Mature Platelets Research Articles

From bench to bedside and back to bench: investigating the role of platelet heterogeneity in coronary artery disease

Abstract Background/Introduction Platelets exhibit considerable heterogeneity in RNA content, size, and thrombogenicity. Our preliminary investigation of the transcriptome of the RNA-rich reticulated platelets(RPs) in healthy donors revealed an enrichment of prothrombotic transcripts compared to mature platelets(MPs). Recently, we validated the prognostic role of elevated RPs at clinical level in a modern cohort of ACS patients treated with potent P2Y12 inhibitors. However, the pathophysiology of RP hyperreactivity remains unclear, particularly its correlation with adverse events and cardiovascular death. Purpose To study the biology of RPs in patients with chronic coronary syndrome(CCS) and to elucidate their role in disease progression. Methods RPs were isolated from peripheral blood of CCS patients(N=20) and compared with MPs. Cell viability and reactivity of RPs were quantified by FACS. Deep transcriptomic profiling was performed using post-sorting bulk RNA sequencing of RPs and MPs and analyzed with ad-hoc bioinformatic pipelines. Proteomic profiling using mass cytometry(CyTOF) was used to validate the transcriptomic findings with single cell resolution. Results FACS analysis confirmed RPs hyperreactivity showing increased P-Selectin expression upon activation with TRAP, ADP and Collagen(Fig. 1A). Total RNA-sequencing detected 2213 differentially regulated genes with an enrichment in RPs of key functional transcript such as, the von Willebrand Factor VWF(p=3.06*10-33), the thromboxane receptor TBXA2R(p=2.07*10-29) and the collagen receptor GP6(p=1.29*10-38, Fig. 1B-C). Gene ontology and gene set enrichment analyses detected an upregulation of relevant categories as "Regulation of platelet activation" and "Platelet aggregation"(Fig. 1D-F). In addition, we detected a novel alternative splicing event on the collagen receptor transcript GP6 upregulated in RPs(Fig. 1G). We detected 33 differentially regulated miRNAs, including miR-409, miR-134 and miR-432, which have been already linked to prothrombotic phenotypes(Fig. 1H). Additionally, we detected an enrichment of circular RNAs in RPs compared to MPs with several circular RNA upregulated in RPs, that have not been described before(Fig. 1I-K). Mass cytometry detected a significant upregulation in RPs of relevant proteins including the collagen receptor GPVI(p=8.98*10-12, Fig.2) and the thrombin receptor PAR1(p=5.21*10-11). Validation assays in an independent cohort detected an upregulation of the PI3K/AKT pathway in RPs which is downstream of the receptor PAR1 and GP6. Conclusion This study represents the first comprehensive multiomic characterization of RPs in CCS patients, providing insights into their prothrombotic phenotype and into their correlation with cardiovascular events. These findings suggest potential avenues for tailored therapeutic interventions targeting the identified upregulated pathways in patients with elevated RPs, warranting further investigation at the clinical level.

Sparse inference of the human haematopoietic system from heterogeneous and partially observed genomic data

Abstract Haematopoiesis is the process of blood cells’ formation, with progenitor stem cells differentiating into mature forms such as white and red blood cells or platelets. While progenitor cells share regulatory pathways involving common nuclear factors, specific networks shape their fate towards particular lineages. This paper analyses the complex regulatory network that drives the formation of mature red blood cells and platelets from their common precursors. Using the latest reverse transcription quantitative real-time PCR genomic data, we develop a dedicated graphical model that incorporates the effect of external genomic data and allows inference of regulatory networks from the high-dimensional and partially observed data.

