Sickle cell disease (SCD) is an inherited hemolytic disorder characterized by a mutation in β-globin, resulting in hemoglobin polymerization, red blood cell rigidity, and a persistently activated endothelium that promotes thromboinflammation. Platelets are recognized as central mediators of this thromboinflammatory milieu, integrating signals from hemostatic and innate immune pathways. In this review, we summarize the disease-specific mechanisms by which platelets are activated in SCD, including GPCR signaling, CLEC-2/GPVI–BTK pathways, and responses to DAMPs such as free heme, and how these processes amplify leukocyte recruitment, inflammasome activation, and vascular occlusion. We highlight emerging platelet-associated biomarkers that correlate with disease severity and acute complications, including platelet-leukocyte aggregates, P-selectin, podoplanin–CLEC-2 signaling, and inflammatory cytokine pathways. Finally, we evaluate clinical trial data targeting platelet pathways, from early aspirin studies to more recent trials of P2Y12 inhibitors and selectin-blocking agents, noting both the promise and limitations of these approaches. Together, these findings underscore the multifaceted contribution of platelets to SCD pathophysiology and point toward ongoing questions regarding whether platelets primarily serve as biomarkers of disease activity or represent modifiable therapeutic targets capable of reducing the clinical burden of SCD.
The risk of venous thromboembolic events (VTE) is elevated in patients with active cancer. Recent antineoplastic therapies, including immunotherapies and targeted therapies may further increase VTE risk, although evidence are limited. In March 2024, we conducted a disproportionality analysis using reporting odds ratios (Reporting-OR) in VigiBase®, the World Health Organization's pharmacovigilance database, to assess the association between 280 antineoplastic therapies approved by the Food and Drug Administration and/or the European Medicines Agency and VTE (symptomatic or not), defined as deep vein thrombosis and/or pulmonary embolism. Reporting-OR were adjusted (aReporting-OR) for case characteristics, including primary tumor site and metastatic status. A total of 37,803 VTE cases associated with at least one antineoplastic therapies were identified. We identified 19 antineoplastic therapies significantly associated with an increased reporting of VTE with nintedanib emerging as a newly recognized association. The strongest associations were observed for lenalidomide (aReporting-OR 3.64; 95%CI 3.47-3.82), bevacizumab (aReporting-OR 3.64; 95%CI 3.43-3.86) and cyproterone (aReporting-OR 3.52; 95%CI 2.55-4.87). The median time to VTE onset was 64 days (27-143), with 81% of VTE events occurring within the first six months after antineoplastic therapy initiation. Future studies should include antineoplastic therapies in VTE risk assessments and evaluate the management of VTE when recurrences occur under while under treatment. ClinicalTrial registration number: NCT04696250.
Erythropoiesis-stimulating agents (ESAs) are commonly used to treat cancer-related anemia by increasing hemoglobin levels and reducing transfusion requirements. However, randomized trials and meta-analyses have linked ESA use to increased thrombotic risk. Contemporary real-world data across diverse cancer populations and ESA formulations remain limited. We conducted a retrospective cohort study using the TriNetX Research Network (January 1, 2015–July 31, 2024) evaluating adult patients with cancer-related anemia. Patients receiving ESAs were propensity score matched 1:1 with non-ESA patients based on demographics, cancer type, and comorbidities. Individuals with prior thrombotic events or anticoagulant use were excluded. Outcomes included incident deep vein thrombosis (DVT), pulmonary embolism (PE), new anticoagulant use, and 1-year survival. Risk differences, relative risks, and Kaplan-Meier survival analyses were performed. After matching, 1,606 patients were included in each cohort, with predominant cancer types including prostate, breast, bladder, colon, and kidney cancer. There was no significant difference in DVT (1.5% vs 2.1%; p=0.19) or PE (0.75% vs 1.1%; p=0.27) between ESA and non-ESA groups. New anticoagulant use was significantly lower in ESA-treated patients (6.3% vs 10.2%; RR 0.52, 95% CI 0.49–0.78; p<0.001). One-year survival was higher in the ESA cohort (73% vs 69%; HR 0.82, 95% CI 0.72–0.94; p=0.004). In this large real-world cohort, ESA use was not associated with increased short-term thrombotic risk and was associated with lower anticoagulant use and improved 1-year survival. These findings suggest that guideline-concordant ESA use may be safe in selected patients with cancer-related anemia.
