Abstract 448: Platelets Contribute to Arterial and Venous Thrombosis in vivo

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Maria Koellnberger ◽  
Marie-Luise von Bruehl ◽  
Wolfgang Bergmeier ◽  
Denisa Wagner ◽  
Nigel Mackman ◽  
...  
Keyword(s):  
Thrombus Formation ◽  
Venous Stasis ◽  
Industrialized Countries ◽  
Cellular Mechanisms ◽  
Venous Thrombus ◽  
Cellular Events ◽  
Promising Strategy ◽  
Novel Model ◽  
Differential Contribution

Background: Arterial (AT) and venous thromboses (VT) are the major causes of morbidity in industrialized countries. While the critical contribution of platelets to AT has been recognized, the molecular and cellular mechanisms that trigger the development of VT are not yet fully understood. Methods: We established a novel model of VT, where thrombus formation occurs after venous stasis in the absence of mechanical endothelial injury. In contrast to published models, our approach closely resembles the triggers that lead to VT in humans. The molecular and cellular events in VT were assessed using intravital and electron microscopy. In particular, we evaluated the differential contribution of platelets (pts) and leukocytes (lcs) to AT and VT. Results: We found that in AT pts adhere rapidly, whereas only few lcs are recruited. The loss of platelet P-Selectin reduced lc accumulation during AT, was associated with thrombus destabilization, but had no effect on pt adhesion/aggregation. The loss of GPIIb led to a complete lack of pt adhesion/aggregation in AT. While AT largely depends on pts, lc adhesion was the prominent initial phenomenon during VT and involved both P-selectin and Mac-1. P-Selectin−/− as well as Mac-1−/− mice showed a massive reduction in lc accumulation. The lack of P-Selectin was accompanied by a reduction of the thrombus size 48 hours after stasis and was only marginally reduced in Mac 1−/− mice, indicating that lc rolling is sufficient to promote VT. The expression of tissue factor by lcs played a pivotal role for VT. Accordingly, mice with reduced TF expression showed a dramatic reduction in venous thrombus size. In addition to TF-bearing lcs, pts were recruited to the vessel wall early in VT. Pt accumulation involved both the fibrinogen and the vWF receptor and was virtually abolished in mice lacking GPIIb or GPIbα. To our surprise, we observed that pts do not accumulate during VT but play a functional role. Reduced pt adhesion in mice lacking either GPIIb, GPIbα, or VWF was associated with a significant reduction in venous thrombus size. Conclusion: Together, we show that VT, unlike AT depends on the concerted action of both lcs and pts. Hence, targeting pt activation and adhesion might provide a promising strategy in the prophylaxis and treatment of VT.

scholarly journals Drosophila Neuronal Injury Follows a Temporal Sequence of Cellular Events Leading to Degeneration at the Neuromuscular Junction

2015 ◽  
Vol 9s2 ◽  
pp. JEN.S25516 ◽  
Author(s):  
Barron L. Lincoln ◽  
Sahar H. Alabsi ◽  
Nicholas Frendo ◽  
Robert Freund ◽  
Lani C. Keller
Keyword(s):  
Neuromuscular Junction ◽  
Model Organism ◽  
Structural Defects ◽  
Cellular Mechanisms ◽  
Post Injury ◽  
Ubiquitinated Proteins ◽  
Cellular Events ◽  
Molecular Events ◽  
Complex Signaling

Neurodegenerative diseases affect millions of people worldwide, and as the global population ages, there is a critical need to improve our understanding of the molecular and cellular mechanisms that drive neurodegeneration. At the molecular level, neurodegeneration involves the activation of complex signaling pathways that drive the active destruction of neurons and their intracellular components. Here, we use an in vivo motor neuron injury assay to acutely induce neurodegeneration in order to follow the temporal order of events that occur following injury in Drosophila melanogaster. We find that sites of injury can be rapidly identified based on structural defects to the neuronal cytoskeleton that result in disrupted axonal transport. Additionally, the neuromuscular junction accumulates ubiquitinated proteins prior to the neurodegenerative events, occurring at 24 hours post injury. Our data provide insights into the early molecular events that occur during axonal and neuromuscular degeneration in a genetically tractable model organism. Importantly, the mechanisms that mediate neurodegeneration in flies are conserved in humans. Thus, these studies have implications for our understanding of the cellular and molecular events that occur in humans and will facilitate the identification of biomedically relevant targets for future treatments.

