ABSTRACT
ABSTRACT: Clinical practice shows that a critical unmet need in the field of thrombosis prevention is the availability of anticoagulant therapy without bleeding risk. Inhibitors against FXIa or FXIIa have been extensively studied because of their low bleeding risk. However, whether these compounds produce synergistic effects has not yet been explored. In this study, analyses of activated partial thromboplastin time in combination with the FXIa inhibitor PN2KPI and the FXIIa inhibitor Infestin4 at different proportions were performed using the SynergyFinder tool identifying synergistic anticoagulation effects. Both an FeCl 3 -induced carotid artery thrombosis mouse model and a transient occlusion of the middle cerebral artery mouse model showed that the combination of PN2KPI and Infestin4, which are 28.57% and 6.25% of the effective dose, respectively, significantly prevents coagulation, and furthermore, dual inhibition does not cause bleeding risk.
Subject(s)
Anticoagulants , Blood Coagulation , Disease Models, Animal , Drug Synergism , Factor XIIa , Factor XIa , Animals , Factor XIa/antagonists & inhibitors , Factor XIa/metabolism , Anticoagulants/pharmacology , Blood Coagulation/drug effects , Male , Factor XIIa/antagonists & inhibitors , Factor XIIa/metabolism , Carotid Artery Thrombosis/prevention & control , Carotid Artery Thrombosis/chemically induced , Carotid Artery Thrombosis/drug therapy , Mice , Infarction, Middle Cerebral Artery/drug therapy , Hemorrhage/chemically induced , Mice, Inbred C57BL , Partial Thromboplastin TimeABSTRACT
Background: Carotid artery thrombosis is the leading cause of stroke. Since there are no apparent symptoms in the early stages of carotid atherosclerosis onset, it causes a more significant clinical diagnosis. Photoacoustic (PA) imaging provides high contrast and good depth information, which has been used for the early detection and diagnosis of many diseases. Methods: We investigated thrombus formation by using 20% ferric chloride (FeCl3) in the carotid arteries of KM mice for the thrombosis model. The near-infrared selenium/polypyrrole (Se@PPy) nanomaterials are easy to synthesize and have excellent optical absorption in vivo, which can be used as PA contrast agents to obtain thrombosis information. Results: In vitro experiments showed that Se@PPy nanocomposites have fulfilling PA ability in the 700 nm to 900 nm wavelength range. In the carotid atherosclerosis model, maximum PA signal enhancement up to 3.44, 4.04, and 5.07 times was observed by injection of Se@PPy nanomaterials, which helped to diagnose the severity of carotid atherosclerosis. Conclusion: The superior PA signal of Se@PPy nanomaterials can identify the extent of atherosclerotic carotid lesions, demonstrating the feasibility of PA imaging technology in diagnosing carotid thrombosis lesion formation. This study demonstrates nanocomposites and PA techniques for imaging and diagnosing carotid thrombosis in vivo.
Subject(s)
Atherosclerosis , Carotid Artery Diseases , Carotid Artery Thrombosis , Nanospheres , Photoacoustic Techniques , Selenium , Thrombosis , Animals , Mice , Polymers , Carotid Artery Thrombosis/chemically induced , Carotid Artery Thrombosis/diagnostic imaging , Photoacoustic Techniques/methods , Pyrroles , Carotid Arteries/diagnostic imaging , Thrombosis/diagnostic imagingSubject(s)
Ad26COVS1/adverse effects , Carotid Artery Thrombosis/chemically induced , Ischemic Stroke/chemically induced , Thrombocytopenia/chemically induced , Thrombosis/chemically induced , Vaccination/adverse effects , Ad26COVS1/administration & dosage , Amaurosis Fugax/chemically induced , Anticoagulants/therapeutic use , Carotid Artery Thrombosis/diagnostic imaging , Carotid Artery Thrombosis/drug therapy , Humans , Ischemic Stroke/diagnostic imaging , Ischemic Stroke/drug therapy , Male , Middle Aged , Recurrence , Syndrome , Thrombocytopenia/diagnosis , Thrombosis/diagnostic imaging , Thrombosis/drug therapyABSTRACT
OBJECTIVE: It is unclear if stopping treatment with dabigatran, a new oral anticoagulant (NOAC), induces a paradoxical rebound prothrombotic state. We investigated if short-term (1-3 days) dabigatran cessation is associated with a higher thrombus volume than expected from a simple reversal of the anticoagulant effect. METHODS: Ten-week-old C57Bl/6 mice (n = 338) received one of the following oral treatments: phosphate-buffered saline (PBS), dabigatran for 7 days with or without 1 to 4 day cessation, and aspirin in either a single dose or daily for 7 days. Some of the animals that ceased dabigatran for 1 to 3 days received single-dose aspirin. Thereafter, we induced FeCl3 -mediated carotid thrombosis in 130 mice, after which we performed micro computed tomography thrombus imaging. The other 208 mice underwent coagulation assays or platelet function tests. As an explorative pilot study, we reviewed the medical records of 18 consecutive patients with NOAC cessation-related cerebral infarction in a large acute stroke cohort. RESULTS: We observed a ~ 40% higher volume of carotid thrombus after dabigatran cessation at 1 to 3 days than after vehicle treatment and showed that this effect could be prevented by single-dose aspirin pretreatment. Dabigatran cessation unduly increased platelet aggregability for 2 days after drug cessation, an effect mediated through thrombin or arachidonic acid, which effect was significantly attenuated by single-dose aspirin pretreatment. In patients, short-term (≤ 3 days) cessation of NOAC therapy, compared with longer-term (≥ 5 days) cessation, tended to be associated with relatively high stroke severity. INTERPRETATION: We provide the first preclinical evidence that a rebound prothrombotic state follows short-term cessation of dabigatran therapy. ANN NEUROL 2021;89:444-458.
