29 Increased expression of annexin A5, via a Kozak polymorphism (-1C T), is associated with a reduced risk of myocardial infarction in young men
29 Increased expression of annexin A5, via a Kozak polymorphism (-1C T), is associated with a reduced risk of myocardial infarction in young men. atomic push microscopy, a method previously used to study the crystallization of annexin A5, to image the effects of monoclonal human being aPL antibodies within the crystal structure of the protein over phospholipid bilayers. In the Ionomycin presence of the aPL monoclonal antibodies (mAbs) and 2-GPI, the major aPL co-factor, constructions presumed to be aPL mAb-antigen complexes were associated with varying examples of disruption to the annexin A5 crystallization pattern on the bilayer. In addition, measurements of prothrombinase activity within the phospholipid bilayers showed the aPL mAbs reduced the anti-coagulant effect of annexin A5 and advertised thrombin generation. These data provide morphological evidence that support the hypothesis that aPL Ionomycin antibodies can disrupt annexin A5 binding to Ionomycin phospholipid membranes and permit increased generation of thrombin. The aPL antibody-mediated disruption of the annexin A5 anticoagulant shield may be an important prothrombotic mechanism in the aPL syndrome. The antiphospholipid (aPL) syndrome is an autoimmune disorder designated by recurrent pregnancy deficits and vascular thrombosis. 1,2 Several mechanism(s) for thrombosis in the aPL syndrome have been proposed, however, the pathophysiology of this condition has remained elusive. 3 Annexin A5 is definitely a potent phospholipid-binding anticoagulant protein previously known by additional titles including placental anticoagulant protein I 4,5 and vascular anti-coagulant 6 (observe Kim and Hajjar 7 for a recent review within the annexin family of proteins). The protein forms two-dimensional (2-D) crystal lattices over anionic phospholipid surfaces 8-10 and shields the phospholipid from availability for phospholipid-dependent coagulation reactions. 11 We previously proposed the hypothesis that aPL antibodies may promote pregnancy deficits and thrombosis by disrupting the annexin A5 anticoagulant shield. 12 The aPL antibody-mediated reduction of annexin A5 has been shown by us via ellipsometry, 13 enzyme-linked immunosorbent assay, 13,14 and by others using circulation cytometry 15 to be caused by displacement of the annexin from the antibodies. However, this has been a subject of controversy since one group, using ellipsometry, asserted that their data unambiguously showed that aPL antibodies are unable to displace annexin V from procoagulant membranes. 16 It would, therefore, be appropriate to use a more direct method to determine whether such displacement could happen. Because the binding of annexin A5 on phospholipid bilayers can be directly imaged by atomic push microscopy (AFM), 10 and because the surface topographies of the crystal forms that bind to the phospholipid bilayers have been characterized, 17 we applied this method to investigate the effects of previously characterized monoclonal human being aPL antibodies 18 within the crystal structure of annexin A5. We provide herein the 1st images of the effects on annexin A5 binding to phospholipid bilayers. We further characterized the aPL monoclonal antibodies (mAbs) by measuring their effects on annexin A5 anticoagulant activity. We found that aPL antibodies can indeed disrupt the crystallization of annexin A5 on phospholipid bilayers and that these antibodies can also reverse the potent anticoagulant effect of this protein. Materials and Methods Preparation and Characterization of Human being mAbs Three aPL mAb IgGs designated Is definitely3, CL1, and CL15, whose characteristics were previously explained 19 (IgG subclasses and light chains are demonstrated in Results and Table 1 ? ), were generated from your peripheral blood mononuclear cells of individuals with the aPL syndrome and were purified by affinity columns as previously explained. 19 The characteristics of these mAbs have been previously explained. 19 By enzyme-linked immunosorbent assay, CL1 and 15 reacted more strongly against human 2-GPI than did Is usually3, which also acknowledged the protein; interestingly, all three mAbs displayed poor reactivity against cardiolipin alone. Two human mAbs (Sigma, St. Louis, MO), from patients with monoclonal gammopathies were used as controls. Table 1. Effects TH of the Human aPL mAbs on Annexin A5 Anticoagulant Activity thead th colspan=”1″ rowspan=”1″ align=”left” valign=”bottom” /th th colspan=”1″ rowspan=”1″ align=”center” valign=”bottom” Control IgG1 /th th colspan=”1″ rowspan=”1″ align=”center” valign=”bottom” Control IgG3 /th th colspan=”1″ rowspan=”1″ align=”center” valign=”bottom” aPL Is usually3 /th th colspan=”1″ rowspan=”1″ align=”center” valign=”bottom” aPL CL1 /th th colspan=”1″ rowspan=”1″ align=”center” valign=”bottom” aPL CL15 /th /thead Immunological classification???? Subclass13333????Light chainThrombin generation (pmol/minute)????Without 2-GPI224200150705537????With 2-GPI338219137610721031 Open in a separate window Proteins Annexin A5 was purified from human placentas according to the method of Funakoshi and colleagues 4 The protein was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis using a previously characterized, affinity-purified monospecific rabbit anti-annexin A5 IgG, as described. 20,21 Human 2-GPI was purified as previously.