DETECTION OF ACTIVATED PLATELETS WITH MONOCLONAL ANTIBODIES

Blood tests reflecting in-vivo activation of platelets are potentially useful in evaluating patients with thrombotic diseases. Recently monoclonal antibodies have been described that react preferentially with activated platelets. We prepared an IgG2b antibody, designated RUU-AP 2.28, that reacted with a 53.000 MW protein that is located in a special subclass of platelet granules in unstimulated platelets and that is exposed on the surface of activated platelets. Increased numbers of platelets that expressed the 2.28 antigen on their surface were observed in patients undergoing cardiopulmonary bypass and in patients with acute deep venous thrombosis. The percentage of RUU-AP 2.28 positive platelets in the circulation was 3,9 ± 2.7 (SD)% in the controls, (n = 20), 24.6 ± 13.5% in patients after cardiopulmonary surgery (n = 10) and 8.5% in patients with acute deep venous thrombosis (n = 2).

In order to detect also earlier stages of platelet activation, such as secretion-independent phenomena, we produced new monoclonal antibodies by fusing spleen cells from Balb/c mice, immunized with thrombin stimulated, paraformaldehyde fixed platelets, with Ag 8653 myeloma cells. As a screening assay we used an ELISA with freshly fixed platelets or fixed thrombin-activated platelets. We detected six monoclonal antibodies (RUU-AP 1-6) specific for thrombin-activated platelets. The results of the ELISA were confirmed by flow cytofluorometry.

None of the antibodies inhibited platelet aggregation induced by ADP, collagen or ristocetin. Ascites of IgGl antibody RUU-AP 3 reacted with normal thrombin-activated platelets but did not react with thrombin-activated platelets from a patient with Glanzmann’s disease. In addition antibody RUU-AP 3 reacted with normal platelets stimulated with 1 pM of ADP. These data suggest that antibody RUU-AP 3 detects a secretion-independent conformational change in the platelet membrane glycoprotein IIb-IIIa complex.