Platelet-reactive Autoantibodies Research Articles

Vincristine-Loaded Platelets Coated with Anti-CD41 MAbs: A New Proposal for the Treatment of Immune Thrombocytopenia

Abstract Objective: Immune thrombocytopenia (ITP) is an autoimmune disorder in which platelet-reactive autoantibodies accelerate the destruction of platelets. Macrophages play a crucial role in ITP through IgG-Fcγ receptor-mediated platelet clearance. Patients responding to initial therapies often troubled with frequent relapses, severe reverse reactions and poor life quantity. Therefore, searching new effective strategies for ITP treatment is in urgent need. Methods: In this study, platelets coated with anti-CD41 antibodies, which can mimic autoantibody-binding platelets in ITP patients, were used as a smart drug delivery system to load vincristine (VCR). Vincristine-loaded platelets coated with anti-CD41 mAbs (CD41-VLP) can selective release VCR in macrophages through IgG-Fcγ receptor-mediated phagocytosis. Scanning electron microscopy (SEM), dynamic light scattering (DLS), and platelet aggregation assays were performed to evaluate the morphological and functional changes of CD41-VLP. The encapsulation efficiency, drug loading and intracellular VCR concentration were determined using high performance liquid chromatography (HPLC). Macrophages were derived from THP-1 induced by phorbol 12-myristate 13-acetate (PMA). The expression of cell differentiation antigen (CD11b and CD14) and was detected by flow cytometry (FCM). The growth inhibition of CD41-VLP on macrophages was detected by Cell Counting Kit-8 (CCK-8) assay and the macrophage apoptosis was quantified by FCM. The possible mechanism of CD41-VLP on macrophages was further investigated by western blotting. Results: platelets coated with anti-CD41 antibodies could load VCR with high drug loading and encapsulation efficiency. There is no significant difference in morphology and function between platelets and CD41-VLP. Meanwhile, VCR was released from CD41-VLP in a pH-triggered behavior and VCR would be rapidly released in an acidic condition. The THP-1 derived macrophages was detected with a strong ability of phagocytosis and a high expression of CD11b and CD14. In the group of macrophages treated with CD41-VLP, intracellular concentration of VCR was significant increased compared with other groups, which demonstrates that Cd41-VLP can selectively deliver VCR to macrophages. In the CD41-VLP group, the growth inhibition rate was also much higher than other groups and the apoptosis rate was significant increased compared with other groups. These results suggest that CD41-VLP can enhance the cytotoxicity of VCR against macrophages. Furthermore, In the CD41-VLP group, the expressions of pro-apoptotic proteins,including Bax, caspase-3 and CyclinB1, were significantly increased and the expression of Bcl-2, anti-apoptotic protein, was decreased. This finding indicates that CD41-VLP might improve the effects of VCR on macrophages through regulating the expression of apoptosis-related proteins. Conclusion: CD41-VLP can improve the cytotoxicity of VCR against macrophages, thus establishing a new targeted therapy for ITP treatment. Disclosures No relevant conflicts of interest to declare.

Pathophysiology of Autoimmune Thrombocytopenia: Current Insight with a Focus on Thrombopoiesis.

Immune thrombocytopenia (ITP) is an autoimmune disease characterized by a significant reduction in the number of circulating platelets which is frequently associated with bleeding. The total count of platelets in the body is finely regulated by the balance between platelet production and destruction. Although the pathogenesis of ITP is still not completely elucidated, it is largely recognized that the low platelet count observed in ITP patients is due to alterations of both mechanisms. An abnormal proliferation of autoreactive T cells leading to the breakdown of immune tolerance to platelet antigens is suggested to be responsible for the up-regulated proliferation of autoantibody producing B cells. Consequently, the immune response induces enhanced T cell-mediated cytotoxicity and antibody-mediated platelet destruction through phagocytosis, complement activation and apoptosis. An additional contribution to the pathophysiology of ITP is given by alterations of thrombopoiesis caused by platelet-reactive autoantibodies or cytotoxic T cells leading to impaired megakaryocyte differentiation and platelet production. All these processes involved in ITP pathophysiology account for the complexity and heterogeneity in the clinical manifestation and therapy responsiveness of this disorder. For this reason, a better understanding of the different underlying mechanisms in ITP is necessary to develop more efficient therapeutic treatments in the future. In this review, we will provide an update on the pathophysiology of ITP with a particular focus on the impact of impaired thrombopoiesis.

