Cytoprotective-selective activated protein C therapy for ischaemic stroke

Summary

Despite years of research and efforts to translate stroke research to clinical therapy, ischaemic stroke remains a major cause of death, disability, and diminished quality of life. Primary and secondary preventive measures combined with improved quality of care have made significant progress. However, no novel drug for ischaemic stroke therapy has been approved in the past decade. Numerous studies have shown beneficial effects of activated protein C (APC) in rodent stroke models. In addition to its natural anticoagulant functions, APC conveys multiple direct cytoprotective effects on many different cell types that involve multiple receptors including protease activated receptor (PAR) 1, PAR3, and the endothelial protein C receptor (EPCR). Application of molecular engineered APC variants with altered selectivity profiles to rodent stroke models demonstrated that the beneficial effects of APC primarily require its cytoprotective activities but not its anticoagulant activities. Extensive basic, preclinical, and clinical research provided a compelling rationale based on strong evidence for translation of APC therapy that has led to the clinical development of the cytoprotectiveselective APC variant, 3K3A-APC, for ischaemic stroke. Recent identification of non-canonical PAR1 and PAR3 activation by APC that give rise to novel tethered-ligands capable of inducing biased cytoprotective signalling as opposed to the canonical signalling provides a mechanistic explanation for how APC-mediated PAR activation can selectively induce cytoprotective signalling pathways. Collectively, these paradigm-shifting discoveries provide detailed insights into the receptor targets and the molecular mechanisms for neuroprotection by cytoprotective-selective 3K3A-APC, which is currently a biologic drug in clinical trials for ischaemic stroke.

Keywords

Stroke - activated protein C - PAR1 - PAR3 - EPCR

• References

• 1 Lozano R, Naghavi M, Foreman K. et al. Global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: a systematic analysis for the Global Burden of Disease Study 2010. Lancet 2012; 380: 2095-2128.
• 2 Go AS, Mozaffarian D, Roger VL. et al. Heart disease and stroke statistics--2014 update: a report from the american heart association. Circulation 2014; 129: e28-e292.
• 3 Murphy SL, Xu J, Kochanek KD. Deaths: final data for 2010. National Vital Statistics Reports 2013; 61: 1-118.
• 4 Thrift AG, Cadilhac DA, Thayabaranathan T. et al. Global stroke statistics. Int J Stroke 2014; 9: 6-18.
• 5 Luengo-Fernandez R, Gray AM, Bull L. et al. Quality of life after TIA and stroke: ten-year results of the Oxford Vascular Study. Neurology 2013; 81: 1588-1595.
• 6 Grossman AW, Broderick JP. Advances and challenges in treatment and prevention of ischaemic stroke. Ann Neurol 2013; 74: 363-372.
• 7 Bivard A, Lin L, Parsonsb MW. Review of Stroke Thrombolytics. J Stroke 2013; 15: 90-98.
• 8 Lees KR, Bluhmki E, von KR. et al. Time to treatment with intravenous alteplase and outcome in stroke: an updated pooled analysis of ECASS, ATLANTIS, NINDS, and EPITHET trials. Lancet 2010; 375: 1695-1703.
• 9 The NINDS t-PA Stroke Study Group. Intracerebral haemorrhage after intravenous t-PA therapy for ischaemic stroke. Stroke 1997; 28: 2109-2118.
• 10 Lackland DT, Roccella EJ, Deutsch AF. et al. Factors influencing the decline in stroke mortality: a statement from the American Heart Association/American Stroke Association. Stroke 2014; 45: 315-353.
• 11 Mazighi M, Chaudhry SA, Ribo M. et al. Impact of onset-to-reperfusion time on stroke mortality: a collaborative pooled analysis. Circulation 2013; 127: 1980-1985.
• 12 Manning NW, Campbell BC, Oxley TJ. et al. Acute ischaemic stroke: time, penumbra, and reperfusion. Stroke 2014; 45: 640-644.
• 13 Tymianski M. Novel approaches to neuroprotection trials in acute ischaemic stroke. Stroke 2013; 44: 2942-2950.
