Procoagulant Role Of Necrotic Platelets Demonstrated Using Novel Platelet Necrosis Marker

Blood ◽  
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.

scholarly journals Thrombin-initiated platelet activation in vivo is vWF independent during thrombus formation in a laser injury model

2007 ◽  
Vol 117 (4) ◽  
pp. 953-960 ◽  
Author(s):  
Christophe Dubois ◽  
Laurence Panicot-Dubois ◽  
Justin F. Gainor ◽  
Barbara C. Furie ◽  
Bruce Furie
Keyword(s):  
Platelet Activation ◽  
Thrombus Formation ◽  
Laser Injury ◽  
Injury Model

Mutation-specific hemostatic variability in mice expressing common type 2B von Willebrand disease substitutions

Blood ◽  
2010 ◽  
Vol 115 (23) ◽  
pp. 4862-4869 ◽  
Author(s):  
Mia Golder ◽  
Cynthia M. Pruss ◽  
Carol Hegadorn ◽  
Jeffrey Mewburn ◽  
Kimberly Laverty ◽  
...  
Keyword(s):  
Ferric Chloride ◽  
Von Willebrand Disease ◽  
Expression Vectors ◽  
Severe Thrombocytopenia ◽  
Normal Platelet ◽  
Injury Model ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Type 2B von Willebrand disease (2B VWD) results from von Willebrand factor (VWF) A1 mutations that enhance VWF-GPIbα binding. These “gain of function” mutations lead to an increased affinity of the mutant VWF for platelets and the binding of mutant high-molecular-weight VWF multimers to platelets in vivo, resulting in an increase in clearance of both platelets and VWF. Three common 2B VWD mutations (R1306W, V1316M, and R1341Q) were independently introduced into the mouse Vwf cDNA sequence and the expression vectors delivered to 8- to 10-week-old C57Bl6 VWF−/− mice, using hydrodynamic injection. The resultant phenotype was examined, and a ferric chloride–induced injury model was used to examine the thrombogenic effect of the 2B VWD variants in mice. Reconstitution of only the plasma component of VWF resulted in the generation of the 2B VWD phenotype in mice. Variable thrombocytopenia was observed in mice expressing 2B VWF, mimicking the severity seen in 2B VWD patients: mice expressing the V1316M mutation showed the most severe thrombocytopenia. Ferric chloride–induced injury to cremaster arterioles showed a marked reduction in thrombus development and platelet adhesion in the presence of circulating 2B VWF. These defects were only partially rescued by normal platelet transfusions, thus emphasizing the key role of the abnormal plasma VWF environment in 2B VWD.

scholarly journals IκB kinase 2 is not essential for platelet activation

2020 ◽  
Vol 4 (4) ◽  
pp. 638-643
Author(s):  
Manuel Salzmann ◽  
Sonja Bleichert ◽  
Bernhard Moser ◽  
Marion Mussbacher ◽  
Mildred Haase ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Active Site ◽  
Bleeding Time ◽  
Thrombus Formation ◽  
Human Platelets ◽  
Iκb Kinase ◽  
Specific Deletion

Abstract Platelets are small anucleate cells that release a plethora of molecules to ensure functional hemostasis. It has been reported that IκB kinase 2 (IKK2), the central enzyme of the inflammatory NF-κB pathway, is involved in platelet activation, because megakaryocyte/platelet-specific deletion of exons 6 and 7 of IKK2 resulted in platelet degranulation defects and prolonged bleeding. We aimed to investigate the role of IKK2 in platelet physiology in more detail, using a platelet-specific IKK2 knockout via excision of exon 3, which makes up the active site of the enzyme. We verified the deletion on genomic and transcriptional levels in megakaryocytes and were not able to detect any residual IKK2 protein; however, platelets from these mice did not show any functional impairment in vivo or in vitro. Bleeding time and thrombus formation were not affected in platelet-specific IKK2-knockout mice. Moreover, platelet aggregation, glycoprotein GPIIb/IIIa activation, and degranulation were unaltered. These observations were confirmed by pharmacological inhibition of IKK2 with TPCA-1 and BMS-345541, which did not affect activation of murine or human platelets over a wide concentration range. Altogether, our results imply that IKK2 is not essential for platelet function.

