Extracellular Protein Disulfide Isomerase Cleaves Allosteric Disulfides in Histidine-Rich Glycoprotein to Regulate Thrombus Formation

Introduction The fine-tuning of thrombus formation is influenced by multiple factors among which extracellular protein disulfide isomerase (PDI) released by activated platelets and endothelial cells plays critical roles. However, the precise mechanisms whereby PDI modulates the kinetics of thrombosis remain elusive. Using mechanism-based kinetic trapping strategy, we identified plasma histidine-rich glycoprotein (HRG) as a substrate of extracellular PDI during thrombus formation. HRG exerts both anticoagulant and procoagulant functions. On one hand, HRG inhibits the contact pathway by binding to activated factor XII (fXIIa); on the other hand, HRG attenuates the anticoagulant activity of antithrombin (AT) by competing with AT binding to endothelial heparan sulfate. Both functions are dependent on zinc ions. In this study, we characterized the effects of PDI-mediated disulfide bond cleavage on HRG functions in the context of thrombosis. Methods Recombinant PDI variant with the C-terminal catalytic Cys of the CGHC motif replaced with Ala (PDI-CA) was used to trap its redox substrates in platelet rich plasma (PRP). Dual fluorescent immunoblotting was utilized to detect the stabilized intermediate complex between PDI-CA and HRG. Differential cysteine alkylation and mass spectrometry was performed using purified plasma HRG to identify the disulfide bonds targeted by PDI. ELISA was performed to determine the effects of PDI treatment on HRG binding to heparin, an analog of endothelial heparan sulfate, and fXIIa. Cell-based ELISA, immunofluorescent imaging, and immunohistochemistry were employed to examine in vitro and in vivo binding of HRG and AT on endothelial cells. HRG-mediated inhibition of fXIIa activity was determined using the chromogenic substrate S-2302. The kinetics of HRG accumulation during thrombus formation were examined using high-speed intravital microscopy in the cremasteric arterioles. The effects of HRG on thrombus formation were examined in the laser injury thrombosis model in the presence (wild-type mice) or absence of fXII (f12-/- mice). Results The trapping mutant PDI-CA, but not variants of endoplasmic protein 57 (ERp57), a close member in the PDI family with similar domain structure, formed disulfide-linked complexes with HRG in PRP. Mass spectrometry showed that PDI cleaves three disulfide bonds, C306-C309, C390-C434 and C409-C410, in the histidine-rich region of HRG that is important for its binding to heparan sulfate and fXIIa. Compared to inert-PDI (PDI-AA), where both catalytic Cys were substituted with Ala, wild-type PDI (PDI-CC) increased HRG binding to heparin in a Zn2+-dependent manner. Plasma treated with PDI-CC had increased HRG binding but decreased AT binding to cultured endothelial cells compared to PDI-AA treated control. Further, PDI-CC increased HRG binding to fXIIa and enhanced its inhibitory effect on fXIIa activity. Following laser injury of cremaster arterioles, plasma HRG accumulates rapidly at the injury site preceding the main platelet signal. When mice were treated with Eptifibatide, an integrin αIIbβ3 antagonist that eliminates platelet deposition and Zn2+release, plasma HRG accumulation at the site of vessel injury was reduced, indicating a critical role of Zn2+ for HRG binding in vivo. Intravenous treatment with a PDI inhibitor, isoquercetin, also inhibited HRG accumulation in the growing thrombus. In addition, following FeCl3-induced carotid injury, PDI inhibition by isoquercetin was found to reduce HRG binding but sustain AT binding on the injured artery as determined by immunohistochemistry. Finally, knockdown of plasma HRG with vivo-siRNA in f12-/- mice attenuated thrombus formation compared to scramble siRNA treatment thus suggesting a procoagulant role of HRG independent of fXIIa. Conclusion PDI cleavage of allosteric disulfide bonds in HRG represents a novel regulatory mechanism that fine-tunes the kinetics of thrombus formation. Our results indicate that at the early stage of thrombosis, PDI promotes HRG binding to endothelial cells to suppress the anticoagulant activity of AT and allow the rapid initiation of thrombosis; at the later stage, PDI reduction of HRG enhances its binding to fXIIa leading to inhibition of fXIIa activity to prevent excessive clot formation. Disclosures Bowley: Pfizer: Current Employment.

