Plasminogen and stroke: more is better

Abstract

It has been well over a century since blood clots formed in test tubes were reported to spontaneously lyse, and in 1893 the term describing this phenomenon, i.e. ‘fibrinolysis’ first appeared in print 1.Dastre A. Fibrinolyse dans le sang.Arch de physiol norm et path. 1893; 5: 661-3Google Scholar. However, the process behind the spontaneous solubilization of fibrin‐rich clots remained unresolved for another 40 years. In 1934, it was revealed that fibrinolysis could be initiated by an entity released from hemolytic streptococci 2.Garner R.L. Tillett W.S. Biochemical studies on the fibrinolytic activity of hemolytic streptococci: I. isolation and characterization of fibrinolysin.J Exp Med. 1934; 60: 239-54Crossref PubMed Google Scholar. The authors referred to this entity as ‘fibrinolysin’ (renamed later as ‘streptokinase’), but the mechanism by which fibrinolysin destroyed clots also remained a mystery until the mid‐1940s, when it was realized that the streptococci‐derived agent required a plasma component 3.Christensen L.R. Streptococcal fibrinolysis: a proteolytic reaction due to a serum enzyme activated by streptococcal fibrinolysin.J Gen Physiol. 1945; 28: 363-83Crossref PubMed Google Scholar. This plasma‐derived cofactor was, in fact, a zymogen that needed to be processed (‘activated’) by fibrinolysin to generate the fibrinolytic activity. Although it was initially referred to as ‘pro‐fibrinolysin’, this name was changed to the more familiar term ‘plasminogen’ (a morphing of ‘plasma’ and ‘zymogen’) in 1946 (reviewed in 4.Macfarlane R.G. Biggs R. Fibrinolysis; its mechanism and significance.Blood. 1948; 3: 1167-87Crossref PubMed Google Scholar). Shortly after this period, the endogenous ‘plasminogen‐activating’ agents, i.e. urokinase and tissue‐type plasminogen activator (t‐PA), were finally discovered 5.Sobel G.W. Mohler S.R. Jones N.W. Dowdy A.B.C. Guest M.M. Urokinase: an activator of plasma profibrinolysin extracted from urine.Am J Physiol. 1952; 171: 768-9Google Scholar, 6.Astrup T. Permin P.M. Fibrinolysis in the animal organism.Nature. 1947; 159: 681-2Crossref PubMed Google Scholar. The identification of the plasminogen activators heralded the beginning of the era of therapeutic thrombolysis. Initial reports indicated clinical benefit when these agents were used, mostly in relation to myocardial infarction (MI). However, there were reports of bleeding complications (some devastating), particularly with respect to the occurrence of intracerebral hemorrhage (ICH). During the 1980s, when t‐PA became the most prevalent thrombolytic agent for MI, ICH was still occurring at a frequency of ~ 1% 7.The TIMI Study Group. Comparison of invasive and conservative strategies after treatment with intravenous tissue plasminogen activator in acute myocardial infarction. Results of the thrombolysis in myocardial infarction (TIMI) phase II trial.N Engl J Med. 1989; 320: 618-27Crossref PubMed Google Scholar. Any thought that thrombolysis could ever be used in patients with ischemic stroke was halted in its tracks because of the overwhelming fear of ICH. After all, if ICH was occurring in patients without any sign of cerebral infarction (i.e. in those with MI, pulmonary embolism, or deep vein thrombosis), how much worse would this become if a cerebral infarct was present? Indeed, in the Oxford Textbook of Medicine, 1983 8.Warlow C.P. Cerebrovascular disease.in: Weatherall DJ Ledingham JGG Warrell DA Oxford Textbook of Medicine. Oxford University Press, London1983: 72Google Scholar, it was stated:‘There is probably little that medical treatment and nothing that vascular surgery can do to alter the immediate prognosis of stroke........Both fibrinolytic drugs and anti‐coagulation increase the risk of intracranial bleeding and should not normally be used.’ Much has changed since the 1980s, and t‐PA‐mediated thrombolysis has finally edged its way into the treatment of patients with ischemic stroke 9.The National Institute of Neurological Disorders and Stroke rt‐PA Stroke Study Group (NINDS). Tissue plasminogen activator for acute ischemic stroke.N Engl J Med. 1995; 333: 1581-7Crossref PubMed Scopus (0) Google Scholar. The risk of ICH is still there, but the risk–benefit ratio is in favor of t‐PA administration. Around the same time that t‐PA was approved for the treatment of ischemic stroke, scientists made the largely serendipitous discovery that t‐PA, and to some extent plasminogen, had unsuspected roles in the brain (reviewed in 10.Samson A.L. Medcalf R.L. Tissue‐type plasminogen activator: a multifaceted modulator of neurotransmission and synaptic plasticity.Neuron. 2006; 50: 673-8Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). Moreover, t‐PA can also increase the permeability of the blood–brain barrier (BBB) 11.Su E.J. Fredriksson L. Geyer M. Folestad E. Cale J. Andrae J. Gao Y. Pietras K. Mann K. Yepes M. Strickland D.K. Betsholtz C. Eriksson U. Lawrence D.A. Activation of PDGF‐CC by tissue plasminogen activator impairs blood–brain barrier integrity during ischemic stroke.Nat Med. 2008; 14: 731-7Crossref PubMed Scopus (0) Google Scholar, 12.Yepes M. Sandkvist M. Moore E.G. Bugge T.H. Strickland D.K. Lawrence D.A. Tissue‐type plasminogen activator induces opening of the blood–brain barrier via the LDL receptor‐related protein.J Clin Invest. 2003; 112: 1533-40Crossref PubMed Scopus (0) Google Scholar, 13.Niego B. Freeman R. Puschmann T.B. Turnley A.M. Medcalf R.L. t‐PA‐specific modulation of a human blood–brain barrier model involves plasmin‐mediated activation of the Rho kinase pathway in astrocytes.Blood. 2012; 119: 4752-61Crossref PubMed Scopus (0) Google Scholar, and this may underlie some of its broader effects in the brain. This effect on the BBB may also provide a plausible explanation of how t‐PA was promoting ICH, even in non‐ischemic stroke scenarios. Mechanistically, t‐PA has a number of options available to increase BBB permeability; t‐PA can bypass plasminogen activation entirely, and instead cleave another key substrate (platelet‐derived growth factor [PDGF]‐CC) 14.Fredriksson L. Li H. Fieber C. Li X. Eriksson U. Tissue plasminogen activator is a potent activator of PDGF‐CC.EMBO J. 2004; 23: 3793-802Crossref PubMed Scopus (0) Google Scholar that, in turn, increases BBB permeability via activation of the tyrosine kinase PDGFR1α receptor and downstream signaling 11.Su E.J. Fredriksson L. Geyer M. Folestad E. Cale J. Andrae J. Gao Y. Pietras K. Mann K. Yepes M. Strickland D.K. Betsholtz C. Eriksson U. Lawrence D.A. Activation of PDGF‐CC by tissue plasminogen activator impairs blood–brain barrier integrity during ischemic stroke.Nat Med. 2008; 14: 731-7Crossref PubMed Scopus (0) Google Scholar. Blockade of PDGF‐CC signaling by the tyrosine kinase inhibitor imatinib (Gleevec) attenuated the capacity of t‐PA to increase BBB permeability in mouse models of ischemic stroke 11.Su E.J. Fredriksson L. Geyer M. Folestad E. Cale J. Andrae J. Gao Y. Pietras K. Mann K. Yepes M. Strickland D.K. Betsholtz C. Eriksson U. Lawrence D.A. Activation of PDGF‐CC by tissue plasminogen activator impairs blood–brain barrier integrity during ischemic stroke.Nat Med. 2008; 14: 731-7Crossref PubMed Scopus (0) Google Scholar and in models of traumatic brain injury (TBI) 15.Su E.J. Fredriksson L. Kanzawa M. Moore S. Folestad E. Stevenson T.K. Nilsson I. Sashindranath M. Schielke G.P. Warnock M. Ragsdale M. Mann K. Lawrence A.L. Medcalf R.L. Eriksson U. Murphy G.G. Lawrence D.A. Imatinib treatment reduces brain injury in a murine model of traumatic brain injury.Front Cell Neurosci. 