Intermittent hypoxia (IH) preconditioning enhances brain resilience, thereby protecting against subsequent ischemic injury, yet its precise mechanisms remain elusive. We tested the novel hypothesis that peripheral paracrine mechanisms mediate IH-induced neuroprotection. A total of 492 C57BL/6J mice were used: 434 young males (2-3 months), 29 young females (2-3 months), and 29 aged males (18 months). A 2-week IH regimen (13% O2, 5-minute intervals, 10 cycles/d) was applied to generate IH-derived plasma (IHP). To test the hypothesis, IHP or normoxic plasma (100 μL/injection) was intravenously administered every 3 days (6 doses total) before distal middle cerebral artery occlusion. For therapeutic evaluation, plasma was administered daily for 3 days after distal middle cerebral artery occlusion or 60-minute transient MCAO, followed by additional doses every 3 days for 6 doses in long-term transient MCAO studies. Infarct volume and neurological deficits were primary outcomes. Candidate circulating mediators were identified via proteomics and validated by antibody-mediated depletion and recombinant protein supplementation. Blood-brain barrier integrity was further examined. Systemic IHP administration protected against acute brain injury after distal middle cerebral artery occlusion, reducing infarct volume and improving sensorimotor performance. Poststroke administration of IHP conferred acute and sustained neuroprotection in transient MCAO, but not distal middle cerebral artery occlusion, improving sensorimotor and cognitive recovery and reducing brain atrophy up to 4 weeks after stroke. Proteomic profiling identified 120 IH-upregulated plasma proteins, notably PF4 (platelet factor 4), a cytokine with potent neuroprotective properties. PF4 immunodepletion abolished IHP-induced neuroprotection, whereas recombinant PF4 replicated these benefits across sex and age. Furthermore, PF4 treatment protected against blood-brain barrier disruption after tMCAO, attenuating IgG extravasation and loss of endothelial expression of zonula occludens-1. These findings identify PF4 as a key paracrine mediator of IH-induced neuroprotection and support the therapeutic potential of both IHP and PF4-based interventions for ischemic stroke.

This article focuses on the central role of antibodies against platelet factor 4 (PF4) in mediating immunothrombosis, from classical heparin-induced thrombocytopenia (HIT) to vaccine-induced immune thrombocytopenia and thrombosis (VITT). The latter condition gained international attention during the rollout of vaccines against SARS-CoV-2. Since then, an increased awareness for anti-PF4 mediated disorders arose and patients were recognized with anti-PF4 disorders occurring without prior heparin or adenoviral vector vaccine exposure. These disorders include various acute and chronic VITT-like conditions, i.e. post-viral VITT, diaplacentally transmitted anti-PF4 antibodies in neonatal stroke, monoclonal gammopathies of thrombotic significance (MGTS) and chronic autoimmune VITT of unknown origin. All anti-PF4 related disorders share key serological and immunopathological features with VITT, such as the formation of immune complexes and platelet activation via the Fcγ receptor IIA (FcγRIIA). Via their activation, platelets form procoagulant, aggregatory and secretory phenotypes shaping their interplay with neutrophils, monocytes, and coagulation factors to amplify thrombotic responses. Integrating recent mechanistic insights, clinical observations and diagnostic developments, this review proposes an updated conceptual framework for anti PF4-related immunothrombosis. We aim to raise awareness among clinicians and researchers, to promote early diagnosis and encourage further translational research towards improved therapeutic strategies in this clinically significant area.

Background and Clinical Significance: Disorders caused by platelet-activating antibodies targeting platelet factor 4 (PF4) are recognized as the cause of severe thrombotic events and are not restricted to heparin-induced thrombocytopenia (HIT). Case Presentation: We report a 67-year-old man with thrombocytopenia and extensive portal-splenic-mesenteric vein thrombosis complicated by intestinal ischemia. Despite intravenous unfractionated heparin (UFH), his condition worsened toward pulmonary embolism, septic shock, and multi-organ failure. Thrombolysis with alteplase was also ineffective. Both thrombophilia testing and autoimmune panels were negative, including those for antiphospholipid syndrome. An anti-PF4 immune thrombotic disorder was hypothesized. Therefore, argatroban was initiated instead of UFH therapy and intravenous immune globulin (IVIG) was administered. The platelet count increased and the patient’s clinical condition progressively improved. An anti-PF4/heparin assay on a blood sample collected before IVIG was highly positive. Platelet activation assays did not demonstrate an increased activation after the addition of heparin (the Heparin-Induced Platelet Activation [HIPA] assay was negative) though increased activation was observed with the addition of PF4 (the PF4-Induced Platelet Activation [PIPA] assay was positive), thus defining a VITT-like syndrome. Conclusions: This case report highlights the crucial function of having adequate laboratory facilities available to disentangle different anti-PF4 disorders for an accurate definition of a specific diagnosis, such as VITT-like syndrome, thereby allowing for the most appropriate therapeutic management of these complex pathological conditions. The clinical suspicion of an anti-PF4 immune disorder should be considered in cases of severe, otherwise unexplained, thrombotic events associated with thrombocytopenia. Specific tests like HIPA and PIPA are essential for definitive diagnosis.

