Tissue Plasminogen Activator: Side Effects and Signaling
- 1. Department of Medicine, Penn State University College of Medicine, US
Lin L, Hu K (2014) Tissue Plasminogen Activator: Side Effects and Signaling. J Drug Des Res 1(1): 1001.
According to the most recent report from American Heart Association, stroke is among the leading causes of death in general population . The predominant form of stroke is the ischemic stroke, which is caused by the clot within a blood vessel that disrupts the blood supply. Currently, the only FDA-approved treatment is tissue-type plasminogen activator (tPA). tPA is a member of the serine protease family that plays a pivotal role in the homeostasis of blood coagulation/fibrinolysis and matrix regulation [2-5]. Generally, tPAconverts plasminogen into active plasmin and activates the fibrinolysis process to dissolve the clot and improve or restore the blood supply. However, besides its beneficial protease activities,the clinical application of tPA has been limited by the harmful side effects that not related to its catalytic function [2,6-11]. In addition, although tPA, as a protease, induces matrix degradation, our investigations demonstrate that tPA promotes tissue fibrosis, a disease condition characterized by excessive matrix accumulation [2,12-15].
The structure and biological function of tPA
tPA is a 69-kDa glycoprotein synthesized within cells and is released as a single chain enzyme. Plasmin subsequently cleaves it into a two-chain form (heavy chain and light chain). There are4 domains within the single-chain tPA: 1) a finger (F) domain, which is homologous to fibronectin; 2) an EGF domain, which is homologous to EGF; 3) two kringle (K) domains; and 4) the catalytic protease (P) domain. The P domain forms the light chain, while the rest F, EGF, and K domains form the heavy chain. The active site responsible for tPA protease activity consists of Histidine 322, Asparagine 371, and Serine 478 . Mutagenesis analysis indicates that single mutation of Serine 478 to Alanine renders tPA catalytically inactive, while its other function remains intact .
Our recent studies demonstrate that tPA is actually a molecule with dual functions [2,12-15]. As a serine protease, tPA plays a pivotal role in the homeostasis of blood coagulation/fibrinolysis and extracellular matrix regulation . As a cytokine, tPA executes multiple actions by binding to its membrane receptors and triggering profound intracellular signaling events [2,12- 15,18].
Signaling of tPA
Although tPA does not have a dedicated and specific receptor, there are at least two known candidates that functionally and biologically serve as tPA receptor: LDL receptor-related protein-1 (LRP-1)  and annexin A2 .
LRP-1-mediated tPA signaling
LRP-1, also known as α2-macroglobulin receptor (α2MR)  or type V TGF-β receptor (TβR-V) , is a member of the LDL receptor family [22,23]. Mature LRP-1 consists of an extracellular 515-kDa α subunit and an 85-kDa β subunit with a transmembrane segment and cytoplasmic tail containing two NPxY motifs and numerous tyrosine residues [22,24,25]. tPA has been shown to bind to the domains II and IV in the extracellular region of LRP-1 [22,26].
