Abstract 14458: Repeated Social Defeat Exaggerates Fibrin-rich Clot Formation in Fecl3 Induced Arterial Thrombosis Mice Model by Enhancing Nets Formation

Background and Objective: Depression is an independent risk factor of cardiovascular disease (CVD). We have recently shown that repeated social defeat (RSD) exaggerates atherosclerosis development by enhancing neutrophil extracellular traps (NETs) formation. Here, we investigated the impact of RSD on arterial thrombosis. Methods and Results: Eight-week-old male WT mice were exposed to RSD by housing with a larger CD-1 mouse in a shared home cage. They were subjected to vigorous physical contact daily for 10 consecutive days. Control mice were housed in the same gage without physical contact. After social interaction test to confirm depressive-like behaviors, defeated mice (19 of 31) and control mice (12 of 14) were underwent arterial injury at 10 wks of age. A filter paper saturated with 10% FeCl3 was applied on the adventitial surface of left carotid artery for 3 min and analyzed 3 hrs later. The volume of thrombi was comparable between the two groups. However, fibrinogen/fibrin-positive areas in immunofluorescent images significantly increased in defeated mice (48.8% vs. 27.8%, p < 0.01). The number of Ly-6G-positive cells in thrombi was markedly higher in defeated mice (878/mm2 vs. 144/mm2, p < 0.05). Further, Ly-6G-positive cells were co-localized with neutrophil elastase, Cit-H3, and CD42b-positive staining. Percentage of CD42b-positive area in thrombi and in vitro platelets aggregations induced by ADP or collagen were comparable between the two groups. Treatment with DNase I completely diminished the exaggerated fibrin-rich clot formation in defeated mice to an extent similar to that in control mice (22.3% vs. 25.7%, p = ns), although the number of Ly-6G-positive cells in thrombi was not affected. We therefore examined the vulnerability to NETs formation induced by thrombin-activated platelets. Flow cytometric analysis showed that in vitro NETs formation assessed by Cit-H3/MPO double-positive cells was significantly higher in defeated mice (20.7% vs 12.5%, p < 0.01). Conclusions: Our findings demonstrate that RSD enhances fibrin-rich clot formation after arterial injury by enhancing NETs formation via platelet-neutrophil interactions, suggesting that NETosis could be a new therapeutic target in depression-related CVD development.

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Repeated social defeat exaggerates fibrin-rich clot formation in FeCl3-induced arterial thrombosis mouse model by enhancing NETs formation via modulation of neutrophil functional properties

European Heart Journal ◽

10.1093/ehjci/ehaa946.3817 ◽

2020 ◽

Vol 41 (Supplement_2) ◽

Author(s):

T Sugimoto ◽

H Yamada ◽

H Kubota ◽

D Miyawaki ◽

M Saburi ◽

...

Keyword(s):