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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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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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Procoagulant Role Of Necrotic Platelets Demonstrated Using Novel Platelet Necrosis Marker

Blood ◽

10.1182/blood.v122.21.3512.3512 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3512-3512

Author(s):

Minh Hua ◽

Leonardo Pasalic ◽

Robert Lindeman ◽

Philip Hogg ◽

Vivien M Chen

Keyword(s):

Platelet Activation ◽

Ferric Chloride ◽

Cyclosporine A ◽

Thrombus Formation ◽

Laser Injury ◽

Agonist Stimulation ◽

Injury Model ◽

Exogenous Calcium

Abstract Strong agonist stimulation generates a platelet subpopulation characterized by phosphatidylserine (PS) exposure, loss of mitochondrial membrane potential and high fibrinogen retention. This population is proposed to be procoagulant, dependent on formation of the mitochondrial permeability transition pore (mPTP) with a distinct role from activated aggregratory platelets. These platelets have features of necrosis. The functional relevance of necrotic platelets in vivo is unknown due to lack of a suitable marker for these platelets. We show that a novel small molecule cellular necrosis marker, GSAO1, labels a procoagulant platelet subpopulation with features of necrosis and use it to explore the functional role of these platelets. We demonstrated using flow cytometry analysis of washed human platelets that fluorescently tagged GSAO labels a subpopulation of P-selectin positive platelets after thrombin and collagen stimulation with features of necrosis: high annexin V binding, calcein loss and dependence on exogenous calcium. This population is not dependent on the intrinsic apoptosis pathway as there was no change with pancaspase inhibition using ZVADFMK prior to dual agonist stimulation (p=0.567, n=5). In contrast, inhibition of mPTP formation through cyclophilin-D inhibition with cyclosporine A significantly inhibited GSAO+ve platelet generation (p<0.001, n=5), confirming dependence on the mitochondrial necrosis pathway. Mass spectrometry analysis of biotin-GSAO labelled proteins from platelets after streptavidin pull down identified thromboxane A synthase (TBXAS-1) as the major binding ligand after dual stimulation. Binding to TBXAS-1 was abrogated by dithiol alkylation, showing the mechanism of retention of GSAO in necrotic platelets is via covalent cross linking of closely-spaced cysteine thiols in the ligand. This allows persistent signal from the probe within the necrotic platelet with no evidence of washout. GSAO+ve platelets correlated with procoagulant potential as measured by peak and endogenous thrombin potential in the calibrated automated thrombogram (CAT) assay. Linear regression analysis showed a significant relationship between % change in GSAO+ve platelets and % change in peak thrombin after treatment with cyclosporine A or in absence of exogenous calcium (R2=0.648, p<0.01), indicating that GSAO identifies a procoagulant subpopulation. In contrast, no relationship was seen between P-selectin and peak thrombin values (R2=0.002). Inhibition of platelet activation by aspirin had no effect on the generation of GSAO+ve platelets indicating a potential uncoupling between platelet activation and necrosis pathways. After establishing that the imaging compound does not affect platelet function and coagulation in vitro, or thrombus formation in vivo, we went on to investigate the presence of GSAO+ve necrotic platelets in thrombus formation in a collagen dependent (ferric chloride) and collagen independent (laser injury) murine model of thrombosis. Confocal intravital imaging of the cremaster arterioles with fluorescent GSAO and tagged-CD42b demonstrated GSAO+ve platelets in the occlusive platelet aggregate after initiation with 10% ferric chloride. The GSAO+ve aggregating platelets specifically colabeled with calcium sensing dye rhodamine 2 indicating high sustained intracellular calcium, consistent with a necrotic phenotype. There was no signal with active site replaced control GSCA. In contrast, the laser injury model showed minimal staining with GSAO three minutes post laser injury. Using a novel platelet necrosis marker, we are able to demonstrate that necrotic platelets are procoagulant and present in the occlusive ferric chloride model and not in the non-occlusive laser injury model of thrombosis. This suggests excess platelet necrosis may be a key driving factor underlying pathological occlusive thrombi. GSAO is a promising tool for understanding factors that potentiate platelet necrosis which may offer attractive anti-thrombotic targets. 1. Park D, Don AS, Massamiri T, et al. Noninvasive imaging of cell death using an hsp90 ligand. J Am Chem Soc. 2011;133(9):2832-2835. Disclosures: No relevant conflicts of interest to declare.

