Use of functional highly purified human platelets for the identification of new proteins of the IPP signaling pathway.
BACKGROUND
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.


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.
Birschmann
Ingvild
I
Institut fuer Klinische Biochemie und Pathobiochemie, Universitaet Wuerzburg, Josef-Schneider-Str 2, 97080 Wuerzburg, Germany. Birschmann@klin-biochem.uni-wuerzburg.de
Mietner
Silke
S
Dittrich
Marcus
M
Pfrang
Julia
J
Dandekar
Thomas
T
Walter
Ulrich
U
D000185:Actinin / Q000097:blood / Q000235:genetics; D048868:Adaptor Proteins, Signal Transducing; D001792:Blood Platelets / Q000378:metabolism*; D001798:Blood Proteins / Q000235:genetics* / Q000302:isolation & purification; D002469:Cell Separation / Q000379:methods; D004268:DNA-Binding Proteins / Q000097:blood / Q000235:genetics; D020869:Gene Expression Profiling; D006683:HLA-DQ Antigens / Q000235:genetics; D006801:Humans; D060588:LIM Domain Proteins; D016256:Lewis X Antigen / Q000097:blood / Q000235:genetics; D008565:Membrane Proteins; D008840:Microfilament Proteins; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014841:von Willebrand Factor / Q000235:genetics; D000096985:Calponins
D000185:Actinin; D048868:Adaptor Proteins, Signal Transducing; D001798:Blood Proteins; D004268:DNA-Binding Proteins; D006683:HLA-DQ Antigens; D060588:LIM Domain Proteins; D016256:Lewis X Antigen; D008565:Membrane Proteins; D008840:Microfilament Proteins; D014841:von Willebrand Factor; C088088:LIMS1 protein, human; C486590:PARVA protein, human; D000096985:Calponins