Subject(s)
Antithrombins/adverse effects , Carotid Artery Thrombosis/diagnostic imaging , Dabigatran/adverse effects , Deprescriptions , Platelet Aggregation/drug effects , Substance Withdrawal Syndrome/blood , Thrombophilia/blood , Aged , Aged, 80 and over , Animals , Antithrombins/pharmacology , Arachidonic Acid/blood , Aspirin/pharmacology , Carotid Artery Thrombosis/chemically induced , Carotid Artery Thrombosis/prevention & control , Cerebral Infarction/diagnostic imaging , Cerebral Infarction/etiology , Cerebral Infarction/physiopathology , Cerebral Infarction/prevention & control , Chlorides/toxicity , Computed Tomography Angiography , Dabigatran/pharmacology , Factor Xa Inhibitors/adverse effects , Female , Ferric Compounds/toxicity , Humans , Ischemic Stroke/diagnostic imaging , Ischemic Stroke/etiology , Ischemic Stroke/physiopathology , Ischemic Stroke/prevention & control , Magnetic Resonance Angiography , Male , Mean Platelet Volume , Mice , Noxae/toxicity , Pilot Projects , Platelet Aggregation Inhibitors/pharmacology , Platelet Count , Pyrazoles/adverse effects , Pyridones/adverse effects , Rivaroxaban/adverse effects , Severity of Illness Index , Substance Withdrawal Syndrome/etiology , Substance Withdrawal Syndrome/prevention & control , Thrombin/metabolism , Thrombophilia/etiology , Thrombophilia/prevention & control , X-Ray MicrotomographyABSTRACT
In spite of current treatment strategies, myocardial infarction and stroke are still major causes of death worldwide. These events are triggered by damage of an atherosclerotic plaque, resulting in occlusive thrombus formation. Mouse studies have significantly contributed to our understanding of the mechanisms of atherogenesis and of thrombosis following plaque injury, but the extent to which the mouse serves as an accurate model of human disease is open to discussion. In this review, we provide a detailed overview and comparison of the described mouse models for atherothrombosis including their (dis)advantages. Herein guidance is provided on how to select a suitable atherothrombosis model for research questions primarily relevant to the field of thrombosis.
Subject(s)
Carotid Artery Thrombosis/etiology , Disease Models, Animal , Mice , Plaque, Atherosclerotic , Animals , Atherosclerosis/chemically induced , Atherosclerosis/genetics , Blood Coagulation , Blood Platelets/metabolism , Blood Platelets/physiology , Carotid Artery Thrombosis/chemically induced , Carotid Artery Thrombosis/metabolism , Chlorides/toxicity , Diet, High-Fat , Ferric Compounds/toxicity , Humans , Ligation , Mice, Knockout, ApoE , Plaque, Atherosclerotic/chemically induced , Plaque, Atherosclerotic/pathology , Ultrasonic WavesABSTRACT
The ferric chloride models of arterial thrombosis are useful tools with which to investigate the cellular and molecular mechanisms that contribute to arterial thrombosis. Recent insights have, however, revealed the complex and multifaceted mechanism by which ferric chloride induces thrombus formation. Here, we discuss the strengths and weaknesses of the ferric chloride models of arterial thrombosis. Particular focus is given to the phenotypes of different knockout mice in the ferric chloride models and how these compare to other models with independent modes of initiation. Further, we discuss the relevance of the ferric chloride models to the human pathology of atherothrombotic disease.
