Real-time in vivo imaging of platelets, tissue factor and fibrin during arterial thrombus formation in the mouse

Falati, Shahrokh; Gross, Peter L.; Merrill-Skoloff, Glenn; Furie, Barbara C.; Furie, Bruce

doi:10.1038/nm782

Cited by 611 publications

(618 citation statements)

References 11 publications

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“…Moreover, the type of activator (tissue factor versus factor XII activator) may constitute an important determinant in interpretation of coagulation kinetics using thromboelastography/thromboelastometry. As of today, the tissue factor pathway is largely acknowledged as the physiological trigger for in vivo generation of a sufficient haemostatic plug [15,16]. Nevertheless, several interesting studies adopted traditional contact activation [17][18][19][20].…”

Section: Introductionmentioning

confidence: 99%

Evaluation of coagulation kinetics using thromboelastometry—methodologic influence of activator and test medium

et al. 2010

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Introduction: Renewed interest has arisen in the use of thromboelastography/thromboelastometry in evaluating coagulation kinetics. The test medium, type of activator and its concentration may influence the interpretation of coagulation kinetics. This study aimed to investigate methodological influences of activator and test medium on thromboelastometric parameters of coagulation kinetics. Methods: Dynamic clot formation was evaluated by thromboelastometry using whole blood (WB), platelet rich plasma or platelet poor plasma employing different concentrations of extrinsic (tissue factor) and contact activator (synthasil) and with variable concentrations of phospholipids. Results: Plasma samples displayed prolonged clot initiation and enhanced clot propagation compared to WB. Clot firmness was markedly reduced in platelet poor plasma as compared to platelet rich plasma and whole blood. Increasing concentration of activator shortened the clot initiation and increased the velocity of clot propagation whereas terminal clot firmness remained unaffected. Platelets accelerated clot propagation and raised clot firmness. Phospholipids shortened the time of clot initiation and increased velocity of propagation, while clot firmness remained unchanged. Conclusion: Our results demonstrate that evaluation of coagulation kinetics using thromboelastometry vary according to the composition of the test medium, type-and concentration of activator, as well as the presence and concentration of phospholipids in the test reagent.Response to Reviewers: Response to reviewers.First and foremost, thank you very much for the positive and constructive review. We have tried our best to answer all questions and revised the manuscript accordingly.Reviewer #1: This is a nice methodological paper about the ROTEM device, which is well written by an experienced group.Minor comments 1) P3 , line 41: physiologically -corrected 2) P4, line 28: significantly -corrected 3) Please indicate molar concentrations of synthasil and Innovin, if possible Unfortunately, it is not possible to use molar concentrations. We are working on a detailed (including molar concentrations and activity) examination of various tissue factor sources, however that reached beyond the scope of the present study. 4) P9: followed -corrected 5) P11, line 38: please add . "in vitro [33] as well as coagulation activation in vivo, when the blood is only re-calcified [PMID: 16420574]" Thank you for this excellent point, we have added accordingly. 6) I am not sure whether I could reproduce the phospholipid preparation, maybe a graphical scheme could be helpfulWe have slightly rephrased 7) As many users of the ROTEM probably will not have the extra software, please consider providing graphical presentations of the CT and /or alpha angle in addition to MaxVel, which is a derived variable CT has been included. We found it too excessive to also show alpha. 8) Refs 21 and 26 appear identical to meWe have corrected the reference list accordingly Reviewer #2: The paper by Sorensen et ...

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Section: Introductionmentioning

confidence: 99%

Evaluation of coagulation kinetics using thromboelastometry—methodologic influence of activator and test medium

et al. 2010

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“…For example, while laser injury to the microvasculature 27,28 or the topical application of ferric chloride in the mouse microcirculation 29 leads to the activation of coagulation, thrombin generation and platelet thrombus formation, these models do not typically induce robust leukocyte recruitment and trafficking responses at the injured vessel wall. Part of this may be related to the study of thrombosis in the arterial circulation, where leukocyte-vessel wall interactions are far less prominent than in venules or veins.…”

