Mechanism Of Platelet Clearance Research Articles

The role of platelet desialylation as a biomarker in primary immune thrombocytopenia: mechanisms and therapeutic perspectives.

Primary immune thrombocytopenia (ITP) is an acquired autoimmune disorder characterized by the destruction of platelets. Although it was long believed that the critical role of autoantibodies in platelet destruction, primarily through the Fc-dependent platelet clearance pathway, recent findings indicate that the significance of the Fc-independent platelet clearance pathway mediated by hepatocytes, thus shedding light on a previously obscure aspect of ITP pathogenesis. Within this context, the desialylation of platelets has emerged as a pivotal biochemical marker. Consequently, targeting platelet desialylation emerges as a novel therapeutic strategy in the pathogenesis of ITP. Notably, prevailing research has largely focused on antiplatelet antibodies and the glycosylation-associated mechanisms of platelet clearance, while comprehensive analysis of platelet desialylation remains scant. In response, we retrospectively discuss the historical progression, inducing factors, generation process, and molecular regulatory mechanisms underlying platelet desialylation in ITP pathogenesis. By systematically evaluating the most recent research findings, we contribute to a comprehensive understanding of the intricate processes involved. Moreover, our manuscript delves into the potential application of desialylation regulatory strategies in ITP therapy, heralding novel therapeutic avenues. In conclusion, this manuscript not only fills a critical void in existing literature but also paves the way for future research by establishing a systematic theoretical framework. By inspiring new research ideas and offering insights into the development of new therapeutic strategies and targeted drugs, our study is poised to significantly advance the clinical management of ITP.

Prevalence of anti-glycoprotein V in patients with primary immune thrombocytopenia

Background Glycoprotein (GP) V is a well-characterized immune target in Varicella-associated and drug-induced thrombocytopenia. Recent studies have focused on the involvement of anti-GP V in the mechanism of platelet clearance as a recent paradigm in the pathophysiology of immune thrombocytopenia (ITP), rather than other anti-GPs like GP IIb/IIIa, GP Ib/IX and less frequently GP Ia/IIa. Objectives This study was conducted to determine the prevalence of anti-GP V in adult ITP Egyptian patients and to detect its impact on the outcome in these patients. Patients and methods This was a cross-sectional study in which, using enzyme-linked immunosorbent assay, the level of serum anti-GP antibodies V was measured in the peripheral blood of 90 patients with primary ITP compared with 20 age-matched and sex-matched normal healthy controls and its relationship with clinical, laboratory parameters, and response to treatment. Results Our study revealed that anti-GP antibodies V level in the cases group was statistically significantly higher as compared with the control group (P=0.005). There was weak statistically significant positive correlation between platelet count and anti-GP V level antibodies in the cases group after treatment with a P value of 0.0548. However, the level of anti-GP V did not show significant correlation with the bleeding score (P=0.123), degree of thrombocytopenia (P=0.967), sex (P=0.067), bone marrow findings (P=0.448), spleen size (P=0.992), or with the response to steroid therapy (P=0.171). Conclusion This study points out that GP V is a frequent immune target in ITP and that the detection of GP-specific autoantibodies being of value in the pathogenesis of ITP. We recommend that further studies including GP V are required before ITP treatment can be tailored according to platelet autoantibody specificity.

Platelet functional abnormalities in pediatric patients with kaposiform hemangioendothelioma/Kasabach-Merritt phenomenon.

