BLOOD COMPONENTS Cryopreservation alters the membrane and cytoskeletal protein profile of platelet microparticles Sarah Raynel,1,2 Matthew P. Padula,2 Denese C. Marks,1 and Lacey Johnson1
BACKGROUND: Cryopreservation of platelets (PLTs) in dimethyl sulfoxide (DMSO) and storage at 2808C extends the PLT shelf life to at least 2 years, allowing greater accessibility in military and rural environments. While cryopreserved PLTs have been extensively characterized, the microparticles formed as a result of cryopreservation are yet to be fully described. STUDY DESIGN AND METHODS: Apheresis PLTs were cryopreserved at 2808C with 5% DMSO and sampled before freezing and after thawing. Microparticle number, size, surface receptor phenotype, and function were assessed by microscopy, flow cytometry, dynamic light scattering, and thrombin-generating capacity. Proteomic changes were examined using twodimensional gel electrophoresis and Western blotting. RESULTS: PLT cryopreservation resulted in a 15-fold increase in the number of microparticles compared to fresh PLTs. The surface receptor phenotype of these microparticles differed to microparticles from fresh PLTs, with more microparticles expressing glycoprotein (GP)IV, GPIIb, and the GPIb-V-IX complex. Cryopreservation drastically altered the abundance of many cytoskeletal proteins in the PLT microparticles, including actin, filamin, gelsolin, and tropomyosin. Despite these changes, PLT microparticles were functional and contributed to phosphatidylserine- and tissue factor– induced thrombin generation. CONCLUSION: This study demonstrates that PLT microparticles formed by cryopreservation are phenotypically distinct from those present before freezing. These differences may be associated with the procoagulant properties of cryopreserved PLTs.
C
ryopreservation of platelets (PLTs) provides a way to extend their shelf life and overcome the logistical issues surrounding fresh, liquid-stored PLTs.1-3 PLT cryopreservation involves the addition of 5% dimethyl sulfoxide (DMSO), followed by removal of the DMSO containing supernatant and freezing at 2808C.1,4 The PLTs are then resuspended in plasma before transfusion. It has been shown that cryopreserved PLTs are more capable of reducing blood loss, when compared to fresh, liquid-stored PLTs.5 The improved clinical effect occurs despite cryopreserved PLTs having a reduced recovery after transfusion.5,6 It is suggested that the improved in vivo effect is due to an altered surface receptor phenotype, and the release of procoagulant mediators,5,7,8 including the formation of phosphatidylserine-expressing microparticles.9 Although cryopreserved PLTs have been comprehensively characterized,4,6-12 the microparticles formed by cryopreservation are yet to be fully assessed. PLT microparticles are known to have many functions; therefore, characterizing the microparticles formed after cryopreservation will enable ABBREVIATIONS: 2DGE 5 two-dimensional gel electrophoresis; DLS 5 dynamic light scattering; GP(s) 5 glycoprotein(s); LC-MS/MS 5 liquid chromatography coupled to tandem mass spectrometry. From 1Research & Development, Australian Red Cross Blood Service, Alexandria, NSW, Australia; and the 2Proteomics Core Facility, University of Technology Sydney, Sydney, NSW, Australia. Address correspondence to: Dr Lacey Johnson, Research and Development, Australian Red Cross Blood Service, 17 O’Riordan Street, Alexandria, NSW, Australia; e-mail: [email protected]. The Australian government funds the Australian Red Cross Blood Service to provide blood, blood products, and services to the Australian community. Received for publication January 28, 2015; revision received April 1, 2015; and accepted April 2, 2015. doi:10.1111/trf.13165 C 2015 AABB V
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an understanding of the role they may play when transfused. As PLT microparticles are anucleate cell fragments that lack DNA and contain very low levels of mRNA and miRNA, proteomics represents an appropriate investigative technique.13,14 As such, the aim of this study was to characterize and compare PLT microparticles before and after cryopreservation.
