In vitro model of annexin A5 crystallization on natural phospholipid bilayers observed by atomic force microscopy.
Annexin A5 is a potent anticoagulant protein with a thrombomodulatory function. It is frequently mentioned with systemic inflammatory autoimmune disease, which share higher vulnerability to cardiovascular diseases. The protein has the ability to bind to membranes containing negatively charged phospholipids in a calcium-dependent manner. The potent anticoagulant properties of the protein are a consequence of this crystallization, which forms the lattice of annexin A5 over phospholipid surface, blocking its availability for coagulation reactions. Crystallization of annexin A5 has been proven on homogeneous synthetic phospholipids. However, the crystallization of annexin A5 on inhomogeneous, naturally derived phospholipid surfaces, in p3 and p6 crystal form, has now been reported for the first time. Atomic force microscopy was chosen for the observation of the crystallization of annexin A5 on different solid supported phospholipid bilayers. In this study model, the optimal results were obtained by using: 0.5 mg/ml lipid vesicles suspension (70% phosphatidylcholine, 30% phosphatidylserine) in HEPES buffer saline (HBS) with 2 mM CaCl(2), large unilamellar vesicles with sizes around 200 nm, 41 degrees C of phase transition temperature and 21 microg/ml of native annexin A5 in HBS with 2 or 20 mM CaCl(2). Results were evaluated by imaging and force measurements. Demonstration that native annexin A5 is able to spontaneously crystallize on naturally derived, inhomogeneous phospholipids is supporting the putative role of annexin A5 crystal structures as possible antithrombotic shield. This in vitro system is probably more appropriate for studying the pathogenetic role of antiphospholipid antibodies.
Irman
Spela
S
Division of Internal Medicine, Department of Rheumatology, University Medical Centre, Vodnikova cesta 61, SI-1000, Ljubljana, Slovenia.
Miha
Skarabot
S
Igor
Musevic
M
Rozman
Blaz
B
Bozic
Borut
B
D017304:Annexin A5 / Q000737:chemistry*; D003460:Crystallization; D005260:Female; D006801:Humans; D008051:Lipid Bilayers / Q000737:chemistry; D018625:Microscopy, Atomic Force / Q000379:methods*; D010743:Phospholipids / Q000737:chemistry*; D010920:Placenta / Q000737:chemistry; D011247:Pregnancy
D017304:Annexin A5; D008051:Lipid Bilayers; D010743:Phospholipids