Endothelial cells (ECs) line the vasculature and integrate hemodynamic, inflammatory, and metabolic cues that are crucial in maintaining vascular health. Endothelial dysfunction is a driver of thrombo-inflammatory conditions including atherosclerosis, thrombosis, stroke, and sepsis-induced multi-organ dysfunction syndrome. Therapies for these conditions that are given systemically fail to target the site of disease and can be associated with off-target side-effects. EC-targeted nanoparticles represent a novel method of concentrating the drug- or gene-based therapy in ECs at the site of disease. Here, we describe studies of EC-directed nanoparticles in thrombo-inflammatory vascular diseases. We discuss challenges in the development of EC-targeted nanoparticle therapies, including issues with biosafety, delivery efficiency, scalability, and clinical translation. To target ECs, nanoparticle surfaces have been coated with ligand-directed antibodies or peptides against EC markers. EC-targeting has also been achieved by coating nanoparticles with cell-mimicking membranes to leverage native homing pathways to sites of vascular injury. Stimuli-responsive nanoparticles have used EC or disease-specific cues to trigger drug release, for instance via reactive oxygen species or shear stress stimuli. EC-targeted nanoparticles extend the therapy circulation time, enhance drug accumulation at target sites, improve therapeutic efficacy, and reduce toxicity. In preclinical studies, EC-targeted nanoparticles have stabilized atherosclerotic plaques, degraded venous thrombi, and reduced cerebral infarct volume. While EC-targeted nanoparticle therapies have not yet been approved for clinical use, advances in nanoparticles design and safety are narrowing the gap between experimental promise and clinical reality.
Thrombocytopenia absent radius (TAR) syndrome is a rare disease characterized by thrombocytopenia that results from defective differentiation of megakaryocyte (MK) precursors. It is caused by mutations in the exon junction complex member, RBM8A, but it is not understood how the deficiency in this gene leads to a defect in megakaryopoiesis. Using TAR patient-derived induced pluripotent stem cell (iPSC) lines and isogenic lines with corrected expression of RBM8A, we compared the phenotypes of derivative hematopoietic progenitor cells (HPC) and MKs. While the generation of HPCs was similar between lines, the proliferation of TAR MKs was significantly reduced. This was accompanied by precocious maturation, differential expression of platelet signaling pathways, and enhanced apoptosis. Despite TAR MK’s enhanced size and markers of maturation, TAR and corrected lines had similar platelet-forming capacity both in vitro and in vivo. Genome-wide gene expression analysis revealed differential gene expression in apoptosis and cell cycle pathways. Splicing analysis showed differential splicing of pathways involved in mRNA splicing as well as platelet degranulation, demonstrating lineage-specific defects. We also identified several candidate targets that could be contributing to the phenotypes observed in this model. Overall, our study shows that the thrombocytopenia in TAR syndrome results from the lack of proliferation and increased cell death of MK progenitors, leading to fewer numbers of mature MKs and platelets.
Immune tolerance induction (ITI) remains the only proven strategy for eradicating factor VIII (FVIII) inhibitors in severe hemophilia A, yet low-dose regimens are associated with prolonged treatment and significant bleeding. Emicizumab provides FVIII-independent hemostatic protection and may permit antigen exposure under conditions of reduced bleeding and inflammation during ITI. We conducted a single-center cohort study with historical controls to evaluate the impact of emicizumab on low-dose ITI outcomes in pediatric patients (<14 years) with severe hemophilia A and high-responding inhibitors undergoing their first low-dose ITI (50 IU/kg 3x/week). Twenty-three patients were included. Patients treated with low-dose ITI alone (LD-ITI) (2010–2020; n=14) were compared with those receiving ITI plus emicizumab prophylaxis (LD-ITI+EMI) (2021–2023; n=9). Complete ITI success occurred in 78% of the LD-ITI+EMI cohort vs. 36% of the LD-ITI (p=0.09). Median time to tolerance was shorter with emicizumab (9 vs. 19 months; p=0.003). Time-to-event analysis confirmed faster tolerance with emicizumab (HR 3.89, p=0.02). Treated bleeds during ITI were drastically reduced in the emicizumab cohort (median ABR 0.0 vs. 5.1; p <0.0001), and a higher bleeding burden was associated with delayed tolerance. No thrombotic complications were observed. Median direct treatment costs were comparable between cohorts. In this pediatric cohort, low-dose ITI combined with emicizumab was associated with faster tolerance achievement, bleeding reduction, and no increase in direct costs. These findings suggest that ITI associated with emicizumab may provide a more efficient and clinically manageable approach to inhibitor eradication in pediatric patients.
