Regulation of Protein Disulfide Isomerase By S-Nitrosylation Controls Its Function during Thrombus Formation

Abstract Protein disulfide isomerase (PDI) is an oxidoreductase that is essential for thrombus formation following vascular injury. Clinical trials testing the efficacy and safety of PDI inhibition in the setting of thrombotic disease are currently underway. Yet while preclinical and clinical trials of PDI in thrombosis have progressed rapidly, the mechanisms by which PDI is regulated in the vasculature and how it mediates thrombosis remain unknown. PDI has an a-b-b'-x-a' domain structure, where the a and a' domains contain a CGHC motif responsible for cleaving and forming disulfide bonds. The active site cysteines within the catalytic CGHC motif that perform oxidoreductive reactions can also undergo S-nitrosylation. We have evaluated the hypothesis that nitric oxide (NO) converts PDI into a nitrosylase and regulates PDI oxidoreductase activity in the vasculature during thrombus formation. Initial studies demonstrated that incubation of recombinant PDI with the NO donor, SNAP, resulted in an 83±1.4% decrease in its reductase activity. A transnitrosylase assay using the NO indicator DAF-FM showed that S-nitrosylated PDI (SNO-PDI) transferred NO into platelets and inhibited platelet aggregation. To define the molecular determinants of PDI nitrosylation activity, we evaluated mutant PDIs containing Cys -> Ala mutations of the CGHC (a domain)/CGHC (a' domain) motifs in the platelet-based transnitrosylase assay. Wild-type PDI (CGHC/CGHC) demonstrated full reductase and nitrosylase activity and the enzymatically dead mutant (AGHA/AGHA) showed neither activity. In contrast, the CGHA/CGHA mutant maintained nitrosylase activity (41±0.23%), but had no reductase activity. This observation suggested that reductase and nitrosylase activities were separable. To further evaluate this supposition, we screened a series of PDI mutants in which intervening sequences of the CGHC domain had been modified. The screen identified CGPC/CGPC as a nitrosylase-biased mutant that showed a 59±2.31% decrease in reductase activity, but a 72±1.83% increase in nitrosylase activity compared to wild-type PDI. Another nitrosylase-biased mutant, CGRC/CGRC, showed a similar activity pattern. Since PDI is prothrombotic and SNO-PDI is antithrombotic, we compared the activity of nitrosylase-biased mutants with wild-type PDI in platelet aggregation studies in the presence of physiological concentrations of GSNO. While wild-type PDI had little effect on platelet aggregation, nitrosylase-biased PDIs such as the CGPC/CGPC and CGRC/CGRC mutant completely inhibited platelet aggregation. These studies show that the prothrombotic oxidoreductase activities of PDI are separable from their antithrombotic nitrosylase activities and that nitrosylase-biased PDI mutants have antiplatelet activity. We next evaluated the effect of PDI nitrosylation on thrombus formation in vivo. Infusion of SNO-PDI into mice inhibited thrombus formation following laser-induced vascular injury of cremaster arterioles. Mice deficient in glutathione-S-nitrosyl reductase (GSNOR) were used to assess the role of endogenous NO in thrombus formation. GSNOR enzymatically reduces GSNO, the main storage form of NO in cells. Platelet accumulation and fibrin formation were hardly detectable in GSNOR-/- mice. Infusion of recombinant WT PDI, but not an enzymatically dead PDI, reversed the defect in platelet accumulation and fibrin generation to levels of WT mice. In order to visualize NO during thrombus formation, the NO-sensitive dye DAF-FM was infused into mice and NO signal in endothelium monitored following laser-induced injury. DAF-FM signal decreased rapidly following laser injury of cremaster arterioles, indicating an activation-induced reduction in endothelial NO in vivo. In conclusion, our studies show that oxidoreductase and nitrosylase activities of PDI are separable and support a model whereby high endothelial NO levels maintain vascular quiescence in part by maintaining PDI as a nitrosylase and blocking its prothrombotic PDI activity. We propose that the reduction of NO levels that occurs with vascular injury or endothelial dysfunction contributes to the conversion of PDI from an anti-thrombotic nitrosylase to a prothrombotic reductase. Disclosures No relevant conflicts of interest to declare.