Linear podosomes display low Cdc42 activity for proplatelet elongation by megakaryocytes

Blood platelets result from differentiation of megakaryocytes (MKs) into the bone marrow. It culminates with the extension of proplatelets (PPT) through medullar sinusoids and release of platelets in the blood stream. Those processes are regulated by contact with the microenvironment mediated by podosomes. We previously demonstrated that contact of megakaryocytes to Collagen I fibers initiated the formation of linear podosomes required for proplatelets extension and release of mature platelets. MKs linear podosomes have the particularity of displaying mechanical pulling activity but, unlike other linear podosomes, they lack the ability of digesting the extracellular matrix (ECM), as we recently demonstrated. The Cdc42 small GTPase is required for actomyosin-dependent maturation of the demarcation membrane system (DMS), a membrane reservoir for PPT and platelets components. Cdc42 is a known protein of the podosomes core, and is instrumental to accurate platelets release into the sinusoids. Indeed, FRET analysis showed that Cdc42 activity was very high and central to DMS formation. Unexpectedly, even though we found the protein in linear podosomes, almost undetectable Cdc42 activity was detected in those structures. This observation suggests that Cdc42 could also act as scaffold to assemble proteins required for PPT formation/elongation along Collagen I fibers in MKs. Eventually, we demonstrated that linear podosomes appear as points of contact between Collagen I fibers and DMS membranes, to mechanically extend PPT along Collagen bundles, independently of Cdc42 activity.

What It Takes To Be a Platelet: Evolving Concepts in Platelet Production.

Platelets are among the most abundant cells within the circulation. Given that the platelet lifespan is 7 to 10 days in humans, a constant production of around 100 billion platelets per day is required. Platelet production from precursor cells called megakaryocytes is one of the most enigmatic processes in human biology. Although it has been studied for over a century, there is still controversy about the exact mechanisms leading to platelet release into circulation. The formation of proplatelet extensions from megakaryocytes into bone marrow sinusoids is the best-described mechanism explaining the origin of blood platelets. However, using powerful imaging techniques, several emerging studies have recently raised challenging questions in the field, suggesting that small platelet-sized structures called buds might also contribute to the circulating platelet pool. How and whether these structures differ from microvesicles or membrane blebs, which have previously been described to be released from megakaryocytes, is still a matter of discussion. In this review, we will summarize what the past and present have revealed about platelet production and whether mature blood platelets might emerge via different mechanisms.

Deep proteome profiling of reticulated platelets in patients with chronic coronary syndrome using mass cytometry

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Else Körner Fresenius Stiftung (EKFS) Background Immature or reticulated platelets (RPs), a subgroup of larger and RNA-rich platelets, which are newly released by megakaryocytes from the bone marrow into blood, have gained rising interest because of the further decryption of RPs’ prothrombotic biology and the potential as biomarkers for predicting adverse events in cardiovascular disease. Purpose The biological features of RPs in patients with chronic coronary syndrome (CCS) are largely unknown. With this project, we aimed to perform the first high resolution proteomic characterisation of reticulated platelets using mass cytometry(CyTOF). Method Two samples of thrombocytes from peripheral blood (non-stimulated, TRAP-stimulated) were prepared from each patient with a custom-made CyTOF-antibody panel of 20 antibodies targeting essential transmembrane proteins(anti-CD9, anti-CD29, anti-CD31, anti-CD36-, anti-CD40, anti-CD41, anti-CD42a, anti-CD42b-, anti-CD47, anti-CD61, anti-CD62P-, anti-CD63, anti-CD69, anti-CD107a, anti-CD154, anti-GPVI, anti-GPIIbIIa complex, anti-Par1, anti-PEAR-1 and the negative control anti-CD3 coupled with different metal isotopes). RPs and mature platelets (MPs) were detected based on their RNA content. Earth mover’s distance (EMD) was assessed as a measure of differential expression. Results CyTOF detected an upregulation of important transmembrane receptors in RPs compared to MPs in quiescent platelets (Fig.1A and B). The collagen receptor GPVI (EMD 33.95), the collagen integrin receptor unit ITGB1 (EMD 32.37), the adhesion protein CD9 (EMD 31.78) and the von Willebrand receptor unit CD42b (EMD 30.08) were upregulated in RPs which all serve as crucial regulators of platelet activation and aggregation. RPs show higher median signal intensities of typical activation markers compared to MPs upon TRAP stimulation (Fig.1C). Conclusion We provide the first proteomic characterization of RPs’ pro-thrombotic profile offering the first biomolecular explanation of their hyperactivity and detrimental role in CCS patients.