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Vascular thiol isomerases

Blood ◽

10.1182/blood-2016-04-636456 ◽

2016 ◽

Vol 128 (7) ◽

pp. 893-901 ◽

Cited By ~ 38

Author(s):

Robert Flaumenhaft ◽

Bruce Furie

Keyword(s):

Endothelial Cells ◽

Vascular Function ◽

Disulfide Bonds ◽

Plasma Membranes ◽

Thrombus Formation ◽

Surface Proteins ◽

Vessel Injury ◽

Multifunctional Enzymes ◽

High Concentrations ◽

Protein Disulfide

Abstract Thiol isomerases are multifunctional enzymes that influence protein structure via their oxidoreductase, isomerase, and chaperone activities. These enzymes localize at high concentrations in the endoplasmic reticulum of all eukaryotic cells where they serve an essential function in folding nascent proteins. However, thiol isomerases can escape endoplasmic retention and be secreted and localized on plasma membranes. Several thiol isomerases including protein disulfide isomerase, ERp57, and ERp5 are secreted by and localize to the membranes of platelets and endothelial cells. These vascular thiol isomerases are released following vessel injury and participate in thrombus formation. Although most of the activities of vascular thiol isomerases that contribute to thrombus formation are yet to be defined at the molecular level, allosteric disulfide bonds that are modified by thiol isomerases have been described in substrates such as αIIbβ3, αvβ3, GPIbα, tissue factor, and thrombospondin. Vascular thiol isomerases also act as redox sensors. They respond to the local redox environment and influence S-nitrosylation of surface proteins on platelets and endothelial cells. Despite our rudimentary understanding of the mechanisms by which thiol isomerases control vascular function, the clinical utility of targeting them in thrombotic disorders is already being explored in clinical trials.

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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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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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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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β2-integrin activity: the role of thiols

Blood ◽

10.1182/blood-2013-03-489872 ◽

2013 ◽

Vol 121 (19) ◽

pp. 3779-3780 ◽

Cited By ~ 2

Author(s):

Alexander Zarbock

Keyword(s):

Disulfide Bonds ◽

Protein Disulfide Isomerase ◽

Neutrophil Recruitment ◽

Regulatory Effect ◽

Disulfide Isomerase ◽

Isomerase Activity ◽

Β2 Integrin ◽

Protein Disulfide

In this issue of Blood, Hahm and colleagues identify the extracellular protein disulfide isomerase (PDI) as an essential regulator of the adhesiveness of the β2-integrin macrophage-1 antigen (Mac-1) on neutrophils.1 In the absence of PDI, Mac-1–dependent neutrophil adhesion and crawling is reduced in vivo. Rescue experiments with exogenous PDI showed that the isomerase activity of extracellular PDI is critical for its regulatory effect on neutrophil recruitment. This intriguing finding suggests that disulfide bonds in Mac-1 regulate integrin activity and neutrophil recruitment.

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Stimulated Platelets but Not Endothelium Generate Thrombin Via a Factor XIIa-Dependent Mechanism Requiring Phosphatidylserine Exposure

Blood ◽

10.1182/blood.v128.22.258.258 ◽

2016 ◽

Vol 128 (22) ◽

pp. 258-258 ◽

Cited By ~ 1

Author(s):

Pavan K. Bendapudi ◽

Karen Deceunynck ◽

Secil Koseoglu ◽

Roelof Hendrik Bekendam ◽

Shauna D Mason ◽

...

