βγ-CAT, a non-lens betagamma-crystallin and trefoil factor complex, induces calcium-dependent platelet apoptosis

Summary

In recent years, it has been reported that apoptosis may occur in platelets and play a role in the clearance of effete platelets. βγ-CAT, a newly identified non-lens βγ-crystallin and trefoil factor complex from frog Bombina maxima skin secretions, caused several in vivo toxic effects on mammals. Through determined haematological parameters of rabbits, it has been found that βγ-CAT significantly reduced the number of platelets in a time-dependent manner. Here, in order to explore the effect of βγ-CAT on platelets, washed platelets were incubated with various concentrations of βγ-CAT for 30 minutes. We found that βγ-CAT induced several apoptosis events in human platelets, including caspase-3 activation, phosphatidylserine (PS) exposure, depolarisation of mitochondrial inner transmembrane potential (ΔΨm), cytochrome c re-lease and strong expression of pro-apoptotic Bax and Bak proteins. However, βγ-CAT did not significantly induce platelet activation as detected by P-selectin surface expression, GPIIb/IIIa activation and platelet aggregation. In addition, we observed that βγ-CAT-induced PS exposure and ΔΨm depolarisation in platelets are Ca²⁺-dependent. Taken together, βγ-CAT can induce Ca²⁺-dependent platelet apoptosis but does not cause platelet activation.

