Proteomic and functional characterisation of platelet microparticle size classes

William L. Dean; Menq J. Lee; Timothy D. Cummins; David J. Schultz; David W. Powell

doi:10.1160/TH09-04-243

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2009; 102(04): 711-718
DOI: 10.1160/TH09-04-243

Platelets and Blood Cells

Schattauer GmbH

Proteomic and functional characterisation of platelet microparticle size classes

William L. Dean

¹Department of Biochemistry and Molecular Biology, University of Louisville, Louisville, Kentucky, USA

,

Menq J. Lee

²Department of Microbiology and Immunology, University of Louisville, Louisville, Kentucky, USA

,

Timothy D. Cummins

¹Department of Biochemistry and Molecular Biology, University of Louisville, Louisville, Kentucky, USA

,

David J. Schultz

³Department of Biology, University of Louisville, Louisville, Kentucky, USA

,

David W. Powell

¹Department of Biochemistry and Molecular Biology, University of Louisville, Louisville, Kentucky, USA

⁴Department of Medicine, University of Louisville, Louisville, Kentucky, USA

› Author Affiliations

Abstract

Summary

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

Keywords

Blood platelets - microparticles - proteomics - platelet activation - transendothelial cell electrical resistance - sphingosine-1-phosphate

Full Text

References

References
1 Wolf P. The nature and significance of platelet products in human plasma. Br J Haematol 1967; 13: 269-288.
2 Heijnen HF, Schiel AE, Fijnheer R. et al. Activated platelets release two types of membrane vesicles; microvesicles by surface shedding and exosomes derived from exocytosis of multivesicular bodies and a-granules. Blood 1999; 94: 3791-3799.
3 Reininger AJ, Heijnen HF, Schumann H. et al. Mechanism of platelet adhesion to vonWillebrand factor and microparticle formation under high shear stress. Blood 2006; 107: 3537-3545.
4 Denzer K, Kleijmeer MJ, Heijnen HFG. et al. Exosome: from internal vesicle of the multivesicular body to intercellular signaling device. J Cell Sci 2000; 113: 3365-3374.
5 Piccin A, Murphy WG, Smith OP. Circulating microparticles: pathophysiology and clinical implications. Blood Rev 2007; 21: 157-171.
6 Flaumenhaft R, James R, Dilks JR. et al Megakaryocyte-derived microparticles: Direct visualization and distinction from platelet-derived microparticles. Blood 2009; 113: 1112-1121.
7 Smalley DM, Root KE, Cho HJ. et al. Proteomic discovery of 21 proteins expressed in human plasmaderived but not platelet-derived microparticles. Thromb Haemost 2007; 97: 67-80.
8 Jin M, Drwal G, Bourgeois T. et al. Distinct proteome features of plasma microparticles. Proteomics 2005; 05: 1940-1952.
9 Gracia BA, Smalley DM, Cho H. et al. The platelet microparticle proteome. J Proteome Res 2005; 04: 1516-1521.
10 Perez-Pujol S, Marker PH, Key NS. Platelet microparticles are heterogeneous and highly dependent on the activation mechanism: studies using a new digital flow cytometer. Cytometry A 2007; 71: 38-45.
11 Biro E, Akkerman JW, Hoek FJ. et al. The phospholipid composition and cholesterol content of platelet derived microparticles: a comparison with platelet membrane fractions. Thromb Haemost 2005; 03: 2754-2763.
12 Sims PJ, Wiedmer T, Esmon CT. et al. Assembly of the platelet prothrombinase complex is linked to vesiculation of the platelet plasma membrane: studies in Scott syndrome, an isolated defect in platelet procoagulent actvitity. J Biol Chem 1989; 264: 17049-17057.
13 Berckmans RJ, Nieuwland R, Tak PP. et al. Cell-derived microparticles in synovial fluid from inflamed arthritic joints support coagulation exclusively via a factor VII-dependent mechanism. Arthritis Rheum 2002; 46: 2857-2866.
14 Nieuwland R, Berckmans RJ, Rotteveel-Eijkman RC. et al. Cell-derived microparticles generated in patients during cardiopulmonary bypass are highly procoagulent. Circulation 1997; 96: 3534-3541.
15 Sinauridze EI, Kireev DA, Popenko NY. et al. Platelet microparticle membranes have 50- to 100-fold higher specific procoagulant activity than activated platelets. Thromb Haemost 2007; 97: 425-434.
16 Merten M, Pakala R, Thiagarajan P, Benedict CR. Platelet microparticles promote platelet interaction with subendothelial matrix in a glycoprotein IIb-IIIadependent mechanism. Circulation 1999; 99: 2577-2582.
17 Dachary-Prigent J, Pasquet JM, Fressinaud E, Toti F, Freyssinet JM, Nurden AT. Aminophospholipid exposure, microvesicularization and abnormal protein tyrosine phosphorylation in the platelets of a patient with Scott syndrome: a study using physiologic agonists and local anaesthetics. Br J Haematol 1997; 99: 959-67.
