scholarly journals Genetic Duplication of Tissue Factor Leads to Partial Specialization of Function in Zebrafish

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 486-486
Author(s):  
Steven Grzegorski ◽  
Divyani Paul ◽  
James H. Morrissey ◽  
Jordan A. Shavit
Keyword(s):  
Tissue Factor ◽  
Complete Loss ◽  
Clot Formation ◽  
Low Flow ◽  
Coagulation Cascade ◽  
Null Alleles ◽  
Prolonged Treatment ◽  
External Fertilization ◽  
Equity Ownership

Tissue factor (TF) is a critical factor for hemostasis in response to tissue injury. Among mouse knockouts of procoagulant factors, those lacking TF have the most severe phenotype, with complete lethality by midgestation. Furthermore, complete loss of TF has never been described in humans. Together, these suggest additional roles in embryonic development beyond coagulation. Zebrafish are a small freshwater teleost fish with a well described hemostatic system, including conservation of the coagulation cascade. Zebrafish are prolific breeders that reproduce through external fertilization, with subsequent rapid and transparent development, allowing studies not possible in mammals. Due to an ancient genomic event, 30-40% of the teleost genome is duplicated, resulting in two TF paralogs (TFa and TFb) with unknown functions. Here we use CRISPR/Cas9 to produce null alleles of TFa and TFb and uncover partial subspecialization of these duplicates. It has been shown previously that both TFs are expressed before the initiation of blood circulation, between 24 and 48 hours post fertilization, yet complete loss of TFa and TFb yielded no gross abnormalities. Embryos and larvae were able to develop normally through juvenile stages but succumbed to hemorrhage by early adulthood at 9 weeks of age. Surprisingly, a single allele of either TFa or TFb was able to rescue survival in the context of complete loss of the other gene. To evaluate for hemostatic effects of TF deficiency, laser-mediated endothelial injury was used in the venous and arterial systems at 3 and 5 days post fertilization (dpf), respectively. Loss of TFb alone at 3 dpf resulted in no observable hemostatic defects. Conversely, loss of TFa led to a 50% increase in the time to venous occlusion (TTO), which was exacerbated by concomitant loss of one allele of TFb. Total TF deficiency led to a complete inability to form occlusive venous thrombi, indicating that both TFs can trigger coagulation but TFa is able to completely compensate for the loss of TFb. Concordant with these data, loss of TFb resulted in transcriptional upregulation of TFa but not vice versa. Interestingly, the roles are reversed in the arterial vasculature. Loss of TFa had no effect, loss of TFb lead to a 60% reduction in the number of occlusive thrombi, and complete deficiency resulted in no arterial occlusion. Combined with the venous results, these data point to differentiated roles of TFa and TFb in the venous and arterial systems. In order to test whether these differences were functional, recombinant TF (rTF) molecules were expressed in E. coli, purified, and incorporated into 80% phosphatidylcholine/20% phosphatidylserine liposomes. Ex vivo tube-tilt clotting assays were performed by using each rTF to activate citrated plasma from lake trout. rTFa triggered stable clot formation within 1-2 minutes of recalcification. rTFb usually failed to induce clot formation, with occasional delayed fibrin thrombi that appeared to be grossly disorganized and were easily disrupted following agitation. Taken with the in vivo data, this hints at an altered kinetic profile, with TFa being a more potent cofactor for factor VIIa in low flow (venous) settings. The laboratory is an artificially safe environment, so a synthetic chemical stress test was performed on 3 dpf larvae. Prolonged treatment with cortisol and epinephrine led to the development of cardiac tamponade in larvae with complete TF deficiency (61%), but similar results were only found at low levels in wild type siblings (2-5%). The same assay in prothrombin mutants also revealed a high rate of tamponade (75%), but lower levels in fibrinogen-deficient larvae (20%). These data suggest an extrahemostatic risk factor for tamponade that is modified by prothrombin and tissue factor levels and is independent of fibrin formation. Our results intimate that TFa and TFb have overlapping procoagulant functions but differential kinetic profiles in venous vs arterial systems. We also find that the duplication provides a layer of quantitative regulation and creates a titratable level for regulation of hemostatic and extrahemostatic roles of TF. Overall, this novel model provides new structural and physiologic information about TF function in vivo, including potential previously unknown roles in perivascular development, cardiovascular stability, remodeling and/or regeneration. Disclosures Morrissey: PrevThro Pharmaceuticals: Equity Ownership; Cayuga Pharmaceuticals: Equity Ownership; Kerafast, Inc: Research Funding; Issued and pending patent applications relating to medical uses of polyphosphate and polyphosphate inhibitors: Patents & Royalties. Shavit:Bayer: Consultancy; Sanofi: Consultancy; Shire/Takeda: Consultancy; Spark Therapeutics: Consultancy; CSL-Behring: Consultancy; Novo Nordisk: Consultancy.

