Protein disulfide isomerase cleaves allosteric disulfides in histidine-rich glycoprotein to regulate thrombosis.

The essence of difference between hemostasis and thrombosis is that the clotting reaction is a highly fine-tuned process. Vascular protein disulfide isomerase (PDI) represents a critical mechanism regulating the functions of hemostatic proteins. Herein we show that histidine-rich glycoprotein (HRG) is a substrate of PDI. Reduction of HRG by PDI enhances the procoagulant and anticoagulant activities of HRG by neutralization of endothelial heparan sulfate (HS) and inhibition of factor XII (FXIIa) activity, respectively. Murine HRG deficiency (Hrg-/-) leads to delayed onset but enhanced formation of thrombus compared to WT. However, in the combined FXII deficiency (F12-/-) and HRG deficiency (by siRNA or Hrg-/-), there is further thrombosis reduction compared to F12-/- alone, confirming HRG's procoagulant activity independent of FXIIa. Mutation of target disulfides of PDI leads to a gain-of-function mutant of HRG that promotes its activities during coagulation. Thus, PDI-HRG pathway fine-tunes thrombosis by promoting its rapid initiation via neutralization of HS and preventing excessive propagation via inhibition of FXIIa.

Gene Dosage of F5 c.3481C>T Stop-Codon (p.R1161Ter) Switches the Clinical Phenotype from Severe Thrombosis to Recurrent Haemorrhage: Novel Hypotheses for Readthrough Strategy.

Inherited defects in the genes of blood coagulation essentially express the severity of the clinical phenotype that is directly correlated to the number of mutated alleles of the candidate leader gene (e.g., heterozygote vs. homozygote) and of possible additional coinherited traits. The F5 gene, which codes for coagulation factor V (FV), plays a two-faced role in the coagulation cascade, exhibiting both procoagulant and anticoagulant functions. Thus, defects in this gene can be predisposed to either bleeding or thrombosis. A Sanger sequence analysis detected a premature stop-codon in exon 13 of the F5 gene (c.3481C>T; p.R1161Ter) in several members of a family characterised by low circulating FV levels and contrasting clinical phenotypes. The propositus, a 29 y.o. male affected by recurrent haemorrhages, was homozygous for the F5 stop-codon and for the F5 c.1691G>A (p.R506Q; FV-Leiden) inherited from the heterozygous parents, which is suggestive of combined cis-segregation. The homozygous condition of the stop-codon completely abolished the F5 gene expression in the propositus (FV:Ag < 1%; FV:C < 1%; assessed by ELISA and PT-based one-stage clotting assay respectively), removing, in turn, any chance for FV-Leiden to act as a prothrombotic molecule. His father (57 y.o.), characterised by severe recurrent venous thromboses, underwent a complete molecular thrombophilic screening, revealing a heterozygous F2 G20210A defect, while his mother (56 y.o.), who was negative for further common coagulation defects, reported fully asymptomatic anamnesis. To dissect these conflicting phenotypes, we performed the ProC®Global (Siemens Helthineers) coagulation test aimed at assessing the global pro- and anticoagulant balance of each family member, investigating the responses to the activated protein C (APC) by means of an APC-sensitivity ratio (APC-sr). The propositus had an unexpectedly poor response to APC (APC-sr: 1.09; n.v. > 2.25), and his father and mother had an APC-sr of 1.5 and 2.0, respectively. Although ProC®Global prevalently detects the anticoagulant side of FV, the exceptionally low APC-sr of the propositus and his discordant severe-moderate haemorrhagic phenotype could suggest a residual expression of mutated FV p.506QQ through a natural readthrough or possible alternative splicing mechanisms. The coagulation pathway may be physiologically rebalanced through natural and induced strategies, and the described insights might be able to track the design of novel treatment approaches and rebalancing molecules.

Apolipoprotein A-IV polymorphisms Q360H and T347S attenuate its endogenous inhibition of thrombosis.