Kaposiform hemangioendothelioma (KHE) is a rare vascular tumor of infancy that is commonly associated with a life-threatening thrombocytopenic condition, Kasabach-Merritt phenomenon (KMP). Platelet CLEC-2, tumor podoplanin interaction is considered the key mechanism of platelet clearance in these patients. Here, we aimed to assess platelet functionality in such patients. Three groups of 6 to 9 children were enrolled: group A with KHE/KMP without hematologic response (HR) to therapy; group B with KHE/KMP with HR; and group C with healthy children. Platelet functionality was assessed by continuous and end point flow cytometry, low-angle light scattering analysis (LaSca), fluorescent microscopy of blood smears, and exvivo thrombi formation. Platelet integrin activation in response to a combination of CRP (GPVI agonist) and TRAP-6 (PAR1 agonist), as well as calcium mobilization and integrin activation in response to CRP or rhodocytin (CLEC-2 agonist) alone, were significantly diminished in groups A and B. At the same time, platelet responses to ADP with or without TRAP-6 were unaltered. Thrombi formation from collagen in parallel plate flow chambers was also noticeably decreased in groups A and B. In silico analysis of these results predicted diminished amounts of CLEC-2 on the platelet surface of patients, which was further confirmed by immunofluorescence microscopy and flow cytometry. In addition, we also noted a decrease in GPVI levels on platelets from group A. In KHE/KMP, platelet responses induced by CLEC-2 or GPVI activation are impaired because of the diminished number of receptors on the platelet surface. This impairment correlates with the severity of the disease and resolves as the patient recovers.

Anti-HLA Class I alloantibodies in platelet transfusion refractoriness: From mechanisms and determinants to therapeutic prospects.

Patients with hematological disorders and severe thrombocytopenia require extensive and iterative platelet transfusion support. In these patients, platelet transfusion refractoriness represents a serious adverse transfusion event with major outcomes for patient care. Recipient alloantibodies against the donor HLA Class I antigens expressed at the cell surface of platelets result in a rapid removal of transfused platelets from the circulation and thus, therapeutic and prophylactic transfusion failure leading to a major bleeding risk. In this case, the only way to support the patient relies on the selection of HLA Class I compatible platelets, an approach restricted by the limited number of HLA-typed donors available and the difficulty of meeting the demand in an emergency. However, not all patients with anti-HLA Class I antibodies develop refractoriness to platelet transfusions, raising the question of the intrinsic characteristics of the antibodies and the immune-mediated mechanisms of platelet clearance associated with a refractory state. In this review, we examine the current challenges in platelet transfusion refractoriness and detail the key features of the antibodies involved that should be considered. Finally, we also provide an overview of future therapeutic strategies.

Rasa3 deficiency minimally affects thrombopoiesis but promotes severe thrombocytopenia due to integrin-dependent platelet clearance.

Platelet homeostasis is dependent on a tight regulation of both platelet production and clearance. The small GTPase Rap1 mediates platelet adhesion and hemostatic plug formation. However, Rap1 signaling is also critical for platelet homeostasis as both Rap1 deficiency and uninhibited Rap1 signaling lead to marked thrombocytopenia in mice. Here, we investigated the mechanism by which deficiency in Rasa3, a critical negative regulator of Rap1, causes macrothrombocytopenia in mice. Despite marked morphological and ultrastructural abnormalities, megakaryocytes in hypomorphic Rasa3hlb/hlb (R3hlb/hlb) or Rasa3–/– mice demonstrated robust proplatelet formation in vivo, suggesting that defective thrombopoiesis is not the main cause of thrombocytopenia. Rather, we observed that R3hlb/hlb platelets became trapped in the spleen marginal zone/red pulp interface, with evidence of platelet phagocytosis by macrophages. Clearance of mutant platelets was also observed in the liver, especially in splenectomized mice. Platelet count and platelet life span in Rasa3-mutant mice were restored by genetic or pharmacological approaches to inhibit the Rap1/talin1/αIIbβ3 integrin axis. A similar pattern of splenic clearance was observed in mice injected with anti-αIIbβ3 but not anti–glycoprotein Ibα platelet-depleting antibodies. In summary, we describe a potentially novel, integrin-based mechanism of platelet clearance that could be critical for our understanding of select inherited and acquired thrombocytopenias.