scholarly journals Tissue factor–positive neutrophils bind to injured endothelial wall and initiate thrombus formation

Blood ◽  
2012 ◽  
Vol 120 (10) ◽  
pp. 2133-2143 ◽  
Author(s):  
Roxane Darbousset ◽  
Grace M. Thomas ◽  
Soraya Mezouar ◽  
Corinne Frère ◽  
Rénaté Bonier ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Tissue Factor ◽  
Blood Coagulation ◽  
Thrombus Formation ◽  
Coagulation Cascade ◽  
Factor Xii ◽  
Long Time ◽  
Platelet Accumulation

AbstractFor a long time, blood coagulation and innate immunity have been viewed as interrelated responses. Recently, the presence of leukocytes at the sites of vessel injury has been described. Here we analyzed interaction of neutrophils, monocytes, and platelets in thrombus formation after a laser-induced injury in vivo. Neutrophils immediately adhered to injured vessels, preceding platelets, by binding to the activated endothelium via leukocyte function antigen-1–ICAM-1 interactions. Monocytes rolled on a thrombus 3 to 5 minutes postinjury. The kinetics of thrombus formation and fibrin generation were drastically reduced in low tissue factor (TF) mice whereas the absence of factor XII had no effect. In vitro, TF was detected in neutrophils. In vivo, the inhibition of neutrophil binding to the vessel wall reduced the presence of TF and diminished the generation of fibrin and platelet accumulation. Injection of wild-type neutrophils into low TF mice partially restored the activation of the blood coagulation cascade and accumulation of platelets. Our results show that the interaction of neutrophils with endothelial cells is a critical step preceding platelet accumulation for initiating arterial thrombosis in injured vessels. Targeting neutrophils interacting with endothelial cells may constitute an efficient strategy to reduce thrombosis.

scholarly journals Monitoring Compound-Related Effects on Coagulability in Rats and Cynomolgus and Rhesus Monkeys by Thrombin Generation Kinetic Measurement

2020 ◽  
Vol 39 (3) ◽  
pp. 207-217
Author(s):  
F. Poitout-Belissent ◽  
D. Culang ◽  
D. Poulin ◽  
R. Samadfan ◽  
S. Cotton ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Thrombin Generation ◽  
In Vivo Studies ◽  
Coagulation Cascade ◽  
Dependent Manner ◽  
Kinetic Assay ◽  
Thrombin Generation Assay ◽  
Coefficients Of Variation