Platelets are small anucleate cells that play a key role in thrombosis and hemostasis. Our group previously identified apolipoprotein A-IV (apoA-IV) as an endogenous inhibitor of thrombosis by competitive blockade of the αIIbß3 integrin on platelets. ApoA-IV inhibition of platelets was dependent on the N-terminal D5/D13 residues, and enhanced with absence of the C-terminus, suggesting it sterically hinders its N-terminal platelet binding site. The C-terminus is also the site of common apoA-IV polymorphisms apoA-IV-1a (T347S) and apoA-IV-2 (Q360H). Interestingly, both are linked with an increased risk of cardiovascular disease, however, the underlying mechanism remains unclear. Here, we generated recombinant apoA-IV and found that the Q360H or T347S polymorphisms dampened its inhibition of platelet aggregation in human platelet-rich plasma and gel-filtered platelets, reduced its inhibition of platelet spreading, and its inhibition of P-selectin on activated platelets. Using an ex vivo thrombosis assay, we found that Q360H and T347S attenuated its inhibition of thrombosis at both high (1800s-1) and low (300s-1) shear rates. We then demonstrate a conserved monomer-dimer distribution among apoA-IV WT, Q360H, and T347S and use protein structure modelling software to show Q360H and T347S enhance C-terminal steric hindrance over the N-terminal platelet-binding site. These data provide critical insight into increased cardiovascular risk for individuals with Q360H or T347S polymorphisms.

Thrombophilia genetic mutations and their relation to disease severity among patients with COVID-19.

OBJECTIVES: Patients with COVID-19 infection appear to develop virus-induced hypercoagulability resulting in numerous thrombotic events. The aim of the present study was to determine the relationship between the thrombophilia genes mutations (prothrombin G20210A, factor V Leiden, and methyltetrahydrofolate reductase (MTHFR)) and the severity of COVID-19 patients. DESIGN: Prospective cross-sectional study. METHOD: One hundred and forty patients (80 adults and 60 children) were included in the current study. They were divided into the severe COVID-19 group and the mild COVID-19 group, with each group comprising 40 adults and 30 children. The patients were assessed for FV R506Q, FV R2H1299R, MTHFR A1298C, MTHFR C677T, and prothrombin gene G20210A polymorphisms. CBC, D-dimer, renal and liver function tests, hs-CRP, ferritin, and LDH were also assessed. Thrombotic events were clinically and radiologically documented. RESULTS: Severe COVID-19 cases were significantly more frequent to have a heterozygous mutation for all the studied genes compared to mild COVID-19 cases (p<0.05 for all). Being mutant to gene FV R506Q carried the highest risk of developing a severe disease course (p<0.0001). Patients with abnormally high D-dimer levels were significantly more frequent to be heterozygous for FV R506Q, FV R2H1299R, and prothrombin gene G20210A (p = 0.006, 0.007, and 0.02, respectively). CONCLUSION: We concluded that there is an evident relationship between severe COVID-19 and inherited thrombophilia. In the current study, FV R506Q gene mutation carried the highest risk of developing a severe COVID-19 disease course.

4'-O-MethylbavachalconeB Targeted 14-3-3ζ Blocking the Integrin ß3 Early Outside-In Signal to Inhibit Platelet Aggregation and Thrombosis.

14-3-3ζ protein, the key target in the regulation and control of integrin ß3 outside-in signaling, is an attractive new strategy to inhibit thrombosis without affecting hemostasis. In this study, 4'-O-methylbavachalconeB (4-O-MB) in Psoraleae Fructus was identified as a 14-3-3ζ ligand with antithrombosis activity by target fishing combined with ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) analysis. The competitive inhibition analysis showed that 4-O-MB targeted 14-3-3ζ and blocked the 14-3-3ζ/integrin ß3 interaction with inhibition constant (Ki) values of 9.98 ± 0.22 µM. Molecular docking and amino acid mutation experiments confirmed that 4-O-MB specifically bound to 14-3-3ζ through LSY9 and SER28 to regulate the 14-3-3ζ/integrin ß3 interaction. Besides, 4-O-MB affected the integrin ß3 early outside-in signal by inhibiting AKT and c-Src phosphorylation. Meanwhile, 4-O-MB could inhibit ADP-, collagen-, or thrombin-induced platelet aggregation function but had no effect on platelet adhesion to collagen-coated surfaces in vivo. Administration of 4-O-MB could significantly inhibit thrombosis formation without disturbing hemostasis in mice. These findings provide new prospects for the antithrombotic effects of Psoraleae Fructus and the potential application of 4-O-MB as lead compounds in the therapy of thrombosis by targeting 14-3-3ζ.