Evaluation of coagulation and platelet activation state and function in heartworm-infected dogs.

Enhanced platelet responses have been demonstrated in heartworm-infected (HWI) dogs; however, the cause and clinical implications of altered platelet function have not been fully elucidated. This study evaluated platelet function in HWI dogs. Anticoagulated whole blood collected from eight HWI and eight uninfected dogs was evaluated using turbidometric platelet aggregometry, a platelet function analyzer (PFA-100), a total thrombus analysis system (T-TAS), tissue factor-activated and tissue plasminogen activator modified thromboelastography (TF- and tPA-TEG), CBC, von Willebrand Factor activity, and fibrinogen concentrations. Platelet activation state and the presence of reticulated platelets were assessed via flow cytometric expression of P-selection (CD-62P) and thiazole orange staining. Platelet aggregation responses to adenosine diphosphate (ADP, 10 μM) or collagen (20 μg/mL), PFA-100 closure times, and T-TAS occlusion times did not differ between groups. TEG values TF-R, tPA-R, TF-K, and TF-LY60 were decreased (P = .025, P = .047, P = .038, P = .025) and TF-MA, tPA-MA, TF-G, tPA-G and TF-alpha angle were increased (P < .04) in HWI dogs. HWI dogs had higher fibrinogen concentrations (465.6 ± 161 mg/dL vs 284.5 ± 38 mg/dL, P = .008) and eosinophil counts (0.686 ± 0.27 × 103/μL vs 0.267 ± 0.20 × 103/μL, P = .003). There was no difference in hematocrit, activation state, or percent of reticulated platelets. Non-activated reticulated platelets exhibited higher CD62P expression compared with mature platelets. Chronic canine heartworm disease was accompanied by hypercoagulability, hyperfibrinogenemia, and decreased fibrinolysis. Enhanced platelet activation was not identified in this group of HWI dogs.

Immature platelets and platelet reactivity in patients with acute ST-segment Elevation Myocardial Infarction using whole blood flow cytometry with SYTO-13 staining

BackgroundReduced effect of antiplatelet therapy has been reported in patients with ST-segment elevation myocardial infarction (STEMI). Multiple factors may concur to explain this, including increased amount of highly reactive immature platelets. ObjectivesTo investigate the association between immature platelets and reactivity determined with multicolour flow cytometry using the SYTO-13 dye in STEMI patients. MethodsWe conducted an observational study of 59 patients with acute STEMI. Blood samples were obtained within 24 h after admission and after loading doses of dual antiplatelet therapy. For comparison, samples were obtained from 50 healthy individuals. Immature platelets and platelet reactivity were investigated using multicolour flow cytometry including the SYTO-13 dye that binds to platelet RNA and thus provides a method for subdividing platelets into immature and mature platelets. Additionally, we assessed platelet aggregation, serum-thromboxane B2 levels and standard immature platelet markers. ResultsImmature platelets were more reactive than mature platelets in both STEMI patients and healthy individuals (p-values < 0.05). STEMI patients had lower platelet aggregation and thromboxane B2 levels than healthy individuals. We found a positive association between automatically determined immature platelet markers and CD63 expression on activated platelets (Spearman's rho: 0.27 to 0.58, p-values < 0.05). ConclusionsOur study shows that immature platelets identified with a multicolour flow cytometric method using the SYTO-13 dye are more reactive than mature platelets in patients with acute STEMI and in healthy individuals. The presence of immature platelets may be important for the overall platelet reactivity, which may have implications for the effect of antiplatelet therapy.

