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Treatment of acute myocardial infarction (AMI) has been with tissue plasminogen activator (tPA) alone since 1987, at an original cost of $2,000 which has risen to $5,000. Over the past decade tPA has been replaced by percutaneous coronary intervention (PCI), an invasive, more time-consuming hospital procedure. The mortality results of the two treatments were similar, but the PCI cost and reimbursement are at least 5-fold higher. At the time, another fibrinolytic regimen was developed which is more effective, but it was ignored, and PCI continued to be the treatment choice and major source of revenue.

Therapeutic fibrinolysis has used tissue plasminogen activator (tPA) since 1987 based on a belief that tPA was responsible for biological fibrinolysis. This belief, however, was belied by clinical experience with tPA showing that it was not an effective fibrinolytic. Comparative clinical trials in almost 100,000 patients with acute myocardial infarction (AMI) failed to show that tPA was unequivocally more effective than streptokinase (SK), an indirect, inefficient, and non-specific plasminogen activator. Instead, it was found that tPA caused significantly more intracranial hemorrhage (ICH) side effects than SK. This disappointing experience led to the abandonment of fibrinolysis and its replacement by percutaneous coronary intervention (PCI) for AMI. However, PCI is a time-consuming, hospital procedure, poorly adapted to salvaging function of an ischemic myocardium, for which success is critically time dependent. Fibrinolysis remains the fastest method available for this and its abandonment is predicated on fibrinolysis and tPA being identical. This assumption, however, is contradicted by evidence that fibrinolysis requires both biological plasminogen activators, and that urokinase plasminogen activator (uPA) is the dominant of the two. This was also documented in a single clinical study in AMI, in which tPA’s fibrinolytic function was found to be analogous to that of the starter in an automobile.

Therapeutic fibrinolysis has used tissue Plasminogen Activator (tPA) alone since 1987,when tPA was first approved for the treatment of Acute Myocardial Infarction (AMI). The use of Tpa was based on the belief that it was responsible for fibrinolysis. However, this assumption should have been put into question from the outset when tPA was found to have the same efficacy as Streptokinase (SK). This was unexpected, since SK has an indirect, less efficient mechanism of action and SK has no fibrin clot affinity, in contrast to tPA. Nevertheless, the 30-day AMI mortality with tPA and SK were identical in the first two trials.

Tissue plasminogen activator (tPA) was approved for therapeutic fibrinolysis in 1987, when it replaced streptokinase (SK) for the treatment of acute myocardial infarction (AMI). It remains the fibrinolytic of choice today although over the last decade fibrinolysis has been replaced by percutaneous coronary intervention (PCI) whenever possible. However, PCI is a technically demanding procedure that is time-consuming whereas for AMI “time is myocardium” and only rapid reperfusion will salvage function of ischemic myocardium. Fibrinolysis is the simplest fastest method, but it requires both plasminogen activators, the second one being urokinase plasminogen activator (uPA), the native form of which is a proenzyme, prouPA. A combination of a 5 mg bolus of tPA followed by a prouPA matches the natural paradigm and was shown to be more effective safer than tPA monotherapy. Activator gene knockout studies also showed that both activators are required, but that uPA rather than tPA was the dominant one, as well as the one neglected for the past 33 years.

Fibrinolysis refers to the natural enzymatic system responsible for dissolving a blood clot or thrombus. Since an intravascular thrombus is responsible for almost all heart attacks and most strokes, interest in fibrinolysis, the only medical treatment, dominated medical interest in the 60’s-90’s. Since that time, the incidence of heart attacks and strokes has not changed much, but interest in fibrinolysis has faded. Instead, interest has shifted to catheter removal of clots, called percutaneous coronary intervention (PCI) which is a time-consuming that delays reperfusion considerably, and is much more costly than fibrinolysis. It is also a cruder treatment that can only remove clots larger than the catheter, but it is very well reimbursed.

Ever since tissue plasminogen activator (tPA) was approved for therapeutic fibrinolysis in 1987, it has been the fibrinolytic of choice. At the same time, it is also recognized that tPA never lived up to its clinical expectations and has more recently been replaced by percutaneous coronary intervention (PCI) as the treatment of choice for acute myocardial infarction (AMI). For other occlusive vascular diseases and for patients in remote areas, tPA remains an essential option. In view of the continued importance of fibrinolysis, it is disappointing that fibrinolysis never evolved beyond what it was when tPA replaced streptokinase (SK) 32 years ago. The endovascular procedure replacement for AMI treatment suffers from being technically demanding, time-consuming, and costly. An untested alternative fibrinolytic paradigm is that of the endogenous, physiological system, which is initiated by tPA but then is followed by the other natural plasminogen activator, urokinase plasminogen activator (uPA). In this combination, it is uPA rather than tPA that has the dominant function. This is also evident from gene knockout studies where deletion of uPA that it has the dominant effect. The fibrinolytic properties of tPA and uPA are complementary so that their combined effect is synergistic, especially when they are administered sequentially starting with tPA. Endogenous fibrinolysis functions without bleeding side effects and is ongoing. This is evidenced by the invariable presence in blood of the fibrin degradation product, D-dimer (normal concentration 110–250 ng/ml). This activator combination, consisting of a mini bolus of tPA followed by a 90-min proUK infusion, was once used to treat 101 AMI patients. Compared with the best of the tPA mega trials, this regimen resulted in an almost a doubling of the infarct artery patency rate and reduced mortality sixfold. To date, a second trial has not yet been done.

