Analysis of Platelet Antigens for Anti-Platelet Antibodies in Itp Using Flow Cytometry

Abstract

Idiopathic thrombocytopenic purpura (ITP) is a syndrome caused by circulating antibodies reactive with the platelet membrane. The antigenic specificity of these antibodies is unknown. We have characterized new monoclonal antibodies that react with a determinant specific to GP Ib and GP Ib- Ia complex, and used flow cytometry to investigate platelets in ITP for antigenic determinants to which autoantibodies are directed. Forty cases of ITP were analyzed in detail by the platelet suspension immunofluorescence test(PSIFT) of von dem Borne et al. The monoclonal antibodies used were 5 against GP Ib-Ia complex (NNKY1-32, NNKY2-5, NNKY2-6, NNKY2-11, NNKY2-18) and 2 against GP lb (NNKY5-4, NNKY5-5). The reactivity of monoclonal antibodies was inhibited by the presence of autoantibody on platelets in some ITP patients. Differences in inhibition were found not only between monoclonal antibodies but also between cases.These results suggest that some ITP patients have circulating antibodies to GP Ib or GP II b - Ia, and that heterogeneous antibodies are present on platelets. Moreover, the presence of these autoantibodies may aggravate or initiate a bleeding tendency.

Periodic Production of Antiplatelet Autoantibody Directed against GPIIIa in Cyclic Thrombocytopenia

“…They were then incubated with NNKY 1-32, NNKY 2-6, which are anti-GPIIb/IIla complex monoclonal antibodies (mAbs) [16], or NNKY 5-5, an anti-GPIb mAh [17], for I h at room temperature (this step was deleted to mea sure the platelet-associated [PA] IgG level). After washing by centrif ugation, the platelets were incubated with fluorescein-labeled goat antihuman IgG (Cappel Products, West Chester, Pa., USA) to deter mine PAlgG levels or with goat antimouse IgG (Kirkcgaard & Perry Laboratories Inc., Gaithersburg, Md., USA) directed against each mAh.…”

“…Similar results were observed when normal control platelets prcincubated with the patient plasma in each phase were substituted for the patient's own plate lets. It has been reported that in some patients with im mune thrombocytopenic purpura, when the reactivity of mAh to patient platelets is decreased, such a patient has autoantibody against the GP which this mAb recognizes [17,20].…”

Are integrin α2β1, glycoprotein Ib and vWf levels correlated with their contributions to platelet adhesion on collagen under high-shear flow?

“…Gi9 is an anti-integrin 2 antibody and reported to inhibit platelet adhesion to collagen but not inhibit collagen-induced platelet aggregation [3]. NNKY5-5 binds to the vWf-binding site of GPIb and inhibits platelet aggregation induced by ristocetin [11]. The effect of the antibody was expressed as the percent of platelet adhesion compared to the control value (without antibody).…”

“…The extent of platelet adhesion was evaluated by two different parameters: surface coverage (%) of platelets at 3 min after the start of the flow and rate of surface coverage calculated as % coverage increase per second (%/s). Blood was also preincubated with Gi9 (10 mg/ml; Antibodies GmbH, Herford, Germany) or NNKY5-5 (10 mg/ml) [11], and platelet adhesion to type I or III collagen was analysed. Gi9 is an anti-integrin 2 antibody and reported to inhibit platelet adhesion to collagen but not inhibit collagen-induced platelet aggregation [3].…”

Von Willebrand factor accelerates platelet adhesion and thrombus formation on a collagen surface in platelet-reduced blood under flow conditions

“…The characteristics of these antibodies were reported before. [30][31][32] All other reagents and buffer components were commercially available with the highest purity. VWF/FVIII concentrate (Confact F) was supplied by Kaketsuken (Kumamoto, Japan), and it was reconstituted by adding distilled water into the concentrate according to the manufacturer's instructions.…”