Vincristine-loaded platelets coated with anti-CD41 mAbs: a new macrophage targeting proposal for the treatment of immune thrombocytopenia.

Immune thrombocytopenia (ITP) is an autoimmune disorder in which platelet-reactive autoantibodies accelerate the destruction of platelets. Macrophages play an important role in ITP through Fc receptor (FcR)-mediated antigen presenting and platelet clearance. In this study, a novel drug delivery system of vincristine-loaded platelets coated with anti-CD41 mAbs (CD41-VCR-PLT, CD41-VLP) was successfully established. The therapeutic effects and safety of CD41-VLP in vitro and in vivo were evaluated, and the possible mechanism was also explored. The results showed that PLT-CD41 could load VCR with high drug loading (DL) and encapsulation efficiency (EE), which were up to 41.16 ± 1.92% and 60.73 ± 2.79%, respectively, where platelets had no obvious morphological or functional changes. CD41-VLP could facilitate vincristine accumulation in macrophages, where the intracellular VCR concentration was 30.72 ± 3.11% at 72 h, which was significantly increased compared with the other groups (P < 0.01), thus inhibiting macrophage cell viability and inducing apoptosis. The cell viability inhibition rate and total apoptosis rate were 73.06 ± 5.26% and 69.70 ± 4.26%, respectively, both much higher than those of the other groups (P < 0.05). In the ITP mouse model, CD41-VLP increased the platelet count in peripheral blood, which was 720 ± 197.98 × 109 L-1, and significantly improved the platelet count compared with that in the VCR group (P < 0.05); moreover, it reduced the systemic toxicity and peripheral neurotoxicity of vincristine. The possible mechanism was that CD41-VLP could precisely target M1 macrophages in spleen and liver tissues through FcγR, thus reducing the platelet destruction caused by M1 macrophages. Therefore, CD41-VLP provides a new targeted therapy for ITP treatment.

Potential Roles of Antiphospholipid Antibodies in Generating Platelet-C4d in Systemic Lupus Erythematosus.

Premature, accelerated onset of atherothrombotic disease is prevalent in patients with systemic lupus erythematosus (SLE). Most, if not all, atherothrombotic diseases are likely to involve platelets and complement. Previously, we discovered that platelets bearing complement activation product C4d (P-C4d) are present in SLE patients, and are significantly associated with antiphospholipid (aPL) antibody positivity and stroke in SLE patients. The goal of the present study was to further elucidate the role of aPL and other platelet-reactive autoantibodies in the generation of P-C4d. To determine the association between P-C4d and aPL antibodies, the serum levels of aPL antibodies and P-C4d of 180 SLE patients were measured by enzyme-linked immunoassays and flow cytometry, respectively. To investigate the role of aPL antibodies, and possibly other autoantibodies as well, in mediating the generation of P-C4d, in vitro 2-step P-C4d induction experiments were performed. The results showed that the presence and levels of aPL antibodies in the serum were specifically elevated in SLE patients with positive P-C4d. The plasma and immunoglobulins purified from SLE patients who were positive for P-C4d and aPL were capable of inducing C4d deposition on normal platelets in vitro. The capacity of SLE plasma in inducing P-C4d appeared to correlate proportionately to the serum aPL levels. Collectively, the results demonstrate that both aPL and other platelet-reactive autoantibodies may participate in mediating the generation of P-C4d in SLE patients.

Autoantibody-mediated generation of platelet-bound C4d in Systemic Lupus Erythematosus (SLE)

