Protein Disulfide Isomerase Is Required for Fibrin Generation and Platelet Thrombus Formation In Vivo.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 292-292 ◽  
Author(s):  
Jaehyung Cho ◽  
Barbara C. Furie ◽  
Shaun R. Coughlin ◽  
Bruce Furie
Keyword(s):  
Tissue Factor ◽  
Platelet Activation ◽  
Thrombus Formation ◽  
Null Mouse ◽  
Fibrin Formation ◽  
Platelet Thrombus ◽  
Wall Injury ◽  
Protein Disulfide ◽  
Dose Dependent

Abstract Thiol isomerases catalyze disulfide oxidation, reduction and isomerization, playing an important role during protein synthesis. Recent studies suggest a role for protein disulfide isomerase (PDI), a prototype of the thiol isomerase family, in platelet function and regulation of tissue factor activity (Essex and Li. Curr Drug Targets. 2006; Chen and Hogg. J Thromb Haemost. 2006). To determine the role of intravascular PDI during thrombus formation, PDI expression, platelet accumulation, and fibrin generation were monitored following laser-induced arteriolar injury in the mouse cremaster muscle by intravital fluorescence microscopy. PDI antigen exhibited a time-dependent increase in the developing thrombus after vessel wall injury and remained associated with the thrombus. Infusion of bacitracin, a non-specific inhibitor of thiol isomerases, into the circulation inhibited platelet thrombus formation and fibrin generation in a dose-dependent manner. Infusion of a function-blocking monoclonal antibody to PDI (RL90) into the circulation of a wild type mouse also resulted in dose-dependent inhibition of platelet accumulation and fibrin generation. To determine whether PDI inhibits fibrin formation by blocking tissue factor activation, or by preventing platelet activation and the development of the membrane surface that is required for assembly of the tenase and the prothrombinase complex in vivo, we explored fibrin formation in mice lacking protease-activated receptor-4 (Par4). Although there is no stable accumulation of platelets and no platelet activation, fibrin formation is normal in the Par4 null mouse (Vandendries et al, Proc Natl Acad Sci USA. 2007), suggesting that fibrin generation in the laser-induced vessel injury model is independent of platelet activation. Infusion of the function-blocking anti-PDI antibody (RL90) into the circulation of a Par4 null mouse prior to vessel wall injury inhibited fibrin generation. These results indicate that PDI is required to generate tissue factor in a form that leads to thrombin generation and fibrin formation during thrombus development and is required for thrombus formation.

scholarly journals Endothelium-derived but not platelet-derived protein disulfide isomerase is required for thrombus formation in vivo

Blood ◽  
2010 ◽  
Vol 116 (22) ◽  
pp. 4665-4674 ◽  
Author(s):  
Reema Jasuja ◽  
Bruce Furie ◽  
Barbara C. Furie
Keyword(s):  
Endothelial Cells ◽  
Protein Disulfide Isomerase ◽  
Vessel Wall ◽  
Thrombus Formation ◽  
In Vivo Studies ◽  
Disulfide Isomerase ◽  
Platelet Thrombus ◽  
Wall Injury ◽  
Protein Disulfide

Protein disulfide isomerase (PDI) catalyzes the oxidation reduction and isomerization of disulfide bonds. We have previously identified an important role for extracellular PDI during thrombus formation in vivo. Here, we show that endothelial cells are a critical cellular source of secreted PDI, important for fibrin generation and platelet accumulation in vivo. Functional PDI is rapidly secreted from human umbilical vein endothelial cells in culture upon activation with thrombin or after laser-induced stimulation. PDI is localized in different cellular compartments in activated and quiescent endothelial cells, and is redistributed to the plasma membrane after cell activation. In vivo studies using intravital microscopy show that PDI appears rapidly after laser-induced vessel wall injury, before the appearance of the platelet thrombus. If platelet thrombus formation is inhibited by the infusion of eptifibatide into the circulation, PDI is detected after vessel wall injury, and fibrin deposition is normal. Treatment of mice with a function blocking anti-PDI antibody completely inhibits fibrin generation in eptifibatide-treated mice. These results indicate that, although both platelets and endothelial cells secrete PDI after laser-induced injury, PDI from endothelial cells is required for fibrin generation in vivo.

