU Leuven, Belgium.

Correspondence to D. Collen, MD, PhD, Center for Molecular and Vascular Biology, Campus Gasthuisberg, O & N, Herestr 49, B-3000 Leuven, Belgium. E-mail desire.collen@med.kuleuven.ac.be.

Key Words: Bench to Bedside • fibrin • myocardial infarction • thrombolysis

	Introduction

Top Introduction Milestones in the Development... A Personal Account of... References

 
Thrombolytic therapy of acute myocardial infarction is based on the premise that coronary artery thrombosis is its proximate cause. Rupture of atheromatous plaque leads to occlusive thrombosis that produces myocardial ischemia and cell necrosis, leading to loss of ventricular function and possibly death.^{1 2} One approach to the treatment of established thrombosis consists of pharmacological dissolution of the blood clot by intravenous infusion of plasminogen activators that activate the fibrinolytic system (Fig 1). The fibrinolytic system includes a proenzyme, plasminogen, which is converted by plasminogen activators to the active enzyme plasmin, which in turn digests fibrin to soluble degradation products. Inhibition of the fibrinolytic system takes place at the level of both the plasminogen activators (mainly by plasminogen activator inhibitor-1) and plasmin (mainly by ₂-antiplasmin).³ Thrombolytic agents that are either approved for clinical use or under clinical investigation in patients with acute myocardial infarction include streptokinase, recombinant tissue-type plasminogen activator (rTPA, prepared either as alteplase or as duteplase), rTPA derivatives such as reteplase and TNK-rTPA, anisoylated plasminogen streptokinase activator complex, two-chain urokinase-type plasminogen activator (UPA), recombinant single-chain UPA (prourokinase), and more recently, recombinant staphylokinase and derivatives. The hypothesis underlying thrombolytic therapy in acute myocardial infarction is that early and sustained recanalization prevents cell death, reduces infarct size, preserves myocardial function, and reduces early and late mortality.

View larger version (22K): [in this window] [in a new window]   Figure 1. Schematic representation of the fibrinolytic system. The proenzyme plasminogen is activated to the active enzyme plasmin by plasminogen activators. Plasmin degrades fibrin into soluble fibrin degradation products. Inhibition of the fibrinolytic system may occur at the level of the plasminogen activators by plasminogen activator inhibitors (PAI) or at the level of plasmin, mainly by 2-antiplasmin. SK indicates streptokinase.

The beneficial effects of thrombolytic therapy in acute myocardial infarction are now well established, and it has become routine treatment; it is given to more than 500 000 patients per year worldwide, but at least three times that number could benefit from this treatment. The biochemical mechanisms of thrombolytic therapy have been elucidated and its clinical application has been developed over the past several decades. The purpose of this review is first, to highlight the milestones in this development and second, to give a personal account of the development of fibrin-selective thrombolytic therapy with rTPA and recombinant staphylokinase.

	Milestones in the Development of Thrombolytic Therapy of Acute Myocardial Infarction

Top Introduction Milestones in the Development... A Personal Account of... References

 
Early Observations
Most of the components of the fibrinolytic system were identified in the 1940s and 1950s. Thrombolytic therapy has been attempted in patients with acute myocardial infarction since the late 1950s,⁴ but no significant progress toward its routine clinical use was made until 1980. Indeed, the prevailing view in those days was that coronary thrombosis was an epiphenomenon resulting from rather than being the underlying cause of acute myocardial infarction. Even the randomized European multicenter trial⁵ in 315 patients, which demonstrated a significantly lower (P<.01) mortality at 6 months with streptokinase (16%) than with glucose (31%) infusion, was unable to change the tide. Lack of understanding of the mechanism of benefit and the fact that the patient group represented only 13.5% of the infarct patients admitted to the 11 participating coronary care units probably explain the lack of impact of this study, notwithstanding its unequivocal statistical significance. Thus, toward the end of the 1970s thrombolytic therapy in acute myocardial infarction was not taken seriously. For a detailed account of historical developments before 1980, the reader is referred to publications by Astrup⁶ and Sherry,⁷ the pioneers of this era.

