Lev PR, Goette NP, Marta RF. Pathophysiological Mechanisms Leading to Low Platelet Count in Immune Thrombocytopenia. J Immunological Sci. (2020); 4(2): 1-7 Journal of Immunological Sciences
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Pathophysiological Mechanisms Leading to Low Platelet Count in Immune Thrombocytopenia Paola Roxana Lev1,2, Nora Paula Goette1, Rosana Fernanda Marta1,2* 1Institute of Medical Research A. Lanari, School of Medicine, University of Buenos Aires, Buenos Aires, Argentina 2Department of Hematology Research, Institute of Medical Research (IDIM), National Scientific and Technical Research Council (CONICET), University of Buenos Aires, Buenos Aires, Argentina
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Article Notes Primary immune thrombocytopenia (ITP) is an autoimmune disorder Received: May 06, 2020 characterized by the decrease in peripheral blood platelet count below 100 Accepted: July 01, 2020 x 109/L, and an increased bleeding risk when thrombocytopenia drops below 9 *Correspondence: 30 x 10 /L. The mechanisms leading to ITP in adults, although not completely Dr. Rosana Fernanda Marta, Institute of Medical Research A. elucidated, involves an imbalance between effector and regulatory cells Lanari, School of Medicine, University of Buenos Aires, Buenos that results in a breakdown of the immune tolerance. Autoantibodies are Aires, Argentina; Email: [email protected] considered the main responsible for thrombocytopenia, although direct T-cell cytotoxic effect and lysis by Complement attachment and activation could also © 2020 Marta RF. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License. contribute to platelet elimination from circulation. In addition to increased peripheral clearance, abnormalities in platelet production also favors platelet Keywords: count reduction. This review is intended to describe some specific knowledge Immune Thrombocytopenia about peripheral and bone marrow mechanisms leading to thrombocytopenia Platelets in adult ITP. Autoantibodies Megakaryopoiesis Thrombopoiesis Apoptosis Introduction Desialylation Megakaryocytes Primary immune thrombocytopenia (ITP) is an acquired Proplatelet formation autoimmune disorder characterized by chronic isolated low platelet 9 1 count (<100 x 10 /L) , triggered by a loss of the immunological system equilibrium that tips the balance toward autoimmunity. Although in the last years the pathophysiology of ITP has been intensively 2,3. Another
studied, this field still remains incompletely understood significant feature of ITP is the heterogeneity observed among aspatients, refractory concerning4. This heterogeneity not only their is likely clinical due profile to the but participation also their ofresponse multiple to pathophysiologicaltreatment, with a small contributing proportion mechanisms, of patients classifiedaffecting individual patients in different degrees. The presence of autoantibodies targeting platelet surface glycoprotein(s) (GP) has been demonstrated in patients with ITP with percentages of patients with positive autoantibodies varying according to different case series5-7. Most of these antibodies are IgG, but IgM and IgA can also be detected, usually in association with IgG8.
and GPIaIIa complex, but, autoantibodies against other GPs (i.e. the Main autoantibody targets are GPIIbIIIa9, against (integrin thrombopoietin αIIbβ3), and/or GPIbIX its receptor c-Mpl10-12 were also detected in some patients. Therefore, humoralvitronectin immunity receptor, has integrin been classically αvβ3) considered the main cause of thrombocytopenia, although other mechanisms such as direct T-cell cytotoxic effect could contribute to platelet elimination as well13,14.