Protein Disulfide Isomerase Inhibitors: A New Class of Antithrombotic Agents

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 369-369 ◽  
Author(s):  
Reema Jasuja ◽  
Freda H. Passam ◽  
Daniel R Kennedy ◽  
Sarah H Kim ◽  
Lotte van Hessem ◽  
...  
Keyword(s):  
Protein Disulfide Isomerase ◽  
Thrombus Formation ◽  
Reductase Activity ◽  
Fibrin Deposition ◽  
Laser Injury ◽  
Injury Model ◽  
Platelet Thrombus ◽  
Protein Disulfide ◽  
Dose Dependent

Abstract Abstract 369 Protein disulfide isomerase (PDI) is a prototypical member of a large family of oxidoreductases that catalyze posttranslational disulfide exchange necessary for proper protein folding. Despite having an ER retention sequence, PDI has been identified at cellular locations outside the ER. PDI is secreted from platelets and endothelial cells upon agonist stimulation or vascular injury. Secreted PDI is essential for platelet thrombus formation and fibrin generation in vivo. Inhibition of PDI with a non specific thiol inhibitor bacitracin A or a specific inhibitory anti-PDI antibody RL90 leads to decreased thrombus formation and fibrin generation in vivo in the laser injury model of thrombosis in mice (Cho J. et al, 2008, J. Clin. Invest. 118:1123; Jasuja R. et al, 2010 Blood116:4665). We screened a 5000 compound library of known bioactive compounds using an insulin reduction assay with turbidimetric end point to identify potent and selective small molecule inhibitors of PDI. The screen identified 18 inhibitory compounds representative of 13 separate chemical scaffolds, including 3 flavonols. Rutin, a glycoside of the flavonol quercetin, was the most effective inhibitor and inhibited PDI reductase activity with an IC50 of 6.1 μM. Inhibition of PDI by rutin was confirmed in an additional fluorescence-based reductase assay using oxidized glutathione coupled to di-eosin (Di-E-GSSG). Rutin specifically inhibited PDI activity and did not affect reductase activity of other thiol isomerases ERp57, ERp72, ERp5, thioredoxin or thioredoxin reductase. PDI inhibition by rutin was fully and rapidly reversible, indicating that rutin does not covalently bind PDI. Evaluation of rutin binding to immobilized PDI using surface plasmon resonance indicated a KD of 2.8 μM. Quercetin-3-glucuronide, an abundant metabolite of rutin found in plasma, demonstrated an IC50 of 5.9 μM (3.5–10.1 μM, 95% confidence interval). Isoquercetin, hyperoside, and datiscin, other flavonols with a 3-O-glycosidic linkage also inhibited PDI reductase activity. Metabolites of rutin that lack a 3-O-glycoside such as tamarixetin, isorhamnetin, diosmetin, or quercetin did not inhibit PDI reductase activity, whether or not they are hydroxylated or methoxylated at the 3' and 4' positions on ring B of the flavonol backbone. Activation of washed human platelets induced by 50 μM AYPGKF, a PAR4 agonist, was reversibly inhibited by rutin in a dose-dependent manner. Rutin effectively blocked fibrin generation from laser activated human umbilical vein endothelial cells bathed in plasma with an IC50of approximately 5 μM and 95 % reduction in fibrin formation at 10 μM rutin (P<0.001). Intravenous infusion of rutin prior to vessel wall injury in a mouse laser injury model of thrombosis showed a dose dependent inhibition of both platelet thrombus formation and fibrin generation in vivo. Platelet thrombus size was reduced by 71% at 0.1 mg/kg and fibrin deposition was inhibited by 68% with an intravenous dose of 0.3 mg/kg. Orally administered rutin also demonstrated antithrombotic activity. However, diosmetin, a non derivatizable form of flavonol that cannot under glycosylation at position 3 of the C ring did not affect platelet thrombus size or fibrin deposition. Infused exogenous recombinant PDI can overcome the inhibitory effect of rutin on thrombus formation. These results indicate that PDI is the relevant antithrombotic target of rutin in vivo. Rutin is well tolerated at concentrations higher than that required to inhibit PDI activity in vivo. Thus, targeting extracellular PDI for antiplatelet and anticoagulant therapy may be a viable approach to prevent thrombosis in a setting of coronary artery disease, stroke and venous thromboembolism. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Accumulation of Tissue Factor into Developing Thrombi In Vivo Is Dependent upon Microparticle P-Selectin Glycoprotein Ligand 1 and Platelet P-Selectin

