scholarly journals Selatogrel, the reversible and specific P2Y12 receptor antagonist, does not interfere with haemostasis

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
M.A Riederer ◽  
L Crescence ◽  
E Caroff ◽  
F Hubler ◽  
L Panicot-Dubois ◽  
...  
Keyword(s):  
Blood Loss ◽  
P2y12 Receptor ◽  
Funding Source ◽  
Wild Type ◽  
Antithrombotic Effect ◽  
Pronounced Increase ◽  
Coronary Syndrome ◽  
Dosing Regimens ◽  
The Stability ◽  
Deficient Mice

Abstract Background Combination of a P2Y12 receptor (P2Y12R) antagonist (clopidogrel, prasugrel, ticagrelor) with aspirin is the recommended standard of care for patients with acute coronary syndrome. Selatogrel is a reversible and potent antagonist of P2Y12R. Interestingly, in an experimental thrombosis model in rat, at equivalent antithrombotic effect, blood loss was lower in the presence of selatogrel, compared with clopidogrel or ticagrelor. Purpose To characterise the lower risk of bleeding previously observed with selatogrel Methods Mechanistic studies were performed to profile laser-induced thrombosis in wild-type and P2Y12 deficient mice with real-time intravital microscopy. Ticagrelor and clopidogrel were used as selatogrel comparators. Results Selatogrel, ticagrelor and clopidogrel dose-dependently inhibited laser-induced platelet thrombus formation. At maximal antithrombotic effect, only small mural platelets aggregates, corresponding to the haemostatic seals, were present. The phenotype of these haemostatic seals depended on the P2Y12R antagonist used. In the presence of clopidogrel or ticagrelor, the stability of haemostatic seals was reduced. In contrast, in the presence of selatogrel, the apparent stability was not disturbed. Moreover, equivalent antithrombotic dosing regimens of ticagrelor and clopidogrel interfered with laser-induced calcium mobilisation in the endothelium, restricted subsequent neutrophil adhesion and thus reduced fibrin-mediated stabilisation of the haemostatic seals in wild type mice. The effects of ticagrelor were also observed in P2Y12R-deficient mice, indicating that the effects are P2Y12R independent and off-target. In contrast, an equivalent antithrombotic dosing regimen of selatogrel did not interfere with the process of haemostasis in wild-type or P2Y12R-deficient mice. The degree of interference with the stability of the haemostatic seals correlated with the blood loss profile. The dosing regimens of clopidogrel and ticagrelor, corresponding to the equivalent antithrombotic effects, induced a more pronounced increase in blood loss than that observed with selatogrel. Conclusion Our data offer a novel mechanistic explanation for the differences in bleeding risk of clopidogrel, ticagrelor and selatogrel. Clopidogrel and ticagrelor were found to interfere with haemostasis due to off-target activities. In contrast, selatogrel did not interfere with haemostasis in wild-type and P2Y12-deficient mice, inferring that the process of haemostasis, as defined by formation of haemostatic seals, is independent of P2Y12R. In addition, our data emphasize that the absence of interference with haemostasis is paramount to preserve the advantage of P2Y12R antagonism. Funding Acknowledgement Type of funding source: Private company. Main funding source(s): Idorsia Pharmaceuticals Ltd. Allschwil, Switzerland

scholarly journals Bone marrow-derived NGFR+ cells regulate arterial remodeling and those poor mobilizations in peripheral blood in acute coronary syndrome predicts plaque progression at the non-targeted lesion

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
S Takashima ◽  
S Usui ◽  
S Matsuura ◽  
C Goten ◽  
O Inoue ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Coronary Syndrome ◽  
Peripheral Blood ◽  
Funding Source ◽  
Arterial Remodeling ◽  
Plaque Progression ◽  
Wild Type ◽  
Plaque Volume ◽  
Coronary Syndrome