Interleukins in Platelet Biology: Unraveling the Complex Regulatory Network.

Interleukins, a diverse family of cytokines produced by various cells, play crucial roles in immune responses, immunoregulation, and a wide range of physiological and pathological processes. In the context of megakaryopoiesis, thrombopoiesis, and platelet function, interleukins have emerged as key regulators, exerting significant influence on the development, maturation, and activity of megakaryocytes (MKs) and platelets. While the therapeutic potential of interleukins in platelet-related diseases has been recognized for decades, their clinical application has been hindered by limitations in basic research and challenges in drug development. Recent advancements in understanding the molecular mechanisms of interleukins and their interactions with MKs and platelets, coupled with breakthroughs in cytokine engineering, have revitalized the field of interleukin-based therapeutics. These breakthroughs have paved the way for the development of more effective and specific interleukin-based therapies for the treatment of platelet disorders. This review provides a comprehensive overview of the effects of interleukins on megakaryopoiesis, thrombopoiesis, and platelet function. It highlights the potential clinical applications of interleukins in regulating megakaryopoiesis and platelet function and discusses the latest bioengineering technologies that could improve the pharmacokinetic properties of interleukins. By synthesizing the current knowledge in this field, this review aims to provide valuable insights for future research into the clinical application of interleukins in platelet-related diseases.

Identification of the Role of SEL1L in Platelet Function with Implications for Atypical Equine Thrombasthenia in Thoroughbred Horses

Atypical Equine Thrombasthenia (AET) is a recessive heritable platelet disorder in horses due to aberrant platelet signaling after thrombin stimulation, preventing platelets from fully activating or efficiently binding to fibrinogen, leading to prolonged bleeding. To date, AET has only been identified in the Thoroughbred horse breed. A prevalence study performed at one breeding farm found that one in every 150 Thoroughbreds was affected. Despite the negative effects on horse health and racing performance, the underlying etiology of this unique platelet disorder is unknown. Here we show that a variant in SEL1L, a gene which encodes a protein known to play a pivotal role in endoplasmic reticulum-associated protein degradation (ERAD) and not previously known to be involved in platelet function, leads to AET. In order to identify the underlying genetic mechanism, we performed whole genome sequencing on five affected, one equivocal, and eleven control Thoroughbred horses. A whole-genome association study was conducted, and associated variants were filtered by determining if they were in or near genes known to be expressed in platelets. This evaluation identified three putative loci: a missense variant in SEL1L (p.Ile604Val), a 1.6kb deletion in a long non-coding RNA ( AL355838.1) and a 73bp intronic deletion in VIPAS39. The long non-coding RNA AL355838.1 did not show expression in horse platelets, and there was no differential expression of SEL1L or VIPAS39 mRNA identified between affected and unaffected individuals. However, a significant difference in SEL1L protein expression was observed in AET-affected horses' platelets. Flow cytometry and immunofluorescence studies demonstrated that SEL1L is located intracellularly in equine platelets, but not in the α-granule, and moves to the surface of the platelet upon activation with thrombin. Although the template bleeding time was normal, platelets from horses that were homozygous for the SEL1L variant showed defective spreading on collagen. To support SEL1L as the causative gene and dissect the underlying mechanisms, we undertook studies in three additional models. Differentiation of human megakaryocytes revealed the presence of two SEL1L protein isoforms, p100 and p38, and both showed increased expression during megakaryopoiesis, although only p100 was delivered to mature platelets. We perfused whole blood from Mx1-Cre +; Sel1l fl/fl conditional knockouts and controls through a collagen-coated microfluidic chamber. We found that significantly fewer platelets from knockout mice bound to the collagen as compared to control siblings. Finally, a knockout zebrafish line was developed by deleting exons 3-20 using CRISPR/Cas9 mediated genome editing. Similar to the mouse, laser-mediated arterial endothelial injury of 5-day old mutant larvae resulted in significantly fewer thrombocytes (the fish platelet equivalent) adhering to the injured vessel wall as compared to wild-type siblings. Overall, these data support the conclusion that the SEL1L missense variant leads to AET, and that SEL1L has a previously undescribed and conserved role in platelet function, particularly the ability of platelets/thrombocytes to adhere to exposed collagen at sites of endothelial injury. These results will allow the Thoroughbred industry to easily diagnose AET-affected horses and make appropriate decisions for their racing and breeding careers. These data also provide an excellent introduction to further investigation into the understudied and potential role of ERAD proteins in human platelet disorders.

