Antiphospholipid Antibodies Impair the Clearance of Apoptotic Platelets.

Background: Thrombocytopenia is frequent in patients with the Antiphospholipid Syndrome (APS). The mechanism(s) that lead to abnormal platelet destruction is not understood. Phosphatidylserine (PS) that is exposed in the outer leaflet of the membrane of aged human platelets (AHP) and β₂glycoprotein I (β₂GPI) mediate their phagocytosis by macrophages without inducing inflammatory or immune responses. We hypothesized that antiphospholipid antibodies (aPL) affect the clearance of AHP and induce immunogenicity of AHP leading to the production of anti-platelet antibodies.

Methods: To examine that question, we studied phagocytosis of AHP by macrophages in the presence of β₂GPI, IgG aPL or control IgG (IgG-NHS). AHP labeled with CM-Orange, were incubated with β₂GPI and aPL IgG or with IgG-NHS, and added to a monolayer of cultured phagocytes. The fluorescent-positive phagocytes (FPPC) were then tested by fluorescence microscopy. Then the cells were trypsinized and analyzed by flow cytometry. We also examined the immunogenicity (production of anti-platelet antibodies) of AHP treated with IgG aPL and with IgG-NHS by immunizing Balb/c mice with AHP and IgG-aPL or IgG-NHS. The patterns of reactivity of the sera of the immunized mice was examined by immunoblot of human platelet lysates.

Results: aPL IgG produced a significantly lower % FPPC compared to the IgG-NHS-treated cells (15.33 ± 5.31 vs 89.0 ± 7.94, respectively). This was confirmed in the flow cytometric studies: IgG aPL produced significantly lower % of FPPC when compared to IgG-NHS-treated platelets (42.49 ±11.77 vs 78.11± 5.43, respectively).. Mice immunized with AHP and aPL IgG produced significantly higher titers of anti-platelet antibodies (as detected by ELISA) when compared to mice immunized with IgG-NHS (p=0.0033). Furthermore, there was a different pattern of reactivity of the sera with respect to recognition of platelet antigens, when sera of immunized mice were analyzed by immunoblot.

Conclusions: The data indicate that aPL impair the clearance of apoptotic platelets and affect their immunogenicity. This may lead to the production of anti-platelet antibodies and thrombocytopenia in APS.