Abstract Premature and accelerated onset of atherothrombotic disease is prevalent in patients with SLE. Most, if not all, atherothrombotic diseases are likely to involve platelets and the complement system in some capacity. Previously, we discovered that platelets bearing complement activation product C4d (P-C4d) are present in approximately 20% of SLE patients and are significantly associated with anti-phospholipid (aPL) antibody positivity and stroke in SLE patients. The goal of the present study was to elucidate the role of aPL and other platelet-reactive autoantibodies in the generation of P-C4d. To determine the association between P-C4d and aPL, the level of P-C4d in 180 SLE patients was measured by flow cytometry. Serum level of aPL, including anti-cardiolipin (aCL) and anti-b2 glycoprotein I (anti-b2GPI) antibodies, in these patients was measured by enzyme-linked immunosorbent assays. To investigate the role of aPL, and possibly other autoantibodies as well, in mediating the generation of P-C4d, in vitro 2-step transfer experiments using normal platelets in combination with plasma or immunoglobulin derived from SLE patients were performed. The results showed that the presence and levels of serum aCL and anti- anti-b2GPI were highly specific for SLE patients with positive P-C4d. The plasma and immunoglobulins purified from SLE patients who were positive for P-C4d and aCL/anti-b2GPI were able to induce C4d deposition on normal platelets in vitro. The capacity of SLE plasma in inducing P-C4d appeared to correlate proportionately to the serum aCL levels. Collectively, the results demonstrate that both aPL, particularly aCL, and other platelet-reactive autoantibodies participate in mediating the generation of P-C4d in SLE patients.

Mechanistic properties of intravenous immunoglobulin in murine immune thrombocytopenia: support for FcγRIIB falls by the wayside

Immune thrombocytopenia (ITP) is an autoimmune disorder characterised by platelet clearance resulting from the production of platelet-reactive autoantibodies. Platelet clearance appears to occur mainly via phagocytosis in the mononuclear phagocytic system, although T-cell-mediated platelet destruction, platelet apoptosis and dysregulation of platelet production can also play a role in disease pathogenesis. One of the most successful treatments for ITP is intravenous immunoglobulin (IVIg), and while it has been used in ITP for over 30 years, its mechanism(s) of action still remain unclear. Animal models of ITP have proven useful in understanding IVIg's immunomodulatory properties, providing a valuable tool to test new mechanistic theories as well as further explore the soundness of older ones. This model has also provided the key evidence that IVIg exerts its effects via activating receptors for IgG Fc, specifically FcγRIII, via formation of IgG dimers or immune complexes. Here, we discuss the validity of one prominent theory of IVIg function, anti-inflammatory activity mediated through the inhibitory Fcγ receptor FcγRIIB, and review evidence to suggest that this theory is not likely valid in the practical sense.

New CNV Regions Identified in ITP Provide Evidence for Genetic Predisposition

Abstract Immune thrombocytopenia (ITP) is an autoimmune disease resulting from inhibition of megakaryopoiesis and destruction of platelets by platelet reactive autoantibodies. Adult-onset ITP usually exhibits a relapsing chronic course and is often associated with other disorders of autoimmune or infectious origin, including systemic lupus erythematosus (SLE), lymphoproliferative diseases, common variable immunodeficiency (CVID) disease, and human immunodeficiency virus (HIV) infection. The exact pathophysiology of ITP has not been fully elucidated. Heterogeneity in disease presentation and in response to immunosuppressive therapy suggests different underlying mechanisms; and thus justifies the investigation of relevant genetic associations. Here we tried to unravel the genetic background of the immune dysfunction in ITP by using SNP array technology. We analyzed the blood samples of 12 adults (6 males and 6 females) with primary refractory ITP, by SNP array approach to identify candidate genomic regions. None of the patients showed clinical and laboratory findings of autoimmune diseases, malignancy or infections. All individuals were genotyped using Illumina Human HumanCytoSNP-12 BeadChip (300K). Whole genome SNP genotyping data were delineated using GenomeStudio software platform. The genotyping data was obtained by further analyzes in terms of copy number variation (CNV). In total we observed 14 CNVs in 12 patients. All CNVs were on autosomal chromosomes; 8 duplications and 6 deletions (gain 2p, 9q, 13q, 14q, 17p, 17q, 20q; loss 5q, 7p, 7q, 15q, 17q, 19q). The rearrangements sizes were between 45.42 Mb and 1.08 Mb. Moreover in five patients the duplications were in the form of mosaic structural genomic rearrangements. These mosaic duplications were found at 17q21.31 (0.2 Mb), 2p23.2 (0.34 Mb), 9q21.2 (10.89 Mb), 9q31.2 (17.77 Mb), 14q13.2 (0.37 Mb), 17p12 (1.46 Mb), 20q11.21 (14.24 Mb). None of the detected CNV regions could be demonstrated in two independent cohorts of 30 individuals with non-hematological disorders and healthy controls. Little is known on the genetic background of immune thrombocytopenia. Our aim was to investigate the impact of CNVs on the pathophysiology of ITP. We found several gains/losses in different chromosomal regions. However, the most striking result of our study was the detection of mosaic patterns that have been reported to most commonly associate with hematological cancers. These mosaic patterns may be associated with clonal expansion of T- or B-cells, leading to the immune dysfunction seen in ITP. Evaluation of the detected regions and their relation to B and T- cell clonality is ongoing. These preliminary results provide early evidence for the presence of predisposing CNVs in adult onset ITP. Disclosures No relevant conflicts of interest to declare.