Modern Era of Coronary Thrombolysis
The modern era of thrombolytic therapy started with the demonstration by DeWood et al² that myocardial infarction in its early stage was invariably associated with thrombotic coronary artery occlusion and the demonstration by Rentrop et al,⁸ following initial work by Chazov et al,⁹ that infusion of streptokinase within the infarct-related coronary artery early after symptom onset induced rapid recanalization. The efficacy of intracoronary streptokinase was quickly confirmed, and evidence accumulated from both experimental animal and clinical studies that timely reopening of a coronary artery led to improved myocardial function. It soon became apparent that widespread application of coronary thrombolysis would depend on the development of simple therapeutic strategies without coronary catheterization.¹⁰ Randomized clinical trials with short-term intravenous streptokinase, initiated by Schröder et al,¹¹ demonstrated moderate but significant potency for coronary artery recanalization and culminated in 1986 in the GISSI trial,¹² which demonstrated a significant overall reduction in mortality with intravenous streptokinase.

In a parallel development, elucidation of biochemical mechanisms that regulate physiological fibrinolysis¹³ led to the concept of fibrin-selective thrombolysis, which fueled the hope that more specific and efficacious thrombolytic agents could be developed. Physiological fibrinolysis appeared to be regulated by specific molecular interactions, which in 1981 were sufficiently understood to allow the following description.¹⁴

Extrinsic plasminogen activator (later called tissue-type plasminogen activator) has a weak affinity for plasminogen in the absence of fibrin (K_M=65 µM) but a much higher affinity in the presence of fibrin (K_M between 0.15 and 1.5 µM). This increased affinity appears to be the result of a "surface assembly" of plasminogen activator and plasminogen on the fibrin surface. In this reaction plasminogen binds to fibrin primarily via specific structures called the "lysine-binding site". Thus one way of regulating fibrinolysis is at the level of plasminogen activation localized at the fibrin surface.

Plasmin is extremely rapidly inactivated by ₂-antiplasmin (k₁~10⁷ M^-1 sec^-1); the half-life of free plasmin in the blood is therefore estimated to be approximately 0.1 sec.

Plasmin with an occupied lysine-binding site is however inactivated 50 times more slowly by ₂-antiplasmin. Reversible blocking of the active site of plasmin with substrate also markedly reduces the rate of inactivation by ₂-antiplasmin. From these findings one can extrapolate that plasmin molecules generated on the fibrin surface, which are bound to fibrin through their lysine-binding sites and involved in fibrin degradation, are protected from rapid inactivation by ₂-antiplasmin. Plasmin released from the fibrin surface would, however, be rapidly inactivated by ₂-antiplasmin. These interactions are schematically visualized in Fig. 2.

View larger version (15K): [in this window] [in a new window]   Figure 2. Schematic visualization of the molecular interactions regulating physiological fibrinolysis. Plasminogen is converted to the proteolytic enzyme plasmin by tissue-type plasminogen activator, but this conversion occurs efficiently only on the fibrin surface, where activator and plasminogen are "assembled." Free plasmin in the blood is very rapidly inactivated by 2-antiplasmin, but plasmin generated at the fibrin surface is partially protected from inactivation. The lysine-binding sites in plasminogen (represented as the "legs" of the animal) are important for the interaction between plasmin(ogen) and fibrin and between plasmin and 2-antiplasmin (reprinted with permission from Reference 14).

These insights into the physiological regulation of fibrinolysis provided the framework for the following concept of fibrin-selective thrombolytic therapy.¹⁴

The molecular model for the regulation of fibrinolysis described above has important consequences for the development of thrombolytic agents. Indeed, the presently available thrombolytic agents streptokinase and urokinase have no specific affinity for fibrin and therefore activate circulating and fibrin-bound plasminogen relatively indiscriminately. Consequently, plasmin formed in circulating blood will initially be neutralized very rapidly by ₂-antiplasmin and be lost for thrombolysis. Once the inhibitor becomes exhausted, residual plasmin will degrade several plasma proteins (fibrinogen, factor V, factor VIII, etc.) and cause a serious bleeding tendency. This may explain why treatment with streptokinase or urokinase has only a limited efficiency and is associated with serious, sometimes life-threatening side effects.

From this reasoning it appears that specific thrombolysis will be possible only if the activation process of plasminogen can be localized at and confined to the fibrin surface. According to the present concepts, this can only be adequately achieved with the use of an activator that, like the physiological activator, adsorbs to the fibrin surface and becomes active in loco.