Subject(s)
Carotid Artery Thrombosis/metabolism , Chlorides/toxicity , Disease Models, Animal , Erythrocytes/metabolism , Ferric Compounds/toxicity , Animals , Anticoagulants/pharmacology , Anticoagulants/therapeutic use , Blood Platelets/pathology , Carotid Artery Thrombosis/chemically induced , Chlorides/metabolism , Ferric Compounds/metabolism , Humans , Mice , Mice, KnockoutABSTRACT
OBJECTIVE: Retinoid X receptors (RXR) are a family of nuclear receptors that play critical roles in the regulation of numerous fundamental biological processes including cell proliferation, differentiation, and death. Earlier studies suggested that treatment with RXR agonists attenuates platelet activation in all adults (male and femal) and mice; however, the underlying molecular mechanisms have remained insufficiently understood. To elaborate further on this issue, we characterized megakaryocyte and platelet-specific RXR knockout mice to study platelet function in vitro and arterial thrombosis in vivo. APPROACH AND RESULTS: First, we identified RXRß as the dominant RXR receptor in mouse platelets, prompting us to generate a megakaryocyte and platelet-specific PF4Cre ;RXRßflox/flox mouse. Second, we studied activation, spreading, and aggregation of platelets from C57Bl/6 wild-type mice (WT), PF4Cre+ ;RXRßflox/flox mice, and PF4Cre- ;RXRßflox/flox littermate controls in the presence or absence of RXR ligands, that is, 9-cis-retinoic acid (9cRA) and methoprene acid (MA). We found that in vitro treatment with RXR ligands attenuates spreading and aggregation of platelets and increases proplatelet particle formation from megakaryocytes (MK). However, these effects are also observed in RXRß-deficient platelets and MKs and are thus independent of RXRß. Third, we investigated arterial thrombus formation in an iron chloride (FeCl3)-induced vascular injury model in vivo, which is also not affected by the absence of RXRß in platelets. CONCLUSIONS: Absence of the most abundant RXR receptor in mouse platelets, RXRß, does not affect platelet function in vitro and thrombus formation in vivo. Furthermore, RXR agonists' mediated effects on platelet function are independent of RXRß expression. Hence, our data do not support a significant contribution of RXRß to arterial thrombosis in mice.
Subject(s)
Blood Platelets/physiology , Carotid Artery Thrombosis/blood , DNA-Binding Proteins/physiology , Animals , Carotid Artery Thrombosis/chemically induced , Chlorides/toxicity , DNA-Binding Proteins/deficiency , DNA-Binding Proteins/genetics , Female , Ferric Compounds/toxicity , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Platelet Activation , Thrombopoiesis/physiologyABSTRACT
BACKGROUND: The homologous plasma proteins prekallikrein and factor XI (FXI) circulate as complexes with high molecular weight kininogen. Although evidence supports an interaction between the prekallikrein-kininogen complexes and vascular endothelium, there is conflicting information regarding FXI binding to endothelium. OBJECTIVE: To study the interaction between FXI and blood vessels in mice. METHODS: C57Bl/6 wild-type or F11-/- mice in which variants of FXI were expressed by hydrodynamic tail vein injection, received intravenous infusions of saline, heparin, polyphosphates, protamine, or enzymes that digest glycosaminoglycans (GAGs). Blood was collected after infusion and plasma was analyzed by western blot for FXI. RESULTS AND CONCLUSIONS: Plasma FXI increased 5- to 10-fold in wild-type mice after infusion of heparin, polyphosphates, protamine, or GAG-digesting enzymes, but not saline. Similar treatments resulted in a much smaller change in plasma FXI levels in rats, and infusions of large boluses of heparin did not change FXI levels appreciably in baboons or humans. The releasable FXI fraction was reconstituted in F11-/- mice by expressing murine FXI, but not human FXI. We identified a cluster of basic residues on the apple 4 domain of mouse FXI that is not present in other species. Replacing the basic residues with alanine prevented the interaction of mouse FXI with blood vessels, whereas introducing the basic residues into human FXI allowed it to bind to blood vessels. Most FXI in mice is noncovalently associated with GAGs on blood vessel endothelium and does not circulate in plasma.