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Endothelium but Not Platelet-Derived Protein Disulfide Isomerase Is Required for Fibrin Generation during Thrombus Formation in Vivo.

Blood ◽

10.1182/blood.v112.11.691.691 ◽

2008 ◽

Vol 112 (11) ◽

pp. 691-691 ◽

Cited By ~ 1

Author(s):

Reema Jasuja ◽

Jaehyung Cho ◽

Bruce Furie ◽

Barbara Furie

Keyword(s):

Endothelial Cells ◽

Protein Disulfide Isomerase ◽

Thrombus Formation ◽

Fibrin Deposition ◽

Wild Type ◽

Disulfide Isomerase ◽

Laser Injury ◽

Injury Model ◽

Protein Disulfide

Abstract We have previously reported that protein disulfide isomerase is required in wild-type mice for platelet thrombus formation and fibrin generation in an in vivo laser injury model of thrombosis (Cho et al. J. Clin. Invest., 2008; 118:1123–31). Fibrin deposition after laser injury to the vessel wall in Par4−/− mice, lacking the G protein-coupled platelet thrombin receptor, is independent of platelets or requires minimal platelet activation or accumulation (Vandendries et al. Proc. Natl. Acad. Sci., 2007; 104:288–92). However, protein disulfide isomerase inhibitors have a dramatic effect on fibrin accumulation in Par4− mice, suggesting that these inhibitors may function by a platelet independent mechanism. Here, we compare the contributions of endothelium and platelet-derived protein disulfide isomerase to fibrin generation in the mouse laser injury model of thrombosis. In vitro studies using cultured human umbilical vein endothelial cells and human aortic endothelial cells show that protein disulfide isomerase can be secreted rapidly into the culture medium from these cells upon thrombin stimulation. Using intravital microscopy, we observe that protein disulfide isomerase is not detectable on the vessel wall prior to laser injury but can be detected on the injured cremaster arteriolar wall and in the developing thrombus very rapidly after laser induced injury in the live mouse. The median integrated fluorescence intensity for protein disulfide isomerase in wild-type mice was compared to wild-type mice injected with 10ug/g mouse of Integrilin, an inhibitor of platelet activation and platelet thrombus formation, and thus, an inhibitor of the contribution of platelet derived protein disulfide isomerase to thrombus formation. Protein disulfide isomerase expression was similar in both treated and untreated animals upto 30 seconds post-laser injury. After 30 seconds, the expression of protein disulfide isomerase in integrilin treated mice was significantly decreased compared to that in untreated mice, indicating that the initial protein disulfide isomerase was derived from the endothelium and later additional protein disulfide isomerase was derived from the platelets following their accumulation in the developing thrombus. Fibrin deposition, a measure of thrombin generation was comparable in wild-type mice that had been treated with Integrilin or treated with a control buffer, suggesting that endothelial-derived protein disulfide isomerase was sufficient for fibrin generation. The rate and amount of fibrin generation was indistinguishable in both groups. Furthermore, inhibition of the protein disulfide isomerase with the function blocking monoclonal antibody RL-90 (3ug/g mouse) eliminated any fibrin deposition in wild-type mice that had been treated with Integrilin. Taken together, these data indicate that endothelium-derived protein disulfide isomerase is necessary to support fibrin deposition in vivo in our laser injury model of thrombus formation. The initial protein disulfide isomerase expressed at the site of injury is derived from endothelial cells but platelets activated at the site of thrombus formation contribute, amplify and sustain protein disulfide isomerase expression.

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Platelet ERp57 Is Required for Hemostasis and Thrombosis.

Blood ◽

10.1182/blood.v120.21.2168.2168 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2168-2168

Author(s):

Lu Wang ◽

Yi Wu ◽

Junsong Zhou ◽

Syed S. Ahmad ◽

Bulent Mutus ◽

...