In addition, Complement activation/fixation capacity is enhanced Page 1 of 7 Lev PR, Goette NP, Marta RF. Pathophysiological Mechanisms Leading to Low Platelet Count in Immune Thrombocytopenia. J Immunological Sci. (2020); 4(2): 1-7 Journal of Immunological Sciences in plasma samples from ITP15,17. Indeed, Complement- mediated platelet destruction has been pointed as another antibodies in triggering this phenomenon. mechanism of platelet elimination, mainly triggered by autoantibodies, suggesting a causal role for these specific In this study, PS externalization was not noticed in anti-platelet autoantibodies, but also observed in some ITP normal platelets incubated with ITP plasmas but it could be patients with no-detectable autoantibodies18. observed by adding normal autologous CD3+ lymphocytes This review summarizes mechanisms leading to to this system, suggesting an antibody-dependent cytotoxic thrombocytopenia in ITP with special focus on peripheral effect. In the same system, anti-platelet antibodies-depleted platelet destruction and impaired platelet production. ITP plasma had no effect on normal platelet apoptosis, while the IgG fraction of ITP plasma retained the apoptotic Increased circulating platelet clearance the involvement of auto-antibodies in the induction of of platelet clearance in ITP is the binding of anti-platelet apoptosis.effect observed when whole plasma was used, confirming autoantibodiesThe first and to their most antigenic commonly glycoprotein accepted targets mechanism that Interestingly, recent work described that autophagy leads to platelet elimination by monocytes/macrophages is decreased in platelets from ITP26 and suggested this reduction could be linked to increased platelet apoptosis. Further studies are warranted to elucidate the interplay liverof the19. reticuloendothelialHowever, additional system pathological through FcγRIIAmechanisms and between autoantibodies, autophagy and apoptosis in this includingFcγRIIIA-mediated platelet apoptosis recognition, and mainly loss of in sialic the spleen acid from and disorder. platelet membrane glycoproteins were described in recent years, that also contribute to platelet clearance from Increased loss of sialic acid from platelet glycoproteins circulation. in ITP Increased platelet apoptosis in ITP In 2015, Li and col.27 Several studies have demonstrated the contribution autoantibodies induced loss described of sialic for theacid firstcapping time of platelet apoptosis to ITP pathogenesis. Winkler and carbohydratesthat plasma from from platelet ITP patients glycoproteins. containing This anti-GPIbIXmechanism col20 showed evidence of platelet apoptosis, including is triggered by the ability of these autoantibodies to caspase 3, 8 and 9 activation, in children with acute ITP, induce platelet activation, which promote neuraminidase which was ameliorated by intravenous immunoglobulin externalization, the ultimate responsible for sialic acid infusion. Concerning adult chronic ITP, increased platelet cleavage from membrane glycoproteins. Platelets bearing phosphatidylserine (PS) exposure, as a marker of platelet 21 22 apoptosis was shown by Catani . the liver by cooperation between macrophage galactose lectinasialoglycoproteins (MGL) and the on C-type their lectin membrane receptor are CLEC4F, clarified both in In 2016, our group described andincreased Alvarez mitochondrial Román membrane depolarization, active caspase 3 and membrane expressed on Kupffer cells, and Ashwell-Morell receptor exposure of PS in platelets from ITP, as markers of apoptosis, while basal platelet activation assessed by PAC-1 binding performed in mice28,29. In addition, activation of the JAK2/ and P-selectin externalization was ruled out as another (AMR) in hepatocytes, as demonstrated by studies possible cause of increased PS expression23. Later, Deng and binding, has been pointed as the mechanism triggering col24 synthesisSTAT3 pathway of thrombopoietin downstream (Tpo),AMR after the main asialoglycoprotein growth factor levels of the proapoptotic molecules Bak and Bax, while of the megakaryocytic lineage30. confirmed these results and showed higher expression the antiapoptotic factor Bcl-xL was decreased. Moreover, In a recent study, our group investigated several the same group demonstrated dysregulation of certain possible causes of thrombocytopenia in a cohort of ITP 25. Cells expressing PS on their membrane are known to be that loss of sialic acid from normal platelet glycoproteins microRNAscleared through related scavenger to apoptosis receptors in platelets in macrophages, from ITP patients, including platelet desialylation. Results showed thus, apoptotic platelets would also be eliminated from GPIIbIIIa ITP autoantibodies, as assessed by the increase circulation by this mechanism. In our cohort of ITP, the inoccurs Ricinus not onlycommunis in the presence of anti-GPIbIX but also anti- inverse relationship found between apoptotic variables and peripheral platelet counts, further reinforces the agglutinin I (RCA-I), recognizing relevance of platelet apoptosis in the development of galactosyl (β-1,4) N-acetylglucosamine (Galβ4GlcNAc) thrombocytopenia in this disorder. Interestingly, platelet exposedand Peanut after agglutinin sialic acid (PNA) loss. which In addition, recognizes desialylation galactosyl apoptosis was observed in all ITP patients carrying auto- could(β-1,3) be concomitantly N-acetylgalactosamine observed with (Galβ3GalNAc), platelet apoptosis, both although this was not a mandatory association31 but was absent in those patients expressing anti-GPIaIIa Manzano and col reported increased apoptosis and loss of antibodies against the glycoproteins GPIIbIIIa and GPIbIX, . Recently,