First-in-Human Study with Preclinical Data of BCL-2/BCL-xL Inhibitor Pelcitoclax in Locally Advanced or Metastatic Solid Tumors.

B-cell lymphoma-extra-large (BCL-xL) regulates apoptosis and is an attractive anticancer therapeutic target. However, BCL-xL inhibition also kills mature platelets, hampering clinical development. Using an innovative prodrug strategy, we have developed pelcitoclax (APG-1252), a potent, dual BCL-2 and BCL-xL inhibitor. Aims of this study were to characterize the antitumor activity and safety of pelcitoclax and explore its underlying mechanisms of action (MOA). Cell line-derived xenograft and patient-derived xenograft (PDX) models were tested to evaluate antitumor activity and elucidate MOA. Subjects (N = 50) with metastatic small-cell lung cancer and other solid tumors received intravenous pelcitoclax once or twice weekly. Primary outcome measures were safety and tolerability; preliminary efficacy (responses every 2 cycles per RECIST version 1.1) represented a secondary endpoint. Pelcitoclax exhibited strong BAX/BAK‒dependent and caspase-mediated antiproliferative and apoptogenic activity in various cancer cell lines. Consistent with cell-based apoptogenic activity, pelcitoclax disrupted BCL-xL:BIM and BCL-xL:PUMA complexes in lung and gastric cancer PDX models. Levels of BCL-xL complexes correlated with tumor growth inhibition by pelcitoclax. Combined with taxanes, pelcitoclax enhanced antitumor activity by downregulating antiapoptotic protein myeloid cell leukemia-1 (MCL-1). Importantly, pelcitoclax was well tolerated and demonstrated preliminary therapeutic efficacy, with overall response and disease control rates of 6.5% and 30.4%, respectively. Most common treatment-related adverse events included transaminase elevations and reduced platelets that were less frequent with a once-weekly schedule. Our data demonstrate that pelcitoclax has antitumor activity and is well tolerated, supporting its further clinical development for human solid tumors, particularly combined with agents that downregulate MCL-1.

The Bone Marrow Is the Primary Site of Thrombopoiesis

Megakaryocytes (MKs) are large cells predominantly residing within the bone marrow, generating thousands of platelets in an elaborate proplatelet formation process. Megakaryocytes, defined by their expression of CD41, have been observed in the bone marrow and distant organs such as the spleen and lung. However, whether these megakaryocytes genuinely contribute to the mature platelet pool remains controversial. Here, we aimed to delineate which organs participate in platelet production, which mechanisms affect platelet release, and how to define alternative tissue-dependent functions of megakaryocytes. Flow cytometry data revealed significantly higher numbers of hematopoietic stem and progenitor cells and megakaryocytes in the murine bone marrow compared to spleens or perfused lungs; megakaryocyte counts were also significantly higher in cryosections of the mouse bone marrow, and also significantly increased in the bone marrow compared to spleen and lungs when analyzed by both live cell bi photon in-vivo imaging and ex vivo 3D volumetric imaging (light-sheet). Moreover, megakaryocytes cultured and differentiated from the bone marrow ex vivo had a higher capacity for proplatelet formation than MKs derived from the spleen or lung. Our analysis of previously published murine and human scRNA-seq datasets supports our observations that only a marginal fraction of megakaryocyte-like cells can be found within the lung and spleen and most likely only marginally contribute to platelet production under a steady state.