• 14 Mosnier LO, Zlokovic BV, Griffin JH. The cytoprotective protein C pathway. Blood 2007; 109: 3161-3172.
• 15 Castellino FJ, Ploplis VA. The protein C pathway and pathologic processes. J Thromb Haemost 2009; 7 (Suppl. 01) 140-145.
• 16 Esmon CT. Inflammation and the activated protein C anticoagulant pathway. Semin Thromb Hemost 2006; 32: 49-60.
• 17 Bouwens EA, Stavenuiter F, Mosnier LO. Mechanisms of anticoagulant and cytoprotective actions of the protein C pathway. J Thromb Haemost 2013; 11: 242-253.
• 18 Rezaie AR. The occupancy of endothelial protein C receptor by its ligand modulates the PAR-1 dependent signalling specificity of coagulation proteases. IUBMB Life 2011; 63: 390-396.
• 19 Russo A, Soh UJ, Trejo J. Proteases Display Biased Agonism at Protease-Activated Receptors: Location matters!. Mol Interv 2009; 9: 87-96.
• 20 Weiler H. Multiple receptor-mediated functions of activated protein C. Hamostaseologie 2011; 31: 185-195.
• 21 Knoebl PN. Severe congenital protein C deficiency: the use of protein C concentrates (human) as replacement therapy for life-threatening blood-clotting complications. Biologics 2008; 2: 285-296.
• 22 Jalbert LR, Rosen ED, Moons L. et al. Inactivation of the gene for anticoagulant protein C causes lethal perinatal consumptive coagulopathy in mice. J Clin Invest 1998; 102: 1481-1488.
• 23 Ishiguro K, Kojima T, Kadomatsu K. et al. Complete antithrombin deficiency in mice results in embryonic lethality. J Clin Invest 2000; 106: 873-878.
• 24 Cui J, Eitzman DT, Westrick RJ. et al. Spontaneous thrombosis in mice carrying the factor V Leiden mutation. Blood 2000; 96: 4222-4226.
• 25 Macko RF, Killewich LA, Fernández JA. et al. Brain-specific protein C activation during carotid artery occlusion in humans. Stroke 1999; 30: 542-545.
• 26 Macko RF, Ameriso SF, Gruber A. et al. Impairments of the protein C system and fibrinolysis in infection-associated stroke. Stroke 1996; 27: 2005-2011.
• 27 Wang L, Tran ND, Kittaka M. et al. Thrombomodulin expression in bovine brain capillaries. Anticoagulant function of the blood-brain barrier, regional differences, and regulatory mechanisms. Arterioscler Thromb Vasc Biol 1997; 17: 3139-3146.
• 28 Malek AM, Jackman RW, Rosenberg RD. et al. Endothelial expression of thrombomodulin is reversibly regulated by fluid shear stress. Circ Res 1994; 74: 852-860.
• 29 Folsom AR, Ohira T, Yamagishi K. et al. Low protein C and incidence of ischaemic stroke and coronary heart disease: the Atherosclerosis Risk in Communities (ARIC) Study. J Thromb Haemost 2009; 7: 1774-1778.
• 30 Zlokovic BV, Griffin JH. Cytoprotective protein C pathways and implications for stroke and neurological disorders. Trends Neurosci 2011; 34: 198-209.
• 31 Danese S, Vetrano S, Zhang L. et al. The protein C pathway in tissue inflammation and injury: pathogenic role and therapeutic implications. Blood 2010; 115: 1121-1130.
• 32 Bock F, Shahzad K, Vergnolle N. et al. Activated protein C based therapeutic strategies in chronic diseases. Thromb Haemost 2014; 111: 610-617.
• 33 Shibata M, Kumar SR, Amar A. et al. Anti-inflammatory, antithrombotic, and neuroprotective effects of activated protein C in a murine model of focal ischaemic stroke. Circulation 2001; 103: 1799-1805.
• 34 Cheng T, Liu D, Griffin JH. et al. Activated protein C blocks p53-mediated apoptosis in ischaemic human brain endothelium and is neuroprotective. Nat Med 2003; 9: 338-342.