Thrombin generation assay (TGA) is a sensitive method for the assessment of the global clotting potential of plasma. This kinetic assay can detect both hypocoagulable and hypercoagulable conditions: delayed or reduced thrombin generation leading to a prolonged clotting time, or induced thrombin activity, shifting the coagulation cascade toward thrombosis. The purpose of this study is to qualify the TGA in nonhuman primates (NHP) and rats for its use during nonclinical in vivo and in vitro studies. Blood was drawn from nonanesthetized animals, and platelet-poor plasma was obtained after double centrifugation; coefficients of variation were <10% for all derived parameters of thrombin generation assessed with 5 pM of tissue factor. Thrombin generation was evaluated in vitro in rat and NHP plasmas with ascending doses of unfractionated heparin (UFH), recombinant tissue factor, and anticoagulant compounds. Thrombin generation was decreased with UFH and anticoagulant compounds, but was increased in the presence of tissue factor, in a dose-dependent manner. In a rat model of inflammation, animals were administered a low dose of lipopolysaccharides. Thrombin generation measurements were decreased 3 hours post-LPS administration with a nadir at 24 hours, while thrombin–antithrombin complexes reached a peak at 8 hours, supporting an earlier production of thrombin. In conclusion, these data demonstrated that TGA can be performed in vitro for screening of compounds expected to have effects on coagulation cascade, and thrombin generation can be measured at interim time points during nonclinical in vivo studies in rats and NHP.

The Effect of Grp78 Inhibition on Tissue Factor Gene Expression, Procoagulant Activity, and Factor Xa Generation.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1924-1924
Author(s):  
Gourab Bhattacharjee ◽  
Jasimuddin Ahamed ◽  
Brian Pedersen ◽  
Amr El-Sheikh ◽  
Cheng Liu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Surface ◽  
Tissue Factor ◽  
Factor Xa ◽  
Cell Types ◽  
Procoagulant Activity ◽  
Coagulation Cascade ◽  
Clotting Factor ◽  
Tf Gene

Abstract In vivo biopanning with phage displayed peptide libraries has generated a group of peptide probes which bind selectively to the surface of atherosclerotic plaque endothelium. The highest affinity peptide, EKO130, binds to the 78 kDa glucose regulated protein (Grp78). Grp78 has been demonstrated to play a role in numerous pathological processes as well as a possible role in the local cell surface regulation of the coagulation cascade. The goal of this study is to determine the role of Grp78 in coagulation including plasma clotting, factor Xa (Xa) generation, and tissue factor (TF) gene expression. siRNA mediated inhibition of Grp78 results in a marked increase in TF gene expression in bEND.3 endothelial cells and RAW macrophage-like cells. Antibody mediated inhibition of cell surface Grp78 results in increased TF procoagulant activity and TF-dependent Xa generation in both the endothelial and macrophage cell types. These studies are consistent with results from another laboratory demonstrating that Grp78 over-expression inhibits TF mediated initiation and support of the coagulation protease cascade. Thus, our work indicates that Grp78 suppresses TF at both the functional and molecular level by inhibiting both its thrombogenic potential and gene expression.

Evidence for a Factor IX-Independent Role for Factor XI in Thrombin Generation

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2244-2244
Author(s):  
Anton Matafonov ◽  
Qiufang Cheng ◽  
Ingrid M. Verhamme ◽  
Obi Umunakwe ◽  
Erik I. Tucker ◽  
...  
Keyword(s):  
Thrombin Generation ◽  
Factor Ix ◽  
Clot Formation ◽  
Coagulation Cascade ◽  
In Vitro Assays ◽  
Dependent Manner ◽  
Vessel Occlusion ◽  
Fibrin Formation ◽  
Equity Ownership ◽  
Deficient Mice

