Merocyanine 540 as a fluorescent probe of altered membrane phospholipid asymmetry in activated whole blood platelets.
BACKGROUND
Platelet activation leads to the loss of a natural asymmetry of membrane phospholipids (PL) and the subsequent exposure of negatively charged PL in platelets with procoagulant activity that can be monitored routinely with annexin V (AN-V).


METHODS
Flow cytometric analysis of merocyanine 540 (MC540) binding may be the alternate choice for the monitoring of platelet procoagulant activity. Due to the increased partition of negatively charged phosphatidylserine (PS) in the membrane outer leaflet of activated platelets, the interaction with MC540 is reduced.


RESULTS
Collagen, which facilitated platelet PL bilayer symmetrization, vastly reduced MC540 fluorescence and augmented AN-V binding to platelets. Such a collagen-induced symmetrization was further augmented in the presence of thrombin receptor-activating peptide (TRAP, SFLLRNPNDKYEPF). In the presence of VO(4) ((-3)) (the inhibitor of aminophospholipid translocase), the rebuilt of membrane asymmetry was attenuated, which resulted in further reduced MC540 fluorescence and enhanced AN-V binding in activated cells. In platelets incubated with thapsigargin, the inhibitor of platelet tubular system Ca(2+) ATP-ase, which elevates intraplatelet Ca(2+) concentration, TRAP increased AN-V and reduced MC540 binding. The chelating of Ca(2+) with EGTA outside of activated platelets reduced AN-V binding, but did not affect MC540-positive platelets. The fluctuations in reduced staining with MC540 paralleled enhanced AN-V binding (r = -0.481, P < 0.01), especially for strong "procoagulant" activating agents.


CONCLUSIONS
(1) MC540 may be used in whole blood flow cytometry for the monitoring of platelet membrane symmetrization as an alternate or compounding method to AN-V. (2) Platelet staining with MC540 is sensitive to the fluctuations in the intraplatelet [Ca(2+)] during platelet activation. (3) Use of MC540 is characterized by improved diagnostic precision and reliability compared with AN-V.
Watala
Cezary
C
Laboratory of Haemostatic Disorders, Medical University of Łódź, Łódź, Poland. cwatala@csk.am.lodz.pl
Waczulikova
Iveta
I
Wieclawska
Boguslawa
B
Rozalski
Marcin
M
Gresner
Peter
P
Gwoździński
Krzysztof
K
Mateasik
Anton
A
Sikurova
Libuśa
L
D000328:Adult; D017304:Annexin A5 / Q000378:metabolism / Q000494:pharmacology; D015415:Biomarkers / Q000032:analysis; D001779:Blood Coagulation Factors / Q000378:metabolism; D001792:Blood Platelets / Q000187:drug effects* / Q000378:metabolism; D002462:Cell Membrane / Q000187:drug effects* / Q000378:metabolism; D003094:Collagen / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004578:Electron Spin Resonance Spectroscopy; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000378:metabolism / Q000494:pharmacology*; D006801:Humans; D008081:Liposomes; D010718:Phosphatidylserines / Q000378:metabolism; D015539:Platelet Activation / Q000187:drug effects*; D011506:Proteins / Q000494:pharmacology; D011744:Pyrimidinones / Q000378:metabolism / Q000494:pharmacology*; D018179:Receptors, Thrombin; D012995:Solubility; D014638:Vanadates / Q000494:pharmacology
D017304:Annexin A5; D015415:Biomarkers; D001779:Blood Coagulation Factors; D005456:Fluorescent Dyes; D008081:Liposomes; D010718:Phosphatidylserines; D011506:Proteins; D011744:Pyrimidinones; D018179:Receptors, Thrombin; C049426:platelet procoagulant factor; D014638:Vanadates; C003954:merocyanine dye; D003094:Collagen