Thrombus formation: direct real-time observation and digital analysis of thrombus assembly in a living mouse by confocal and widefield intravital microscopy

Celi, Alessandro; Merrill-Skoloff, Glenn; Gross, Peter L.; Falati, Shahrokh; Sim, Derek; Flaumenhaft, Robert; Furie, Bruce

doi:10.1046/j.1538-7836.2003.t01-1-00033.x

Cited by 112 publications

(85 citation statements)

References 8 publications

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“…Finally, for each frame, the integrated fluorescence intensity was calculated as per following equation: integrated fluorescence intensity = sum Intensity of signal -(mean of the maximal background intensity × area of the signal). This calculation was performed for all frames in each thrombus and plotted versus time to provide the kinetics of thrombus formation (8,27,48,49). For multiple fluorescence channels, calculations of background were made independently for each channel.…”

Section: Figurementioning

confidence: 99%

Protein disulfide isomerase inhibitors constitute a new class of antithrombotic agents

Jasuja

Passam²,

Kennedy³

et al. 2012

J. Clin. Invest.

265

345

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Thrombosis, or blood clot formation, and its sequelae remain a leading cause of morbidity and mortality, and recurrent thrombosis is common despite current optimal therapy. Protein disulfide isomerase (PDI) is an oxidoreductase that has recently been shown to participate in thrombus formation. While currently available antithrombotic agents inhibit either platelet aggregation or fibrin generation, inhibition of secreted PDI blocks the earliest stages of thrombus formation, suppressing both pathways. Here, we explored extracellular PDI as an alternative target of antithrombotic therapy. A high-throughput screen identified quercetin-3-rutinoside as an inhibitor of PDI reductase activity in vitro. Inhibition of PDI was selective, as quercetin-3-rutinoside failed to inhibit the reductase activity of several other thiol isomerases found in the vasculature. Cellular assays showed that quercetin-3-rutinoside inhibited aggregation of human and mouse platelets and endothelial cell-mediated fibrin generation in human endothelial cells. Using intravital microscopy in mice, we demonstrated that quercetin-3-rutinoside blocks thrombus formation in vivo by inhibiting PDI. Infusion of recombinant PDI reversed the antithrombotic effect of quercetin-3-rutinoside. Thus, PDI is a viable target for small molecule inhibition of thrombus formation, and its inhibition may prove to be a useful adjunct in refractory thrombotic diseases that are not controlled with conventional antithrombotic agents.

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Section: Figurementioning

confidence: 99%

Protein disulfide isomerase inhibitors constitute a new class of antithrombotic agents

Jasuja

Passam²,

Kennedy³

et al. 2012

J. Clin. Invest.

265

345

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show abstract

“…38,39 Briefly, each mouse was anesthetized using an intraperitoneal injection of pentobarbitol sodium (80 mg/kg; Abbott Laboratories) and maintained with the same anesthetic delivered via a catheterized jugular vein, as needed. Microvessels were studied using an Olympus BX61WI microscope (Olympus) equipped with a 40ϫ/0.8 numeric aperture water-immersion objective lens.…”