With the development of TPA for thrombolytic therapy, this hypothesis could be subjected to testing. Initially, two coronary patency studies supported the higher efficacy of fibrin-selective rTPA over non–fibrin-selective streptokinase,^{15 16} but two subsequent megatrials^{17 18} could not confirm that this translated into a mortality benefit. This apparent discrepancy between the results of smaller mechanistic studies and clinical outcome questioned the validity of the "open-artery hypothesis"¹⁹ and led, in an increasingly cost-conscious environment, to acrimonious debates without much substance (eg, see References 20 through 24). Finally, the GUSTO trial²⁵ and its angiographic substudy²⁶ revisited the open-artery hypothesis and conclusively established that brisk (TIMI 3 flow), early, and persistent coronary artery recanalization is the primary determinant of clinical benefit.²⁷

Toward Improved Thrombolytic Therapy
Currently available thrombolytic agents have several important limitations. At best, TIMI 3 flow within 90 minutes is obtained in somewhat over 50% of patients, acute coronary reocclusion occurs in roughly 10% of patients, anterograde coronary flow requires on average 45 minutes or more, intracerebral bleeding occurs in 0.3% to 0.7%, and the residual mortality is at least 50% of that without thrombolytic treatment.

At least three complementary approaches to improve thrombolytic therapy have become apparent: (1) earlier and accelerated treatment to reduce the duration of ischemia, (2) the use of plasminogen activators with increased thrombolytic potency and/or specific thrombolytic activity to enhance coronary thrombolysis, and (3) the use of more specific and potent anticoagulant and antiplatelet agents to accelerate recanalization and prevent reocclusion.²⁸

Because there is compelling evidence from most clinical trials that mortality reduction is greatest in patients treated soon after the onset of symptoms, early recanalization must remain the main objective of pharmacologically induced coronary thrombolysis. Continued and intensified education of the public, paramedical personnel, and physicians together with the development of rapid and efficient triage systems are essential to achieve these goals. Improvements in this area may well turn out to be the most difficult to achieve.

Several attempts to increase the efficacy of plasminogen activators or reduce their clearance have been undertaken. In these efforts, rTPA (Fig 3) has most frequently served as the template. rTPA domain deletion mutants that lack the finger (F), epidermal growth factor (E), and/or first kringle (K₁) domains have a substantially reduced plasma clearance that is, however, often associated with a reduced specific thrombolytic activity, resulting in an unchanged or only marginally improved thrombolytic efficacy.²⁹ On the basis of our present understanding of molecular mechanisms of fibrinolysis, domain deletion and substitution mutants of TPA will not constitute superior thrombolytic agents, although recent clinical experience with one such compound was relatively promising.³⁰ The combination triple mutant of rTPA, TNK-rTPA, with threonine 103 substituted for asparagine (introducing a glycosylation site), asparagine 117 substituted with glutamine (eliminating the high mannose glycosylation site), and Lys²⁹⁶-His-Arg-Arg²⁹⁹ replaced by alanine (increased zymogenicity and resistance to plasminogen activator inhibitor-1), constitutes an interesting second-generation product. Relative to rTPA, it appears to have a threefold to fivefold reduced clearance, intact specific activity, and a threefold to eightfold higher thrombolytic potency in animal models.^{31 32}

View larger version (45K): [in this window] [in a new window]   Figure 3. Schematic representation of the primary structure of TPA. The amino acids are represented by their single-letter symbols, and black bars indicate disulfide bonds. The active site residues His 322, Asp 371, and Ser 478 are indicated with an asterisk. The arrow indicates the plasmin cleavage site for conversion of single-chain to two-chain TPA.

The vampire bat (Desmodus rotundus) salivary plasminogen activator is homologous with human TPA but lacks the second kringle domain and the plasmin cleavage site for conversion to a two-chain form. It is more fibrin selective than human TPA in experimental animal models³³ but has not yet been tested in humans. Single-chain UPA also displays some degree of fibrin specificity, but at present there is not much evidence that it will outperform any of the currently approved thrombolytic agents in patients with acute myocardial infarction. Several chimeric plasminogen activators, consisting of various portions of TPA and UPA, have been constructed in an effort to combine the mechanisms of fibrin selectivity of both molecules, as reviewed elsewhere.²⁹ However, the thrombolytic properties and fibrin specificity of these chimeras are usually similar but not superior to those of the parent molecules. Murine monoclonal anti-human fibrin antibodies conjugated with plasminogen activators or recombinant fusion proteins of single-chain anti-fibrin antibodies with single-chain UPA appear to have significantly increased in vivo thrombolytic activities in animal models.³⁴ Their potential clinical value remains to be evaluated.

