- Email: [email protected]
Platelet Autoantibodies
CHAPTER
Platelet Autoantibodies
61
Boris Shenkman,1 Nurit Rosenberg,1 and Yulia Einav2 1Sheba
Medical Center, Tel-Hashomer, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel, 2Holon Institute of Technology, Holon, Israel
Historical notes In the mid-16th century, Amatus Lusitanus described an exanthema in a disease called “flea-like without fever.” Lazarus Riverius (1658) observed bleedings that come out at the nose. A hundred years later, in 1735, Paul Gottlieb Werlhof reported a disease called “morbus maculosus hemorrhagicus.” In 1808, Robert Willan described various types of purpura. Joseph Denys found in 1887 that purpura was associated with low platelet count. Name Kaznelson (1916) hypothesized that the spleen was the site of platelet destruction and performed the first splenectomy in a thrombocytopenia patient. First evidence for humoral factors causing thrombocytopenia was shown in 1951 by William Harrington, who transfused plasma from immune thrombocytopenia (ITP) patients into normal volunteers, which was followed by a rapid fall in platelet counts. The immune nature of the disease was suspected when the factor absorbed by platelets was present in the immunoglobulin (Ig)G-rich plasma fraction (Shulman, 1965). Since the 1970s, the identification of platelet antigens led to definition of specific platelet autoantibodies causing thrombocytopenia.
Platelet autoantigens Definition and characterization Platelet autoantigens are defined by antibodies that react with the patient’s own platelets and with platelets from normal individuals. The development of thrombocytopenia may be idiopathic (ITP) and druginduced. The antigenic targets of platelet autoantibodies are common surface glycoproteins (GPs) as GPIIb/IIIa, the fibrinogen receptor, and GPIb/IX, the von Willebrand factor (vWF) receptor. Other GPs, such as GPIV, GPVI, or GPIa/IIa, though rare, can be involved. Patients who lack a specific platelet antigen can develop antiplatelet antibodies directed against the deficient GP following administration of random platelets or during pregnancy.
Idiopathic thrombocytopenia ITP is caused by an autoimmune response to components of the platelet surface, mainly GPs, leading to platelets coated with antibodies to be removed by the spleen and resulting in thrombocytopenia. Alternatively, these antibodies can mediate destruction of platelets by the monocyte-macrophage Autoantibodies. http://dx.doi.org/10.1016/B978-0-444-56378-1.00061-7 Copyright © 2014 Elsevier B.V. All rights reserved.
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system, as well as suppression of megakaryocyte proliferation and maturation [1]. Multiple targets of autoantibodies have been found among patients with chronic ITP, most of them recognized as the platelet membrane GPIIb/IIIa (integrin αIIbβ3) and the GPIb/IX complex. Platelet-associated anti-αIIbβ3 antibodies are frequently bound to cation-dependent conformational antigens and did not react with αvβ3, suggesting that the target epitopes localized mainly on αIIb. Furthermore, ITP was shown to be placed on three blades in the N-terminal portion of the β-propeller domain and show clonality [2]. Platelet-associated anti-αIIbβ3 antibodies typically recognize conformational epitopes rather than linear epitopes. Therefore, the autoantigen requires the retention of major conformations in αIIbβ3. In human immunodeficiency virus (HIV)-associated ITP, the target epitopes appeared to be localized to the 49–66 residues of β3 subunits, and some other autoantibodies have been shown to bind to the disulfide-rich region of β3 integrin that consists of EGFs and β-tail domains [3]. There are few data on epitopes on GPIb/IX, and those that have been identified localize to GPIbα amino acids 333–341, beyond the vWF-binding site. In rare cases, the autoantibodies affect the platelet function in vivo, causing acquired Glanzmann thrombasthenia, if the epitope is localized to integrin αIIbβ3, or acquired Bernard Soulier syndrome, if it is on GPIb/IX complex. ITP can be triggered by viral infection due to the fact that molecular mimicry between viral and platelet GPs can cause cross reactivity. Such mechanism can be involved in ITP secondary to HIV infection or associated with Helicobacter pylori. Other mechanisms of self-antigen recognition are formation of cryptic epitope and epitope spreading: exposure of new epitopes due to chemical/ bacterial modification of the native GPs or due to the processing of platelet GPs by presenting cells [4].