Abstract Background In our previous 5-year cohort study, we demonstrated that low gene expression of nerve growth factor receptor (NGFR) in peripheral leucocytes in acute coronary syndrome (ACS) predicted repetitive coronary interventions at the de novo lesions. An NGFR-positive cell has been demonstrated to reside in bone marrow (BM) stromal fraction and to be increased in peripheral blood mononuclear cell (MNCs) fraction in patients with ischemic heart disease. Purpose To investigate whether the BM-NGFR+ cell is associated with arterial remodeling and the relationship between the levels of peripheral NGFR+ cells after ACS and coronary plaque progression in an experimental and prospective clinical study. Methods and results In an experimental study, 8-week-old C57B6/J wild type male mice were subjected to irradiation with 9.6 Gy and transplantation with BM (BMT) isolated from GFP-transgenic NGFR wild type (WT) or knock-out (KO) mice at day 1. Four weeks after BMT, the right carotid artery was ligated for 4 weeks. Induced neointimal area was increased (p<0.05), where cells under apoptosis were decreased (p<0.05) in NGFR-KO-BMT group compared to WT-BMT group (n=4). NGFR+ cells were not detected in wild type sham-operated artery, whereas in the ligated artery in WT-BMT group NGFR+ cells assembled in the developed neointima and exclusively presented double positive with GFP, but absent in NGFR-KO-BMT group (p<0.05, n=4). In a clinical study, thirty patients with ACS who underwent primary percutaneous coronary intervention (PCI) were enrolled. The peripheral blood sample was collected on days 0, 3 and 7, and 9 months follow-up and the number of NGFR+MNCs were measured by flowcytometric analysis. The plaque volume at non-targeted coronary lesion (non-TL:>5 mm proximal or distal to the implanted stents) were quantitatively analysed using gray-scale intravascular ultrasound (IVUS) and Q-IVUS™ software at the acute phase and 9 months follow-up. The number of NGFR+MNCs in peripheral blood was 1.5-fold increased at day 3 (0.064±0.056%) compared to day 0 (0.042±0.030%) (p<0.05). The change in normalized total plaque volume (TAVN) at non-TL at 9 months was negatively correlated with the number of NGFR+MNCs at day 0 (r=−0.51), day 3 (r=−0.51) and 9 months (r=−0.59) after ACS (p<0.05). Multiple regression analysis showed that NGFR+MNCs at day 0 (β=−0.48, p=0.01) and CRP (β=−0.53, P<0.01) are independent factors associating with TAVN change at non-TL at 9 months, regardless of LDL-cholesterol control level. ROC analysis revealed that NGFR+MNCs <0.049 at day 0 predicted the increase of TAVN with AUC 0.78; sensitivity 0.82 and specificity 0.67. Conclusions Bone marrow-derived peripheral NGFR+ cells negatively regulate arterial remodeling through appropriate apoptosis of neointimal cells and the peripheral level of NGFR+ cells in ACS predicts plaque progression at the non-targeted lesion. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): KAKENHI

Monocyte-Derived Microparticles Improve Hemostasis in Factor VIIIDeficient Mice: Exploring the Role of PSGL-1/P-Selectin.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3386-3386
Author(s):  
Peter L. Gross ◽  
Nima Vaezzadeh ◽  
Lori Ivicic ◽  
Ran Ni ◽  
Bruno Esposito ◽  
...  
Keyword(s):  
Blood Loss ◽  
High Speed ◽  
Calcium Ionophore ◽  
Ionophore A23187 ◽  
Wild Type ◽  
Spontaneous Bleeding ◽  
Platelet Accumulation ◽  
Deficient Mice ◽  
Tail Clip