• 35 Griffin JH, Zlokovic BV, Fernández JA. Activated protein C: potential therapy for severe sepsis, thrombosis, and stroke. Semin Hematol 2002; 39: 197-205.
• 36 Guo H, Liu D, Gelbard H. et al. Activated protein C prevents neuronal apoptosis via protease activated receptors 1 and 3. Neuron 2004; 41: 563-572.
• 37 Liu D, Cheng T, Guo H. et al. Tissue plasminogen activator neurovascular toxicity is controlled by activated protein C. Nat Med 2004; 10: 1379-1383.
• 38 Cheng T, Petraglia AL, Li Z. et al. Activated protein C inhibits tissue plasminogen activator-induced brain haemorrhage. Nat Med 2006; 12: 1278-1285.
• 39 Zlokovic BV, Zhang C, Liu D. et al. Functional recovery after embolic stroke in rodents by activated protein C. Ann Neurol 2005; 58: 474-477.
• 40 Wildhagen KC, Lutgens E, Loubele ST. et al. The structure-function relationship of activated protein C. Lessons from natural and engineered mutations. Thromb Haemost 2011; 106: 1034-1045.
• 41 Rezaie AR. Regulation of the protein C anticoagulant and antiinflammatory pathways. Curr Med Chem 2010; 17: 2059-2069.
• 42 Bae JS, Yang L, Manithody C. et al. Engineering a disulfide bond to stabilize the calcium binding loop of activated protein C eliminates its anticoagulant but not protective signalling properties. J Biol Chem 2007; 282: 9251-9259.
• 43 Han MH, Hwang SI, Roy DB. et al. Proteomic analysis of active multiple sclerosis lesions reveals therapeutic targets. Nature 2008; 451: 1076-1081.
• 44 Mosnier LO, Gale AJ, Yegneswaran S. et al. Activated protein C variants with normal cytoprotective but reduced anticoagulant activity. Blood 2004; 104: 1740-1745.
• 45 Mosnier LO, Yang XV, Griffin JH. Activated protein C mutant with minimal anticoagulant activity, normal cytoprotective activity, and preservation of thrombin activable fibrinolysis inhibitor-dependent cytoprotective functions. J Biol Chem 2007; 282: 33022-33033.
• 46 Gupta A, Gerlitz B, Richardson MA. et al. Distinct Functions of Activated Protein C Differentially Attenuate Acute Kidney Injury. J Am Soc Nephrol 2009; 20: 267-277.
• 47 Harmon S, Preston RJ, Ainle FN. et al. Dissociation of activated protein C functions by elimination of protein S cofactor enhancement. J Biol Chem 2008; 283: 30531-30539.
• 48 Ni Ainle F, O’Donnell JS, Johnson JA. et al. Activated protein C N-linked glycans modulate cytoprotective signalling function on endothelial cells. J Biol Chem 2011; 286: 1323-1330.
• 49 Preston RJ, Ajzner E, Razzari C. et al. Multifunctional specificity of the protein C/activated protein C GLA domain. J Biol Chem 2006; 281: 28850-28857.
• 50 Yang L, Bae JS, Manithody C. et al. Identification of a specific exosite on activated protein C for interaction with protease activated receptor 1. J Biol Chem 2007; 282: 25493-25500.
• 51 Mosnier LO, Zampolli A, Kerschen EJ. et al. Hyper-antithrombotic, non-cyto-protective Glu149Ala-activated protein C mutant. Blood 2009; 113: 5970-5978.
• 52 Guo H, Wang Y, Singh I. et al. Species-dependent neuroprotection by activated protein C mutants with reduced anticoagulant activity. J Neurochem 2009; 109: 116-124.
• 53 Kerschen EJ, Fernandez JA, Cooley BC. et al. Endotoxemia and sepsis mortality reduction by non-anticoagulant activated protein C. J Exp Med 2007; 204: 2439-2448.