2015; 9: 385Crossref Scopus (32) Google Scholar. On the other hand, formation of t‐PA–plasminogen activator inhibitor‐1 complexes was also shown to increase BBB opening in models of TBI, via LDL receptor signaling 16.Sashindranath M. Sales E. Daglas M. Freeman R. Samson A.L. Cops E.J. Beckham S. Galle A. McLean C. Morganti‐Kossmann C. Rosenfeld J.V. Madani R. Vassalli J.D. Su E.J. Lawrence D.A. Medcalf R.L. The tissue‐type plasminogen activator–plasminogen activator inhibitor 1 complex promotes neurovascular injury in brain trauma: evidence from mice and humans.Brain. 2012; 135: 3251-64Crossref PubMed Scopus (0) Google Scholar, whereas a requirement for plasminogen has been shown to be important in this process in some models 17.Suzuki Y. Nagai N. Umemura K. Collen D. Lijnen H.R. Stromelysin‐1 (MMP‐3) is critical for intracranial bleeding after t‐PA treatment of stroke in mice.J Thromb Haemost. 2007; 5: 1732-9Crossref PubMed Scopus (0) Google Scholar, but not in others 12.Yepes M. Sandkvist M. Moore E.G. Bugge T.H. Strickland D.K. Lawrence D.A. Tissue‐type plasminogen activator induces opening of the blood–brain barrier via the LDL receptor‐related protein.J Clin Invest. 2003; 112: 1533-40Crossref PubMed Scopus (0) Google Scholar. Considering the broader role of the fibrinolytic system, the role of plasma versus brain‐derived plasminogen in ischemic stroke outcome may not be so obvious. Also, what would be the expected outcome if levels of plasma plasminogen were dramatically altered in a mouse model of ischemic stroke? In the study by Singh et al. published in this issue of JTH 18.Singh S. Houng A.K. Wang D. Reed G.L. Physiologic variations in blood plasminogen levels affect outcomes after acute cerebral thromboembolism in mice: a pathophysiologic role for microvascular thrombosis.J Thromb Haemost. 2016; 14: 1822-32Abstract Full Text Full Text PDF Scopus (0) Google Scholar, a series of elegant experiments were undertaken to evaluate changes in endogenous plasminogen levels in a mouse thromboembolic model of ischemic stroke. The degree of cerebral infarction, BBB breakdown and matrix metalloproteinase (MMP) expression/activation were evaluated in plg−/− mice, plg−/+ mice (expressing 50% plasminogen), and plg+/+ mice. In some experiments, plg−/− and plg+/+ mice were supplemented intravenously with additional plasminogen, restoring normal plasma (not brain) plasminogen levels to plg−/− mice, and also producing supraphysiologic levels (approximately two‐fold greater) to the plasma compartment in plg+/+ mice. Plasminogen levels vary widely in human plasma (range up to eight‐fold 19.Tait R.C. Walker I.D. Conkie J.A. Islam S.I. McCall F. Mitchell R. Davidson J.F. Plasminogen levels in healthy volunteers – influence of age, sex, smoking and oral contraceptives.Thromb Haemost. 1992; 68: 506-10Crossref PubMed Scopus (0) Google Scholar), hence, the question of the effect of plasma plasminogen levels on stroke outcome is highly relevant. Singh et al. indicated that the plasma level of plasminogen correlated negatively with infarct size; indeed, infarct volumes in plg−/− mice were 100% greater than in plg+/+ mice. Consistent with a need for plasma‐derived plasminogen, supplementation of plasma plasminogen to plg−/− mice reduced the degree of cerebral infarction to a similar degree as seen in plg+/+ mice. Hence, it seemed that the beneficial effect of plasminogen was simply a reflection of a greater capacity to generate plasmin in plasma and remove the offending macrovascular thrombus introduced into the middle cerebral artery; indeed, this was confirmed by clot lysis assays with plasma from plg−/−, plg+/− and plg+/+ mice, and with plasma from plg+/+ mice with supraphysiologic levels of plasminogen, the latter showing the highest lytic activity. Cerebral ischemia is also known to be associated with the formation of microvascular thrombi. Singh et al. evaluated fibrin deposition in the brains of mice, and confirmed that microvascular thrombosis was indeed occurring following cerebral ischemia; this was most prominent in plg−/− mice, but was