Delayed-onset of procoagulant signalling revealed by kinetic analysis of COAT platelet formation

Lorenzo Alberio; Catherine Ravanat; Béatrice Hechler; Pierre H. Mangin; François Lanza; Christian Gachet

doi:10.1160/TH16-09-0711

Thrombosis and Haemostasis, Inhaltsverzeichnis

Thromb Haemost 2017; 117(06): 1101-1114
DOI: 10.1160/TH16-09-0711

Cellular Haemostasis and Platelets

Schattauer GmbH

Delayed-onset of procoagulant signalling revealed by kinetic analysis of COAT platelet formation

Lorenzo Alberio

¹UMR S949 INSERM, Établissement Français du Sang Grand Est, University of Strasbourg, Strasbourg, France

²Division of Hematology and Central Hematology Laboratory, Centre Hospitalier Universitaire Vaudois, University of Lausanne, Lausanne, Switzerland

,

Catherine Ravanat

¹UMR S949 INSERM, Établissement Français du Sang Grand Est, University of Strasbourg, Strasbourg, France

,

Béatrice Hechler

¹UMR S949 INSERM, Établissement Français du Sang Grand Est, University of Strasbourg, Strasbourg, France

,

Pierre H. Mangin

¹UMR S949 INSERM, Établissement Français du Sang Grand Est, University of Strasbourg, Strasbourg, France

,

François Lanza

¹UMR S949 INSERM, Établissement Français du Sang Grand Est, University of Strasbourg, Strasbourg, France

,

Christian Gachet

¹UMR S949 INSERM, Établissement Français du Sang Grand Est, University of Strasbourg, Strasbourg, France

› Institutsangaben

Abstract

Summary

The combined action of collagen and thrombin induces the formation of COAT platelets, which are characterised by a coat of procoagulant and adhesive molecules on their surface. Although recent work has started to highlight their clinical relevance, the exact mechanisms regulating the formation of procoagulant COAT platelets remain unclear. Therefore, we employed flow cytometry in order to visualise in real time surface and intracellular events following simultaneous platelet activation with convulxin and thrombin. After a rapid initial response pattern characterised by the homogenous activation of the fibrinogen receptor glycoprotein IIb/IIIa in all platelets, starting with a delay of about 2 minutes an increasing fraction transforms to procoagulant COAT platelets. Their surface is characterised by progressive loss of PAC-1 binding, expression of negative phospholipids and retention of α-granule von Willebrand factor. Intracellular events in procoagulant COAT platelets are a marked increase of free calcium into the low micromolar range, concomitantly with early depolarisation of the mitochondrial membrane and activation of caspase-3, while non-COAT platelets keep the intracellular free calcium in the nanomolar range and maintain an intact mitochondrial membrane. We show for the first time that the flow-cytometrically distinct fractions of COAT and non-COAT platelets differentially phosphorylate two signalling proteins, PKCα and p38MAPK, which may be involved in the regulation of the different calcium fluxes observed in COAT versus non-COAT platelets. This study demonstrates the utility of concomitant cellular and signalling evaluation using flow cytometry in order to further dissect the mechanisms underlying the dichotomous platelet response observed after collagen/thrombin stimulation.

Supplementary Material to this article is available online at www.thrombosis-online.com.

Keywords

Platelets - procoagulant - COAT - surface - intracellular signalling - calcium - phosphorylation - apoptosis - flow cytometry