Drug-dependent epitopes Drug-dependent autoantibodies bind to specific epitopes on platelet surface GPs only in the presence of the sensitizing drug (or food) [5]. It has been proposed that the sensitizing drugs typically contain charged and/or hydrophobic regions that bind noncovalently and reversibly to both: the antibody and the platelet GPs. In this model, the drug interacts to improve the “fit” between naturally occurring weak antibodies against platelet GPs, commonly GPIIb/IIIa and GPIb/IX/V complex, and such antibodies were reported for quinine and quinidine [6]. Since quinine contains significant hydrophobic elements, another explanation could be that it binds to and stabilizes denatured or non-native conformations in platelet receptors, some of which are recognized as foreign and thereby initiate specific immune response. Arginine-glycine-aspartic acid (RGD)-mimetic platelet inhibitors, such as tirofiban or eptifibatide, induce structural changes in integrin αIIbβ3, leading to exposure of new epitopes recognized by the drug-specific antibodies. Abciximab can induce thrombocytopenia due to antibodies against the murine structural elements or the neoepitopes exposed upon structural changes. Another type of epitope can be a complex of a drug with regular platelet protein as shown for heparin with platelet factor 4 (PF4); the antibody is directed against heparin–PF4 complex, leading to platelet activation by the FcγRIIA found on the platelet surface. Some drugs are only the trigger for the immune response, like penicillin, which acts as a hapten, or gold salts and procaine amide that perturb the immune response in such a way that drug-independent antibodies specific for a platelet membrane GP are produced.
Platelet autoantibodies
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Alloantigens Platelet-specific alloantigens result from genetic polymorphisms in genes encoding platelet membrane GP designated as human platelet antigens (HPA) [7]. These alloantigens have been defined by immune sera from females who gave birth to infants with neonatal thrombocytopenia (NAIT) and numbered in the order of their discovery. The molecular basis for 27 HPAs have been resolved and most of them identified as a single amino acid substitution caused by single nucleotide polymorphism in a gene coding for a platelet membrane GP (see www.ebi.ac.uk/ipd/hpa/index.html). Fourteen HPAs are located in integrin β3, four in integrin αIIb, and the rest in integrin α2, the GPIb, and CD109. HPA-1 is most frequently involved in NAIT (about 80% of the cases), followed by HPA-5 (5–15%) and HPA-3. In Asian people, NAIT can also be associated with the HPA-4 or CD36 null phenotype. Analysis of HPA-1a and 1b by molecular dynamics indicate that the proline in position 33 introduces flexibility in the Plexins, Semaphorins, and Integrins (PSI), I-epidermal growth factor (EGF)-1, and I-EGF-2 domains of the β3 structure, thus substitution of leucine to proline at position 33 can alter the structure of the PSI domain as well as the I-EGF-1 and I-EGF-2 domains. This finding describes how a change of a single amino acid can create a conformational epitope rather than linear one [8].
Genetics Genetic tendency may contribute to the development of ITP and NAIT. HLA class I and class II have been reported to be involved in ITP with an increased frequency of HLA Aw32, DRw2, and DRB1*0410 alleles and lower frequency of HLA-DRB1*11 and -DQB1*03 alleles in ITP patients compared to healthy control. Polymorphisms of FcγRIIIa and FcγRIIa were also reported to contribute to susceptibility to ITP and influence the effectiveness of medications [9]. The frequency of NAIT in Caucasian people is lower than would be expected, as only 10% of mothers homozygous for HPA-1b and exposed to HPA-1a platelets during pregnancy become immunized. The risk and severity of the alloimmunization is increased in the presence of two HLA alleles: DRB3*01:01 and DRB4*01:01 in the mother, and it also reduced the success of a preventive IgG treatment.
Platelet autoantibodies Definition The majority of platelet antibodies in ITP are of the IgG and/or IgM classes, although IgA and IgE have also been described. The IgG class is responsible for the interaction between antibody-bound platelets and macrophages of the reticuloendothelial system. Alternatively, antibody-sensitized platelets can be removed from the circulating blood by complement-mediated lysis. Platelets contain low affinity FcγRIIA receptors capable of binding both antibodies and immune complexes containing IgG. The clear role of the FcγRIIA receptors in ITP has been attributed to platelet clearance by macrophages. This interaction leads directly to the question of platelet function. In contrast to ITP, platelet destruction in HIV patients is usually caused by nonspecific binding of immune complexes and complement to the platelets. However, in a part of these patients, antibodies against GPIIb/IIIa have been found. Thrombocytopenia may also reflect crossreactivity with antibodies directed against HIV antigens.
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Pathogenic role In most cases of immune-related thrombocytopenia, platelet function remains. However, in certain cases, antibodies against specific GPs, though considered as rare cases, have been shown to suppress platelet function. In other cases, antibodies may enhance platelet function, contributing to thrombotic complications [10]. This usually occurs in heparin-induced thrombocytopenia with thrombosis, antiphospholipid syndrome, and following abciximab administration. All antibodies may activate platelets via Fc receptors. ITP antibodies cause substantial inhibitory effect on megakaryopoiesis, including reduction in the number of megakaryocytes produced as well as maturation, induction of apoptosis, and reduction of pro-platelet formation. These effects were mainly observed in the presence of anti-GPIb/IX antibodies. In contrast, treatment of megakaryocytes with GPIIb/IIIa antibodies had no detectable effect on proplatelet formation.