A 7‐Year Case of Furosemide‐Induced Immune Thrombocytopenia

Immune thrombocytopenia has been attributed to many causes. Several drugs have been implicated as culprits in causing drug-induced thrombocytopenia. Although the mechanism for this type of thrombocytopenia is not well understood, at least three types of antibodies appear to be involved: drug-dependent antibodies, hapten-dependent antibodies, and drug-induced platelet-reactive autoantibodies. In this report, we describe a case in which furosemide was identified as the probable cause of drug-induced thrombocytopenia in an 84-year-old man with chronic symptomatic idiopathic thrombocytopenia for seven years before discovery. The patient's platelet count and daily furosemide dose, both intravenous and oral, were documented throughout his medical history. A dose-dependent change in platelet count was observed in association with the furosemide dose. His platelet count increased on discontinuation of furosemide and beginning of torsemide. Several months after discontinuation of furosemide, his platelet count increased to a 9-year high of 206 × 10³/mm³ from a low of 36 × 10³/mm³ while receiving furosemide therapy. Based on the observations of this case report, clinicians should more readily consider furosemide as a potential cause of thrombocytopenia.

Detection of expression of IL-18 and its binding protein in Egyptian pediatric immune thrombocytopenic purpura

Immune thrombocytopenic purpura (ITP) is an autoimmune disorder, characterized by dysfunctional cellular immunity including the presence of activated platelet specific autoreactive T cells that recognize and respond to autologous platelet antigens. Autoreactive T cells drive the generation of platelet reactive autoantibodies by B cells as well as T-cytotoxic cell-mediated lysis of platelets. Interleukin-18 (IL-18) is a mediator of T helper type 1 cell responses synergistically with IL-12 that initiate and promote host defense and inflammation. IL-18 has a specific binding protein (IL-18BP) which belongs to the immunoglobulin superfamily. In the present study, serum level and messenger RNA( mRNA) expression of IL-18 as well as IL-18BP mRNA expression were measured in peripheral blood mononuclear cells (PBMNCs) of 100 Egyptian pediatric patients with ITP (70 acute and 30 chronic). In addition to this, we recruited 80 healthy volunteers in order to investigate the possible association between the imbalance of IL-18 and IL-18 BP expressions and the pathogenesis of ITP. IL-18 serum level and mRNA expression were not elevated in cases more than in the control group, but IL-18 mRNA was higher in chronic cases when compared to the acute ones (p = 0.031) and there was a good negative correlation between the platelet count and serum IL-18. IL-18 BP m-RNA was slightly elevated in cases more than in the control group (95% Confidence interval = 1.15–2.01). Our results were not supportive for previous findings of elevated IL18/BP mRNA ratio in ITP patients. This could be referred to the fact that autoimmune diseases are complex genetic disorders, therefore further studies on polymorphisms affecting IL-18 gene expression as well as kinetics of IL-18 expression are required to evaluate the role of interleukin 18 and its binding protein in the pathogenesis of ITP.