Subject(s)
Endothelium, Vascular/metabolism , Factor XI/metabolism , Glycosaminoglycans/blood , Animals , Binding Sites , Carotid Artery Thrombosis/blood , Carotid Artery Thrombosis/chemically induced , Chlorides/toxicity , Factor XI/chemistry , Factor XI Deficiency/blood , Ferric Compounds/toxicity , Heparin/pharmacology , Humans , Kininogens/blood , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Models, Molecular , Papio , Prekallikrein/metabolism , Protein Binding , Protein Conformation , Rats , Rats, Sprague-Dawley , Recombinant Proteins/metabolism , Sequence Alignment , Species Specificity , Static ElectricityABSTRACT
Sulfated polysaccharides from marine algae have high potential as promising candidates for marine drug development. In this study, a homogeneous sulfated polysaccharide from the marine green alga Monostroma nitidum, designated MS-1, was isolated using water extraction and anion-exchange and size-exclusion chromatography. Results of chemical and spectroscopic analyses showed that MS-1 mainly consisted of â3)-α-l-Rhap-(1â and â2)-α-l-Rhap-(1â residues, with additional branches consisting of 4-linked ß-d-xylose, 4-/6-linked d-glucose, terminal ß-d-glucuronic acid, and 3-/2-linked α-l-rhamnose. Sulfate ester groups substituted mainly at C-2/C-4 of â3)-α-l-Rhap-(1â and C-4 of â2)-α-l-Rhap-(1â residues, slightly at C-2 of terminal ß-d-glucuronic residues. MS-1 exhibited strong anticoagulant activity in vitro and in vivo as evaluated by the activated partial thromboplastin time and thrombin time assays, and significantly decreased platelet aggregation. The anticoagulant activity mechanism of MS-1 was mainly attributed to strong potentiation thrombin by heparin cofactor-II, and it also hastened thrombin and coagulation factor Xa inhibitions by potentiating antithrombin-III. MS-1 possessed markedly thrombolytic activity evaluated by plasminogen activator inhibitior-1, fibrin degradation products, and D-dimer levels using rats plasma, and recanalization rate by FeCl3-induced carotid artery thrombosis in mice. MS-1 exhibited strong antithrombotic activity in vitro and in vivo evaluated by the wet weighs and lengths of thrombus, and thrombus occlusion time by electrically-induced carotid artery thrombosis in rats. These results suggested that MS-1 could be a promising marine drug for prevention and therapy of thromboembolic disease.
Subject(s)
Anticoagulants/pharmacology , Blood Coagulation/drug effects , Chlorophyta/chemistry , Fibrinolytic Agents/pharmacology , Polysaccharides/pharmacology , Sulfates/pharmacology , Animals , Carotid Artery Thrombosis/blood , Carotid Artery Thrombosis/chemically induced , Humans , Male , Mice , Platelet Aggregation/drug effects , Polysaccharides/chemistry , Polysaccharides/isolation & purification , Rats , Rats, Sprague-Dawley , Sulfates/chemistry , Sulfates/isolation & purificationABSTRACT
Occlusive arterial thrombosis leading to cerebral ischemic stroke and myocardial infarction contributes to ~13 million deaths every year globally. Here, we have translated a vascular injury model from a small animal into a large animal (canine), with slight modifications that can be used for pre-clinical screening of prophylactic and thrombolytic agents. In addition to the surgical methods, the modified protocol describes the step-by-step methods to assess carotid artery canalization by angiography, detailed instructions to process both the brain and carotid artery for histological analysis to verify carotid canalization and cerebral hemorrhage, and specific parameters to complete an assessment of downstream thromboembolic events by utilizing magnetic resonance imaging (MRI). In addition, specific procedural changes from the previously well-established small animal model necessary to translate into a large animal (canine) vascular injury are discussed.
Subject(s)
Carotid Artery Thrombosis/chemically induced , Chlorides/adverse effects , Ferric Compounds/adverse effects , Vascular System Injuries/chemically induced , Animals , Disease Models, Animal , Dogs , Humans , MaleABSTRACT
Essentials Microbe-dependent production of trimethylamine N-oxide (TMAO) contributes to thrombosis risk. The impact of host flavin monooxygenase 3 (FMO3) modulation on platelet function is unknown. Genetic manipulation of FMO3 in mice alters systemic TMAO levels and thrombosis potential. Genetic manipulation of FMO3 is associated with alteration of gut microbial community structure. SUMMARY: Background Gut microbes play a critical role in the production of trimethylamine N-oxide (TMAO), an atherogenic metabolite that impacts platelet responsiveness and thrombosis potential. Involving both microbe and host enzymatic machinery, TMAO generation utilizes a metaorganismal pathway, beginning with ingestion of trimethylamine (TMA)-containing dietary nutrients such as choline, phosphatidylcholine and carnitine, which are abundant in a Western diet. Gut microbial TMA lyases use these nutrients as substrates to produce TMA, which upon delivery to the liver via the portal circulation, is converted into TMAO by host hepatic flavin monooxygenases (FMOs). Gut microbial production of TMA is rate limiting in the metaorganismal TMAO pathway because hepatic FMO activity is typically in excess. Objectives FMO3 is the major FMO responsible for host generation of TMAO; however, a role for FMO3 in altering platelet responsiveness and thrombosis potential in vivo has not yet been explored. Methods The impact of FMO3 suppression (antisense oligonucleotide-targeting) and overexpression (as transgene) on plasma TMAO levels, platelet responsiveness and thrombosis potential was examined using a murine FeCl3 -induced carotid artery injury model. Cecal microbial composition was examined using 16S analyses. Results Modulation of FMO3 directly impacts systemic TMAO levels, platelet responsiveness and rate of thrombus formation in vivo. Microbial composition analyses reveal taxa whose proportions are associated with both plasma TMAO levels and in vivo thrombosis potential. Conclusions The present studies demonstrate that host hepatic FMO3, the terminal step in the metaorganismal TMAO pathway, participates in diet-dependent and gut microbiota-dependent changes in both platelet responsiveness and thrombosis potential in vivo.