Kinetics of Mature Platelet Fraction as a Tool for Predicting the Time Course of Shiga Toxin-Producing Escherichia coli-Associated Hemolytic Uremic Syndrome (STEC-HUS)

Kinetics of Mature Platelet Fraction as a Tool for Predicting the Time Course of Shiga Toxin-Producing Escherichia coli-Associated Hemolytic Uremic Syndrome (STEC-HUS)

Anagrelide alleviates myocardial ischaemia–reperfusion injury by inhibiting reticulated platelets

Ischaemia–reperfusion injury (IRI) has been a major challenge during acute coronary syndrome (ACS) with reperfusion therapy. But despite consistent therapeutic improvements in terms of optimized reperfusion strategies and novel antithrombotic medications, IRI still remains highly prevalent. Platelets play a pivotal role in the IRI. Expression ofreticulated platelets (RPs), is found among patients with ACS, which is associated with increased activity and contributes to an impaired response to antiplatelet drugs. RPs might play a more important role in IRI than mature platelets, regarded as a potential therapeutic target for myocardial IRI. Anagrelide, an antithrombopoietic drug indicating for essential thrombocythemia, decreases newly formed platelets by inhibiting the proliferation and differentiation of megakaryocytes in bone marrow. We hypothesize that RPs play a major role in platelet-mediated IRI and that anagrelide alleviates IRI primarily by reducing the number of RPs in the peripheral circulation. To investigate this hypothesis, we have designed animal experiments and clinical trials. Ultimately, we have thoroughly examined potential limitations in confirming this hypothesis and have put forth strategies to effectively address and mitigate these challenges.

Age-Dependent Surface Receptor Expression Patterns in Immature Versus Mature Platelets in Mouse Models of Regenerative Thrombocytopenia.

Aging is a multifaceted process that unfolds at both the individual and cellular levels, resulting in changes in platelet count and platelet reactivity. These alterations are influenced by shifts in platelet production, as well as by various environmental factors that affect circulating platelets. Aging also triggers functional changes in platelets, including a reduction in RNA content and protein production capacity. Older individuals and RNA-rich immature platelets often exhibit hyperactivity, contributing significantly to pathologic conditions such as cardiovascular diseases, sepsis, and thrombosis. However, the impact of aging on surface receptor expression of circulating platelets, particularly whether these effects vary between immature and mature platelets, remains largely unexplored. Thus, we investigated the expression of certain surface and activation receptors on platelets from young and old mice as well as on immature and mature platelets from mouse models of regenerative thrombocytopenia by flow cytometry. Our findings indicate that aged mice show an upregulated expression of the platelet endothelial cell adhesion molecule-1 (CD31), tetraspanin-29 (CD9), and Toll-like receptor 2 (TLR2) compared to their younger counterparts. Interestingly, when comparing immature and mature platelets in both young and old mice, no differences were observed in mature platelets. However, immature platelets from young mice displayed higher surface expression compared to immature platelets from old mice. Additionally, in mouse models of regenerative thrombocytopenia, the majority of receptors were upregulated in immature platelets. These results suggest that distinct surface receptor expressions are increased on platelets from old mice and immature platelets, which may partially explain their heightened activity and contribute to an increased thrombotic risk.

Retraction.

Retraction: 'De novo protein synthesis in mature platelets: a consideration for transfusion medicine', P. Schubert and D.V. Devine, Vox Sanguinis, 10.1111/j.1423-0410.2010.01333.x. The above article from Vox Sanguinis, published online on July 14, 2010 in Wiley Online Library (http://onlinelibrary.wiley.com/), has been retracted by agreement between the authors, the journal Editor in Chief, Dr. Miquel Lozano, and John Wiley and Sons. The retraction has been agreed due to unattributed overlap between this article and the following article published in Seminars in Thrombosis and Hemostasis, "Change in protein phenotype without a nucleus: translational control in platelets", by A. S. Weyrich, S. Lindemann, N.D. Tolley, L.W. Kraiss, D.A. Dixon, T.M. Mahoney, S.P. Prescott, T.M. McIntyre, G.A. Zimmerman, Volume 30(4), 2004, pages 491-8.