Abstract Introduction: Microparticles derived from leukocytes contribute to fibrin formation at thrombi in vivo and factor VIII-deficient (FVIII) mice treated with an agent that elevates their microparticles have decreased bleeding. A novel therapy for hemophilia patients with inhibitors is needed. We evaluated whether microparticles generated in vitro could improve hemostasis in FVIII mice. Methods: Mouse CD11b positive monocytes, isolated by MACS, or cultured monocytic cells (WEHI274.1) were treated with the calcium ionophore A23187. The resulting microparticles (isolated by differential centrifugation, and defined as CD18 positive events less than 1 μm diameter) or PIPES buffer were infused intravenously into FVIII-deficient mice (B6.129S4-F8tm1Kaz) or control wild type B6.129 mice prior to evaluation. The amount of platelets in laser-generated thrombi in cremaster muscle arterioles was evaluated using high-speed intravital fluorescence microscopy. The amount of hemoglobin shed from a 2 mm tail tip amputation measured blood loss. Results: Infusion of MPs at doses above 1000/g resulted in the death of wild type mice; FVIII-deficient mice tolerated MPs at doses up to 4000/g. Blood loss after tail clip in FVIII-deficient mice was 6-fold higher than blood loss from wild type mice. Blood loss after tail clip in FVIII-deficient mice was reduced to normal after the infusion of MPs at concentrations as low as 400/g. MPs, at 400/g, from CD11b positive cells isolated from wild type, FVIII-deficient mice or PSGL-1-deficient mice all similarly reduced blood loss after tail clip in FVIII-deficient mice. The biological half life of MP effect on tail-bleeding was 3 hours. Platelet accumulation in cremaster arteriolar thrombi was impaired in FVIII-deficient mice. Infusion of MPs at a concentration of 1000/g normalized platelet accumulation, but infusion of MPs at a lower concentration (400/g) did not. Conclusion: Abnormal hemostasis in FVIII-deficient mice can be temporarily reversed by the infusion of in vitro generated monocyte-derived MPs, including MPs derived from monocytes from FVIII-deficient or PSGL-1-deficient mice. The dose whereby MPs normalize FVIII-deficient mice is different between the hemostasis and thrombosis models. To explore whether P-selectin at injuries is required for the effect of MPs, we have generated by cross-breeding FVIII/P-selectin double deficient mice. These mice are born at expected mendelian frequency. Two of 20 male FVIII/P-selectin double deficient mice had spontaneous bleeding at 8 weeks of age, one in the thigh and one from the ear. FVIII/P-selectin double deficient mice also have prolonged tail bleeding times, which will serve as a model for testing the P-selectin targeting of microparticles.

scholarly journals Is “one size fits all” anti-aggregation really effective? Variability in the response to P2Y12 receptor inhibitors in obese patients

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
J Playan Escribano ◽  
D Vivas Balcones ◽  
L.M Lugo Gavidia ◽  
J.C Gomez Polo ◽  
A.L Marcano Fernandez ◽  
...  
Keyword(s):  
Platelet Function ◽  
Ex Vivo ◽  
Statistical Significance ◽  
P2y12 Receptor ◽  
Funding Source ◽  
Obese Patients ◽  
Platelet Function Tests ◽  
P2y12 Inhibitors ◽  
Coronary Syndrome ◽  
Function Tests

Abstract Background Different “ex vivo” studies have shown both a greater platelet activation and higher rates of resistance to clopidogrel in obese patients. Although there is less evidence, less prasugrel activity has also been observed in these patients. Our aim was to study the variability of the response to clopidogrel, ticagrelor and prasugrel in obese patients, defined as a body mass index ≥30. Methods Prospective, multicenter, observational, pharmacodynamic study, conducted in a Spanish population of patients with an acute coronary syndrome (ACS) treated with percutaneous coronary intervention (PCI) and double anti-aggregation with acetylsalicylic acid and a P2Y12 receptor inhibitor. Platelet function tests were performed the morning after the ICP and 30 days after it, including: 1) VerifyNow P2Y12 assay; 2) multiple electrode aggreometry (Multiplate); and 3) VASP analysis. Results Of the total patients included (988), 300 were obese (30.3%). The obese group was younger (62.8±12 years vs 64.9±12), had a higher incidence of arterial hypertension (76.3% vs. 56.7%), diabetes mellitus (35% vs. 27.5%); and lower incidence of chronic kidney disease (7.7% vs. 17%). There were no differences in the acute phase (day 1 after PCI) in the pharmacodynamic response to any of the P2Y12 inhibitors used. After 30 days, greater platelet aggregation (decreased response) was documented in obese patients treated with prasugrel according to VASP tests (PRI in non-obese 23.9±13% vs. 30.4±14.7% in obese, p 0.035) and MEA (area under the aggregation units curve in non-obese 251.7±104.1 vs 320±166.7 in obese, p 0.007) and a numerical trend with VerifyNow. A trend in the same direction was also observed in patients treated with clopidogrel that did not reach statistical significance with all the platelet function tests used. No differences were observed in the ticagrelor group. Conclusion Obese patients with an ACS treated with PCI have a worse response to thienopyridines than non-obese patients in the maintenance phase of antiaggregant treatment, while the response to ticagrelor is not affected by obesity. Completing the clinical follow-up proposed by the registry is necessary to know if these differences have an implication in cardiovascular events. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Fondo de Investigaciones Sanitarias (FIS)

