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Genome-wide platelet transcriptomics is increasingly used to uncover new aspects of platelet biology and as a diagnostic and prognostic tool. Nevertheless, platelet isolation methods for transcriptomic studies are not standardized, introducing challenges for cross-study comparisons, data integration, and replication. In this prospective multicenter study, called "Standardizing Platelet Transcriptomics for Discovery, Diagnostics, and Therapeutics in the Thrombosis and Hemostasis Community (STRIDE)" by the International Society on Thrombosis and Haemostasis Scientific and Standardization Committees, we assessed how 3 of the most commonly used platelet isolation protocols influence metrics from next-generation bulk RNA sequencing and functional assays. Compared with washing alone, more stringent removal of leukocytes by anti-CD45 beads or PALL filters resulted in a sufficient quantity of RNA for next-generation sequencing and similar quality of RNA sequencing metrics. Importantly, stringent removal of leukocytes resulted in the lower relative expression of known leukocyte-specific genes and the higher relative expression of known platelet-specific genes. The results were consistent across enrolling sites, suggesting that the techniques are transferrable and reproducible. Moreover, all 3 isolation techniques did not influence basal platelet reactivity, but agonist-induced integrin αIIbβ 3 activation is reduced by anti-CD45 bead isolation compared with washing alone. In conclusion, the isolation technique chosen influences genome-wide transcriptional and functional assays in platelets. These results should help the research community make informed choices about platelet isolation techniques in their own platelet studies., Competing Interests: Declaration of competing interests M.T.R. reports patents pending or issued on using platelet transcriptomics. All other authors have no conflicts to declare., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Threonine tyrosine kinase (TTK) and polo-like kinase 1 (PLK1) are common essential kinases that collaborate in activating the spindle assembly checkpoint (SAC) at the kinetochore, ensuring appropriate chromosome alignment and segregation prior to mitotic exit. Targeting of either TTK or PLK1 has been clinically evaluated in cancer patients; however, dual inhibitors have not yet been pursued. Here we present the in vitro and in vivo characterization of a first in class, dual TTK/PLK1 inhibitor (BAL0891)., Methods: Mechanism of action studies utilized biochemical kinase and proteomics-based target-engagement assays. Cellular end-point assays included immunoblot- and flow cytometry-based cell cycle analyses and SAC integrity evaluation using immunoprecipitation and immunofluorescence approaches. Anticancer activity was assessed in vitro using cell growth assays and efficacy was evaluated, alone and in combination with paclitaxel and carboplatin, using mouse models of triple negative breast cancer (TNBC)., Results: BAL0891 elicits a prolonged effect on TTK, with a transient activity on PLK1. This unique profile potentiates SAC disruption, forcing tumor cells to aberrantly exit mitosis with faster kinetics than observed with a TTK-specific inhibitor. Broad anti-proliferative activity was demonstrated across solid tumor cell lines in vitro . Moreover, intermittent intravenous single-agent BAL0891 treatment of the MDA-MB-231 mouse model of TNBC induced profound tumor regressions associated with prolonged TTK and transient PLK1 in-tumor target occupancy. Furthermore, differential tumor responses across a panel of thirteen TNBC patient-derived xenograft models indicated profound anticancer activity in a subset (~40%). Using a flexible dosing approach, pathologically confirmed cures were observed in combination with paclitaxel, whereas synergy with carboplatin was schedule dependent., Conclusions: Dual TTK/PLK1 inhibition represents a novel approach for the treatment of human cancer, including TNBC patients, with a potential for potent anticancer activity and a favorable therapeutic index. Moreover, combination approaches may provide an avenue to expand responsive patient populations., Competing Interests: EZ, NF-G, FB, PM, KB, AG, MR, MS and HL were all previous employees of Basilea Pharmaceutica International Ltd, Allschwil. AB, KL, LK and LB are current employees of Basilea Pharmaceutica International Ltd, Allschwil. MS, AB, KL, LK and HL are also Basilea Pharmaceutica International Ltd, Allschwil, stockholders. HL is also a consultant for SillaJen Inc. NW-S, HR and GZ are employees of Oncolines BV and previous Netherlands Translational Research Center NTRC B.V. employees. JM, D-VP and RB are employees of Crossfire Oncology BV formerly Netherlands Translational Research Center NTRC B.V. The studies presented in this manuscript were funded by Basilea Pharmaceutica International Ltd, Allschwil. The funder contributed to study design, as well as the collection, analysis, and interpretation of the results., (Copyright © 2024 Zanini, Forster-Gross, Bachmann, Brüngger, McSheehy, Litherland, Burger, Groner, Roceri, Bury, Stieger, Willemsen-Seegers, de Man, Vu-Pham, van Riel, Zaman, Buijsman, Kellenberger and Lane.)