MATERIALS AND METHODS This study had appropriate ethics approval and all blood donations were from eligible, voluntary donors. Leukoreduced (
BACKGROUND: Cryopreservation of platelets (PLTs) in dimethyl sulfoxide (DMSO) and storage at 2808C extends the PLT shelf life to at least 2 years, allowing greater accessibility in military and rural environments. While cryopreserved PLTs have been extensively characterized, the microparticles formed as a result of cryopreservation are yet to be fully described. STUDY DESIGN AND METHODS: Apheresis PLTs were cryopreserved at 2808C with 5% DMSO and sampled before freezing and after thawing. Microparticle number, size, surface receptor phenotype, and function were assessed by microscopy, flow cytometry, dynamic light scattering, and thrombin-generating capacity. Proteomic changes were examined using twodimensional gel electrophoresis and Western blotting. RESULTS: PLT cryopreservation resulted in a 15-fold increase in the number of microparticles compared to fresh PLTs. The surface receptor phenotype of these microparticles differed to microparticles from fresh PLTs, with more microparticles expressing glycoprotein (GP)IV, GPIIb, and the GPIb-V-IX complex. Cryopreservation drastically altered the abundance of many cytoskeletal proteins in the PLT microparticles, including actin, filamin, gelsolin, and tropomyosin. Despite these changes, PLT microparticles were functional and contributed to phosphatidylserine- and tissue factor– induced thrombin generation. CONCLUSION: This study demonstrates that PLT microparticles formed by cryopreservation are phenotypically distinct from those present before freezing. These differences may be associated with the procoagulant properties of cryopreserved PLTs.
C
ryopreservation of platelets (PLTs) provides a way to extend their shelf life and overcome the logistical issues surrounding fresh, liquid-stored PLTs.1-3 PLT cryopreservation involves the addition of 5% dimethyl sulfoxide (DMSO), followed by removal of the DMSO containing supernatant and freezing at 2808C.1,4 The PLTs are then resuspended in plasma before transfusion. It has been shown that cryopreserved PLTs are more capable of reducing blood loss, when compared to fresh, liquid-stored PLTs.5 The improved clinical effect occurs despite cryopreserved PLTs having a reduced recovery after transfusion.5,6 It is suggested that the improved in vivo effect is due to an altered surface receptor phenotype, and the release of procoagulant mediators,5,7,8 including the formation of phosphatidylserine-expressing microparticles.9 Although cryopreserved PLTs have been comprehensively characterized,4,6-12 the microparticles formed by cryopreservation are yet to be fully assessed. PLT microparticles are known to have many functions; therefore, characterizing the microparticles formed after cryopreservation will enable ABBREVIATIONS: 2DGE 5 two-dimensional gel electrophoresis; DLS 5 dynamic light scattering; GP(s) 5 glycoprotein(s); LC-MS/MS 5 liquid chromatography coupled to tandem mass spectrometry. From 1Research & Development, Australian Red Cross Blood Service, Alexandria, NSW, Australia; and the 2Proteomics Core Facility, University of Technology Sydney, Sydney, NSW, Australia. Address correspondence to: Dr Lacey Johnson, Research and Development, Australian Red Cross Blood Service, 17 O’Riordan Street, Alexandria, NSW, Australia; e-mail: [email protected]. The Australian government funds the Australian Red Cross Blood Service to provide blood, blood products, and services to the Australian community. Received for publication January 28, 2015; revision received April 1, 2015; and accepted April 2, 2015. doi:10.1111/trf.13165 C 2015 AABB V
TRANSFUSION 2015;55;2422–2432 2422 TRANSFUSION Volume 55, October 2015
CRYOPRESERVED PLT MICROPARTICLES
an understanding of the role they may play when transfused. As PLT microparticles are anucleate cell fragments that lack DNA and contain very low levels of mRNA and miRNA, proteomics represents an appropriate investigative technique.13,14 As such, the aim of this study was to characterize and compare PLT microparticles before and after cryopreservation.
MATERIALS AND METHODS This study had appropriate ethics approval and all blood donations were from eligible, voluntary donors. Leukoreduced (