Platelets are small blood cells, historically viewed principally as mediators of hemostasis and thrombosis. However, it is now well established that platelets are pivotal modulators of the inflammatory response, establishing the concepts of thromboinflammation and immunothrombosis. Here we propose the novel term ‘thromborepair’ to describe how platelets promote tissue repair by facilitating leukocyte extravasation, resolving tissue inflammation and coordinating fibrosis and angiogenesis. This review aims to discuss these processes and stimulate further exploration of platelet-based therapies to promote tissue repair.
The FVIII B-domain, a large and heavily glycosylated region, is crucial for FVIII secretion, though its structural and functional roles remain incompletely understood. While the B-domain is dispensable for cofactor activity, prior research hints at multiple, yet unverified, functional roles. Here, we employed an integrative hybrid approach to generate detailed structural models of glycosylated and furin-cleaved full-length FVIII (Gly/FC-FL-FVIII) that include the B-domain, utilizing AlphaFold2 (AF2) predictions. These models were further refined and validated with biophysical data from atomic force microscopy (AFM) and cryo-electron microscopy (cryo-EM) of recombinant and plasma-derived FL-FVIII proteins. Additionally, we modeled a FL-FVIII-VWF complex with both the B-domain and D′-D3 domains (FL-FVIII-VWF-D′-D3) to investigate B-domain interactions during intracellular processing. Our results suggest that the B-domain functions largely as an independent domain encircling the FVIII core. AFM studies revealed that VWF tails are associated with globular FVIII structures. Structural analysis indicated that B-domain glycosylation enhances stability, supports proper folding, and promotes efficient trafficking through the ER-Golgi pathway. We propose that, within the Golgi, the B-domain may unfold around FVIII via furin cleavages, facilitating essential interactions with partners such as VWF. Our models also suggest that the B-domain shields key FVIII epitopes, potentially reducing immune interference and inhibiting premature activation. This study presents a novel structural model of the FVIII B-domain and full-length FVIII, offering critical insights into previously unexplored aspects of B-domain function and its essential role in FVIII biology.
Acquired hemophilia A (AHA) is a rare, autoimmune disease that leads to a severe bleeding diathesis. The efficacy of emicizumab use in the inpatient management of AHA has been recognized but widespread inpatient use remains limited due to its high cost. To evaluate cost-effectiveness of up-front emicizumab use in the inpatient management of AHA in the US. We built a Markov simulation to examine the cost-effectiveness of administering emicizumab with concurrent recombinant factor VIIa (rFVIIa) versus rFVIIa alone (standard of care, SOC) in older individuals newly diagnosed with AHA and hospitalized for bleeding control. Model outcomes included direct costs (medication use and hospital stay) and utilities associated with bleeding and nonbleeding health states (measured using quality-adjusted life days, QALDs). The analysis was conducted over a 20-day hospitalization horizon with a health system perspective. We conducted deterministic and probabilistic sensitivity analyses (PSA), capturing uncertainty across parameters over 10,000 Monte Carlo simulations. The addition of up-front emicizumab to SOC vs SOC alone yielded lower total direct cost ($248,934 vs. $569,038) and more QALDs (15.92 vs. 14.72). The addition of up-front emicizumab to SOC was the dominant strategy. The duration of daily rFVIIa use had the largest impact on the incremental net monetary benefit. In a PSA, emicizumab was cost-effective in 100% of simulations. The addition of up-front emicizumab to SOC is less costly and more effective, even at current emicizumab pricing. Our results provide strong economic and clinical justification to consider up-front emicizumab use in AHA management.
Patients with hereditary hemorrhagic telangiectasia (HHT) experience recurrent epistaxis. Doxycycline has been proposed as a possible treatment, although its efficacy remains controversial. Whether the most common genotypes (activin receptor-like kinase 1, endoglin, and SMAD4) contribute to a differential response has not been investigated. In this study, we evaluate the effectiveness of doxycycline among the different HHT genotypes. A retrospective cohort study was conducted at the University of Florida's Hereditary Hemorrhagic Telangiectasia Center. Forty-one adult patients (aged ≥18 years) with HHT, diagnosed by Curacao criteria and genetic testing, were classified as responders and nonresponders based on the minimal clinically important change between pre- and posttreatment epistaxis severity scores (ESS). Hemoglobin and hematocrit values were also collected to assess treatment response. Overall, the cohort was 61% female and 90.2% White, with a mean age of 58.1 years; 26 responders had a decrease (P < .0001) in ESS from a baseline median of 4.6 (interquartile range [IQR], 3.3) to posttreatment ESS of 2.4 (IQR, 1.9). The median baseline ESS for the total cohort was 4.4 (IQR, 3.3), and after a mean follow-up of 4.1 months, it significantly (P < .0001) decreased to an ESS of 3.3 (IQR, 3.2). The mean hemoglobin and hematocrit values did not exhibit significant changes. The relationship between genotype and doxycycline response was not statistically significant. Patients with HHT treated with doxycycline showed an overall reduction in epistaxis severity, which did not appear to be associated with genotype. Doxycycline may be a safe, effective, and accessible treatment option for epistaxis in HHT.