Antithrombotic activity of dermatan sulfate in heparin cofactor II-deficient mice

Blood ◽  
2004 ◽  
Vol 104 (13) ◽  
pp. 3965-3970 ◽  
Author(s):  
Cristina P. Vicente ◽  
Li He ◽  
Mauro S. G. Pavão ◽  
Douglas M. Tollefsen
Keyword(s):  
Carotid Artery ◽  
Dermatan Sulfate ◽  
Wild Type ◽  
Antithrombotic Effect ◽  
Porcine Skin ◽  
Heparin Cofactor Ii ◽  
Thrombotic Occlusion ◽  
Antithrombotic Activity ◽  
Occlusion Time ◽  
Deficient Mice

Abstract Heparin cofactor II (HCII) is a plasma protein that inhibits thrombin rapidly in the presence of dermatan sulfate or heparin. We previously reported that the time to thrombotic occlusion of the carotid artery after photochemical injury was shorter in HCII-deficient mice than in wild-type control animals. In this paper, we describe the antithrombotic activity of dermatan sulfate in wild-type and HCII-deficient mice. Intravenous administration of porcine skin dermatan sulfate induced a dose-dependent prolongation of the carotid artery occlusion time in HCII+/+ mice that was not observed in HCII-/- animals. Pharmacokinetic studies suggested that porcine skin dermatan sulfate expresses antithrombotic activity after being transferred from the plasma to sites in the vessel wall. Using invertebrate dermatan sulfate preparations, we showed that N-acetylgalactosamine-4-O-sulfate residues are required for the HCII-dependent antithrombotic effect. Furthermore, the invertebrate dermatan sulfates, which have higher charge densities than mammalian dermatan sulfate, slightly prolonged the thrombotic occlusion time of HCII-/- mice. These results indicate that HCII mediates the antithrombotic effect of porcine skin dermatan sulfate after injury to the carotid arterial endothelium in mice, whereas more highly charged dermatan sulfates possess weak antithrombotic activity independent of HCII. (Blood. 2004;104:3965-3970)

scholarly journals Human Factor VIII Expression and Normalization of Bleeding Following AAV Gene Therapy in a Double Knockout Mouse Model of Hemophilia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3239-3239
Author(s):  
Stuart Bunting ◽  
Lening Zhang ◽  
Lin Xie ◽  
Sherry Bullens ◽  
Rajeev Mahimkar ◽  
...  
Keyword(s):  
Blood Loss ◽  
Factor Viii ◽  
Knockout Mouse ◽  
Bleeding Time ◽  
Wild Type ◽  
Double Knockout ◽  
Post Dose ◽  
Knockout Mouse Model ◽  
Protein Levels ◽  
Deficient Mice