• 54 Wang Y, Sinha RK, Mosnier LO. et al. Neurotoxicity of the anticoagulant-selective E149A-activated protein C variant after focal ischaemic stroke in mice. Blood Cells Mol Dis 2013; 51: 104-108.
• 55 Walker CT, Marky AH, Petraglia AL. et al. Activated protein C analog with reduced anti-coagulant activity improves functional recovery and reduces bleeding risk following controlled cortical impact. Brain Res 2010; 1347: 125-131.
• 56 Guo H, Singh I, Wang Y. et al. Neuroprotective activities of activated protein C mutant with reduced anticoagulant activity. Eur J Neurosci 2009; 29: 1119-1130.
• 57 Wang Y, Thiyagarajan M, Chow N. et al. Differential neuroprotection and risk for bleeding from activated protein C with varying degrees of anticoagulant activity. Stroke 2008; 40: 1864-1869.
• 58 Wang Y, Zhao Z, Chow N. et al. Activated protein C analog protects from ischaemic stroke and extends the therapeutic window of tissue-type plasminogen activator in aged female mice and hypertensive rats. Stroke 2013; 44: 3529-3536.
• 59 Wang Y, Zhang Z, Chow N. et al. An activated protein C analog with reduced anticoagulant activity extends the therapeutic window of tissue plasminogen activator for ischaemic stroke in rodents. Stroke 2012; 43: 2444-2449.
• 60 Bir N, Lafargue M, Howard M. et al. Cytoprotective-selective activated protein C attenuates Pseudomonas aeruginosa-induced lung injury in mice. Am J Respir Cell Mol Biol 2011; 45: 632-641.
• 61 Chen B, Friedman B, Whitney MA. et al. Thrombin activity associated with neuronal damage during acute focal ischaemia. J Neurosci 2012; 32: 7622-7631.
• 62 Chen B, Cheng Q, Yang K. et al. Thrombin mediates severe neurovascular injury during ischaemia. Stroke 2010; 41: 2348-2352.
• 63 Coughlin SR. Protease-activated receptors in hemostasis, thrombosis and vascular biology. J Thromb Haemost 2005; 3: 1800-1814.
• 64 Mosnier LO, Sinha RK, Burnier L. et al. Biased agonism of protease-activated receptor 1 by activated protein C caused by non-canonical cleavage at Arg46. Blood 2012; 120: 5237-5246.
• 65 Soh UJ, Trejo J. Activated protein C promotes protease-activated receptor-1 cytoprotective signalling through beta-arrestin and dishevelled-2 scaffolds. Proc Natl Acad Sci USA 2011; 108: E1372-E1380.
• 66 Canto I, Soh UJ, Canto I. et al. Allosteric modulation of protease-activated receptor signalling. Mini Rev Med Chem 2012; 12: 804-811.
• 67 Reiter E, Ahn S, Shukla AK. et al. Molecular mechanism ofαααααααααααααααα agonism at seven-transmembrane receptors. Annu Rev Pharmacol Toxicol 2012; 52: 179-197.
• 68 Wisler JW, Xiao K, Thomsen AR. et al. Recent developments in biased agonism. Curr Opin Cell Biol 2014; 27C: 18-24.
• 69 McLaughlin JN, Patterson MM, Malik AB. Protease-activated receptor-3 (PAR3) regulates PAR1 signalling by receptor dimerisation. Proc Natl Acad Sci USA 2007; 104: 5662-5667.
• 70 Burnier L, Mosnier LO. Novel mechanisms for activated protein C cytoprotective activities involving non-canonical activation of protease-activated receptor 3. Blood 2013; 122: 807-816.
• 71 Madhusudhan T, Wang H, Straub BK. et al. Cytoprotective signalling by activated protein C requires protease activated receptor-3 in podocytes. Blood 2012; 119: 874-883.
• 72 Bae JS, Rezaie AR. Protease activated receptor 1 (PAR-1) activation by thrombin is protective in human pulmonary artery endothelial cells if endothelial protein C receptor is occupied by its natural ligand. Thromb Haemost 2008; 100: 101-109.