Aspirin and heparin have a limited impact on the speed of coronary thrombolysis and the resistance to lysis and do not consistently prevent reocclusion. Because aspirin is a nonselective inhibitor of the synthesis of both proaggregatory and antiaggregatory prostaglandins and heparin is ineffective for the inhibition of clot-associated thrombin, more specific inhibitors of platelet aggregation or coagulation might constitute better conjunctive agents for thrombolytic therapy in acute myocardial infarction (reviewed in Reference 35). Specific reduction of platelet aggregation is currently being explored in clinical trials, with monoclonal antibodies or synthetic peptides against the platelet GP IIb/IIIa receptor, among others. Another approach is the use of selective inhibitors of coagulation factors (eg, thrombin, factor Xa, factor VIIa), including hirudin and its derivatives or synthetic inhibitors. Some of these agents have been shown to be more effective than aspirin and/or heparin for the prevention of arterial thrombosis, the acceleration of arterial recanalization, and the prevention of early and delayed reocclusion after reflow. Unfortunately, most of these combinations also produce a substantial lengthening of the bleeding time, which may be suggestive of an increased bleeding risk.

In conclusion, the beneficial effects of thrombolytic therapy in acute myocardial infarction are now well established, but the limited efficacy and potentially life-threatening side effects of the current thrombolytic strategies remain a problem. Optimized thrombolytic therapy will eventually most likely consist of administration of potent fibrin-selective plasminogen activators in conjunction with specific anticoagulant and/or antiplatelet agents.

Recombinant Staphylokinase, a Potent Fibrin-Selective Thrombolytic Agent
Staphylokinase, a 136-amino-acid protein produced by certain strains of Staphylococcus aureus, was shown more than 4 decades ago to have profibrinolytic properties. Its in vitro fibrinolytic properties were evaluated in the 1950s and 1960s and its in vivo thrombolytic properties in dogs in 1964 and 1986 (reviewed in Reference 36). The in vivo results were most discouraging, comprising limited thrombolytic potency, extensive fibrinogen breakdown, and bleeding, whereupon interest in the development of staphylokinase as a thrombolytic agent faded away. In retrospect, however, these studies were misleading because the dog appears to be unusually sensitive to systemic fibrinolytic activation with staphylokinase.

The staphylokinase gene was cloned in the 1980s, and its biochemical and biological properties were reevaluated (reviewed in Reference 36). The mechanism of activation of plasminogen by staphylokinase bears similarities to that of streptokinase, but it differs in some essential aspects. Like streptokinase, staphylokinase forms a 1:1 stoichiometric complex with plasminogen, but unlike the streptokinase-plasminogen complex, the staphylokinase-plasminogen complex is inactive and requires conversion to staphylokinase-plasmin to expose the active site and become a potent plasminogen activator (K_m=7 µmol/L, k_cat=1.5 s^-1). The staphylokinase-plasmin complex is rapidly neutralized by ₂-antiplasmin (second-order rate constant >10⁶ L · mol^-1 · s^-1), whereas the streptokinase-plasmin(ogen) complex is not. The inhibition rate, however, is >100-fold reduced in the presence of fibrin. Furthermore, staphylokinase is released from the staphylokinase-plasmin complex following its inhibition by ₂-antiplasmin and is recycled to other plasminogen molecules.

These molecular interactions between staphylokinase, plasminogen, ₂-antiplasmin, and fibrin endow the molecule with a unique mechanism of fibrin selectivity in a plasma milieu. In the absence of fibrin, no activation of plasminogen by staphylokinase occurs, most likely because ₂-antiplasmin prevents the generation of active staphylokinase-plasmin complex. At the fibrin surface traces of plasmin are present, which form an active staphylokinase-plasmin complex that is bound to fibrin via the lysine binding sites of the plasmin molecule and protected from rapid inhibition by ₂-antiplasmin. After digestion of the fibrin clot, the staphylokinase-plasmin complex is released and inhibited and further plasminogen activation interrupted.

The thrombolytic potency and fibrin selectivity of staphylokinase has been confirmed in vitro, in several animal models, and in patients (reviewed in Reference 36). In a recent randomized study versus alteplase in 100 patients with acute myocardial infarction, recombinant staphylokinase was shown to be at least as potent and significantly more fibrin selective than rTPA.³⁷ However, staphylokinase is a heterologous protein that induces antibody formation and resistance to repeated administration.