Therapeutic fibrinolysis has been dominated by the experience with tissue-type plasminogen activator (t-PA), which proved little better than streptokinase in acute myocardial infarction. In contrast, endogenous fibrinolysis, using one-thousandth of the t-PA concentration, is regularly lysing fibrin and induced Thrombolysis In Myocardial Infarction flow grade 3 patency in 15% of patients with acute myocardial infarction. This efficacy is due to the effects of t-PA and urokinase plasminogen activator (uPA). They are complementary in fibrinolysis so that in combination, their effect is synergistic. Lysis of intact fibrin is initiated by t-PA, and uPA activates the remaining plasminogens. Knockout of the uPA gene, but not the t-PA gene, inhibited fibrinolysis. In the clinic, a minibolus of t-PA followed by an infusion of uPA was administered to 101 patients with acute myocardial infarction; superior infarct artery patency, no reocclusions, and 1% mortality resulted. Endogenous fibrinolysis may provide a paradigm that is relevant for therapeutic fibrinolysis.

Thrombosis is a major contributor to disability and mortality worldwide and is estimated to be the cause of one in four deaths [1]. It is the pathology responsible for triggering acute myocardial infarction (AMI) and ischemic stroke, and is the primary cause of venous thromboembolism. The pathogenesis has been well-studied starting with Virchow more than 150 years ago [2], and its prevention by anti-thrombotic drugs has made much progress, culminating most recently in the approval of new oral anticoagulants which are specific inhibitors of thrombin or factor Xa. By contrast, treatment by thrombolysis, the only pharmacological means available, has remained at a near standstill since Genentech first applied for FDA approval of tissue plasminogen activator (tPA) in 1983. A replacement for streptokinase (SK) had been long-sought, but after early successes in AMI, tPA use has since been on the decline. Instead, percutaneous coronary intervention (PCI) has become the treatment of choice in AMI, despite its technical, time-consuming, and costly demands. Pre-treatment with tPA to hasten coronary reperfusion, so-called facilitated PCI, was found to increase PCI complications including intracranial hemorrhage (ICH) making the two incompatible [3]. In ischemic stroke, tPA has been estimated to be used in only 2–5 % of patients over-all [4], and in pulmonary embolism, bleeding and stroke complications limited its use to the minority of patients with hemodynamic decompensation [5]. The recent trend in stroke has also begun to favor the use of endovascular devices with three major trials in ischemic stroke published in the first 2 months of 2015 in the NEJM [6–8]. In this same period, a clinical trial of catheter-directed ultrasound was also published which showed a significant acceleration of the thrombolysis rate by this method [9]. These device reperfusion techniques require bringing the patient to a qualified facility which inevitably consumes precious time for conditions in which “minutes are myocardium or neurons.” An optimal treatment for these time-sensitive conditions can only be one that can be brought to the patient and is easily and safely administered. The resort to alternative methods is a reflection on the inadequacy of current thrombolysis, a problem that was foretold by the results with tPA in major clinical trials.

Thrombolysis with tissue plasminogen activator (tPA) has been a disappointment and has now been replaced by an endovascular procedure whenever possible. Nevertheless, thrombolysis remains the only means by which circulation in a thrombosed artery can be restored rapidly. In contrast to tPA monotherapy, endogenous fibrinolysis uses both tPA and urokinase plasminogen activator (uPA), whose native form is a proenzyme, prouPA. This combination is remarkably effective as evidenced by the fibrin degradation product, D-dimer, which is invariably present in plasma. The two activators have complementary mechanisms of plasminogen activation and are synergistic in combination. Since tPA initiates fibrinolysis when released from the vessel wall and prouPA is in the blood, they induce fibrinolysis sequentially. It was postulated that this may be more effective and fibrin-specific. The hypothesis was tested in a model of clot lysis in plasma in which a clot was first exposed to tPA for 5 min, washed and incubated with prouPA. Lysis was compared with that of clots incubated with both activators simultaneously. The sequential combination was almost twice as effective and caused less fibrinogenolysis than the simultaneous combination (p

In the interesting study by Zhou et al [1] of a TAFI inhibitor, DS1040, the authors state (p 962, col 1, para 3) that “When plasmin cleaves fibrin … [it] exposes C-terminal lysine residues [that] can bind both plasminogen and tPA.” While this is technically correct, it is functionally misleading, as is also Figure 1 showing tPA binding to lysines. The reason is that the lysine affinity of tPA is the same as that of plasminogen [2]. This article is protected by copyright. All rights reserved.