In the numerous organ injury models including brain, liver, and kidneys, tPA expression is up-regulated [6,12,13,27]. Our recent work demonstrated that myeloid cells are the major source of the endogenous tPA induction in the diseased organs . The myeloid-derived tPA interacts with the LRP-1 on various cells to initiate cell type-specific signaling to modulate cellular processes and cell differentiation. Our previous work demonstrated that LRP-1 on fibroblasts mediates multiple tPA signaling cascades to promote fibroblast activation, transdifferentiation, growth and survival, leading to renal fibrosis: 1) tPA induces matrix metalloproteinases(MMPs) production to initiate the epithelial mesenchymal transition (EMT) through LRP-1-mediate MAPK pathway  ; 2) tPA promotes the survival, proliferation, and interstitial accumulation of fibroblasts in the diseased kidneys through p90RSK-mediated Bad or GSK3β signaling [2,14]. We also showed that tPA induces the phosphorylation of LRP-1 Tyr 4507, which is indispensable to tPA-mediated fibroblast proliferation ; 3) tPA promotes myofibroblast activation by activating LRP-1-mediated β1 integrin/ integrin-linked kinase (ILK) signaling . In macrophages, Cao, et al demonstrated that genetic inactivation of integrin Mac-1, tPA, PAI-1 or LRP1 abrogates LPS-induced peritoneal macrophage efflux, and tPA forms complex with LRP-1, Mac-1, and PAI-1 to promote macrophage migration . We recently found that LRP-1 mediates tPA-induced macrophage motility through activation of FAK and Rac-1 signaling . In the brain, LRP-1-mediated MMP-9 induction in human cerebral microvascular endothelial cells has been considered as one of the cellular mechanisms of tPA-induced neurotoxic side effects , tPA has been shown to induce cerebral vascular LRP-1-mediated opening of blood-brain barrier (6), and tPA and LRP-1 have been shown to mediate ischemia-induced NF-κB activation . tPA also activates Rac1 and controls LRP-1-mediated Schwann cells migration . In neurons, LRP-1 mediates tPA-induced Trk receptor phosphorylation and activation of downstream Akt and Erk pathway, leading to neurite outgrowth .
A2-mediated tPA signaling
Annexin A2 is a member of the Ca2+- and phospholipidbinding protein family. Annexin A2 has been identified as a major membrane receptor of tPA on endothelial , microglia cells , and other cancer cells  ; and is implicated in mediating certain signal transductions [9,34,36]. tPA has been shown to bind to the hexapeptide LCKLSL (residues 7–12) in the N terminus of annexin A2 . Recently, we demonstrated that tPA induces the aggregation of annexin A2 and integrin CD11b on macrophages and the subsequently activation of ILK pathway, leading to NFκB activation (15). Activation of NF-κB signaling contributes to tPA-mediated macrophage migration . In addition, the finger domain of tPA has been shown to bind to annexin A2 and induce microglial activation to cause brain injury (20). Annexin A2 on the pancreatic cancer cells has also been shown to be responsible for tPA-induced cell proliferation .
Although the direct in vivo investigations towards tPA cytokine functions are still missing, numerous studies clearly indicate that the side effects and toxicity of tPA are likely mediated by its protease-independent cytokine functions. Previous report that plasminogen activator inhibitor type 1 (PAI-1)-derived hexapeptide (EEIIMD) blocks tPA signaling and reduces the neurotoxic effects of tPA without compromising its fibrinolytic activity  strongly supports the above view, and lays a strong foundation for future development of therapeutic strategies targeting tPA side effects and toxicity.
This work was supported by grants from NIH NIDDK [1R01DK102624], American Heart Association [14GRNT20380289, 10SDG3900029, and 09BGIA2100010], and Barsumian Trust .
1. Go AS, Mozaffarian D, Roger VL, Benjamin EJ, Berry JD, Blaha MJ, et al. Heart disease and stroke statistics--2014 update: a report from the American Heart Association. Circulation. 2014; 129; 28-292
2. Hu K, Lin L, Tan X, Yang J, Bu G, Mars WM, et al. tPA protects renal interstitial fibroblasts and myofibroblasts from apoptosis. J Am Soc Nephrol. 2008; 19: 503-514.
3. Mars WM, Zarnegar R, Michalopoulos GK. Activation of hepatocyte growth factor by the plasminogen activators uPA and tPA. Am J Pathol. 1993; 143: 949-958.
4. Yee JA,Yan L, Dominguez JC, Allan EH, Martin TJ. Plasminogen-dependent activation of latent transforming growth factor beta (TGF beta) by growing cultures of osteoblast-like cells. J Cell Physiol. 1993; 157: 528-534.
5. 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- 3802.
6. Yepes M, Sandkvist M, Moore EG, Bugge TH, Strickland DK, Lawrence DA. Tissue-type plasminogen activator induces opening of the blood-brain barrier via the LDL receptor-related protein. J Clin Invest. 2003; 112: 1533-1540.