Pathogenic mechanisms contributing to thrombocytopenia in patients with systemic lupus erythematosus

Summary Systemic lupus erythematosus (SLE) is an autoimmune condition developing thrombocytopenia in about 10–15% of cases, however, mechanisms leading to low platelet count were not deeply investigated in this illness. Here we studied possible causes of thrombocytopenia, including different mechanisms of platelet clearance and impairment in platelet production. Twenty-five SLE patients with and without thrombocytopenia were included. Platelet apoptosis, assessed by measurement of loss of mitochondrial membrane potential, active caspase 3 and phosphatidylserine exposure, was found to increase in thrombocytopenic patients. Plasma from 67% SLE patients (thrombocytopenic and non-thrombocytopenic) induced loss of sialic acid (Ricinus communis agglutinin I and/or Peanut agglutinin binding) from normal platelet glycoproteins. Concerning platelet production, SLE plasma increased megakaryopoiesis (evaluated using normal human cord blood CD34+ hematopoietic progenitors), but inhibited thrombopoiesis (proplatelet count). Anti-platelet autoantibody depletion from SLE plasma reverted this inhibition. Overall, abnormalities were more frequently observed in thrombocytopenic than non-thrombocytopenic SLE patients and in those with active disease (SLEDAI≥5). In conclusion, platelet clearance due to apoptosis and desialylation, and impaired platelet production mainly due to inhibition of thrombopoiesis, could be relevant mechanisms leading to thrombocytopenia in SLE. These findings could provide a rational basis for the choice of proper therapies to correct platelet counts in these patients.

Calcium Ion Chelation Preserves Platelet Function During Cold Storage.

Platelet transfusion is a life-saving therapy to prevent or treat bleeding in patients with thrombocytopenia or platelet dysfunction. However, for >6 decades, safe and effective strategies for platelet storage have been an impediment to widespread use of platelet transfusion. Refrigerated platelets are cleared rapidly from circulation, precluding cold storage of platelets for transfusion. Consequently, platelets are stored at room temperature with an upper limit of 5 days due to risks of bacterial contamination and loss of platelet function. This practice severely limits platelet availability for transfusion. This study is to identify the mechanism of platelet clearance after cold storage and develop a method for platelet cold storage. Approach and Results: We found that rapid clearance of cold-stored platelets was largely due to integrin activation and apoptosis. Deficiency of integrin β3 or caspase-3 prolonged cold-stored platelets in circulation. Pretreatment of platelets with EGTA, a cell impermeable calcium ion chelator, reversely inhibited cold storage-induced platelet activation and consequently prolonged circulation of cold-stored platelets. Moreover, transfusion of EGTA-treated, cold-stored platelets, but not room temperature-stored platelets, into the mice deficient in glycoprotein Ibα significantly shortened tail-bleeding times and diminished blood loss. Integrin activation and apoptosis is the underlying mechanism of rapid clearance of platelets after cold storage. Addition of a cell impermeable calcium ion chelator to platelet products is potentially a simple and effective method to enable cold storage of platelets for transfusion.

P1838Thrombocytopenia under ECMO and Shear-induced shedding of platelet receptor Glycoprotein-(GP)Ialpha