2003 ◽  
Vol 197 (11) ◽  
pp. 1585-1598 ◽  
Author(s):  
Shahrokh Falati ◽  
Qingde Liu ◽  
Peter Gross ◽  
Glenn Merrill-Skoloff ◽  
Janet Chou ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Blood Coagulation ◽  
Thrombus Formation ◽  
Recipient Mouse ◽  
Wild Type ◽  
Activated Platelets ◽  
Injury Model ◽  
Platelet Thrombus ◽  
Flowing Blood

Using a laser-induced endothelial injury model, we examined thrombus formation in the microcirculation of wild-type and genetically altered mice by real-time in vivo microscopy to analyze this complex physiologic process in a system that includes the vessel wall, the presence of flowing blood, and the absence of anticoagulants. We observe P-selectin expression, tissue factor accumulation, and fibrin generation after platelet localization in the developing thrombus in arterioles of wild-type mice. However, mice lacking P-selectin glycoprotein ligand 1 (PSGL-1) or P-selectin, or wild-type mice infused with blocking P-selectin antibodies, developed platelet thrombi containing minimal tissue factor and fibrin. To explore the delivery of tissue factor into a developing thrombus, we identified monocyte-derived microparticles in human platelet–poor plasma that express tissue factor, PSGL-1, and CD14. Fluorescently labeled mouse microparticles infused into a recipient mouse localized within the developing thrombus, indicating that one pathway for the initiation of blood coagulation in vivo involves the accumulation of tissue factor– and PSGL-1–containing microparticles in the platelet thrombus expressing P-selectin. These monocyte-derived microparticles bind to activated platelets in an interaction mediated by platelet P-selectin and microparticle PSGL-1. We propose that PSGL-1 plays a role in blood coagulation in addition to its known role in leukocyte trafficking.

scholarly journals Tannic Acid Inhibits Protein Disulfide Isomerase, Platelet Activation and Thrombus Formation

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2418-2418
Author(s):  
Li Zhu
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Tannic Acid ◽  
Protein Disulfide Isomerase ◽  
Thrombus Formation ◽  
Platelet Surface ◽  
Disulfide Isomerase ◽  
Protein Disulfide

Abstract Tannic acid (TA) was a polyphenol that harbors anti-oxidant capacity. A recent report implied that surface coating with TA might blunt thrombosis via altering the structure of fibrinogen. However, the effect of TA on platelet function and in vivo thrombus formation has not been reported. In this study, we showed that TA inhibits PDI activity and attenuates platelet activation. To explore the effects of TA on platelet aggregation, gel-filtered human platelets from healthy human donors were pretreated with TA (10/30/50 μM) or vehicle (0.9% sodium chloride) before being stimulated by various agonists. Turbidity analyses on a Chronolog aggregometer showed that TA dose-dependently inhibited platelet aggregation induced by thrombin, SFLLRN, GYQGQV, collagen, CRP, U46619, and ristocetin. Next, we employed flow cytometry (FACS) to determine the role of TA in platelet activation, including α-granule secretion and integrin activation. Pretreatment of platelets with TA led to significant reductions in surface P-selectin expression and soluble fibrinogen binding, supporting the inhibition of diverse platelet activation pathways. Supportively, platelet spreading on immobilized fibrinogen was significantly suppressed by TA treatment. In addition, cell viability assay with Almar blue agent showed no detrimental impact of TA on the survival of platelets. To ask whether the antiplatelet role of TA might be translated into an antithrombotic efficacy, we tested the effect of TA in both ex vivo and in vivo thrombosis models. Calcein-labeled human whole blood was perfused through microfluidic channels coated with collagen, and adherent platelets were visualized under a fluorescent microscopy. However, treatment with TA suppressed the number of adherent platelets under flow conditions. Moreover, in laser-induced mouse cremaster muscle arteries, administration of TA (5mg/kg) significantly reduced the size of forming thrombi compared with the vehicle. Verification of bleeding risk using tail truncation assay indicated no prolongation of bleeding time in mice receiving TA. Thus, TA shows an antiplatelet effect and may also attenuate thrombus formation. To gain a mechanistic insight to the role of TA in platelet function, we performed a molecular docking screen of the structure of TA and platelet surface proteins using the Autodock Vina software, which displayed the binding of TA with protein disulfide isomerase at the enzymatic active center. We then measured the impact of TA on PDI reductase activity with the dieosin glutathione disulfide assay in vitro (di-GSSG), showing that TA significantly inhibited PDI activity in a concentration-dependent manner. The results were verified in platelets using the 3-(N-Maleimidylpropionyl) biocytin (MPB) labeling, which showed that TA abrogated thrombin-stimulated free thiol formation on platelet surface. Supportively, FACS demonstrated that TA significantly suppressed the binding of fluorescent-labeled PDI to Mn2+-activated platelet integrin β3. Taken together, our findings demonstrated that TA inhibits PDI activity and may become a novel antithrombotic agent. Disclosures No relevant conflicts of interest to declare.