Page 2 of 7 Lev PR, Goette NP, Marta RF. Pathophysiological Mechanisms Leading to Low Platelet Count in Immune Thrombocytopenia. J Immunological Sci. (2020); 4(2): 1-7 Journal of Immunological Sciences sialic acid in platelets from non-responsive ITP patients32. through the endothelial bone marrow barrier, platelets are Interestingly, although a direct correlation was found released from their tips into the bloodstream. between caspase activation and desialylation in the whole population included in the study (ITP patients who did Megakaryopoiesis in ITP not need treatment for at least six months, ITP patients Early independent works from Chang38 and McMillan39 responding to agonists of thrombopoietin receptors, studied the effect of the addition of 10% of ITP plasma to and non-responders), levels of caspase activity between the culture medium of normal hematopoietic progenitors non-responders and thrombopoietin receptor agonists- induced to differentiate into megakaryocytes. Their responders were not different, supporting the notion that results showed that plasma from ITP patients impaired apoptosis and desialylation are not necessarily related. megakaryocyte development, pointing to autoantibodies In addition, decreased platelet surface glycans in non- responders ITP patients was accompanied by a decrease effect. Unlike these previous studies that used mononuclear in T regulatory lymphocyte population suggesting a link cellsdirected39 or to20-30% GPIbIX pure and CD34+ GPIIbIIIa progenitors as responsible38 as source for this of between this platelet abnormality and immune imbalance. normal hematopoietic precursors, we reproduced these The observation that anti-GPIIbIIIa autoantibodies can experiments using at least 93% pure CD34+ hematopoietic also induce loss of sialic acid from platelet glycoproteins progenitors isolated from normal human cord blood. In 33. By blocking the these conditions, megakaryopoiesis was not inhibited by ITP samples. Moreover, the expression of CD42b, a wasloss recentlyis at least confirmed partially due by Marini to crosslinking and col of anti-GPIIbIIIa marker of megakaryocyte maturation, was increased in the FcγRIIA signaling, authors demonstrated that sialic acid presence of 45% of the ITP plasmas, while megakaryocyte 31 release of sialidase from the intracellular compartments of size and ploidy were normal . These results are in line with platelets.autoantibodies to FcγRIIA, which is responsible for the the known concept that the number of megakaryocytes in bone marrow from ITP patients is normal or increased40,41. Concerning circulating Tpo levels in ITP, it was suggested Besides, although it was described that circulating that they were lower than expected for the degree of levels of thrombopoietin are low for the degree of thrombocytopenia34. In the late ´90s, when Tpo synthesis by thrombocytopenia in ITP, as mentioned before, they seem the liver was supposed to be constant, the cumulative mass of megakaryocytes and platelets were considered as the 35,36 in bone marrow. main determinant of Tpo levels sufficient to maintain a proper megakaryocyte population Participation of apoptosis during megakaryocyte and least in part, responsible for Tpo synthesis,. Nowadays, differences assuming in platelet production plateletthat binding desialylation of desialylated levels plateletsamong patients to hepatic with AMR ITP is, and at the fact that some of them do not display increased platelet lineage performed on bone marrow samples from ITP desialylation, could account for variations in Tpo levels patientsReports are describing controversial. apoptosis Uçar inand the col megakaryocytic42 found no differences in the number of megakaryocytes and their in modulating circulating Tpo levels in ITP should be apoptotic status between ITP and control children; testedamong by patients comparing population. these variables The influence in a large of desialylation ITP cohort. Houwerzijl et al43 found ultrastructural abnormalities compatible with apoptosis in adult ITP, and a recent described to regulate hepatic Tpo homeostasis37. In this study described a decrease of megakaryocyte apoptosis regard,In addition, ITP otherautoantibodies factors such targeting as platelet this glycoprotein GPIbα were in bone marrow biopsies of ITP subjects compared to could interfere with this mechanism. Therefore, further healthy controls44. Although these discrepant results are investigations are needed to fully understand the regulation intriguing and would encourage researchers to continue of Tpo