Abstract Abstract 2244 In the widely used activated partial thromboplastin time (aPTT) assay, fibrin formation is induced by a series of sequential activations of the plasma protease zymogens factor (f) XII, fXI, fIX, fX and prothrombin, in that order. Conversion of prothrombin to the protease α-thrombin results in fibrin formation. α-Thrombin also enhances its own generation through activation of the cofactors fV and fVIII. While the linear sequence of reactions in the aPTT implies that loss of any single protease should have a comparable deleterious effect on the system, it is recognized that complete deficiency of a protein near the start of the sequence (e.g. fXII or fXI) results in greater aPTT prolongation than deficiency of proteins further down the sequence (e.g. fIX). This implies that proteases activated early in the process have multiple plasma substrates. For example, fXIa was recently reported to activate fVIII and fV (JTH 8;1532:2010), in addition to its role in fIX activation. Here, we present evidence that fXIa contributes to α-thrombin generation in the absence of fIX through activation of fX and/or fV. We noted that an anti-fXI antibody (O1A6) prolonged the aPTT of plasma from a patient with severe hemophilia B (fIX antigen undetectable) or plasma immunodepleted of fIX. This observation held even when an anti-fIX antibody was added to neutralize potential traces of fIX. Addition of activated fXI (fXIa - 3 nM) directly to fIX-deficient recalcified plasmas induced clot formation, and the time to clot formation was prolonged by O1A6. To further exclude the possibility that traces of fIX were contributing to thrombin generation, we confirmed the results using plasma from mice with combined complete deficiencies of fXII, fXI, and fIX. We tested the capacity of fXIa to cleave/activate fX and fV, the protease zymogen and cofactor, respectively, immediately downstream of fIX in the coagulation cascade. FX, the zymogen of the protease fXa, is evolutionarily related to fIX. SDS-PAGE analysis confirmed that fXIa cleaves fX. FX cleaved by fXIa demonstrated fXa activity in a chromogenic substrate assay, and converted prothrombin to α-thrombin in the presence of fVa and phospholipid. As previously reported, fXIa readily cleaved fV. The cleavage pattern differed from that generated by α-thrombin, however, formation of the fVa light chain was clearly evident. In a plasma clotting assay designed to measure either fXa or fVa activity, fX or fV pre-incubated with fXIa significantly shortened the clotting time of fIX-deficient plasma, while fX or fV pre-incubated with vehicle did not. In thrombin generation assays, fXIa (1.25 to 15 nM) induced thrombin generation in fIX-deficient plasma supplemented with anti-fIX antibody in a concentration dependent manner. FXIa did not induce thrombin generation in plasma lacking fV, or in fIX-deficient plasma containing the fXa inhibitor apixaban. This indicates that fXIa is working at the level of fX/fV in this assay, and is not directly converting prothrombin to α-thrombin. A recombinant variant of fXIa lacking the major fIX-binding exosite (fXIaPKA3, J Biol Chem 1996;271:29023) demonstrated a marked defect, compared to wild type fXIa (fXIaWT), in its capacity to induce thrombin generation in normal plasma. However, in fIX-deficient plasma, fXIaPKA3 and fXIaWT are comparable in their ability to enhance thrombin generation, supporting the premise that fXIa is acting through activation of fX and/or fV in the absence of fIX. Previously, we observed that fXI deficient mice and fIX deficient mice are comparably resistant to carotid artery thrombosis induced by exposure of the vessel to ferric chloride, despite having very different propensities to bleed. The animals were uniformly resistant to thrombosis with 5% FeCl3, and some were resistant at 7.5% FeCl3. All experienced vessel occlusion with 10% FeCl3. This is consistent with fXIa contributing to thrombosis in this model through fIX activation. However, we observed that some mice with combined fIX and fXI deficiency were resistant to FeCl3 concentrations up to 12.5%, implying that fXIa was contributing to thrombosis in a fIX-independent manner, as well. These results are consistent with those from the in vitro assays described above, and support the hypothesis that fXIa contributes to thrombin generation through fIX-dependent and fIX-independent processes. Disclosures: Tucker: Aronora, LLC: Employment, Equity Ownership. Gruber:Aronora, LLC: Consultancy, Equity Ownership.

scholarly journals New players in haemostasis and thrombosis

2014 ◽  
Vol 111 (04) ◽  
pp. 570-574 ◽  
Author(s):  
Julia Geddings ◽  
Nigel Mackman
Keyword(s):  
Tissue Factor ◽  
Mouse Models ◽  
Cell Damage ◽  
Membrane Vesicles ◽  
Coagulation Cascade ◽  
Factor Xii ◽  
Nucleic Acid Binding ◽  
Inorganic Polyphosphate ◽  
Extracellular Rna