Staphylokinase (SakSTAR variant³⁸ ) was found to contain three nonoverlapping immunodominant epitopes, two of which could be eliminated, albeit with partial inactivation of the molecule, by site-directed mutagenesis of clusters of two or three charged amino acids to alanine.³⁹ The variants SakSTAR.M38 (with Lys35, Glu38, Lys74, Glu75, and Arg77 substituted by Ala) and SakSTAR.M89 (with Lys74, Glu75, Arg77, Glu80, and Asp82 substituted by Ala) were found to be thrombolytically active and induce significantly less antibody formation than the wild-type molecule in animal models and patients with peripheral arterial occlusion,⁴⁰ suggesting that it will be possible to develop nonimmunogenic staphylokinase variants.

In summary, the available evidence suggests that recombinant staphylokinase is a potent, highly fibrin-selective thrombolytic agent. Larger clinical studies to determine its safety and clinical value for thrombolytic therapy in patients with acute myocardial infarction would appear to be warranted.

	A Personal Account of the Development of rTPA and Recombinant Staphylokinase

Top Introduction Milestones in the Development... A Personal Account of... References

 
From the above milestones, it might appear that the development of the concept of fibrin selectivity and its clinical achievement with rTPA and (more preliminarily) recombinant staphylokinase were the result of a logical sequence of biochemical, experimental animal, and clinical studies. However, progress has been decisively influenced by a few serendipitous observations and very simple experiments. Because I had the good fortune to be involved in several of these events, I will attempt to recall them chronologically.

Early Studies
During the course of my thesis work on the turnover of iodine-labeled plasminogen in humans⁴¹ in the Laboratory of Blood Coagulation of the Faculty of Medicine of the University of Leuven, Belgium, in 1969, a marked discrepancy was found between the rapid disappearance of functional plasminogen and the much slower disappearance of radioisotope from the blood in patients undergoing streptokinase treatment.⁴² Gel filtration of serial plasma samples revealed the in vivo formation of a main labeled inactive compound with M_r 150 000, probably resulting from reaction of generated plasmin with a plasma protease inhibitor. After purification of the complex by affinity chromatography on lysine-Sepharose and characterization, the inhibitor appeared to be an unidentified plasma protein, which is now called ₂-antiplasmin.⁴³

This serendipitous discovery of ₂-antiplasmin in 1974, which was discovered simultaneously and independently by two other groups as well,^{44 45} was followed by a series of systematic studies on its biochemical and kinetic properties, initially primarily in collaboration with B. Wiman from Stockholm, Sweden, during his stay in Leuven in 1977 and 1978^{46 47} and subsequently with H.R. Lijnen from Leuven, Belgium, and W.E. Holmes from San Francisco, Calif (reviewed in Reference 48). These studies, in conjunction with earlier observations on the enhancing effect of fibrin on the activation of plasminogen by TPA, allowed the formulation of a hypothetical molecular model for the regulation of physiological fibrinolysis⁴⁹ that after the subsequent isolation and characterization of TPA could be subjected to testing.¹⁴ In this model thrombolysis required selective activation of plasminogen at the fibrin surface, out of reach of the potent circulating ₂-antiplasmin.

On the basis of observations by E. Reich et al⁵⁰ from Rockefeller University, New York, NY, on the correlation between the malignant phenotype of tumor cells and secretion of plasminogen activators, the effect of plasma on the fibrinolytic activity generated by malignant cells was studied in 1976 in collaboration with A. Billiau from the Laboratory of Virology in Leuven, Belgium. It was found that plasma inhibited the fibrinolytic activity of malignant cells and that this inhibition required the presence of ₂-antiplasmin.⁵¹ Our hypothesis was that ₂-antiplasmin might react not only with plasmin but possibly also with the "malignant plasminogen activators" associated with tumor cells. To study this further, an enriched source of such malignant proteases became necessary. Dr G. Barlow from Abbott Laboratories, North Chicago, Ill, provided us with conditioned medium of a human melanoma cell line that he had acquired via Dr Reich's laboratory, which appeared to be suitable for our initial studies.⁵¹ To obtain larger amounts of conditioned medium for purification of the "malignant plasminogen activator," the cell line itself, known as the Bowes melanoma cell line, was obtained from Dr D. Rifkin from New York University Medical Center around the end of 1978. This was a most fortunate choice because, although at that time the relation between malignant plasminogen activators and the physiological activators was neither established nor suspected, it is now known that most malignant cell lines secrete UPA. Thus, by chance the very first cell line we studied turned out to be the one that probably is still the best known natural producer of TPA (see below).