A clot that obstructs blood flow triggers the onset of most heart attacks and 85% of strokes, the leading causes of death and disability worldwide. Reestablishing blood flow rapidly limits damage to the heart or brain, saves lives, and can restore well-being. Since “time is heart or brain,” treatment must be initiated rapidly, which means by a readily available method. Fibrinolysis is the only therapy able to fulfill these criteria but the current method using tissue Plasminogen Activator (tPA) monotherapy is not sufficiently safe to be administered without pre-testing nor is it sufficiently effective to be very useful. Although anticoagulants have evolved, especially in recent years, the same thrombolytic has been used since 1987, when tPA monotherapy was approved for the treatment of Acute Myocardial Infarction (AMI) and subsequently also for ischemic stroke.

Experience with thrombolysis for 25 years has been essentially limited to tissue plasminogen activator (tPA). Therefore, tPA has been perceived as synonymous with all thrombolysis. However, tPA has a unique mode of action different from that of urokinase plasminogen activator (uPA), whose native form is prouPA. At the pharmacological concentrations, both physiological activators induced unanticipated effects that were also different. Reperfusion with tPA is almost twice as effective as streptokinase, the former thrombolytic standard. Nevertheless, three mega-trials with a total of 94,740 patients were required to show a significant difference in acute myocardial infarction. Surprisingly, intracranial hemorrhage was also significantly more with tPA. These paradoxical findings can be explained by tPA’s rethrombosis rate undermining its efficacy, and lysis of hemostatic fibrin causing tPA bleeding. By contrast, prouPA has a low rethrombosis rate and it spares hemostatic fibrin by virtue of its different mechanism of fibrin-bound plasminogen activation. Unfortunately, these potential advantages are out of reach in practice since at high therapeutic concentrations in blood, prouPA is vulnerable to non-specific conversion to uPA, a non-specific plasminogen activator that induces a hemorrhagic diathesis. A single-site mutant, M5, was designed to correct this prouPA problem. In vitro and in vivo studies showed that M5 clot lysis was more effective than prouPA or tPA and was accompanied by a sparing of hemostatic fibrin. When M5 administration was preceded by a bolus of plasma C1-inhibitor, it further improved the plasma stability of M5 and permitted a maximum clot lysis rate to be achieved without fibrinogenolysis.

Therapeutic fibrinolysis has been dominated by the experience with tissue-type plasminogen activator (t-PA), which proved little better than streptokinase in acute myocardial infarction. In contrast, endogenous fibrinolysis, using one-thousandth of the t-PA concentration, is regularly lysing fibrin and induced Thrombolysis In Myocardial Infarction flow grade 3 patency in 15% of patients with acute myocardial infarction. This efficacy is due to the effects of t-PA and urokinase plasminogen activator (uPA). They are complementary in fibrinolysis so that in combination, their effect is synergistic. Lysis of intact fibrin is initiated by t-PA, and uPA activates the remaining plasminogens. Knockout of the uPA gene, but not the t-PA gene, inhibited fibrinolysis. In the clinic, a minibolus of t-PA followed by an infusion of uPA was administered to 101 patients with acute myocardial infarction; superior infarct artery patency, no reocclusions, and 1% mortality resulted. Endogenous fibrinolysis may provide a paradigm that is relevant for therapeutic fibrinolysis.

An anticlotting activity present in rabbit blood following the administration of small amounts of thrombin is described. The substrate for this activity was found to be related to the vitamin-K dependent clotting factors. The activity was labile in blood but relatively stable after it was isolated from plasma of thrombin treated animals. Oxy-genation of blood was found to accelerate its inactivation. A method for the rapid chromatographic isolation of the activity from small amounts of plasma is described. The method is considered sufficiently practical to be utilized clinically. The anti-clotting activity is considered similar to that which can be elaborated in vitro from thrombin activation of isolated prothrombin complex as previously described by others. A physiologic role for the activity is proposed.

A blood clot or thrombus triggers the onset of most vascular diseases, like stroke or heart attack. Thrombolysis is the only treatment that can restore blood flow rapidly and easily. Unfortunately, the standard thrombolytic, tissue plasminogen activator (tPA), has proven inadequate and is being replaced by invasive endovascular procedures, which are time consuming and limited in their availability in relation to the scope of the problem. Historically, when tPA clinical trials began, it was not recognized sufficiently that without the other natural plasminogen activator, prourokinase (proUK), thrombolysis by tPA was seriously compromised. The reason is that the 2 activators have complementary mechanisms of action in fibrinolysis, making their combination a requirement for optimal efficacy and synergy. Biological fibrinolysis also uses both activators, explaining why such low endogenous concentrations are sufficient. A low-dose sequential combination of tPA and proUK was tested in acute myocardial infarction, where it was exceptionally effective and safe. Because native proUK at pharmacological doses was vulnerable to spontaneous conversion to urokinase, jeopardizing safety, a site-directed mutant was developed that improved proUK's plasma stability fivefold without interfering with its mode of action. Mini-bolus tPA followed by low-dose proUK infusion is a simple, safe, effective, and promising first-line treatment of acute thrombotic disorders.