Functional Properties ◽

Arterial Thrombosis ◽

Social Defeat ◽

Arterial Injury ◽

Physical Contact ◽

Clot Formation ◽

Positive Staining ◽

Double Positive ◽

The Impact

Abstract Background and objective Depression is an independent risk factor of cardiovascular disease (CVD). We have recently shown that repeated social defeat (RSD) precipitates depressive-like behaviors in apoE−/− mice and exaggerates atherosclerosis development by enhancing neutrophil extracellular traps (NETs) formation. Here, we investigated the impact of RSD on arterial thrombosis. Methods and results Eight-week-old male WT mice were exposed to RSD by housing with a larger CD-1 mouse in a shared home cage. They were subjected to vigorous physical contact daily for 10 consecutive days. Control mice were housed in the same gage without physical contact. After social interaction test to confirm depressive-like behaviors, defeated mice (19 of 31) and control mice (12 of 14) were underwent arterial injury at 10 wks of age. A filter paper saturated with 10% FeCl3 was applied on the adventitial surface of left carotid artery for 3 min and analyzed 3 hrs later. The volume of thrombi was comparable between the two groups. However, fibrinogen/fibrin-positive areas in immunofluorescent images significantly increased in defeated mice (27.8% vs. 48.8%, p<0.01). The number of Ly-6G-positive cells in thrombi was markedly higher in defeated mice (144/mm2 vs. 878/mm2, p<0.05). Further, Ly-6G-positive cells were almost accumulated at the inner surface of injured artery, which were co-localized with neutrophil elastase, Cit-H3, and CD41-positive staining. Treatment with DNase I completely diminished the exaggerated fibrin-rich clot formation in defeated mice to an extent similar to that in control mice (25.7% vs. 22.3%, p = ns), without affecting the volume of thrombi and accumulation of Ly-6G-positive cells. Given that platelet aggregations induced by ADP or collagen were comparable between the two groups, neutrophil functional properties primarily contribute to the exaggerated fibrin-rich clot formation in defeated mice. We then examined neutrophil subset and vulnerability to NETs formation. At 3 hrs after FeCl3 application, the numbers of immature neutrophils (Ly6Glo/+CXCR2-) were comparable between the two groups in both bone marrow (BM) and peripheral blood (PB). In contrast, the number of PB mature neutrophils (Ly6G+CXCR2+) was markedly higher in defeated mice than control mice (580±68 /μl vs. 1265±114, p<0.01). We next examined in vitro NETs formation upon PMA in BM mature neutrophils by FACS and nucleic acid staining. The percentage of double-positive cells (Cit-H3, MPO) was significantly higher in defeated mice (7.5% vs. 10.2%, p<0.05), as well as SYTOX green-positive cells expelling DNA fibers (8.1% vs. 11.8%, p<0.05). Conclusions Our findings demonstrate for the first time that repeated social defeat enhances fibrin-rich clot formation after arterial injury by enhancing NETs formation via modulation of neutrophil functional properties, suggesting that NETosis could be a new therapeutic target in depression-related CVD development. Funding Acknowledgement Type of funding source: None

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P740Repeated social defeat exaggerates fibrin-rich clot formation in FeCl3-induced arterial thrombosis mice model by enhancing NETs formation

European Heart Journal ◽

10.1093/eurheartj/ehz747.0343 ◽

2019 ◽

Vol 40 (Supplement_1) ◽

Author(s):

T Sugimoto ◽

H Yamada ◽

H Kubota ◽

D Miyawaki ◽

S Motoyama ◽

...

Keyword(s):

Arterial Thrombosis ◽

Social Defeat ◽

Arterial Injury ◽

Physical Contact ◽

Clot Formation ◽

Positive Staining ◽

Mice Model ◽

Cross Sectional ◽

Atherosclerosis Development ◽

The Impact

Abstract Background and objective Depression is an independent risk factor of cardiovascular disease (CVD). We have recently shown that repeated social defeat (RSD) precipitates depressive-like behaviorsin apoE−/− mice and exaggerates atherosclerosis development by enhancing neutrophil extracellular traps (NETs) formation (BBRC 2018; 500:490). Here, we investigated the impact of RSD on arterial thrombosis. Methods and results Eight-week-old male WT mice were exposed to RSDby housing with a larger CD-1 mouse in a shared home cage. They were subjected to vigorous physical contact daily for 10 consecutive days. Control mice were housed in the same gage without physical contact. After social interaction testto confirm depressive-like behaviors, defeated mice (19 of 31) and control mice (12 of 14) were underwent arterial injury at 10 wks of age. A filter paper saturated with 10% FeCl3was applied on the adventitial surface of left carotid artery for 3 min and analyzed 3 hrs later. The volume of thrombi calculated by summing8–15 frozen cross-sectional images, each separated by 200 μm, was comparable between the 2 groups. However, fibrinogen/fibrin-positive areas in immunofluorescent images were significantly increased in defeated mice (27.8% vs. 48.8%, Control vs. Defeat, P<0.01).The numberof Ly-6G-positive cells in thrombi was markedly higher in defeated mice (144/mm2 vs. 878/mm2, Control vs. Defeat, P<0.05). Further, Ly-6G-positive cells were almost accumulated at the inner surface of injured artery, which were co-localized with neutrophil elastase, Cit-H3, and CD41-positive staining. Treatment with DNase Icompletely diminished the exaggerated fibrin-rich clot formation in defeated miceto a similar extent of control mice (25.7% vs. 22.3%, Control vs. Defeat, P= NS), while the volume of thrombi and number of Ly-6G-positive cells in thrombi were comparable between the 2 groups even afterDNase I treatment. Platelet aggregations induced by ADP or collagen were comparable between the 2 groups, suggesting that NETs formation primarily contributes to the exaggerated fibrin-rich clot formation in defeated mice. Conclusions Our findings demonstrate for the first time that repeated social defeat enhances fibrin-rich clot formation after arterial injury by enhancing NETs formation, suggesting that NETosis could be a new therapeutic target in depression-related CVD development.