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Referenzen

References
1 Heemskerk JW, Mattheij NJ, Cosemans JM. Platelet-based coagulation: different populations, different functions. J Thromb Haemost 2013; 11: 2-16.
2 Mazepa M, Hoffman M, Monroe D. Superactivated platelets: thrombus regulators, thrombin generators, and potential clinical targets. Arterioscler Thromb Vasc Biol 2013; 33: 1747-1752.
3 Bevers EM, Comfurius P, Zwaal RF. Platelet procoagulant activity: physiological significance and mechanisms of exposure. Blood Rev 1991; 5: 146-154.
4 Dachary-Prigent J, Freyssinet JM, Pasquet JM. et al. Annexin V as a probe of aminophospholipid exposure and platelet membrane vesiculation: a flow cytometry study showing a role for free sulfhydryl groups. Blood 1993; 81: 2554-2565.
5 Dachary-Prigent J, Pasquet JM, Nurden AT. Simultaneous detection of changes in cytoplasmic Ca(2+), aminophospholipid exposure and micro-vesiculation in activated platelets. Platelets 1997; 8: 405-412.
6 Alberio L, Safa O, Clemetson KJ. et al. Surface expression and functional characterization of alpha-granule factor V in human platelets: effects of ionophore A23187, thrombin, collagen, and convulxin. Blood 2000; 95: 1694-1702.
7 Polgar J, Clemetson JM, Kehrel BE. et al. Platelet activation and signal transduction by convulxin, a C-type lectin from Crotalus durissus terrificus (tropical rattlesnake) venom via the p62/GPVI collagen receptor. J Biol Chem 1997; 272: 13576-13583.
8 Dale GL, Friese P, Batar P. et al. Stimulated platelets use serotonin to enhance their retention of procoagulant proteins on the cell surface. Nature 2002; 415: 175-179.
9 Dale GL. Coated-platelets: an emerging component of the procoagulant response. J Thromb Haemost 2005; 3: 2185-2192.
10 Kempton CL, Hoffman M, Roberts HR. et al. Platelet heterogeneity: variation in coagulation complexes on platelet subpopulations. Arterioscler Thromb Vasc Biol 2005; 25: 861-866.
11 Rukoyatkina N, Begonja AJ, Geiger J. et al. Phosphatidylserine surface expression and integrin alpha IIb beta 3 activity on thrombin/convulxin stimulated platelets/particles of different sizes. Br J Haematol 2009; 144: 591-602.
12 Topalov NN, Kotova YN, Vasil’ev SA. et al. Identification of signal transduction pathways involved in the formation of platelet subpopulations upon activation. Br J Haematol 2012; 157: 105-115.
13 Choo HJ, Saafir TB, Mkumba L. et al. Mitochondrial calcium and reactive oxygen species regulate agonist-initiated platelet phosphatidylserine exposure. Arterioscler Thromb Vasc Biol 2012; 32: 2946-2955.
14 Daskalakis M, Colucci G, Keller P. et al. Decreased generation of procoagulant platelets detected by flow cytometric analysis in patients with bleeding diathesis. Cytometry B 2014; 86: 397-409.
15 Prodan CI, Stoner JA, Dale GL. Lower Coated-Platelet Levels Are Associated With Increased Mortality After Spontaneous Intracerebral Hemorrhage. Stroke 2015; 46: 1819-1825.
16 Colucci G, Stutz M, Rochat S. et al. The effect of desmopressin on platelet function: a selective enhancement of procoagulant COAT platelets in patients with primary platelet function defects. Blood 2014; 123: 1905-1916.
17 Cazenave JP, Ohlmann P, Cassel D. et al. Preparation of washed platelet suspensions from human and rodent blood. Methods Mol Biol 2004; 272: 13-28.
18 Monteiro MD, Goncalves MJ, Sansonetty F. et al. Flow cytometric analysis of calcium mobilization in whole-blood platelets. Curr Protoc Cytom 2003; 9: 9-20.
19 Tsien RY, Pozzan T, Rink TJ. Calcium homeostasis in intact lymphocytes: cytoplasmic free calcium monitored with a new, intracellularly trapped fluorescent indicator. J Cell Biol 1982; 94: 325-334.
20 Merritt JE, McCarthy SA, Davies MP. et al. Use of fluo-3 to measure cytosolic Ca2+ in platelets and neutrophils. Loading cells with the dye, calibration of traces, measurements in the presence of plasma, and buffering of cytosolic Ca2+. Biochem J 1990; 269: 513-519.
21 Goto S, Tamura N, Ishida H. et al. Dependence of platelet thrombus stability on sustained glycoprotein IIb/IIIa activation through adenosine 5’-diphosphate receptor stimulation and cyclic calcium signaling. J Am Coll Cardiol 2006; 47: 155-162.
22 June CH, Moore JS. Measurement of intracellular ions by flow cytometry. Curr Protoc Immunol 2004; 5: 5.