Report on the 15th International Society of Blood Transfusion platelet immunology workshop

The aim of the 15th ISBT Platelet Immunology Workshop was to evaluate the detection of free platelet-reactive autoantibodies from ITP patients by the use of a standardized MAIPA protocol, to compare sensitivity and specificity of antibody detection for anti-HPA-1a and serologically difficult-to-assess antibodies against HPA-3, to identify whether anti-HPA-1a titration results can be compared between laboratories, and to evaluate HPA genotyping methods. Workshop materials were shipped from the organizing laboratory in Giessen, Germany. Thirty laboratories from 19 countries participated. Results for the detection of autoantibodies differed greatly between the laboratories and no consensus was reached for one of the two sera. Detection and titration of antibodies against HPA-1a, in contrast, gave largely congruent results. Serologically difficult-to-assess antibodies recognizing HPA-3a and HPA-3b were not detected by many laboratories. For genotyping, good agreement was achieved. Detection of HPA-1a antibodies, titration of anti-HPA-1a, and HPA genotyping are well performed in most participating laboratories. The workshop has identified two specific areas with room and need for improvement: the detection of autoantibodies and the detection of HPA-3 alloantibodies. Recommendations of the Working Party on techniques that can help to overcome these problems are desirable.

Drug-Induced Immune Thrombocytopenia

Thrombocytopenia can have several causes, including the use of certain drugs. The mechanism behind drug-induced thrombocytopenia is either a decrease in platelet production (bone marrow toxicity) or an increased destruction (immune-mediated thrombocytopenia). In addition, pseudothrombocytopenia, an in vitro effect, has to be distinguished from true drug-induced thrombocytopenia. This article reviews literature on drug-induced immune thrombocytopenia, with the exception of thrombo-haemorrhagic disorders such as thrombotic thrombocytopenic purpura and heparin-induced thrombocytopenia and thrombosis. A literature search in PubMed combined with a check of the reference lists of all the retrieved articles resulted in 108 articles relevant to the subject. The drug classes that are most often associated with drug-induced immune thrombocytopenia are cinchona alkaloid derivatives (quinine, quinidine), sulfonamides, NSAIDs, anticonvulsants, disease modifying antirheumatic drugs and diuretics. Several other drugs are occasionally described in case reports of thrombocytopenia; an updated review of these case reports can be found on the internet. A small number of epidemiological studies, differing largely in the methodology used, describe incidences in the magnitude of 10 cases per 1 000 000 inhabitants per year. No clear risk factors could be identified from these studies. The underlying mechanism of drug-induced immune thrombocytopenia is not completely clarified, but at least three different types of antibodies appear to play a role (hapten-dependent antibodies, drug-induced, platelet-reactive auto-antibodies and drug-dependent antibodies). Targets for drug-dependent antibodies are glycoproteins on the cell membrane of the platelets, such as glycoprotein (GP) Ib/IX and GPIIb/IIIa. Diagnosis of drug-induced immune thrombocytopenia may consist of identifying clinical symptoms (bruising, petechiae, bleeding), a careful evaluation of the causal relationship of the suspected causative drug, general laboratory investigation, such as total blood count and peripheral blood smear (to rule out pseudothrombocytopenia), and platelet serology tests. The sensitivity of these tests is dependent on factors such as the concentration of the drug in the test and the potential sensitisation of the patient by metabolites instead of the parent drug. Drug-induced immune thrombocytopenia can be treated by withholding the causative drug and, in severe cases associated with bleeding, by platelet transfusion. Although drug-induced thrombocytopenia is a relatively rare adverse drug reaction, its consequences may be severe. Therefore it is important to extend our knowledge on this subject. Future research should focus on the identification of potential risk factors, as well as the exact mechanism underlying drug-induced thrombocytopenia.

Mechanisms of action of therapeutics in idiopathic thrombocytopenic purpura.

Idiopathic thrombocytopenic purpura (ITP) is a common immune disorder caused by platelet-reactive autoantibodies. Antibody-coated platelets are cleared more rapidly from the circulation, often in the spleen, than they can be replaced by compensatory stimulation of platelet production in the bone marrow. In some patients, platelet production is depressed as well. ITP in adults does not generally remit spontaneously, and most patients require treatment to prevent bleeding at one time or another. Therapy with corticosteroids, danazol, intravenous immune globulin, anti-D antibody, and several other agents inhibits clearance of the antibody-coated platelets but is rarely curative. Most patients will sustain a hemostatic response after splenectomy, although relapses may occur at any time. Patients may be more responsive to these same modalities after splenectomy, but treatment with an immunosuppressant that inhibits T- and B-cell function and cooperation, including azathioprine, cyclophosphamide, cyclosporine, mycophenolate mofetil, or anti-CD20, may be required. Antiviral therapy is useful in patients with HIV or hepatitis C infection, but no consensus has been reached as to the efficacy of antibiotics to eradicate Helicobacter pylori. Promising results have been seen in several patients treated with a modified thrombopoietin. It may be possible to design therapeutics that exploit the apparent restricted immunoglobulin gene usage by antiplatelet antibodies, perhaps in the form of engineered anti-idiotypic antibodies or other compounds that specifically target autoantibody-producing B cells. Rationale therapy awaits a more thorough understanding of autoantibody production.