Abstract Background Several mechanisms are suspected to thrombocytopenia under Extracorporeal Membrane Oxygenation (ECMO) such as platelet-consumption or sepsis. Shedding of glycoprotein-(GP)Ibα is a recently identified mechanism of platelet clearance. ECMO generates high shear stress forces that could impact GPIbα-shedding. We hypothesized that ECMO continuous-flow devices could directly induce thrombocytopenia through shear-induced GPIbα-shedding. Aims Determine if ECMO induce GpIb-shedding in vitro and in vivo and determinates the kinetic evolution of platelet-count and GpIb-shedding after patient's implantation. Methods Platelet GPIbα-shedding was first investigated in vitro using a high-shear pump loop model. Plasma with normal platelet count (plasma-NPC) was obtained by dilution of platelet-rich plasma obtained from healthy donors in fresh-frozen-plasma. Samples were collected before and after (5, 30, 60 and 180 min) perfusion at 37°C of plasma-NPC at intermediate and high speed (2.6 and 3.6 L min–1 respectively, n=4 each). Platelet count and GPIbα-shedding were next investigated in 20 ECMO patients before/after implantation (WITECMO trial) and in 20 healthy volunteers. The geometric mean-fluorescence-intensity (gMFI) of platelet GPIbα (PE-staining) and GPIX (FITC-staining) was measured with a Navios flow cytometer (Beckman Coulter, Miami, FL). Results are expressed as GPIbα/GPIX gMFI-ratio. Results A significant time-dependent loss of GPIbα/GPIX gMFI-ratio was already apparent after 30 min in vitro and was significantly more pronounced at high-speed compared to intermediate-speed (pANOVA<0.001 and p<0.01 at 180 min respectively). GPIbα/GPIX gMFI-ratio was significantly increased in ECMO patients compared to healthy subjects 1- and 24-hour after implantation (p<0.001). A significantly lower platelet count was observed 1 hour after ECMO implantation (−23% vs baseline, p<0.01) with a further significant decrease at 24-hours (−53% vs baseline, p<0.0001). Figure 1. A. Significant time-dependent loss of platelet GPIbα/GPIX gMFI-ratio (pANOVA <0.001) assessed by flow-cytometry after 30 min of perfusion at 3.6 L/min with a high-shear continuous-flow device in vitro. B. Representative experiment showing the apparition of a platelet sub-population with loss of GPIbα expression after 30 min of perfusion at 3.6 L/minwith a high-shear continuous-flow device in vitro.

Desialylation of platelets induced by Von Willebrand Factor is a novel mechanism of platelet clearance in dengue.

Thrombocytopenia and platelet dysfunction are commonly observed in patients with dengue virus (DENV) infection and may contribute to complications such as bleeding and plasma leakage. The etiology of dengue-associated thrombocytopenia is multifactorial and includes increased platelet clearance. The binding of the coagulation protein von Willebrand factor (VWF) to the platelet membrane and removal of sialic acid (desialylation) are two well-known mechanisms of platelet clearance, but whether these conditions also contribute to thrombocytopenia in dengue infection is unknown. In two observational cohort studies in Bandung and Jepara, Indonesia, we show that adult patients with dengue not only had higher plasma concentrations of plasma VWF antigen and active VWF, but that circulating platelets had also bound more VWF to their membrane. The amount of platelet-VWF binding correlated well with platelet count. Furthermore, sialic acid levels in dengue patients were significantly reduced as assessed by the binding of Sambucus nigra lectin (SNA) and Maackia amurensis lectin II (MAL-II) to platelets. Sialic acid on the platelet membrane is neuraminidase-labile, but dengue virus has no known neuraminidase activity. Indeed, no detectable activity of neuraminidase was present in plasma of dengue patients and no desialylation was found of plasma transferrin. Platelet sialylation was also not altered by in vitro exposure of platelets to DENV nonstructural protein 1 or cultured DENV. In contrast, induction of binding of VWF to glycoprotein 1b on platelets using the VWF-activating protein ristocetin resulted in the removal of platelet sialic acid by translocation of platelet neuraminidase to the platelet surface. The neuraminidase inhibitor oseltamivir reduced VWF-induced platelet desialylation. Our data demonstrate that excessive binding of VWF to platelets in dengue results in neuraminidase-mediated platelet desialylation and platelet clearance. Oseltamivir might be a novel treatment option for severe thrombocytopenia in dengue infection.

In Vitro Monocyte/Macrophage Phagocytosis Assay for the Prediction of Drug-Induced Thrombocytopenia.