Abstract Hemophilia A is an X-linked bleeding disorder caused by mutations in the gene encoding the Factor VIII coagulation protein (FVIII). Bleeding episodes in patients are reduced by prophylactic therapy or treated acutely using recombinant or plasma derived FVIII. Recently, Nathwani et al demonstrated in preclinical and clinical studies sustained expression of coagulation factor IX using AAV8 technology to deliver the human gene to the liver, driven by a liver specific promoter. The same group demonstrated FVIII expression in mice and primates using a modified B-domain truncated form of FVIII delivered in an AAV8 capsid. We have made an AAV5 construct containing a B-domain deleted FVIII gene (AAV5-SQ) with a liver specific promoter and evaluated it in a double knockout mouse model of hemophilia. The double knockout mice (DKO) were created by crossing factor VIII deficient mice with RAG2 deficient mice (RAG2 KO). The RAG2 KO mice lacked the ability to mount an adaptive immune response thereby allowing sustained expression of a human protein without the development of an antibody response. Eight week old male DKOs were randomly distributed into three groups, twenty per group, and treated via a single IV injection with either vehicle, or AAV5-SQ at 2 x 1013 or 1 x 1014 vg/kg. C57BL/6J mice comprised a fourth group and were treated with vehicle via a single IV injection to demonstrate wild type bleeding times and blood loss. Bleeding times and blood loss were assessed in these mice 8 weeks post-dose, at 16 weeks of age. In addition, forty 16 week old DKO mice were randomly divided into two groups and treated with a single IV injection of rhFVIII-SQ protein (rhSQ, Xyntha®) at either 50 or 200 IU/kg. Bleeding times were assessed in these mice 30 minutes post-dose, at 16 weeks of age. Eight weeks post dosing with either AAV5-SQ or vehicle, the tail bleeding time and blood loss were measured following transection of the tip of the tail for evaluation of the functional efficacy of AAV5-SQ gene therapy. Wild-type mice receiving vehicle had a mean of 0.040 ± 0.073 g blood loss and 5.11 ± 5.61 min bleeding time. DKO mice treated with vehicle had a mean blood loss and bleeding time of 0.741 ± 0.207 g and 28.96 ± 1.40 min, respectively. Mice receiving AAV5-SQ at 2x1013 vg/kg showed significantly reduced blood loss (0.387 ± 0.384 g, p=0.0008 vs DKO+ vehicle; p=0.0003 vs WT) and bleeding time (17.12 ± 11.58 min, p=0.00005 vs DKO+ vehicle; p=0.0013 vs WT) while 1x1014 vg/kg AAV5-SQ treatment corrected blood loss and bleeding times to wild-type levels (0.104 ± 0.203 g [p=0.192 vs WT, p= 5.49x10-12 vs DKO + vehicle] and 5.58 ± 9.32 mins [p=0.847 vs WT, p= 1.75x-13 vs DKO + vehicle], respectively). The effect of AAV5-SQ treatment on blood loss and bleeding time was comparable to the effects of rhSQ. DKO mice receiving 50 IU/kg of rhSQ had a mean blood loss and bleeding time of 0.492 ± 0.297 g and 18.14 ± 9.39 min, respectively, which was not significantly different from mice receiving AAV5-SQ at 2x1013 (p=0.343 for blood loss, p=0.760 for bleeding time). DKO mice receiving 200 IU/kg of rhSQ had a mean blood loss and bleeding time of 0.134 ± 0.191 g and 4.29 ± 6.16 min, respectively, which was not significantly different from mice receiving AAV5-SQ at 1x1014 (p=0.635 for blood loss, p=0.608 for bleeding time). In a separate experiment, 4 groups of DKO mice, n=10 per group, were injected with either vehicle, AAV5-SQ at 2x1013, AAV5-SQ at 2x1014 vg/kg or rhSQ at 50 IU/kg. Blood was collected 8 weeks after AAV5-SQ treatment or 5 and 30 min after rhSQ for evaluation of plasma hFVIII-SQ protein levels and activity. Expressed hFVIII-SQ levels were measured by electrochemiluminescence assay. Factor VIII-SQ protein levels at 2x1013 vg/kg AAV5-SQwere 46.8±44.0 ng/ml and 355±166ng/ml at 2x1014 vg/kg. At 50 IU/kg of rhSQ the plasma protein levels were 79.1±11.3 ng/ml at 5 min and 44.7±16.6 ng/ml at 30min post dosing. Western blot analysis of the plasma from these mice showed the expressed protein to be similar in size to rhSQ. In summary, AAV5-SQ injected into DKO hemophilic mice resulted in a dose dependent expression of B-domain deleted FVIII protein and a corresponding correction of bleeding time and blood loss. At the highest dose tested complete correction was achieved. Similar corrections in bleeding were observed at approximately the same plasma levels of FVIII protein produced either endogenously by AAV5-SQ or following exogenous administration of B-domain deleted FVIII. Disclosures Bunting: BioMarin Pharmaceutical: Employment. Zhang:BioMarin Pharmaceutical: Employment. Xie:BioMarin Pharmaceutical: Employment. Bullens:BioMarin Pharmaceutical: Employment. Mahimkar:BioMarin Pharmaceutical: Employment. Fong:BioMarin Pharmaceutical: Employment. Sandza:BioMarin Pharmaceutical: Employment. Colosi:BioMarin Pharmaceutical: Employment. Long:BioMarin Pharmaceutical: Employment. Vehar:BioMarin Pharmaceutical: Employment. Carter:BioMarin Pharmaceutical: Employment.