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Repeated Social Defeat Exaggerates Fibrin-Rich Clot Formation by Enhancing Neutrophil Extracellular Trap Formation via Platelet–Neutrophil Interactions

Cells ◽

10.3390/cells10123344 ◽

2021 ◽

Vol 10 (12) ◽

pp. 3344

Author(s):

Takeshi Sugimoto ◽

Hiroyuki Yamada ◽

Naotoshi Wada ◽

Shinichiro Motoyama ◽

Makoto Saburi ◽

...

Keyword(s):

Flow Cytometric Analysis ◽

Social Defeat ◽

Arterial Injury ◽

Physical Contact ◽

Clot Formation ◽

Neutrophil Extracellular Trap ◽

Positive Staining ◽

Antibody Treatment ◽

Extracellular Trap ◽

The Impact

Depression is an independent risk factor for cardiovascular disease (CVD). We have previously shown that repeated social defeat (RSD) exaggerates atherosclerosis development by enhancing neutrophil extracellular trap (NET) formation. In this study, we investigated the impact of RSD on arterial thrombosis. Eight-week-old male wild-type mice (C57BL/6J) were exposed to RSD by housing with larger CD-1 mice in a shared home cage. They were subjected to vigorous physical contact daily for 10 consecutive days. After confirming depression-like behaviors, mice underwent FeCl3-induced carotid arterial injury and were analyzed after 3 h. Although the volume of thrombi was comparable between the two groups, fibrin(ogen)-positive areas were significantly increased in defeated mice, in which Ly-6G-positive cells were appreciably co-localized with Cit-H3-positive staining. Treatment with DNase I completely diminished exaggerated fibrin-rich clot formation in defeated mice. Flow cytometric analysis showed that neutrophil CD11b expression before FeCl3 application was significantly higher in defeated mice than in control mice. In vitro NET formation induced by activated platelets was significantly augmented in defeated mice, which was substantially inhibited by anti-CD11b antibody treatment. Our findings demonstrate that RSD enhances fibrin-rich clot formation after arterial injury by enhancing NET formation, suggesting that NET can be a new therapeutic target in depression-related CVD.

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Disruption of GAS6-MER Pathway Leads to Defects in Physiological Clearance of Expelled Nuclei from Erythroblasts by Bone Marrow Macrophages

Blood ◽

10.1182/blood.v112.11.499.499 ◽

2008 ◽

Vol 112 (11) ◽

pp. 499-499

Author(s):

Linda Kadi ◽

Laurent Burnier ◽

Rocco Sugamele ◽

Peter Carmeliet ◽

Greg Lemke ◽

...

Keyword(s):