Impact of allele-selective silencing of von Willebrand factor in mice based on a single nucleotide allelic difference in von Willebrand factor.

INTRODUCTION: Von Willebrand factor (VWF) plays a pathophysiological role in hemostatic disorders. Partial inhibition of the VWF gene through small interfering RNA (siRNA)-mediated allele-selective silencing could be a promising therapeutic strategy. For von Willebrand disease, allele-selectively inhibiting dominant-negative VWF-alleles might ameliorate the phenotype. For thrombotic disorders, partial VWF reduction can lower thrombotic risk, while avoiding bleeding. Previously, we demonstrated the feasibility of Vwf-silencing in homozygous C57BL/6J (B6) or 129S1/SvImJ (129S) mice. The present study investigated allele-selective Vwf-silencing in a complex heterozygous setting of crossed B6 and 129S mice and its subsequent hemostatic impact. MATERIALS AND METHODS: Heterozygous B6.129S mice were treated with siRNAs targeting Vwf expressed from either B6- (siVwf.B6) or 129S-alleles (siVwf.129S). Plasma VWF and lung Vwf mRNA were determined. siVwf.B6-treated B6.129S mice were subjected to ferric chloride-induced mesenteric vessel thrombosis and tail-bleeding. RESULTS: In B6.129S mice, siVwf.B6 reduced Vwf mRNA of the targeted B6-allele by 72% vs. only 12% of the non-targeted 129S-allele (41% total mRNA reduction), lowering plasma VWF by 46%. Oppositely, siVwf.129S reduced Vwf mRNA by 45%, now selectively inhibiting the 129S-allele over the B6-allele (58% vs. 9%), decreasing plasma VWF by 43%. The allele-selective VWF reduction by siVwf.B6 coincided with decreased thrombus formation in mesenteric arterioles, without prolonging tail-bleeding times. CONCLUSIONS: This study demonstrates the feasibility of allele-selective Vwf-silencing in a heterozygous setting, achieving a controlled close to 50% reduction of plasma VWF. The observed thromboprotection and absence of prolonged bleeding times underline the potential of allele-selective Vwf-silencing as a therapeutic strategy in hemostatic disorders.

Impact of CALR and JAK2V617F Mutations on Clinical Course and Disease Outcomes in Essential Thrombocythemia: A Multicenter Retrospective Study in Turkish Patients.

Objective: In this study, we investigated the effects of calreticulin (CALR) and JAK2V617F mutational status on clinical course and disease outcomes in Turkish patients with essential thrombocythemia (ET). Materials and Methods: Seventeen centers from Türkiye participated in the study and CALR- and JAK2V617F-mutated ET patients were evaluated retrospectively. Results: A total of 302 patients were included, of whom 203 (67.2%) and 99 (32.8%) were JAK2V617F- and CALR-positive, respectively. CALR-mutated patients were significantly younger (51 years vs. 57.5 years, p=0.03), with higher median platelet counts (987x109/L vs. 709x109/L, p<0.001) and lower median hemoglobin levels (13.1 g/dL vs. 14.1 g/dL, p<0.001) compared to JAK2V617F-mutated patients. Thromboembolic events (TEEs) occurred in 54 patients (17.9%), 77.8% of which were arterial. Compared to CALR mutation, JAK2V617F was associated with a higher risk of thrombosis (8.1% vs. 22.7%, p=0.002). Rates of transformation to myelofibrosis (MF) and leukemia were 4% and 0.7%, respectively, and these rates were comparable between JAK2V617F- and CALR-mutated cases. The estimated overall survival (OS) and MF-free survival of the entire cohort were 265.1 months and 235.7 months, respectively. OS and MF-free survival durations were similar between JAK2V617F- and CALR-mutated patients. Thrombosis-free survival (TFS) was superior in CALR-mutated patients compared to JAK2V617F-positive patients (5-year TFS: 90% vs. 71%, respectively; p=0.001). Age at diagnosis was an independent factor affecting the incidence of TEEs. Conclusion: In our ET cohort, CALR mutations resulted in higher platelet counts and lower hemoglobin levels than JAK2V617F and were associated with younger age at diagnosis. JAK2V617F was strongly associated with thrombosis and worse TFS. Hydroxyurea was the most preferred cytoreductive agent for patients with high thrombosis risk.

Analysis of genes characterizing chronic thrombosis and associated pathways in chronic thromboembolic pulmonary hypertension.