SummaryThe blood coagulation cascade is essential for haemostasis, but excessive activation can cause thrombosis. Importantly, recent studies have identified factors that contribute to thrombosis but not haemostasis. These include factor XII (FXII), tissue factor-positive microparticles (MPs) and neutrophil extracellular traps (NETs). Studies have shown that FXII plays a role in thrombosis but not haemostasis. FXII is activated in vivo by a variety of negatively-charged polyphosphates, which include extracellular RNA, DNA and inorganic polyphosphate (PolyP) that are released during cell damage and infection. These findings have led to the development of nucleic acid-binding polymers as a new class of anticoagulant drug. Other studies have analysed the role of MPs in experimental thrombosis. MPs are small membrane vesicles released from activated or apoptotic cells. We and others have found that tissue factor-positive MPs enhance thrombosis in mouse models and are elevated in the plasma of pancreatic cancer patients. Finally, NETs have been shown to contribute to experimental venous thrombosis in mouse models and are present in human thrombi. NETs are composed of chromatin fibers that are released from neutrophils undergoing cell death. NETs can capture platelets and increase fibrin deposition. The recent advances in our understanding of the factors contributing to thrombosis in animal models provide new opportunities for the development of safer anticoagulant drugs.

Reintroduction of the Rat for Experimental Subarachnoid Hemorrhage with Accelerated Clot Formation: A Low Mortality Model with Persistent Clots as a Precondition for Studies in Vasospasm

2018 ◽  
Vol 79 (05) ◽  
pp. 424-433 ◽  
Author(s):  
Ulrich Budde ◽  
Ralf Middendorff ◽  
Gerd Manthei ◽  
Andre Kemmling ◽  
Bastian Tiemann ◽  
...  
Keyword(s):  
Subarachnoid Hemorrhage ◽  
Tissue Factor ◽  
Rat Model ◽  
Animal Studies ◽  
Clot Formation ◽  
Delayed Effects ◽  
In Vivo Testing ◽  
Experimental Subarachnoid Hemorrhage

Background Cerebral vasospasm as a delayed, possibly treatable sequel of subarachnoid hemorrhage (SAH) is a focus of experimental animal research. For this purpose, the rat is not a good model because of the difficulty creating a stable subarachnoid clot that persists > 1 to 2 days and could induce vasospasm. Only in rat models with a high mortality of ∼ 50% or more can SAH and its effects be investigated. Therefore, other animals than rodents are used for investigating the delayed effects of SAH. Only animal studies addressing the acute effects of SAH use rats. Objective We designed a model that allows intensive clot formation combined with low mortality to facilitate studies on the delayed effects of experimental SAH, for example, delayed vasospasm or other alterations of vessels. Methods After in vitro acceleration of the clotting process in the rats' blood by tissue factor and preliminary in vivo testing, we induced a SAH by injecting blood together with tissue factor in 22 rats. We analyzed clot expansion, length of clot persistence, chronic alterations, and histologic changes. Results The injection of blood supplemented by tissue factor led to persistent voluminous blood clots in the subarachnoid space close to the large arteries. Despite the pronounced SAH, all animals survived, allowing investigation of delayed SAH effects. All animals killed within the first 7 days after surgery had extensive clots; in some animals, the clots remained until postoperative day 12. During further clot degradation connective tissue appeared, possibly as a precursor of SAH-related late hydrocephalus. Conclusion The injection of blood together with tissue factor significantly improves SAH induction in the rat model. This rat model allows studying delayed SAH effects as found in humans.

scholarly journals Analysis of blood clotting with the total thrombus analysis system in healthy dogs

2021 ◽  
pp. 104063872199186
Author(s):  
Tomoko Iwanaga ◽  
Ryuji Fukushima ◽  
Tomoka Nagasato ◽  
Ikuro Maruyama ◽  
Naoki Miura
Keyword(s):  
Blood Flow ◽  
Platelet Function ◽  
Whole Blood ◽  
Fibrin Clot ◽  
Clot Formation ◽  
Coagulation Cascade ◽  
Test Pressure ◽  
Analysis System ◽  
The Right

