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Liver-resident CD8+ T cells can play critical roles in the control of pathogens, including Plasmodium and hepatitis B virus. Paradoxically, it has also been proposed that the liver may act as the main place for the elimination of CD8+ T cells at the resolution of immune responses. We hypothesized that different adhesion processes may drive residence versus elimination of T cells in the liver. Specifically, we investigated whether the expression of asialo-glycoproteins (ASGPs) drives the localization and elimination of effector CD8+ T cells in the liver, while interactions with platelets facilitate liver residence and protective function. Using murine CD8+ T cells activated in vitro, or in vivo by immunization with Plasmodium berghei sporozoites, we found that, unexpectedly, inhibition of ASGP receptors did not inhibit the accumulation of effector cells in the liver, but instead prevented these cells from accumulating in the spleen. In addition, enforced expression of ASGP on effector CD8+ T cells using St3GalI-deficient cells lead to their loss from the spleen. We also found, using different mouse models of thrombocytopenia, that severe reduction in platelet concentration in circulation did not strongly influence the residence and protective function of CD8+ T cells in the liver. These data suggest that platelets play a marginal role in CD8+ T cell function in the liver. Furthermore, ASGP-expressing effector CD8+ T cells accumulate in the spleen, not the liver, prior to their destruction.

Summary Background. Uterine leiomyosarcoma is a rare aggressive smooth muscle cancer with poor survival rates. RNA Polymerase I (Pol I) activity is elevated in many cancers supporting tumour growth and prior studies in uterine leiomyosarcoma revealed enlarged nucleoli and upregulated Pol I activity-related genes. This study aimed to investigate the anti-tumour potential of CX-5461, a Pol I transcription inhibitor currently being evaluated in clinical trials for several cancers, against the human uterine leiomyosarcoma cell line, SK-UT-1. Methods. SK-UT-1 was characterised using genome profiling and western blotting. The anti-tumour effects of CX-5461 were investigated using cell proliferation assays, expression analysis using qRT-PCR, and BrdU/PI based cell cycle analysis. Results. Genetic analysis of SK-UT-1 revealed mutations in TP53, RB1, PTEN, APC and TSC1 & 2, all potentially associated with increased Pol I activity. Protein expression analysis showed dysregulated p53, RB1 and c-Myc. CX-5461 treatment resulted in an anti-proliferation response, G2 phase cell-cycle arrest and on-target activity demonstrated by reduced ribosomal DNA transcription. Conclusions. SK-UT-1 was confirmed as a representative model of uterine leiomyosarcoma and CX-5461 has significant potential as a novel adjuvant for this rare cancer.

PDF file, 430K, 4T1.2 subcutaneous tumor growth in platelet-deficient mice.

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PDF file, 320K, Tissue factor expression in B16F1 melanoma and 4T1.2 breast cancer cell lines.

PDF file, 905K, Antibody-mediated NK-cell depletion.

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 × 10

Background In critical conditions such as sepsis, severe trauma, COVID-19 and non-COVID acute respiratory failure, hypofibrinolysis is associated with multi-organ dysfunction syndrome and death. The mechanisms underpinning hypofibrinolysis may include reduced tissue plasminogen activator (t-PA) and/or plasmin effect due to elevated inhibitor levels, reduced expression and/or exhaustion. This study in critically ill patients with hypofibrinolysis aimed to evaluate the ability of t-PA and plasminogen supplementation to restore fibrinolysis assessed by bedside viscoelastic testing (VET). Methods Prospective observational and interventional studies were undertaken in 28 critically ill patients identified as hypercoagulant and hypofibrinolytic using standard ClotPro VET. Hypercoagulation was defined as above normal values for clot amplitude on the EX-test (tissue factor (TF) activated coagulation) or FIB-test (TF activated coagulation with platelet inhibition). Hypofibrinolysis was defined as a clot lysis time > 300 seconds on the TPA-test (TF activated coagulation with t-PA accelerated fibrinolysis). In experimental VET, repeat TPA-tests were spiked with additional t-PA and/or plasminogen and the effect on lysis time determined. In a hypofibrinolytic patient, alteplase was administered intravenously over a 24-hr period with standard ClotPro VET repeated frequently throughout to monitor the effect on coagulation and fibrinolysis. Results In the ex-vivo studies, distinct response groups emerged with increased fibrinolysis observed following (i) additional t-PA supplementation only, or (ii) combined plasminogen and t-PA supplementation. A baseline TPA-test lysis time of > 1000 sec associated with the latter group. In the interventional study, alteplase administered as a 2-hr bolus (25 mg) followed by a 22-hr infusion (1 mg/hr) resulted in a gradual reduction in serial TPA-test lysis times. Conclusions ClotPro viscoelastic testing, the associated TPA-test and the novel spiked ex-vivo assays may be utilised to (i) investigate the potential mechanisms of hypofibrinolysis, (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 hypofibrinolysis has the potential to increase treatment benefit, whilst minimising adverse events in hypofibrinolytic critically ill patients. Trial Registration: VETtiPAT ARF, a clinical trial evaluating the use of ClotPro-guided tissue plasminogen activator (alteplase) administration in hypofibrinolytic patients with acute respiratory failure is ongoing (ClinicalTrials.gov NCT05540834, registered 15 September 2022, retrospectively registered).