Advanced Flow Cytometry Using the SYTO-13 Dye for the Assessment of Platelet Reactivity and Maturity in Whole Blood

Newly produced immature platelets are larger, contain higher amounts of residual RNA, and are more reactive than mature platelets. Flow cytometry using the SYTO-13 dye is a method for the subdivision of immature platelets from mature platelets based on the labelling of intracellular platelet RNA, enabling the simultaneous investigation of the reactivity of each platelet population. This method provides detailed information on several aspects of platelet physiology using a combination of platelet surface markers and agonists. Currently, no standardized protocol exists across laboratories. Here, we describe a flow cytometry protocol in detail to investigate platelet reactivity and its relation to platelet maturity. We analyzed 20 healthy individuals with the protocol and compared the platelet subpopulation with the highest SYTO-13 labelling (in the first quintile, "SYTO-high") corresponding to the most immature platelets (highest RNA content) with the platelet subpopulation with the lowest SYTO-13 labelling (in the fifth quintile, "SYTO-low") corresponding to the mature platelets with the lowest RNA content. SYTO-high platelets had overall significantly increased platelet reactivity compared with that of SYTO-low platelets. The presented method may be a valuable research tool for the analysis of platelet reactivity and its relation to platelet maturity.

Proteomic landscapes of inherited platelet disorders with different etiologies

BackgroundInherited platelet disorders (IPDs) are a heterogeneous group of rare diseases that are caused by the defects in early megakaryopoiesis, proplatelet formation, and/or mature platelet function. Although genomic sequencing is increasingly used to identify genetic variants underlying IPD, this technique does not disclose resulting molecular changes that impact platelet function. Proteins are the functional units that shape platelet function; however, insights into how variants that cause IPDs impact platelet proteomes are limited. ObjectivesThe objective of this study was to profile the platelet proteomics signatures of IPDs. MethodsWe performed unbiased label-free quantitative mass spectrometry (MS)–based proteome profiling on platelets of 34 patients with IPDs with variants in 13 ISTH TIER1 genes that affect different stages of platelet development. ResultsIn line with the phenotypical heterogeneity between IPDs, proteomes were diverse between IPDs. We observed extensive proteomic alterations in patients with a GFI1B variant and for genetic variants in genes encoding proteins that impact cytoskeletal processes (MYH9, TUBB1, and WAS). Using the diversity between IPDs, we clustered protein dynamics, revealing disrupted protein-protein complexes. This analysis furthermore grouped proteins with similar cellular function and location, classifying mitochondrial protein constituents and identifying both known and putative novel alpha granule associated proteins. ConclusionsWith this study, we demonstrate a MS–based proteomics perspective to IPDs. By integrating the effects of IPDs that impact different aspects of platelet function, we dissected the biological contexts of protein alterations to gain further insights into the biology of platelet (dys)function.

New functions of C3G in platelet biology: Contribution to ischemia-induced angiogenesis, tumor metastasis and TPO clearance.