Keyword(s):

Platelet Aggregation ◽

Platelet Function ◽

Protein Disulfide Isomerase ◽

Conflicts Of Interest ◽

Thrombus Formation ◽

Platelet Glycoprotein ◽

Platelet Surface ◽

Disulfide Isomerase ◽

Protein Disulfide

Abstract Abstract 2168 Several members of the protein disulfide isomerase family of enzymes are important in platelet function and in thrombosis. Platelet protein disulfide isomerase (PDI) has been shown to have an important role in platelet function but is reported to not be required for thrombus formation in vivo. A novel platelet PDI called ERp57 mediates platelet aggregation but its role in thrombus formation is unknown. To determine the specific role of platelet-derived ERp57 in hemostasis and thrombosis we generated a megakaryocyte/platelet specific knockout. Despite normal platelet counts and platelet glycoprotein expression, mice with ERp57-deficient platelets had prolonged tail-bleeding times and thrombus occlusion times, and defective activation of the αIIbβ3 integrin and platelet aggregation. The aggregation defect was corrected by addition of exogenous ERp57 implicating surface ERp57 in platelet aggregation. Platelet surface ERp57 protein and activity increased substantially with platelet activation. We conclude that platelet-derived ERp57 is required for hemostasis and thrombosis and platelet function. Disclosures: No relevant conflicts of interest to declare.

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

Blood ◽

10.1182/blood-2018-99-114418 ◽

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.

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Novel anti-thrombotic agent for modulation of protein disulfide isomerase family member ERp57 for prophylactic therapy

Scientific Reports ◽

10.1038/srep10353 ◽

2015 ◽

Vol 5 (1) ◽

Cited By ~ 9

Author(s):

Guozhen Cui ◽

Luchen Shan ◽

Lin Guo ◽

Ivan Keung Chu ◽

Guohui Li ◽

...

Keyword(s):

Platelet Aggregation ◽

Protein Disulfide Isomerase ◽

Thrombus Formation ◽

Myocardial Infarct ◽

Redox Activity ◽

Acute Myocardial Infarct ◽

Disulfide Isomerase ◽

Protein Disulfide

Abstract Protein disulfide isomerase (PDI) family members including PDI and ERp57 emerge as novel targets for anti-thrombotic treatments, but chemical agents with selectivity remain to be explored. We previously reported a novel derivative of danshensu (DSS), known as ADTM, displayed strong cardioprotective effects against oxidative stress-induced cellular injury in vitro and acute myocardial infarct in vivo. Herein, using chemical proteomics approach, we identified ERp57 as a major target of ADTM. ADTM displayed potent inhibitory effects on the redox activity of ERp57, inhibited the adenosine diphosphate (ADP)-induced expressions of P-selectin and αIIbβ3 integrin and disrupted the interaction between ERp57 and αIIbβ3. In addition, ADTM inhibited both arachidonic acid (AA)-induced and ADP-induced platelet aggregation in vitro. Furthermore, ADTM significantly inhibited rat platelet aggregation and thrombus formation in vivo. Taken together, ADTM represents a promising candidate for anti-thrombotic therapy targeting ERp57.

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Endobrevin/VAMP-8-Mediated Dense Granule Release Is Required for Efficient Thrombus Formation in Vivo.

Blood ◽

10.1182/blood.v112.11.1836.1836 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1836-1836

Author(s):

Price S. Blair ◽

Qiansheng Ren ◽

Gwenda J. Graham ◽

James R. Dilks ◽

Sidney W. Whiteheart ◽

...

Keyword(s):