Studies on Human TPA
During initial efforts in February 1979 to purify the plasminogen activator from the Bowes melanoma culture fluid, the following very simple experiment was performed, with significant consequences. When conditioned cell culture medium was mixed with fibrinogen and the mixture clotted, the activator, unlike urokinase, remained associated with the clot, from which it could be recovered with potassium thiocyanate. This observation suggested that the melanoma plasminogen activator was similar or identical to the physiological activator in blood now called TPA and that the Bowes melanoma cell line represented a potential plentiful source of TPA. Efforts to purify TPA using methods previously described by Wallén,⁵² such as chromatography on fibrin-Sepharose, lysine-Sepharose, arginine-Sepharose, and butyl-Sepharose, revealed not only that the melanoma activator behaved like TPA on chromatography but also that extensive losses occurred by adsorption to glass, gels, dialysis tubing, and ultrafilters. Although some progress was made with the purification, no homogeneous product was obtained by the summer of 1979.

In October 1979, D.C. Rijken from Leiden, Netherlands, joined our group. He had developed a method for the purification of the plasminogen activator from human uterus in which adsorption of the activator to surfaces was prevented by the use of the detergent Tween 80. We applied a simplified version of his purification procedure to the melanoma cell culture fluid and by the end of November 1979 obtained purified melanoma plasminogen activator, which was homogeneous by sodium dodecyl/sulfate–polyacrylamide gel electrophoresis and which was immunologically indistinguishable from the uterine plasminogen activator.⁵³ The purification procedure was scaled up, and 2 to 3 g of pure melanoma TPA was produced between 1980 and 1983.⁵⁴ This material has allowed us to study the biochemical, biological, and thrombolytic properties of TPA, initiate collaborations with several other research groups, and clear the way for the subsequent rapid development of rTPA as a thrombolytic agent.

O. Matsuo from Myasaki, Japan, who joined our laboratory in September 1979 for 1 year, took part in studies to establish the specific thrombolytic effect of TPA in plasma⁵⁵ and the in vivo thrombolytic effect in rabbits with experimental pulmonary embolism.⁵⁶ M. Hoylaerts, a Belgian biochemist, studied the kinetics of plasminogen activation by TPA and the role of fibrin.⁵⁷ These results, in conjunction with previous observations on the inhibition of plasmin by ₂-antiplasmin, led to the formulation of the concept of fibrin selectivity in 1981.¹⁴ To evaluate the thrombolytic and pharmacokinetic properties of thrombolytic agents, a simple quantitative model in rabbits with experimental jugular vein thrombosis was developed⁵⁸ that has since been used extensively by many investigators in the field. The first administration of TPA in humans was performed in May 1981 by W. Weimar from Rotterdam, Netherlands. Two renal transplant patients with renal vein thrombosis were successfully treated by intravenous infusion of only 5 or 7.5 mg TPA of melanoma origin over 24 hours.⁵⁹ In retrospect, these minidoses of TPA were probably effective because the clot architecture in these uremic patients may have been more fragile.

At the Fibrinolysis Congress in Malmö, Sweden, in June 1980, where our first results on TPA were presented, I was approached for collaboration by D. Pennica of the Department of Molecular Biology of Genentech Inc, South San Francisco, Calif. This led to the cloning and expression of the TPA gene.⁶⁰ Building on the successful cloning and expression of TPA and the demonstration that rTPA was indistinguishable from melanoma TPA with respect to kinetic properties, turnover in vivo, fibrin specificity, and thrombolytic properties,⁶¹ Genentech made a profound commitment to developing rTPA as a thrombolytic drug. Rapid progress was possible due to collaborations with several laboratories in different countries and the antecedent demonstration of the thrombolytic potential of natural TPA in patients with acute myocardial infarction.⁶² The first rTPA was administered with FDA approval in the United States in February 1984, less than 3 years after the first use of natural TPA in humans and the expression of rTPA.

