A recombinant signalling-selective activated protein C that lacks anticoagulant activity is efficacious and safe in cutaneous wound preclinical models
Corresponding Author
Ruilong Zhao MBBS, PhD
Sutton Laboratory, Kolling Institute of Medical Research, Sydney, New South Wales, Australia
Correspondence
Ruilong Zhao, Sutton Laboratory, Kolling Institute, The University of Sydney, 10 Westbourne Street, St Leonards, Sydney, NSW 2065, Australia.
Email: [email protected]
Search for more papers by this authorMeilang Xue PhD
Sutton Laboratory, Kolling Institute of Medical Research, Sydney, New South Wales, Australia
Search for more papers by this authorHaiyan Lin MPhil
Sutton Laboratory, Kolling Institute of Medical Research, Sydney, New South Wales, Australia
Search for more papers by this authorMargaret Smith PhD
Raymond Purves Laboratory, Kolling Institute of Medical Research, Sydney, New South Wales, Australia
Search for more papers by this authorHelena Liang PhD
Sutton Laboratory, Kolling Institute of Medical Research, Sydney, New South Wales, Australia
Search for more papers by this authorHartmut Weiler PhD
Department of Physiology, Blood Research Institute, Milwaukee, Wisconsin, USA
Search for more papers by this authorJohn H. Griffin PhD
Department of Molecular Medicine, The Scripps Research Institute, San Diego, California, USA
Search for more papers by this authorChristopher J. Jackson PhD
Sutton Laboratory, Kolling Institute of Medical Research, Sydney, New South Wales, Australia
Search for more papers by this authorCorresponding Author
Ruilong Zhao MBBS, PhD
Sutton Laboratory, Kolling Institute of Medical Research, Sydney, New South Wales, Australia
Correspondence
Ruilong Zhao, Sutton Laboratory, Kolling Institute, The University of Sydney, 10 Westbourne Street, St Leonards, Sydney, NSW 2065, Australia.
Email: [email protected]
Search for more papers by this authorMeilang Xue PhD
Sutton Laboratory, Kolling Institute of Medical Research, Sydney, New South Wales, Australia
Search for more papers by this authorHaiyan Lin MPhil
Sutton Laboratory, Kolling Institute of Medical Research, Sydney, New South Wales, Australia
Search for more papers by this authorMargaret Smith PhD
Raymond Purves Laboratory, Kolling Institute of Medical Research, Sydney, New South Wales, Australia
Search for more papers by this authorHelena Liang PhD
Sutton Laboratory, Kolling Institute of Medical Research, Sydney, New South Wales, Australia
Search for more papers by this authorHartmut Weiler PhD
Department of Physiology, Blood Research Institute, Milwaukee, Wisconsin, USA
Search for more papers by this authorJohn H. Griffin PhD
Department of Molecular Medicine, The Scripps Research Institute, San Diego, California, USA
Search for more papers by this authorChristopher J. Jackson PhD
Sutton Laboratory, Kolling Institute of Medical Research, Sydney, New South Wales, Australia
Search for more papers by this authorAbstract
Various preclinical and clinical studies have demonstrated the robust wound healing capacity of the natural anticoagulant activated protein C (APC). A bioengineered APC variant designated 3K3A-APC retains APC's cytoprotective cell signalling actions with <10% anticoagulant activity. This study was aimed to provide preclinical evidence that 3K3A-APC is efficacious and safe as a wound healing agent. 3K3A-APC, like wild-type APC, demonstrated positive effects on proliferation of human skin cells (keratinocytes, endothelial cells and fibroblasts). Similarly it also increased matrix metollaproteinase-2 activation in keratinocytes and fibroblasts. Topical 3K3A-APC treatment at 10 or 30 μg both accelerated mouse wound healing when culled on Day 11. And at 10 μg, it was superior to APC and had half the dermal wound gape compared to control. Further testing was conducted in excisional porcine wounds due to their congruence to human skin. Here, 3K3A-APC advanced macroscopic healing in a dose-dependent manner (100, 250 and 500 μg) when culled on Day 21. This was histologically corroborated by greater collagen maturity, suggesting more advanced remodelling. A non-interference arm of this study found no evidence that topical 3K3A-APC caused either any significant systemic side-effects or any significant leakage into the circulation. However the female pigs exhibited transient and mild local reactions after treatments in week three, which did not impact healing. Overall these preclinical studies support the hypothesis that 3K3A-APC merits future human wound studies.