To date, coagulation tests are unable to reflect in vivo coagulation status in the same system, including platelet function, fibrin clot formation, and whole blood flow. The Total Thrombus Analysis System (T-TAS), which is a microfluidic assay that simulates conditions in vivo, measures whole blood flow at defined shear rates under conditions designed to assess platelet function (PL-chip) or coagulation and fibrin clot formation (AR-chip). The T-TAS records occlusion start time, occlusion time, and area under the curve. We evaluated this test in healthy control dogs. We also investigated the effect in vivo of acetylsalicylic acid (ASA), and the effect in vitro of an anticoagulation drug (dalteparin; low-molecular-weight heparin; LMWH). The CV of the AUC of both chips was good (CVs of 6.45% [PL] and 1.57% [AR]). The inhibition of platelet function by ASA was evident in the right-shift in the PL test pressure curve. The right-shift in the AR test pressure curves showed that the administration of LMWH inhibited both platelets and the coagulation cascade. The T-TAS may be useful in the evaluation of canine blood coagulation.

Increased Factor VIII Levels Have a Thrombogenic Effect In Vitro and In Vivo.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3623-3623
Author(s):  
Mia Golder ◽  
Erin Burnett ◽  
Jeff Mewburn ◽  
David Lillicrap
Keyword(s):  
Factor Viii ◽  
Tissue Factor ◽  
Calcium Chloride ◽  
Hemophilia A ◽  
Coagulation Cascade ◽  
In Vitro Test ◽  
Fviii Activity ◽  
Observation Period

Abstract The procoagulant co-factor, factor VIII (FVIII), plays a crucial role in the intrinsic blood coagulation cascade. Epidemiologic studies have established a causal association between elevated FVIII levels and venous thrombosis incidence, whereas no such association has been confirmed with arterial thrombosis. Importantly, no objective in vitro or in vivo examinations of this association have previously been performed to establish a mechanistic role for elevated FVIII levels and thrombogenicity. To establish the in vitro thrombogenic effect of elevated FVIII activity, platelet-poor C57Bl/6 hemophilia A mouse plasma was spiked with recombinant human FVIII (r-huFVIII) to FVIII levels 100% and 250% that of normal plasma. Physiological concentrations of tissue factor and calcium chloride were added to the plasma to stimulate the formation of thrombin-antithrombin (TAT) complexes. Over 25 minutes, at 5-minute intervals, the plasma was sub-sampled, and the TAT complex concentration measured using an ELISA. The results showed that as FVIII concentration was increased, the rate of formation of TAT complexes was increased and the maximum concentration of complex was increased. As a second in vitro test of thrombogenicity, FVIII deficient human plasma was spiked with r-huFVIII to FVIII levels similar to those in the TAT experiments. Thromboelastography (TEG) was performed on the samples following the addition of physiological concentrations of tissue factor and calcium chloride. As FVIII levels were increased, the time to initial fibrin formation decreased significantly, in a negative logarithmic manner. As well, the speed with which the clot formed increased significantly, in a logarithmic manner. Although the clot strength (MA) of the FVIII spiked samples differed significantly to the 0% FVIII samples, there was no significant change in MA as FVIII levels were elevated to levels greater than 100%, likely as a result of the platelet poor nature of the samples. To examine the effect of increasing FVIII levels in vivo, fluorescence intravital microscopy was used to visualize the arterioles of the cremaster muscle. Circulating platelets were labeled in vivo with rhodamine 6G (200ng/mL). C57Bl/6 normal mice (n=5), C57Bl/6 hemophillia A mice (n=5), and C57Bl/6 mice hemophilia A mice whose FVIII levels were elevated to 200% (n=3) through an intravenous infusion of r-huFVIII were examined. Arterioles were injured for 3 minutes with 10% ferric chloride soaked filter paper and observed for 40 minutes. None of the hemophilic mice arterioles occluded in the observation period, whereas the normal mice occluded at 25 minutes and the mice with 200% FVIII activity occluded at 20 minutes. Fluorescence analysis revealed that the normal mice and those with 200% FVIII activity had stable platelet accumulation, as there was little variation in the fluorescence intensity over time, but a gradual, and persistent, increase in overall intensity. In contrast, there was strong variation in the accumulation of platelets within the injured arterioles of the hemophilic mice and no persistence in the intensity of fluorescence throughout the observation period. Together, these in vitro and in vivo data indicate that elevated FVIII levels produce a thrombogenic effect that increases with FVIII elevations. However, it is necessary to further examine this relationship to determine whether the thrombogenicity of FVIII is proportional to the FVIII increase and whether the thrombogenicity is affected by the duration of FVIII elevation.