Subject(s)
Blood Platelets/physiology , Gastrointestinal Microbiome/physiology , Liver/enzymology , Methylamines/metabolism , Oxygenases/physiology , Thrombophilia/enzymology , Animals , Carotid Artery Thrombosis/blood , Carotid Artery Thrombosis/chemically induced , Carotid Artery, Common , Chlorides/toxicity , Ferric Compounds/toxicity , Gene Knockdown Techniques , Humans , Mice , Mice, Inbred C57BL , Oligonucleotides, Antisense/pharmacology , Oxygenases/antagonists & inhibitors , Oxygenases/genetics , Platelet-Rich Plasma , Ribotyping , Risk , Thrombophilia/microbiology , TransgenesABSTRACT
A thrombus (blood clot) is formed in injured vessels to maintain the integrity of vasculature. However, obstruction of blood vessels by thrombosis slows blood flow, leading to death of tissues fed by the artery and is the main culprit of various life-threatening cardiovascular diseases. Herein, we report a rationally designed nanomedicine that could specifically image obstructed vessels and inhibit thrombus formation. On the basis of the physicochemical and biological characteristics of thrombi such as an abundance of fibrin and an elevated level of hydrogen peroxide (H2O2), we developed a fibrin-targeted imaging and antithrombotic nanomedicine, termed FTIAN, as a theranostic system for obstructive thrombosis. FTIAN inhibited the generation of H2O2 and suppressed the expression of tumor necrosis factor-alpha (TNF-α) and soluble CD40 ligand (sCD40L) in activated platelets, demonstrating its intrinsic antioxidant, anti-inflammatory, and antiplatelet activity. In a mouse model of ferric chloride (FeCl3)-induced carotid thrombosis, FTIAN specifically targeted the obstructive thrombus and significantly enhanced the fluorescence/photoacoustic signal. When loaded with the antiplatelet drug tirofiban, FTIAN remarkably suppressed thrombus formation. Given its thrombus-specific imaging along with excellent therapeutic activities, FTIAN offers tremendous translational potential as a nanotheranostic agent for obstructive thrombosis.
Subject(s)
Carotid Artery Thrombosis/diagnostic imaging , Carotid Artery Thrombosis/drug therapy , Fibrin/metabolism , Fibrinolytic Agents/therapeutic use , Fluorescent Dyes/chemistry , Hydrogen Peroxide/metabolism , Nanoparticles/chemistry , Animals , Boronic Acids/chemistry , CD40 Ligand/metabolism , Carotid Artery Thrombosis/chemically induced , Carotid Artery Thrombosis/metabolism , Cell Survival/drug effects , Chlorides , Drug Carriers , Drug Liberation , Endothelial Cells/cytology , Endothelial Cells/drug effects , Ferric Compounds , Fibrinolytic Agents/chemistry , Humans , Lipopeptides/chemistry , Mice , Optical Imaging , Polymers , RAW 264.7 Cells , Theranostic Nanomedicine , Thrombosis/diagnostic imaging , Thrombosis/drug therapy , Thrombosis/metabolism , Tirofiban/chemistry , Tirofiban/therapeutic use , Tumor Necrosis Factor-alpha/metabolismABSTRACT
Tick saliva is a rich source of antihemostatic compounds. We amplified a cDNA from the salivary glands of the tropical bont tick (Amblyomma variegatum) using primers based on the variegin sequence, which we previously identified as a novel thrombin inhibitor from the same tick species. The transcript encodes a precursor protein comprising a signal peptide and 5 repeats of variegin-like sequences that could be processed into multiple short peptides. These peptides share 31 to 34% identity with variegin. Here, we structurally and functionally characterized one of these peptides named "avathrin." Avathrin is a fast, tight binding competitive inhibitor with an affinity of 545 pM for thrombin and is 4 orders of magnitude more selective towards thrombin than to the other serine proteases of the coagulation cascade. The crystal structure of thrombin-avathrin complex at 2.09 Å revealed that avathrin interacts with the thrombin active site and exosite-I. Although avathrin is cleaved by thrombin, the C-terminal cleavage product continues to exert prolonged inhibition. Avathrin is more potent than hirulog-1 in a murine carotid artery thrombosis model. Such precursor proteins that could be processed into multiple thrombin inhibiting peptides appear to be widespread among Amblyomminae, providing an enormous library of molecules for development as potent antithrombotics.-Iyer, J. K., Koh, C. Y., Kazimirova, M., Roller, L., Jobichen, C., Swaminathan, K., Mizuguchi, J., Iwanaga, S., Nuttall, P. A., Chan, M. Y., Kini, R. M. Avathrin: a novel thrombin inhibitor derived from a multicopy precursor in the salivary glands of the ixodid tick, Amblyomma variegatum.
