ABSTRACT Background Glycoprotein (GP)VI is involved in platelet activation, procoagulant phenotype development and reactive oxygen species (ROS) production. Patients with diabetes have altered platelet reactivity and abnormal clot structure, contributing to elevated thrombosis risk. The role of GPVI in clot formation in hyperglycemia is under-investigated. Objectives To compare the effect of GPVI-deficiency on ROS production and ex vivo and in vivo clot formation in normo- vs hyperglycemic mice. Methods Wild-type (WT) and GPVI-deficient (GPVI−/−) mice had access to hyperglycemic (30% sucrose) or normoglycaemic (standard) diet for 8-weeks. ROS and mitochondrial activity (CS) were measured in isolated platelets. Clot contraction was performed with whole blood. Thrombus formation in vivo was assessed by inferior vena cava (IVC) ligation. Results Hyperglycemia increased ROS and CS activity in WT but not GPVI−/− mice. Clot weight was decreased in GPVI−/− vs WT mice in normoglycaemia, but these effects were lost in hyperglycemia. Clot weight after IVC ligation was reduced in GPVI−/− vs WT mice in normoglycaemia, with the inverse observed in hyperglycemia. Conclusions Our data suggest that clot formation is affected differentially by absence of GPVI in normo- vs hyperglycemia, supporting a role for GPVI in thrombosis under normoglycaemic conditions only.

ABSTRACT Thrombocytopenia 4 (THC4) is a rare autosomal dominant inherited thrombocytopenia caused by pathogenic variants in CYCS, the gene encoding cytochrome c. Although CYCS-related thrombocytopenia is well characterized as a mild, non-syndromic quantitative platelet disorder, few families have been described worldwide. Recently, the missense variant c.274A > G (p.Arg92Gly) was reported in a single family. Here, we describe an additional multi-generational family from the Middle East carrying the same variant, thereby providing independent confirmation of pathogenicity and expanding the available phenotypic data. Seven affected individuals exhibited stable, moderate thrombocytopenia (55–88 × 109/L) with normal platelet size and morphology, normal platelet aggregation responses, and no syndromic features. One newborn presented with petechiae and grade 1 intracranial hemorrhage but recovered fully. Whole-exome sequencing identified p.Arg92Gly as the only variant segregating with disease; Sanger sequencing confirmed heterozygosity in all affected members. Structural modeling demonstrated loss of stabilizing hydrogen bonds involving a highly conserved residue within the C-terminal helical region of cytochrome c, likely impairing local structural stability. Importantly, one affected individual tolerated long-term aspirin and clopidogrel therapy following an ischemic stroke without bleeding complications—an observation not previously reported in THC4. This Brief Report strengthens the association between CYCS p.Arg92Gly and inherited thrombocytopenia and provides clinically important data regarding antiplatelet therapy safety in this rare condition.

ABSTRACT Defects in platelet secretion contribute to hemorrhagic disorders and coagulopathies, primarily link to the biogenesis of lysosome-related organelles (LROs), namely alpha granules (AGs) and dense granules (DGs). While deficiencies in the biogenesis of lysosome-related organelles complex-1 (BLOC-1) are known to impair DG formation and platelet function, the distinct roles of individual BLOC-1 subunits remain unresolved. Here, we investigated BLOS1, a shared subunit of BLOC-1 and BLOC-1-related complex (BORC) encoded by BLOC1S1. Through comparative analysis of platelet-specific Bloc1s1-conditional knockout (Bloc1s1-cKO) mice and sdy mice (deficiency in BLOC-1 subunit dysbindin), we found that Bloc1s1-cKO platelets exhibited a 50% reduction in DG content compared to sdy mice, indicating BLOS1 deficiency causes moderate DG biogenesis defects. Consistently, agonist-induced DG secretion in Bloc1s1-cKO mice was moderately impaired relative to the severe defects in sdy mice. Distinct steady-state levels of granule proteins were observed between the two mutants. These findings suggest that dysbindin (only in BLOC-1) and BLOS1 (a shared BLOC-1 and BORC subunit) regulate LRO biogenesis and function in a different manner.