Bone Marrow ◽

Peripheral Blood ◽

Fetal Liver ◽

Specific Gene ◽

Positive Staining ◽

Morphological Examination ◽

Double Positive ◽

Blood Island

Abstract Late in erythropoiesis, nuclei are expelled from erythroblasts and 2×1011 anucleated new red blood cells are daily delivered in the peripheral blood. Expelled nuclei expose phosphatidylserine (PS) on their surface, which is used as an “eat me” signal for their engulfment by macrophages located in the blood island. The two PS opsonins, milk-fatglobule EGF8 (MFG-E8) and Growth arrest-specific gene 6 product (GAS6) together with their respective receptors αvβ5/αvβ3 and TAM (TYRO3, AXL and MER), are involved in the phagocytosis of apoptotic cells, but their role in the phagocytosis of expelled nuclei from erythroblasts is not determined. Because fetal liver and bone marrow macrophages do not express MFG-E8, the GAS6-MER pathway might constitute a crucial pathway for the engulfment of nuclei expelled from erythroblasts. To test this hypothesis, we isolated nuclei from late-stage erythroblasts from spleens of phlebotomized mice, and studied nuclei internalization capacity of bone marrow derived macrophages (BMDM) from mice deficient either in GAS6 (GAS6−/−), AXL (AXL−/−) or TYRO3 (TYRO3−/−), or lacking MER kinase domain (MERkd). Released nuclei were identified by flow cytometry according to their size and their double positive staining for the erythroid lineage marker Ter119 and Annexin V for PS. Purity of the preparation was checked by morphological examination of cytospin preparations. In vitro phagocytosis assays show that GAS6−/− BMDM cleared 30% less nuclei than wild-type (WT) BMDM. We observed a slight decrease of internalization capacity for AXL−/− BMDM, whereas TYRO3−/− BMDM engulfed the nuclei as efficiently as WT BMDM. In contrast, MER deficiency nearly abolished nuclei phagocytosis. AXL−/−/TYRO3−/− and AXL−/−/MERkd BMDM were tested and did not show any cumulative effects when compared to WT and single knockouts. We also investigated the signalling pathway downstream of MER in BMDM. In particular, we assessed the expression of the activated form of Rac1, which is crucial for the cytoskeletal reorganization in phagocytosis. Activation of Rac1 after the initiation of the phagocytosis was delayed for 45 minutes in MERkd as compared to WT BMDM. In vivo, we found an accumulation of nuclei in MERkd mice 4 days post phlebotomy, when erythropoiesis is increased in response to anemia. Nuclei circulated in the blood of MERkd mice at a level of 0.08 ± 0.042 G/L and were identified on peripheral blood smears of these mice whereas they were undetectable in the blood of WT mice. We demonstrated an increase of a double labelled Ter119/AnnexinV population corresponding to nuclei in BM (2-fold) and spleen (1.5-fold) of MERkd mice as compared to WT mice. The augmentation of this double labelled population in the MERkd mice translated the phenotype of splenomegaly of these mice. Hematocrit and reticulocyte levels were comparable between WT and MERkd as previously reported (JCI118:583–596, 2008). Thus, MER was critical for in vitro phagocytosis of nuclei from erythroblasts whereas the role of AXL and TYRO3 appeared to be negligible. GAS6 binding to nuclei exposing PS on their surface might form a bridge between PS and MER receptor on BMDM, allowing nuclei clearance. In vivo, the absence of MER caused an accumulation of nuclei in BM and spleen and their appearance in circulating blood due to their inefficient elimination during erythropoietic response to anemia. In conclusion, we postulate that GAS6 and its receptor MER were involved in late erythropoiesis when nuclei are expelled from the erythroblasts and engulfed by BMDM in the blood island, through Rac1 activation.

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HIF 1 Alpha: A Suitable Target for Multiple Myeloma

Blood ◽

10.1182/blood.v118.21.2901.2901 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2901-2901 ◽

Cited By ~ 2

Author(s):

Giulia Perrone ◽

Enrica Borsi ◽

Carolina Terragna ◽

Sandra Durante ◽

Marina Martello ◽

...

Keyword(s):