Background: Inherited bleeding, thrombotic, and platelet disorders (BTPDs) are a heterogeneous set of diseases, many of which are very rare globally. Over the past 5 decades, the genetic basis of some of these disorders has been identified, and recently, high-throughput sequencing has become the primary means of identifying disease-causing genetic variants., Objectives: Knowledge of the clinical validity of a gene-disease relationship is essential to provide an accurate diagnosis based on results of diagnostic gene panel tests and inform the construction of such panels. The Scientific and Standardization Committee for Genetics in Thrombosis and Hemostasis undertook a curation process for selecting 96 TIER1 genes for BTPDs. The purpose of the process was to evaluate the evidence supporting each gene-disease relationship and provide an expert-reviewed classification for the clinical validity of genes associated with BTPDs., Methods: The Clinical Genome Resource (ClinGen) Hemostasis/Thrombosis Gene Curation Expert Panel assessed the strength of evidence for TIER1 genes using the semiquantitative ClinGen gene-disease clinical validity framework. ClinGen Lumping and Splitting guidelines were used to determine the appropriate disease entity or entities for each gene, and 101 gene-disease relationships were identified for curation., Results: The final outcome included 68 Definitive (67%), 26 Moderate (26%), and 7 Limited (7%) classifications. The summary of each curation is available on the ClinGen website., Conclusion: Expert-reviewed assignment of gene-disease relationships by the ClinGen Hemostasis/Thrombosis Gene Curation Expert Panel facilitates accurate molecular diagnoses of BTPDs by clinicians and diagnostic laboratories. These curation efforts can allow genetic testing to focus on genes with a validated role in disease., Competing Interests: Declaration of competing interests The following authors are employee, trainee, or consultant for a commercial laboratory offering genetic testing related to disorders of hemostasis/thrombosis: L.B., K.F., P.G., C.K., J.P.B., M.S., and J.Z. The following authors have made substantial contributions to the literature on gene-disease relationships relevant to this work: L.B., K.D., K.F., P.G., C.K., M.L., K.M., P.M., N.M., J.P.B., N.S., and K.Z. Additionally, K.F. chairs the Scientific and Standardization Committee for Genetics in Thrombosis and Hemostasis of the International Society on Thrombosis and Hemostasis. The other authors declare no conflicts of interest., (Copyright © 2023 International Society on Thrombosis and Haemostasis. All rights reserved.)

Introduction: Method choice is an important component of quality abortion care and qualitative research suggests that abortion stigma can influence provider preference and provision of abortion methods. This study is the first to explore the relationships between abortion providers' method preferences, their provision of medication or instrumentation abortion or both methods, and abortion stigma., Methods: We conducted secondary analysis of a survey of United Kingdom (UK) abortion providers (N = 172) to describe and compare providers' self-reported method preferences and provision. We used multinomial logistic regression to assess the association between method preference and provider experiences of abortion stigma (measured using a revised Abortion Provider Stigma Scale (APSS)), adjusting for relevant provider and facility characteristics., Results: Almost half (52%) of providers reported that they only provided medication abortion care, while 5% only provided instrumentation abortion care and 43% provided both methods. Most (62%) preferred to provide both methods while 32% preferred to provide only medication abortion and 6% only instrumentation abortion. There was no significant difference in revised APSS scores by provider method preference or provision., Discussion: Most surveyed UK abortion providers prefer to offer both methods, but over half only provide medication abortion. This may reflect patients' preferences for medication abortion, and health system and legal constraints on instrumentation abortion. Addressing these systemic constraints on method provision could expand patient choice. Providers' method preference was not significantly associated with provider stigma but future research should consider the influence of structural stigma on method provision at the health system level., (© 2024 The Authors. Perspectives on Sexual and Reproductive Health published by Wiley Periodicals LLC on behalf of University of Ottawa.)