Interindividual variability in response to low-dose aspirin (acetylsalicylic acid; ASA) contributes to residual platelet activation and thrombotic risk. Platelet microRNAs (miRNAs) may modulate ASA pharmacodynamics, but their mechanistic role remains unclear. We investigated whether platelet miR-144-5p regulates cyclooxygenase 1/2 (COX-1/2) expression and modulates ASA response in patients with cardiometabolic disease. Two hundred ASA- treated patients were stratified into tertiles of serum thromboxane B2 (sTXB2) slope, an ex vivo marker of COX-1 recovery. Platelet and extracellular vesicle (EV) miR-144-5p levels were quantified by quantitative reverse transcription polymerase chain reaction and correlated with biochemical and clinical variables. Functional studies were performed in megakaryocytic (DAMI cells) and endothelial (human umbilical vein endothelial cells [HUVECs]) cells transfected with miR-144-5p mimics or inhibitors to assess COX-1/2 regulation. Patients with poor ASA response (third tertile of sTXB2 slope) exhibited reduced platelet miR-144-5p (P = .027) and higher COX-1 mRNA/protein levels (P ≤ .027), whereas miR-144-5p was enriched in platelet-derived EVs. Platelet miR-144-5p was inversely correlated with sTXB2 (ρ = -0.235, P = .007) and COX-1/2 mRNA expression. In DAMI cells, ASA exposure upregulated miR-144-5p cells; in addition, miR-144-5p overexpression suppressed, and inhibition enhanced, COX-1 protein levels. Similar effects were observed in HUVECs, where miR-144-5p downregulated both COX-1 and COX-2. Among our patients, platelet miR-144-5p levels were lower in those with obesity, dyslipidemia, and metabolic dysfunction-associated steatotic liver disease, conditions known to affect aspirin response. Platelet miR-144-5p directly regulates COX-1/2 expression in vitro and is associated with interindividual variability in ASA response. Low platelet miR-144-5p level identifies patients with accelerated COX-1 recovery and residual thromboxane generation, providing a potential mechanistic framework for precision antiplatelet therapy and miRNA-targeted interventions.
Aspirin treatment irreversibly inhibits cyclooxygenase activity, blocking thromboxane A2 (TxA2) synthesis and reducing thromboxane-prostanoid (TP) receptor-induced platelet activation. Aspirin resistance refers to the failure of regular aspirin doses to produce the expected antiplatelet effect. Aspirin-induced bleeding complications are frequently observed in patients receiving excessive doses or combined antiplatelet and anticoagulant therapy. Although selective TP receptor antagonists have been tested clinically, they are ineffective due to persistently high prostaglandin levels, causing unexpected signaling effects in platelets and other cell types. Hydroxyflavonoids are bioactive phytonutrients with antithrombotic effects, inhibiting TxA2-mediated platelet signaling and preventing abnormal platelet activation in cardiovascular diseases without major side effects. Using a recently published TP receptor crystal structure and docking studies with different flavonoid classes, including genistein and tectorigenin, we found that flavonoids occupy similar ligand-binding surface of TxA2, thereby preventing TxA2 binding. To evaluate our bioinformatics data, a mutant TP receptor variant was overexpressed in HEK293 cells, deleting interacting amino acids with hydroxyflavonoids, and stimulated with the TxA2 analog U46619. Intracellular Ca2+ responses were measured in Fura-2-labeled cells. Amino acid substitutions in the extracellular domain, which did not influence U46619 binding, abolished hydroxyflavonoid-mediated inhibition of the TP receptor. These results suggest that hydroxyflavonoids directly bind to and inhibit the TP receptor. Using aggregometry and flow chamber assays, we confirmed that genistein and tectorigenin synergize with aspirin to inhibit platelet aggregation and thrombus growth in human and mouse platelets. Whether combined genistein-aspirin therapy improves antiplatelet responses in aspirin-resistant patients requires further investigation.
