C1q binding to platelets induced by monoclonal antibodies and immune complexes-a flow cytometric analysis

In recent years there has been a growing interest in measurement of platelet activation. Fluorescence-activated flow cytometry has a high sensitivity and allows the study of whole blood. Platelet-bound fibrinogen, thrombospondin, the alpha-granule protein GMP 140 (CD62) and the fibrinogen receptor have been used as markers for platelet activation utilising fluorescence-labelled antibodies. However, when a labelled antibody used in flow cytometry reacts with the antigen, an immune complex is formed. We have shown that immune complexes containing mouse or rabbit IgG, but not chicken IgG, causes platelet activation. Several monoclonal antibodies directed towards platelet glycoprotein receptors also activate platelets. Suggested potential mechanisms have been direct receptor activation, FcR mediated activation and complement activation. We demonstrate that the activation in platelet-rich plasma by the monoclonal anti-GPIb antibody AN51 could not be prevented by antibodies towards the Fc-receptor, but the antibody induced an extensive binding of C1q. The findings suggest that the complement system has an important role in platelet activation. We would suggest that monoclonal antibodies intended for use in measuring platelet activation should be screened for induction of C1q and C5 binding to the platelets. Chicken antibodies are favourable for the measurement of platelet bound plasma proteins as they do not induce platelet activation.