Background: In hemophilia and von Willebrand disease, the degree of alteration of laboratory assays correlates with bleeding manifestations. Few studies have assessed the predictive value for bleeding of laboratory assays in patients with inherited platelet function disorders (IPFDs)., Objectives: To assess whether there is an association between platelet function assay results and bleeding history, as evaluated by the International Society on Thrombosis and Haemostasis (ISTH) bleeding assessment tool (BAT)., Methods: Centers participating in the international ISTH-BAT validation study were asked to provide results of the diagnostic assays employed for the patients they enrolled, and the association with the individual patients' bleeding score (BS) was assessed., Results: Sixty-eight patients with 14 different IPFDs were included. Maximal amplitude of platelet aggregation was significantly lower in patients with a pathologic BS and correlated inversely with the BS, a finding largely driven by the subgroup of patients with Glanzmann thrombasthenia and CalDAG-GEFI deficiency; after their exclusion, TRAP-induced aggregation remained significantly lower in patients with a pathologic BS. Bleeding time was significantly more prolonged in patients with a high BS than in those with a normal BS (27.1 ± 6.2 minutes vs 15.1 ± 10.6 minutes; P  < .01). Reduced α-granule content was significantly more common among patients with a pathologic BS than among those with a normal BS (80% vs 20%; P  < .05). Receiver operating characteristic curve analysis revealed a significant discriminative ability of all the aforementioned tests for pathologic BS ( P  < .001), also after exclusion of patients with Glanzmann thrombasthenia and CalDAG-GEFI deficiency., Conclusion: This study shows that altered platelet laboratory assay results are associated with an abnormal ISTH-BAT BS in IPFD., (© 2023 The Author(s).)

Platelet-type von Willebrand disease (PT-VWD) is an inherited platelet disorder. It is characterized by macrothrombocytopenia and mucocutaneous bleeding, of variable severity, due to gain-of-function variants of GP1BA conferring to glycoprotein Ibα (GPIbα) enhanced affinity for von Willebrand factor (VWF). The bleeding tendency is conventionally attributed to thrombocytopenia and large VWF-multimer depletion. However, while some indications suggest that platelet dysfunction may contribute to the bleeding phenotype, no information on its characteristics and causes are available. The aim of the present study was to characterize platelet dysfunction in PT-VWD and shed light on its mechanism. Platelets from a PT-VWD patient carrying the p.M239V variant, and from PT-VWD mice carrying the p.G233V variant, showed a remarkable platelet function defect, with impaired aggregation, defective granule secretion and reduced adhesion under static and flow conditions. VWFbinding to GPIbα is known to trigger intracellular signaling involving Src-family kinases (SFK). We found that constitutive phosphorylation of the platelet SFK Lyn induces a negative-feedback loop downregulating platelet activation through phosphorylation of PECAM1 on Tyr686 and that this is triggered by the constitutive binding of VWF to GPIbα. These data show, for the first time, that the abnormal triggering of inhibitory signals mediated by Lyn and PECAM1 may lead to platelet dysfunction. In conclusion, our study unravels the mechanism of platelet dysfunction in PT-VWD caused by deranged inhibitory signaling. This is triggered by the constitutive binding of VWF to GPIbα which may significantly contribute to the bleeding phenotype of these patients.

