anti-P1A1 Antibody Research Articles

Reactivity of autoantibodies from chronic ITP patients with recombinant glycoprotein Ilia peptides

Chronic immune thrombocytopenia is an autoimmune disorder characterized by destructive thrombocytopenia due to the formation of autoantibodies against platelet-associated antigens. Most antiplatelet autoantibodies react with either the platelet glycoprotein IIb/IIIa or Ib/IX complex, whereas some plasma autoantibodies react with glycoprotein IIIa. Previous studies from our laboratory suggested that most platelet-associated autoantibodies to platelet GPIIb/IIIa, which bind to the intact complex, bind much less avidly to the EDTA-dissociated complex, suggesting that the epitopes were complex-dependent. To evaluate this further we have studied the binding of platelet-associated autoantibody and plasma auto- and alloantibody eluates to large recombinant GPIIIa peptides: peptide 1 (GPIIIa Gly1-Val200); peptide 2 (GPIIIa Arg150-Glu400); peptide 3 (GPIIIa Lys350-Asp550); peptide 4 (GPIIIa Asn450-Val700) and peptide 5 (GPIIIa Trp715-Thr762, cytoplasmic fragment). Of the 33 platelet-associated antibody eluates tested, all bound avidly to the GPIIb/IIIa complex, but only one showed significant binding (>3 SD above control values) to one of the immobilized peptides (peptide 3). Conversely, antibodies known to bind to specific regions of GPIIIa (murine monoclonal antibody, anti-LIBS2; plasma autoantibody against the GPIIIa cytoplasmic fragment and anti-P1A1 antibody) all bound avidly to the GPIIIa peptide containing the appropriate epitope. Based on these and our previous results, we conclude that platelet-associated antibodies from chronic ITP patients rarely bind to epitopes localized to GPIIIa alone.

The effect of intravenous immunoglobulin on placental transfer of a platelet-specific antibody: anti-P1A1.

The isolated perfused lobule of human placenta was used as an in-vitro model to study the effect of intravenous immunoglobulin (IVGG) on the placental transfer of a human platelet-specific antibody (anti-P1A1). Normal human IgG was shown to transfer from the maternal to the fetal circulation of the placental model after a lag period of 2-3 h. IVGG also transferred across the placenta but only after a longer lag period (3-4 h) than normal human IgG at the same concentration, which suggests that IVGG may contain a factor that inhibits the transfer of its own component IgG. The sensitive Western immunoblotting technique was used to demonstrate progressive transfer of anti-P1A1 antibody to the fetal circulation after a 2-3 h lag period. When IVGG and anti-P1A1 antibody were added simultaneously to the maternal circulation, the transfer of platelet-specific antibody was strongly inhibited by IVGG. The inhibitory effect of IVGG on anti-P1A1 antibody transfer was consistent for three different batches of the same IVGG product (Sandoglobulin). These studies provide the first scientific data to support the use of IVGG to inhibit antiplatelet antibody transfer as part of the antenatal management of neonatal alloimmune thrombocytopenia.

Complement activation in vitro by antiplatelet antibodies in chronic immune thrombocytopenic purpura.

Many chronic ITP patients have increased amounts of platelet-associated IgG, C3, C4 and C9, suggesting in vivo complement activation. In this study, we assessed the ability of various antiplatelet antibodies (APA) to activate and deposit complement proteins and to cause platelet lysis in vitro. Platelet sensitization with rabbit APA, anti-P1A1 antibody (one patient), anti-HLA antibody (two patients) and ITP autoantibodies (four patients) resulted in the deposition of C4 and C3 onto platelets in an amount proportional to the quantity of antibody-containing sera used to sensitize the platelets. Although C9 deposition onto platelets could not be quantitatively demonstrated on platelets sensitized with ITP serum, platelet lysis (51Cr release) was noted after incubation with each of three ITP sera and complement. When compared, ITP autoantibodies, anti-HLA and anti-P1A1 antibodies activated complement to a similar degree. We conclude that some autoantibodies in chronic ITP activate the classical complement pathway. The demonstration of in vitro platelet lysis by autoantibodies and complement suggests that in vivo platelet lysis may occur in some chronic ITP patients.