Simultaneous with the pursuit of rTPA in the laboratory, the thrombolytic potential of natural TPA had become increasingly apparent. B. Sobel from St Louis, Mo, had initiated a collaboration in late 1981 in which our two groups explored the use of TPA for lysis of coronary artery thrombosis in canine myocardial infarction.⁶³ Intravenous infusion of human TPA purified from melanoma cell culture fluid resulted in prompt recanalization of an occluded coronary artery without inducing systemic activation of the fibrinolytic system. Subsequently, these observations were extended to rTPA in collaborative studies with F. Van de Werf from Leuven, Belgium, and B. Sobel⁶⁴ and a concurrent study with H.K. Gold from Boston, Mass.⁶⁵

The first use of rTPA in humans was within the framework of a multicenter, blinded, randomized, placebo-controlled trial performed in 50 patients between February 11 and June 20, 1984.⁶⁶ This study provided the foundation for the design of several subsequent studies with rTPA in patients with acute myocardial infarction—among others the TIMI, ECSG, TAMI, GISSI, and ISIS studies—carried out in both the United States and Europe, which have recently been reviewed in detail.⁶⁷ These developments culminated in the GUSTO trial and its angiographic substudy,^{25 26} which conclusively established the potential and limitations of rTPA for thrombolytic therapy in acute myocardial infarction.

Studies on Staphylokinase
For reasons described above and detailed elsewhere,³⁶ there was little or no interest in staphylokinase in the 1980s except for two groups, which had cloned and expressed the staphylokinase gene^{68 69} and partially characterized recombinant staphylokinase. The limited information available on the in vitro and in vivo properties of staphylokinase suggested that it was quite uninteresting and at best similar to streptokinase. My interest in it was aroused during a discussion with O. Matsuo in Tokyo in 1989 at the Congress of the International Society on Thrombosis and Haemostasis. He had performed in vitro experiments in human plasma in which staphylokinase, like TPA in experiments carried out in Leuven in 1980,⁵⁵ dissolved a ¹²⁵I-fibrin–labeled plasma clot submerged in plasma without breakdown of the plasma fibrinogen.⁷⁰ This unexpected finding resulted in a collaboration with O. Matsuo, with the involvement of H.R. Lijnen, on the mechanism of action of staphylokinase, which confirmed and extended Matsuo's observations,^{71 72 73} although they could not be translated into the superior thrombolytic potency of staphylokinase over streptokinase in rabbits and hamsters.⁷⁴

Because of the uncertainty about continued supply of recombinant staphylokinase from Japan, we decided to produce it locally⁷⁵ for continued evaluation of its biochemical and thrombolytic properties. A series of studies was carried out on the gene structure,³⁸ interaction with plasminogen and inhibition by ₂-antiplasmin,^{76 77 78} and fibrinolytic potency of staphylokinase in plasma in vitro and animal models in vivo,^{79 80} as reviewed elsewhere.³⁶ These studies allowed us to elucidate the unique mechanism of fibrin selectivity of staphylokinase as summarized above and led to a pilot study in five patients with acute myocardial infarction.^{81 82} Infusion of 10 mg recombinant staphylokinase induced rapid coronary artery recanalization in four patients in the absence of any measurable systemic plasminogen activation or fibrinogen degradation.

It quickly became obvious that our local method for production of recombinant staphylokinase would be unsuitable for large-scale production. Therefore, a collaboration was set up with the former group of D. Behnke at the Institute for Molecular Biotechnology in Jena, Germany, who had developed a high-yield expression system in Escherichia coli yielding mature, soluble recombinant staphylokinase.⁸³ This material was produced and used for further biochemical studies^{84 85 86 87} and initial clinical evaluation in patients with acute myocardial infarction³⁷ and peripheral arterial occlusion.⁸⁸ The technology is presently being adapted at the Hans Knoll Institute in Jena, Germany, to yield "good manufacturing practice" material for clinical evaluation. To develop staphylokinase for clinical use, Thromb-X NV, a spin-off company of the University of Leuven, was constituted in 1991, which obtained licenses from the Center for Molecular and Vascular Biology of the University of Leuven, Belgium, and via Medac GmbH of Hamburg, Germany, from the Institute of Molecular Biotechnology and the Hans Knoll Institute in Jena, Germany.

In a parallel development, the immunogenicity of staphylokinase has been studied^{89 90} and efforts have been undertaken to reduce it by protein engineering techniques. Initial studies resulted in the development of engineered mutants of recombinant staphylokinase with a reduced antibody-inducing capacity.^{39 40} A comprehensive analysis of the molecule using site-specific and random mutagenesis techniques will allow identification of potential nonimmunogenic variants with high probability.

Although at present it is difficult to assess the clinical value of recombinant staphylokinase in patients with acute myocardial infarction, in the light of the concept of fibrin selectivity and the open-artery hypothesis it might well represent a potent, safe, and possibly less expensive thrombolytic agent.