Phagocytosis of platelets by monocytes and macrophages is a primary mechanism of platelet clearance in vivo and has been increasingly implicated in playing an important role in thrombocytopenia mediated by monoclonal antibodies intended for therapeutic purposes. In the present article, we describe an in vitro flow cytometry assay to assess the effect of antibody-mediated platelet phagocytosis by monocytes. Freshly isolated platelets were labeled with a fluorescent probe, 5-chloromethylfluorescein diacetate (CMFDA) and then co-cultured with isolated peripheral blood mononuclear cells (PBMCs) from the same donor in the presence of increasing concentrations of a monoclonal antibody drug. After incubation, an increase in CMFDA fluorescence intensity of CD14 positive monocytes was evaluated by flow cytometry as an assessment for drug-mediated platelet phagocytosis by monocytes. The assay has been evaluated using both human and cynomolgus monkey cells for the prediction of drug-induced thrombocytopenia. © 2019 by John Wiley & Sons, Inc.

Dimerization of glycoprotein Ibα is not sufficient to induce platelet clearance

ESSENTIALS: Many anti-glycoprotein (GP)Ibα antibodies induce platelet clearance in a dimer-dependent manner. Characterization of monoclonal antibodies that bind the mechanosensitive domain (MSD) of GPIbα. An anti-MSD antibody binds two copies of GPIbα in platelets but does not induce platelet clearance. The prevailing clustering model of GPIbα signaling is incorrect or needs revision. The mechanism of platelet clearance is not clear. Many antibodies binding the membrane-distal ligand-binding domain of glycoprotein (GP)Ibα induce rapid clearance of platelets and acute thrombocytopenia, which requires the bifurcated antibody structure. It was thought that binding of these antibodies induced lateral dimerization or clustering of GPIbα in the plasma membrane, which leads to downstream signaling and platelet clearance. However, many antibodies targeting GPIbβ and GPIX, which are associated with GPIbα in the GPIb-IX complex, do not induce platelet clearance, which is in contradiction to the clustering model. To test whether dimerization or clustering of GPIbα is sufficient to transmit the signal that leads to platelet clearance. We have recently raised several mAbs targeting the mechanosensitive domain (MSD) of GPIbα. Binding of these anti-MSD antibodies was characterized with biochemical methods. Their ability to stimulate platelets and induce platelet clearance in mice was assessed. Infusion of anti-MSD antibodies does not cause thrombocytopenia in mice. These antibodies show no detectable effects on platelet activation and aggregation in vitro. Further biochemical investigation showed that the anti-MSD antibody 3D1 binds two copies of GPIbα on the platelet surface. Therefore, lateral dimerization of GPIbα induced by antibody binding is not sufficient to initiate GPIb-IX signaling and induce platelet clearance. Our results suggest that a factor other than or in addition to clustering of GPIbα is required to induce platelet clearance.

Novel mechanisms of platelet clearance and thrombopoietin regulation.

The human body produces and removes 10 platelets daily to maintain a normal steady-state platelet count. Platelet production must be tightly regulated to avoid spontaneous bleeding or arterial occlusion and organ damage. Multifaceted and complex mechanisms control platelet removal and production in physiological and pathological conditions. This review will focus on different mechanisms of platelet clearance, with focus on the biological significance of platelet glycans. The Ashwell-Morrell receptor (AMR) recognizes senescent, desialylated platelets under steady state conditions. Desialylated platelets and the AMR are the physiological ligand-receptor pair regulating hepatic thrombopoietin (TPO) mRNA production, resolving the longstanding mystery of steady state TPO regulation. The AMR-mediated removal of desialylated platelets regulates TPO synthesis in the liver by recruiting JAK2 and STAT3 to increase thrombopoiesis. Inhibition of TPO production downstream of the hepatic AMR-JAK2 signaling cascade could additionally contribute to the thrombocytopenia associated with JAK1/2 treatment, which is clinically used in myeloproliferative neoplasms.

Current methods of assessing platelet function: relevance to transfusion medicine

Current methods of assessing platelet function: relevance to transfusion medicine