GATA2 is a transcription factor with key roles in hematopoiesis. Germline GATA2 gene variants have been associated with several inherited and acquired hematologic disorders, including myelodysplastic syndromes. Among the spectrum of GATA2 deficiency- associated manifestations thrombosis has been reported in 25% of patients, but the mechanisms are unknown. GATA2 was shown to be involved in endothelial nitric oxide synthase (eNOS) regulation and vascular development. We assessed eNOS expression and angiogenesis in patients with GATA2 deficiency. Platelets and blood outgrowth endothelial cells (BOEC) from GATA2 variant carriers showed impaired NO production and reduction of eNOS mRNA and protein expression and of eNOS activity. GATA2 binding to the eNOS gene was impaired in BOEC from GATA2-deficient patients, differently from control BOEC. GATA2 deficiency BOEC showed also defective angiogenesis, which was completely restored by treatment with the NO-donor Snitroso- N-acetylpenicillamine (SNAP). Atorvastatin, but not resveratrol, largely restored eNOS expression, NO biosynthesis and neoangiogenesis in GATA2-deficient BOEC by a mechanism involving increased expression of the eNOS transcription factor AP-1/c-JUN, replacing GATA2 when the latter is inactive. Our results unravel a possible thrombogenic mechanism of GATA2 mutations, definitely establish the regulation of eNOS by GATA2 in endothelial cells and show that endothelial angiogenesis is strictly dependent on the eNOS/NO axis. Given the ability of atorvastatin to restore NO production and angiogenesis by GATA2-deficient endothelial cells, the preventive effect of atorvastatin on thrombotic events and possibly on other clinical manifestations of the syndrome related to deranged angiogenesis should be explored in patients with GATA2 deficiency in an ad hoc designed clinical trial.

Gain-of-function (GOF) variants in GP1BA cause platelet-type von Willebrand disease (PT-VWD), a rare inherited autosomal dominant bleeding disorder characterized by enhanced platelet GPIbα to von Willebrand factor (VWF) interaction, and thrombocytopenia. To date, only 6 variants causing PT-VWD have been described, 5 in the C-terminal disulfide loop of the VWF-binding domain of GPIbα and 1 in the macroglycopeptide. GOF GP1BA variants generate a high-affinity conformation of the C-terminal disulfide loop with a consequent allosteric conformational change on another region of GPIbα, the leucine-rich-repeat (LRR) domain. We identified a novel GP1BA variant (p.Arg127Gln) affecting the LRR5 domain of GPIbα in a boy with easy bruising and laboratory test results suggestive of PT-VWD. We thus aimed to investigate the impact of the p.Arg127Gln variant on GPIbα affinity for VWF and GPIbα structure. Chinese hamster ovary cells expressing p.Arg127Gln GPIbα showed increased binding of VWF induced by ristocetin and enhanced tethering on immobilized VWF as compared with cells expressing wild-type GPIbα. Surface plasmon resonance confirmed that p.Arg127Gln enhances the binding affinity of GPIbα for VWF. Hydrogen-deuterium exchange mass spectrometry showed that p.Arg127Gln of LRR, while having little effect on the dynamics of the LRR locally, enhances the conformational dynamics of the GPIbα C-terminal disulfide loop structure. Our data demonstrate for the first time that GOF variants outside the GPIbα C-terminal disulfide loop may be pathogenic and that aminoacidic changes in the LRR may cause allosterically conformational changes in the C-terminal disulfide loop of GPIbα, inducing a conformation with high affinity for VWF., (© 2022 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Background: Circulating dysfunctional factor IX (FIX) might modulate distribution of infused FIX in hemophilia B (HB) patients. Recurrent substitutions at FIX activation sites (R191-R226, >300 patients) are associated with variable FIX activity and antigen (FIXag) levels., Objectives: To investigate the (1) expression of a complete panel of missense mutations at FIX activation sites and (2) contribution of F9 genotypes on the FIX pharmacokinetics (PK)., Methods: We checked FIX activity and antigen and activity assays in