Regional citrate anticoagulation (RCA) improves the biocompatibility of hemodialysis (HD) by reducing activation of complement, leukocytes, and platelets. Despite the new possibility of expanded hemodialysis (HDx), RCA and unfractionated heparin (UFH) have not been compared in this dialysis modality. This study compared the biocompatibility and efficacy of RCA versus full-dose UFH during HDx. In a randomized cross-over trial, 32 chronic HD patients each underwent two HDx sessions with a Theranova dialyzer: one with RCA and one with UFH. Biocompatibility was assessed by plasma complement factor 3a (C3a), myeloperoxidase (MPO), and platelet factor 4 (PF4) measured pre-HDx, after 15 min, and at session end. In a subset of patients (n = 5), scanning electron microscopy (SEM) visualized cell adhesion and clotting on dialyzer fibers. Efficacy outcomes were overall clearances and reduction ratios (RR) for creatinine, urea, and β2-microglobulin. MPO and C3a increased after 15 min of HDx only with UFH (p < 0.001). In the available PF4 data, a significant increase after 15 min of HDx occurred only with UFH (p = 0.02). SEM showed minimal cell adhesion with both anticoagulants. Overall clearances and RRs of creatinine, urea, and β2-microglobulin did not differ. RCA was well tolerated, with no significant electrolyte disturbances. RCA during HDx provided superior biocompatibility over UFH, eliminating complement, granulocyte, and platelet activation without improving dialysis efficacy.

Antibody-mediated multicellular pathophysiology of heparin-induced thrombocytopenia and vaccine-induced thrombotic thrombocytopenia: the dynamic roles of platelets, neutrophils, endothelial cells, and monocytes.

Coronavirus disease 2019 (COVID-19) affected billions of individuals globally, with symptoms ranging from isolated blood clotting to severe acute hypoxemic respiratory failure requiring intensive respiratory support ventilators. Those with advanced chronic kidney disease (CKD stage 5) were at high risk of severe disease faced a particularly heightened risk of severe illness. Inflammation and associated immune-thrombotic events in CKD stage 5 have attracted increasing attention, yet remain poorly understood. In this prospective cohort study, we examined and compared neutrophil extracellular traps (NETs) and low-density granulocytes (LDGs) in heathy controls (n = 15), CKD stage 5 patients without COVID-19 (n = 15), patients with COVID-19 (n = 15), and CKD stage 5 patients with COVID-19 (n = 90). Serum citrullinated histone H3 (CitH3) and related gene expression (PAD4, ERK1, PKC), NET complexes, platelet factor 4 (PF4), von Willebrand factor (vWF), RANTES, and specific cytokines were quantified. Proof-of-concept experiments were conducted to assess the role of lipopolysaccharide-LDG complexes in NETs detected in COVID-19 plasma. NETs complexes were significantly higher in COVID-19 cases, and more so in patients with CKD stage 5D, and those who died. Disease severity was directly correlated with NETs complex levels (p = 0.016). CitH3 and gene expression levels were not correlated with advanced CKD stage, while levels of components that initiate NETs formation were higher in patients with COVID-19 and in CKD stage 5 patients. In conclusion, NET activation could partially explain the thrombotic manifestations in patients with COVID-19 and in CKD stage 5 patients.

Journal Club: exercise and hypoxia differentially influence platelet activation of brain-derived neurotrophic factor and platelet factor 4.