SummaryEndothelial progenitor cells (EPC) displaya unique ability to repair vascular injury and promote neovascularization although the underlying molecular mechanisms remain poorly understood. Urokinase-type plasminogen activator (uPA) and its receptor (uPAR) play a critical role in cell migration and angiogenesis by facilitating proteolysis of extracellular matrix.The aim of the present study was to characterize the uPA/uPAR-dependent proteolytic potential of EPC outgrown from human umbilical cord blood and to analyze its contribution to their angiogenic properties in vitro. Cells derived from EPC (EPDC), presenting typical features of late outgrowth endothelial cells, were compared to mature endothelial cells, represented by human umbilical vein endothelial cells (HUVEC). Using quantitative flow cytometry, enzyme-linked immunosorbent assays and zymography, we demonstrated that EPDC displayed higher levels of uPA and uPAR. In conditioned culture media, uPA-dependant proteolytic activity was also found to be significantly increased in EPDC.This activity was paralleled bya higher secretion of pro-metalloproteinase-2 (pro-MMP-2). Inhibition of EPDC-associated uPA by monoclonal antibodies that block either uPA activity or receptor binding, significantly reduced proliferation, migration and capillary like tube formation. Moreover, tumor necrosis factoralpha and vascular endothelial growth factor,known to be locally secreted in ischemic areas, further increased the proteolytic potential of EPDC by up-regulating uPA and uPAR expression respectively.The EPDC response to these factors was found to be more pronounced than that of HUVEC. In conclusion, these findings indicated that EPDC are characterized by high intrinsic uPA/uPAR-dependent proteolytic potential that could contribute to their invasive and angiogenic behaviour.

The interesting article on the antifibrinolytic effect of Factor XIII by Rijken et al [1] was predicated on the assumption that fibrinolysis is mediated only by tPA, since that is what was studied. However, there is evidence that this common assumption should be put into question. In a study published almost thirty years ago, we showed that whereas spontaneous lysis of platelet poor plasma clots was mediated by tPA, that of platelet-rich plasma clots, with which this article was concerned, could not be abolished by tPA antibody, but instead was abolished by the addition of uPA antibody. This article is protected by copyright. All rights reserved.

Signal transduction by urokinase-type plasminogen activator (u-PA) bound to its cell receptor has been well established. In the present study, we found, for the first time to our knowledge, that u-PA promotes its own synthesis by endothelial cells and monocytes. This phenomenon was characterized and shown to involve the u-PA receptor (u-PAR) pathway. The finding may be of general importance, since most cells that express u-PAR also produce u-PA. Human umbilical vein endothelial cells (HUVECs), U937 monocytes, and human peripheral blood monocytes (PFMCs) were incubated with diisopropylfluorophosphate (DFP)-pretreated u-PA, the amino-terminal fragment (ATF) of u-PA, or the kringle domain. A threefold up-regulation of u-PA secretion and synthesis by u-PA or ATF was found. The predominant effect was expressed in HUVECs, in which u-PA mRNA was also up-regulated. The u-PA kringle domain had no effect on u-PA synthesis, leading to the conclusion that the EGF domain was responsible. This was also consistent with the additional finding that the u-PAR, to which the EGF domain binds, was necessary for the up-regulation. The results indicate that u-PA up-regulates itself via its EGF domain and u-PAR. The possibilities that the results were related to displacement of receptor-bound u-PA or the blocking of u-PA incorporation into the cells were excluded. A modest up-regulation of u-PAR was also associated with this phenomenon.

The enzymatic and fibrinolytic properties of glycosylated and nonglycosylated recombinant human pro-urokinase (pro-UK) produced in yeast Pichia pastoris were characterized and compared with those of Escherichia coli and mammalian cell-derived pro-UK. Among the five different forms of pro-UK, the yeast glycosylated pro-UK was activated by plasmin with the lowest catalytic efficiency (kcat/Km). The yeast glycosylated urokinase (UK) also had the highest Km in its activation of Glu-plasminogen, and had a substantially lower fibrinolytic activity than the other four forms. These findings suggest that the poly-mannose on Asn-302 of yeast glycosylated pro-UK interfered with its activation by plasmin and its binding interaction with plasminogen. By contrast to plasminogen, the activation of the small synthetic substrate, S2444, was comparable for all five forms of recombinant UK. It is concluded that the glycosyl residue on pro-UK/UK is functionally important and modulates its activatability and its catalytic efficiency against its natural substrate. Therefore, pro-UK from different expression systems cannot be assumed to have comparable fibrinolytic activities.

It has been well documented that Lp(a) binds noncovalently to fibrin or human umbilical vein endothelial cells. This binding is to lysines and is inhibited by lysine analogues such as epsilon-aminocaproic acid (EACA). In the present study, Lp(a) (0.006-0.6 microM) binding to immobilized fibrin and endothelial cells was evaluated by ELISA with an anti-Lp(a) antibody. A significant portion (approximately 65%) of the Lp(a) was found to resist dissociation by EACA (0.2 M). The EACA resistant binding of Lp(a) was time and concentration dependent. The addition of EDTA to the incubation mixture had no effect, thereby excluding cross-linking by transglutaminase as a mechanism. This portion of Lp(a) was also resistant to dissociation by acid (0.1 N HCl), 0.1% SDS, 1 M benzamidine, Tris-HCl (1 M, pH 12), or DTT (5 mM), but it was washed off by 0.1 N NaOH (which did not remove the immobilized fibrin). This suggested that the Lp(a) was covalently linked by an ester bond. Covalent binding was inhibited when Lp(a) was mildly oxidized by BioRad Enzymobeads, which may explain why it escaped recognition in experiments with radiolabeled Lp(a). Covalent binding was attenuated when Lp(a) was pretreated with DFP suggesting that the serine residue in the pseudo active site of Lp(a) was involved. Lp(a) also bound covalently to immobilized BSA, indicating some nonspecificity. However, binding to BSA was almost 3-fold less than to fibrin, suggesting that lysine binding may facilitate covalent binding. A similar proportion of EACA resistant binding of Lp(a) was found with endothelial cells. In conclusion, the findings demonstrate a novel, covalent binding by Lp(a) which is kringle independent and is postulated to involve the pseudo protease domain of Lp(a). This property may contribute to the deposition of Lp(a) on endothelial surfaces and its colocalization with fibrin in atheromas.