	Acknowledgments

 
I have had the good fortune to work since 1965 in a research-oriented environment in the Laboratory of Blood Coagulation and the subsequent Center for Thrombosis and Vascular Research (presently the Center for Molecular and Vascular Biology) of the University of Leuven. I am indebted to its former director, Prof M. Verstraete, for his continued support of my research, even when it was outside his own field of expertise. I was also most fortunate to have been able to collaborate with many young Belgian and foreign MDs and PhDs and several qualified laboratory technicians with long-standing experience: some are mentioned above, and others can be identified as coauthors in the reference list. Furthermore, the present development of rTPA and staphylokinase could not have been achieved without collaborations with many investigators from both academia and industry.

With a background as an MD and a PhD in chemistry, my role in these developments has been primarily to integrate biochemical, experimental animal, and clinical concepts and techniques and maintain continuity between collaborations and projects. Still, several of the events with an impact on progress in our understanding were fortuitous observations, such as the identification of ₂-antiplasmin via its in vivo complex formation with plasmin, the identification of the Bowes melanoma cell plasminogen activator as TPA via its affinity for fibrin, and the observation that staphylokinase had been discounted primarily because of initial selection of an inadequate species for in vivo experiments. The fact that the concept of fibrin selectivity as a basic mechanism of thrombolytic efficacy appears to be upheld across the boundaries of biochemical, physiological, and clinical disciplines is personally most gratifying.

	Footnotes

 
Dr Collen is a party to a royalty-bearing licensing agreement on recombinant tissue-type plasminogen activator between the University of Leuven and Genentech Inc, South San Francisco, Calif, and has an equity interest in Thromb-X NV, a spin-off company of the University of Leuven involved in the development of recombinant staphylokinase for thrombolytic therapy. Neither of these companies nor the University has any control over the statements made in this review.

	References

Top Introduction Milestones in the Development... A Personal Account of... References

 
1. Davies MJ, Thomas AC. Plaque fissuring: the cause of acute myocardial infarction, sudden ischaemic death, and crescendo angina. Br Heart J. 1985;53:363-373. [Free Full Text]

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54. Collen D, Rijken DC, Van Damme J, Billiau A. Purification of human tissue-type plasminogen activator in centigram quantities from human melanoma cell culture fluid and its conditioning for use in vivo. Thromb Haemost. 1982;48:294-296. [Medline] [Order article via Infotrieve]

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77. Silence K, Collen D, Lijnen HR. Interaction between staphylokinase, plasmin(ogen) and ₂-antiplasmin: recycling of staphylokinase after neutralization of the plasmin-staphylokinase complex by ₂-antiplasmin. J Biol Chem. 1993;268:9811-9816. [Abstract/Free Full Text]

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80. Collen D, De Cock F, Stassen JM. Comparative immunogenicity and thrombolytic properties toward arterial and venous thrombi of streptokinase and recombinant staphylokinase in baboons. Circulation. 1993;87:996-1006. [Abstract/Free Full Text]

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86. Schlott B, Hartmann M, Gührs KH, Birch-Hirschfeld E, Gase A, Vettermann S, Collen D, Lijnen HR. Functional properties of recombinant staphylokinase variants obtained by site-specific mutagenesis of methionine-26. Biochim Biophys Acta. 1994;1204:235-242. [Medline] [Order article via Infotrieve]

87. Lijnen HR, De Cock F, Van Hoef B, Schlott B, Collen D. Characterization of the interaction between plasminogen and staphylokinase. Eur J Biochem. 1994;224:143-149. [Medline] [Order article via Infotrieve]

88. Vanderschueren S, Stockx L, Wilms G, Lacroix H, Verhaeghe R, Vermylen J, Collen D. Thrombolytic therapy of peripheral arterial occlusion with recombinant staphylokinase. Circulation. 1995;92:2050-2057. [Abstract/Free Full Text]

89. Declerck PJ, Vanderschueren S, Billiet J, Moreau H, Collen D. Prevalence and induction of circulating antibodies against recombinant staphylokinase. Thromb Haemost. 1994;71:129-133. [Medline] [Order article via Infotrieve]

90. Vanderschueren SMF, Stassen JM, Collen D. On the immunogenicity of recombinant staphylokinase in patients and in animal models. Thromb Haemost. 1994;72:297-301.[Medline] [Order article via Infotrieve]

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