plasma and after recombinant expression of FIX variants and performed an analysis of infused FIX PK parameters in patients (n = 30), mostly enrolled in the F9 Genotype and PK HB Italian Study (GePKHIS; EudraCT ID2017-003902-42)., Results: The variable FIXag amounts and good relation between biosynthesis and activity of multiple R191 variants results in graded moderate-to-mild severity of the R191C>L>P>H substitutions. Recombinant expression may predict the absence in the HB mutation database of the benign R191Q/W/K and R226K substitutions. Equivalent changes at R191/R226 produced higher FIXag levels for R226Q/W/P substitutions, as also observed in p.R226W female carrier plasma. Pharmacokinetics analysis in patients suggested that infused FIX Alpha distribution and Beta elimination phases positively correlated with endogenous FIXag levels. Mean residence time was particularly prolonged (79.4 h, 95% confidence interval 44.3-114.5) in patients (n = 7) with the R191/R226 substitutions, which in regression analysis were independent predictors (β coefficient 0.699, P = .004) of Beta half-life, potentially prolonged by the increasing over time ratio between endogenous and infused FIX., Conclusions: FIX activity and antigen levels and specific features of the dysfunctional R191/R226 variants may exert pleiotropic effects both on HB patients' phenotypes and substitutive treatment., (© 2021 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis.)

β1-Tubulin plays a major role in proplatelet formation and platelet shape maintenance, and pathogenic variants in TUBB1 lead to thrombocytopenia and platelet anisocytosis (TUBB1-RT). To date, the reported number of pedigrees with TUBB1-RT and of rare TUBB1 variants with experimental demonstration of pathogenicity is limited. Here, we report 9 unrelated families presenting with thrombocytopenia carrying 6 β1-tubulin variants, p.Cys12LeufsTer12, p.Thr107Pro, p.Gln423*, p.Arg359Trp, p.Gly109Glu, and p.Gly269Asp, the last of which novel. Segregation studies showed incomplete penetrance of these variants for platelet traits. Indeed, most carriers showed macrothrombocytopenia, some only increased platelet size, and a minority had no abnormalities. Moreover, only homozygous carriers of the p.Gly109Glu variant displayed macrothrombocytopenia, highlighting the importance of allele burden in the phenotypic expression of TUBB1-RT. The p.Arg359Trp, p.Gly269Asp, and p.Gly109Glu variants deranged β1-tubulin incorporation into the microtubular marginal ring in platelets but had a negligible effect on platelet activation, secretion, or spreading, suggesting that β1-tubulin is dispensable for these processes. Transfection of TUBB1 missense variants in CHO cells altered β1-tubulin incorporation into the microtubular network. In addition, TUBB1 variants markedly impaired proplatelet formation from peripheral blood CD34+ cell-derived megakaryocytes. Our study, using in vitro modeling, molecular characterization, and clinical investigations provides a deeper insight into the pathogenicity of rare TUBB1 variants. These novel data expand the genetic spectrum of TUBB1-RT and highlight a remarkable heterogeneity in its clinical presentation, indicating that allelic burden or combination with other genetic or environmental factors modulate the phenotypic impact of rare TUBB1 variants., (© 2021 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

The Rubber Foot Illusion (RFI) is an illusion in which one is made to feel that a model foot is their own through synchronous visuo-tactile stimulation. Previous research suggests that the conditions the RFI can be elicited under are similar to those of the Rubber Hand Illusion (RHI). However, it was unknown if the RFI could be elicited by synchronous movement of a participant's foot and a model foot. To examine this, we developed the Moving Rubber Foot Illusion (mRFI) and compared participants' experience of it to the RFI. The results of this study suggests that the RFI can be elicited through synchronous movement, and results in more proprioceptive drift than a static variant of the RFI. More work is needed to better understand the mechanisms underlying the mRFI.