• 73 Deane R, LaRue B, Sagare AP. et al. Endothelial protein C receptor-assisted transport of activated protein C across the mouse blood-brain barrier. J Cereb Blood Flow Metab 2009; 29: 25-33.
• 74 Zhong Z, Ilieva H, Hallagan L. et al. Activated protein C therapy slows ALS-like disease in mice by transcriptionally inhibiting SOD1 in motor neurons and microglia cells. J Clin Invest 2009; 119: 3437-3449.
• 75 Abu FR, Nassar T, Yarovoi S. et al. Blood-brain barrier permeability and tPA-mediated neurotoxicity. Neuropharmacology 2010; 58: 972-980.
• 76 Jin R, Yang G, Li G. Molecular insights and therapeutic targets for blood-brain barrier disruption in ischaemic stroke: critical role of matrix metalloproteinases and tissue-type plasminogen activator. Neurobiol Dis 2010; 38: 376-385.
• 77 Gorbacheva LR, Storozhevykh TP, Pinelis VG. et al. Activated protein C via PAR1 receptor regulates survival of neurons under conditions of glutamate excitotoxicity. Biochemistry 2008; 73: 717-724.
• 78 Gorbacheva L, Pinelis V, Ishiwata S. et al. Activated protein C prevents gluta-mate- and thrombin-induced activation of nuclear factor-kappaB in cultured hippocampal neurons. Neuroscience 2010; 165: 1138-1146.
• 79 Thiyagarajan M, Fernandez JA, Lane SM. et al. Activated protein C promotes neovascularisation and neurogenesis in postischaemic brain via protease-activated receptor 1. J Neurosci 2008; 28: 12788-12797.
• 80 Wang Y, Zhao Z, Chow N. et al. Activated protein C analog promotes neurogenesis and improves neurological outcome after focal ischaemic stroke in mice via protease activated receptor 1. Brain Res 2013; 1507: 97-104.
• 81 Guo H, Zhao Z, Yang Q. et al. An activated protein C analog stimulates neuronal production by human neural progenitor cells via a PAR1-PAR3-S1PR1-Akt pathway. J Neurosci 2013; 33: 6181-6190.
• 82 Maggio N, Itsekson Z, Ikenberg B. et al. The anticoagulant activated protein C (aPC) promotes metaplasticity in the hippocampus through an EPCRPAR1-S1P1 receptors dependent mechanism. Hippocampus. 2014 Epub ahead of print.
• 83 O’Collins VE, Macleod MR, Donnan GA. et al. 1, 026 experimental treatments in acute stroke. Ann Neurol 2006; 59: 467-477.
• 84 Bernard GR, Vincent JL, Laterre PF. et al. Efficacy and safety of recombinant human activated protein C for severe sepsis. N Engl J Med 2001; 344: 699-709.
• 85 Ranieri VM, Thompson BT, Barie PS. et al. Drotrecogin Alfa (Activated) in Adults with Septic Shock. N Engl J Med 2012; 366: 2055-2064.
• 86 Bernard GR, Macias WL, Joyce DE. et al. Safety assessment of drotrecogin alfa (activated) in the treatment of adult patients with severe sepsis. Crit Care 2003; 7: 155-163.
• 87 Bruckner M, Lasarzik I, Jahn-Eimermacher A. et al. High dose infusion of activated protein C (rhAPC) fails to improve neuronal damage and cognitive deficit after global cerebral ischaemia in rats. Neurosci Lett 2013; 551: 28-33.
• 88 Williams PD, Zlokovic BV, Griffin JH. et al. Preclinical safety and pharmacokinetic profile of 3K3A-APC, a novel, modified activated protein C for ischaemic stroke. Curr Pharm Des 2012; 18: 4215-4222.
• 89 Lyden P, Levy H, Weymer S. et al. Phase 1 Safety, Tolerability and Pharmacokinetics of 3K3A-APC in Healthy Adult Volunteers. Curr Pharm Des 2013; 19: 7479-7485.
• 90 Mather T, Oganessyan V, Hof P. et al. The 2.8 Å crystal structure of Gla-domainless activated protein C. EMBO J 1996; 15: 6822-6831.