PURPOSE: In chronic thromboembolic pulmonary hypertension (CTEPH), fibrosis of thrombi in the lumen of blood vessels and obstruction of blood vessels are important factors in the progression of the disease. Therefore, it is important to explore the key genes that lead to chronic thrombosis in order to understand the development of CTEPH, and at the same time, it is beneficial to provide new directions for early identification, disease prevention, clinical diagnosis and treatment, and development of novel therapeutic agents. METHODS: The GSE130391 dataset was downloaded from the Gene Expression Omnibus (GEO) public database, which includes the full gene expression profiles of patients with CTEPH and Idiopathic Pulmonary Arterial Hypertension (IPAH). Differentially Expressed Genes (DEGs) of CTEPH and IPAH were screened, and then Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) functional enrichment analyses were performed on the DEGs; Weighted Gene Co-Expression Network Analysis (WGCNA) to screen the key gene modules and take the intersection genes of DEGs and the key module genes in WGCNA; STRING database was used to construct the protein-protein interaction (PPI) network; and cytoHubba analysis was performed to identify the hub genes. RESULTS: A total of 924 DEGs were screened, and the MEturquoise module with the strongest correlation was selected to take the intersection with DEGs A total of 757 intersecting genes were screened. The top ten hub genes were analyzed by cytoHubba: IL-1B, CXCL8, CCL22, CCL5, CCL20, TNF, IL-12B, JUN, EP300, and CCL4. CONCLUSION: IL-1B, CXCL8, CCL22, CCL5, CCL20, TNF, IL-12B, JUN, EP300, and CCL4 have diagnostic and therapeutic value in CTEPH disease, especially playing a role in chronic thrombosis. The discovery of NF-κB, AP-1 transcription factors, and TNF signaling pathway through pivotal genes may be involved in the disease progression process.

Pulmonary thromboembolism associated with hereditary antithrombin III deficiency: A case report.

BACKGROUND: Thrombophilia is a coagulation disorder closely associated with venous thromboembolism. Hereditary antithrombin III (AT III) deficiency is a type of genetic thrombophilia. In China, genetic thrombophilia patients mainly suffer from deficiencies in AT III, protein S, and protein C. Multiple mutations in the serpin family C member 1 (SERPINC1) can affect AT III activity, resulting in thrombosis. CASE PRESENTATION: This case presented a 17-year-old adolescent female who developed lower extremity venous thrombosis and subsequently pulmonary embolism (PE) following a right leg injury. A missense mutation in gene SERPINC1 of c.331 T > C, p.S111P was detected on the patient, resulting in a decreased AT III activity and an elevated risk of thrombosis. The patient received anticoagulation treatment for approximately 5 months. During follow-up, the blood clot gradually dissolved, and there have been no recurrent thrombotic events reported thus far. DISCUSSION: Hereditary AT deficiency can be classified into two types based on the plasma levels of the enzymatic activity and antigen. Type I is a quantitative defect, while Type II is a qualitive defect. Until 2021, 486 SERPINC1 gene mutations have been registered, more than 18% of which are point mutations. The SERPINC1 mutation c.331 T > C in was firstly reported in 2017, which was classified into type I AT III deficiency. CONCLUSION: Hereditary thrombophilia is a coagulation disorder with a high omission diagnostic rate. Minor mutations in the SERPINC1 gene can also lead to hereditary AT III deficiency, which in turn can cause PE. We emphasized the importance of etiological screening for hereditary thrombophilia in venous thromboembolism patients without obvious high-risk factors. Long-term anticoagulation treatment and avoidance of potential thrombosis risk factors are critical for such patients.

Impact of tissue factor expression and administration routes on thrombosis development induced by mesenchymal stem/stromal cell infusions: re-evaluating the dogma.