CONFLICT OF INTEREST STATEMENT
Part of this work was funded by ZZ-Biotech, a company currently seeking FDA approval to conduct a human clinical trial in wound healing. CJJ, JHG and MX are shareholders in ZZ-Biotech. CJJ and JHG are advisors for ZZ-Biotech.
Open Research
DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.
Supporting Information
| Filename | Description |
|---|---|
| wrr13148-sup-0001-Supinfo01.pptxPowerPoint 2007 presentation , 38.8 KB | Supplementary 1. Semi-quantitative histological scores of mouse wounds. |
| wrr13148-sup-0002-Supinfo02.pptxPowerPoint 2007 presentation , 35.1 KB | Supplementary 2. Semi-quantitative immunohistochemical scores of pig wounds. |
Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.
REFERENCES
- 1Mammen EF, Thomas WR, Seegers WH. Activation of purified prothrombin to autoprothrombin I or autoprothrombin II (platelet cofactor II or autoprothrombin II-A). Thromb Diath Haemorrh. 1960; 5: 218-249.
- 2Baker WF Jr, Bick RL. Treatment of hereditary and acquired thrombophilic disorders. Semin Thromb Hemost. 1999; 25(4): 387-406.
- 3Mosnier LO, Zlokovic BV, Griffin JH. The cytoprotective protein C pathway. Blood. 2007; 109(8): 3161-3172.
- 4John C, Xue M. Anti-inflammatory actions of the anticoagulant, activated protein C. In: A Nagal, ed. Inflammatory Diseases—A Modern Perspective. InTech; 2011: 42-74.
10.5772/25608 Google Scholar
- 5Xue M, Campbell D, Jackson CJ. Protein C is an autocrine growth factor for human skin keratinocytes. J Biol Chem. 2007; 282(18): 13610-13616.
- 6Xue M, Thompson P, Kelso I, Jackson C. Activated protein C stimulates proliferation, migration and wound closure, inhibits apoptosis and upregulates MMP-2 activity in cultured human keratinocytes. Exp Cell Res. 2004; 299(1): 119-127.
- 7Brueckmann M, Marx A, Weiler HM, et al. Stabilization of monocyte chemoattractant protein-1-mRNA by activated protein C. Thromb Haemost. 2003; 89(1): 149-160.
- 8Uchiba M, Okajima K, Oike Y, et al. Activated protein C induces endothelial cell proliferation by mitogen-activated protein kinase activation in vitro and angiogenesis in vivo. Circ Res. 2004; 95(1): 34-41.
- 9Xue M, Chow SO, Dervish S, Chan YK, Julovi SM, Jackson CJ. Activated protein C enhances human keratinocyte barrier integrity via sequential activation of epidermal growth factor receptor and Tie2. J Biol Chem. 2011; 286(8): 6742-6750.
- 10Feistritzer C, Riewald M. Endothelial barrier protection by activated protein C through PAR1-dependent sphingosine 1-phosphate receptor-1 crossactivation. Blood. 2005; 105(8): 3178-3184.
- 11Minhas N, Xue M, Fukudome K, Jackson CJ. Activated protein C utilizes the angiopoietin/Tie2 axis to promote endothelial barrier function. FASEB J. 2010; 24(3): 873-881.
- 12Jackson CJ, Xue M, Thompson P, et al. Activated protein C prevents inflammation yet stimulates angiogenesis to promote cutaneous wound healing. Wound Repair Regener. 2005; 13(3): 284-294.
- 13Julovi SM, Xue M, Dervish S, Sambrook PN, March L, Jackson CJ. Protease activated receptor-2 mediates activated protein C-induced cutaneous wound healing via inhibition of p38. Am J Pathol. 2011; 179(5): 2233-2242.
- 14Whitmont K, Reid I, Tritton S, et al. Treatment of chronic leg ulcers with topical activated protein C. Arch Dermatol. 2008; 144(11): 1479-1483.
- 15Wijewardena A, Vandervord E, Lajevardi SS, Vandervord J, Jackson CJ. Combination of activated protein C and topical negative pressure rapidly regenerates granulation tissue over exposed bone to heal recalcitrant orthopedic wounds. Int J Low Extrem Wounds. 2011; 10(3): 146-151.
- 16Kapila S, Reid I, Dixit S, et al. Use of dermal injection of activated protein C for treatment of large chronic wounds secondary to pyoderma gangrenosum. Clin Exp Dermatol. 2014; 39(7): 785-790.
- 17Wijewardena A, Lajevardi SS, Vandervord E, et al. Activated protein C to heal pressure ulcers. Int Wound J. 2016; 13(5): 986-991.
