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Abstract Background Fibrinolysisis is essential for vascular blood flow maintenance and is triggered by endothelial and platelet release of tissue plasminogen activator (t-PA). In certain critical conditions, e.g. sepsis, acute respiratory failure (ARF) and trauma, the fibrinolytic response is reduced and may lead to widespread thrombosis and multi-organ failure. The mechanisms underpinning fibrinolysis resistance include reduced t-PA expression and/or release, reduced t-PA and/or plasmin effect due to elevated inhibitor levels, increased consumption and/or clearance. This study in critically ill patients with fibrinolysis resistance aimed to evaluate the ability of t-PA and plasminogen supplementation to restore fibrinolysis with assessment using point-of-care ClotPro viscoelastic testing (VET). Methods In prospective, observational studies, whole-blood ClotPro VET evaluation was carried out in 105 critically ill patients. In 32 of 58 patients identified as fibrinolysis-resistant (clot lysis time > 300 s on the TPA-test: tissue factor activated coagulation with t-PA accelerated fibrinolysis), consecutive experimental whole-blood VET was carried out with repeat TPA-tests spiked with additional t-PA and/or plasminogen and the effect on lysis time determined. In an interventional study in a patient with ARF and fibrinolysis resistance, the impact of a 24 h intravenous low-dose alteplase infusion on coagulation and fibrinolysis was prospectively monitored using standard ClotPro VET. Results Distinct response groups emerged in the ex vivo experimental VET, with increased fibrinolysis observed following supplementation with (i) t-PA only or (ii) plasminogen and t-PA. A baseline TPA-test lysis time of > 1000 s was associated with the latter group. In the interventional study, a gradual reduction (25%) in serial TPA-test lysis times was observed during the 24 h low-dose alteplase infusion. Conclusions ClotPro viscoelastic testing, the associated TPA-test and the novel experimental assays may be utilised to (i) investigate the potential mechanisms of fibrinolysis resistance, (ii) guide corrective treatment and (iii) monitor in real-time the treatment effect. Such a precision medicine and personalised treatment approach to the management of fibrinolysis resistance has the potential to increase treatment benefit, while minimising adverse events in critically ill patients. Trial registration: VETtiPAT-ARF, a clinical trial evaluating ClotPro-guided t-PA (alteplase) administration in fibrinolysis-resistant patients with ARF, is ongoing (ClinicalTrials.gov NCT05540834 ; retrospectively registered September 15th 2022).

Abstract Platelet transfusions are not always available for bleeding in severe thrombocytopenia, as storage outside of major centers is limited by their short shelf‐life. Data are lacking to support alternative available blood products; however, additional fibrinogen has been shown to enhance clot formation in vitro. To test the hypothesis that cryoprecipitate supplementation could improve clot formation in severe thrombocytopenia, eight hematological malignancy patients with platelet counts under 10 × 109/L each had 10 units of apheresis cryoprecipitate transfused prior to planned prophylactic platelet transfusions. The primary endpoint of thromboelastometry amplitude at 20 min increased by a mean of 5.1 mm (p

The ability of platelets to promote carcinoma and melanoma progression has been thoroughly studied and occurs in numerous ways. In contrast, the effect of platelets on sarcomas, tumors arising from mesenchymal cells, has received very little attention. This study was undertaken to simultaneously compare the effects of platelets on murine and human sarcomas and carcinomas. In contrast to their effect on carcinomas, platelets inhibited the invasion of some murine- and all human sarcomas tested in vitro. Further invasion studies with TGFβ treatment only partially recapitulated the results seen with whole platelets. In a spontaneous tumor growth and lung metastasis model, platelets promoted 4T1 mammary carcinoma metastasis but not MCA-1 fibrosarcoma metastasis. Gene expression analysis of the platelet-promoted MDA-MB-231 breast carcinoma, and the platelet-inhibited HT1080 fibrosarcoma cell lines revealed that exposure of MDA-MB-231 to platelets, resulted in upregulation of oncogenes and EMT-associated genes whereas in HT1080 a tumor-suppressor gene was significantly upregulated. Thus, this study has revealed a potential diametrically opposing effect of platelets on mesenchymal and epithelial cancers, a finding that warrants further investigation.

The survival rate of patients with osteosarcoma (OS) has not improved over the last 30 years. Mutations in the genes TP53, RB1 and c-Myc frequently occur in OS and enhance RNA Polymerase I (Pol I) activity, thus supporting uncontrolled cancer cell proliferation. We therefore hypothesised that Pol I inhibition may be an effective therapeutic strategy for this aggressive cancer. The Pol I inhibitor CX-5461 has demonstrated therapeutic efficacy in different cancers in pre-clinical and phase I clinical trials; thus, the effects were determined on ten human OS cell lines. Following characterisation using genome profiling and Western blotting, RNA Pol I activity, cell proliferation and cell cycle progression were evaluated in vitro, and the growth of TP53 wild-type and mutant tumours was measured in a murine allograft model and in two human xenograft OS models. CX-5461 treatment resulted in reduced ribosomal DNA (rDNA) transcription and Growth 2 (G2)-phase cell cycle arrest in all OS cell lines. Additionally, tumour growth in all allograft and xenograft OS models was effectively suppressed without apparent toxicity. Our study demonstrates the efficacy of Pol I inhibition against OS with varying genetic alterations. This study provides pre-clinical evidence to support this novel therapeutic approach in OS.

Histones, proteins that bind DNA, are toxic for pathogens outside cells but can also cause multi-organ damage as seen in sepsis. Here the authors develop small negatively charged molecules that can be used as histone antidotes, and show that they improve the phenotype in mouse models with histone-related pathologies.

