The diagnosis of idiopathic immune thrombocytopenia remains a clinical diagnosis based on the exclusion of other causes of immune and nonimmune thrombocytopenia. Measurement of platelet-associated Ig (PAIg), while sensitive, is nonspecific for the diagnosis of immune thrombocytopenia. Published experience of antigen capture assays (including monoclonal antibody immobilization of platelet antigens or MAIPA) suggest a high sensitivity and specificity (70% to 80%) in selected groups of patients. In a prospective evaluation of 158 patients with thrombocytopenia from all causes, we report a sensitivity of 51% and specificity of 80% for direct MAIPA assays. MAIPA was considerably better in discriminating immune from nonimmune thrombocytopenia than two assays of PAIgG. Antiplatelet antibodies detected by MAIPA were more frequently directed against the glycoprotein (GP) IIb/IIIa than the GP Ib/IX complex. Our experience suggests that MAIPA assays are useful in the laboratory assessment of thrombocytopenia, should be performed before therapy, and that some patients with ‘nonimmune’ thrombocytopenia may have genuine antiplatelet antibodies.
A new monoclonal antibody (MoAb), SZ 2, reactive with the human platelet glycoprotein Ib complex has been produced by the hybridoma technique. SZ 2 immunoprecipitated the components of the glycoprotein Ib complex, glycoprotein Ib and glycoprotein IX, from Triton-X-100- solubilized, periodate-labeled platelets. Western blot analysis indicated that the epitope for SZ 2 was on the alpha-subunit of glycoprotein Ib. Scatchard analysis of SZ 2 binding to formaldehyde- fixed, washed platelets revealed a single class of binding sites with Kd = 6.6 +/- 3.3 X 10(-10) mol/L and 15,200 +/- 4,100 binding sites per platelet (mean +/- SD, n = 10). Intact antibody and its purified (Fab')2 fragments not only inhibited the ristocetin-dependent binding of von Willebrand factor to platelets and ristocetin-induced platelet agglutination but also inhibited platelet aggregation induced by Type I collagen and platelet-activating factor (PAF). SZ 2 inhibited platelet serotonin and beta-thromboglobulin release in response to these stimuli and also platelet thromboxane A2 formation in response to ristocetin and collagen. SZ 2 was without effect on platelet aggregation or release in response to other platelet stimuli such as ADP, thrombin, or arachidonic acid. The inhibition by SZ 2 of collagen- and PAF-induced platelet aggregation is surprising in that Bernard-Soulier syndrome platelets, which lack the glycoprotein Ib complex, respond normally to both these stimuli. SZ 2 was unreactive toward Bernard-Soulier syndrome platelets, as evaluated by fluorescence-associated cell sorting, and had no effect on the collagen- and PAF-induced aggregation of Bernard- Soulier syndrome platelets. The combined results suggest that the inhibition by SZ 2 of collagen- and PAF-induced aggregation of normal platelets is steric and are consistent with the glycoprotein Ib complex and the platelet collagen and PAF receptor(s) being adjacent in the human platelet plasma membrane.
Two new murine monoclonal antibodies, AK 1 and SZ 1, reactive with the human platelet glycoprotein (GP) Ib-IX complex have been produced by the hybridoma technique. Both AK 1 and SZ 1 immunoprecipitated the GP Ib-IX complex from Triton X-100-solubilized, periodate-labeled platelets. With trypsinized, labeled platelets, AK 1, SZ 1, and FMC 25 (epitope on GP IX) immunoprecipitated a membrane-bound proteolytic fragment of the GP Ib-IX complex consisting of GP IX and an congruent to 25,000 mol wt remnant of the alpha-chain of GP lb disulfide-linked to the beta-subunit. Unexpectedly, although AK 1 and SZ 1 immunoprecipitated purified GP Ib-IX complex, neither antibody immunoprecipitated the individual components of this complex, GP Ib or GP IX. When GP Ib and GP IX were recombined, however, AK 1 and SZ 1 again immunoprecipitated the reformed complex, strongly suggesting that both antibodies were recognizing an epitope present only on the intact complex. Cross-blocking studies indicated that AK 1 and SZ 1 recognized a very similar or identical epitope that was proximal to the epitope for FMC 25. Both AK 1 and SZ 1 bound to a similar number of binding sites (congruent to 25,000) on intact platelets as monoclonal antibodies directed against either GP lb or GP IX. The combined data suggests that GP lb and GP IX are fully complexed in the intact platelet membrane.
The diagnosis of idiopathic immune thrombocytopenia remains a clinical diagnosis based on the exclusion of other causes of immune and nonimmune thrombocytopenia. Measurement of platelet-associated Ig (PAIg), while sensitive, is nonspecific for the diagnosis of immune thrombocytopenia. Published experience of antigen capture assays (including monoclonal antibody immobilization of platelet antigens or MAIPA) suggest a high sensitivity and specificity (70% to 80%) in selected groups of patients. In a prospective evaluation of 158 patients with thrombocytopenia from all causes, we report a sensitivity of 51% and specificity of 80% for direct MAIPA assays. MAIPA was considerably better in discriminating immune from nonimmune thrombocytopenia than two assays of PAIgG. Antiplatelet antibodies detected by MAIPA were more frequently directed against the glycoprotein (GP) IIb/IIIa than the GP Ib/IX complex. Our experience suggests that MAIPA assays are useful in the laboratory assessment of thrombocytopenia, should be performed before therapy, and that some patients with ‘nonimmune’ thrombocytopenia may have genuine antiplatelet antibodies.
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