Two thirds of metastatic osteosarcoma patients die within 5 years of diagnosis. Improved experimental models of osteosarcoma metastasis will facilitate the development of more effective therapies. Intravenous cancer cell injection can produce lung metastases in nude mice, but this "experimental metastasis" technique has been predominantly applied to a single osteosarcoma cell line (143B) and required injection of 1-2 million cells. Using two human osteosarcoma cell lines, we discovered that transient Natural Killer cell depletion dramatically enhanced the efficiency of experimental pulmonary osteosarcoma metastasis. This technique for modeling osteosarcoma metastasis may enable the identification of better treatments for this aggressive cancer.

Background Collagen and fibrin engagement and activation of glycoprotein (GP) VI induces proteolytic cleavage of the GPVI ectodomain generating shed soluble GPVI (sGPVI). Collagen-mediated GPVI shedding requires intracellular signalling to release the sGPVI, mediated by A Disintegrin And Metalloproteinase 10 (ADAM10); however, the precise mechanism by which fibrin induces GPVI shedding remains elusive. Plasma sGPVI levels are elevated in patients with coagulopathies, sepsis, or inflammation and can predict onset of sepsis and sepsis-related mortality; therefore, it is clinically important to understand the mechanisms of GPVI shedding under conditions of minimal collagen exposure. Objectives Our aim was to characterize mechanisms by which fibrin-GPVI interactions trigger GPVI shedding. Methods Platelet aggregometry, sGPVI ELISA, and an ADAM10 fluorescence resonance energy transfer assay were used to measure fibrin-mediated platelet responses. Results Fibrin induced αIIbβ3-independent washed platelet aggregate formation, GPVI shedding, and increased ADAM10 activity, all of which were insensitive to pre-treatment with inhibitors of Src family kinases but were divalent cation- and metalloproteinase-dependent. In contrast, treatment of washed platelets with other GPVI ligands, collagen, and collagen-related peptide caused αIIbβ3-dependent platelet aggregation and GPVI release but did not increase constitutive ADAM10 activity. Conclusions Fibrin engages GPVI in a manner that differs from other GPVI ligands. Inclusion of polyanionic molecules disrupted fibrin-induced platelet aggregate formation and sGPVI release, suggesting that electrostatic charge may play a role in fibrin/GPVI engagement. It may be feasible to exploit this property and specifically disrupt GPVI/fibrin interactions whilst sparing GPVI/collagen engagement.Fibrin engages GPVI in a manner that differs from other GPVI ligands. Inclusion of polyanionic molecules disrupted fibrin-induced platelet aggregate formation and sGPVI release, suggesting that electrostatic charge may play a role in fibrin/GPVI engagement. It may be feasible to exploit this property and specifically disrupt GPVI/fibrin interactions whilst sparing GPVI/collagen engagement.