T cells and cytokines The type 1 T-cell profile promotes cell-mediated cytotoxicity and immunoglobulin production. Increased expression of genes related to cytotoxic mediators has been found in T cells of ITP patients [4]. This is accompanied by increased levels of some cytokines, such as interleukin (IL)-2, interferon-γ, and IL-10. T1/T2 ratio is higher in ITP and is inversely correlated with the platelet count, as opposed to a T2-cytokine response in patients in remission or treated with intravenous immunoglobulin. An elevated level of transforming growth factor (TGF)-β, a potent immunosuppressive cytokine, has been found in ITP during remission. This might be a viable approach for tolerance induction. T-cell-mediated immunity and increase of cytokine levels in serum lead to tolerance failure followed by autoantibodies production, platelet destruction, and, in some cases, to megakaryocyte suppression. Levels of CXCL10 are higher and the levels of CXCL5, CCL5, and CD40L are lower in ITP patients with platelet count less than 50 × 109/L compared with healthy controls [11]. In vitro, concentrations of these cytokines in the supernatants of platelet suspensions are proportional to platelet numbers. The authors conclude that these cytokines are mainly platelet-derived, confirming a role of platelets in immune responses and inflammation.
Methods of detection Various techniques have been developed to detect platelet autoantibodies to facilitate the diagnosis of ITP. The measurement of direct platelet-associated immunoglobulins by flow cytometry or highly sensitive enzyme-linked immunosorbent assay (ELISA) is considered a sensitive tool for ITP diagnosis. However, the specificity of these assays is too low because they cannot differentiate platelet-GP-specific antibodies from nonplatelet-specific antibodies and therefore are not defined in the diagnostic algorithm of ITP. In contrast, assay of serum antibodies that bind to normal platelets by fluorescence flow cytometry is more specific but less sensitive. Taking into account that antibodies are directed to one of the membrane GPs, several laboratories proposed a variety of antigen-specific assays, such as immunoblot techniques, radio-immunobead immunoprecipitation, immunobead assay, and monoclonal antibody-specific immobilization of antibodies (MAIPA) [12]. Despite possessing high specificity (78–93%) to confirm the diagnosis of ITP, their sensitivity (49–66%) was not high enough to exclude
Clinical utility
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the diagnosis of ITP when results were negative. Additionally, these assays carried certain methodologic or practical limitations, so none of them is currently approved to be applicable for the routine diagnosis of ITP. Nevertheless, the GP-specific platelet autoantibodies assays continue to be used. For NAIT diagnosis, HPA genotyping of neonate and its parents is recommended as well as GP-specific antibodies in the serum of the mother. Cross-match of maternal serum with the father’s platelets also can be considered.
Clinical utility Idiopathic thrombocytopenia The term ITP was used to refer to idiopathic or immune thrombocytopenic purpura. However, now the disease cannot be considered idiopathic. In addition, many patients do not have purpura at the time of diagnosis. Therefore, the term ITP now refers to immune thrombocytopenia. The annual incidence of ITP is 5.5 per 100,000 people. ITP is defined as a platelet count of less than 100 × 109/L with no evidence of leukopenia and anemia. ITP can be primary or secondary. Primary ITP accounts for the majority of cases where other conditions are absent. Thus, a very meticulous clinical history is required. Secondary ITP appears after infections, such as Helicobacter pylori, hepatitis C, and HIV. Other cases of thrombocytopenia include systemic lupus erythematosus, Wiskott-Aldrich syndrome, antiphospholipid syndrome, chronic lymphocytic leukemia, and association with drugs (e.g., heparin, abciximab). In children, the presentation of ITP is acute with severe thrombocytopenia and underlying conditions, unlike adults where most of the cases with ITP are diagnosed incidentally on routine blood test. The diagnosis of ITP during pregnancy can sometimes be undistinguished from pregnancy-induced thrombocytopenia, and unless a nonpregnant platelet count is available, diagnosis is not feasible. Maternal ITP rarely causes serious thrombocytopenia and bleeding in the fetus or neonate; however, instrumental delivery should be avoided. The bleeding manifestation in ITP is in correlation with the degree of thrombocytopenia. Usually bleeding becomes evident when platelet counts are less than 20 × 109/L unless platelet function is impaired by antibodies. Epistaxis, menorrhagia, and in some cases hematuria can occur. Intracranial hemorrhage usually presents in young people when platelet level is less than 10 × 109/L. Aspiration of bone marrow is not indicated in patients with typical presentation of ITP and under the age of 60 years.
Alloimmune thrombocytopenia NAIT has a frequency of 1–4 per 1000 live births. Unlike hemolytic disease, thrombocytopenia in the fetus can occur during first pregnancy when maternal antibodies (IgG subclass) are directed against platelet epitopes inherited by the father and transferred through the placenta. About 30–40% of neonates born to immunized women develop severe thrombocytopenia. The diagnosis of NAIT is suspected in otherwise healthy infants with unexpected petechiae, purpura, and even with intracranial hemorrhage, which appears early during pregnancy or during labor. With subsequent pregnancies, the degree of thrombocytopenia becomes more severe. The diagnosis is confirmed by genotyping maternal and paternal platelets as well as the newborn or the fetus. The genotype by amniocentesis is helpful in some cases, as it will define whether the mother needs treatment.