The Evaluation of a Prothrombotic Risk in a Human Fibrinogen Concentrate

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4064-4064
Author(s):  
Gerhard Dickneite ◽  
Christine Joch ◽  
Garrett E. Bergman
Keyword(s):  
Thrombus Formation ◽  
Factor Vii ◽  
Venous Stasis ◽  
Preclinical Model ◽  
Thromboembolic Events ◽  
Postmarketing Surveillance ◽  
Fibrinogen Concentrate ◽  
Human Fibrinogen ◽  
Spontaneous Reports

Abstract Fibrinogen is a circulating coagulant protein which is converted to polymerizable fibrin by thrombin. The human fibrinogen concentrate (FC) (CSL Behring) has been used for the treatment of bleeding in fibrinogen deficiency. In this investigation we investigated the prothrombotic potential of FC using the in-vivo, Wessler venous stasis assay in rabbits Thrombogenicity in clinical use was assessed in a postmarketing drug surveillance review. Forty-five rabbits were randomized to receive either placebo or 40 mg/kg of one of four different lots of FC, injected into a marginal ear vein. Thirty seconds thereafter, a segment of the right jugular vein (1.5 – 2.0 cm) was ligated. After 15 minutes the segment was removed and evaluated for thrombus formation according to a score system from 0 –3. Thrombogenic materials, such as activated prothrombin complexes, have consistently shown high scores of thrombus formation in this assay. The mean score in the FC treated rabbits (0.85 ± 1.04) was not different from that in the placebo-treated rabbits (0.8 ± 0.84). Postmarketing data was comprised of spontaneous reports to a postmarketing surveillance system for Adverse Drug Reports (ADR). The number of ADR reports was expressed relative to the number of estimated single standard doses. The estimated dose per patient was adjusted to the degree of bleeding (between one to eight gram). Between January 01, 1986 and April 30, 2008 in total 983,195 g of Haemocomplettan® P had been distributed. Very few reports (n=9) of thromboembolic events have been reported throughout postmarketing surveillance, corresponding to one case report for every 13,655 treatments of 8 grams FC. The reported thromboembolic events occurred in patients with congenital afibrinogenemia or acquired hypofibrinogenemia. In most cases, patients had additional risk factors (e.g. concomitant administration of blood products prothrombin complex concentrate, platelet concentrates, activated factor VII, etc.). Although a causal relationship to administration of FC could not be definitely excluded, the occurrence of thromboembolic events is very rare. From congruent results of both a classical preclinical model of thrombogenicity and analysis of postmarketing data, FC appears to have a very low risk of thrombogenicity.

scholarly journals C1-esterase inhibitor treatment: preclinical safety aspects on the potential prothrombotic risk

2014 ◽  
Vol 112 (11) ◽  
pp. 960-971 ◽  
Author(s):  
Elmar Raquet ◽  
Marc Nolte ◽  
Frauke May ◽  
Jochen Müller-Cohrs ◽  
Jenny Björkqvist ◽  
...  
Keyword(s):  
Thrombus Formation ◽  
Venous Stasis ◽  
High Dose ◽  
In Vivo Models ◽  
C1 Esterase Inhibitor ◽  
Activity Measurements ◽  
High Doses ◽  
Esterase Inhibitor