Cancer-associated thrombosis (CAT), encompassing both venous thromboembolism and arterial thrombosis, contributes to up to 14% of cancer-related mortality and remains difficult to treat due to the bleeding risks of conventional anticoagulants. Protein disulfide isomerase (PDI) and its family member ERp57 (PDIA3) are thiol isomerases that regulate both arterial and venous thrombosis and are also upregulated in tumors, where they promote growth, metastasis, and immune evasion. Here, we evaluated the therapeutic potential of two thiol isomerase inhibitors-isoquercetin (ISOQ), a selective PDI inhibitor, and zafirlukast (ZAF), a broad-spectrum inhibitor of thiol isomerases such as PDI and ERp57-individually and in combination, in a xenograft model of ovarian cancer. ISOQ inhibited both platelet aggregation and Factor Xa generation induced by tumor cells and significantly suppressed tumor growth, thromboinflammatory markers, and expression of tissue factor, VEGF, TMEM176B, and PD-L1. ISOQ also potentiated standard cisplatin/gemcitabine chemotherapy. Notably, the combination of low-dose ISOQ plus ZAF achieved ≥ 80% inhibition of key tumor-associated markers at one-third the monotherapy dose and outperformed either agent alone. These findings support ISOQ and ZAF as promising agents for the treatment of cancer and CAT and establish thiol isomerase inhibition as a strategy to simultaneously target thrombosis, tumor progression, and immune escape.

Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a new disorder that emerged in the wake of coronavirus disease 2019 (COVID-19) vaccination. It is a rare but life-threatening condition that requires an aggressive course of treatment to improve patient outcomes. Currently, the diagnosis of VITT relies on a comprehensive panel of criteria, including a history of recent vaccination, platelet count, positive enzyme linked immunosorbent assays (ELISA)result for a closely related thrombotic disorder, heparin-induced thrombocytopenia (HIT), and platelet factor 4 (PF4)-dependent functional assays. The study describes a technically simple antigenic assay designed to detect autoimmune antibodies associated with VITT. It is first shown that cross-linked platelet factor 4 (c-PF4) represents an antigenic target specific for VITT/VITT-like Abs. This antigenic target is then incorporated into a microfluidic electrochemical biosensor and specific and sensitive detection of VITT/VITT-like antibodies is demonstrated in a fully automated manner while using microliter volumes of patient sera. Fifty-five serum samples are tested using the microfluidic electrochemical biosensor and demonstrated high accuracy for the detection of VITT sera and VITT-like monoclonal gammopathy of thrombotic ignificance (MGTS)antibodies compared to healthy controls and HIT patients. It is envisioned that the microfluidic electrochemical biosensor will be used in combination with other clinical criteria to enable timely, sensitive, and specific diagnosis of VITT.

Nanoparticles (NPs) designed for intracellular delivery must first navigate the cell-surface glycocalyx before reaching the plasma membrane for internalization. Here, we hypothesized that the glycocalyx can both hinder NP uptake via a barrier effect and enhance uptake by providing recognition sites for corona proteins. To dissect these opposing mechanisms, we prepared NPs with plasma protein coronas either enriched or depleted in heparin-binding proteins (HBPs), along with model coronas containing selected HBPs or non-HBPs. Biophysical assays confirmed strong heparin interactions for HBP-rich NPs, whereas HBP-poor NPs showed weak or no binding. To assess the role of corona-glycocalyx interactions in NP uptake, we used glycocalyx-depleted cells, a chemical inhibitor, and heparin and antithrombin competition assays. We found that canonical HBPs within the protein corona, including antithrombin, apolipoprotein E, and platelet factor 4, significantly enhanced NP surface retention and internalization through protein-glycocalyx interactions. In contrast, HBP-poor NPs showed weak or no interactions with the glycocalyx and, correspondingly, reduced uptake. Significantly, these findings also extended to physiologically derived coronas from control and dyslipidemic sera, with the latter producing HBP-enriched coronas that bound more strongly to heparin and promoted more efficient glycocalyx-dependent NP uptake. These findings highlight the underappreciated role of the glycocalyx in actively engaging with coronal HBPs to drive efficient NP uptake. This insight underscores the need to expand corona engineering beyond membrane receptor interactions, incorporating strategies that optimize glycocalyx interactions for more effective NP delivery.