7. Nagai N, Yamamoto S, Tsuboi T, Ihara H, Urano T, Takada Y, et al. Tissue-type plasminogen activator is involved in the process of neuronal death induced by oxygen-glucose deprivation in culture. J Cereb Blood Flow Metab. 2001; 21: 631-634.
8. Welling TH , Huber TS, Messina LM, Stanley JC. Tissue plasminogen activator increases canine endothelial cell proliferation rate through a plasmin-independent, receptor-mediated mechanism. J Surg Res. 1996; 66: 36-42.
9. Ortiz-Zapater E , Peiró S, Roda O, Corominas JM, Aguilar S, Ampurdanés C, Real FX. Tissue plasminogen activator induces pancreatic cancer cell proliferation by a non-catalytic mechanism that requires extracellular signal-regulated kinase 1/2 activation through epidermal growth factor receptor and annexin A2. Am J Pathol. 2007; 170: 1573-1584.
10. Wang X , Asahi M, Lo EH. Tissue type plasminogen activator amplifies hemoglobin-induced neurotoxicity in rat neuronal cultures. Neurosci Lett. 1999; 274: 79-82.
11. Armstead WM, Nassar T, Akkawi S, Smith DH, Chen XH, Cines DB, et al. Neutralizing the neurotoxic effects of exogenous and endogenous tPA. Nat Neurosci. 2006; 9: 1150-1155.
12. Hu K, Wu C, Mars WM, Liu Y. Tissue-type plasminogen activator promotes murine myofibroblast activation through LDL receptor-related protein 1-mediated integrin signaling. J Clin Invest. 2007; 117: 3821-3832.
13. Hu K, Yang J, Tanaka S, Gonias SL, Mars WM, Liu Y. Tissue-type plasminogen activator acts as a cytokine that triggers intracellular signal transduction and induces matrix metalloproteinase-9 gene expression. J Biol Chem. 2006; 281: 2120-2127.
14. Lin L, Bu G, Mars WM, Reeves WB, Tanaka S, Hu K. tPA activates LDL receptor-related protein 1-mediated mitogenic signaling involving the p90RSK and GSK3beta pathway. Am J Pathol. 2010; 177: 1687-1696.
15. Lin L, Wu C, Hu K. Tissue plasminogen activator activates NF-κB through a pathway involving annexin A2/CD11b and integrin-linked kinase. J Am Soc Nephrol. 2012; 23: 1329-1338.
16. Vivien D, Gauberti M, Montagne A, Defer G, Touzé E. Impact of tissue plasminogen activator on the neurovascular unit: from clinical data to experimental evidence. J Cereb Blood Flow Metab. 2011; 31: 2119- 2134.
17. Olson ST, Swanson R, Day D, Verhamme I, Kvassman J, Shore JD. Resolution of Michaelis complex, acylation, and conformational change steps in the reactions of the serpin, plasminogen activator inhibitor-1, with tissue plasminogen activator and trypsin. Biochemistry. 2001; 40: 11742-11756.
18. Shi Y, Mantuano E, Inoue G, Campana WM, Gonias SL. Ligand binding to LRP1 transactivates Trk receptors by a Src family kinase-dependent pathway. Sci Signal. 2009; 2: ra18.
19. Bu G, Williams S, Strickland DK, Schwartz AL. Low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor is an hepatic receptor for tissue-type plasminogen activator. Proc Natl Acad Sci U S A. 1992; 89: 7427-7431.
20. Siao CJ, Tsirka SE. Tissue plasminogen activator mediates microglial activation via its finger domain through annexin II. J Neurosci. 2002; 22: 3352-3358. 21.Huang SS, Ling TY, Tseng WF, Huang YH, Tang FM, Leal SM, et al. Cellular growth inhibition by IGFBP-3 and TGF-beta1 requires LRP-1. FASEB J. 2003; 17: 2068-2081.