C3G is a Rap1 guanine nucleotide exchange factor that controls platelet activation, aggregation, and the release of α-granule content. Transgenic expression of C3G in platelets produces a net proangiogenic secretome through the retention of thrombospondin-1. In a physiological context, C3G also promotes megakaryocyte maturation and proplatelet formation, but without affecting mature platelet production. The aim of this work is to investigate whether C3G is involved in pathological megakaryopoiesis, as well as its specific role in platelet mediated angiogenesis and tumor metastasis. Using megakaryocyte-specific C3G knockout and transgenic mouse models, we found that both C3G overexpression and deletion promoted platelet-mediated angiogenesis, induced by tumor cell implantation or hindlimb ischemia, through differential release of proangiogenic and antiangiogenic factors. However, only C3G deletion resulted in a higher recruitment of hemangiocytes from the bone marrow. In addition, C3G null expression enhanced thrombopoietin (TPO)-induced platelet production, associated with reduced TPO plasma levels. Moreover, after 5-fluorouracil-induced platelet depletion and rebound, C3G knockout mice showed a defective return to homeostatic platelet levels, indicating impaired platelet turnover. Mechanistically, C3G promotes c-Mpl ubiquitination by inducing Src-mediated c-Cbl phosphorylation and participates in c-Mpl degradation via the proteasome and lysosome systems, affecting TPO internalization. We also unveiled a positive role of platelet C3G in tumor cell-induced platelet aggregation, which facilitated metastatic cell homing and adhesion. Overall, these findings revealed that C3G plays a crucial role in platelet-mediated angiogenesis and metastasis, as well as in platelet level modulation in response to pathogenic stimuli.

Gemtuzumab Ozogamicin Treatment Results in Decreased Proliferation and Differentiation of Human Megakaryocytes but Does Not Inhibit Mature Platelet Function.

Mylotarg (Gemtuzumab ozogamicin [GO]), an antibody drug conjugate comprising a CD33-directed antibody linked to calicheamicin, is approved for use in certain acute myeloid leukemia patients. Following reports of prolonged thrombocytopenia and hemorrhagic events in a subset of patients, a detailed series of in vitro and ex vivo studies was performed at the request of regulators, both to look at the effects of GO on platelet production and to determine whether treatment with GO was likely to affect platelet aggregation under a variety of conditions. Treatment with GO resulted in cellular cytotoxicity and/or decreased differentiation during human megakaryocyte development. However, GO did not impair platelet aggregation under the experimental conditions evaluated. Ultimately, the effect of GO on megakaryocyte development observed in our studies was determined to have no impact on the risk-benefit assessment in the intended patient population, as thrombocytopenia is a known side effect of GO, and monitoring of platelet counts in patients is already strongly recommended.

The polymorphic landscape analysis of GATA1 exons uncovered the genetic variants associated with higher thrombocytopenia in dengue patients

The current study elucidated an association between gene variants and thrombocytopenia through the investigation of the exonic polymorphic landscape of hematopoietic transcription factor—GATA1 gene in dengue patients. A total of 115 unrelated dengue patients with dengue fever (DF) (N = 91) and dengue hemorrhagic fever (DHF) (N = 24) were included in the study. All dengue patients were confirmed through detection of NS1 antigen, IgM, and IgG antibodies against the dengue virus. Polymerase chain reaction using specific primers amplified the exonic regions of GATA1 while Sanger sequencing and chromatogram analyses facilitated the identification of variants. Variants G>A (at chX: 48792009) and C>A (at chX: 4879118) had higher frequency out of 13 variants identified (3 annotated and 10 newly recognized). Patients carrying either nonsynonymous or synonymous variants had significantly lower mean values of platelets compared to those harboring the reference nucleotides (NC_000023.11). Further analyses revealed that the change in amino acid residue leads to the altered three-dimensional structure followed by interaction with neighboring residues. Increased stability of the protein due to substitution of serine by asparagine (S129N at chX: 48792009) may cause increased rigidity followed by reduced structural flexibility which may ultimately disturb the dimerization (an important prerequisite for GATA1 to perform its biological activity) process of the GATA1 protein. This, in turn, may affect the function of GATA1 followed by impaired production of mature platelets which may be reflected by the lower platelet counts in individuals with such variation. In summary, we have identified new variants within the GATA1 gene which were found to be clinically relevant to the outcome of dengue patients and thus, have the potential as candidate biomarkers for the determination of severity and prognosis of thrombocytopenia caused by dengue virus. However, further validation of this study in a large number of dengue patients is warranted.Trial Registration: number SLCTR/2019/037.