Role of Protein Disulfide Isomerase In Thrombus Formation In a Collagen-Induced Pathway of Thrombus Formation

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 345-345
Author(s):  
Lola Bellido Martin ◽  
Bruce Furie ◽  
Barbara C. Furie
Keyword(s):  
Platelet Activation ◽  
Ferric Chloride ◽  
Protein Disulfide Isomerase ◽  
Thrombus Formation ◽  
Disulfide Isomerase ◽  
Platelet Thrombus ◽  
Platelet Accumulation ◽  
Bacitracin A ◽  
Protein Disulfide

Abstract Abstract 345 We have demonstrated that platelet activation in vivo can take place via two pathways, one initiated by the generation of thrombin and the other initiated by the exposure of collagen on the injured vessel wall (Furie & Furie, 2008). In our laser-induced model of thrombosis, in which the endothelium is activated but intact, platelet activation by thrombin dominates and collagen is not required. In the widely used ferric chloride model of thrombosis the endothelium is denuded exposing collagen which leads to initial platelet activation. Using our laser-induced thrombosis model we previously demonstrated that protein disulfide isomerase is expressed on the vessel wall and within the platelet thrombus at the site of injury. Both bacitracin A, a non-specific inhibitor of thiol isomerases, and an inhibitory antibody specific for protein disulfide isomerase (RL90) block platelet thrombus formation and fibrin generation. Here we extend our study of the role of protein disulfide isomerase in thrombus formation to the ferric chloride model of thrombosis. We used intravital fluorescence microscopy in mouse arterioles exposed to filter paper saturated with 10% ferric chloride for 3 minutes. Protein disulfide isomerase, detected with a non-inhibitory polyclonal anti-protein disulfide isomerase antibody, accumulated in ferric chloride-induced platelet thrombi in cremaster arterioles. Bacitracin A (5 mg/mouse) delayed initiation of thrombus formation in mesenteric arterioles. Median time to initial platelet accumulation increased from 1 min in the absence of inhibitor to 4 min in the presence of inhibitor. In 4 out of 8 mice treated with 7 mg of bacitracin A platelet accumulation was completely inhibited. Similarly, bacitracin A prolonged the time to occlusion of ferric chloride-injured arterioles. Less than 50% of injured arterioles in mice treated with 5 mg of bacitracin A and only 25% of injured arterioles in mice treated with 7 mg of bacitracin A occluded after 30 minutes compared to a 100% of arterioles occluded in control saline treated mice. Pretreatment of mice with RL90 at 0.3, 1 or 3 μg/g mouse delayed the appearance of the first aggregates of platelets. Median time to initial platelet accumulation was prolonged from 0.6 min in the presence of isotype-matched control antibody (1 ug/g mouse) to 1.5 min in the presence of 0.3 mg/g mouse of RL90. Platelet accumulation was not observed in 1 out of 7 animals treated with RL90 at 1 mg/g mouse and in 3 out of 7 animals treated with RL90 at 3 mg/g mouse. RL90 also inhibited fibrin deposition after ferric chloride injury. Minimal fibrin was detected in the presence of RL90 at 1 μg/g mouse while fibrin appeared rapidly in mice treated with a control antibody. These data indicate that PDI is a component of a general regulatory pathway for initiation of thrombus formation. Disclosures: No relevant conflicts of interest to declare.