Diagnosis of immune thrombocytopenia (ITP) and prediction of response to therapy remain significant and constant challenges in hematology. In patients who present with ITP, the platelet count is frequently used as a surrogate marker for disease severity, and so often determines the need for therapy. Although there is a clear link between thrombocytopenia and hemostasis, a direct correlation between the extent of thrombocytopenia and bleeding symptoms, especially at lower platelet counts is lacking. Thus, bleeding in ITP is heterogeneous, unpredictable, and nearly always based on a multitude of risk factors, beyond the platelet count. The development of an evidence-based, validated risk stratification model for ITP treatment is a major goal in the ITP community and this review discusses new laboratory approaches to evaluate the various pathobiologies of ITP that may inform such a model.

Platelet-neutrophil aggregates (PNAs) facilitate neutrophil activation and migration and could underpin the recruitment of neutrophils to the pancreas during type 1 diabetes (T1D) pathogenesis. PNAs, measured by flow cytometry, were significantly elevated in the circulation of autoantibody-positive (Aab+) children and new-onset T1D children, as well as in pre-T1D (at 4 weeks and 10–12 weeks) and T1D-onset NOD mice, compared with relevant controls, and PNAs were characterized by activated P-selectin+ platelets. PNAs were similarly increased in pre-T1D and T1D-onset NOD isolated islets/insulitis, and immunofluorescence staining revealed increased islet-associated neutrophil extracellular trap (NET) products (myeloperoxidase [MPO] and citrullinated histones [CitH3]) in NOD pancreata. In vitro, cell-free histones and NETs induced islet cell damage, which was prevented by the small polyanionic drug methyl cellobiose sulfate (mCBS) that binds to histones and neutralizes their pathological effects. Elevated circulating PNAs could, therefore, act as an innate immune and pathogenic biomarker of T1D autoimmunity. Platelet hyperreactivity within PNAs appears to represent a previously unrecognized hematological abnormality that precedes T1D onset. In summary, PNAs could contribute to the pathogenesis of T1D and potentially function as a pre-T1D diagnostic.

Abstract Dichloroacetate (DCA) is an investigational drug targeting the glycolytic hallmark of cancer by inhibiting pyruvate dehydrogenase kinases (PDK). It is metabolized by GSTZ1, which has common polymorphisms altering enzyme or promoter activity. GSTZ1 is also irreversibly inactivated by DCA. In the first clinical trial of DCA in a hematological malignancy, DiCAM (DiChloroAcetate in Myeloma), we have examined the relationship between DCA concentrations, GSTZ1 genotype, side effects, and patient response. DiCAM recruited seven myeloma patients in partial remission. DCA was administered orally for 3 months with a loading dose. Pharmacokinetics were performed on day 1 and 8. Trough and peak concentrations of DCA were measured monthly. GSTZ1 genotypes were correlated with drug concentrations, tolerability, and disease outcomes. One patient responded and two patients showed a partial response after one month of DCA treatment, which included the loading dose. The initial half‐life of DCA was shorter in two patients, correlating with heterozygosity for GSTZ1*A genotype, a high enzyme activity variant. Over 3 months, one patient maintained DCA trough concentrations approximately threefold higher than other patients, which correlated with a low activity promoter genotype (−1002A, rs7160195) for GSTZ1. This patient displayed the strongest response, but also the strongest neuropathy. Overall, serum concentrations of DCA were sufficient to inhibit the constitutive target PDK2, but unlikely to inhibit targets induced in cancer. Promoter GSTZ1 polymorphisms may be important determinants of DCA concentrations and neuropathy during chronic treatment. Novel dosing regimens may be necessary to achieve effective DCA concentrations in most cancer patients while avoiding neuropathy.

Bioluminescent tumor cell lines are used extensively in vivo to monitor tumor growth and metastasis but rarely used in vitro to follow tumor cell behavior. Tumor cell migration is frequently studied in vitro using transwell assays, however, current methods do not permit the co-incubation of tumor cells with different stromal cell types for analysis of the effects of intercellular cross-talk on tumor cell migration. We describe a novel migration assay using bioluminescent tumor cell lines that is rapid, accurate, and permits the study of the effects of tumor cell-stromal cell interactions on tumor cell migratory behavior.

Heme oxygenase-1 is critical for iron recycling during red blood cell turnover, whereas its impact on steady-state erythropoiesis and red blood cell lifespan is not known. We show here that in 8- to 14-week old mice, heme oxygenase-1 deficiency adversely affects steady-state erythropoiesis in the bone marrow. This is manifested by a decrease in Ter-119+-erythroid cells, abnormal adhesion molecule expression on macrophages and erythroid cells, and a greatly diminished ability to form erythroblastic islands. Compared with wild-type animals, red blood cell size and hemoglobin content are decreased, while the number of circulating red blood cells is increased in heme oxygenase-1 deficient mice, overall leading to microcytic anemia. Heme oxygenase-1 deficiency increases oxidative stress in circulating red blood cells and greatly decreases the frequency of macrophages expressing the phosphatidylserine receptor Tim4 in bone marrow, spleen and liver. Heme oxygenase-1 deficiency increases spleen weight and Ter119+-erythroid cells in the spleen, although α4β1-integrin expression by these cells and splenic macrophages positive for vascular cell adhesion molecule 1 are both decreased. Red blood cell lifespan is prolonged in heme oxygenase-1 deficient mice compared with wild-type mice. Our findings suggest that while macrophages and relevant receptors required for red blood cell formation and removal are substantially depleted in heme oxygenase-1 deficient mice, the extent of anemia in these mice may be ameliorated by the prolonged lifespan of their oxidatively stressed erythrocytes.