synthesis in ITP as well as in physiologic conditions. the investigations, bone marrow samples form ITP are not Impaired platelet production easily available because bone marrow examination is not generally needed to diagnose ITP. Beyond megakaryopoiesis, data about the participation megakaryopoiesisPlatelet production and thrombopoiesis. is the final step Megakaryopoiesis of a complex of apoptotic events during normal physiologic proplatelet initiatesseries of when events undifferentiated involving two wellCD34+ defined hematopoietic processes: formation and platelet production has also been progenitor commits to the megakaryocytic lineage and ends controversial. While several studies support that localized when megakaryocytes reach their full maturational state. apoptotic events are needed for mature megakaryocytes to rearrange their cytoplasmic architecture and proceed cytoplasmic megakaryocytic reorganization, proplatelet with proplatelet extension and platelet release, more extensionNext, the and process branching identified and, as after thrombopoiesis these proplatelets involves go recent information hold the notion that apoptosis must be
Page 3 of 7 Lev PR, Goette NP, Marta RF. Pathophysiological Mechanisms Leading to Low Platelet Count in Immune Thrombocytopenia. J Immunological Sci. (2020); 4(2): 1-7 Journal of Immunological Sciences restrained during proplatelet formation, and only when utoantibodies impair megakaryocyte adhesion platelets have been released, the remaining naked nuclei and spreading to vWf, and preclude Src phosphorylation undergoes apoptosis (reviewed by McArthur45). downstreamGPIbIX a this receptor. Similarly, investigations evaluating the effect of ITP The main raison détre of megakaryocytes is platelet plasma on mature megakaryocyte apoptosis have led to production, and, as a consequence of autoantibody binding discordant results. Yang et al46 described lower expression of tumor necrosis factor-related apoptosis-inducing ligand, is inhibited 47,51. The participation of autoantibodies in higher expression of Bcl-xL and lower platelet release toabnormal GPIIbIIIa thrombopoiesis and/or GPIbIX complexes, was proved proplatelet by reproducing formation the in normal megakaryocytes incubated with ITP plasma, pointing to decreased apoptosis as a contributing factor its reversion when ITP plasma was immunodepleted from to reduced platelet production. In our hands, normal autoantibodiesinhibitory effect by of incubation ITP plasma with using normal purified platelets. IgG, and by mature megakaryocytes incubated for 48 hours with 10% In addition to the decrease in the number of ITP plasma did not suffer caspase 3-7 activation, and the megakaryocytes producing proplatelets, morphological number of megakaryocytes displaying nuclear picnosis changes were observed in the architecture of proplatelets was slightly increased. However, proplatelet formation was generated in the presence of ITP plasma. Most of the 47. Then, patient´s samples induced wider and shorter proplatelets, although the number of megakaryocytes in bone marrow bearing lower numbers of bifurcation points and tips, fromsignificantly ITP is decreasednormal or as elevated,will be discussed abnormalities later in the and overall, less complex than those observed when apoptotic pathways of mature megakaryocytes could not megakaryocytes were cultured with the addition of normal be ruled out as possible factors leading to a lower number plasma47. Considering that megakaryocytes must elongate of megakaryocytes capable of producing proplatelets. their proplatelets through the bone marrow endothelial Indeed, further work is necessary to clarify this topic. barrier in order to release platelets into the bloodstream, Altered megakaryocyte functionality and impaired these morphological proplatelet features could hinder thrombopoiesis thrombopoiesis. Altogether, these results could imply that, in addition to the lower number of megakaryocytes Other important players participating in the proper capable of producing proplatelets in ITP, each proplatelet- functionality of megakaryocytes are extracellular matrix bearing megakaryocyte would be unable to produce a proteins present in the bone marrow milieu. Megakaryocyte proper number of platelets. roles in megakaryocytic physiology. Two of these essential The osteoblastic niche of bone marrow is a type I interaction with different matrix proteins play specific collagen-enriched environment. Megakaryocyte binding and von Willebrand factor (vWf). Main receptors for these to collagen through GPIaIIa complex inhibits proplatelet molecules present in the vascular niche are fibrinogen production52, precluding premature platelet release within that recognizes vWf. In addition to their key role in platelet this compartment. Our in vitro experiments demonstrated ligands are GPIIbIIIa, which binds both proteins, and GPIbIX, in megakaryocyte development and platelet production. containing anti-GPIaIIa autoantibodies, induced a Prooffunction, of their GPIIbIIIa participation and GPIbIXV in these complexes events are are the involved deep decreasethat plasma in andmegakaryocyte purified IgG adhesion fractions and from spreading ITP patients on abnormalities observed in macrothrombocytopenia due type I collagen along with lower MLC2 phosphorylation to mutations leading to partial activation of GPIIbIIIa48, downstream GPIaIIa50. In agreement with the fact that MLC2 phosphorylation is a key downstream effector mediating syndrome 49. GPIaIIa-induced negative regulation of thrombopoiesis53, and mutations in GPIbIXV, responsible for Bernard-Soulier these autoantibodies interfered with normal inhibition Grodzielski and col50 demonstrated that plasma and of proplatelet formation on type I collagen, allowing megakaryocytes to extend proplatelets even on a type with adhesion and spreading of megakaryocytes. 