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Transplant-induced alloimmune thrombocytopenia can occur due to residual HPA-1 mismatched host cells or donor cells following bone marrow and solid organ transplantation. A clinical history in this case can help in making the diagnosis. Passive alloimmune thrombocytopenia results in bleeding following infusion of antibodies within the transfused blood product, resulting in destruction of host platelets. To date, only HPA-1 and HPA-5b have been implicated for such phenomenon. Acute and severe post-transfusion purpura occurs 7 days after transfusion of blood product containing platelets. The alloantibodies are usually anti-HPA-1a, destroying not only donor platelets but also the host platelets. The diagnosis is confirmed by demonstrating antibodies in a patient’s serum directed against donor specific antigens. Transfusion refractoriness is a failure of increase in platelet count following platelet transfusion as a result of antibodies against HLA, and rarely against another epitope, which develops after previous transfusions. The presence of alloantibodies leads to rapid destruction of transfused platelets. A similar phenomenon occurs in Glanzmann thrombasthenia patients who were heavily transfused.
Pseudo-thrombocytopenia The prevalence rate of pseudo-thrombocytopenia (PTCP) was reported as 0.07–0.20%. Ethylenedi aminetetraacetic acid (EDTA) is usually used as an anticoagulant in blood cell counts and sometimes induces platelet clumping, which results in artificially low platelet counts. In such situations, the automated counter reported lower platelet count than the actual count since platelet clumps are read as leukocytes. On microscopic examination of blood film, platelet clumps are observed and confirm the diagnosis. The EDTA-initiated antibodies do not appear to have any clinical implication but can be transferred through the placenta, leading to PTCP in the neonate. In contrast to EDTA, platelet clumping is absent and platelet count is normal using sodium citrate or heparin. Platelet-leukocyte satellitism is a phenomenon that is similar to pseudo-thrombocytopenia and stems from antibodies that react with platelet GP and Fcγ receptor of leukocytes, creating a rosette around the periphery of leukocytes when observed by microscope [13]. Ex vivo platelet clumping should be considered in patients with acute viral infections, particularly in hepatitis infections. Failure to identify PTCP may result in a clinical problem like unnecessary diagnostic tests, glucocorticoid therapy, and platelet transfusion.
Take-home messages • I mmune response against platelet surface antigens results in antiplatelet antibodies production, which is the basis for most immune thrombocytopenias: idiopathic, drug-induced, post-transfusion, and neonatal alloimmune, as well as refractoriness to random donor platelets. • The antiplatelet antibodies are mainly IgG, mostly against GPIIb/IIIa and GPIb/IX. The autoantibody production is under the control of T-helper cells and cytokines. • Involvement of HLA class I and II was reported in ITP and NAIT development, explaining the hereditary tendency of the immune diseases in some of the cases. • EDTA sometimes induces platelet clumping, which results in artificially low platelet count. This phenomenon named as PTCP. Ex vivo platelet clumping may occur in patients with acute viral infections.
References
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References [1] Stasi R. Pathophysiology and therapeutic options in primary immune thrombocytopenia. Blood Transfus 2011;9:262–73. [2] Kiyomizu K, Kashiwagi H, Nakazawa T, Tadokoro S, Honda S, Kanakura Y, et al. Recognition of highly restricted regions in the β-propeller domain of αIIb by platelet-associated anti-αIIbβ3 autoantibodies in primary immune thrombocytopenia. Blood 2012;120:1499–509. [3] Beardsley DJS, Tang C, Chen BG, Lamborn C, Gomes E, Srimatkandada V. The disulphide-rich region of platelet GPIIIa contains hydrophilic peptide sequences that bind anti-GPIIIa autoantibodies from patients with immune thrombocytopenic purpura (ITP). Biophys Chem 2003;105:503–15. [4] Zhou B, Zhao H, Yang RC, Han ZC. Multi-dysfunctional pathophysiology in ITP. Clin Rev Oncol/Hematol 2005;54:107–16. [5] George JN, Aster RH. Drug-induced thrombocytopenia: pathogenesis, evaluation, and management. Hematology Am Soc Hematol Educ Program 2009:153–8. [6] Peterson JA, Nelson TN, Kanack AJ, Aster RH. Fine specificity of drug-dependent antibodies reactive with a restricted domain of platelet GPIIIA. Blood 2008;111:1234–9. [7] Salama A. Alloimmune thrombocytopenias. J Pediatr Hematol Oncol 2003;25:S39–41. [8] Jallu V, Poulain P, Fuchs PFJ, Kaplan C, de Brevern AG. Modeling and molecular dynamics of HPA-1a and 1b polymorphisms: effects on the structure of the β3 subunit of the αIIbβ3 integrin. PLoS ONE 2012;7:e47304. [9] Eyada TK, Farawela HM, Khorshied MM, Shaheen IA, Selim NM, Khalifa IA. FcγRIIa and FcγRIIIa genetic polymorphisms in a group of pediatric immune thrombocytopenic purpura in Egypt. Blood Coagul Fibrinol 2012;23:64–8. [10] Kekomaki R. Platelet function and immune response. J Pediatr Hematol Oncol 2003;25:S19–23. [11] Feng X, Scheinberg P, Samsel L, Rios O, Chen J, McCoy JP, et al. Decreased plasma cytokines are associated with low platelet counts in aplastic anemia and immune thrombocytopenic purpura. J Thromb Haemost 2012;10:1616–23. [12] McMillan R. Antiplatelet antibodies in chronic adult immune thrombocytopenic purpura: assays and epitopes. J Pediatr Hematol Oncol 2003;25:S57–61. [13] Diz-Kucukkaya R, Gushiken FC, Lopez JA. Thrombocytopenia. In: Lichtman MA, Beutler E, Kipps TJ, Seligsohn U, Kaushansky K, Prchal JT, editors. Williams Hematology, vol. 110. New York, McGraw-Hill Medical Publishers; 2006. p. 1749–84.