23 Ehrenberg B, Montana V, Wei MD. et al. Membrane potential can be determined in individual cells from the nernstian distribution of cationic dyes. Biophys J 1988; 53: 785-794.
24 Floryk D, Houstek J. Tetramethyl rhodamine methyl ester (TMRM) is suitable for cytofluorometric measurements of mitochondrial membrane potential in cells treated with digitonin. Biosci Rep 1999; 19: 27-34.
25 Li P, Nijhawan D, Wang X. Mitochondrial activation of apoptosis. Cell 2004; 116 (Suppl. 02) S57-59.
26 Kulkarni S, Jackson SP. Platelet factor XIII and calpain negatively regulate integrin alphaIIbbeta3 adhesive function and thrombus growth. J Biol Chem 2004; 279: 30697-30706.
27 Suzuki J, Umeda M, Sims PJ. et al. Calcium-dependent phospholipid scrambling by TMEM16F. Nature 2010; 468: 834-838.
28 Mattheij NJ, Braun A, van Kruchten R. et al. Survival protein anoctamin-6 controls multiple platelet responses including phospholipid scrambling, swelling, and protein cleavage. FASEB J 2016; 30: 727-737.
29 Remenyi G, Szasz R, Friese P. et al. Role of mitochondrial permeability transition pore in coated-platelet formation. Arterioscler Thromb Vasc Biol 2005; 25: 467-471.
30 Mattheij NJ, Gilio K, van Kruchten R. et al. Dual mechanism of integrin alphaIIbbeta3 closure in procoagulant platelets. J Biol Chem 2013; 288: 13325-13336.
31 Mattheij NJ, Swieringa F, Mastenbroek TG. et al. Coated platelets function in platelet-dependent fibrin formation via integrin alphaIIbbeta3 and transglutaminase factor XIII. Haematologica 2016; 101: 427-436.
32 Abaeva AA, Canault M, Kotova YN. et al. Procoagulant platelets form an alpha-granule protein-covered „cap“ on their surface that promotes their attachment to aggregates. J Biol Chem 2013; 288: 29621-29632.
33 Szasz R, Dale GL. Thrombospondin and fibrinogen bind serotonin-derivatized proteins on COAT-platelets. Blood 2002; 100: 2827-2831.
34 Munnix IC, Cosemans JM, Auger JM. et al. Platelet response heterogeneity in thrombus formation. Thromb Haemost 2009; 102: 1149-1156.
35 Yakimenko AO, Verholomova FY, Kotova YN. et al. Identification of different proaggregatory abilities of activated platelet subpopulations. Biophys J 2012; 102: 2261-2269.
36 Alberio L, Friese P, Clemetson KJ. et al. Collagen response and glycoprotein VI function decline progressively as canine platelets age in vivo. Thromb Haemost 2002; 88: 510-516.
37 Jobe SM, Wilson KM, Leo L. et al. Critical role for the mitochondrial permeability transition pore and cyclophilin D in platelet activation and thrombosis. Blood 2008; 111: 1257-1265.
38 Arachiche A, Kerbiriou-Nabias D, Garcin I. et al. Rapid procoagulant phosphatidylserine exposure relies on high cytosolic calcium rather than on mitochondrial depolarization. Arterioscler Thromb Vasc Biol 2009; 29: 1883-1889.
39 Schoenwaelder SM, Yuan Y, Josefsson EC. et al. Two distinct pathways regulate platelet phosphatidylserine exposure and procoagulant function. Blood 2009; 114: 663-666.
40 Jackson SP, Schoenwaelder SM. Procoagulant platelets: are they necrotic?. Blood 2010; 116: 2011-2018.
41 Versteeg HH, Heemskerk JW, Levi M. et al. New fundamentals in hemostasis. Physiol Rev 2013; 93: 327-358.
42 Harper MT, Molkentin JD, Poole AW. Protein kinase C alpha enhances sodium-calcium exchange during store-operated calcium entry in mouse platelets. Cell Calcium 2010; 48: 333-340.
43 Adam F, Kauskot A, Rosa JP. et al. Mitogen-activated protein kinases in hemostasis and thrombosis. J Thromb Haemost 2008; 6: 2007-2016.
44 Siljander P, Farndale RW, Feijge MA. et al. Platelet adhesion enhances the glyco-protein VI-dependent procoagulant response: Involvement of p38 MAP kinase and calpain. Arterioscler Thromb Vasc Biol 2001; 21: 618-627.
45 Pasquet JM, Dachary-Prigent J, Nurden AT. Microvesicle release is associated with extensive protein tyrosine dephosphorylation in platelets stimulated by A23187 or a mixture of thrombin and collagen. Biochem J 1998; 333: 591-599.
46 Nofer JR, Noll C, Feuerborn R. et al. Low density lipoproteins inhibit the Na+/H+ antiport in human platelets via activation of p38MAP kinase. Biochem Biophys Res Commun 2006; 340: 751-757.
47 Rosskopf D. Sodium-hydrogen exchange and platelet function. J Thromb Thrombolysis 1999; 8: 15-24.

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