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.

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.

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

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

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

ML359, a Small Molecule Inhibitor of Protein Disulfide Isomerase That Prevents Thrombus Formation and Inhibits Oxidoreductase but Not Transnitrosylase Activity

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2880-2880
Author(s):  
Pavan K Bendapudi ◽  
Roelof H Bekendam ◽  
Lin Lin ◽  
Mingdong Huang ◽  
Bruce Furie ◽  
...  
Keyword(s):  
Small Molecule ◽  
Protein Disulfide Isomerase ◽  
Thrombus Formation ◽  
Oxidoreductase Activity ◽  
Disulfide Isomerase ◽  
Linker Region ◽  
Laser Injury ◽  
Molecule Inhibitor ◽  
Injury Model ◽  
Protein Disulfide

Abstract Vascular thiol isomerases comprise a family of enzymes including protein disulfide isomerase (PDI), ERp5, and ERp57 that are important in the process of thrombus formation. PDI is secreted at sites of vascular injury, and antibody-mediated PDI inhibition prevents thrombus formation in a mouse laser injury model. Our group has previously reported on the discovery of the small molecule PDI inhibitors quercetin-3-rutinoside and ML359. Identified as part of a high-throughput screen, ML359 is a second-generation PDI inhibitor that selectively blocks PDI oxidoreductase activity with approximately ten-fold the potency of quercetin-3-rutinoside. To better understand the mechanism of allosteric modulation of PDI by small molecules, we evaluated the association of ML359 with isolated domains of PDI, determined the effects of ML359 on a variety of PDI functions, and compared the activity of ML359 to that of quercetin-3-rutinoside. PDI is composed of four thioredoxin-like domains and an x-linker region in the sequence a-b-b’-x-a’. Major substrate binding is thought to occur in the b-b’ region while the a and a’ domains contain catalytically active cysteine motifs (CGHC) that mediate the oxidoreducase, nitrosylase, and thiol isomerase functions of PDI. In order to identify potential binding sites of ML359 on PDI, we constructed and expressed the domain fragments a, ab, abb’, b’xa’, and a’. These fragments were tested in the presence of 10 µM ML359 using an insulin turbidometric assay that measures the oxidoreductase activity of PDI. ML359 demonstrated full inhibition of oxidoreductase activity when full-length PDI and the b’xa’ fragment were used whereas no inhibition was observed with the other fragments assayed. These results are consistent with docking studies showing that ML359 likely binds in a pocket formed at the b’x interface. In contrast, when the same experiment was performed in the presence of 30 µM of quercetin-3-rutinoside, inhibition was only noted with full-length PDI and the abb’ and b’xa’ fragments, suggesting that binding was dependent on the b’ and not the x-linker region. To determine if ML359 has differential effects on the oxidoreductase and nitrosylase functions of PDI, we utilized a platelet-based assay in which fluorescence intensity stemming from the NO-sensitive intracellular dye DAF-FM was measured as an indicator of PDI-mediated translocation of NO from the extracellular surface into the cytosol (transnitrosylation). While quercetin-3-rutinoside potently inhibited PDI-mediated transnitrosylation activity, ML359 had no effect. These results are consistent with the idea that the transnitrosylase and oxidoreducase functions of PDI are separable and inhibition of either is specific to the small molecule used. We evaluated the ability of ML359 to inhibit thrombosis in a mouse laser injury model. Intravital microscopy was used to follow thrombus formation in mouse cremaster arterioles after laser-induced vascular injury. Infusion of ML359 resulted in inhibition of thrombus formation, in contrast to thrombosis seen after infusion of vehicle alone. In summary, ML359 is a second generation small molecule inhibitor of PDI that likely binds at the b’x interface of the enzyme. Furthermore, ML359 is able to selectively inhibit PDI oxidoreductase activity without affecting transnitrosylase activity. ML359 may prove a useful molecular probe to better understand the different functions of PDI relative to thrombus formation in vivo. Disclosures No relevant conflicts of interest to declare.