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Boris Shenkman,1 Nurit Rosenberg,1 and Yulia Einav2 1Sheba
Medical Center, Tel-Hashomer, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel, 2Holon Institute of Technology, Holon, Israel
Historical notes In the mid-16th century, Amatus Lusitanus described an exanthema in a disease called “flea-like without fever.” Lazarus Riverius (1658) observed bleedings that come out at the nose. A hundred years later, in 1735, Paul Gottlieb Werlhof reported a disease called “morbus maculosus hemorrhagicus.” In 1808, Robert Willan described various types of purpura. Joseph Denys found in 1887 that purpura was associated with low platelet count. Name Kaznelson (1916) hypothesized that the spleen was the site of platelet destruction and performed the first splenectomy in a thrombocytopenia patient. First evidence for humoral factors causing thrombocytopenia was shown in 1951 by William Harrington, who transfused plasma from immune thrombocytopenia (ITP) patients into normal volunteers, which was followed by a rapid fall in platelet counts. The immune nature of the disease was suspected when the factor absorbed by platelets was present in the immunoglobulin (Ig)G-rich plasma fraction (Shulman, 1965). Since the 1970s, the identification of platelet antigens led to definition of specific platelet autoantibodies causing thrombocytopenia.
Platelet autoantigens Definition and characterization Platelet autoantigens are defined by antibodies that react with the patient’s own platelets and with platelets from normal individuals. The development of thrombocytopenia may be idiopathic (ITP) and druginduced. The antigenic targets of platelet autoantibodies are common surface glycoproteins (GPs) as GPIIb/IIIa, the fibrinogen receptor, and GPIb/IX, the von Willebrand factor (vWF) receptor. Other GPs, such as GPIV, GPVI, or GPIa/IIa, though rare, can be involved. Patients who lack a specific platelet antigen can develop antiplatelet antibodies directed against the deficient GP following administration of random platelets or during pregnancy.
Idiopathic thrombocytopenia ITP is caused by an autoimmune response to components of the platelet surface, mainly GPs, leading to platelets coated with antibodies to be removed by the spleen and resulting in thrombocytopenia. Alternatively, these antibodies can mediate destruction of platelets by the monocyte-macrophage Autoantibodies. http://dx.doi.org/10.1016/B978-0-444-56378-1.00061-7 Copyright © 2014 Elsevier B.V. All rights reserved.
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CHAPTER 61 Platelet Autoantibodies
system, as well as suppression of megakaryocyte proliferation and maturation [1]. Multiple targets of autoantibodies have been found among patients with chronic ITP, most of them recognized as the platelet membrane GPIIb/IIIa (integrin αIIbβ3) and the GPIb/IX complex. Platelet-associated anti-αIIbβ3 antibodies are frequently bound to cation-dependent conformational antigens and did not react with αvβ3, suggesting that the target epitopes localized mainly on αIIb. Furthermore, ITP was shown to be placed on three blades in the N-terminal portion of the β-propeller domain and show clonality [2]. Platelet-associated anti-αIIbβ3 antibodies typically recognize conformational epitopes rather than linear epitopes. Therefore, the autoantigen requires the retention of major conformations in αIIbβ3. In human immunodeficiency virus (HIV)-associated ITP, the target epitopes appeared to be localized to the 49–66 residues of β3 subunits, and some other autoantibodies have been shown to bind to the disulfide-rich region of β3 integrin that consists of EGFs and β-tail domains [3]. There are few data on epitopes on GPIb/IX, and those that have been identified localize to GPIbα amino acids 333–341, beyond the vWF-binding site. In rare cases, the autoantibodies affect the platelet function in vivo, causing acquired Glanzmann thrombasthenia, if the epitope is localized to integrin αIIbβ3, or acquired Bernard Soulier syndrome, if it is on GPIb/IX complex. ITP can be triggered by viral infection due to the fact that molecular mimicry between viral and platelet GPs can cause cross reactivity. Such mechanism can be involved in ITP secondary to HIV infection or associated with Helicobacter pylori. Other mechanisms of self-antigen recognition are formation of cryptic epitope and epitope spreading: exposure of new epitopes due to chemical/ bacterial modification of the native GPs or due to the processing of platelet GPs by presenting cells [4].