Liver-resident CD8+ T cells can play critical roles in the control of pathogens including Plasmodium and hepatitis B virus. Paradoxically, it has also been proposed that the liver may act as the main place for the elimination of CD8+ T cells at the resolution of immune responses. We hypothesized that different adhesion processes may drive residence versus elimination of T cells in the liver. Specifically, we investigated whether the expression of asialoglycoproteins (ASGPs) drives the localization and elimination of effector CD8+ T cells in the liver, while interactions with platelets facilitates liver-residence and protective function. Using murine CD8+ T cells activated in vitro, or in vivo by immunization with Plasmodium berghei sporozoites, we found that, unexpectedly, inhibition of ASGP receptors did not inhibit the accumulation of effector cells in the liver, but instead prevented these cells from accumulating in the spleen. Additionally, enforced expression of ASGP on effector CD8+ T cells using St3GalI deficient cells lead to their loss from the spleen. We also found, using different mouse models of thrombocytopenia, that severe reduction in platelet concentration in circulation did not strongly influence the residence and protective function of CD8+ T cells in the liver. These data suggest that platelets play a marginal role in CD8+ T cell function in the liver. Furthermore, ASGP-expressing effector CD8+ T cells accumulate in the spleen, not the liver, prior to their destruction.

Uterine leiomyosarcoma is a rare aggressive smooth muscle cancer with poor survival rates. RNA Polymerase I (Pol I) activity is elevated in many cancers supporting tumour growth and prior studies in uterine leiomyosarcoma revealed enlarged nucleoli and upregulated Pol I activity-related genes. This study aimed to investigate the anti-tumour potential of CX-5461, a Pol I transcription inhibitor currently being evaluated in clinical trials for several cancers, against the human uterine leiomyosarcoma cell line, SK-UT-1.SK-UT-1 was characterised using genome profiling and western blotting. The anti-tumour effects of CX-5461 were investigated using cell proliferation assays, expression analysis using qRT-PCR, and BrdU/PI based cell cycle analysis.Genetic analysis of SK-UT-1 revealed mutations in TP53, RB1, PTEN, APC and TSC12, all potentially associated with increased Pol I activity. Protein expression analysis showed dysregulated p53, RB1 and c-Myc. CX-5461 treatment resulted in an anti-proliferation response, G2 phase cell-cycle arrest and on-target activity demonstrated by reduced ribosomal DNA transcription.SK-UT-1 was confirmed as a representative model of uterine leiomyosarcoma and CX-5461 has significant potential as a novel adjuvant for this rare cancer.

Many materials have been engineered and commercialized as hemostatic agents. However, there is still a gap in the availability of hemostats that offer biocompatibility and biodegradability in combination with effective hemostatic properties. Cellulose nanofibers are investigated as hemostatic materials with most studies focusing on oxidized cellulose-derived hemostats. The recent studies demonstrate that by optimizing the morphological properties of nonoxidized cellulose nanofibers (CNFs) enhanced hemostasis is achieved. Herein, the hemostatic and wound-healing properties of CNFs with optimized morphology using two forms, gel, and sponge is investigated. In vitro thromboelastometry studies demonstrate that CNFs reduce clotting time by 68% (±SE 2%) and 88% (±SE 5%) in gel and sponge forms, respectively. In an in vivo murine liver injury model, CNFs significantly reduce blood loss by 38% (±SE 10%). The pH-neutral CNFs do not damage red blood cells, nor do they impede the proliferation of fibroblast or endothelial cells. Subcutaneously-implanted CNFs show a foreign body reaction resolving with the degradation of CNFs on histological examination and there is no scarring in the skin after 8 weeks. Demonstrating superior hemostatic performance in a variety of forms, as well as biocompatibility and biodegradability, CNFs hold significant potential for use in surgical and first-aid environments.

Hemorrhage remains a significant cause of morbidity and mortality following trauma and during complex surgeries. A variety of nanomaterials, including oxidized cellulose nanofibers (OCNFs), have been studied to overcome the disadvantages of current commercial topical hemostats. However, the relationship between nano-structural characteristics and hemostatic efficacy of non-oxidized cellulose nanofibers (CNFs) has not been elucidated. Herein, we present the first report of the correlation between structure and hemostatic performance of CNFs. In vitro thromboelastometry studies on CNFs, synthesized by ball-milling, showed that there is an optimum balance point between the aspect ratio (AR) and specific surface area (SSA) of nanofibers in terms of their maximum contribution to platelet function and plasma coagulation. The optimized CNFs with high SSA (17 m2/g) and a high AR (166) shortened normal whole blood clotting time by 68 %, outperforming cellulose-based hemostats. Additionally, CNFs reduced clotting time in platelet-deficient blood (by 80 %) and heparinized blood (by 54 %).

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.

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 TGF�� 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.