Thrombin-Dependent Platelet Activation Is VWF-Independent during Thrombus Formation In Vivo.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1510-1510
Author(s):  
Christophe Dubois ◽  
Laurence Panicot-Dubois ◽  
Justin F. Gainor ◽  
Barbara C. Furie ◽  
Bruce Furie
Keyword(s):  
Platelet Activation ◽  
Thrombus Formation ◽  
Specific Inhibitor ◽  
Calcium Mobilization ◽  
Wild Type ◽  
Platelet Surface ◽  
Platelet Thrombus ◽  
Platelet Accumulation ◽  
Kinetics Of

Abstract Adhesion to and activation of platelets at an injured vessel wall are critical events in the formation of a thrombus. Calcium mobilization is one marker of platelet activation. Of different agonists capable of activating platelets in vitro, thrombin, collagen and vWF have been described to induce calcium mobilization, leading to the formation of aggregates. Using high speed digital multichannel intravital microscopy, we characterized calcium mobilization during platelet activation and thrombus formation in genetically modified mice. The kinetics of platelet activation and accumulation after laser-induced injury in cremaster muscle arterioles of living mice were analyzed. In wild type mice, platelets adhered and accumulated rapidly at the site of laser-induced injury. Thrombi increased in size, reached a maximum size at 90–120 sec, decreased in size and then stabilized within 3 to 4 min post-injury. In vWF−/− mice, the kinetics of platelet accumulation followed the same pattern as in wild type mice. However, a significant albeit modest reduction in the size of each thrombus was observed in these genetically deficient mice in comparison with wild type mice. By ranking the thrombi by size, we observed that 40% of the thrombi formed in vWF−/− mice were present in the quadrant containing the smallest thrombi versus 18% for the wild type mice. Only 8% of the thrombi formed in vWF−/− mice were distributed in the quadrant containing the largest thrombi versus 32% for the wild type mice. In wild type mice treated with lepirudin, a specific inhibitor of thrombin activity, a small early accumulation of platelets was observed at about 16 sec whereas in untreated wild type mice this early accumulation is often obscured by subsequent thrombin-mediated platelet accumulation and activation. However, at the time of maximal thrombus size in wild-type mice, platelet accumulation in wild type mice was more than ten-fold greater than in wild type mice treated with lepirudin. The kinetics of platelet accumulation were similar in FcRγ−/− mice lacking GPVI, GPVI-depleted mice and wild type mice. Furthermore, depletion of GPVI from the platelet surface of vWF−/− mice or platelets of wild type mice treated with lepirudin did not alter the kinetics of platelet accumulation in those mice. By monitoring calcium mobilization per platelet engaged in the growing thrombus, we observed that elevated calcium levels in each platelet were similar in FcRγ−/−, GPVI depleted, vWF−/− and wild type mice. However in wild type mice treated with lepirudin, platelet calcium mobilization was almost completely inhibited in comparison with those observed in wild type mice. Our results indicate that thrombin is the major agonist leading to platelet activation after laser-induced injury. Collagen, as previously reported (Dubois, Blood.2006;107:3902) does not play a role in platelet thrombus formation after laser injury and, based on data reported here, does not play a role in platelet activation in this model. vWF is important for the growth of the platelet thrombus but is not required for initial platelet accumulation or platelet activation in vivo in this thrombosis model. The platelet agonist or ligand responsible for initial early platelet accumulation after laser-induced injury is unknown, and does not require GPVI, thrombin or vWF.

Rapid Activation of Unstimulated Endothelial Cells Containing Tissue Factor Following Laser-Induced Injury as Monitored Via Calcium Mobilization.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1786-1786 ◽  
Author(s):  
Ben T. Atkinson ◽  
Cynthia A. Kos ◽  
Barbara C. Furie ◽  
Bruce Furie
Keyword(s):  
Endothelial Cells ◽  
Plasma Membrane ◽  
Intracellular Calcium ◽  
Tissue Factor ◽  
Vessel Wall ◽  
Thrombus Formation ◽  
Fibrin Formation ◽  
Thrombosis Model ◽  
Calcium Elevation