Vascular Injury ◽

Thrombus Formation ◽

Dense Granule ◽

Wild Type ◽

Platelet Accumulation ◽

Induced Aggregation ◽

Kinetics Of ◽

Granule Release

Abstract Individuals whose platelets lack dense core or alpha-granules suffer varying degrees of abnormal bleeding, implying that granule cargo contributes to hemostasis. Despite these clinical observations, little is known regarding the effects of impaired platelet granule secretion on thrombus formation in vivo. The release of cargo from platelet granules requires a group of membrane proteins called SNAREs (Soluble NSF Attachment Protein Receptors) that mediate fusion of granule membranes to the plasma membrane and open canalicular system. Endobrevin/VAMP-8 is the primary vesicular-SNARE (v-SNARE) responsible for efficient release of dense core and a-granule contents. To evaluate the importance of VAMP-8-mediated secretion on the kinetics of thrombus formation in vivo, we measured platelet accumulation following laser-induced vascular injury in VAMP-8−/− mice. Three different phases of thrombus formation - initiation, maximal accumulation, and stabilized platelet accumulation - were tested. Analysis of initial thrombus formation from wild-type and VAMP-8−/− mice showed that average platelet accumulation in VAMP- 8−/− mice was 23% of accumulation in wild-type mice (P=0.009) at 30 sec following injury. There was a trend towards smaller maximal thrombus size in VAMP-8−/− mice, but the difference was not statistically significant (P=0.1). Average stabilized platelet accumulation at 180 sec in VAMP-8−/− mice was 40% of wild-type mice (P=0.05). Thus, thrombus formation is delayed and decreased in VAMP-8−/− mice, but not absent. Dense granule release occurs more rapidly than alpha-granule release, which does not occur for 2–3 min following laser-induced vascular injury. Agonist-induced dense granule release from VAMP-8−/− platelets is defective. To directly evaluate the role of dense granule release on the kinetics of thrombus formation, we assessed thrombus formation in the mouse model of Hermansky-Pudlak syndrome, ruby-eye, which lack dense granules. Thrombus formation following laser-induced vascular injury was nearly abolished in ruby-eye mice such that maximal platelet accumulation was 15% that of wild-type mice. In vitro, the thrombin doses required to induce irreversible aggregation in wild-type, VAMP-8−/−, and ruby-eye platelets were 25 mU, 50 mU, and 150 mU, respectively. Incubation with apyrase had little effect on thrombin-induced aggregation of VAMP-8−/− or ruby-eye platelets. In contrast, incubation of wild-type platelets with apyrase reduced their thrombin sensitivity compared to that of ruby-eye platelets. Supplementation with a substimulatory ADP concentration reversed the thrombin-induced aggregation defect in VAMP-8−/− and ruby-eye mice. Thus, defective ADP release is the primary abnormality leading to impaired aggregation in VAMP-8−/− and ruby-eye mice. Tail bleeding times were assessed in VAMP- 8−/− mice to evaluate the role of VAMP-8 in hemostasis. In contrast to ruby-eye mice, which have a markedly prolonged bleeding time, tail bleeding times in VAMP-8−/− mice were not significantly prolonged compared to those in wild-type mice. These results demonstrate the importance of VAMP-8 and dense granule release in the initial phases of thrombus formation and validate the distal platelet secretory machinery as a potential target for anti-platelet therapies.

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Extracellular Protein Disulfide Isomerase Regulates Vitronectin during the Initiation of Thrombus Formation

Blood ◽

10.1182/blood.v128.22.15.15 ◽

2016 ◽

Vol 128 (22) ◽

pp. 15-15

Author(s):

Chao Fang ◽

Sheryl R. Bowley ◽

Barbara C. Furie ◽

Bruce Furie

Keyword(s):