BACKGROUND: Hyperactive coagulation might cause dangerous complications such as portal vein thrombosis and pulmonary embolism after mesenchymal stem/stromal cell (MSC) therapy. Tissue factor (TF), an initiator of the extrinsic coagulation pathway, has been suggested as a predictor of this process. METHODS: The expression of TF and other pro- and anticoagulant genes was analyzed in xeno- and serum-free manufactured MSCs. Furthermore, culture factors affecting its expression in MSCs were investigated. Finally, coagulation tests of fibrinogen, D-dimer, aPPTs, PTs, and TTs were measured in patient serum after umbilical cord (UC)-MSC infusions to challenge a potential connection between TF expression and MSC-induced coagulant activity.  RESULTS: Xeno- and serum-free cultured adipose tissue and UC-derived MSCs expressed the highest level of TF, followed by those from dental pulp, and the lowest expression was observed in MSCs of bone marrow origin. Environmental factors such as cell density, hypoxia, and inflammation impact TF expression, so in vitro analysis might fail to reflect their in vivo behaviors. MSCs also expressed heterogeneous levels of the coagulant factor COL1A1 and surface phosphatidylserine and anticoagulant factors TFPI and PTGIR. MSCs of diverse origins induced fibrin clots in healthy plasma that were partially suppressed by an anti-TF inhibitory monoclonal antibody. Furthermore, human umbilical vein endothelial cells exhibited coagulant activity in vitro despite their negative expression of TF and COL1A1. Patients receiving intravenous UC-MSC infusion exhibited a transient increase in D-dimer serum concentration, while this remained stable in the group with intrathecal infusion. There was no correlation between TF expression and D-dimer or other coagulation indicators. CONCLUSIONS: The study suggests that TF cannot be used as a solid biomarker to predict MSC-induced hypercoagulation. Local administration, prophylactic intervention with anticoagulation drugs, and monitoring of coagulation indicators are useful to prevent thrombogenic events in patients receiving MSCs. Trial registration NCT05292625. Registered March 23, 2022, retrospectively registered, https://www. CLINICALTRIALS: gov/ct2/show/NCT05292625?term=NCT05292625&draw=2&rank=1 . NCT04919135. Registered June 9, 2021, https://www. CLINICALTRIALS: gov/ct2/show/NCT04919135?term=NCT04919135&draw=2&rank=1 .

Clonal Hematopoiesis and Cardiovascular Risk: Atherosclerosis, Thrombosis, and beyond.

Acquired mutations that lead to clonal hematopoiesis have emerged as a new and potent risk factor for atherosclerotic cardiovascular disease and other cardiovascular conditions. Human sequencing studies and experiments in mouse models provide compelling evidence supporting that this condition, particularly when driven by specific mutated genes, contributes to the development of atherosclerosis by exacerbating inflammatory responses. The insights gained from these studies are paving the way for the development of new personalized preventive care strategies against cardiovascular disease. Furthermore, available evidence also suggests a potential relevance of these mutation in the context of thrombosis, an area requiring thorough investigation. In this review, we provide an overview of our current understanding of this emerging cardiovascular risk factor, focusing on its relationship to atherosclerosis and thrombosis.

A Chromosome-Level Genome Assembly of the Non-Hematophagous Leech Whitmania pigra (Whitman 1884): Identification and Expression Analysis of Antithrombotic Genes.

Despite being a non-hematophagous leech, Whitmania pigra is widely used in traditional Chinese medicine for the treatment of antithrombotic diseases. In this study, we provide a high quality genome of W. pigra and based on which, we performed a systematic identification of the potential antithrombotic genes and their corresponding proteins. We identified twenty antithrombotic gene families including thirteen coagulation inhibitors, three platelet aggregation inhibitors, three fibrinolysis enhancers, and one tissue penetration enhancer. Unexpectedly, a total of 79 antithrombotic genes were identified, more than a typical blood-feeding Hirudinaria manillensis, which had only 72 antithrombotic genes. In addition, combining with the RNA-seq data of W. pigra and H. manillensis, we calculated the expression levels of antithrombotic genes of the two species. Five and four gene families had significantly higher and lower expression levels in W. pigra than in H. manillensis, respectively. These results showed that the number and expression level of antithrombotic genes of a non-hematophagous leech are not always less than those of a hematophagous leech. Our study provides the most comprehensive collection of antithrombotic biomacromolecules from a non-hematophagous leech to date and will significantly enhance the investigation and utilization of leech derivatives in thrombosis therapy research and pharmaceutical applications.

Clinical Features and Long-Term Outcomes of a Pan-Canadian Cohort of Adolescents and Young Adults with Myeloproliferative Neoplasms: A Canadian MPN Group Study.