Extracellular histones have been shown to play an important pathogenic role in many diseases, primarily through their cytotoxicity toward nucleated cells and their ability to promote platelet activation with resultant thrombosis and thrombocytopenia. In contrast, little is known about the effect of extracellular histones on erythrocyte function. We demonstrate in this study that histones promote erythrocyte aggregation, sedimentation, and using a novel in vitro shear stress model, we show that histones induce erythrocyte fragility and lysis in a concentration-dependent manner. Furthermore, histones impair erythrocyte deformability based on reduced passage of erythrocytes through an artificial spleen. These in vitro results were mirrored in vivo with the injection of histones inducing anemia within minutes of administration, with a concomitant increase in splenic hemoglobin content. Thrombocytopenia and leukopenia were also observed. These findings suggest that histones binding to erythrocytes may contribute to the elevated erythrocyte sedimentation rates observed in inflammatory conditions. Furthermore, histone-induced increases in red blood cell lysis and splenic clearance may be a significant factor in the unexplained anemias seen in critically ill patients.

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.

Extracellular histones in neutrophil extracellular traps (NETs) or in chromatin from injured tissues are highly pathological, particularly when liberated by DNases. We report the development of small polyanions (SPAs) (~0.9–1.4 kDa) that interact electrostatically with histones, neutralizing their pathological effects. In vitro, SPAs inhibited the cytotoxic, platelet-activating and erythrocyte-damaging effects of histones, mechanistic studies revealing that SPAs block disruption of lipid-bilayers by histones. In vivo, SPAs significantly inhibited sepsis, deep-vein thrombosis, and cardiac and tissue-flap models of ischemia-reperfusion injury (IRI), but appeared to differ in their capacity to neutralize NET-bound versus free histones. Analysis of sera from sepsis and cardiac IRI patients supported these differential findings. Further investigations revealed this effect was likely due to the ability of certain SPAs to displace histones from NETs, thus destabilising the structure. Finally, based on our work, a non-toxic SPA that inhibits both NET-bound and free histone mediated pathologies was identified for clinical development., 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.

The asymmetric distribution of phospholipids in the plasma/organellar membranes is generated and maintained through phospholipid flippases in resting cells, but becomes disrupted in apoptotic cells and activated platelets, resulting in phosphatidylserine (PS) exposure on the cell surface. Stable PS exposure during apoptosis requires inactivation of flippases to prevent PS from being reinternalized. Here we show that flippase ATP8A1 is highly expressed in both murine and human platelets, but is not present in the plasma membrane. ATP8A1 is cleaved by the cysteine protease calpain during apoptosis, and the cleavage is prevented indirectly by caspase inhibition, involving blockage of calcium influx into platelets and subsequent calpain activation. In contrast, in platelets activated with thrombin and collagen and exposing PS, ATP8A1 remains intact. These data reveal a novel mechanism of flippase cleavage and suggest that flippase activity in intracellular membranes differs between platelets undergoing apoptosis and activation.

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.

The significant role of platelets in the protection of tumour cells from immune attack and shear forces and the promotion of tumour cell extravasation from the bloodstream in the process of haematogenous metastasis have been extensively studied. The role of platelets, and in particular platelet membranes, in the promotion of a more metastatic phenotype in tumour cells is a more recent and, therefore, less well-recognised area of research. This review article summarises studies that have focused on the impact of tumour cell interactions with platelets and platelet membranes on tumour cell behaviour in vitro and in vivo. Furthermore, the gene expression changes that occur within tumour cells following contact with platelet membranes are also extensively reviewed. Overall, the interaction of platelet membranes with tumour cells results in a more invasive phenotype and the promotion of epithelial to mesenchymal transition with our own genetic studies revealing that matrix metalloproteinase-1, plasminogen activator inhibitor-1 and interleukin-8 are globally upregulated in a range of tumour cell lines.