Autoantibodies Against the Platelet Glycoprotein Ilb/IIIa Complex in Patients With Chronic ITP

Chronic idiopathic thrombocytopenic purpura (ITP) is caused by an antibody reactive with platelet-associated antigens. The present studies provide direct evidence that some patients with chronic ITP have autoantibodies against the platelet glycoprotein (GP) IIb/IIIa complex. Microtiter wells, coated with a monoclonal antibody (2G12) specific for GPIIb/GPIIIa were reacted with GPIIb/GPIIIa contained in a platelet extract. Control wells containing the same antibody were reacted with a cell extract containing no GPIIb/GPIIIa. After washing, the wells were reacted with patient or control plasma, and IgG binding was detected using 125I-Fab2-anti-human IgG. Assay values were expressed as binding ratios (cpm GPIIb/GPIIIa wells/cpm control wells). Plasma from 5 of 56 patients with chronic ITP had ratios (1.36-3.14) greater than 3 standard deviations above the mean (+/- SD) of control plasmas--0.93 +/- 0.12. Elevated values were also noted in two patients with anti-P1A1 antibody (ratios greater than 30) and in one patient with Hodgkin's disease and an ITP-like syndrome (ratio 1.53). Normal values were noted in 34 patients with a variety of immune and nonimmune diseases. Plasma from two of the positive ITP patients was reacted with 125I-surface-labeled platelets and, after solubilization, the IgG and bound antigen were precipitated with Staph-A. Autoradiographs from SDS-PAGE electrophoresis of the Staph-A-bound proteins shows two radioactive bands consistent in size with GPIIb and GPIIIa.

Autoantibodies against the platelet glycoprotein IIb/IIIa complex in patients with chronic ITP

Chronic idiopathic thrombocytopenic purpura (ITP) is caused by an antibody reactive with platelet-associated antigens. The present studies provide direct evidence that some patients with chronic ITP have autoantibodies against the platelet glycoprotein (GP) IIb/IIIa complex. Microtiter wells, coated with a monoclonal antibody (2G12) specific for GPIIb/GPIIIa were reacted with GPIIb/GPIIIa contained in a platelet extract. Control wells containing the same antibody were reacted with a cell extract containing no GPIIb/GPIIIa. After washing, the wells were reacted with patient or control plasma, and IgG binding was detected using 125I-Fab2-anti-human IgG. Assay values were expressed as binding ratios (cpm GPIIb/GPIIIa wells/cpm control wells). Plasma from 5 of 56 patients with chronic ITP had ratios (1.36–3.14) greater than 3 standard deviations above the mean (+/- SD) of control plasmas--0.93 +/- 0.12. Elevated values were also noted in two patients with anti-P1A1 antibody (ratios greater than 30) and in one patient with Hodgkin's disease and an ITP-like syndrome (ratio 1.53). Normal values were noted in 34 patients with a variety of immune and nonimmune diseases. Plasma from two of the positive ITP patients was reacted with 125I-surface-labeled platelets and, after solubilization, the IgG and bound antigen were precipitated with Staph-A. Autoradiographs from SDS- PAGE electrophoresis of the Staph-A-bound proteins shows two radioactive bands consistent in size with GPIIb and GPIIIa.

Identification of human megakaryocytes derived from pure megakaryocytic colonies (CFU-M), megakaryocytic-erythroid colonies (CFU-M/E), and mixed hemopoietic colonies (CFU-GEMM) by antibodies against platelet associated antigens.

Pure megakaryocytic colonies, megakaryocytic-erythroid colonies and mixed hemopoietic colonies can be cultured from human bone marrow under appropriate culture conditions. Human plasma and mercaptoethanol support the growth for these different types of hemopoietic colonies. However, the addition of medium conditioned by leucocytes in the presence of phytohemagglutinin (PHA-LCM), as a source of thrombopoietin, is required for the formation of megakaryocytic colonies or megakaryocytes within mixed colonies. Megakaryocytes were identified by their typical morphological appearance in culture. Pure megakaryocytic colonies, megakaryocytic-erythroid colonies and mixed colonies were plucked by micropipette and analysed by the PAP-slide technique using antibodies to human factor VIII-related protein or serum derived from a patient with posttransfusion purpura; this particular serum demonstrated anti-P1A1 antibody activity. These antibodies might provide an excellent probe to identify megakaryocytic progeny from committed and non-committed hemopoietic progenitors, facilitating studies of early events in megakaryopoiesis.

