Proteomic and functional characterisation of platelet microparticle size classes.
Activated platelets release large lipid-protein complexes termed microparticles. These platelet microparticles (PMP) are composed of vesicular fragments of the plasma membrane and alpha-granules. PMP facilitate coagulation, promote platelet and leukocyte adhesion to the subendothelial matrix, support angiogenesis and stimulate vascular smooth muscle proliferation.


OBJECTIVE
PMP were separated into 4 size classes to facilitate identification of active protein and lipid components. PMP were obtained from activated human platelets and separated into 4 size classes by gel filtration chromatography. Proteins were identified using 2-dimensional, liquid chromatography tandem mass spectrometry. Functional effects on platelets were determined using the PFA-100 and on endothelial cells by measuring transendothelial cell electrical resistance. PMP size classes differed significantly in their contents of plasma membrane receptors and adhesion molecules, chemokines, growth factors and protease inhibitors. The two smallest size classes (3 and 4) inhibited collagen/adenosine-diphosphate-mediated platelet thrombus formation, while fractions 2 and 4 stimulated barrier formation by endothelial cells. Heat denaturation blocked the effect of fraction 4 on endothelial cell function, but not fraction 2 implying that the active component in fraction 4 is a protein and in fraction 2 is a heat-stable protein or lipid but not sphingosine-1-phosphate. Proteomic and functional analysis of PMP size fractions has shown that PMP can be separated into different size classes that differ in protein components, protein/lipid ratio, and functional effects on platelets and endothelial cells. This analysis will facilitate identification of active components in the PMP and clarify their involvement in diseases such as atherosclerosis and cancer.
Dean
William L
WL
Department of Biochemistry and Molecular Biology, University of Louisville School of Medicine Louisville, KY 40292, USA. bill.dean@louisville.edu
Lee
Menq J
MJ
Cummins
Timothy D
TD
Schultz
David J
DJ
Powell
David W
DW
D001777:Blood Coagulation; D001792:Blood Platelets / Q000276:immunology / Q000378:metabolism* / Q000648:ultrastructure; D002458:Cell Fractionation; D055252:Cell-Derived Microparticles / Q000145:classification / Q000378:metabolism* / Q000648:ultrastructure; D002478:Cells, Cultured; D017097:Electric Impedance; D042783:Endothelial Cells / Q000276:immunology / Q000378:metabolism* / Q000473:pathology; D005260:Female; D006487:Hemostasis; D006801:Humans; D013058:Mass Spectrometry; D053141:Organelle Size; D011247:Pregnancy; D040901:Proteomics; D014470:Umbilical Cord / Q000473:pathology