scholarly journals Improved coagulation and haemostasis in haemophilia with inhibitors by combinations of superFactor Va and Factor VIIa

2016 ◽  
Vol 115 (03) ◽  
pp. 551-561 ◽  
Author(s):  
Andrew J. Gale ◽  
John H. Griffin ◽  
Laurent O. Mosnier ◽  
Vikas Bhat ◽  
Annette von Drygalski
Keyword(s):  
Blood Loss ◽  
Thrombin Generation ◽  
Factor Viia ◽  
Clot Lysis ◽  
Wild Type ◽  
Therapeutic Concentration ◽  
Low Concentration ◽  
Normal Human ◽  
Supplementary Material ◽  
Deficient Mice

SummaryBypassing inhibitors in haemophilia patients is limited to activated (a) Factor(F)VII products. We introduced “FVa activity augmentation” as another bypassing strategy and studied effects of an engineered FVa variant designated superFVa. Procoagulant and clot stabilising properties of superFVa and recombinant human (rh)FVIIa, either alone or in combination, were studied in thrombin generation and clot lysis assays in normal human plasma (NHP) with or without anti-FVIII inhibitors, in haemophilia plasma, and in FVIII-deficient mice or in wild-type mice with anti-FVIII inhibitors. SuperFVa was as effective as rhFVIIa to improve thrombin generation or clot lysis. Furthermore, procoagulant effects were significantly enhanced when these compounds were combined. RhFVIIa at 40 nM (a therapeutic concentration) improved thrombin generation mildly, but markedly improved thrombin generation when combined with a low concentration (e. g. 3 nM) of superFVa. In clot lysis studies, the concentration of rhFVIIa to normalise clot lysis times could be reduced by 100-fold (e. g. from 40 nM to 0.4 nM) when combined with a low concentration (0.37 nM) of superFVa. In haemostasis studies of FVIII-deficient mice, blood loss was dose-dependently reduced by either superFVa or rhFVIIa. SuperFVa (200 U/kg) corrected mean blood loss indistinguishably from rhFVIII. Blood loss correction by rhFVIIa was greatly improved when combined with superFVa. Similar blood loss correction results were observed for therapies in wild-type mice after infusion with anti-FVIII inhibitors. Thus, superFVa may be an effective procoagulant agent in the setting of haemophilia with inhibitors and it merits further evaluation for new bypassing strategies.Supplementary Material to this article is available online at www.thrombosis-online.com.

scholarly journals Deletion of the SNARE vti1b in Mice Results in the Loss of a Single SNARE Partner, Syntaxin 8

2003 ◽  
Vol 23 (15) ◽  
pp. 5198-5207 ◽  
Author(s):  
Vadim Atlashkin ◽  
Vera Kreykenbohm ◽  
Eeva-Liisa Eskelinen ◽  
Dirk Wenzel ◽  
Afshin Fayyazi ◽  
...  
Keyword(s):  
Amino Acid Identity ◽  
Snare Complex ◽  
Snare Proteins ◽  
Multivesicular Bodies ◽  
Wild Type ◽  
Late Endosomes ◽  
The Stability ◽  
Deficient Mice