Section: Cremaster Laser Injury Modelmentioning

confidence: 99%

Monocyte-bound PF4 in the pathogenesis of heparin-induced thrombocytopenia

Rauova

Hirsch

Greene

et al. 2010

Blood

144

160

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Heparin-induced thrombocytopenia (HIT)is a life-and limb-threatening thrombotic disorder that develops after exposure to heparin, often in the setting of inflammation. We have shown previously that HIT is associated with antibodies to complexes that form between platelet factor 4 and glycosaminoglycan (GAG) side chains on the surface of platelets. However, thrombosis can occur in the absence of thrombocytopenia. We now show that platelet factor 4 binds to monocytes and forms antigenic complexes with their surface GAG side chains more efficiently than on platelets likely due to differences in GAG composition. Binding to monocytes is enhanced when the cells are activated by endotoxin. Monocyte accumulation within developing arteriolar thrombi was visualized by situ microscopy. Monocyte depletion or inactivation in vivo attenuates thrombus formation induced by photochemical injury of the carotid artery in a modified murine model of HIT while paradoxically exacerbating thrombocytopenia. These studies demonstrate a previously unappreciated role for monocytes in the pathogenesis of arterial thrombosis in HIT and suggest that therapies targeting these cells might provide an alternative approach to help limit thrombosis in this and possibly other thrombotic disorders that occur in the setting of inflammation. IntroductionPlatelet factor 4 (PF4) is a cationic chemokine with high affinity for unfractionated heparin (UFH) and other large, negatively charged molecules. 1 PF4 is stored in platelet ␣-granules, released upon activation, when it then binds rapidly to glycosaminoglycan (GAG) side chains expressed on the surface of platelets 2 and other vascular cells, with little remaining free in the circulation. 3 Heparin-induced thrombocytopenia (HIT) is an iatrogenic complication of heparin therapy caused by antibodies that recognize complexes of human (h) PF4 with heparin or other GAGs. 4,5 In solution, formation of antigenic complexes between PF4 and heparin is critically dependent on their molar ratio, with loss of antibody binding when the optimal ratio is disrupted by an excess of either component. 6,7 Antigen formation on the platelet surface also follows a bell-shaped curve as PF4 concentration is increased, with maximal binding of antibody seen at an exogenous PF4 concentration of 50 g/mL. 8 Chondroitin sulfates (CSs) are the predominant GAG side chains expressed on platelets. 9,10 We have shown that the binding of the HIT-like monoclonal antibody KKO 11 to platelets is abrogated by chondroitinase ABC, 8 indicating that HIT antibodies bind to PF4/CS complexes on this cell type. Therapeutic concentrations of UFH disrupt antibody binding in part by eluting PF4 from the platelet surface, which reduces formation of antigenic complexes and the potential for platelet activation 8 through platelet Fc␥RIIA. 12 Thus, variation in the expression of platelet-derived PF4 (or CS) might help to explain why only a small percentage of patients who generate antibodies to PF4/UFH develop HIT. 13 Although it has been generally accepte...

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“…Microvessel data were obtained using an Olympus AX microscope with a 60× 0.9 NA water immersion objective. The fluorescence microscopy system has previously been described (47). Digital images were captured with a Cooke Sensicam CCD camera in 640 × 480-pixel format.…”

Section: Figurementioning

confidence: 99%

Bile salt–dependent lipase interacts with platelet CXCR4 and modulates thrombus formation in mice and humans

Panicot‐Dubois¹,

Thomas²,

Furie³

et al. 2007

J. Clin. Invest.

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Bile salt-dependent lipase (BSDL) is an enzyme involved in the duodenal hydrolysis and absorption of cholesteryl esters. Although some BSDL is transported to blood, the role of circulating BSDL is unknown. Here, we demonstrate that BSDL is stored in platelets and released upon platelet activation. Because BSDL contains a region that is structurally homologous to the V3 loop of HIV-1, which binds to CXC chemokine receptor 4 (CXCR4), we hypothesized that BSDL might bind to CXCR4 present on platelets. In human platelets in vitro, both BSDL and a peptide corresponding to its V3-like loop induced calcium mobilization and enhanced thrombin-mediated platelet aggregation, spreading, and activated α IIb β 3 levels. These effects were abolished by CXCR4 inhibition. BSDL also increased the production of prostacyclin by human endothelial cells. In a mouse thrombosis model, BSDL accumulated at sites of vessel wall injury. When CXCR4 was antagonized, the accumulation of BSDL was inhibited and thrombus size was reduced. In BSDL -/-mice, calcium mobilization in platelets and thrombus formation were attenuated and tail bleeding times were increased in comparison with those of wild-type mice. We conclude that BSDL plays a role in optimal platelet activation and thrombus formation by interacting with CXCR4 on platelets.

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Thrombus formation: direct real-time observation and digital analysis of thrombus assembly in a living mouse by confocal and widefield intravital microscopy

Cited by 112 publications

References 8 publications

Protein disulfide isomerase inhibitors constitute a new class of antithrombotic agents

Protein disulfide isomerase inhibitors constitute a new class of antithrombotic agents

Monocyte-bound PF4 in the pathogenesis of heparin-induced thrombocytopenia

Bile salt–dependent lipase interacts with platelet CXCR4 and modulates thrombus formation in mice and humans

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