It was recently proposed that hydrophobic interactions control the active conformation of serine proteases in the trypsin family (Hedstrom et al. (1996) Biochemistry 35, 4515-23) rather than a charge interaction with Asp next to the active site Ser, as formerly believed. In the present study, certain site-directed mutants of the serine protease zymogen pro-urokinase (pro-UK) and its two-chain enzymatic derivative urokinase (UK) were characterized. The results provide information on the structure-function of the catalytic domain of pro-UK/UK, which is relevant to this controversy. Mutations at Asp355(c194), which eliminated its charge, induced a 6250-fold reduction in the catalytic activity of UK. By contrast, reducing the hydrophobicity at the neoterminal Ile159(c16) of UK had relatively little effect. However, when both the hydrophobicity and the size of the side chain were reduced by a glycine substitution at this position, a major reduction (9090-fold) in the catalytic efficiency of UK occurred. This effect was related to the smaller side chain increasing the cavity and the flexibility of the N-terminus and thereby interfering with its charge interaction with Asp355(c194). A similar mechanism, rather than a change in hydrophobicity, is believed also to explain the reduction in the stabilization energy of the activation domain observed in a trypsin mutant by Hedstrom et al. (1996). Although hydrophobic interaction facilitated the charge interaction with Asp355(c194), the latter was the primary force which stabilized the active conformation of UK. The charge interaction with Asp355(c194) was also found to be the principal determinant of the intrinsic catalytic activity of single-chain pro-UK. Additionally, the findings confirmed that the KM of pro-UK for its natural substrate was significantly lower than that of UK. Since this same phenomenon was also seen with each of the mutants, the substrate binding pocket of these single-chain zymogens was better formed than that of their two-chain, enzymatic derivatives.

SummaryAntiserum from rabbits immunised with pure human fibrinogen was affinity purified on immobilised fibrin fragment E (FFE). This FFE antibody (Ab) induced significant growth inhibition of a human cancer xenograft in mice and suppression of tumour angiogenesis, leaving no formed vessels and only CD31-staining endothelial fragments in place. Tubule formation of HUVEC on MatrigelTM was also significantly inhibited by FFE Ab. Since MatrigelTM is fibrin-free, this effect implicated a different FFE Ab binding site than FFE. Flow cytometry of HUVEC showed that FFE Ab bound to HUVEC, but with a broad range of 55–98 %. Immunofluorescent staining of HUVEC explained this range, since FFE Ab was seen not to bind to human umbilical vein endothelial cells (HUVEC) directly but instead to a matrix protein variably adherent to HUVEC. This protein was identified as fibronectin (FN) by appearance, staining with FN Ab, and by a FN knockdown study. Neither HUVEC nor matrix reacted with fibrin D-dimer (DD) Ab. Immunofluorescent stains of HUVEC matrix with FFE and FN Ab’s showed that these Ab’s bound to the same epitopes on FN, as also seen on Western blots of purified FN. These findings indicate the presence of an antigenic determinant in fibrinogen/FFE that is homologous with an epitope(s) in FN recognised by FFE Ab, and critical for angiogenesis in this xenograft. The FN epitope(s) remains to be identified, but the present findings can be used for the selection of the appropriate clones from mice immunised with fibrinogen which can facilitate this identification, and which may also be of clinical use.

Gene knockout mice studies indicate that urokinase-type plasminogen activator (u-PA) is importantly involved in fibrinolysis, but its physiologic mechanism of action remains poorly understood. We postulated that platelets may be involved in this mechanism, as they carry a novel receptor for u-PA and a portion of the single-chain u-PA (scu-PA) intrinsic to blood is tightly associated with platelets. Therefore, plasminogen activation by platelet-associated u-PA was studied. When washed platelets were incubated with plasminogen, no plasmin was generated as detected by plasmin synthetic substrate (S2403) hydrolysis; however, after the addition of thrombin, but not other agonists, platelet-dependent plasminogen activation occurred. Plasminogen activation was surface-related, being inhibited by blocking platelet fibrinogen receptors or by preventing plasminogen binding to the thrombin-activated platelet surface. U-PA was identified as the only plasminogen activator responsible and enrichment of platelets with exogenous scu-PA significantly augmented plasminogen activation. These findings appeared paradoxical because thrombin inactivates scu-PA. Indeed, zymograms showed inactivation of scu-PA during the first hour of incubation with even the lowest dose of thrombin used (1 u/mL). However, this was followed by a thrombin dose-dependent (1 to 10 u/mL) partial return of u-PA activity. Reactivation of u-PA was not due to the direct action of thrombin, but required platelets and was found to be related to a platelet lysosomal thiol protease, consistent with cathepsin C. In conclusion, a new pathway of plasminogen activation by platelet-associated endogenous or exogenous scu-PA was demonstrated, which is specifically triggered by thrombin activation of platelets. These findings may help explain u-PA–mediated physiological fibrinolysis and have implications for therapeutic thrombolysis with scu-PA.