Childhood immune thrombocytopenic purpura

Childhood immune thrombocytopenic purpura (ITP) is acute and generally seasonal in nature, suggesting that infectious or environmental agents may trigger the immune response to produce platelet-reactive autoantibodies 4 to 8 weeks following an infection. In general, the patient is well apart from the diffuse bruising and petechiae indicative of a profound thrombocytopenia. Over a period of 6 months, the thrombocytopenia resolves in approximately 85% of children, while the remaining 15% with persistent platelet consumption are designated as chronic ITP patients. The peak age of acute ITP is 2 to 5 years of age, a period when children experience the greatest frequency of viral infections. Children with the chronic form of ITP mirror the adult phenotype, in that females predominate, and there is no seasonal fluctuation of the disease. Evidence from our laboratory suggests that the activated platelet itself may play a role in perpetuating autoantibody production and immune dysregulation associated with ITP. Current data on lymphocyte studies and cytokine alterations noted in response to the variety of regimens used in children with ITP suggest that acute ITP is accompanied by autoantibodies to GPIb and a cytokine profile that is proinflammatory in nature. Early recognition of the immune dysregulation driving acute versus chronic ITP will distinguish those children who might benefit from immunotherapy versus those who will recover without therapeutic intervention.

Identification of the platelet glycoprotein IIb/IIIa complex as a target antigen in primary biliary cirrhosis-associated autoimmune thrombocytopenia: Evidence that platelet-reactive autoantibodies can also bind to the mitochondrial antigen M2

A 67-year-old woman with a 4-year history of primary biliary cirrhosis (PBC) unexpectedly developed autoimmune thrombocytopenia. The platelet-bound IgG antibody was eluted from the patient's platelets to determine the platelet target antigen. The autoantibodies were found to precipitate the platelet glycoprotein complex IIb/IIIa of autologous and allogeneic platelets. A further precipitate of 70 kDa was detectable under reducing conditions. In addition, platelet-reactive antibodies bound to the 70 kDa mitochondrial antigen M2. No cross-absorption studies were performed to confirm that a single antibody reacted with both antigens. Computer analysis of published peptide sequences of the mitochondrial protein and the platelet GPIIb/IIIa complex showed partial amino acid sequence homology suggesting the possibility of a common antibody binding site. These findings suggest a relationship between the development of autoimmune thrombocytopenia in PBC and the underlying liver disease.

Cyclic thrombocytopenia of apparent autoimmune etiology

Serial studies were performed in two patients with cyclic thrombocytopenia to investigate the pathogenesis of this disorder. Mean life span of autologous platelets when platelet levels were declining was subnormal (2.4 and 0.8 days), and megakaryocytes were abundant in the bone marrow during thrombocytopenia. Megakaryocyte colony- stimulating activity could not be detected in the serum of either patient at any point of their cycles. In each patient, total platelet- associated IgG varied inversely with platelet levels. Surface platelet- associated IgG was measured only in patient 2 and was significantly elevated (greater than 1,280 IgG molecules per platelet) at all stages of the cycle, even during thrombocytosis. However, the highest values were observed during thrombocytopenia. Platelet-bindable IgG in plasma declined to normal immediately before platelet levels began to rise. IgG eluted from the platelets of this patient reacted strongly with autologous and homologous platelets in contrast to a “mock eluate” prepared from platelets of a normal subject. The eluate from the patient's platelets reacted strongly with immobilized autologous and homologous glycoprotein IIb/IIIa complex and weakly with GPIb but not with isolated GPIIIa alone. In each patient the decline in platelet levels was significantly delayed following administration of intravenous gamma globulin 0.4 g/kg body weight for five days. These findings suggest that platelet-reactive autoantibodies are of pathogenic significance in some patients with cyclic thrombocytopenia.