Abstract The induction of thrombus formation in vivo through a laser-induced injury to the vessel wall in the living mouse has demonstrated the rapid expression of tissue factor antigen, thrombin generation and fibrin formation during thrombus formation. This thrombosis model is dominated by a tissue factor-dependent mechanism of platelet activation and fibrin formation. Specifically, this model does not lead to exposure of detectable sub-endothelial collagen, and the growth of the resulting thrombus is not dependent upon collagen-induced signaling. In light of prior in vivo results suggesting the rapid expression of active tissue factor on the vessel wall, we have determined if endothelial cells are activated in response to the injury induced by a pulsed laser. Laser-induced activation of individual cultured endothelial cells and cell populations were monitored through elevation of intracellular calcium and fluorescence microscopy. Cultured human umbilical vein endothelial cells (HUVECs) were loaded with the calcium-sensitive dye Fluo-4 and subjected to a single pulse of the nitrogen dye-tuned laser. HUVEC activation, was characterized by rapid elevation of intracellular calcium, with a sustained peak observed within 10 sec. Comparable calcium elevation could be achieved by exogenous addition of either ADP (10 μM) or thrombin (1 U/ml). Although reduced, significant laser-induced elevation of intracellular calcium remained when cells were bathed in calcium-free media, thus suggesting that both calcium influx and calcium mobilization play a part in the total calcium elevation observed. In addition, targeting of single cells within a confluent culture of endothelial cells initiated calcium elevation of the targeted cell and was followed by a wave of calcium elevation in surrounding cells. These results imply either a release of secondary mediators or cell-cell communication. Using both isolated and confluent cultured HUVECs, we performed widefield immunofluorescence and differential interference contrast microscopy to detect tissue factor (TF) in unstimulated HUVECs using an anti-TF antibody of high affinity and specificity. Tissue factor was not detected in intact endothelial cells but was localized to abundant small granules within the cytoplasm in Triton X-100-permeabilized cultured HUVECs. The J82 bladder carcinoma cell line, which constitutively expresses TF on the plasma membrane, was used as a positive control for surface expression of TF and an isotype-matched non-immune antibody used as a negative control. These results indicate the presence of a preformed intracellular pool of TF within HUVECs that under resting conditions is not detectable on the cell surface. Given the rapid time course of TF expression and platelet accumulation in vivo following laser-induced injury of the endothelium, any TF expression on the vessel wall must be preformed and not derived via endothelial protein synthesis. Although to date, we have not been able to detect TF antigen on the plasma membrane of laser-activated HUVECs it is possible that the amount of TF antigen is below the limits of sensitivity of immunodetection. Alternatively, TF, like P-selectin, may be rapidly recycled from the plasma membrane to the cell interior. Nonetheless, we suspect that tissue factor is translocated to the endothelial cell surface following cell activation and that preformed endothelial cell tissue factor, following laser-induced injury of the endothelium, plays a critical role in thrombin generation and fibrin formation in this thrombosis model.

scholarly journals Protein disulfide isomerase capture during thrombus formation in vivo depends on the presence of β3 integrins

Blood ◽  
2012 ◽  
Vol 120 (3) ◽  
pp. 647-655 ◽  
Author(s):  
Jaehyung Cho ◽  
Daniel R. Kennedy ◽  
Lin Lin ◽  
Mingdong Huang ◽  
Glenn Merrill-Skoloff ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Protein Disulfide Isomerase ◽  
Thrombus Formation ◽  
Wild Type ◽  
Disulfide Isomerase ◽  
Vessel Injury ◽  
Arteriolar Wall ◽  
Platelet Thrombus ◽  
Wall Injury ◽  
Protein Disulfide

Abstract Extracellular protein disulfide isomerase (PDI) is required for platelet thrombus formation and fibrin generation after arteriolar wall injury in live mice. PDI is secreted from platelets and endothelial cells on cellular activation, but the mechanism of capture of secreted PDI within the injured vasculature is unknown. We establish that, like the endothelial β3 integrin αVβ3, the platelet integrin αIIbβ3 binds PDI. PDI also binds to recombinant β3. Using intravital microscopy, we demonstrate that PDI accumulation at the site of laser-induced arteriolar wall injury is markedly reduced in β3-null (β3−/−) mice, and neither a platelet thrombus nor fibrin is generated at the vessel injury site. The absence of fibrin after vascular injury in β3−/− mice is because of the absence of extracellular PDI. To evaluate the relative importance of endothelial αVβ3 versus platelet αIIbβ3 or αVβ3, we performed reciprocal bone marrow transplants on wild-type and β3−/− mice. PDI accumulation and platelet thrombus formation were markedly decreased after vessel injury in wild-type mice transplanted with β3−/− bone marrow or in β3−/− mice transplanted with wild-type bone marrow. These results indicate that both endothelial and platelet β3 integrins contribute to extracellular PDI binding at the vascular injury site.