Urokinase-type plasminogen activator (u-PA) was found to induce monocyte adhesion through a u-PA receptor (u-PAR)-mediated cAMP-dependent signal transduction pathway (J. Biol. Chem.270, 30282–30285, 1995). In the present study, the effects of kininogen, kallikrein, factor XII, and plasminogen on u-PA-induced monocyte adhesion were examined since these proteins are abundant in plasma and closely related to u-PA in fibrinolysis and inflammation. Monocyte adhesion to a standard plastic surface by u-PA was shown to be inhibited by the activated, two-chain forms of kininogen (HKa) and kallikrein. The latter occurred only at higher, though physiological, concentrations and was dependent on its catalytic activity. Monocyte adhesion was promoted by factor XII and plasminogen via a noncatalytic mechanism. The findings indicated that u-PA-induced monocyte adhesion was downregulated by HKa and kallikrein and upregulated by factor XII and plasminogen at physiological concentrations. Therefore, these contact system proteins may be important modulators of u-PA-induced monocyte adhesion, a process which is involved in many pathophysiological events.

Endogenous urokinase-type plasminogen activator (u-PA) has been identified in platelet membrane, and platelets have been shown to take up exogenous high molecular weight u-PA from the ambient medium. In this report, the mechanism of the association of u-PA with platelets was investigated using recombinant, single chain u-PA. When gel filtered human platelets were incubated with radiolabeled u-PA, the u- PA was found to specifically and saturably bind to the resting platelets in a dose-dependent manner. Unlabeled u-PA and the amino terminal fragment of u-PA inhibited 125I-u-PA binding to platelets with a mean IC50 of 65 and 58 nmol/L, respectively. A single saturable binding site in intact resting platelets was found with a mean kd of 43 +/- 25 nmol/L and 2263 +/- 809 sites per platelet. In contrast to resting platelets, 125I-u-PA did not bind to thrombin-induced platelets. Western blotting studies, using a monoclonal or a polyclonal antibody specific for the u-PA cell-surface receptor (u- PAR), failed to show evidence of u-PAR in resting platelets, whereas, u-PAR was found at approximately 54 and approximately 48 kD on U937 monocytes, which served as a positive control. Ligand blotting of platelet membrane and of U937 cell proteins with 125I-u-PA revealed a u-PA binding protein of approximately 70 kD in the platelets and one of approximately 54 kD in the U937 cells. Complexion of u-PA with a platelet membrane protein was also shown by gel filtration of a mixture of u-PA and platelet membrane proteins. A u-PA complex was further shown by enzyme-linked immunosorbent assay when microtiter plates were coated with platelet membrane proteins, and this complex formation was shown to be dose-dependent and saturable with an apparent kd of 17 nmol/L. It was concluded that platelet membrane contains a specific, high affinity u-PA-binding protein that is distinct from u-PAR.

Concern about the risks of cardiovascular illness in women using combined oral contraceptives (OC) containing the progestagens desogestrel and gestodene prompted two studies of data from the UK General Practice Research Database. We compared the risks of certain cardiovascular illnesses in otherwise healthy women exposed to one of three OCs containing < 35 micrograms oestrogen plus levonorgestrel, desogestrel, or gestodene. In the first study, based on some 470 general practices, there were 15 cases of unexpected idiopathic cardiovascular death among 303,470 women who were current users of one of the study OCs. The estimated incidence rates were 8/184,536 (4.3 per 100,000) woman-years at risk for users of combined OCs containing levonorgestrel, 2/135,567 (1.5 per 100,000) for desogestrel users, and 5/105,201 (4.8 per 100,000) for gestodene users. The relative risk (RR) estimates were 0.4 (95% CI 0.1-2.1) and 1.4 (CI 0.5-4.5) for desogestrel and gestodene, respectively, compared with levonorgestrel. In the second study, derived from some 370 general practices, there were 80 cases of nonfatal venous thromboembolism (VTE) in a cohort of 238,130 otherwise healthy women. The incidence rates of VTE per 100,000 woman-years at risk were 16.1 for levonorgestrel users, 29.3 for desogestrel, and 28.1 for gestodene. The adjusted RR estimates from the cohort analysis were 1.9 (1.1-3.2) and 1.8 (1.0-3.2) for desogestrel and gestodene users, respectively, compared with users of levonorgestrel. In a nested case-control analysis the adjusted matched RR estimates were 2.2 (1.1-4.4) and 2.1 (1.0-4.4) for desogestrel and gestodene users, respectively, compared with users of levonorgestrel. The excess risk for nonfatal VTE associated with the new generation of combined OCs containing low-dose oestrogen and the progestagens desogestrel or gestodene compared with levonorgestrel is estimated to be 16 per 100,000 woman-years.