Drug-dependent epitopes Drug-dependent autoantibodies bind to specific epitopes on platelet surface GPs only in the presence of the sensitizing drug (or food) [5]. It has been proposed that the sensitizing drugs typically contain charged and/or hydrophobic regions that bind noncovalently and reversibly to both: the antibody and the platelet GPs. In this model, the drug interacts to improve the “fit” between naturally occurring weak antibodies against platelet GPs, commonly GPIIb/IIIa and GPIb/IX/V complex, and such antibodies were reported for quinine and quinidine [6]. Since quinine contains significant hydrophobic elements, another explanation could be that it binds to and stabilizes denatured or non-native conformations in platelet receptors, some of which are recognized as foreign and thereby initiate specific immune response. Arginine-glycine-aspartic acid (RGD)-mimetic platelet inhibitors, such as tirofiban or eptifibatide, induce structural changes in integrin αIIbβ3, leading to exposure of new epitopes recognized by the drug-specific antibodies. Abciximab can induce thrombocytopenia due to antibodies against the murine structural elements or the neoepitopes exposed upon structural changes. Another type of epitope can be a complex of a drug with regular platelet protein as shown for heparin with platelet factor 4 (PF4); the antibody is directed against heparin–PF4 complex, leading to platelet activation by the FcγRIIA found on the platelet surface. Some drugs are only the trigger for the immune response, like penicillin, which acts as a hapten, or gold salts and procaine amide that perturb the immune response in such a way that drug-independent antibodies specific for a platelet membrane GP are produced.
Platelet autoantibodies
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Alloantigens Platelet-specific alloantigens result from genetic polymorphisms in genes encoding platelet membrane GP designated as human platelet antigens (HPA) [7]. These alloantigens have been defined by immune sera from females who gave birth to infants with neonatal thrombocytopenia (NAIT) and numbered in the order of their discovery. The molecular basis for 27 HPAs have been resolved and most of them identified as a single amino acid substitution caused by single nucleotide polymorphism in a gene coding for a platelet membrane GP (see www.ebi.ac.uk/ipd/hpa/index.html). Fourteen HPAs are located in integrin β3, four in integrin αIIb, and the rest in integrin α2, the GPIb, and CD109. HPA-1 is most frequently involved in NAIT (about 80% of the cases), followed by HPA-5 (5–15%) and HPA-3. In Asian people, NAIT can also be associated with the HPA-4 or CD36 null phenotype. Analysis of HPA-1a and 1b by molecular dynamics indicate that the proline in position 33 introduces flexibility in the Plexins, Semaphorins, and Integrins (PSI), I-epidermal growth factor (EGF)-1, and I-EGF-2 domains of the β3 structure, thus substitution of leucine to proline at position 33 can alter the structure of the PSI domain as well as the I-EGF-1 and I-EGF-2 domains. This finding describes how a change of a single amino acid can create a conformational epitope rather than linear one [8].
Genetics Genetic tendency may contribute to the development of ITP and NAIT. HLA class I and class II have been reported to be involved in ITP with an increased frequency of HLA Aw32, DRw2, and DRB1*0410 alleles and lower frequency of HLA-DRB1*11 and -DQB1*03 alleles in ITP patients compared to healthy control. Polymorphisms of FcγRIIIa and FcγRIIa were also reported to contribute to susceptibility to ITP and influence the effectiveness of medications [9]. The frequency of NAIT in Caucasian people is lower than would be expected, as only 10% of mothers homozygous for HPA-1b and exposed to HPA-1a platelets during pregnancy become immunized. The risk and severity of the alloimmunization is increased in the presence of two HLA alleles: DRB3*01:01 and DRB4*01:01 in the mother, and it also reduced the success of a preventive IgG treatment.