22. Herz J, Strickland DK. LRP: a multifunctional scavenger and signaling receptor. J Clin Invest. 2001; 108: 779-784.
23. Lillis AP, Van Duyn LB, Murphy-Ullrich JE, Strickland DK. LDL receptor-related protein 1: unique tissue-specific functions revealed by selective gene knockout studies. Physiol Rev. 2008; 88: 887-918.
24. Hussain MM. Structural, biochemical and signaling properties of the low-density lipoprotein receptor gene family. Front Biosci. 2001; 6: D417-428.
25. Strickland DK, Ranganathan S. Diverse role of LDL receptor-related protein in the clearance of proteases and in signaling. J Thromb Haemost. 2003; 1: 1663-1670.
26. Obermoeller-McCormick LM, Li Y, Osaka H, FitzGerald DJ, Schwartz AL, Bu G. Dissection of receptor folding and ligand-binding property with functional minireceptors of LDL receptor-related protein. J Cell Sci. 2001; 114: 899-908.
27. Wang YF, Tsirka SE, Strickland S, Stieg PE, Soriano SG, Lipton SA,. Tissue plasminogen activator (tPA) increases neuronal damage after focal cerebral ischemia in wild-type and tPA-deficient mice. Nat Med. 1998; 4: 228-231.
28. Lin L , Jin Y2, Mars WM3, Reeves WB1, Hu K4. Myeloid-Derived TissueType Plasminogen Activator Promotes Macrophage Motility through FAK, Rac1, and NF-ÎºB PathwayAm J Pathol. 2014; 184: 2757-2767.
29. Cao C , Lawrence DA, Li Y, Von Arnim CA, Herz J, Su EJ, et al. Endocytic receptor LRP together with tPA and PAI-1 coordinates Mac-1- dependent macrophage migration. EMBO J. 2006; 25: 1860-1870.
30. Wang X , Lee SR, Arai K, Lee SR, Tsuji K, Rebeck GW, Lo EH. Lipoprotein receptor-mediated induction of matrix metalloproteinase by tissue plasminogen activator. Nat Med. 2003; 9: 1313-1317.
31. Zhang X, Polavarapu R, She H, Mao Z, Yepes M. Tissue-type plasminogen activator and the low-density lipoprotein receptor-related protein mediate cerebral ischemia-induced nuclear factor-kappaB pathway activation. Am J Pathol. 2007; 171: 1281-1290.
32. Mantuano E1, Jo M, Gonias SL, Campana WM. Low density lipoprotein receptor-related protein (LRP1) regulates Rac1 and RhoA reciprocally to control Schwann cell adhesion and migration. J Biol Chem. 2010; 285: 14259-14266.
33. Cesarman, G. M., Guevara, C. A., and Hajjar, K. A. An endothelial cell receptor for plasminogen/tissue plasminogen activator (t-PA). II. Annexin II-mediated enhancement of t-PA-dependent plasminogen activation.J Biol Chem, 1994 ;269: 21198-21203.
34. Siao CJ, Tsirka SE. Tissue plasminogen activator mediates microglial activation via its finger domain through annexin II. J Neurosci. 2002; 22: 3352-3358.
35. Kim J, Hajjar KA. Annexin II: a plasminogen-plasminogen activator co-receptor. Front Biosci. 2002; 7: d341-348.
36. Babbin BA,Parkos CA, Mandell KJ, Winfree LM, Laur O, Ivanov AI, et al. Annexin 2 regulates intestinal epithelial cell spreading and wound closure through Rho-related signaling. Am J Pathol. 2007; 170: 951- 966.
37. Hajjar KA, Mauri L, Jacovina AT, Zhong F, Mirza UA, Padovan JC, et al. Tissue plasminogen activator binding to the annexin II tail domain. Direct modulation by homocysteine. J Biol Chem. 1998; 273: 9987- 9993.