Platelet autoantigen-autoantibody-monocyte interaction was studied by utilization of a specific monoclonal antibody (MoAb) 10E5 to trap and immobilize the GPIIb-GPIIIa complex on microtiter plates. Peripheral blood mononuclear cells (PBMC) or purified monocytes formed distinct morphologic clusters after incubation with immobilized antigen for 18 hours at 37 degrees C. PBMC of 18 and 19 patients with autoimmune thrombocytopenic purpura (ATP) formed 48 +/- 6.8 (SEM) clusters/well compared with 7.4 +/- 1.0 for control subjects, P less than .001. The number of clusters per well correlated inversely and exponentially with platelet count, r = -.8, n = 21, indicating that the GPIIb-GPIIIa autoantigen is pathophysiologically relevant. Binding of ATP PBMC to immobilized GPIIb-GPIIIa could be inhibited by F(ab')2 fragments of immunoglobulin (Ig) G of ATP patients, indicating that monocyte IgG bound to autoantigen by its F(ab')2 domain. Optimal cluster formation could be obtained with normal monocytes if preincubated with ATP IgG but not with F(ab')2 fragments of ATP IgG, indicating that ATP IgG binds to monocytes by its Fc domain. Armed monocytes (ie, normal monocytes preincubated with ATP IgG) bound to immobilized autoantigen 5.8-fold greater than normal monocytes incubated with immobilized autoantigen opsonized with ATP IgG. Armed monocyte adhesion could be inhibited 81% from 18.9 +/- 1.6 to 3.6 +/- 0.5 clusters/well by prior fixation with 0.1% formalin, whereas fixation of IgG before arming of monocytes was not inhibitory. MoAb MM41, directed against the alpha m- chain of the Mac-1 adhesive protein receptor of monocytes, inhibited cluster formation by 79%. Thus, (1) armed monocyte interaction with autoantigen is considerably more effective than monocyte interaction with opsonized autoantigen; (2) armed monocyte interaction requires specific F(ab')2-antigen recognition; and (3) monocyte-autoantigen interaction requires a secondary nonimmunologic adhesive event.

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