Multiple Myeloma ◽

Solid Tumors ◽

Cell Lines ◽

Conflicts Of Interest ◽

Critical Role ◽

Flow Cytometric Analysis ◽

Parp Cleavage ◽

Survival Assay ◽

The Impact

Abstract Abstract 2901 Hypoxia-inducible factor-1 alpha (HIF1 α) is a transcription factor that plays a critical role in survival and angiogenesis. In solid tumors, elevated expression of HIF-1 α, in response to hypoxia or activation of growth factor pathways, is associated with tumor proliferation, metastasis, and drug resistance and correlated with poor prognosis. In contrast to solid tumors, the role of HIF1 α in hematological malignancies is not completely known. In particular in multiple myeloma (MM) HIF1 α has been suggested to be constitutively expressed and HIF1 α knockdown cell lines have shown higher sensitivity to standard chemotherapy, suggesting a role in the pathophysiology of MM. In the present study, we explored the effect of EZN2968, an antisense oligonucleotide against HIF1 α, as a molecular target in MM. We showed, using real time PCR, and Western blotting analysis, that the expression of HIF1 α in several MM cell lines (MM1S, U266, OPM2, RPMI8226) is detectable under conditions of normoxia or hypoxia and is increased in the presence of growth stimuli (IL-6 and stroma cells). The immunofluorescence analysis suggested that the protein is ubiquitously present in both the cytosol and nucleus. To evaluate the specificity of the oligonucleotide for the target, we tested whether EZN2968 was able to induce a selective and stable down-modulation of HIF1 α mRNA and protein expression. We confirmed that the downmodulation was lasting in a long term culture experiment (up to 96 hours) either in normoxic or hypoxic conditions, and did not affect the expression of other family members of hypoxia inducible transcription factors (HIF2 α). We next explored the effects of EZN-2968 on the growth and survival of MM cells. Using an MTT colorimetric survival assay, we showed that, after 48 hours of culture in the presence of the HIF1 α inhibitor (20μM), MM1.S and U266 cell lines exhibited a reduction of 30% of viability compared to untreated cells, while RPMI8226 of 15%. AnnexinV/PI staining revealed that EZN-2968 (20μM) increased, after 48 hours of culture, the percentage of PI+ cells compared to the control, suggesting a disruption on membrane permeability. In addition, immunoblotting revealed PARP cleavage as early as 24 hours. Evaluation of cell cycle profile, by flow cytometric analysis, showed an increase of the sub-G0/G1 population from 3.5% to 30 %, after 48 hour of exposure to EZN-2968. To evaluate if the impact on cell viability was irreversible, we performed a cell death commitment assays. MM1S cells were incubated with EZN2968 (20 μM) for 24 to 96 hours, following incubation in drug-free medium for additional 24 to 72 hours. MTT colorimetric survival assay showed that EZN-2968 treatment for as early as 24h resulted in commitment to death in all cell lines tested. To evaluate the effect of microenvironment, MM cells treated with EZN2968 were exposed to IL-6 and stroma cells for additional 24 hours. EZN2968 overcame the proliferative effect induced by cytokines. We next evaluated the impact of EZN-2968 on purified CD138+ cells from MM patients with advanced MM. MTT colorimetric survival assay showed a reduction of cells viability of 30% after 24 hours of incubation. In addition we observed a low sensitivity of PBMCs and CD34+cells, derived from healthy donors, to EZN-2968 treatment suggesting that EZN-2968 has selective in vitro activity against MM cells. Evaluation of gene expression profiling modulation induced by EZN 2968 is on going. In summary, our results suggests that the inhibition of HIF1 α activity can be used as an attractive therapeutic target for MM patients and provide the rationale for clinical evaluation of HIF inhibitors. Disclosures: No relevant conflicts of interest to declare.

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Glycyrrhetinic acid-induced apoptosis in thymocytes: impact of 11β-hydroxysteroid dehydrogenase inhibition

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.1999.277.4.e624 ◽

1999 ◽

Vol 277 (4) ◽

pp. E624-E630 ◽

Cited By ~ 4

Author(s):

Hiroshi Horigome ◽

Atsushi Horigome ◽

Masato Homma ◽

Toshihiko Hirano ◽

Kitaro Oka

Keyword(s):

