INTRODUCTION: Identification of the full content of platelet proteins and their mRNAs would be helpful for further studies of human platelet function. For this purpose, proteomic as well as transcriptomic methods (SAGE and qRT-PCR) can be utilized, but the purity of the platelet samples studied is crucial. Here we report the development of a new, effective, and efficient technique for purification of human platelets from washed apheresis platelet concentrates and whole blood. MATERIALS AND METHODS: Methods used are a combination of differential and gradient centrifugation steps. The level of purification was determined by nephelometry, FACS, and PCR. RESULTS: We could show that even the P2Y purinoceptor 12 (P2Y(12)) receptor, which undergoes rapid homologous desensitization, was still functional after the purification procedure. The presence of PINCH (particularly interesting new Cys-His protein) and alpha-parvin, which constitute the IPP (ILK-PINCH-parvin) complex together with the integrin-linked kinase (ILK), has been predicted in platelets by proteomic analysis. We could confirm this observation with our purified platelets. Detection of these proteins is an example of the application of this purification protocol that can be used for the verification of proteins postulated by high-throughput studies. CONCLUSIONS: The procedure for obtaining purified platelets described here provides an essential, much-needed tool for the comprehensive investigation of platelet proteins and functions.
INTRODUCTION: Identification of the full content of platelet proteins and their mRNAs would be helpful for further studies of human platelet function. For this purpose, proteomic as well as transcriptomic methods (SAGE and qRT-PCR) can be utilized, but the purity of the platelet samples studied is crucial. Here we report the development of a new, effective, and efficient technique for purification of human platelets from washed apheresis platelet concentrates and whole blood. MATERIALS AND METHODS: Methods used are a combination of differential and gradient centrifugation steps. The level of purification was determined by nephelometry, FACS, and PCR. RESULTS: We could show that even the P2Y purinoceptor 12 (P2Y(12)) receptor, which undergoes rapid homologous desensitization, was still functional after the purification procedure. The presence of PINCH (particularly interesting new Cys-His protein) and alpha-parvin, which constitute the IPP (ILK-PINCH-parvin) complex together with the integrin-linked kinase (ILK), has been predicted in platelets by proteomic analysis. We could confirm this observation with our purified platelets. Detection of these proteins is an example of the application of this purification protocol that can be used for the verification of proteins postulated by high-throughput studies. CONCLUSIONS: The procedure for obtaining purified platelets described here provides an essential, much-needed tool for the comprehensive investigation of platelet proteins and functions.
Authors: Maria T Aatonen; Tiina Ohman; Tuula A Nyman; Saara Laitinen; Mikaela Grönholm; Pia R-M Siljander Journal: J Extracell Vesicles Date: 2014-08-06
Authors: Nóra Homoródi; Emese G Kovács; Sarolta Leé; Éva Katona; Amir H Shemirani; Gizella Haramura; László Balogh; Zsuzsanna Bereczky; Gabriella Szőke; Hajna Péterfy; Róbert G Kiss; István Édes; László Muszbek Journal: J Transl Med Date: 2016-03-15 Impact factor: 5.531