Due to platelet storage lesions (PSL), transfused platelets are unable to function properly in the prevention and treatment of bleeding in critically ill patients. It is a traditional assumption that PSL is closely related to platelet activation during storage because of the exposure of CD62P, phosphatidylserine (PS), etc. In this standpoint, activated platelets in vitro cannot be reactivated in vivo to exert their hemostatic function and exposed PS accelerates platelet clearance. Therefore, reducing platelet activation is helpful to alleviate PSL. Diannexin is the dimer of annexin that has a higher affinity for PS. CD39 is an ADP hydrolase produced by the vascular endothelium. As a result, we construct CD39-Diannexin (CD39-DA) fusion protein and hypothesize that CD39-DA can reduce platelet activation during storage to alleviate PSL. CD39-DA can bind to the exposed PS on the surface of stored platelets by immunofluorescence. Compared to the control groups, CD39-DA reserves part of stored platelets’ aggregation function confirmed by platelet aggregation assay, induced by AA, ADP and collagen. Additionally, CD39-DA reduces lactic dehydrogenase (LDH) levels and CD62P-positive events after three-day storage. Interestingly, we preliminarily discover that CD39-DA may reduce stored platelets’ apoptosis and increase aggregatory platelets after activation by thrombin, collagen and calcium, which is marked by GSAO. In conclusion, we confirm that CD39-DA can alleviate PSL by reducing platelet activation.

One of the leading thrombocytopenia occasions in childhood is immune thrombocytopenia (ITP). Small number of these patients do not respond to treatment methods known to be effective for ITP such as corticosteroids, intravenous immunoglobulin and thrombopoietin receptor agonists (eltrombopag, romiplostim, etc.) and have serious bleeding symptoms. We present a case in which a molecular genetic analysis was performed due to challenging responses to medical treatment for severe systemic bleeding symptoms which were similar to ITP. A hemizygous variant was detected in the GATA1 gene and a homozygous variant in the ATP2A1 gene for Brody myopathy. Eltrombopag, romiplostim, IVIG and corticosteroid were applied to resistant thrombocytopenia of the patient without knowing the cause. Patient had response in platelet counts in some occasions. This situation attracted our attention after the detection of this GATA variant in the etiopathogenesis of thrombocytopenia. Further investigations should be performed for much more rare causes of thrombocytopenia in patients with refractory thrombocytopenia attacks.

Immune thrombocytopenia (ITP) is an autoimmune disease with isolated platelet count decrease. A subset of patients responds inferiorly to the first-line therapies including glucocorticoid and intravenous immunoglobulins (IVIG), of which the underlying mechanisms have not been fully elucidated. We first found that expression of miR-28-5p was obviously increased in complete responders, and decreased to substantially low levels in partial or non-responders. In the passive ITP model, upregulation of miR-28-5p by injecting agomir slightly improved thrombocytopenia, and obviously inhibited the Rap1b gene expression. Luciferase reporter assay demonstrated there was significant decrease of luciferase activity in 293T cells co-transfected with miR-28-5p mimics and plasmids with Rap1b wide-type sequence. Upregulation of miR-28-5p, and downregulation of Rap1b played a favorable role in reducing B cell infiltration in the marginal zone of spleen in mice. However, miR-28-5p exhibited no significant influence on megakaryocyte maturation in ITP both in vitro and in vivo studies. Finally, we confirmed that miR-28-5p upregulation was associated with superior early treatment response in ITP, and possibly functioned by targeting Rap1b gene to inhibit humoral immunity.

Immune-checkpoint blockades (ICBs) are now used in early-stage diseases like triple-negative breast cancer (TNBC). While effective, they can cause severe toxicities. We report the first case of life-threatening immune thrombocytopenia (ITP) induced by pembrolizumab during neoadjuvant chemo-immunotherapy for early TNBC. A 42-year-old woman with early-stage TNBC developed grade 4 thrombocytopenia, diagnosed as ITP, after 107 days of pembrolizumab treatment. She required intensive care unit (ICU) admission and high-dose steroids, and intravenous immunoglobulin therapy, leading to a rapid recovery. ITP is a rare but potentially fatal complication of immunotherapy, with an incidence of less than 1% and a mortality rate of up to 20% in affected patients. Immediate recognition and steroid therapy are critical, as platelet transfusion is usually ineffective. Diagnosis is often delayed due to its similarity to chemotherapy-induced marrow toxicity. Immunotherapy-induced ITP generally contraindicates further use of the treatment. ITP, although uncommon, is a serious complication of immunotherapy requiring immediate intervention. The growing use of immunotherapy necessitates increased awareness of its potential toxicities among healthcare providers.