less prominent in plg−/+ mice, and was reduced further in plg+/+ mice. Hence, there was a significant inverse relationship between plasminogen levels and fibrin deposition following cerebral ischemia. To explore the influence of microvascular thrombosis on outcome, mice with varying levels of plasminogen were treated with the thrombin inhibitor argatroban to prevent microvascular thrombosis from occurring following ischemic stroke. Argatroban had no effect on the ex vivo‐generated macrovascular thrombus; however, argatroban not only reduced the extent of microvascular thrombosis in plg−/− mice to the levels seen in plg+/+ mice, but also decreased the extent of infarction. Hence, plasminogen was having a protective effect in the ischemic brain not only by improving the clearance of macrovascular thrombi and restoring reperfusion, but also by reducing the formation of microvascular thrombi. Curiously, Singh et al. found that there was also a dose‐dependent effect of plasminogen on the extent of BBB breakdown, with plg−/− mice having marked localized increases in cerebral vessel permeability. The extent of BBB permeability further decreased in plg+/− mice, and was even lower in plg+/+ mice. Also, reconstitution of plasminogen in plg−/− mice reduced the degree of BBB opening following cerebral ischemia. So how does a reduction in plasminogen levels translate into an increase in BBB breakdown? This might appear to be counterintuitive, as increases in t‐PA levels are known to be associated with increases in BBB permeability. If at least part of this effect of t‐PA was driven by plasmin, then the opposite result would have been expected in the plg−/− mice. Singh et al. also evaluated changes in the levels of MMP‐3 and MMP‐9. MMP‐3 and MMP‐9 levels were significantly increased in the brains of plg−/− mice. MMP‐9 expression was mostly localized to cerebral microvessels following ischemic stroke. Whereas MMP‐9 itself has been associated with the promotion of BBB permeability 20.Turner R.J. Sharp F.R. Implications of MMP9 for blood brain barrier disruption and hemorrhagic transformation following ischemic stroke.Front Cell Neurosci. 2016; 10: 56Crossref PubMed Scopus (247) Google Scholar, the majority of the MMP‐9 detected in the brain was in its pro‐form, and therefore not active. It remains to be determined how plasminogen deficiency increases BBB permeability in this model of ischemic stroke. Could this simply be a consequence of ischemia and microvascular thrombosis causing generalized degeneration of BBB integrity? Or is it possible that, in the absence of plasminogen, endogenous t‐PA could have better access to other substrates linked to BBB disruption (e.g. PDGF‐CC) that actively open the BBB? However, as this study did not evaluate the effect of t‐PA‐induced thrombolysis in the thromboembolic stroke model, it remains to be determined whether changes in plasma plasminogen levels improve stroke outcome following thrombolysis. This is important, as t‐PA administration to patients with ischemic stroke carries an even greater risk of ICH. Although enhanced thrombolysis would be predicted, further increases in plasmin levels caused by exogenous t‐PA may also have unexpected consequences, e.g. on MMP activation and on BBB permeability. Nonetheless, what is most interesting here is that changes in plasminogen levels within the physiologic range impact on stroke outcome, at least in mice. This now begs for a clinical study to determine whether this is reflected in humans; could it be that individuals with lower plasminogen levels are at a greater risk of developing ischemic stroke? The finding that increases in plasma plasminogen levels within the physiologic range are of benefit in a thrombotic model of ischemic stroke is a very interesting observation that should prompt further research in this area. The author states that he has no conflicts of interest.