Objectives . The present study was designed to test the efficacy and safety of a sequential combination of recombinant tissue-type plasminogen activator (rt-PA) and pro-urokinase in patients with acute myocardial infarction. Background . Efforts continue to identify a thrombolytic regimen that induces rapid, complete and sustained coronary artery patency in acute myocardial infarction. The two endogenous plasminogen activators rt-PA and pro-urokinase have been shown experimentally to induce fibrinolysis by sequential and complementary mechanisms. As a result, certain combinations of these activators have been found to be synergistic in vitro and in vivo. Methods . In a multicenter observational study with core facilities for angiographic and laboratory analysis, 101 patients with acute myocardial infarction were enrolled and gives a low dose bolus of rt-PA (5 to 10 mg) followed by a 90-min infusion of pro-urokinase (40 mg/h). All patients received intravenous heparin and oral aspirin. Coronary angiography was performed in all patients at 90 min. Results . Angiography at 90 min showed the infarct-related artery to be patent (Thrombolysis in Myocardial Infarction [TIMI] grade 2 or 3 flow) in 77% of patients, and 60% achieved TIMI grade 3 flow. At one center, angiography was repeated at 24 h to detect a possible reocclusion. All 28 patients with a patent infarct-related artery at 90 min had patency at 24 h (82% achieved TIMI grade 3 flow). Treatment was well tolerated, with bleeding complications essentially confined to arterial puncture site hematomas. There was only one in-hospital death. Conclusions . A sequential combination of low dose rt-PA and reduced-dose pro-urokinase produced a high TIMI 3 patency rate, was well tolerated and was associated with a low reocclusion rate.

Summary In a previous study it was shown that about 20% of the endogenous pro-urokinase (pro-UK) in whole blood was associated with platelets and that there was uptake of pro-UK by platelets from the ambient plasma or buffer. In the present study, the association of urokinase-type plasminogen activator (u-PA) with platelets was further characterized. Isolated platelet suspensions were exposed to high and low molecular weight u-PA or tissue plasminogen activator (t-PA), then washed and analyzed by SDS gel electrophoresis and zymography. Only the high molecular weight u-PA was found associated with platelets and this association resisted acid washing, aggregation by thrombin, or osmotic swelling of platelets to obliterate the open canalicular system. Ultrasonic disruption of platelets and isolation of the membrane portion indicated that u-PA was associated with the membrane. The catalytic site of u-PA remained available on the platelet surface as evidenced by its inhibition by the specific inhibitor glutamyl-glycyl-arginyl chloromethylketone (GGAck). Uptake by platelets, but not red cells, of pro-UK from whole blood was demonstrated, and this was detectable at physiological concentrations of pro-UK suggesting that endogenous u-PA in platelets may originate from the ambient plasma. A Western blot of platelet membrane proteins with antibody to u-PA receptor (u-PAR) showed no evidence of u-PAR. It was postulated that u-PA binds to a platelet membrane protein distinct from u-PAR, via the A-chain of u-PA, and that this interaction becomes stabilized resisting dissociation.

SummaryFactor XII has long been implicated in the intrinsic pathway of fibrinolysis, but the mechanism by which it triggers plasminogen activation and targets fibrinolysis has not been established. In the present study, the assembly and function of activated Factor XII (F.XIIa), prourokinase (pro-u-PA), high molecular weight kininogen (H-kininogen), and prekallikrein on human umbilical vein endothelial cells (HUVEC) was investigated. 125I-prekallikrein was shown to bind to HUVEC via receptor-bound H-kininogen in the presence of 50 μM ZnCl2. After the addition of F.XIIa, 78% of the 125I-prekallikrein initially bound to HUVEC was converted to 125I-kallikrein. However, only 6% of the HUVEC-bound 125I-pro-u-PA was thereby activated. This discrepancy was shown to be related to rapid dissociation (>50% within 15 min) of prekallikrein/kallikrein, but not pro-u-PA, from HUVEC. Increasing the level of cell-bound kallikrein increased the portion of cell-bound pro-u-PA activated, indicating that their co-localization was important for this pathway. Finally, F.XIIa was shown to trigger plasminogen activation on HUVEC via this pathway. This assembly of reactants on the endothelium suggests a mechanism whereby local fibrinolysis may be triggered by blood coagulation.

SummarySafety and efficacy of the thrombolytic agent pro-urokinase (pro-UK) in the treatment of deep vein thrombosis of the lower limbs (DVT) have been investigated in an open, uncontrolled, pilot study. Fifteen patients were infused with 800.000 IU (5 mg)/h of pro-UK over 24 h (120 mg), together with unfractionated heparin adjusted to maintain the activated partial thromboplastin time between 1.5 and 2.5 times the basal value. Efficacy was assessed comparing venographic changes in the 11 evaluable limbs before and after pro-UK infusion. The Marder score decreased from a median pre-thrombolysis value of 28 (range 4-40) to 16 (3-38) (p