- 18Whitmont K, McKelvey KJ, Fulcher G, et al. Treatment of chronic diabetic lower leg ulcers with activated protein C: a randomised placebo-controlled, double-blind pilot clinical trial. Int Wound J. 2015; 12(4): 422-427.
- 19Ranieri VM, Thompson BT, Barie PS, et al. Drotrecogin alfa (activated) in adults with septic shock. N Engl J Med. 2012; 366(22): 2055-2064.
- 20Mosnier LO, Gale AJ, Yegneswaran S, Griffin JH. Activated protein C variants with normal cytoprotective but reduced anticoagulant activity. Blood. 2004; 104(6): 1740-1744.
- 21Lyden P, Pryor KE, Coffey CS, et al. Final results of the RHAPSODY trial: a multi-center, phase 2 trial using a continual reassessment method to determine the safety and tolerability of 3K3A-APC, a recombinant variant of human activated protein C, in combination with tissue plasminogen activator, mechanical thrombectomy or both in moderate to severe acute ischemic stroke. Ann Neurol. 2019; 85(1): 125-136.
- 22Mukherjee P, Lyden P, Fernandez JA, et al. 3K3A-activated protein C variant does not interfere with the plasma clot lysis activity of Tenecteplase. Stroke. 2020; 51(7): 2236-2239.
- 23 Macquarie University A, Zz Biotech LLC. 3K3A-APC for Treatment of Amyotrophic Lateral Sclerosis (ALS). 2022.
- 24Jaffe EA, Nachman RL, Becker CG, Minick CR. Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria. J Clin Invest. 1973; 52(11): 2745-2756.
- 25Prystowsky JH, Clevenger CV, Zheng ZS. Inhibition of ornithine decarboxylase activity and cell proliferation by ultraviolet B radiation in EGF-stimulated cultured human epidermal keratinocytes. J Invest Dermatol. 1993; 101(1): 54-58.
- 26Herron GS, Banda MJ, Clark EJ, Gavrilovic J, Werb Z. Secretion of metalloproteinases by stimulated capillary endothelial cells. II. Expression of collagenase and stromelysin activities is regulated by endogenous inhibitors. J Biol Chem. 1986; 261(6): 2814-2818.
- 27Li W, Zheng X, Gu JM, et al. Extraembryonic expression of EPCR is essential for embryonic viability. Blood. 2005; 106(8): 2716-2722.
- 28 Group FDAWHCF. Guidance for industry: chronic cutaneous ulcer and burn wounds-developing products for treatment. Wound Repair Regener. 2001; 9(4): 258-268.
- 29Zhang J, Dong J, Gu H, et al. CD9 is critical for cutaneous wound healing through JNK signaling. J Investig Dermatol. 2012; 132(1): 226-236.
- 30Devendra A, Niranjan KC, Swetha A, Kaveri H. Histochemical analysis of collagen reorganization at the invasive front of oral squamous cell carcinoma tumors. J Investig Clin Dent. 2018; 9(1):1-8.
- 31Varghese F, Bukhari AB, Malhotra R, De A. IHC profiler: an open source plugin for the quantitative evaluation and automated scoring of immunohistochemistry images of human tissue samples. PLoS One. 2014; 9(5):e96801.
- 32Nguyen M, Arkell J, Jackson CJ. Activated protein C directly activates human endothelial gelatinase A. J Biol Chem. 2000; 275(13): 9095-9098.
- 33Sullivan TP, Eaglstein WH, Davis SC, Mertz P. The pig as a model for human wound healing. Wound Repair Regener. 2001; 9(2): 66-76.
- 34Strzepa A, Pritchard KA, Dittel BN. Myeloperoxidase: a new player in autoimmunity. Cell Immunol. 2017; 317: 1-8.
- 35van der Veen BS, de Winther MP, Heeringa P. Myeloperoxidase: molecular mechanisms of action and their relevance to human health and disease. Antioxid Redox Signal. 2009; 11(11): 2899-2937.
- 36Reinke JM, Sorg H. Wound repair and regeneration. Eur Surg Res. 2012; 49(1): 35-43.
- 37Li J, Chen J, Kirsner R. Pathophysiology of acute wound healing. Clin Dermatol. 2007; 25(1): 9-18.
- 38Calabrese EJ. Hormetic mechanisms. Crit Rev Toxicol. 2013; 43(7): 580-606.
- 39Owen SC, Doak AK, Ganesh AN, et al. Colloidal drug formulations can explain “bell-shaped” concentration–response curves. ACS Chem Biol. 2014; 9(3): 777-784.