A Role for Bile Salt-Dependent Lipase in Platelet Activation and in Thrombus Formation in Vivo.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3526-3526 ◽  
Author(s):  
Laurence Panicot-Dubois ◽  
Christophe Dubois ◽  
Barbara C. Furie ◽  
Bruce Furie ◽  
Dominique Lombardo
Keyword(s):  
Bile Salt ◽  
Platelet Activation ◽  
Thrombus Formation ◽  
Pancreatic Acinar Cells ◽  
Cremaster Muscle ◽  
Terminal Domain ◽  
Laser Injury ◽  
Phosphatidylserine Exposure

Abstract Bile Salt Dependent Lipase (BSDL) is an enzyme secreted by pancreatic acinar cells. BSDL, in the presence of primary bile salts, participates in the hydrolysis of dietary lipid esters in the duodenum lumen. This 105 kDa N and O-glycosylated protein has been detected in the plasma of normal subjects. Recent in vitro and in vivo studies demonstrated that pancreatic BSDL reaches the blood via transcytosis through enterocytes. Other studies showed that pancreatic human BSDL is captured by human umbilical vein endothelial cells and induces the proliferation of smooth muscle cells in vitro at BSDL concentrations found in blood, suggesting that this enzyme may play a role in hemostasis and thrombosis. However the specific role of circulating BSDL is unknown. The goal of this study was to determine the possible involvement of circulating BSDL in thrombus formation. We investigated the participation of circulating mouse BSDL in thrombus formation using widefield intravital microscopy in the cremaster muscle of living mice. Thrombi were formed following laser injury of the vessel wall of an arteriole in the cremaster muscle. Pancreatic mouse BSDL, a 74 kDa glycoprotein, was detected using several antibodies directed against either the whole human BSDL (pAbL64, pAbL32) or a peptide based on a sequence in the N-terminal domain of BSDL (Ser326-Thr350; pAbAntipeptide). Mouse and human BSDL share about 80% sequence homology, the main difference localized in the C-terminal domain, which is truncated to the mouse BSDL compared with the human enzyme. All the antibodies are able to specifically recognize the mouse pancreatic BSDL. Using antibodies pAbL64, pAbL32, or pAbAntipeptide we observed specific accumulation of circulating mouse BSDL into the growing thrombus. The circulating BSDL co-localized with platelets present in the thrombus. These results suggest that circulating BSDL is involved in thrombus formation in vivo. In order to determine if BSDL plays a role in platelet activation and aggregation, we performed in vitro studies on human washed platelets. BSDL increased both the amount of phosphatidylserine exposure on the surface of platelets and the activation of αIIbβ3 induced by thrombin. These results indicate that this enzyme can amplify the activation of platelets in vitro. While BSDL alone cannot induce the aggregation of platelets, this enzyme significantly increases the amount of platelet aggregation induced by SFLLRN peptide or thrombin. Altogether, these data suggeste that circulating BSDL participates in the thrombus formation after laser injury of the arterial wall and can amplify both the activation of platelets and the phosphatidylserine exposure, increasing the thrombotic response after vessel injury. This mechanism may be operative in the development of venous thromboembolic disease in pancreatic cancer.

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

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 691-691 ◽  
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.

CLEC-2 is an essential platelet-activating receptor in hemostasis and thrombosis

Blood ◽  
2009 ◽  
Vol 114 (16) ◽  
pp. 3464-3472 ◽  
Author(s):  
Frauke May ◽  
Ina Hagedorn ◽  
Irina Pleines ◽  
Markus Bender ◽  
Timo Vögtle ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Thrombus Formation ◽  
Antibody Treatment ◽  
Cellular Activation ◽  
Primary Hemostasis ◽  
Arterial Thrombus ◽  
Normal Adhesion

Abstract Damage to the integrity of the vessel wall leads to exposure of the subendothelial extracellular matrix (ECM), triggering platelet activation and aggregation. This process is essential for primary hemostasis but it may also lead to arterial thrombosis. Although the mechanisms underlying platelet activation on the ECM are well explored, it is less clear which receptors mediate cellular activation in a growing thrombus. Here we studied the role of the recently identified C-type lectin-like receptor 2 (CLEC-2) in this process. We show that anti–CLEC-2 antibody treatment of mice leads to complete and highly specific loss of CLEC-2 in circulating platelets for several days. CLEC-2–deficient platelets displayed normal adhesion under flow, but subsequent aggregate formation was severely defective in vitro and in vivo. As a consequence, CLEC-2 deficiency was associated with increased bleeding times and profound protection from occlusive arterial thrombus formation. These results reveal an essential function of CLEC-2 in hemostasis and thrombosis.

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.

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

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 11-12
Author(s):  
Shuai Chen ◽  
Xu-Lin Xu ◽  
Joyce Chiu ◽  
Sheryl Bowley ◽  
Yi Wu ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Heparan Sulfate ◽  
Disulfide Bonds ◽  
Thrombus Formation ◽  
Anticoagulant Activity ◽  
Laser Injury ◽  
Protein Disulfide ◽  
Kinetics Of

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.

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.

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.

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