SummaryClots formed from platelet rich plasma were found to be lysed more readily by low concentrations of pro-urokinase (pro-UK) than clots formed from platelet poor plasma. This was not a non-specific effect since the reverse occurred with tissue plasminogen activator. A mechanical explanation due to platelet-mediated clot retraction was excluded by experiments in which retraction was inhibited with cyto-chalasin B. Therefore, a platelet-mediated enzymatic mechanism was postulated to explain the promotion of fibrinolysis. Casein autography of isolated platelets revealed a ≈ 90 kDa band of activity which comigrated with plasma prekallikrein (PK)/kallikrein, a known activator of pro-UK. Furthermore, treatment of platelets with plasma PK activator (PPA), consisting essentially of factor XIIa, induced activation of pro-UK and of chromomgenic substrate for kallikrein (S-2302). This activity corresponded to approximately 40-200 pM kallikrein per 10 8 washed and gel filtered platelets per ml. The activation of pro-UK by PPA-pretreated platelets was dose-dependent and inhibited by soybean trypsin inhibitor but not by bdellin, a specific inhibitor of plasmin, nor by the corn inhibitor of factor XIIa. Kinetic analysis of pro-UK activation by kallikrein showed promotion of the reaction by platelets. The KM of the reaction was reduced by platelets by ≈ 7-fold, while the kcat was essentially unchanged. In conclusion, PK was shown to be tightly associated with platelets where it can be activated by factor XIIa during clotting. The activation of pro-UK by platelet-bound kallikrein provides an explanation for the observed platelet mediated promotion of pro-UK-induced clot lysis. Since pro-UK and plasminogen have also been shown to be associated with platelets, the present findings suggest a mechanism by which the factor Xlla-dependent intrinsic pathway of fibrinolysis can be localized and targeted to a thrombus.

A single-site mutant (M5) of native urokinase plasminogen activator (prouPA) induces effective thrombolysis in dogs with venous or arterial thrombosis with a reduction in bleeding complications compared to tPA. This effect, related to inhibition of two-chain M5 (tcM5) by plasma C1-inhibitor (C1I), thereby preventing non-specific plasmin generation, was augmented by the addition of exogenous C1I to plasma in vitro. In the present study, tPA, M5 or placebo +/- C1I were administered in two rat stroke models. In Part-I, permanent MCA occlusion was used to evaluate intracranial hemorrhage (ICH) by the thrombolytic regimens. In Part II, thromboembolic occlusion was used with thrombolysis administered 2 h later. Infarct and edema volumes, and ICH were determined at 24 h, and neuroscore pre (2 h) and post (24 h) treatment. In Part I, fatal ICH occurred in 57% of tPA and 75% of M5 rats. Adjunctive C1I reduced this to 25% and 17% respectively. Similarly, semiquantitation of ICH by neuropathological examination showed significantly less ICH in rats given adjunctive C1I compared with tPA or M5 alone. In Part-II, tPA, M5, and M5+C1I induced comparable ischemic volume reductions (>55%) compared with the saline or C1I controls, indicating the three treatments had a similar fibrinolytic effect. ICH was seen in 40% of tPA and 50% of M5 rats, with 1 death in the latter. Only 17% of the M5+C1I rats showed ICH, and the bleeding score in this group was significantly less than that in either the tPA or M5 group. The M5+C1I group had the best Benefit Index, calculated by dividing percent brain salvaged by the ICH visual score in each group. In conclusion, adjunctive C1I inhibited bleeding by M5, induced significant neuroscore improvement and had the best Benefit Index. The C1I did not compromise fibrinolysis by M5 in contrast with tPA, consistent with previous in vitro findings.

Lipoprotein(a) [Lp(a)] has been postulated to inhibit fibrinolysis due to its structural homology to plasminogen. Indeed, it has been reported that Lp(a) competitively inhibits the promotion by fibrin of tissue plasminogen activator (t-PA)-catalyzed plasminogen activation. However, it has also been reported that this inhibition is uncompetitive. No studies have been published, to our knowledge, of the effect of Lp(a) on prourokinase (pro-UK)-catalyzed plasminogen activation. Plasminogen activation by pro-UK or a plasmin-resistant mutant pro-UK was previously shown to be promoted by fibrin fragment E2, whereas that by t-PA is promoted by fragment D. Therefore, the influence of Lp(a) on the kinetics of these two reactions was examined. When Lp(a) was added (90-600 nM), no change in the rate of plasmin generation by Ala158-pro-UK was observed. Consistent with this, immobilized Lp(a) also failed to bind to fragment E2, whereas it did bind to D dimer. When t-PA-catalyzed plasminogen activation in the presence of D dimer was measured, uncompetitive inhibition by Lp(a) was found, but only at low concentrations of D dimer (< 0.5 microM) or t-PA (0.05 nM). At higher concentrations of D dimer and t-PA, instead of inhibition, Lp(a) induced a 2.4-fold promotion of plasminogen activation. Similarly, Lp(a) enhanced (up to 2.5-fold) plasminogen binding to immobilized fibrin in both buffer and plasma milieus at the physiological concentration of plasminogen (2.0 microM). In conclusion, Lp(a) had no effect on plasminogen activation by pro-UK and induced only limited inhibition of activation by t-PA.(ABSTRACT TRUNCATED AT 250 WORDS)