Keywords

Platelet apoptosis - non-lens βγ-crystallins - trefoil factor - βγ-CAT - Ca²⁺

• References

• 1 Munnix IC, Cosemans JM, Auger JM. et al. Platelet response heterogeneity in thrombus formation. Thromb Haemost 2009; 102: 1149-1156.
• 2 Danial NN, Korsmeyer SJ. Cell death: critical control points. Cell 2004; 116: 205-219.
• 3 Schulze-Osthoff K, Walczak H, Droge W. et al. Cell nucleus and DNA fragmentation are not required for apoptosis. J Cell Biol 1994; 127: 15-20.
• 4 Lopez JJ, Redondo PC, Salido GM. et al. N,N,N‘,N‘-tetrakis(2-pyridylmethyl)ethylenediamine induces apoptosis through the activation of caspases-3 and –8 in human platelets. A role for endoplasmic reticulum stress. J Thromb Haemost 2009; 7: 992-999.
• 5 van der Wal DE, Du VX, Lo KS. et al. Platelet apoptosis by cold-induced glycoprotein Ibalpha clustering. J Thromb Haemost 2010; 8: 2554-2562.
• 6 Lopez JJ, Salido GM, Pariente JA. et al. Thrombin induces activation and translocation of Bid, Bax and Bak to the mitochondria in human platelets. J Thromb Haemost 2008; 6: 1780-1788.
• 7 Leytin V, Allen DJ, Mykhaylov S. et al. Thrombin-triggered platelet apoptosis. J Thromb Haemost 2006; 4: 2656-2663.
• 8 Lin KH, Hsiao G, Shih CM. et al. Mechanisms of resveratrol-induced platelet apoptosis. Cardiovasc Res 2009; 83: 575-585.
• 9 Mason KD, Carpinelli MR, Fletcher JI. et al. Programmed anuclear cell death delimits platelet life span. Cell 2007; 128: 1173-1186.
• 10 Zhang H, Nimmer PM, Tahir SK. et al. Bcl-2 family proteins are essential for platelet survival. Cell Death Differ 2007; 14: 943-951.
• 11 Augereau O, Rossignol R, DeGiorgi F. et al. Apoptotic-like mitochondrial events associated to phosphatidylserine exposure in blood platelets induced by local an-aesthetics. Thromb Haemost 2004; 92: 104-113.
• 12 Bertino AM, Qi XQ, Li J. et al. Apoptotic markers are increased in platelets stored at 37 degrees C. Transfusion 2003; 43: 857-866.
• 13 Perrotta PL, Perrotta CL, Snyder EL. Apoptotic activity in stored human platelets. Transfusion 2003; 43: 526-535.
• 14 Gwozdz AM, Leung R, Wang H. et al. Calpain inhibition by calpeptin does not prevent APLT activity reduction in PS-exposing platelets, but calpeptin has independent pro-apoptotic effects. Thromb Haemost 2010; 103: 1218-1227.
• 15 Wolf BB, Goldstein JC, Stennicke HR. et al. Calpain functions in a caspase-independent manner to promote apoptosis-like events during platelet activation. Blood 1999; 94: 1683-1692.
• 16 Vanags DM, Orrenius S, Aguilar-Santelises M. Alterations in Bcl-2/Bax protein levels in platelets form part of an ionomycin-induced process that resembles apoptosis. Br J Haematol 1997; 99: 824-831.
• 17 Rand ML, Wang H, Bang KW. et al. Procoagulant surface exposure and apoptosis in rabbit platelets: association with shortened survival and steady-state senescence. J Thromb Haemost 2004; 2: 651-659.
• 18 Pereira J, Soto M, Palomo I. et al. Platelet aging in vivo is associated with activation of apoptotic pathways: studies in a model of suppressed thrombopoiesis in dogs. Thromb Haemost 2002; 87: 905-909.
• 19 Jaenicke R, Slingsby C. Lens crystallins and their microbial homologs: structure, stability, and function. Crit Rev Biochem Mol Biol 2001; 36: 435-499.
• 20 Ogawa M, Takahashi TC, Takabatake T. et al. Isolation and characterization of a gene expressed mainly in the gastric epithelium, a novel member of the ep37 family that belongs to the betagamma-crystallin superfamily. Dev Growth Differ 1998; 40: 465-473.
• 21 Ray ME, Wistow G, Su YA. et al. AIM1, a novel non-lens member of the betagamma-crystallin superfamily, is associated with the control of tumorigenicity in human malignant melanoma. Proc Natl Acad Sci USA 1997; 94: 3229-3234.
• 22 Thim L, May FE. Structure of mammalian trefoil factors and functional insights. Cell Mol Life Sci 2005; 62: 2956-2973.
• 23 Hauser F, Roeben C, Hoffmann W. xP2, a new member of the P-domain peptide family of potential growth factors, is synthesized in Xenopus laevis skin. J Biol Chem 1992; 267: 14451-14455.
• 24 Hauser F, Hoffmann W. xP1 and xP4. P-domain peptides expressed in Xenopus laevis stomach mucosa. J Biol Chem 1991; 266: 21306-21309.
• 25 Zhang J, Zhang Y, Wan SG. et al. Bm-TFF2, a trefoil factor protein with platelet activation activity from frog Bombina maxima skin secretions. Biochem Biophys Res Commun 2005; 330: 1027-1033.
• 26 Taupin D, Podolsky DK. Trefoil factors: initiators of mucosal healing. Nat Rev Mol Cell Biol 2003; 4: 721-732.
• 27 Liu SB, He YY, Zhang Y. et al. A novel non-lens betagamma-crystallin and trefoil factor complex from amphibian skin and its functional implications. PLoS One 2008; 3: e1770.
• 28 Aravind P, Mishra A, Suman SK. et al. The betagamma-crystallin superfamily contains a universal motif for binding calcium. Biochemistry 2009; 48: 12180-12190.
• 29 Hunter SE, Clarke IN, Kelly DC. et al. Cloning and nucleotide sequencing of the Clostridium perfringens epsilon-toxin gene and its expression in Escherichia coli. Infect Immun 1992; 60: 102-110.
• 30 Qian JQ, Liu SB, He YY. et al. Acute toxicity of betagamma-CAT, a naturally existing non-lens betagamma-crystallin and trefoil factor complex from frog Bombina maxima skin secretions. Toxicon 2008; 52: 22-31.
• 31 Riedl SJ, Shi Y. Molecular mechanisms of caspase regulation during apoptosis. Nat Rev Mol Cell Biol 2004; 5: 897-907.
• 32 Schoenwaelder SM, Yuan Y, Josefsson EC. et al. Two distinct pathways regulate platelet phosphatidylserine exposure and procoagulant function. Blood 2009; 114: 663-666.
• 33 Kroemer G, Reed JC. Mitochondrial control of cell death. Nat Med 2000; 6: 513-519.
• 34 Muhlestein JB. Effect of antiplatelet therapy on inflammatory markers in atherothrombotic patients. Thromb Haemost 2010; 103: 71-82.
• 35 Bergmeier W, Schulte V, Brockhoff G. et al. Flow cytometric detection of activated mouse integrin alphaIIbbeta3 with a novel monoclonal antibody. Cytometry 2002; 48: 80-86.
• 36 Redondo PC, Salido GM, Pariente JA. et al. Dual effect of hydrogen peroxide on store-mediated calcium entry in human platelets. Biochem Pharmacol 2004; 67: 1065-1076.
• 37 Bevins CL, Zasloff M. Peptides from frog skin. Annu Rev Biochem 1990; 59: 395-414.
• 38 Clarke BT. The natural history of amphibian skin secretions, their normal functioning and potential medical applications. Biol Rev Camb Philos Soc 1997; 72: 365-379.
• 39 Zhang Y, Yu G, Xiang Y. et al. Bm-TFF2, a toad trefoil factor, promotes cell migration, survival and wound healing. Biochem Biophys Res Commun 2010; 398: 559-564.
• 40 He YY, Liu SB, Lee WH. et al. Melanoma cell growth inhibition by betagammaCAT, which is a novel non-lens betagamma-crystallin and trefoil factor complex from frog Bombina maxima skin. Toxicon 2008; 52: 341-347.
• 41 Tan KT, Lip GY. The potential role of platelet microparticles in atherosclerosis. Thromb Haemost 2005; 94: 488-492.
• 42 Shcherbina A, Remold-O’Donnell E. Role of caspase in a subset of human platelet activation responses. Blood 1999; 93: 4222-4231.
• 43 Stegner D, Nieswandt B. Platelet receptor signaling in thrombus formation. J Mol Med 2010; 89: 109-121.
• 44 Leytin V, Allen D, Mutlu A. et al. Platelet activation and apoptosis are different phenomena: evidence from the sequential dynamics and the magnitude of responses during platelet storage. British journal of haematology 2008; 142: 494-497.
• 45 Leytin V, Allen DJ, Lyubimov E. et al. Higher thrombin concentrations are required to induce platelet apoptosis than to induce platelet activation. Br J Haematol 2007; 136: 762-764.
• 46 Wang Z, Li S, Shi Q. et al. Calmodulin antagonists induce platelet apoptosis. Thromb Res 2010; 125: 340-350.
• 47 Heon Seo K, Ko HM, Kim HA. et al. Platelet-activating factor induces up-regulation of antiapoptotic factors in a melanoma cell line through nuclear factor-kappaB activation. Cancer Res 2006; 66: 4681-4686.
• 48 Schubert P, Devine DV. De novo protein synthesis in mature platelets: a consideration for transfusion medicine. Vox Sang 2010; 99: 112-122.
• 49 Rizzuto R, Pinton P, Ferrari D. et al. Calcium and apoptosis: facts and hypotheses. Oncogene 2003; 22: 8619-8627.