Myeloproliferative neoplasms (MPNs) are a group of chronic hematologic malignancies that lead to morbidity and early mortality due to thrombotic complications and progression to acute leukemia. Clinical and mutational risk factors have been demonstrated to predict outcomes in patients with MPNs and are used commonly to guide therapeutic decisions, including allogenic stem cell transplant, in myelofibrosis. Adolescents and young adults (AYA, age ≤45 years) comprise less than 10% of all MPN patients and have unique clinical and therapeutic considerations. The prevalence and clinical impact of somatic mutations implicated in myeloid disease has not been extensively examined in this population. We conducted a retrospective review of patients evaluated at eight Canadian centers for MPN patients diagnosed at ≤45 years of age. In total, 609 patients were included in the study, with median overall survival of 36.8 years. Diagnosis of prefibrotic or overt PMF is associated with the lowest OS and highest risk of AP/BP transformation. Thrombotic complications (24%), including splanchnic circulation thrombosis (9%), were frequent in the cohort. Mutations in addition to those in JAK2/MPL/CALR are uncommon in the initial disease phase in our AYA population (12%); but our data indicate they may be predictive of transformation to post-ET/PV myelofibrosis.

A genetic association study of circulating coagulation factor VIII and von Willebrand factor levels.

ABSTRACT: Coagulation factor VIII (FVIII) and its carrier protein von Willebrand factor (VWF) are critical to coagulation and platelet aggregation. We leveraged whole-genome sequence data from the Trans-Omics for Precision Medicine (TOPMed) program along with TOPMed-based imputation of genotypes in additional samples to identify genetic associations with circulating FVIII and VWF levels in a single-variant meta-analysis, including up to 45 289 participants. Gene-based aggregate tests were implemented in TOPMed. We identified 3 candidate causal genes and tested their functional effect on FVIII release from human liver endothelial cells (HLECs) and VWF release from human umbilical vein endothelial cells. Mendelian randomization was also performed to provide evidence for causal associations of FVIII and VWF with thrombotic outcomes. We identified associations (P < 5 × 10-9) at 7 new loci for FVIII (ST3GAL4, CLEC4M, B3GNT2, ASGR1, F12, KNG1, and TREM1/NCR2) and 1 for VWF (B3GNT2). VWF, ABO, and STAB2 were associated with FVIII and VWF in gene-based analyses. Multiphenotype analysis of FVIII and VWF identified another 3 new loci, including PDIA3. Silencing of B3GNT2 and the previously reported CD36 gene decreased release of FVIII by HLECs, whereas silencing of B3GNT2, CD36, and PDIA3 decreased release of VWF by HVECs. Mendelian randomization supports causal association of higher FVIII and VWF with increased risk of thrombotic outcomes. Seven new loci were identified for FVIII and 1 for VWF, with evidence supporting causal associations of FVIII and VWF with thrombotic outcomes. B3GNT2, CD36, and PDIA3 modulate the release of FVIII and/or VWF in vitro.

Cardiovascular risk in a contemporary cohort of patients with myeloproliferative neoplasms'.

BACKGROUND: Myeloproliferative neoplasms (MPNs) are a group of disorders of clonal haemopoiesis associated with an inherent risk of arterial and venous thrombotic complications. The prevalence of thrombotic complications and the impact of cardiovascular risk factors (CVRFs) in contemporary patient cohorts within the current era of MPN treatments have not been completely defined. OBJECTIVES: We aim to characterise the cardiovascular risk of patients with MPN by identifying the prevalence of CVRFs and describing the pattern of thrombotic events. We also aim to utilise the QRISK3 algorithm, which is a validated model used to estimate an individual's risk of developing cardiovascular disease, to further phenotype this cohort of patients. METHODS: We perform a retrospective analysis on a single-centre cohort of 438 patients with MPN. RESULTS: MPN patients continue to carry a high burden of vascular morbidity with a prevalence of arterial thrombotic events in 15.8 % (69/438) and venous thrombotic events in 13.2 % (58/438) of the cohort. The novel use of the QRISK3 algorithm, which showed a mean score of 13.7 % across the MPN population, provides further evidence to suggest an increased cardiovascular risk in MPN patients. CONCLUSION: With an increased risk of cardiovascular disease in patients with MPN, we propose an integrated approach between primary and specialised healthcare services using risk stratification tools such as QRISK3, which will allow aggressive optimisation of CVRFs to prevent thrombosis and reduce the overall morbidity and mortality in patients with MPN.