- 40Puliafito A, Hufnagel L, Neveu P, et al. Collective and single cell behavior in epithelial contact inhibition. Proc Natl Acad Sci U S A. 2012; 109(3): 739-744.
- 41Makela M, Larjava H, Pirila E, et al. Matrix metalloproteinase 2 (gelatinase A) is related to migration of keratinocytes. Exp Cell Res. 1999; 251(1): 67-78.
- 42Wang Y, Thiyagarajan M, Chow N, et al. Differential neuroprotection and risk for bleeding from activated protein C with varying degrees of anticoagulant activity. Stroke. 2009; 40(5): 1864-1869.
- 43Guo H, Singh I, Wang Y, et al. Neuroprotective activities of activated protein C mutant with reduced anticoagulant activity. Eur J Neurosci. 2009; 29(6): 1119-1130.
- 44Zhao R, Lin H, Bereza-Malcolm L, Clarke E, Jackson CJ, Xue M. Activated protein C in cutaneous wound healing: from bench to bedside. Int J Mol Sci. 2019; 20(4):1-20.
- 45Xue M, Smith MM, Little CB, Sambrook P, March L, Jackson CJ. Activated protein C mediates a healing phenotype in cultured tenocytes. J Cell Mol Med. 2009; 13(4): 749-757.
- 46Wang H, Wang P, Liang X, et al. Down-regulation of endothelial protein C receptor promotes preeclampsia by affecting actin polymerization. J Cell Mol Med. 2020; 24(6): 3370-3383.
- 47Wang Q, Yang H, Zhuo Q, Xu Y, Zhang P. Knockdown of EPCR inhibits the proliferation and migration of human gastric cancer cells via the ERK1/2 pathway in a PAR-1-dependent manner. Oncol Rep. 2018; 39(4): 1843-1852.
- 48Xue M, Dervish S, Chan B, Jackson CJ. The endothelial protein C receptor is a potential stem cell marker for epidermal keratinocytes. Stem Cells. 2017; 35(7): 1786-1798.
- 49Ramirez KP, Witherden D, Ruf W, Havran WL. Endothelial protein C receptor regulates skin gamma delta T cell functions. J Immunol. 2016; 196:59.4.
- 50Riewald M, Petrovan RJ, Donner A, Mueller BM, Ruf W. Activation of endothelial cell protease activated receptor 1 by the protein C pathway. Science. 2002; 296(5574): 1880-1882.
- 51Nowak D, Popow-Wozniak A, Raznikiewicz L, Malicka-Blaszkiewicz M. Actin in the wound healing process. Postepy Biochem. 2009; 55(2): 138-144.
- 52Darby I, Skalli O, Gabbiani G. Alpha-smooth muscle actin is transiently expressed by myofibroblasts during experimental wound healing. Lab Invest. 1990; 63(1): 21-29.
- 53Esmon CT. Inflammation and the activated protein C anticoagulant pathway. Semin Thromb Hemost. 2006; 32(Suppl 1): 49-60.
- 54Nagaraja S, Wallqvist A, Reifman J, Mitrophanov AY. Computational approach to characterize causative factors and molecular indicators of chronic wound inflammation. J Immunol. 2014; 192(4): 1824-1834.
- 55Riewald M, Ruf W. PAR1-signaling by activated protein C in cytokine perturbed endothelial cells is distinct from thrombin signaling. JBiolChem. 2005; 280: 19808-19814.
- 56Guo H, Liu D, Gelbard H, et al. Activated protein C prevents neuronal apoptosis via protease activated receptors 1 and 3. Neuron. 2004; 41(4): 563-572.
- 57Burnier L, Mosnier LO. Novel mechanisms for activated protein C cytoprotective activities involving noncanonical activation of protease-activated receptor 3. Blood. 2013; 122(5): 807-816.
- 58Connolly AJ, Suh DY, Hunt TK, Coughlin SR. Mice lacking the thrombin receptor, PAR1, have normal skin wound healing. Am J Pathol. 1997; 151(5): 1199-1204.
- 59Xue M, Lin H, Zhao R, Fryer C, March L, Jackson CJ. Activated protein C protects against murine contact dermatitis by suppressing protease-activated receptor 2. Int J Mol Sci. 2022; 23(1):1-15.
- 60Minhas N, Xue M, Jackson CJ. Activated protein C binds directly to Tie2: possible beneficial effects on endothelial barrier function. Cell Mol Life Sci. 2017; 74(10): 1895-1906.
- 61Fish EN. The X-files in immunity: sex-based differences predispose immune responses. Nat Rev Immunol. 2008; 8(9): 737-744.
Citing Literature