SummaryHuman plasma-derived C1-esterase inhibitor (C1–INH) is an efficacious and safe treatment for hereditary angioedema. However, thrombotic events in subjects treated with C1–INH at recommended or offlabel, high doses have been reported. In this study, we addressed the potential prothrombotic risk of C1–INH treatment in high doses using a non-clinical rabbit model. Following intravenous infusion of C1–INH to rabbits at doses up to 800 IU/kg, the exposure and the pharmacodynamic efficacy of C1–INH in rabbits were confirmed by activity measurements of C1-esterase, and coagulation factors XIa and XIIa, respectively. Potential prothrombotic effects were assessed following induction of venous and arterial thrombosis using in vivo models of venous and arterial stasis, complemented by various in vitro assays of coagulation markers. Administration of C1–INH at doses up to 800 IU/ kg did not potentiate thrombus formation during venous stasis. In contrast, inhibition of arterial occlusion was observed upon C1–INH administration when compared with isotonic saline treatment, indicating antithrombotic rather than prothrombotic activity of high dose C1–INH treatment in vivo. This was further confirmed in vitro by decreased thrombin generation, increased activated partial thromboplastin time, clotting time and clot formation time, and inhibition of platelet aggregation. No relevant changes in fibrinolysis or in the levels of thrombin-antithrombin complexes, and prothrombin fragment 1+2 were observed upon high dose C1–INH treatment. The data suggest that treatment of healthy rabbits with high doses of C1–INH could potentially inhibit coagulation and thrombus formation rather than induce a prothrombotic risk.

Ultra-low-dose opioid antagonists to enhance opioid analgesia

2006 ◽  
Vol 2 (5) ◽  
pp. 295 ◽  
Author(s):  
Paul Sloan, MD ◽  
Scott Hamann, PhD, MD
Keyword(s):  
Low Dose ◽  
Neuronal Cell ◽  
Opioid Antagonists ◽  
Cellular Mechanisms ◽  
Opioid Agonist ◽  
History Of ◽  
Primary Focus ◽  
Novel Model

This article will review decades of science contributing to current interest in opioid excitatory pharmacology. A long history of clinical confusion provided the stimulus for recent, detailed in vivo and in vitro investigations of the neuropharmacologic mechanisms involved in analgesic and hyperalgesic actions of opioid agonists and antagonists. Following the discovery of central nervous system opioid excitatory-hyperalgesic processes in animals, detailed neuronal cell culture experiments established opioid receptor/G protein/adenylate cyclase neurobiochemical mechanisms for bimodal inhibitory versus excitatory actions of opioids. Once this novel model was available to explain the cellular mechanisms responsible for the duality of opioid actions, clinical translation of this technology began to emerge, with a primary focus on selective antagonism of opioid excitatory actions with concomitant low-dose opioid antagonists. Encouraging results from recent animal and clinical studies will be discussed as further evidence that therapeutic pain management may be improved through enhancement of opioid agonist analgesia by cotreatment with ultra-low-dose opioid antagonists that selectively attenuate opioid-mediated hyperalgesia.

Plasmin Generation Plays different Roles in the Formation and Removal of Arterial and Venous Thrombus in Mice

2002 ◽  
Vol 87 (01) ◽  
pp. 98-104 ◽  
Author(s):  
Osamu Kozawa ◽  
Kiyotaka Okada ◽  
Shigeru Ueshima ◽  
Osamu Matsuo ◽  
Toshihiko Uematsu ◽  
...  
Keyword(s):  
Carotid Artery ◽  
Bleeding Time ◽  
Jugular Vein ◽  
Thrombus Formation ◽  
Endothelial Injury ◽  
Wild Type ◽  
Venous Thrombus ◽  
Vascular Patency ◽  
The Times

SummaryThe role of plasminogen (Plg) and α2-antiplasmin (α2-AP) in vascular thrombolysis in vivo was investigated in mice deficient in plasminogen (Plg−/−) or α2-AP (α2-AP−/−) or their wild type (PAI-1+/+, α2AP+/+). A thrombus was induced in the murine carotid artery or the internal jugular vein by endothelial injury. Blood flow was continuously monitored for 90 min and for 6 h 30 min after the initiation of endothelial injury. The times to occlusion by the developing thrombus in the carotid artery and the jugular vein of wild type mice were 12 ± 1.8 and 7.2 ± 1.9 min, respectively. The arterial thrombus formation in α2AP−/− mice was indistinguishable from the one in wild type mice, whereas the time to occlusion in Plg−/− was significantly shortened to 5.9 ± 1.7 min. Vascular patency after spontaneous reperfusion was markedly improved in α2-AP−/− mice. On the contrary, arterial patency in Plg−/− mice was aggravated. In venous thrombus formation, the time to occlusion in α2-AP−/− mice was significantly prolonged (27.1 ± 5.2 min), whereas in Plg−/− it was slightly shortened to 6.5 ± 2.5 min. Vascular patency after spontaneous reperfusion was also improved in α2-AP−/− mice, but not in Plg−/− mice. Histological observations using SEM indicated that fibrin nets were firmly fixed on the injured area in Plg−/− mice, but not in α2-AP−/− mice. The tail bleeding time was not different in any type of mice. However, re-bleeding time using a template bleeding device was significantly prolonged in α2-AP−/− as compared with that of wild type mice. In conclusion, lack of plasminogen markedly reduces the antithrombotic activities in vivo, whereas α2-AP plays a more important role in the formation and removal of venous thrombus in mice. Consequently, the inhibition of α2-AP could be a useful tool for the therapy of venous thrombosis and the prevention of re-thrombus formation.