ABSTRACT SNARE proteins participate in recognition and fusion of membranes. A SNARE complex consisting of vti1b, syntaxin 8, syntaxin 7, and endobrevin/VAMP-8 which is required for fusion of late endosomes in vitro has been identified recently. Here, we generated mice deficient in vti1b to study the function of this protein in vivo. vti1b-deficient mice had reduced amounts of syntaxin 8 due to degradation of the syntaxin 8 protein, while the amounts of syntaxin 7 and endobrevin did not change. These data indicate that vti1b is specifically required for the stability of a single SNARE partner. vti1b-deficient mice were viable and fertile. Most vti1b-deficient mice were indistinguishable from wild-type mice and did not display defects in transport to the lysosome. However, 20% of the vti1b-deficient mice were smaller. Lysosomal degradation of an endocytosed protein was slightly delayed in hepatocytes derived from these mice. Multivesicular bodies and autophagic vacuoles accumulated in hepatocytes of some smaller vti1b-deficient mice. This suggests that other SNAREs can compensate for the reduction in syntaxin 8 and for the loss of vti1b in most mice even though vti1b shows only 30% amino acid identity with its closest relative.

scholarly journals VWF-FVIII Interactions Influence Hemostatic Thrombus Stability in Murine Models of Hemophilia Α and Type 2N VWD

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1403-1403
Author(s):  
Laura L Swystun ◽  
Courtney Dwyer ◽  
Kate Nesbitt ◽  
Kassandra Hebert ◽  
David Lillicrap
Keyword(s):  
Blood Loss ◽  
Research Funding ◽  
Bleeding Time ◽  
Thrombus Formation ◽  
Wild Type ◽  
Bleeding Events ◽  
Experimental Conditions ◽  
Total Blood ◽  
Tail Vein ◽  
Deficient Mice

Abstract von Willebrand factor (VWF) and factor VIII (FVIII) circulate in the plasma as a non-covalent complex. VWF can influence FVIII activity by stabilizing FVIII plasma levels and transporting FVIII to the site of thrombus formation. We have previously generated a murine model of type 2N VWD through hydrodynamic expression of the severe R816W VWF variant that exhibits a 90% decrease in FVIII-binding ability and a failure to stabilize endogenous FVIII when expressed in a VWF-deficient mouse. Intravital studies of arteriole platelet thrombus formation demonstrated that FVIII knockout and type 2N VWD mice produced smaller, less stable thrombi. Aim: In this study, we assess the contribution of VWF and FVIII interactions to the formation and stability of hemostatic thrombi in a murine tail vein transection (TVT) bleeding model. Method: Under isoflurane anaesthesia the left lateral tail vein was transected using standardized measuring and transection templates (provided by Novo Nordisk, described in detail in Johansen et al. Hemophilia. 2016). Bleeding time was observed over the course of 60 minutes. Blood was collected into saline for hemoglobin quantification. For experimental conditions, 300 U/Kg wild type or type 2N (R816W) VWF was administered IV to VWF KO mice 2 hours prior to TVT in order to allow for endogenous FVIII stabilization. Results: Consistent with previous reports, VWF (44.15 min; p<0.0001) and FVIII-deficient mice (45 min; p<0.0001) exhibited a significantly prolonged total bleeding time relative to normal mice (4.6 min). Infusions of plasma-derived murine VWF (FVIII-free) into VWF deficient mice have demonstrated that wild type (WT) VWF is capable of restoring FVIII:C levels to ~75% 2 hours post-infusion, while the R816W variant does not influence FVIII stabilization and levels remain at ~15%. Mice infused with WT VWF had a shorter bleeding time (16.2 min) relative to mice infused with the severe type 2N VWD variant (44.67 minutes, p<0.0001). For FVIII KO mice, the primary bleeding time (1.53 min), defined as the period of time until first bleeding cessation, was not different from normal mice (1.2 min) but significantly extended for VWF KO mice (35.97 min, p<0.0001). Primary bleeding time was markedly reduced for VWF KO mice infused with WT VWF (4.97 min, p=0.07) confirming the restoration of primary hemostasis by infusion of murine plasma-derived VWF. Hemoglobin quantification confirmed that FVIII (243.8 mg, p<0.0001) and VWF KO mice (198.8 mg, p<0.0001) had increased blood loss compared to normal mice (9.91 mg). Similarly, VWF KO mice infused with type 2N VWF (308.04 mg) had increased blood loss compared to mice infused with WT VWF (73.78 mg, p=0.0008). Total blood loss and bleeding times were positively correlated across all groups (r2=0.625, p<0.0001). Thrombus stability was characterized by the frequency of spontaneous re-bleeding events. 11% of normal mice experienced one or more spontaneous re-bleeding events during the course of injury, while 100% of FVIII deficient mice experienced spontaneous re-bleeding (p=0.0004). A similar trend was observed for VWF KO mice infused with WT VWF (0%) and type 2N VWF (66.7%, p=0.021). Conclusions: These studies suggest that in the murine TVT model of physiological hemostasis, the initial hemostatic thrombus formation is predominantly driven by platelet plug formation and is VWF-dependent. In contrast, thrombus stability over the course of the model is reliant on the processes responsible for fibrin formation, and is FVIII-dependent. This suggests that patients with type 2N VWD and hemophilia A may have bleeding that is the result of impaired stabilization of the primary platelet plug. Disclosures Lillicrap: Baxalta: Research Funding; Biogen-Idec: Research Funding; Bayer: Research Funding; Octapharma: Research Funding.