Sickle cell disease (SCD) is characterized by chronic intravascular hemolysis and depletion of the heme scavenger hemopexin (HPX), generating a high-heme milieu that may increase susceptibility to inflammatory organ injury. Sepsis is a leading cause of acute kidney injury (AKI), yet the contribution of hemolysis-derived free heme to polymicrobial sepsis-associated AKI in SCD remains poorly defined. Using humanized SCD mice, we tested whether augmenting heme clearance via HPX mitigates septic AKI. Low-grade cecal ligation and puncture induced exaggerated systemic inflammation, elevated circulating heme and ferritin, and marked reductions in glomerular filtration rate (GFR) in sickling (hemoglobin SS [HbSS]) mice compared with nonsickling (HbAA) control mice. Septic SS mice also exhibited enhanced renal nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 inflammasome activation, increased oxidative stress, and tubular injury, consistent with heme-driven inflammatory and cytotoxic signaling. Acute administration of purified human HPX reduced circulating heme and ferritin, attenuated cytokine responses, and preserved GFR, indicating that restoration of heme buffering in SCD interrupts kidney injury pathways during septic stress. To achieve sustained heme control, we restored HPX expression using liver-directed delivery of adeno-associated virus serotype 8 (AAV8). AAV8-HPX restored hepatic HPX, reduced basal and sepsis-induced plasma heme levels, conferred protection against kidney dysfunction and tubular stress, and improved survival without evidence of hepatotoxicity. Collectively, these findings identify free heme as a central mediator linking hemolysis to sepsis-associated AKI in SCD, and establish that HPX augmentation via protein replacement or gene therapy is a mechanistically targeted, potentially translatable therapeutic strategy to mitigate heme-driven tissue injury and improve outcomes in SCD-associated sepsis.
Thrombomodulin (THBD) is an endothelial membrane protein that has anticoagulant and anti-inflammatory actions, inhibits fibrinolysis, and modulates innate immunity and complement activation. We report the characterization of a novel THBD mutation, C256S, homozygously expressed in an adolescent boy. The patient presented with a wide range of symptoms, including life-threating epistaxis, thrombotic microangiopathy, massive intraoperative bleeding, venous thromboses, and skin necrosis. THBD-C256S is predicted to prevent the formation of a critical disulfide bond within epidermal growth factor-like region 1. The mutant was nonfunctional when expressed in a zebra fish model. In patient-derived endothelial cells, THBD-C256S exhibited impaired glycosylation, increased intracellular retention, and reduced cell surface levels of THBD. Expression of the mutant led to decreased levels of other endothelial proteins, including platelet endothelial cell adhesion molecule-1 and vascular endothelial-cadherin. Our data suggest that misfolding of THBD-C256S disrupts normal THBD functions leading to the broad constellation of clinical abnormalities.
Over recent decades, numerous experimental models have been developed to investigate arterial thrombosis and evaluate the efficacy of antithrombotic therapies. A major limitation of these models lies in the biphasic dynamics of a thrombus, which prevents assessment of its destabilization/disaggregation/thrombolysis after pharmacological treatment. In this study, we developed a stable thrombosis model based on the external application of 2 pieces of 7.5% FeCl3-saturated Whatman papers to the carotid artery. Histological analysis and transmission electron microscopy revealed no exposure of the subendothelial matrix. Intravital microscopy identified platelets as the major cell type adhering to the injured vessel wall, with plateau-type kinetics of thrombus formation throughout the experiment. Doppler probe measurements detected the arrest of blood flow at 14 ± 3 minutes, when the thrombus reached its maximum area. Intravital and histological studies further showed that thrombus composition changed over time after injury. At 45 minutes, thrombi were enriched in red blood cells, with a shift in the platelet-fibrin composition toward fibrin predominance compared with thrombi at 15 minutes. To evaluate the efficacy of known antithrombotic agents in this model, inhibitors were injected 15 minutes after initiation of thrombosis. Video microscopy revealed that 0.4 mg/mL cangrelor (P2Y12 inhibitor) or 4 mg/mL GR 144053 trihydrochloride (αIIbβ3 blocker) induced complete disaggregation, whereas 50 IU/mL heparin or 4 mg/mL recombinant tissue plasminogen activator induced destabilization of the thrombus. In contrast, 3.2 mg/mL aspirin, 40 IU/mL unfractionated heparin, or 4 mg/mL hirudin had no effect on a preformed thrombus. Altogether, this model constitutes a valuable platform for assessing therapeutic strategies aimed at stabilizing or dissolving established thrombi.