Pan-Selectin Antagonist GMI-1070 Affects Biomarkers of Adhesion, Activation and the Coagulation Cascade in Sickle Cell Adults At Steady State

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 87-87
Author(s):  
John L. Magnani ◽  
Frans A Kuypers ◽  
John T. Patton ◽  
Sandra K Larkin ◽  
Lori Styles ◽  
...  
Keyword(s):  
Steady State ◽  
Tissue Factor ◽  
Sickle Cell ◽  
Research Funding ◽  
Thrombus Formation ◽  
Small Sample ◽  
Coagulation Cascade ◽  
Loading Dose ◽  
Employment Equity ◽  
Equity Ownership

Abstract Abstract 87 Introduction: Engagement of selectins by their ligands leads to cellular activation and adhesion and plays a role in thrombus formation. In sickle cell disease (SCD), the selectins underlie vaso-occlusion, which results in vaso-occlusive crisis (VOC). In SCD patients high levels of soluble E-selectin (sEsel) are associated with increased mortality (Kato, BJH 2005). In addition, SCD patients exhibit chronic activation of the coagulation cascade and of leukocytes. Previously, we showed in animal models of SCD VOC that pan-selectin antagonist GMI-1070 reduced arrested RBC/WBC aggregates and improved blood flow and survival. In a Phase 1 study of SCD adults at steady state (not in VOC), GMI-1070 inhibited neutrophil activation and platelet/neutrophil aggregate formation and increased circulating neutrophils. Herein we report on the effect of GMI-1070 on biomarkers of monocyte, endothelial cell, and coagulation cascade activation; and on the effect of hydroxycarbamide (hydroxyurea or HU) on these biomarkers for patients on this trial. Methods: SCD adults at steady state (n=15) received an IV loading dose of GMI-1070 (20 mg/kg) and a second dose ten hours later (10 mg/kg). Safety and PK were reported elsewhere. HU use was noted. Biomarker blood samples were drawn prior to treatment, and at 4, 8, 24 and 48 hrs after the loading dose. Analytes measured included: soluble adhesion molecules sEsel, soluble P-selectin (sPsel) and intracellular adhesion molecule-1 (ICAM-1) by multiplex ELISA; tissue factor and thrombin-antithrombin complexes (TF and TAT) by ELISA; and, surface expression of monocyte b2 integrins MAC-1 & LFA-1; and platelet-monocyte aggregates (PMA) by flow cytometry. Expression levels were compared against pre-treatment, and stratified by HU use. Analysis was by ANOVA F-test from mixed effects model. Data are reported for 11 subjects (biomarkers) and 15 subjects (neutrophils). Results: Soluble adhesion markers were reduced after 8 hrs (sEsel), or 4 and 8 hrs (sPsel; ICAM-1). Tissue factor (TF) was reduced at 4 and 8 hrs. Thrombin-antithrombin complex (TAT) levels and expression of MAC-1 and LFA-1 were reduced at all time points. The percentage of PMA was reduced at 8 hrs. (Table) When HU use was considered (HU–No; HU–Yes), the levels of sEsel, sPsel, ICAM-1, TF, TAT, MAC-1, and neutrophil counts were higher and more variable at baseline in the HU-No group, significantly so for ICAM-1 (p=0.048) and MAC-1 (p=0.001). After GMI-1070, significant reduction from baseline was seen in both groups: in the HU-No group for ICAM-1, TF, PMA, LFA-1, and in the HU-Yes group for sEsel, MAC-1, TF, TAT. Neutrophil counts increased at 24 hours in the HU-No group only (p=0.001). Conclusion: In this small sample of adults with SCD at steady state, selectin inhibition by treatment with GMI-1070 resulted in reduction in soluble adhesion markers, leukocyte activation, PMA, and markers of coagulation activation. This suggests that selectin inhibition affects downstream processes in vivo, continuing after plasma clearance of >97% of the drug. This may represent interference in the processes that lead to VOC in SCD. A phase 2 study is underway to further explore the effect of this experimental drug on these markers and in the treatment of VOC. Disclosures: Magnani: GlycoMimetics: Employment, Equity Ownership. Kuypers:GlycoMimetics: Research Funding. Patton:GlycoMimetics: Employment. Larkin:GlycoMimetics: Research Funding. Styles:GlycoMimetics: Research Funding. DeCastro:GlycoMimetics: Research Funding. Telen:GlycoMimetics: Research Funding. Wun:GlycoMimetics: Research Funding. Thackray:GlycoMimetics: Employment, Equity Ownership.