Platelet autoantibodies Definition The majority of platelet antibodies in ITP are of the IgG and/or IgM classes, although IgA and IgE have also been described. The IgG class is responsible for the interaction between antibody-bound platelets and macrophages of the reticuloendothelial system. Alternatively, antibody-sensitized platelets can be removed from the circulating blood by complement-mediated lysis. Platelets contain low affinity FcγRIIA receptors capable of binding both antibodies and immune complexes containing IgG. The clear role of the FcγRIIA receptors in ITP has been attributed to platelet clearance by macrophages. This interaction leads directly to the question of platelet function. In contrast to ITP, platelet destruction in HIV patients is usually caused by nonspecific binding of immune complexes and complement to the platelets. However, in a part of these patients, antibodies against GPIIb/IIIa have been found. Thrombocytopenia may also reflect crossreactivity with antibodies directed against HIV antigens.
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CHAPTER 61 Platelet Autoantibodies
Pathogenic role In most cases of immune-related thrombocytopenia, platelet function remains. However, in certain cases, antibodies against specific GPs, though considered as rare cases, have been shown to suppress platelet function. In other cases, antibodies may enhance platelet function, contributing to thrombotic complications [10]. This usually occurs in heparin-induced thrombocytopenia with thrombosis, antiphospholipid syndrome, and following abciximab administration. All antibodies may activate platelets via Fc receptors. ITP antibodies cause substantial inhibitory effect on megakaryopoiesis, including reduction in the number of megakaryocytes produced as well as maturation, induction of apoptosis, and reduction of pro-platelet formation. These effects were mainly observed in the presence of anti-GPIb/IX antibodies. In contrast, treatment of megakaryocytes with GPIIb/IIIa antibodies had no detectable effect on proplatelet formation.
T cells and cytokines The type 1 T-cell profile promotes cell-mediated cytotoxicity and immunoglobulin production. Increased expression of genes related to cytotoxic mediators has been found in T cells of ITP patients [4]. This is accompanied by increased levels of some cytokines, such as interleukin (IL)-2, interferon-γ, and IL-10. T1/T2 ratio is higher in ITP and is inversely correlated with the platelet count, as opposed to a T2-cytokine response in patients in remission or treated with intravenous immunoglobulin. An elevated level of transforming growth factor (TGF)-β, a potent immunosuppressive cytokine, has been found in ITP during remission. This might be a viable approach for tolerance induction. T-cell-mediated immunity and increase of cytokine levels in serum lead to tolerance failure followed by autoantibodies production, platelet destruction, and, in some cases, to megakaryocyte suppression. Levels of CXCL10 are higher and the levels of CXCL5, CCL5, and CD40L are lower in ITP patients with platelet count less than 50 × 109/L compared with healthy controls [11]. In vitro, concentrations of these cytokines in the supernatants of platelet suspensions are proportional to platelet numbers. The authors conclude that these cytokines are mainly platelet-derived, confirming a role of platelets in immune responses and inflammation.
Methods of detection Various techniques have been developed to detect platelet autoantibodies to facilitate the diagnosis of ITP. The measurement of direct platelet-associated immunoglobulins by flow cytometry or highly sensitive enzyme-linked immunosorbent assay (ELISA) is considered a sensitive tool for ITP diagnosis. However, the specificity of these assays is too low because they cannot differentiate platelet-GP-specific antibodies from nonplatelet-specific antibodies and therefore are not defined in the diagnostic algorithm of ITP. In contrast, assay of serum antibodies that bind to normal platelets by fluorescence flow cytometry is more specific but less sensitive. Taking into account that antibodies are directed to one of the membrane GPs, several laboratories proposed a variety of antigen-specific assays, such as immunoblot techniques, radio-immunobead immunoprecipitation, immunobead assay, and monoclonal antibody-specific immobilization of antibodies (MAIPA) [12]. Despite possessing high specificity (78–93%) to confirm the diagnosis of ITP, their sensitivity (49–66%) was not high enough to exclude
Clinical utility
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the diagnosis of ITP when results were negative. Additionally, these assays carried certain methodologic or practical limitations, so none of them is currently approved to be applicable for the routine diagnosis of ITP. Nevertheless, the GP-specific platelet autoantibodies assays continue to be used. For NAIT diagnosis, HPA genotyping of neonate and its parents is recommended as well as GP-specific antibodies in the serum of the mother. Cross-match of maternal serum with the father’s platelets also can be considered.