18 Toti F, Satta N, Fressinaud E. et al. Scott syndrome, characterized by impaired transmembrane migration of procoagulent phosphatidylserine and hemorrhagic complications, is an inherited disorder. Blood 1996; 87: 1409-15.
19 Kim HK, Song KS, Chung JH. et al. Platelet microparticles induce angiogenesis in vitro. Br J Haematol 2004; 124: 376-384.
20 Weber A, Koppen HO, Schror K. Platelet-derived microparticles stimuilate coronary artery smooth muscle cell mitogenesis by a PDGF-independent mechanism. Thromb Res 2000; 98: 461-466.
21 Boulanger CM, Amabile N, Tedgui A. Circulating Microparticles: a potential prognosis marker for atherosclerotic vascular disease. Hypertension 2006; 48: 180-186.
22 Preston RA, Jy W, Jimenez JJ. et al. Effects of severe hypertension on endothelial and platelet microparticles. Hypertension 2003; 41: 211-217.
23 van der Zee PM, Biró E, Ko Y. et al. P-Selectin and CD63-exposing platelet microparticles reflect platelet activation in peripheral arterial disease and myocardial infarction. Clin Chem 2006; 52: 657-664.
24 Diehl P, Nagy F, Sossong V. et al. Increased levels of circulating microparticles in patients with severe aortic valve stenosis. Thromb Haemost 2008; 99: 711-719.
25 Barry OP, Pratico D, Lawson JA. et al. Transcellular activiation of platelets and endothelial cells by bioactive lipids in platelet microparticles. J Clin Invest 1997; 99: 2118-2127.
26 Pfister SL. Role of Platelet Microparticles in the production of thromboxane by rabbit pulmonary artery. Hypertension 2004; 43: 428-433.
27 Barry OP, Kazanietz MG, Praticò D. et al. Arachidonic acid in platelet microparticles up-regulates cyclooxygenase-2-dependent prostaglandin formation via a protein kinaseC/mitogen-activated protein kinase-dependent pathway. J Biol Chem 1999; 274: 7545-7556.
28 Alvarex SE, Milstien S, Spiegel S. Acutocrine and paracrine roles of sphingosine-1-phosphate. Trends Endocrinol Metab 2007; 18: 300-307.
29 Sano T, Baker D, Virag T. et al. Multiple Mechanisms linked to platelet activation results in lysophosphatidic acid and sphingosine-1-phosphate generaion in blood. J Biol Chem 2002; 277: 21197-21206.
30 Yatomi Y, Ohmori T, Rile G. et al. Sphingosine1-phosphate as a major bioactive lysophospholipid that is released from platelets and interacts with endothelial cells. Blood 2000; 96: 3431-3438.
31 Kobayashi N, Nishi T, Hirata T. et al. Sphingosine1-phosphate is released from the cytosol of rat platelets in a carrier-mediated manner. J Lipid Res 2006; 47: 614-621.
32 Bozulic LD, Malik MT, Powell DW. et al. Plasma membrane Ca2+-ATPase associates with the actin cytoskeleton via CLP36 in human platelets. Thromb Haemost 2007; 87: 587-597.
33 Bartlett GR. Phosphorus Assay in Column Chromatography. J Biol Chem 1959; 234: 466-468.
34 Powell DW, Merchant ML, Link AJ. Discovery of regulatory molecular events and biomarkers using 2D capillary chromatography and mass spectrometry. Expert Rev Proteomics 2006; 03: 63-74.
35 Powell DW, Weaver CM, Jennings JL. et al. Cluster analysis of mass spectrometry data reveals a novel component of SAGA. Mol Cell Biol 2004; 24: 7249-7259.
36 Yoon HT, Yoo HS, Shin BK. et al. Improved fluorescent determination method of cellular sphingoid bases in high-performance liquid chromatography. Arch Pharm Res 1999; 22: 294-299.
37 Caligan TB, Peters K, Ou J. et al. A high-performance liquid chromatographic method to measure sphingosine-1-phosphate and related compounds from sphingosine kinase assays and other biological samples. Anal Biochem 2000; 281: 36-44.
38 Kundu SK, Heilmann EJ, Sio R. et al. Description of an in vitro platelet function analyzer, PFA-100. Semin Thromb Hemost 1995; 21: 106-112.
39 Heilmann EJ, Kundu SK, Sio R. et al. Comparison of four commercial citrate blood collection systems for platelet function analysis by the PFA-100 system. Thromb Res 1997; 87: 159-164.
40 Keese CR, Wegener J, Walker SR. et al. Electrical wound-healing assay for cells in vitro. Proc Natl Acad Sci USA 2001; 101: 1554-1559.
41 Lee JF, Zeng Q, Ozaki H. et al. Dual roles of tight junction-associated protein, zonula occuledens-1, in sphingosine 1 phosphate-mediated endothelial chemotaxis and barrier integrity. J Biol Chem 2006; 281: 29190-29200.
42 Guidotti G. Membrane Proteins. Annu Re. Biochem 1972; 41: 731-752.
43 Davis MD, Clemens JJ, Macdonald TL. et al. Sphingosine-1-phosphate analogs as receptor antagonists. J Biol Chem 2005; 280: 9833-9841.
44 Moebius J, Zahedi RP, Lewandrowski U. et al. The human platelet membrane proteome reveals several new potential membrane proteins. Mol Cell Prot 2005; 04: 1754-1761.