Dna Cleavage ◽

Flow Cytometric Analysis ◽

Intraperitoneal Administration ◽

Hydroxysteroid Dehydrogenase ◽

Glycyrrhetinic Acid ◽

Immunocytochemical Staining ◽

Double Positive ◽

Corticosterone Concentration

It has been proposed that glycyrrhetinic acid (GA) enhances endogenous glucocorticoid (GC) action by suppressing the metabolism of the steroid. We show here that marked involution of the thymus occurred within 24 h of a single intraperitoneal administration of GA in mice. Thymocytes from mice treated with GA exhibited DNA cleavage and mitochondrial transmembrane potential disruption, as demonstrated with agarose gel electrophoresis and flow cytometric analysis. Immunocytochemical staining revealed that CD4+CD8+double positive cells markedly decreased after GA treatment. In contrast to GA in vivo, GA in vitro did not induce apoptosis of cultured thymocytes. These findings suggest that the apoptosis-inducing effect of GA on thymocytes is due to its indirect action. Because GA has been known to inhibit 11β-hydroxysteroid dehydrogenase (11β-HSD), we measured the enzyme activity in major organs and endogenous corticosterone concentration after GA treatment. The results showed a significant decrease of 11β-HSD activity ( P < 0.0001) and an increase in serum corticosterone concentration ( P< 0.005). We concluded that the inhibition of hepatic 11β-HSD activity by GA has a serious effect on GC metabolism, which results in a significant elevation of systemic GC levels. Apoptosis of thymocytes occurred as a consequence of the elevation in the level of endogenous corticosterone.

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Periadventitial atRA citrate-based polyester membranes reduce neointimal hyperplasia and restenosis after carotid injury in rats

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00914.2013 ◽

2014 ◽

Vol 307 (10) ◽

pp. H1419-H1429 ◽

Cited By ~ 11

Author(s):

Elaine K. Gregory ◽

Antonio R. Webb ◽

Janet M. Vercammen ◽

Megan E. Flynn ◽

Guillermo A. Ameer ◽

...

Keyword(s):

High Performance ◽

Neointimal Hyperplasia ◽

Arterial Injury ◽

Macrophage Infiltration ◽

Appreciable Effect ◽

Balloon Injury ◽

Neointimal Formation ◽

The Impact

Oral all-trans retinoic acid (atRA) has been shown to reduce the formation of neointimal hyperplasia; however, the dose required was 30 times the chemotherapeutic dose, which already has reported side effects. As neointimal formation is a localized process, new approaches to localized delivery are required. This study assessed whether atRA within a citrate-based polyester, poly(1,8 octanediolcitrate) (POC), perivascular membrane would prevent neointimal hyperplasia following arterial injury. atRA-POC membranes were prepared and characterized for atRA release via high-performance liquid chromatography with mass spectrometry detection. Rat adventitial fibroblasts (AF) and vascular smooth muscle cells (VSMC) were exposed to various concentrations of atRA; proliferation, apoptosis, and necrosis were assessed in vitro. The rat carotid artery balloon injury model was used to evaluate the impact of the atRA-POC membranes on neointimal formation, cell proliferation, apoptosis, macrophage infiltration, and vascular cell adhesion molecule 1 (VCAM-1) expression in vivo. atRA-POC membranes released 12 μg of atRA over 2 wk, with 92% of the release occurring in the first week. At 24 h, atRA (200 μmol/l) inhibited [3H]-thymidine incorporation into AF and VSMC by 78% and 72%, respectively (* P = 0.001), with negligible apoptosis or necrosis. Histomorphometry analysis showed that atRA-POC membranes inhibited neointimal formation after balloon injury, with a 56%, 57%, and 50% decrease in the intimal area, intima-to-media area ratio, and percent stenosis, respectively ( P = 0.001). atRA-POC membranes had no appreciable effect on apoptosis or proliferation at 2 wk. Regarding biocompatibility, we found a 76% decrease in macrophage infiltration in the intima layer ( P < 0.003) in animals treated with atRA-POC membranes, with a coinciding 53% reduction in VCAM-1 staining ( P < 0.001). In conclusion, perivascular delivery of atRA inhibited neointimal formation and restenosis. These data suggest that atRA-POC membranes may be suitable as localized therapy to inhibit neointimal hyperplasia following open cardiovascular procedures.