Effect of anti-P1A1 antibody on human platelets. I. The role of complement

We studied the interaction of complement with human platelets. Complement was activated by IgG anti-P1A1 antibody obtained from 3 patients with the post-transfusion purpura syndrome. We used a heparin- plasma buffer system that permits complement activation and also preserves platelet function. With this system complement activation was efficient, and platelet immune alteration was extensive. Anti-P1A1 antibody was effective only in the presence of complement, in which case both platelet lysis and serotonin release (release reaction) in the absence of lysis were observed. Platelet lysis, as assessed by 51Cr loss, required 10-fold more antibody than was necessary to induce platelet aggregation and release of 14C-serotonin. This platelet release reaction required an intact classic complement sequence through C6. The extent of platelet serotonin release parallelled the depletion of C1 and C4 from platelet-rich plasma. Concentrations of antibody insufficient to induce platelet aggregation and serotonin release could still activate C1 and deposit increased C3 on the platelet surface. These studies demonstrated that complement activation by anti-P1A1 antibody can alter human platelets in a nonlytic system. Several phases of complement-mediated human platelet alteration are possible, depending on the concentration of anti-P1A1 antibody.

Effect of Anti-P1A1 Antibody on Human Platelets. I. The Role of Complement

We studied the interaction of complement with human platelets. Complement was activated by IgG anti-P1A1 antibody obtained from 3 patients with the post-transfusion purpura syndrome. We used a heparin-plasma buffer system that permits complement activation and also preserves platelet function. With this system complement activation was efficient, and platelet immune alteration was extensive. Anti-PiA1 antibody was effective only in the presence of complement, in which case both platelet lysis and serotonin release (release reaction) in the absence of lysis were observed. Platelet lysis, as assessed by 51Cr loss, required 10-fold more antibody than was necessary to induce platelet aggregation and release of 14C-serotonin. This platelet release reaction required an intact classic complement sequence through C6. The extent of platelet serotonin release paralleled the depletion of C1 and C4 from platelet-rich plasma. Concentrations of antibody insufficient to induce platelet aggregation and serotonin release could still activate C1 and deposit increased C3 on the platelet surface. These studies demonstrated that complement activation by anti-P1A1 antibody can alter human platelets in a nonlytic system. Several phases of complement-mediated human platelet alteration are possible, depending on the concentration of anti-PlA1 antibody.

Effect of anti-P1A1 antibody on human platelets. II. Mechanism of the complement-dependent release reaction

We studied the mechanism by which complement activated by anti-P1A1 antibody elicits the platelet release reaction. Anti-P1A1 antibody mediates its action through the classic complement pathway, and its effect depends on the concentration of IgG antibody on the platelet surface. At relatively high concentrations of anti-P1A1 antibody the release reaction was mediated by a mechanism in part independent of extracellular ADP and metabolic energy and inhibited by only high concentrations of PGE1. However, at lower concentrations of anti-P1A1 antibody the release reaction was dependent on metabolic energy and ADP, and the concentration of PGE1 required to inhibit platelet release was similar to that required to inhibit ADP-induced release. The cyclooxygenase inhibitor acetylsalicylic acid inhibited the release reaction at all nonlytic antibody levels studied. None of the agents studied inhibited the induction of platelet lysis by very high concentrations of anti-P1A1 antibody, and no effect of antibody on platelet 14C-serotonin uptake was observed at antibody concentrations that did not mediate direct in vitro alteration. These studies suggest the possible use of pharmacologic agents in modifying some complement-mediated platelet alterations.

Effect of Anti-P1A1 Antibody on Human Platelets. II. Mechanism of the Complement-Dependent Release Reaction

We studied the mechanism by which complement activated by anti-P1A1 antibody elicits the platelet release reaction. Anti-P1A1 antibody mediates its action through the classic complement pathway, and its effect depends on the concentration of IgG antibody on the platelet surface. At relatively high concentrations of anti-PiA1 antibody the release reaction was mediated by a mechanism in part independent of extracellular ADP and metabolic energy and inhibited by only high concentrations of PGE. However, at lower concentrations of anti-P1A1 antibody the release reaction was dependent on metabolic energy and ADP, and the concentration of PGE, required to inhibit platelet release was similar to that required to inhibit ADP-induced release. The cyclooxygenase inhibitor acetylsalicylic acid inhibited the release reaction at all nonlytic antibody levels studied. None of the agents studied inhibited the induction of platelet lysis by very high concentrations of anti-PiA1 antibody, and no effect of antibody on platelet 14C-serotonin uptake was observed at antibody concentrations that did not mediate direct in vitro alteration. These studies suggest the possible use of pharmacologic agents in modifying some complement-mediated platelet alterations.