Subject(s)
Ixodidae/metabolism , Peptides/pharmacology , Salivary Glands/metabolism , Thrombin/antagonists & inhibitors , Amino Acid Sequence , Animals , Arthropod Proteins , Carotid Artery Thrombosis/chemically induced , Carotid Artery Thrombosis/drug therapy , Cattle , Chlorides/toxicity , Cloning, Molecular , Female , Ferric Compounds/toxicity , Fibrinogen/metabolism , Humans , Kallikreins/metabolism , Male , Mice , Mice, Inbred C57BL , Nymph , Salivary Glands/chemistry , Trypsin/metabolismABSTRACT
UNLABELLED: Essentials Microembolic signal (MES) is an independent predictor of stroke risk in patients. A rabbit model of cerebral microembolic signals was established. Therapeutic efficacy was demonstrated for aspirin and clopidogrel on microembolic signals. Potential translational value of this preclinical model of MES was demonstrated. SUMMARY: Objectives Cerebral microembolic signals (MESs) detected by transcranial Doppler (TCD) ultrasound constitute an independent predictor of stroke risk and prognosis. The aim of this study was to develop a novel preclinical model of MESs to facilitate translational research. Methods A clinical TCD ultrasound machine was used to detect MESs in the cerebral circulation of New Zealand White rabbits. Technical feasibility was assessed for the measurement of MESs in the middle cerebral artery (MCA) by TCD. FeCl3 -induced carotid arterial thrombosis was optimized for the generation of endogenous microemboli. Ascending doses of two antithrombotic agents (aspirin and clopidogrel) were evaluated individually and in combination for their effects on both arterial thrombosis and MESs in a 30% FeCl3 -induced carotid arterial thrombosis model, along with ex vivo functional assays. Results Dose-dependent FeCl3 -induced arterial thrombosis studies showed that 30% FeCl3 resulted in the most consistent and reproducible MESs in the MCA (3.3 ± 0.7 MESs h(-1) ). Ascending-dose studies showed that the effective doses for 50% inhibition (ED50 ) of thrombus formation, based on integrated blood flow and thrombus weight, respectively, were 3.1 mg kg(-1) and 4.2 mg kg(-1) orally for aspirin, and 0.3 mg kg(-1) and 0.28 mg kg(-1) orally for clopidogrel. The ED50 values for MES incidence were 12.7 mg kg(-1) orally for aspirin, and 0.25 mg kg(-1) orally for clopidogrel. Dual treatment with aspirin (5 mg kg(-1) ) and clopidogel (0.3 mg kg(-1) ) resulted in significant reductions in cerebral MESs (P < 0.05) as compared with monotherapy with either agent. Conclusions Our study demonstrated the successful establishment of the MES model in rabbits, and it may provide translational value for MESs and ischemic stroke research.
Subject(s)
Aspirin/therapeutic use , Intracranial Embolism/drug therapy , Stroke/drug therapy , Ticlopidine/analogs & derivatives , Animals , Carotid Artery Thrombosis/chemically induced , Carotid Artery Thrombosis/drug therapy , Chlorides , Clopidogrel , Disease Models, Animal , Drug Evaluation, Preclinical , Ferric Compounds , Fibrinolytic Agents/therapeutic use , Intracranial Embolism/physiopathology , Male , Middle Cerebral Artery/physiopathology , Platelet Aggregation , Rabbits , Stroke/complications , Ticlopidine/therapeutic use , Translational Research, Biomedical , Ultrasonography , Ultrasonography, DopplerABSTRACT
BACKGROUND: Platelets are central to the process of hemostasis, rapidly aggregating at sites of blood vessel injury and acting as coagulation nidus sites. On interaction with the subendothelial matrix, platelets are transformed into balloonlike structures as part of the hemostatic response. It remains unclear, however, how and why platelets generate these structures. We set out to determine the physiological relevance and cellular and molecular mechanisms underlying platelet membrane ballooning. METHODS AND RESULTS: Using 4-dimensional live-cell imaging and electron microscopy, we show that human platelets adherent to collagen are transformed into phosphatidylserine-exposing balloonlike structures with expansive macro/microvesiculate contact surfaces, by a process that we termed procoagulant spreading. We reveal that ballooning is mechanistically and structurally distinct from membrane blebbing and involves disruption to the platelet microtubule cytoskeleton and inflation through fluid entry. Unlike blebbing, procoagulant ballooning is irreversible and a consequence of Na(+), Cl(-), and water entry. Furthermore, membrane ballooning correlated with microparticle generation. Inhibition of Na(+), Cl(-), or water entry impaired ballooning, procoagulant spreading, and microparticle generation, and it also diminished local thrombin generation. Human Scott syndrome platelets, which lack expression of Ano-6, also showed a marked reduction in membrane ballooning, consistent with a role for chloride entry in the process. Finally, the blockade of water entry by acetazolamide attenuated ballooning in vitro and markedly suppressed thrombus formation in vivo in a mouse model of thrombosis. CONCLUSIONS: Ballooning and procoagulant spreading of platelets are driven by fluid entry into the cells, and are important for the amplification of localized coagulation in thrombosis.
