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.