Background: Thrombocytopenia occurs reasonably frequently with the underlying causes being numerous and requiring time to delineate. Patients may present with active bleeding or low platelet counts may be detected incidentally. Treatments are varied, refractory cases are not uncommon, and in those who do respond, relapses occur. The clinician's principal concern is to limit the risk of a life-threatening hemorrhage, however, the platelet count is an unreliable predictor of bleeding risk. Adult patients presenting with platelet counts < 20 x 109/L present the greatest safety dilemma for clinicians. Previous studies using ROTEM in adult ITP patients with platelet counts < 60 x 109/L and paediatric cases with platelet counts < 30 x 109/L demonstrated that clot firmness parameters correlated with bleeding score1. Our aim, therefore, was to perform ROTEM analysis on patients with a platelet count Methods: Patients referred to the Haematology Department of The Canberra Hospital with thrombocytopenia were consented to the study. Blood was collected into Na-citrate tubes and analysed within 4 hr of collection. Each patient had an EXTEM and FibTEM assay performed on a ROTEM Delta machine. The measurement of fibrinogen levels was commenced part-way through the study. Data was analysed using PRISM software. Results: Twenty-five blood samples were analysed from 21 patients (17 primary ITP, 2 secondary ITP, 1 AML, 1 sepsis), 7 samples were treatment naive. Platelet count ranged from 0-17 x 109/L (mean 7.1 +/- 5.7) Even in this severely thrombocytopenic cohort, platelet count was a poor predictor of bleeding risk, as was the fibrin clot amplitude measured with FibTEM. In comparison, the calculated value for platelet contribution to clot formation (EXTEM A10 minus FibTEM A10) plotted against the platelet count demonstrated that subjects with a bleeding phenotype had a reduced platelet contribution to clot formation in comparison to their non-bleeding counterparts (Figure). Conclusions: ROTEM has the capacity to detect platelet dysfunction associated with bleeding in severely thrombocytopenic patients. Further studies are underway to elucidate determinants of platelet dysfunction such as specific anti-glycoprotein antibodies that may inhibit platelet function. Reference #1: Lindsey A. Greene et al., British Journal of Haematology 166: 592-600 (2014) Figure Disclosures D'Rozario: Alexion: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Introduction Paroxysmal nocturnal haemoglobinuria (PNH) is a rare acquired clonal haematopoietic stem cell disorder characterised by haemolytic anaemia, thrombosis and bone marrow failure. Thrombosis is considered a major cause for high morbidity and mortality in PNH patients (Hillmen P et al N Eng J Med 1995). However, the underlying mechanisms of thrombosis in PNH are still poorly understood. Various factors including defective interactions between the complement system, platelets and coagulation systems have been proposed (Peacock-Young B et al Haematologica 2017). Platelet function and clot formation in this disorder have not been comprehensively evaluated. Here we describe standard and novel techniques to evaluate platelet function in blood from PNH patients, to elucidate the underlying pro-thrombotic mechanisms. Methods Whole blood was collected from five PNH patients and compared to same-day healthy donor (HD) controls. The samples were collected at trough for those patients who were on eculizumab (Complement C5 inhibitor used in the treatment of PNH). Surface levels of platelet adhesion proteins and activation markers P-selectin and phosphatidylserine (PS) were assessed using flow cytometry. Plasma soluble GPVI (a platelet-specific activation marker) and cytokine levels were measured by ELISA. Clot formation was assessed by viscoelastic testing (ROTEM). Adhesion of platelets to collagen under flow in a whole blood assay was evaluated by Digital Holographic Microscopy (DHM) and thrombus height, surface area and volume quantified using custom-built MatLab-based software. Results Clinical characteristics of PNH patients were highly variable: two patients had history of thrombosis, three patients were on eculizumab, four patients were thrombocytopenic ( Conclusion Analysis of these PNH patients with highly variable clinical characteristics did not identify a unifying platelet lesion. DHM could detect and quantify parameters of small thrombi in real time, in PNH patient samples and these data were consistent with enhanced thrombogenic potential in PNH patients. Mechanisms beyond platelet activation that contribute to increased thrombosis in these patients and the impact of eculizumab therapy on thrombotic propensity need to be explored further. Disclosures D'Rozario: Alexion: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Introduction Neutrophil extracellular traps (NETs) are released from neutrophils following activation by pathogens and/or tissue injury and exhibit anti-microbial activity. Uncontrolled formation of NETs can, however, become pathogenic, particularly via their associated histones, and induce endothelial cell death, systemic vascular obstruction, multiple organ failure and initiate coagulation by both activating platelets and damaging erythrocytes such that they become pro-thrombotic. Study Objectives Recently we discovered an additional effect of histones on erythrocyte, which is to enhance their fragility when they are subjected to shear stress associated with blood flow through the vascular system and spleen in particular. Our laboratory has synthesised a family of small polyanions (SPAs) which have been screened for their ability to neutralise the pathogenic effects of histones and NETs. Methods We have used a novel mechanically-induced shear stress method as well as an in vitro spleen filtration model to investigate the effects of mammalian histones ± SPAs, on RBC fragility. The in vitro findings were mirrored in vivo with the injection of histones and SPAs in mice. Results Our results revealed that free histones bind to erythrocytes with high affinity and render erythrocytes susceptible to lysis by shear stress in a dose-dependent manner and some SPAs could totally inhibit or reverse this phenomenon. Conclusions Based on these data we propose that erythrocytes can neutralise the damaging effects of histones in the circulation and also transport them to the spleen for disposal. Overload of this disposal system could cause the unexplained anaemia associated with sepsis, cancer and other pathological conditions.