Keyword(s):

Endothelial Cells ◽

Thrombin Generation ◽

Thrombus Formation ◽

Specific Inhibitor ◽

Unexpected Finding ◽

Factor Xii ◽

Fibrin Formation ◽

Platelet Accumulation

Abstract To inhibit pathological thrombus formation without impairing hemostasis is the holy grail of anticoagulant therapy. Recent data from animal models have indicated that factor XII (FXII) may be a promising new antithrombotic target that is particularly intriguing due to the longstanding clinical observation that severe congenital FXII deficiency is not associated with a bleeding diathesis in humans. FXII is thought to participate in thrombus formation after being activated in high shear arterial environments. FXIIa then initiates downstream activation of the contact pathway, culminating in thrombin generation. However, the relevant cell surface for FXII activation remains unclear. Here we compare the role of platelets versus endothelial cells in FXII activation and study the function of surface phospholipids in this process. To explore the effect of FXII inhibition on thrombus formation in vivo, we used antibody X210-C01, a novel human IgG1 developed using phage-display technology that blocks both mouse and human FXIIa. Using a mouse laser injury model of arterial thrombosis, we showed that X210-C01 inhibited both fibrin formation and platelet accumulation at sites of vascular injury. Plasma removed from animals after completion of these experiments was used to quantify the concentrations of X210-C01 achieved in vivo at a given dose. FXII inhibition was somewhat more potent in preventing platelet accumulation (IC50 dose = 27 mg/kg, R2=0.93) than fibrin formation (IC50 dose = 43 mg/kg, R2=0.95). Importantly, treatment with X210-C01 at 100 mg/kg did not prolong bleeding times or increase total blood loss in a tail bleeding assay. To evaluate the mechanism underlying our in vivo observations, we studied the differential role of FXII in thrombin generation by stimulated platelets and endothelium. X210-C01 did not globally inhibit SFLLRN-induced platelet aggregation or granule release. We next performed a fluorogenic thrombin generation assay (TGA) using human platelets treated with the peptide agonist SFLLRN. X210-C01 inhibited platelet-based thrombin generation in a dose-dependent fashion, whereas anti-tissue factor (TF) and anti-factor VIIa (FVIIa) antibodies did not. By contrast, in a similar TGA using SFLLRN-stimulated endothelial cells, X210-C01 had no effect, while anti-TF antibodies abrogated thrombin generation. These results indicate that stimulated endothelium generates thrombin by a mechanism distinct from that of platelets. FXII is known to be activated in vitro by anionic surfaces. Because phosphatidylserine (PS) is a negatively-charged phospholipid expressed on the surface of stimulated platelets, we reasoned that PS may serve as the platelet-based activator of FXII. To test this hypothesis, we used lactadherin, a potent and specific inhibitor of PS, in the platelet-based TGA and showed that PS blockade inhibited platelet-based thrombin generation at concentrations as low as 10 nM. We then used a chromogenic FXIIa activity assay to test the ability of PS-containing liposomes to activate FXII. Liposomes containing 80% phosphatidylcholine (PC) and 20% PS (PC-PS 80/20) failed to activate FXII at concentrations as high as 100 µM. In this assay, SFLLRN-stimulated platelets led to significantly greater FXII activation than either resting platelets or the PC-PS 80/20 liposomes tested. In summary, we have made the unexpected finding that thrombin generation on the surface of stimulated platelets proceeds by a FXIIa-dependent pathway and does not require FVIIa or TF. By contrast, thrombin generation on endothelium requires TF but not FXIIa. Additionally, surface PS is necessary but not sufficient for platelet-based FXII activation and thrombin generation, pointing to the involvement of a second platelet component. Further studies will be directed towards investigating the in vivo role of platelet-based FXII activation in arterial thrombus formation. Disclosures Mason: Shire Pharmaceuticals: Employment. Kenniston:Shire Pharmaceuticals: Employment.

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