Comparison of the In Vivo Anticoagulant Properties of Standard Heparin and the Highly Selective Factor Xa Inhibitors Antistasin and Tick Anticoagulant Peptide (TAP) in a Rabbit Model of Venous Thrombosis

1991 ◽  
Vol 65 (03) ◽  
pp. 257-262 ◽  
Author(s):  
George P Vlasuk ◽  
Denise Ramjit ◽  
Tsuneo Fujita ◽  
Christopher T Dunwiddie ◽  
Elka M Nutt ◽  
...  
Keyword(s):  
Venous Thrombosis ◽  
Thrombus Formation ◽  
Rabbit Model ◽  
Factor Xa ◽  
Clot Formation ◽  
Factor Xa Inhibitors ◽  
Venous Stasis ◽  
Thrombosis Model ◽  
Tick Anticoagulant Peptide

SummaryAn in vivo thromboplastin (TP)-induced venous stasis thrombosis model in rabbits was used to compare the efficacy of standard heparin with the selective factor Xa inhibitors, recombinant tick anticoagulant peptide (rTAP) and recombinant antistasin (rATS), in prophylactic prevention of thrombus formation. Heparin significantly reduced TP-induced clot formation at doses of 55 and 100 U kg−1 h−1 yielding clot weights of 9 ± 4 and 6 ± 2%, respectively. Clot formation was significantly decreased by i.v. infusions of rTAP at doses of 21, 37 and 64 Μg kg−1 min−1 resulting in normalized clot weights of 13 ± 3, 8 ± 2 and 2 ± 1%, respectively. rATS was approximately 10-fold more potent than rTAP, reducing normalized clot weights to 16 ± 5, 2 ± 1 and 1 ± 0.8% at rATS doses of 1.25, 2.5 and 5.0 Μg kg−1 min−1, respectively. These data suggest that factor Xa-mediated inhibition of coagulation with rTAP and rATS is as effective as conventional anticogulant treatment with heparin in preventing venous thrombosis.

Abstract 9389: Thrombus Formation Processes are Dependent on Endothelial Injuries: Examined by in vivo Two-photon Molecular Imaging

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Satoshi Nishimura
Keyword(s):  
Imaging System ◽  
Thrombus Formation ◽  
Cellular Mechanisms ◽  
Cell Dynamics ◽  
Tlr4 Signaling ◽  
Two Photon Microscopy ◽  
Two Photon ◽  
Inflammatory Status ◽  
Thrombotic Diseases

Aim: The cellular mechanisms of thrombotic diseases associated with cardiovascular events remains unclear, largely because of an inability to visualize thrombus formation. Thus, we developed in vivo imaging technique based on single- and multi-photon microscopy and laser injuries technique to revealed the multicellular processes during thrombus development. Methods: We visualized the cell dynamics including single platelet behavior, and assessed dynamic cellular interplay in two thrombosis models using two photon microscopy to CAG-eGFP mice (Figure a, b). Results: First, we visualized that rapidly developing thrombi composed of discoid platelets without EC disruption was triggered by ROS induced by laser irradiation (Figure c). In this model, thrombus consisted by discoid platelet aggregations without leukocyte recruitment and not affected by Gr-1 antibody. The second model is, thrombus with EC disruption. High power laser induced EC erosion and extravasations of circulating leukocytes with thrombus development. Inflammatory cytokine, adhesion molecules dynamically control these two processes, and Gr-1 antibody significantly suppressed these steps. (Figure d) Leukocyte was immediately recruited into the subendothelial layers with bleeding and hemostatic reactions, and TLR4 signaling contributed to these steps. Pretreatmet of LPS or ischemic procedures markedly enhanced these steps. Thrombus included calcium activated cores and deformed platelets. Immigrated leukocyte also showed the increase of intracellular calcium. Summary: These results indicated that endothelial function, especially inflammatory status, determined the thrombotic reaction. Leukocyte also contributed with TLR4 signaling. In sum, using our imaging system can be a powerful tool to analyze thrombus formation and evaluate the therapeutic strategies.