Cyclic Thrombocytopenia of Apparent Autoimmune Etiology

Serial studies were performed in two patients with cyclic thrombocytopenia to investigate the pathogenesis of this disorder. Mean life span of autologous platelets when platelet levels were declining was subnormal (2.4 and 0.8 days), and megakaryocytes were abundant in the bone marrow during thrombocytopenia. Megakaryocyte colony-stimulating activity could not be detected in the serum of either patient at any point of their cycles. In each patient, total platelet-associated IgG varied inversely with platelet levels. Surface platelet-associated IgG was measured only in patient 2 and was significantly elevated (greater than 1,280 IgG molecules per platelet) at all stages of the cycle, even during thrombocytosis. However, the highest values were observed during thrombocytopenia. Platelet-bindable IgG in plasma declined to normal immediately before platelet levels began to rise. IgG eluted from the platelets of this patient reacted strongly with autologous and homologous platelets in contrast to a "mock eluate" prepared from platelets of a normal subject. The eluate from the patient's platelets reacted strongly with immobilized autologous and homologous glycoprotein IIb/IIIa complex and weakly with GPIb but not with isolated GPIIIa alone. In each patient the decline in platelet levels was significantly delayed following administration of intravenous gamma globulin 0.4 g/kg body weight for five days. These findings suggest that platelet-reactive autoantibodies are of pathogenic significance in some patients with cyclic thrombocytopenia.

In vitro fixation of C3d and C5b-9 on platelets by human platelet reactive antibodies.

Most platelet-reactive autoantibodies and alloantibodies are not able to fix complement in vitro. However, exceptions have been found. These antibodies are usually characterized by the conventional platelet complement fixation test. A recently developed competitive enzyme immunoassay for quantitation of platelet-associated immunoglobulins and a modification thereof allowed the quantitative study of fixation of C3d and the membrane attack complex (C5b-9) on platelets by HLA antibodies, human platelet autoantibodies, and drug-dependent antibodies (ddab). The highest amounts of both complement products were fixed through ddabs, whereas autoantibodies only showed moderate complement fixation. This enzyme immunoassay is a valuable tool for the characterization of the complement-fixing properties of platelet-reactive antibodies.

Monoclonal Antibody-Specific Immobilization of Platelet Antigens (MAIPA): A New Tool for the Identification of Platelet-Reactive Antibodies

The analysis of sera containing different platelet-reactive antibodies, eg, autoantibodies, platelet-specific alloantibodies like anti-PIA1, -PIA2, -Baka, and HLA antibodies, is still difficult. Recently, monoclonal antibodies against major platelet membrane constituents (glycoproteins IIb/IIIa and Ib and HLA class I molecule) have become available. In this report we describe a new assay that takes advantage of these highly specific reagents to investigate selectively platelet reactive antibodies against epitopes on different glycoproteins. The reliability and specificity of this assay is demonstrated with known platelet-reactive autoantibodies and alloantibodies (anti-PIA1, -Baka, -Pen). The discovery of a PIA2 antibody in a serum of a polytransfused patient underscores the efficiency of this technique. Possible applications of this assay are discussed in detail.

Monoclonal antibody--specific immobilization of platelet antigens (MAIPA): a new tool for the identification of platelet-reactive antibodies

The analysis of sera containing different platelet-reactive antibodies, eg, autoantibodies, platelet-specific alloantibodies like anti-PIA1, - PIA2, -Baka, and HLA antibodies, is still difficult. Recently, monoclonal antibodies against major platelet membrane constituents (glycoproteins IIb/IIIa and Ib and HLA class I molecule) have become available. In this report we describe a new assay that takes advantage of these highly specific reagents to investigate selectively platelet reactive antibodies against epitopes on different glycoproteins. The reliability and specificity of this assay is demonstrated with known platelet-reactive autoantibodies and alloantibodies (anti-PIA1, -Baka, - Pen). The discovery of a PIA2 antibody in a serum of a polytransfused patient underscores the efficiency of this technique. Possible applications of this assay are discussed in detail.