Clinical utility Idiopathic thrombocytopenia The term ITP was used to refer to idiopathic or immune thrombocytopenic purpura. However, now the disease cannot be considered idiopathic. In addition, many patients do not have purpura at the time of diagnosis. Therefore, the term ITP now refers to immune thrombocytopenia. The annual incidence of ITP is 5.5 per 100,000 people. ITP is defined as a platelet count of less than 100 × 109/L with no evidence of leukopenia and anemia. ITP can be primary or secondary. Primary ITP accounts for the majority of cases where other conditions are absent. Thus, a very meticulous clinical history is required. Secondary ITP appears after infections, such as Helicobacter pylori, hepatitis C, and HIV. Other cases of thrombocytopenia include systemic lupus erythematosus, Wiskott-Aldrich syndrome, antiphospholipid syndrome, chronic lymphocytic leukemia, and association with drugs (e.g., heparin, abciximab). In children, the presentation of ITP is acute with severe thrombocytopenia and underlying conditions, unlike adults where most of the cases with ITP are diagnosed incidentally on routine blood test. The diagnosis of ITP during pregnancy can sometimes be undistinguished from pregnancy-induced thrombocytopenia, and unless a nonpregnant platelet count is available, diagnosis is not feasible. Maternal ITP rarely causes serious thrombocytopenia and bleeding in the fetus or neonate; however, instrumental delivery should be avoided. The bleeding manifestation in ITP is in correlation with the degree of thrombocytopenia. Usually bleeding becomes evident when platelet counts are less than 20 × 109/L unless platelet function is impaired by antibodies. Epistaxis, menorrhagia, and in some cases hematuria can occur. Intracranial hemorrhage usually presents in young people when platelet level is less than 10 × 109/L. Aspiration of bone marrow is not indicated in patients with typical presentation of ITP and under the age of 60 years.
Alloimmune thrombocytopenia NAIT has a frequency of 1–4 per 1000 live births. Unlike hemolytic disease, thrombocytopenia in the fetus can occur during first pregnancy when maternal antibodies (IgG subclass) are directed against platelet epitopes inherited by the father and transferred through the placenta. About 30–40% of neonates born to immunized women develop severe thrombocytopenia. The diagnosis of NAIT is suspected in otherwise healthy infants with unexpected petechiae, purpura, and even with intracranial hemorrhage, which appears early during pregnancy or during labor. With subsequent pregnancies, the degree of thrombocytopenia becomes more severe. The diagnosis is confirmed by genotyping maternal and paternal platelets as well as the newborn or the fetus. The genotype by amniocentesis is helpful in some cases, as it will define whether the mother needs treatment.
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CHAPTER 61 Platelet Autoantibodies
Transplant-induced alloimmune thrombocytopenia can occur due to residual HPA-1 mismatched host cells or donor cells following bone marrow and solid organ transplantation. A clinical history in this case can help in making the diagnosis. Passive alloimmune thrombocytopenia results in bleeding following infusion of antibodies within the transfused blood product, resulting in destruction of host platelets. To date, only HPA-1 and HPA-5b have been implicated for such phenomenon. Acute and severe post-transfusion purpura occurs 7 days after transfusion of blood product containing platelets. The alloantibodies are usually anti-HPA-1a, destroying not only donor platelets but also the host platelets. The diagnosis is confirmed by demonstrating antibodies in a patient’s serum directed against donor specific antigens. Transfusion refractoriness is a failure of increase in platelet count following platelet transfusion as a result of antibodies against HLA, and rarely against another epitope, which develops after previous transfusions. The presence of alloantibodies leads to rapid destruction of transfused platelets. A similar phenomenon occurs in Glanzmann thrombasthenia patients who were heavily transfused.
Pseudo-thrombocytopenia The prevalence rate of pseudo-thrombocytopenia (PTCP) was reported as 0.07–0.20%. Ethylenedi aminetetraacetic acid (EDTA) is usually used as an anticoagulant in blood cell counts and sometimes induces platelet clumping, which results in artificially low platelet counts. In such situations, the automated counter reported lower platelet count than the actual count since platelet clumps are read as leukocytes. On microscopic examination of blood film, platelet clumps are observed and confirm the diagnosis. The EDTA-initiated antibodies do not appear to have any clinical implication but can be transferred through the placenta, leading to PTCP in the neonate. In contrast to EDTA, platelet clumping is absent and platelet count is normal using sodium citrate or heparin. Platelet-leukocyte satellitism is a phenomenon that is similar to pseudo-thrombocytopenia and stems from antibodies that react with platelet GP and Fcγ receptor of leukocytes, creating a rosette around the periphery of leukocytes when observed by microscope [13]. Ex vivo platelet clumping should be considered in patients with acute viral infections, particularly in hepatitis infections. Failure to identify PTCP may result in a clinical problem like unnecessary diagnostic tests, glucocorticoid therapy, and platelet transfusion.
Take-home messages • I mmune response against platelet surface antigens results in antiplatelet antibodies production, which is the basis for most immune thrombocytopenias: idiopathic, drug-induced, post-transfusion, and neonatal alloimmune, as well as refractoriness to random donor platelets. • The antiplatelet antibodies are mainly IgG, mostly against GPIIb/IIIa and GPIb/IX. The autoantibody production is under the control of T-helper cells and cytokines. • Involvement of HLA class I and II was reported in ITP and NAIT development, explaining the hereditary tendency of the immune diseases in some of the cases. • EDTA sometimes induces platelet clumping, which results in artificially low platelet count. This phenomenon named as PTCP. Ex vivo platelet clumping may occur in patients with acute viral infections.
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