Subject(s)
Blood Platelets/ultrastructure , Acetazolamide/pharmacology , Actomyosin/metabolism , Amides/pharmacology , Animals , Anoctamins , Blood Coagulation Disorders/blood , Blood Platelets/drug effects , Blood Platelets/metabolism , Carotid Artery Thrombosis/blood , Carotid Artery Thrombosis/chemically induced , Carotid Artery Thrombosis/drug therapy , Cell Adhesion , Cell Membrane/drug effects , Cell Membrane/metabolism , Cell Membrane/ultrastructure , Cell Shape/drug effects , Cell Shape/physiology , Cell Size/drug effects , Cell-Derived Microparticles , Chlorides/metabolism , Collagen , Cytochalasin D/pharmacology , Heterocyclic Compounds, 4 or More Rings/pharmacology , Humans , Mice , Microtubules/drug effects , Phospholipid Transfer Proteins/deficiency , Phospholipid Transfer Proteins/physiology , Pyridines/pharmacology , Sodium/metabolism , Thrombin/biosynthesis , Thrombosis/prevention & control , Water/metabolismABSTRACT
Severe thrombosis and its ischemic consequences such as myocardial infarction, pulmonary embolism and stroke are major worldwide health issues. The ferric chloride injury is now a well-established technique to rapidly and accurately induce the formation of thrombi in exposed veins or artery of small and large diameter. This model has played a key role in the study of the pathophysiology of thrombosis, in the discovery and validation of novel antithrombotic drugs and in the understanding of the mechanism of action of these new agents. Here, the implementation of this technique on a mesenteric vessel and carotid artery in mice is presented. The method describes how to label circulating leukocytes and platelets with a fluorescent dye and to observe, by intravital microscopy on the exposed mesentery, their accumulation at the injured vessel wall which leads to the formation of a thrombus. On the carotid artery, the occlusion caused by the clot formation is measured by monitoring the blood flow with a Doppler probe.
Subject(s)
Carotid Artery Thrombosis/chemically induced , Chlorides/administration & dosage , Disease Models, Animal , Ferric Compounds/administration & dosage , Mesentery/blood supply , Thrombosis/chemically induced , Animals , Carotid Artery Thrombosis/pathology , Male , Mice , Mice, Inbred C57BL , Microscopy, Fluorescence/methods , Thrombosis/pathologyABSTRACT
Antiphospholipid (aPL)/anti-ß2-glycoprotein I (ß2GPI) antibodies are considered to play a pivotal pathogenic role in antiphospholipid syndrome (APS) by inducing an intracellular signaling and procoagulant/proinflammatory phenotype that leads to thrombosis. There is increasing evidence that Toll-like receptor 4 (TLR4) could serve as an important molecule for anti-ß2GPI recognition on target cells. However, few studies have focused on the effects of TLR4 in in vivo models. Here, we investigated the role of TLR4 in the pathogenic effects of aPL/anti-ß2GPI more precisely using TLR4-intact (C3H/HeN) and TLR4-defective (C3H/HeJ) mice. C3H/HeN and C3H/HeJ mice were injected with either IgG isolated from patient with APS (IgG-APS) or epitope-specific anti-ß2GPI purified from ß2GPI peptide-immunized rabbits. We found that, following anti-ß2GPI injections and vascular injury, thrombus formation in both the carotid artery and femoral vein was markedly reduced in C3H/HeJ mice when compared with C3H/HeN mice. IgG-APS or anti-ß2GPI-induced carotid artery and peritoneal macrophage tissue factor activity/expression was significantly lesser in C3H/HeJ than in C3H/HeN mice. Furthermore, the IgG-APS or anti-ß2GPI induced expression of VCAM-1, ICAM-1, and E-selectin in the aorta and of IL-1ß, IL-6, and TNF-α in peritoneal macrophages of C3H/HeJ mice was also significantly reduced compared to C3H/HeN mice. Together, these data suggest that TLR4 is involved in the pathogenic effects of aPL/anti-ß2GPI antibodies in vivo.