Abstract 806: Protein Disulfide Isomerase (PDI) signals Tissue Factor (TF)-dependent Fibrin formation in vivo

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Marie-Luise von Bruehl ◽  
Lenka Grahl ◽  
Ildiko Konrad ◽  
Michael Lorenz ◽  
Christian Schulz ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Arterial Thrombosis ◽  
Protein Disulfide Isomerase ◽  
Disulfide Isomerase ◽  
Intravascular Coagulation ◽  
Disulfide Formation ◽  
Fibrin Formation ◽  
Protein Disulfide

Background: Pathological fibrin formation is a predominant cause for arterial thrombosis, triggering myocardial infarction and stroke. Tissue factor (TF), which is expressed by vascular endothelium and cell-derived microparticles, is a central trigger of intravascular fibrin generation. In intact blood vessels, TF is only minimally active, and the coagulation system is down-regulated. In contrast, TF is rapidly stimulated following endothelial disruption. However, the molecular mechanisms that trigger fibrin formation via regulation of TF activation are largely undefined. Methods and Results: Here, we have identified a novel pathway that leads to TF activation in vitro and contributes to intravascular coagulation in vivo. We show that the protein disulfide isomerase (PDI) induces disulfide formation of the Cys186/Cys209 pair located in the extracellular domain of TF. In vitro, PDI-mediated disulfide formation led to profound activation of TF. In vivo, we found that PDI is not present in the intact vessel wall, but becomes abundantly expressed following vessel damage. We show that injured smooth muscle cells and adherent/activated platelets are the major cellular sources of PDI at sites of vascular injury. We then assessed fibrin formation following injury of the common carotid artery using intravital microscopy. We observed that PDI-blockade strongly reduced fibrin formation by approximately 45– 60%. Likewise, intravenous infusion of PDI significantly enhanced TF-dependent fibrin formation. Together, this implies that PDI initiates coagulation in vitro and also during arterial thrombosis in vivo. Notably, PDI did not act on platelets to trigger intravascular coagulation. Correspondingly, PDI blockade also inhibited fibrin formation, when platelet adhesion was prevented using a function-blocking anti-GPVI antibody. Conclusion: These findings reveal for the first time in vivo that the thiol isomerase PDI is a major molecular trigger of blood coagulation, which is exposed after vessel injury and converts TF into its functionally active form. Hence, PDI represents a promising novel target for the treatment of pathologic fibrin formation in patients with thrombotic disorders.

Effects of NO-Donors on Thrombus Formation and Microcirculation in Cerebral Vessels of the Rat

1996 ◽  
Vol 76 (01) ◽  
pp. 111-117 ◽  
Author(s):  
Yasuto Sasaki ◽  
Junji Seki ◽  
John C Giddings ◽  
Junichiro Yamamoto
Keyword(s):  
Blood Flow ◽  
Thrombus Formation ◽  
Dependent Manner ◽  
Red Cell ◽  
Cell Velocity ◽  
Red Cell Velocity ◽  
The Mean ◽  
Wall Shear ◽  
Dose Dependent

SummarySodium nitroprusside (SNP) and 3-morpholinosydnonimine (SIN-1), are known to liberate nitric oxide (NO). In this study the effects of SNP and SIN-1 on thrombus formation in rat cerebral arterioles and venules in vivo were assessed using a helium-neon (He-Ne) laser. SNP infused at doses from 10 Μg/kg/h significantly inhibited thrombus formation in a dose dependent manner. This inhibition of thrombus formation was suppressed by methylene blue. SIN-1 at a dose of 100 Μg/kg/h also demonstrated a significant antithrombotic effect. Moreover, treatment with SNP increased vessel diameter in a dose dependent manner and enhanced the mean red cell velocity measured with a fiber-optic laser-Doppler anemometer microscope (FLDAM). Blood flow, calculated from the mean red cell velocity and vessel diameters was increased significantly during infusion. In contrast, mean wall shear rates in the arterioles and venules were not changed by SNP infusion. The results indicated that SNP and SIN-1 possessed potent antithrombotic activities, whilst SNP increased cerebral blood flow without changing wall shear rate. The findings suggest that the NO released by SNP and SIN-1 may be beneficial for the treatment and protection of cerebral infarction

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