Cyclophilin A is a ligand for RAGE in thrombo-inflammation.

AIMS: Cyclophilin A (CyPA) induces leucocyte recruitment and platelet activation upon release into the extracellular space. Extracellular CyPA therefore plays a critical role in immuno-inflammatory responses in tissue injury and thrombosis upon platelet activation. To date, CD147 (EMMPRIN) has been described as the primary receptor mediating extracellular effects of CyPA in platelets and leucocytes. The receptor for advanced glycation end products (RAGE) shares inflammatory and prothrombotic properties and has also been found to have similar ligands as CD147. In this study, we investigated the role of RAGE as a previously unknown interaction partner for CyPA. METHODS AND RESULTS: Confocal imaging, proximity ligation, co-immunoprecipitation, and atomic force microscopy were performed and demonstrated an interaction of CyPA with RAGE on the cell surface. Static and dynamic cell adhesion and chemotaxis assays towards extracellular CyPA using human leucocytes and leucocytes from RAGE-deficient Ager-/- mice were conducted. Inhibition of RAGE abrogated CyPA-induced effects on leucocyte adhesion and chemotaxis in vitro. Accordingly, Ager-/- mice showed reduced leucocyte recruitment and endothelial adhesion towards CyPA in vivo. In wild-type mice, we observed a downregulation of RAGE on leucocytes when endogenous extracellular CyPA was reduced. We furthermore evaluated the role of RAGE for platelet activation and thrombus formation upon CyPA stimulation. CyPA-induced activation of platelets was found to be dependent on RAGE, as inhibition of RAGE, as well as platelets from Ager-/- mice showed a diminished activation and thrombus formation upon CyPA stimulation. CyPA-induced signalling through RAGE was found to involve central signalling pathways including the adaptor protein MyD88, intracellular Ca2+ signalling, and NF-κB activation. CONCLUSION: We propose RAGE as a hitherto unknown receptor for CyPA mediating leucocyte as well as platelet activation. The CyPA-RAGE interaction thus represents a novel mechanism in thrombo-inflammation.

One thousand patients with essential thrombocythemia: the Florence-CRIMM experience.

We describe 1000 patients with essential thrombocythemia seen at the Center Research and Innovation of Myeloproliferative Neoplasms (CRIMM), Florence, Italy, between 1980 and 2023: median age 59 years (18-95), females 65%, JAK2/CALR/MPL-mutated 66%/19%/4%, triple-negative (TN) 11%. Extreme thrombocytosis (ExT, platelets ≥1000 × 109/L) in 16%, leukocytosis (leukocytes >11 × 109/L) in 16%, and at least one cardiovascular risk factor in 52% of cases. JAK2-mutated patients were older (median 62 years) and CALR-mutated and TN (53 years for both) younger (p < 0.001). Female gender clustered with TN (76%) and JAK2 (67%) vs CALR (46%) mutations (p < 0.001). ExT clustered with CALR (type-2 more than type-1), TN and MPL, and leukocytosis with JAK2 mutation (p < 0.001). In multivariable analysis, risk factors for arterial thrombosis-free survival were age ≥60 years (HR 2.0; p < 0.001) and JAK2 mutation (HR 1.3; p = 0.02) with borderline significance for male gender (p = 0.08) and cardiovascular risk factors (p = 0.08); for venous thrombosis-free survival, JAK2 mutation (HR 1.9; p = 0.03) with borderline significance for venous thrombosis history (p = 0.07); for overall survival, older age (p < 0.001), male gender (HR 1.9; p < 0.001), absolute neutrophil count (ANC) ≥ 8 × 109/L (HR 1.8; p = 0.01), absolute lymphocyte count (ALC) < 1.7 × 109/L (HR 1.2; p = 0.03); for myelofibrosis-free survival, CALR mutation (HR 2.7; p < 0.001, particularly for CALR type 1/1-like, HR 3.3) and MPL mutation (HR 3.9; p = 0.001); for leukemia-free survival, older age (p = 0.03). Cytoreductive therapy appeared to mitigate both venous (HR 0.3; p = 0.01) and arterial thrombosis (HR 4; p = 0.04); there was a trend for aspirin in preventing arterial thrombosis recurrence. The current study provides real-world observations in essential thrombocythemia, representing a valid source document for interpreting current literature and planning future studies.