Subject(s)
Antibodies, Antiphospholipid/immunology , Antiphospholipid Syndrome/immunology , Carotid Artery Thrombosis/immunology , Toll-Like Receptor 4/immunology , Venous Thrombosis/immunology , Animals , Carotid Artery Thrombosis/chemically induced , Cell Adhesion , Chlorides/toxicity , Disease Models, Animal , E-Selectin/metabolism , Femoral Vein , Ferric Compounds/toxicity , Immunoglobulin G/immunology , Inflammation Mediators/immunology , Intercellular Adhesion Molecule-1/metabolism , Interleukin-1beta/immunology , Interleukin-6/immunology , Lipopolysaccharides , Mice , Toll-Like Receptor 4/metabolism , Tumor Necrosis Factor-alpha/immunology , Vascular Cell Adhesion Molecule-1/metabolism , Venous Thrombosis/chemically induced , beta 2-Glycoprotein I/immunologyABSTRACT
Cigarette smoking is a major risk factor for acute coronary thrombosis. In fact, both active/first-hand smoke and passive/second-hand smoke exposure are known to increase the risk of coronary thrombosis. Although recently a new risk has been identified and termed third-hand smoke (THS), which is the residual tobacco smoke contaminant that remains after a cigarette is extinguished, it remains to be determined whether it can also enhance the risk of thrombogenesis, much like first-hand smoke and second-hand smoke. Therefore, the present studies investigated the impact of THS exposure in the context of platelet biology and related disease states. It was found that THS-exposed mice exhibited an enhanced platelet aggregation and secretion responses as well as enhanced integrin GPIIb-IIIa activation. Furthermore, it was found that THS exposure shortens the tail bleeding time and the occlusion time in a model of thrombosis. Thus, our data demonstrate for the first time (at least in mice) that THS exposure increases the risk of thrombosis-based disease states, which is attributed, at least in part, to their hyperactive platelets.
Subject(s)
Carotid Artery Thrombosis/chemically induced , Hemostasis/drug effects , Inhalation Exposure/adverse effects , Platelet Aggregation/drug effects , Tobacco Products/adverse effects , Tobacco Smoke Pollution/adverse effects , Animals , Carotid Artery Thrombosis/blood , Hemostasis/physiology , Mice , Mice, Inbred C57BL , Platelet Aggregation/physiologyABSTRACT
AIMS: Thymic stromal lymphopoietin (TSLP) plays an important role in inflammatory diseases and is over-expressed in human atherosclerotic artery specimens. The present study investigated the role of TSLP in platelet activation and thrombosis models in vitro and in vivo, as well as the underlying mechanism and signaling pathway. METHODS AND RESULTS: Western blotting and flow cytometry demonstrated that the TSLP receptor was expressed on murine platelets. According to flow cytometry, platelet stimulation with TSLP induced platelet degranulation and integrin αIIbß3 activation. A TSLPR deficiency caused defective platelet aggregation, defective platelet secretion and markedly blunted thrombus growth in perfusion chambers at both low and high shear rates. TSLPR KO mice exhibited defective carotid artery thrombus formation after exposure to FeCl3. TSLP increased Akt phosphorylation, an effect that was abrogated by the PI3K inhibitors wortmannin and LY294002. The PI3K inhibitors further diminished TSLP-induced platelet activation. TSLP-mediated platelet degranulation, integrin αIIbß3 activation and Akt phosphorylation were blunted in platelets that lacked the TSLP receptor. CONCLUSION: This study demonstrated that the functional TSLPR was surface-expressed on murine platelets. The inflammatory cytokine TSLP triggered platelet activation and thrombus formation via TSLP-dependent PI3K/Akt signaling, which suggests an important role for TSLP in linking vascular inflammation and thrombo-occlusive diseases.
Subject(s)
Blood Platelets/metabolism , Cytokines/pharmacology , Immunoglobulins/metabolism , Platelet Activation/drug effects , Platelet Aggregation/drug effects , Receptors, Cytokine/metabolism , Signal Transduction/drug effects , Androstadienes/pharmacology , Animals , Blood Platelets/drug effects , Carotid Artery Thrombosis/chemically induced , Carotid Artery Thrombosis/metabolism , Carotid Artery Thrombosis/pathology , Chlorides/toxicity , Chromones/pharmacology , Disease Models, Animal , Ferric Compounds/toxicity , Humans , Immunoglobulins/deficiency , Immunoglobulins/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Morpholines/pharmacology , P-Selectin/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Phosphoinositide-3 Kinase Inhibitors , Phosphorylation/drug effects , Platelet Glycoprotein GPIIb-IIIa Complex/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Receptors, Cytokine/deficiency , Receptors, Cytokine/genetics , Wortmannin , Thymic Stromal LymphopoietinABSTRACT
A 73-year-old woman who suddenly developed left hemiparesis was admitted to our hospital. Ultrasonography on admission showed a free-floating thrombus (FFT) attached to an ulcerative plaque in the right common carotid artery. The FFT almost disappeared during treatment with intravenous anticoagulation therapy for acute stroke, but it reappeared when the therapy was discontinued. She underwent endarterectomy on day 13, after which she was free from stroke recurrence.