47 specifically the IgG fraction from ITP patients interfere I collagen-coated surface the presence of anti-GPIaIIa autoantibodies could lead to pair depending on the type of autoantibody present. premature proplatelet production. These in findingsthe osteoblastic suggest niche, that Anti-GPIIbIIIaThis interference autoantibodies is specific forinhibit each megakaryocytic receptor-ligand hindering platelet release within the bloodstream. and signal transduction downstream this receptor, as Along with these abnormalities, Zeng and col.54 added interaction with fibrinogen, reducing adhesion, spreading autoantibodies targeting the vitronectin receptor, to the Abnormal glycoprotein function was also observed by list of factors responsible for the interference between impaireddemonstrated PAC-1 by binding decreased to megakaryocytes β3 subunit phosphorylation. incubated with megakaryocyte adhesion to the vascular niche, and ITP plasmas bearing these autoantibodies47. Similarly, anti- potentially contributing to impair platelet production in ITP.
Page 4 of 7 Lev PR, Goette NP, Marta RF. Pathophysiological Mechanisms Leading to Low Platelet Count in Immune Thrombocytopenia. J Immunological Sci. (2020); 4(2): 1-7 Journal of Immunological Sciences
In addition to platelets, megakaryocyte glycoproteins Conclusions are also target for autoantibody desialylation. Unlike what The growing amount of knowledge concerning was observed in platelets, loss of sialic acid from normal pathophysiological abnormalities in ITP and results that megakaryocytes was only found in the presence of ITP evaluated several of these mechanisms in individual 31. Marini patients, show that mechanisms of thrombocytopenia are and col.33 demonstrated that desialylation induced by ITP plasma containing anti-GPIbIX autoantibodies multifactorial, and frequently involve concomitant defects megakaryocyte adhesion to the bone marrow extracellular in platelet production and clearance. One of the main autoantibodies (either anti GPIIbIIIa or GPIbIX) inhibits conclusions of our work concerning platelet production differentiation and reduced ability to extend proplatelets. is that ITP autoantibodies are more harmful on inhibiting Thus,matrix besides proteins increasing fibrinogen platelet and vWf, clearance, leading to desialylation limited cell thrombopoiesis than affecting megakaryopoiesis. has also been established as a mechanism of impaired platelet production. dependent mechanism, platelet apoptosis and desialylation areRegarding additional platelet and clearance,independent apart processes from the contributing classical Fc- Overall, these studies demonstrate that ITP autoantibodies a rational for the use of combination therapy, including depending on their target proteins, interfering with normal drugsto lower with platelet different count mechanisms (Figure 1). of These action, findings in patients provide with megakaryocyticmainly affect specific functions and differentwithin the intracellular bone marrow pathways, and highly refractory disease. leading to decreased platelet production.
Figure 1: Mechanisms leading to low platelet count in Immune Thrombocytopenia. This graphical abstract includes mechanisms published in references 19, 24, 37 and 40. Impaired platelet production is likely due to abnormal megakaryocytic function, including altered interaction with extracellular matrix proteins from bone marrow and thrombopoiesis (reduced number of proplatelet-producing megakaryocytes and abnormal proplatelet structure), induced by autoantibody binding to megakaryocytic glycoproteins. Although autoantibody-induced abnormalities were also observed during megakaryopoiesis, they seem to contribute in lesser extent to the reduction in platelet production. Enhanced peripheral platelet clearance could be related to monocyte/ macrophage phagocytosis induced by binding of platelet-autoantibody complex to Fcγ receptors IIA and IIIA and phosphatidylserine expressed on apoptotic platelets that binds to scavenger receptors. In addition, desialylated platelets could be clarified by cooperation between Kupffer cells and hepatocytes in the liver.
Page 5 of 7 Lev PR, Goette NP, Marta RF. Pathophysiological Mechanisms Leading to Low Platelet Count in Immune Thrombocytopenia. J Immunological Sci. (2020); 4(2): 1-7 Journal of Immunological Sciences
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