Rotational atherectomy (RA) is an established technique for modifying heavily calcified and fibrotic coronary artery lesions. Despite its efficacy, the use of a high-speed rotating burr can provoke platelet activation and endothelial injury, thereby increasing thrombotic risk, promoting inflammation, and impairing vascular healing. This study investigated the effects of RA and its procedural characteristics on endothelial function and platelet activation by assessing circulating biomarkers. We prospectively analyzed 34 patients undergoing elective RA at a tertiary center. Blood samples were obtained before and 12-24 h after the procedure. Plasma levels of soluble E-selectin, soluble intercellular adhesion molecule-1 (ICAM-1), platelet factor 4 (PF4), P-selectin, and cluster of differentiation 40 ligand (CD40L) were measured. The study population had a mean age of 71 ± 8.9 years, and 73.8% were male. Cardiovascular comorbidities were prevalent, including diabetes (61.9%), hypertension (92.9%), hypercholesterolemia (42.9%), heart failure (45.2%), atrial fibrillation (21.4%), prior PCI (81%), and prior CABG (11.9%). RA significantly increased levels of P-selectin (55.5 ± 26.1 vs. 68.9 ± 26.5, p < 0.001), CD40L (2261.3 ± 2489.9 vs. 3602.0 ± 2428.5, p = 0.01), and PF4 (6054.7 ± 5751.8 vs. 10,877.6 ± 4979.7, p < 0.001). Moreover, mean burr speed correlated with CD40L elevation, while burr-to-artery ratio correlated with E-selectin increase (all p < 0.05). RA induces significant platelet activation and endothelial injury, with biomarker changes suggesting correlation with procedural parameters. These findings highlight the biological impact of RA and may inform strategies to optimize the safety of complex PCI.

Platelet factor 4 regulates hematopoietic stem cell aging.

SummaryThe adenoviral vector-based AstraZeneca and Janssen COVID-19 vaccines have been associated with rare cases of thrombosis, believed to be triggered, among other factors, by vector binding to the blood protein platelet factor 4 (PF4). To identify vectors with lower thrombosis risk, we screened 50 natural and hexon-modified adenoviruses (Ads). Unlike the applied COVID-19 vaccines and most tested vectors, Ad34 and Ad80, as well as Ad5 vectors with deleted or chemically shielded hexon hyper-variable region 1 (HVR1), did not bind to PF4. Furthermore, interactions with PF4 substantially modified Ad5 infectivity in various immortalized and primary cells, suggesting that PF4 may influence existing vector tropism. Finally, HVR1-deleted Ad5 and Ad34 vectors expressing SARS-CoV-2 spike S1 domain were tested as vaccine candidates in mice and induced robust cellular immune responses. Therefore, the identified PF4 non-binding vectors may represent safe and efficient candidates for clinical applications.

Aberrant proteostasis in alveolar type 2 epithelial cells (AEC2s) contributes to idiopathic pulmonary fibrosis (IPF), but the role of the ubiquitin-proteasome system (UPS) is unclear. Here, we show that UPS disruption in AEC2s amplifies profibrotic signaling to macrophages through macrophage migration inhibitory factor (MIF) family proteins in several models. Modeling UPS disruption with an AEC2-specific cullin 3 (Cul3) deletion produced spontaneous fibrosis in a physiological aging mouse model and exacerbated fibrosis in the bleomycin-induced lung injury model. This was accompanied by expansion of transitional epithelial states and increased MIF and MIF-2 in lung tissue and bronchoalveolar lavage fluid (BALF) in the model. Global or conditional AEC2-specific deletions of Mif or Mif-2 attenuated fibrosis in the bleomycin-treated mouse model, as did conditional deletions of Cd74, the cognate receptor for MIF and MIF-2, in C-X3-C motif chemokine receptor 1 (Cx3cr1)-expressing and platelet factor 4 (Pf4)-expressing cells. Pharmacological inhibition of MIF attenuated fibrosis in bleomycin-treated and transforming growth factor-β1 (TGFB1) transgenic mouse models and in ex vivo human precision-cut lung slices treated with fibrotic cocktail. In study participants with IPF, BALF MIF was elevated compared with that in study participants without IPF. In participants with IPF, BALF MIF greater than 4000 picograms per milliliter was associated with increased mortality compared with participants with IPF with lower MIF. Together, these findings define a UPS-sensitive epithelial-macrophage signaling connection and identify MIF-CD74 cross-talk as a potential therapeutic target in fibrotic lung disease.