scholarly journals The P2Y2 Nucleotide Receptor Mediates Monocyte Tissue Factor Expression and Endotoxemia Death in Mice

2021 ◽  
Author(s):  
Qianman Peng ◽  
Shenqi Qian ◽  
Saud Alqahtani ◽  
Peter Panizzi ◽  
Jianzhong Shen
Keyword(s):  
Tissue Factor ◽  
Bone Marrow Cells ◽  
Lethal Dose ◽  
Atp Release ◽  
Coagulation Cascade ◽  
Nucleotide Receptors ◽  
Human Coronary Artery ◽  
Tf Gene ◽  
Stimulation Of

Recently we reported that in human coronary artery endothelial cells, activation of the P2Y2 receptor (P2Y2R) induces up-regulation of tissue factor (TF), a vital initiator of the coagulation cascade. However, others have shown that monocyte TF is more critical than endothelial TF in provoking a pro-thrombotic state. Thus, we aimed to study whether monocytes express the P2Y2R, its role in controlling TF expression, and its relevance in vivo. RT-PCR and receptor activity assays revealed that among the eight P2Y nucleotide receptors, the P2Y2 subtype was selectively and functionally expressed in human monocytic THP-1 cells and primary monocytes. Stimulation of the cells by ATP or UTP dramatically increased TF protein expression, which was abolished by AR-C118925, a selective P2Y2R antagonist, or by siRNA silencing the P2Y2R. In addition, UTP or ATP treatment induced a rapid accumulation of TF mRNA preceded with an increased TF pre-mRNA, indicating enhanced TF gene transcription. In addition, stimulation of the monocyte P2Y2R significantly activated ERK1/2, JNK, p38, and Akt, along with their downstream transcription factors including c-Jun, c-Fos, and ATF-2, whereas blocking these pathways respectively, all significantly suppressed P2Y2R-mediated TF expression. Furthermore, we found that LPS triggered ATP release and TF expression, the latter of which was suppressed by apyrase or P2Y2R blockage. Importantly, P2Y2R-null mice were more resistant than wild-type mice in response to a lethal dose of LPS, accompanied by much less TF expression in bone marrow cells. These findings demonstrate for the first time that the P2Y2R mediates TF expression in human monocytes through mechanisms involving ERK1/2, JNK, p38, and AKT, and that P2Y2R deletion protects the mice from endotoxemia-induced TF expression and death, highlighting monocyte P2Y2R may be a new drug target for the prevention and/or treatment of relevant thrombotic disease.

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