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MULTICENTRE
STUDIES
BINDING OF β2GP1 AND OTHER PHOSPHOLIPID COFACTOR PROTEINS TO
PHOSPHOLIPIDS: ROLE OF CALCIUM AND OF ANTIBODIES: UNDERSTANDING THE
ANTICOAGULANT PARADOX?
COORDINATORS
Amiral J, Vissac AM
HYPHEN-BIOMED, Zac de Neuville Université- -95000-Neuville sur Oise,
France.
PROJECT
β2GP1 dependent Anti-Phospholipid
Antibodies (APA) can be anticoagulant in vitro and associated with Thrombosis
in vivo. This paradox is not totally explained. We studied the binding of β2GP1 to various Phospholipid (plp)
mixtures (PS, PE, PC, Clp and Phosphatidic Acid), without or with calcium. β2GP1 binding to anionic plps is much
lower in presence of Ca++. Polyclonal anti- β2GP1 antibodies highly enhanced the
binding of β2GP1 to plps (> x10), inasmuch the protein affinity for plps was low.
There was a direct relationship between the binding of β2GP1 to plps, its concentration and
the antibody concentration. β2GP1 bound as a complex with antibodies forming
a large size network. This observation strengthens the role of β2GP1 antibodies for increasing the
protein binding to anionic plps. The amount of antibodies bound via this
mechanism is dependent on the β2GP1 concentration. When antibodies are in
excess, there is a hook effect, and the amount of bound protein antibody
complex decreases. Our finding allows developing assays with a higher clinical
significance. The plp mixture is coated on the Elisa plate and there is no
saturation with animal serum. The assayed plasma is diluted in presence of
human β2GP1 and Ca++. If antibodies are present, the plp surface favours the
formation of an antibody complex, which is measured with a tag second antibody
(or alternatively by measuring the bound β2GP1). The low binding of β2GP1 to anionic plps in presence of
Ca++ and its dramatic enhancement by β2GP1 antibodies contributes to
understand the anticoagulant paradox, and the lack of correlation between LA
activity (clotting assays) and immunological testing (usually performed without
Ca++). This observation suggests a mechanism for the pathological effects of
antibodies in vivo: they enhance binding of β2GP1 to low affinity plps, onto they
form antibody-protein complexes, targeting the immune system harmful effect.
In order to check if this mechanism
was also involved for the other phospholipid cofactor proteins (Annexin V,
Prothrombin, etc…), studies on their binding to various Phospholipid (plp)
surfaces) were performed. Highly purified human proteins and well characterized
plps were used. Analyses were performed without or with Calcium. Dose dependent
binding of these proteins to plp surfaces was established. Interestingly,
binding affinity for plps was Calcium dependent for Annexin V, but in a lesser
extend for Prothrombin. These patterns were consistent with protein affinity
binding to plps. We then studied the effect of monoclonal or polyclonal
antibodies on protein binding to plps. We established assay methods using plps
coated micro Elisa plates saturated with only bovine serum albumin and
Poly-Ethylene-Glycol. The plp cofactor protein, in buffer (without or with
Ca++) or in diluted plasma, was incubated with variable antibody
concentrations. Bound components were evidenced with a tag second antibody,
which was either an anti-protein cofactor or an anti-Ig. This protocol allowed
measuring bound proteins and bound antibodies. Antibodies inhibited in a dose
dependent manner the binding to high affinity plp surfaces and dramatically
enhanced the binding to low affinity ones. There was a hook effect, the maximum
binding being dependent on both the protein and the antibody concentration. In
presence of the appropriate antibody concentration binding to low affinity plp
surfaces was at least equivalent to that to high affinity plp surfaces in the
absence of antibodies. This mechanism is similar to that observed for β2GP1.
These
preliminary data demonstrate how antibodies can target important amounts of
protein (plp cofactor) antibody complexes to low affinity plp surfaces, thus
focusing the harmful effects of the immune system to these sites. This
mechanism can be important for pathological complications in patients with plp
dependent antibodies
For additional
information and applications, please e-mail: mailto:jamiral@hyphen-biomed.com
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Last updated: 17 November 2005 |
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