The prometastatic role of platelets has long been recognized with proposed mechanisms of action including shielding tumor cells from natural killer (NK) cell destruction and aiding endothelial attachment and extravasation of tumor cells with platelet P-selectin being implicated in these processes. However, many aspects of the prometastatic function of platelets remain unclear. In this study, we used mouse models of metastatic breast cancer and melanoma to investigate the platelet effect, focusing on organ specificity, the relationship with NK cells and the relative importance of platelet-derived versus endothelial-derived P-selectin. We found that platelets promote lung metastasis in the absence of NK cells in both acute and spontaneous metastasis models. In addition, the prometastatic action of platelets was found to be organ specific, clearly enhancing lung metastasis but not affecting B16F1 liver metastasis, in fact, liver metastasis was enhanced in the absence of platelets. Furthermore, the profound antimetastatic activity of NK cells was equally effective in the presence or absence of platelets and chronologically distinct from the prometastatic role of platelets. Finally, it was shown that endothelial-derived P-selectin is just as important as platelet-derived P-selectin in promoting lung metastasis and also plays an important role in liver metastasis. Taken together, our findings help clarify the roles of platelets, NK cells and P-selectin in metastasis, and they identify P-selectin as an attractive therapeutic target for preventing metastasis in multiple organs. Cancer Res; 72(18); 4662–71. ©2012 AACR.

The promotion of tumour metastasis by platelets may occur through several mechanisms including the induction of a more metastatic phenotype in tumour cells and assisted extravasation of circulating tumour cells. Whilst the mechanisms underlying platelet-assisted extravasation have been extensively studied, much less attention has been paid to the mechanisms underlying platelet promotion of an aggressive phenotype within a tumour cell population. Herein, we demonstrate in vitro that MDA-MB-231 breast carcinoma cells incubated with washed thrombin-activated platelet membranes adopt a Matrigel-degrading phenotype in a dose- and contact time-dependent manner. The same phenotypic change was observed with three other human tumour cell lines of diverse anatomical origin. Moreover, tumour cell lines that had been cultured with washed thrombin-activated platelet membranes had a greater metastatic capacity when injected into mice. This in vivo effect was reliant upon a co-incubation period of >2 h implying a mechanism involving more than platelet membrane binding that occurred within 5 min. Upon further investigation it was found that simultaneous blocking of the platelet-membrane proteins P-selectin and GPIIb/IIIa prevented interactions between platelet membranes and MDA-MB-231 cells but also significantly reduced the ability of tumour cells to degrade Matrigel. These results confirm that platelets induce a more aggressive phenotype in tumour cells but also identify the platelet proteins involved in this effect. P-selectin and GPIIb/IIIa also play a role in assisting tumour cell extravasation and, thus, are ideal targets for the therapeutic intervention of both stages of platelet-assisted metastasis.

The significant role of platelets and P-selectin in assisting tumor cell metastasis to the lungs has been frequently reported and reviewed. However, evidence recently has come to light on other pro-metastatic mechanisms of platelets beyond that of tumor cell protection from immune cell attack and aiding extravasation, such as promoting epithelial to mesenchymal transition in tumor cells and conveying signals from the primary tumor to distant tissues that optimize conditions for metastasis. Moreover, the role of platelets and selectins in hematogenous metastasis to frequently targeted organs other than the lungs has been less well examined. This review aims to summarize the literature on the roles of platelets in all stages of the metastatic process and to examine the participation of platelets and selectins in hematogenous metastasis to the lungs, liver, bone, and brain. In the light of the available evidence, potential therapeutic avenues for the control of metastasis are also discussed.