Appropriate platelet function determines both the perioperative haemostasis and the risk of postoperative thrombotic complications in patients undergoing branched endovascular repair (bEVAR) of thoracoabdominal aortic aneurysm (TAAA). We aimed to assess the effect of bEVAR on platelet function and the predictive value of preoperative platelet function for postoperative bleeding. We measured platelet function using impedance aggregometry and total thrombus-formation analysis system in 50 consecutive patients, with TAAA undergoing elective bEVAR. After bEVAR, platelet reactivity was assessed using ASPI test, ADP test and TRAP test and thrombus size decreased, whereas time to clot formation increased, compared to baseline (p ≤ .042 for all). Preoperative platelet reactivity in the TRAP test was lower in patients who experienced post-operative bleeding, defined as ≥3 red blood cell units transfusion, compared to those who did not (p = .038). Baseline hemoglobin level <13 g/dl and TRAP test result ≤29.5 AUC increased the odds of bleeding by 5.4-fold and 6.8-fold, respectively, independent of other clinical variables. We conclude that in patients with TAAA undergoing bEVAR, platelet reactivity and platelet-rich thrombus formation decreased directly after the operation. Preoperative hemoglobin level and platelet reactivity in the TRAP test were independent predictors of postoperative bleeding complications.

Light transmission aggregometry (LTA) is a method used to investigate platelet functions in platelet-rich plasma (PRP), notably when screening for platelet disorders. Various national guidelines and recommendations help in setting up the LTA test in specialized laboratories. However, due to the nature of the sample matrix and its subsequent specificities, more accurate positions are needed to achieve LTA accreditation according to the standard NF EN ISO 15 189. We reviewed guidelines and recommendations as they can be useful in the accreditation process, and we conducted a survey on LTA practice among members of the Société Française de Thrombose et d’Hémostase (SFTH) in 2021. We formulated 28 proposals, which have been approved by vote within the SFTH. All aspects to take into consideration for the proper conduct of LTA assays and their accreditation have been covered. Notably, preanalytical, analytical and postanalytical aspects are depicted, including blood sampling, PRP preparation, instruments, agonists, performance assessment, personnel training and data interpretation. This document, essentially representing a French position paper on the current recommendations and subsequent proposals for LTA accreditation, might prove useful also outside France for relevant laboratories and auditors involved in LTA accreditation.

Soluble CLEC-2 is anticipated to have various clinical applications as a novel biomarker for in vivo platelet activation, assessable using plasma obtained through routine sampling procedures. While sCLEC-2 has been measured using ELISA, we have developed a highly sensitive chemiluminescence enzyme immunoassay (CLEIA) reagent that can yield results in approximately 19 minutes. This study aims to assess its fundamental performance and explore the molecular forms of sCLEC-2 measured in patient samples. We examined the sensitivity, precision, linearity, influence of endogenous substances, residual platelets, as well as the correlation with the ELISA method, for the sCLEC-2 CLEIA reagent. The CLEIA method demonstrated sufficient sensitivity for levels observed in healthy donors, and its basic performance was satisfactory. It exhibited a strong correlation with the previously described ELISA method, with reference ranges that did not significantly differ from the ELISA data. The sCLEC-2 reference range for males was significantly higher than that for females. Since it is known that sCLEC-2 exists in shed form and microparticle form, we investigated molecular forms of sCLEC-2 measured by the CLEIA in in vitro-activated platelets and in patients’ plasma using gel filtration. It is considered that the CLEIA method shows significantly stronger reactivity with the shed form compared to the microparticle form. Studies using gel filtration of patient samples also suggest that the shed form is being primarily measured. The sCLEC-2 CLEIA reagent exhibits robust performance and is promising for clinical applications.