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In vitro factor XIII supplementation increases clot firmness in Rotation Thromboelastometry (ROTEM®)

Thrombosis and Haemostasis ◽

10.1160/th09-12-0858 ◽

2010 ◽

Vol 104 (08) ◽

pp. 385-391 ◽

Cited By ~ 73

Author(s):

Lars Asmis ◽

Burkhardt Seifert ◽

Donat Spahn ◽

Oliver Theusinger ◽

Werner Baulig

Keyword(s):

Factor Xiii ◽

Clot Formation ◽

Intensive Care Patients ◽

Essential Parameter ◽

Α Angle ◽

The Impact ◽

Clot Formation Time ◽

Clot Stability ◽

Maximum Clot Firmness

SummaryFactor XIII (F XIII) is an essential parameter for final clot stability. The purpose of this study was to determine the impact of the addition of factor (F)XIII on clot stability as assessed by Rotation Thromboelastometry (ROTEM®). In 90 intensive care patients ROTEM® measurements were performed after in vitro addition of F XIII 0.32 IU, 0.63 IU, 1.25 IU and compared to diluent controls (DC; aqua injectabile) resulting in approximate F XIII concentrations of 150, 300 and 600%. Baseline measurements without any additions were also performed. The following ROTEM® parameters were measured in FIBTEM and EXTEM tests: clotting time (CT), clot formation time (CFT), maximum clot firmness (MCF), maximum lysis (ML), maximum clot elasticity (MCE) and α-angle (αA). Additionally, laboratory values for FXIII, fibrinogen (FBG), platelets and haematocrit were contemporaneously determined. In the perioperative patient population mean FBG concentration was elevated at 5.2 g/l and mean FXIII concentration was low at 62%. The addition of FXIII led to a FBG concentration-dependent increase in MCF both in FIBTEM and EXTEM. Mean increases in MCF (FXIII vs. DC) of approximately 7 mm and 6 mm were observed in FIBTEM and EXTEM, respectively. F XIII addition also led to decreased CFT, increased αA, and reduced ML in FIBTEM and EXTEM. In vitro supplementation of FXIII to supraphysiologic levels increases maximum clot firmness, accelerates clot formation and increases clot stability in EXTEM and FIBTEM as assayed by ROTEM® in perioperative patients with high fibrinogen and low FXIII levels.

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Impact of fibrinogen carbamylation on fibrin clot formation and stability

Thrombosis and Haemostasis ◽

10.1160/th16-09-0704 ◽

2017 ◽

Vol 117 (05) ◽

pp. 899-910 ◽

Cited By ~ 18

Author(s):

Stéphane Jaisson ◽

Philippe Gillery ◽

Carsten Scavenius ◽

Endy Spriet ◽

Anne Nyhaug ◽

...

Keyword(s):

Kidney Disease ◽

Mechanical Stability ◽

Inflammatory Diseases ◽

Plasma Concentrations ◽

Fibrin Clot ◽

Clot Formation ◽

Cross Linking ◽

Post Translational Modification ◽

The Impact

SummaryCarbamylation is a non-enzymatic post-translational modification induced upon exposure of free amino groups to urea-derived cyanate leading to irreversible changes of protein charge, structure and function. Levels of carbamylated proteins increase significantly in chronic kidney disease and carbamylated albumin is considered as an important biomarker indicating mortality risk. High plasma concentrations and long half-life make fibrinogen a prime target for carbamylation. As aggregation and cross-linking of fibrin monomers rely on lysine residues, it is likely that carbamylation impacts fibrinogen processing. In this study we investigated carbamylation levels of fibrinogen from kidney disease patients as well as the impact of carbamylation on fibrinogen cleavage by thrombin, fibrin polymerisation and cross-linking in vitro. In conjunction, all these factors determine clot structure and stability and thus control biochemical and mechanical properties. LC-MS/MS analyses revealed significantly higher homocitrulline levels in patient fibrinogen than in fibrinogen isolated from control plasma. In our in vitro studies we found that although carbamylation does not affect thrombin cleavage per se, it alters fibrin polymerisation kinetics and impairs cross-linking and clot degradation. In addition, carbamylated fibrin clots had reduced fiber size and porosity associated with decreased mechanical stability. Using mass spectroscopy, we discovered that N-terminally carbamylated fibrinopeptide A was generated in this process and acted as a strong neutrophil chemoattractant potentially mediating recruitment of inflammatory cells to sites of fibrin(ogen) turnover. Taken together, carbamylation of fibrinogen seems to play a role in aberrant fibrin clot formation and might be involved in haemostatic disorders associated with chronic inflammatory diseases.