Transmembrane lipid transporters are believed to establish and maintain phospholipid asymmetry in biological membranes; however, little is known about the in vivo function of the specific transporters involved. Here, we report that developing erythrocytes from mice lacking the putative phosphatidylserine flippase ATP11C showed a lower rate of PS translocation in vitro compared with erythrocytes from wild-type littermates. Furthermore, the mutant mice had an elevated percentage of phosphatidylserine-exposing mature erythrocytes in the periphery. Although erythrocyte development in ATP11C-deficient mice was normal, the mature erythrocytes had an abnormal shape (stomatocytosis), and the life span of mature erythrocytes was shortened relative to that in control littermates, resulting in anemia in the mutant mice. Thus, our findings uncover an essential role for ATP11C in erythrocyte morphology and survival and provide a new candidate for the rare inherited blood disorder stomatocytosis with uncompensated anemia.

In patients with coronary heart disease and a broad range of cholesterol levels, cholesterol-lowering therapy reduces the risk of coronary events, but the effects on mortality from coronary heart disease and overall mortality have remained uncertain.In a double-blind, randomized trial, we compared the effects of pravastatin (40 mg daily) with those of a placebo over a mean follow-up period of 6.1 years in 9014 patients who were 31 to 75 years of age. The patients had a history of myocardial infarction or hospitalization for unstable angina and initial plasma total cholesterol levels of 155 to 271 mg per deciliter. Both groups received advice on following a cholesterol-lowering diet. The primary study outcome was mortality from coronary heart disease.Death from coronary heart disease occurred in 8.3 percent of the patients in the placebo group and 6.4 percent of those in the pravastatin group, a relative reduction in risk of 24 percent (95 percent confidence interval, 12 to 35 percent; P0.001). Overall mortality was 14.1 percent in the placebo group and 11.0 percent in the pravastatin group (relative reduction in risk, 22 percent; 95 percent confidence interval, 13 to 31 percent; P0.001). The incidence of all cardiovascular outcomes was consistently lower among patients assigned to receive pravastatin; these outcomes included myocardial infarction (reduction in risk, 29 percent; P0.001), death from coronary heart disease or nonfatal myocardial infarction (a 24 percent reduction in risk, P0.001), stroke (a 19 percent reduction in risk, P=0.048), and coronary revascularization (a 20 percent reduction in risk, P0.001). The effects of treatment were similar for all predefined subgroups. There were no clinically significant adverse effects of treatment with pravastatin.Pravastatin therapy reduced mortality from coronary heart disease and overall mortality, as compared with the rates in the placebo group, as well as the incidence of all prespecified cardiovascular events in patients with a history of myocardial infarction or unstable angina who had a broad range of initial cholesterol levels.

Acute myelocytic leukemia (AML) is a malignant disease characterized by the proliferation of immature myelocytic precursor cells causing the disruption of normal bone marrow function. Many chromosomal aberrations have been described in AML including translocations, inversions, deletions, and additions. Here we describe a novel deletion of chromosome 1, del(1)(p34p36) in a case of AML, French-American-British classification M1, in a previously healthy 33-year-old male. This isolated cytogenetic abnormality occurred in 33% of the myeloblasts examined at diagnosis. Subsequent cytogenetic analyses conducted on marrow following induction and consolidation therapy demonstrated a normal male karyotype in all cells examined. The patient remains in clinical and hematological remission 22 months following diagnosis. The presence of 1p abnormalities in AML and other malignancies is reviewed, as are candidate tumor suppressor genes in the 1p34 approximately p36 region. The implications of chromosome 1p abnormalities on clinical outcome are also discussed.

The less common chronic B-cell lymphomas include hairy cell leukemia, hairy cell leukemia variant and splenic lymphoma with villous lymphocytes. These disease entities can sometimes cause a diagnostic dilemma; however, immunophenotypic markers have been identified as disease specific and scoring systems have been proposed to assist the process. This study reports a case of a chronic B-cell lymphoma with long cytoplasmic projections which does not fit into any of the published disease categories based upon a combination of clinical and morphological features and immunophenotyping. Furthermore, this case featured a combination of cytogenetic abnormalities not previously described in the published literature in association with a B-cell lymphoproliferative disorder.