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confidence: 99%

Thrombin-dependent intravascular leukocyte trafficking regulated by fibrin and the platelet receptors GPIb and PAR4

et al. 2015

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Thrombin is a central regulator of leukocyte recruitment and inflammation at sites of vascular injury, a function thought to involve primarily endothelial PAR cleavage. Here we demonstrate the existence of a distinct leukocyte-trafficking mechanism regulated by components of the haemostatic system, including platelet PAR4, GPIba and fibrin. Utilizing a mouse endothelial injury model we show that thrombin cleavage of platelet PAR4 promotes leukocyte recruitment to sites of vascular injury. This process is negatively regulated by GPIba, as seen in mice with abrogated thrombin-platelet GPIba binding (hGPIba D277N ). In addition, we demonstrate that fibrin limits leukocyte trafficking by forming a physical barrier to intravascular leukocyte migration. These studies demonstrate a distinct 'checkpoint' mechanism of leukocyte trafficking involving balanced thrombin interactions with PAR4, GPIba and fibrin. Dysregulation of this checkpoint mechanism is likely to contribute to the development of thromboinflammatory disorders.

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“…The animal was immobilized on the platform of a fluorescence microscope and the formation of thrombi monitored in vivo [35]. In addition, several variations of intravital microscopic methods have been applied [34], including the method of Falati et al [36], who infused coagulation-specific fluorescently labeled antibodies into mice and used confocal and widefield microscopy to study thrombosis in the cremaster muscle microcirculation. While these and other elegant and powerful systems for detecting thrombi in vivo reviewed by Day et al [34] are very useful, they require more expensive and complicated equipment, as well as more technical expertise, than the ferric chloride/Doppler blood flow system used in this work.…”

Section: Discussionmentioning

confidence: 99%

Engineered human soluble calcium-activated nucleotidase inhibits coagulation in vitro and thrombosis in vivo

Yang

Kirley

2008

Thrombosis Research

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Human soluble calcium-activated nucleotidase (human SCAN) is a homologue of the salivary anticoagulant apyrases injected by insects into their hosts to allow blood feeding. However, the human enzyme, unlike its insect counterparts, does not efficiently hydrolyze the platelet agonist, ADP. By site-directed mutagenesis, two mutant human SCANs were constructed and expressed in bacteria. Following refolding from inclusion bodies and purification, these enzymes were assessed for anticoagulant and anti-thrombotic efficacy. These engineered proteins include both active site mutations and a dimer interface mutation to increase the stability and ADPase activity of the modified human nucleotidase. The ADPase activity of these mutants increased more than ten fold. The E130Y/ K201M/E216M SCAN mutant efficiently inhibited platelet aggregation in vitro. In addition, the E130Y/K201M/T206K/T207E/E216M mutant inhibited jugular vein thrombosis in the murine ferric chloride-induced model of thrombosis, as assessed by laser Doppler blood flow measurements. The bed bug insect homologue of human SCAN was also expressed and purified, and used in these in vivo experiments as a benchmark to assess the therapeutic potential of the engineered human enzymes. The most active modified human enzyme was able to completely inhibit the thrombosis induced by ferric chloride at roughly double the protein dose used for the bed bug enzyme. Thus, for the first time, we show that an engineered form of this human protein is efficacious in an in vivo model of thrombosis, demonstrating that suitably modified human SCAN enzymes have therapeutic potential as anti-coagulant and anti-thrombotic therapeutic agents. This suggests their utility in future treatment strategies for thrombotic cardiovascular diseases, including myocardial infarctions and ischemic strokes. Keywordscalcium-activated nucleotidase; ferric chloride-induced thrombosis; aggregometry; platelets; anticoagulant proteins; laser Doppler blood flow Cardiovascular and cerebrovascular diseases continue to be the leading causes of death in the developed world, with more than 450,000 patients in the United States afflicted by stroke annually [1]. The platelet is a major contributor to occlusive thrombus formation in acute Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Conflict of Interest StatementThere are no conflicts of interest to declare. The sole funding for this study was obtained via NIH grant HL72882 to T.L.K. Mammals express a protein homologous to the nucleotidases found in the saliva of bloodsucking insects. In insects, these soluble pro...

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Real-time in vivo imaging of platelets, tissue factor and fibrin during arterial thrombus formation in the mouse

Cited by 611 publications

References 11 publications

Evaluation of coagulation kinetics using thromboelastometry—methodologic influence of activator and test medium

Evaluation of coagulation kinetics using thromboelastometry—methodologic influence of activator and test medium

Thrombin-dependent intravascular leukocyte trafficking regulated by fibrin and the platelet receptors GPIb and PAR4

Engineered human soluble calcium-activated nucleotidase inhibits coagulation in vitro and thrombosis in vivo

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