Endothelial Cells ◽

Protein Disulfide Isomerase ◽

Vessel Wall ◽

Plasma Sample ◽

Thrombus Formation ◽

Disulfide Isomerase ◽

Platelet Accumulation ◽

Protein Disulfide ◽

Kinetic Trapping

Abstract Protein disulfide isomerase (PDI), secreted by platelets and endothelial cells upon vascular injury, is required for thrombus formation. However, the precise mechanism by which PDI regulates thrombosis remains elusive. Using PDI variants that form stable mixed disulfide complexes with their substrates, we performed kinetic trapping experiment in platelet rich plasma and identified multiple substrate proteins for PDI, including vitronectin. Importantly, when using variants of endoplasmic reticulum protein 57 (ERp57), a thiol isomerase that has a similar domain structure as PDI and is also important for thrombus formation, the trapping mutants of ERp57 do not interact with vitronectin. This result has demonstrated the substrate specificity of PDI during our kinetic trapping experiment. Further study using polyethylene glycol (PEG)-based gel mobility shift assay combined with mass spectrometry has identified the redox reaction between PDI and vitronectin occurs on two disulfide bonds Cys 137-161 and Cys 274-453 in the hemopexin-like domains of plasma vitronectin. Vitronectin, as a substrate of extracellular PDI, has been shown to be important for thrombus formation. Vitronectin null mice have reduced platelet accumulation and fibrin deposition in the cremaster arterioles following laser injury. Vitronectin null mice also have significantly prolonged large-vessel thrombosis in the carotid artery using the ferric chloride thrombosis model. Using intravital microscopy we showed that vitronectin rapidly accumulates in a growing thrombus following vessel injury. When mice are treated with eptifibatide to eliminate platelet accumulation, we still observe significant amount of vitronectin accumulation on the vessel wall in the absence of platelet thrombus. This observation was further confirmed using confocal intravital microscopy. After 3D reconstruction of a growing thrombus in mouse cremaster arteriole, vitronectin was identified to locate primarily on the CD31 stained vessel wall. These combined studies suggest that plasma-derived vitronectin and not platelet-derived vitronectin is the primary substrate of PDI. Our study further showed that the indispensable role of vitronectin to a growing thrombus depends on extracellular PDI. Native plasma vitronectin does not bind to αvβ3 or αIIbβ3-integrins on endothelial cells and platelets. On solid phase binding assay, plasma sample pre-treated with wild-type PDI showed significantly increased binding of vitronectin to its ligand αvβ3 or αIIbβ3-integrins. However, this increase was not observed in plasma pre-treated with dead-mutant PDI or ERp57. In addition, using immunofluorescent staining, PDI treated plasma sample also showed significantly increased binding of vitronectin to activated human umbilical vein endothelial cells (HUVECs) and this binding was abrogated by RGD peptides or an αvβ3 blocking antibody. The critical role of extracellular PDI for the regulation of vitronectin in a growing thrombus was further confirmed in our in vivo studies. When mice were treated with quecetin-3-rutinoside or two different inhibitory antibodies that selectively block PDI activity, the accumulation of vitronectin and platelets was significantly reduced. These combined results demonstrate that extracellular PDI regulates vitronectin in a growing thrombus to promote platelet accumulation and fibrin generation. In summary, our studies have revealed a novel regulatory mechanism during the initiation of thrombus formation. Under normal physiologic conditions in the absence of secreted PDI, thrombus formation is suppressed and maintains a quiescent, patent vasculature. The release of PDI during vascular injury serves as a novel regulatory switch that allows activation of proteins, including vitronectin, which are critical for the following platelet accumulation and fibrin generation. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

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Platelet protein disulfide isomerase is required for thrombus formation but not for hemostasis in mice

Blood ◽

10.1182/blood-2013-03-492504 ◽

2013 ◽

Vol 122 (6) ◽

pp. 1052-1061 ◽

Cited By ~ 94

Author(s):

Kyungho Kim ◽

Eunsil Hahm ◽

Jing Li ◽

Lisa-Marie Holbrook ◽

Parvathy Sasikumar ◽

...

Keyword(s):

Protein Disulfide Isomerase ◽

Thrombus Formation ◽

Integrin Signaling ◽

Disulfide Isomerase ◽

Platelet Protein ◽

Key Points ◽

Platelet Accumulation ◽

Protein Disulfide ◽

Inside Out ◽

Αiibβ3 Integrin

Key Points Platelet PDI regulates αIIbβ3 integrin activation without affecting platelet activation and inside-out integrin signaling. Platelet PDI is essential for platelet accumulation but not for fibrin generation and hemostasis in mice.

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

Blood ◽

10.1182/blood-2010-04-278184 ◽

2010 ◽

Vol 116 (22) ◽

pp. 4665-4674 ◽

Cited By ~ 94

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.

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

Blood ◽

10.1182/blood.v116.21.345.345 ◽

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.

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Protein Disulfide Isomerase Inhibitors: A New Class of Antithrombotic Agents

Blood ◽

10.1182/blood.v118.21.369.369 ◽

2011 ◽

Vol 118 (21) ◽

pp. 369-369 ◽

Cited By ~ 2

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.

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