Background Sarcomas are cancers that arise from tissues of mesenchymal origin and there has been limited improvement in treatments over the last 30 years. The Warburg effect is a widespread metabolic phenotype of cancer, where glycolysis is favoured despite the presence of oxygen. Dichloroacetate (DCA) is a pyruvate dehydrogenase kinase (PDK) inhibitor in clinical use that can reverse the Warburg effect, inhibiting growth and enhancing apoptosis in a range of cancers. We have investigated its effectiveness against sarcoma cells in vitro and in vivo. Materials and methods Three cell lines (mouse fibrosarcoma S180, mouse osteosarcoma K7M2 and human fibrosarcoma HT1080-luc2) were examined for cell viability after DCA treatment in vitro (neutral red uptake assay), alone and in combination with doxorubicin. In vivo, K7M2 cells were injected s.c. into Rag1 -/- mice (2 sites per mouse, 7-9 mice per group) and established tumours were treated with DCA in the drinking water (0, 0.5, 1.0 and 1.5 g/L, delivering 0, 70, 125 and 165 mg/kg/day, respectively). Tumour growth was monitored with callipers. Plasma was collected on d1 and d15 for measurement of DCA levels (LC-MS). Results DCA significantly reduced the total viable cell number after 48 h of treatment in the mouse sarcoma lines (~15% at 0.5 mM DCA, and 30-40% at 5 mM DCA), however HT1080-luc2 cells showed on ly a1 0% reduction in cell number with 5 mM DCA. There was no morphological indication of apoptosis, suggesting DCA was decreasing proliferation. Chronic treatment of the mouse cells (5 mM DCA for 2 weeks) resulted in significantly slower growth rates as measured over 48 h (7 and 13% total cell number compared to untreated S180 and K7M2 cells, respectively). DCA did not synergise with doxorubicin but was additive at lower concentrations of doxorubicin. In vivo ,1 .0 and 1.5 g/L DCA significantly reduced tumour growth (33.0 and 33.1% reduction in tumour size on d13, p=0.001 and 0.01 respectively). Plasma DCA was undetectable on d1 of treatment, but by d15, 1.0 and 1.5 g/L DCA delivered 2-44 uM. Tumour DCA concentrations were also measured and found to be in the range of 25-470 uM, much lower than those typically used in in vitro studies. Conclusions DCA was effective against an in vivo sarcoma model, with tumour DCA concentrations in the micromolar range. These concentrations are achievable clinically, thus DCA warrants further investigation for sarcoma treatment.

Objective The factors determining platelet and erythrocyte lifespan are not completely understood, despite extensive study. The lack of success may be attributed to the methods used to measure lifespan kinetics, all of which require processing of cells prior to analysis, and the inconsistent and potentially inappropriate use of mathematical models for data analysis. The aims of this study were to establish an in vivo platelet and erythrocyte labeling method using carboxyfluorescein diacetate succinimidyl ester (CFSE), determine the most appropriate mathematical model for lifespan analysis, and apply both to the study of factors that control platelet and erythrocyte lifespans. Materials and Methods Control, c-mpl knockout (KO), and Bcl-X L mutant mice were injected with CFSE and platelet and erythrocyte fluorescence followed over time. Datasets were analyzed using linear, exponential, multiple-hit, and lognormal mathematical models. Results In vivo CFSE labeling of platelets and erythrocytes requires no postcollection processing, proved stable, nontoxic, nonimmunogenic, and the lifespans were highly reproducible. Mathematical modeling revealed the lognormal model gave a robust fit to control and extreme datasets when either extrinsic or intrinsic factors determined lifespan. Using these methods, platelet lifespans were found to be significantly shortened in thrombopoietin-receptor–deficient mice independent of blood loss, and the antiapoptotic protein Bcl-X L was shown to play a role in prolonging erythrocyte lifespans. Conclusions The simultaneous study of platelet and erythrocyte lifespans using in vivo CFSE labeling with lognormal modeling yielded insight into common intrinsic and extrinsic platelet and erythrocyte lifespan determinants and provides an improved methodology for use in this field of research.

Mutations in the human sulphate transporter gene, DTDST, have been implicated in several diseases. Analysis of affected patients has linked disease symptoms to faulty sulphate transporter activity. We have reproduced two of these mutations in SHST1, a homologous member of the family isolated from the tropical legume, Stylosanthes hamata. Both mutations significantly reduce sulphate transport activity of SHST1. These results indicate that conserved residues between distinct members of the family may share essential roles in structure or function. The results also suggest that putative helix 9 may be important for stability and/or trafficking of SHST1 to the plasma membrane.