scholarly journals Discovery of an intrinsic tenase complex inhibitor: Pure nonasaccharide from fucosylated glycosaminoglycan

2015 ◽  
Vol 112 (27) ◽  
pp. 8284-8289 ◽  
Author(s):  
Longyan Zhao ◽  
Mingyi Wu ◽  
Chuang Xiao ◽  
Lian Yang ◽  
Lutan Zhou ◽  
...  
Keyword(s):  
Adverse Effects ◽  
Bleeding Risk ◽  
Selective Inhibition ◽  
Selective Inhibitor ◽  
Enzyme Complex ◽  
Venous Thrombus ◽  
Promising Strategy ◽  
Rate Limiting ◽  
Coagulation Pathway

Selective inhibition of the intrinsic coagulation pathway is a promising strategy for developing safer anticoagulants that do not cause serious bleeding. Intrinsic tenase, the final and rate-limiting enzyme complex in the intrinsic coagulation pathway, is an attractive but less explored target for anticoagulants due to the lack of a pure selective inhibitor. Fucosylated glycosaminoglycan (FG), which has a distinct but complicated and ill-defined structure, is a potent natural anticoagulant with nonselective and adverse activities. Herein we present a range of oligosaccharides prepared via the deacetylation–deaminative cleavage of FG. Analysis of these purified oligosaccharides reveals the precise structure of FG. Among these fragments, nonasaccharide is the minimum fragment that retains the potent selective inhibition of the intrinsic tenase while avoiding the adverse effects of native FG. In vivo, the nonasaccharide shows 97% inhibition of venous thrombus at a dose of 10 mg/kg in rats and has no obvious bleeding risk. This nonasaccharide may therefore serve as a novel promising anticoagulant.

Recombinant Desulphatohirudin (CGP 39393) Anticoagulant and Antithrombotic Properties In Vivo

1989 ◽  
Vol 61 (01) ◽  
pp. 077-080 ◽  
Author(s):  
M D Talbot ◽  
J Ambler ◽  
K D Butler ◽  
V S Findlay ◽  
K A Mitchell ◽  
...  
Keyword(s):  
Partial Thromboplastin Time ◽  
Thrombus Formation ◽  
Activated Partial Thromboplastin Time ◽  
Venous Stasis ◽  
Thrombin Inhibitor ◽  
Antithrombotic Activity ◽  
Biotechnology Product ◽  
Acceptable Range ◽  
Antithrombotic Effects

SummaryThe effects of the newly available biotechnology product, recombinant desulphatohirudin (CGP 39393) have been investigated in rats. This highly potent and selective thrombin inhibitor exhibited marked anticoagulant properties with controllable titration of anticoagulant effect, as measured by activated partial thromboplastin time (APTT), up to nearly four times control values. Furthermore, CGP 39393 exhibited impressive antithrombotic activity in vivo. In an arteriovenous shunt model of thrombus formation on a cotton-thread, the compound was capable of complete inhibition of thrombus development (ED50 = 0.3 mg/kg i.v. and 1.0 mg/kg s.c.). Venous stasis thrombosis was also highly susceptible to inhibition by CGP 39393 (ED50 = 0.01 mg/kg i.v. and 0.45 mg/kg s.c.). Comparison of the anticoagulant and antithrombotic activities of the compound shows that potent antithrombotic effects (83-97% inhibition in the rat shunt model) are achieved within the generally acceptable range of anticoagulation. These results suggest a clear potential for this new agent in the clinical treatment of thrombotic disease.

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