Different effects of chronic helicobactor pylori infection on parietal and ECL cells between wild type and gastrin-deficient mice

2001 ◽  
Vol 120 (5) ◽  
pp. A728-A728
Author(s):  
D CHEN ◽  
L FRIISHANSEN ◽  
X WANG ◽  
C ZHAO ◽  
H WALDUM ◽  
...  
Keyword(s):  
Wild Type ◽  
Ecl Cells ◽  
Helicobactor Pylori ◽  
Deficient Mice

scholarly journals InsP7is a small-molecule regulator of NUDT3-mediated mRNA decapping and processing-body dynamics

2020 ◽  
Vol 117 (32) ◽  
pp. 19245-19253 ◽  
Author(s):  
Soumyadip Sahu ◽  
Zhenzhen Wang ◽  
Xinfu Jiao ◽  
Chunfang Gu ◽  
Nikolaus Jork ◽  
...  
Keyword(s):  
Stress Responses ◽  
Messenger Rna ◽  
Control Process ◽  
Stem Cell Differentiation ◽  
Wild Type ◽  
Inositol Pyrophosphate ◽  
Body Dynamics ◽  
Processing Body ◽  
The Stability

Regulation of enzymatic 5′ decapping of messenger RNA (mRNA), which normally commits transcripts to their destruction, has the capacity to dynamically reshape the transcriptome. For example, protection from 5′ decapping promotes accumulation of mRNAs into processing (P) bodies—membraneless, biomolecular condensates. Such compartmentalization of mRNAs temporarily removes them from the translatable pool; these repressed transcripts are stabilized and stored until P-body dissolution permits transcript reentry into the cytosol. Here, we describe regulation of mRNA stability and P-body dynamics by the inositol pyrophosphate signaling molecule 5-InsP7(5-diphosphoinositol pentakisphosphate). First, we demonstrate 5-InsP7inhibits decapping by recombinant NUDT3 (Nudix [nucleoside diphosphate linked moiety X]-type hydrolase 3) in vitro. Next, in intact HEK293 and HCT116 cells, we monitored the stability of a cadre of NUDT3 mRNA substrates following CRISPR-Cas9 knockout ofPPIP5Ks(diphosphoinositol pentakisphosphate 5-kinases type 1 and 2, i.e.,PPIP5KKO), which elevates cellular 5-InsP7levels by two- to threefold (i.e., within the physiological rheostatic range). ThePPIP5KKO cells exhibited elevated levels of NUDT3 mRNA substrates and increased P-body abundance. Pharmacological and genetic attenuation of 5-InsP7synthesis in the KO background reverted both NUDT3 mRNA substrate levels and P-body counts to those of wild-type cells. Furthermore, liposomal delivery of a metabolically resistant 5-InsP7analog into wild-type cells elevated levels of NUDT3 mRNA substrates and raised P-body abundance. In the context that cellular 5-InsP7levels normally fluctuate in response to changes in the bioenergetic environment, regulation of mRNA structure by this inositol pyrophosphate represents an epitranscriptomic control process. The associated impact on P-body dynamics has relevance to regulation of stem cell differentiation, stress responses, and, potentially, amelioration of neurodegenerative diseases and aging.

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