Chromogenic anti-Xa assay is currently used in the management of patients on unfractionated heparin (UFH). It has been shown that inter-assay variability in anti-Xa levels can be explained in part by the presence or absence of dextran sulfate (DXS) in the reagents. DXS has the ability to dissociate UFH from neutralizing proteins, including platelet factor 4 (PF4).Investigate whether PF4 plasma levels along with the presence/absence of DXS in anti-Xa reagents are associated with variations in UFH anti-Xa levels in different clinical situations.In the prospective multicenter study DEXHEP-NCT04700670, critically ill patients on UFH therapy (four groups) were recruited. Blood was collected into citrate and CTAD tubes. Chromogenic anti-Xa levels were assessed using seven reagent/analyzer combinations including two without DXS. Plasma PF4 was measured by ELISA (Zymutest-PF4-Hyphen-Biomed).A total of 144 patients were analyzed: average PF4 levels in citrate plasma samples were consistently higher than in CTAD ones (206 vs. 46 ng/mL, p < 10-4), regardless of the patient group. Using a linear mixed-effect model, we found a significant effect of both DXS and PF4 on anti-Xa level, with a significant interaction term (p < 10-4). Considering the 0.3 to 0.7 IU/mL therapeutic range, agreement between anti-Xa values (Liquid-anti-Xa/DXS-free vs. Biophen-LRT/DXS) was observed in roughly two-thirds of the patients.PF4 levels slightly affects anti-Xa levels, the use of CTAD tubes minimizing the effect. However, PF4 levels do not fully explain the differences of anti-Xa levels observed in the presence or absence of DXS, which has a greater effect. Anti-Xa assays require better standardization.

Elevated level of platelet factor 4 in follicular fluid is associated with polycystic ovary syndrome.

Platelet Factor 4 (PF4), also known as CXCL4, is a CXC chemokine crucial for hemostasis, inflammation, and immune responses. Under physiological conditions PF4 assembles into asymmetric tetramers (31.2kDa) that are dimers of dimers with highly flexible N-terminal regions. PF4 tetramers play a central role in prothrombotic autoimmune conditions, such as heparin-induced thrombocytopenia (HIT), as well as vaccine-induced immune thrombocytopenia and thrombosis (VITT). Here, we report the resonance assignments of 1H, 15N, and 13C nuclei for wild-type asymmetric PF4 tetramers using TROSY-based triple resonance NMR experiments. We also used Nz-exchange spectroscopy to identify peaks split by slow-exchange between two distinct conformational states caused by the asymmetry of PF4 tetramers. Our NMR assignments establish a foundation for future investigations into the structural dynamics and functional mechanisms of PF4 as well as its pathological role in anti-PF4 disorders.

A pediatric case of anti-PF4 antibody-induced cerebral venous sinus thrombosis and thrombocytopenia following adenovirus infection: a literature review.

To evaluate the effect of varying centrifugal force and spin times on platelet, leukocyte, erythrocyte, and growth factor concentrations in canine platelet-rich plasma (PRP) prepared with a new PRP separating device system (Autologous Conditioned Plasma [ACP] Max System; Arthrex Inc). 12 healthy client-owned dogs were included in this experimental study. The study was performed from July to October 2023 and assessed PRP composition following different centrifugation protocols. Whole blood was collected from 6 healthy client-owned dogs and processed with 4 first-spin protocols: 2,420 or 2,696 X g for 6 or 9 minutes. A standardized second spin cycle (315.5 X g for 5 minutes) was then performed. Complete blood counts were performed to quantify platelet, leukocyte, and erythrocyte concentrations in whole blood and PRP. Growth factors in PRP were quantified with ELISAs for PDGF-ββ, FGF, TGF-β1, IGF-1, VEGF, and IL-1RA. All spin protocols reduced erythrocyte and leukocyte concentrations and increased platelet concentrations compared to whole blood. Platelet concentration was significantly increased compared to whole blood with an initial spin cycle of 2,420 X g. The 2,420 X g for 9 minutes protocol yielded a significant increase in PDGF-ββ. Transforming growth factor β1 was significantly increased across all protocols. The ACP Max System effectively produced PRP with reduced erythrocytes and leukocytes and increased platelet concentrations. Among the protocols tested, 2,420 X g for 9 minutes most effectively enhanced platelet and PDGF-ββ concentrations. Centrifugation settings impact the cellular and growth factor composition of the final PRP product. Clinical efficacy of variable PRP products needs further evaluation.

Biomarkers of in vivo platelet activation in coronary artery disease: a systematic review and meta-analysis: communication from the SSC of the ISTH.