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Leukemia Stem/Progenitor Cells From AML Patients Treated With The Multi-Kinase Inhibitor TG02 Demonstrate Increased Proliferation and Are Sensitized To Chemotherapeutic Agents

Blood ◽

10.1182/blood.v122.21.3892.3892 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3892-3892 ◽

Cited By ~ 2

Author(s):

Monica L. Guzman ◽

Wen Xie ◽

Jeanne P. De Leon ◽

Francis Burrows ◽

Eric J. Feldman ◽

...

Keyword(s):

Progenitor Cells ◽

Peripheral Blood ◽

Colony Formation ◽

Kinase Inhibitor ◽

Chemotherapeutic Agents ◽

Positive Staining ◽

Annual Meeting Abstract ◽

The Impact

Abstract TG02 is a multi-kinase inhibitor that targets cyclin-dependent kinases (CDKs), ERK5, JAK2, and Flt3. In vitro studies of TG02 have shown robust induction of apoptosis in both acute myeloid leukemia (AML) cell lines and primary cells (Goh et al, 2011). Leukemia stem cells (LSCs) comprise a largely quiescent, highly chemotherapy-resistant cell population and are believed to initiate and maintain AML, as well as contribute to its poor prognosis. Thus, we sought to investigate the impact of TG02 on LSCs collected from patients with relapsed/refractory AML enrolled in a phase I dose escalation trial. Patients ≥ 18 years with advanced hematological malignancies or newly diagnosed AML pts ≥ 65 years unfit for intensive therapy were enrolled onto daily (A) and intermittent (B, 5 days on 2 days off X 2 weeks) schedules in 28-day cycles. Pts had acceptable organ function and ECOG PS 0-2. Dose levels were 10 - 70 mg on arm A and 30 - 150 mg on arm B. We evaluated immunophenotypically defined leukemia stem and progenitor cells (LSPCs) by flow cytometry, cell cycle status and colony forming assays. A total of 16 patients were evaluated with treatment doses ranging from 10-150 mg of TG02. Clinically, treatment with TG02 did not have an effect in AML tumor burden, and most patients at our center only received one cycle of treatment (Roboz et al ASCO 2012 Annual Meeting Abstract #6557, J Clin. Oncol. 30, 2012). However, we found that 8 patient samples showed increased LSPCs in both the bone marrow and peripheral blood. Interestingly, we observed an increase in LSPC cell proliferation, as determined by Ki-67 positive staining. AML colony forming assays also showed increased colony formation (n=5) after one cycle of treatment, which suggests an increase in the frequency of LSPCs. The increase in colony formation in peripheral blood samples suggests mobilization of LSPCs from the marrow into the circulation. Thus, we hypothesized that exposure to TG02 in vivo may result in sensitization to other chemotherapeutic agents, such as Ara-C. We evaluated the effects of Ara-C and other chemotherapeutics, such as vincristine, in primary AML cells obtained from patients before and after treatment with TG02. We found that in vivo exposure to TG02 resulted in significantly increased sensitivity to Ara-C in vitro in 3 out of 4 samples tested Together, our data suggest that TG02 induces an effect in LSCs resulting in increased proliferation and, thus, sensitization to other chemotherapeutic drugs, such as Ara-C. Importantly, although no patients at our center receiving single agent TG02 met the criteria for an objective response, by performing correlative studies in association with the clinical trial, we found the TG02 has a marked effect in AML LSCs that could potentially be exploited by combining it with other agents. Disclosures: Burrows: Tragara Pharmaceuticals: Employment, Equity Ownership. Feldman:Tragara Pharmaceuticals: Consultancy.

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