Identification of critical antigen-specific mechanisms in the development of immune thrombocytopenic purpura in mice

Abstract

The pathogenic effects of antiplatelet antibodies were investigated in mice. Monoclonal antibodies (mAbs) of different immunoglobulin G subclass directed against mouse GPIIbIIIa, GPIIIa, GPIbα, GPIb-IX, GPV, and CD31 were generated and characterized biochemically. MAbs against GPIb-IX, GPV, CD31, and linear epitopes on GPIIIa had mild and transient effects on platelet counts and induced no spontaneous bleeding. Anti-GPIbα mAbs induced profound irreversible thrombocytopenia (< 3% of normal) by Fc-independent mechanisms but only had minor effects on hematocrits. In contrast, injection of intact mAbs, but not F(ab)2 fragments, against conformational epitopes on GPIIbIIIa, induced irreversible thrombocytopenia, acute systemic reactions, hypothermia, decreased hematocrits, and a paradoxical loss of surface GPIIbIIIa on platelets in vivo, the latter suggesting the formation of platelet-derived microparticles. Blockage of platelet-activating factor receptors inhibited the acute reactions, but not thrombocytopenia, loss of GPIIbIIIa, and decreases in hematocrits. Repeated injections of low doses of anti-GPIIbIIIa antibodies resulted in profound thrombocytopenia and bleeding, whereas no acute systemic reactions were observed. These data strongly suggest that the identity of the target antigen recognized by antiplatelet antibodies determines the mechanisms of platelet destruction and the severity of bleeding in mice, the latter depending on previously unrecognized anti-GPIIbIIIa-specific inflammatory mechanisms.

Flow cytometric detection of activated mouse integrin αIIbβ3 with a novel monoclonal antibody

“…We tested whether JON/A PE can be used for such studies. For that purpose, CRP (1 μg/ml) was added to washed platelets or diluted whole blood for 10 min and the samples were stained subsequently with JON/A PE in combination with p0p6 FITC (anti‐GPIX; 11). Platelets in whole blood were identified by their light scatter characteristics (forward scatter [FSC]) and Fl1 positivity (Fig.…”

Modulation of platelet caspases and life‐span by anti‐platelet antibodies in mice

“…A recent study using a large number of anti‐platelet mAbs injected in mice (2) demonstrates that the effects of anti‐platelet antibody are highly dependent upon the epitope‐recognized, rather than upon the isotype or IgG subclass. Our results with three anti‐β2 integrin mAb, which bind similarly with platelets, are in accord with this concept, since two of these antibodies (2E6 and H‐35) were neutral in term of kinetics, while one M 18.2 did raise platelet counts.…”

“…Antibodies which react with platelets are known to induce some of the thrombocytopenia observed during auto‐immune diseases in humans or mice (1). A wide variety of anti‐platelet monoclonal antibodies have been reported to induce thrombocytopenia, notably anti‐GPIb and GPIIbIIIa (2). Some of these mAb exert a thrombocytopenia by targeting platelets via their Fc‐receptors in the reticulo‐endothelial system, while others appear to act by undefined Fc‐independent mechanisms (1, 2).…”

“…A wide variety of anti‐platelet monoclonal antibodies have been reported to induce thrombocytopenia, notably anti‐GPIb and GPIIbIIIa (2). Some of these mAb exert a thrombocytopenia by targeting platelets via their Fc‐receptors in the reticulo‐endothelial system, while others appear to act by undefined Fc‐independent mechanisms (1, 2). In vitro , antibodies recognizing platelet membrane proteins have been reported to act as platelet agonists for human platelets, resulting in aggregation and/or degranulation.…”

Dominant negative Gfi1b mutations cause moderate thrombocytopenia and an impaired stress thrombopoiesis associated with mild erythropoietic abnormalities in mice

“…1 In GFI1B-RT, mutations in GFI1B lead to either the production of truncated proteins lacking the last two zinc fingers or to the disruption of the first zinc finger alone and cause mild to moderate bleeding diathesis, macrothrombocytopenia, an α -granule deficiency in platelets for most but not all cases and is also often associated with anisocytosis and poikilocytosis. [2][3][4][5][6][7][8][9][10][11][12][13] GFI1B-RT was first associated with the gray platelet syndrome (GPS), or BDPLT4, caused by mutation in the NBEAL2 gene. 14 Although these two forms of thrombocytopenia share a variety of features such as enlarged platelets with reduced α -granule content and abnormal megakaryocytes (MKs), 6,14,15 they also exhibit significant differences, such as a higher variability in α-granule deficiency in GFI1B-RT, differences in platelet aggregation function, autosomal recessive (NBEAL2) versus autosomal dominant (GFI1B) transmission and GFI1B-specific red blood cells (RBC) defects.…”