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

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

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

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

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 93-93
Author(s):  
Roelof H Bekendam ◽  
Gopal Srila ◽  
Pavan K Bendapudi ◽  
James R Dilks ◽  
Lin Lin ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Vascular Injury ◽  
Protein Disulfide Isomerase ◽  
Thrombus Formation ◽  
Reductase Activity ◽  
Wild Type ◽  
Disulfide Isomerase ◽  
Platelet Accumulation ◽  
Protein Disulfide

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.

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

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

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

scholarly journals Hirudin interruption of heparin-resistant arterial thrombus formation in baboons

Blood ◽  
1991 ◽  
Vol 77 (5) ◽  
pp. 1006-1012 ◽  
Author(s):  
AB Kelly ◽  
UM Marzec ◽  
W Krupski ◽  
A Bass ◽  
Y Cadroy ◽  
...  
Keyword(s):  
Bleeding Time ◽  
Thrombus Formation ◽  
Dependent Manner ◽  
Fibrin Deposition ◽  
Recombinant Hirudin ◽  
Arterial Thrombus ◽  
Dependent Inhibition ◽  
Antithrombotic Effects ◽  
Dose Dependent

Abstract To determine the role of thrombin in high blood flow, platelet- dependent thrombotic and hemostatic processes we measured the relative antithrombotic and antihemostatic effects in baboons of hirudin, a highly potent and specific antithrombin, and compared the effects of heparin, an antithrombin III-dependent inhibitor of thrombin. Thrombus formation was determined in vivo using three relevant models (homologous endarterectomized aorta, collagen-coated tubing, and Dacron vascular graft) by measuring: (1) platelet deposition, using gamma camera imaging of 111In-platelets; (2) fibrin deposition, as assessed by the incorporation of circulating 125I-fibrinogen; and (3) occlusion. The continuous intravenous infusion of 1, 5, and 20 nmol/kg per minute of recombinant hirudin (desulfatohirudin) maintained constant plasma levels of 0.16 +/- 0.03, 0.79 +/- 0.44, and 3.3 +/- 0.77 mumol/mL, respectively. Hirudin interrupted platelet and fibrin deposition in a dose-dependent manner that was profound at the highest dose for all three thrombogenic surfaces and significant at the lowest dose for thrombus formation on endarterectomized aorta. Thrombotic occlusion was prevented by all doses studied. In contrast, heparin did not inhibit either platelet or fibrin deposition when administered at a dose that maximally prolonged clotting times (100 U/kg) (P greater than .1), and only intermediate effects were produced at 10-fold that dose (1,000 U/kg). Moreover, heparin did not prevent occlusion of the test segments. Hirudin inhibited platelet hemostatic function in concert with its antithrombotic effects (bleeding times were prolonged by the intermediate and higher doses). By comparison, intravenous heparin failed to affect the bleeding time at the 100 U/kg dose (P greater than .5), and only minimally prolonged the bleeding time at the 1,000 U/kg dose (P less than .05). We conclude that platelet-dependent thrombotic and hemostatic processes are thrombin-mediated and that the biologic antithrombin hirudin produces a potent, dose-dependent inhibition of arterial thrombus formation that greatly exceeds the minimal antithrombotic effects produced by heparin.

Platelet ERp57 Is Required for Hemostasis and Thrombosis.

Blood ◽  
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.

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

Reduction of Phosphatidylserine Exposure through Copper Chelation Leads to Reduced Fibrin Deposition After Laser Induced Vascular Injury In Vivo.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3049-3049
Author(s):  
Reema Jasuja ◽  
Hans-Ulrich Pauer ◽  
Regan T Baird ◽  
Bruce Furie ◽  
Barbara Furie
Keyword(s):  
Thrombus Formation ◽  
Annexin V ◽  
Fold Increase ◽  
Fibrin Deposition ◽  
Copper Chelation ◽  
Prothrombin Fragment ◽  
Laser Injury ◽  
Phosphatidylserine Exposure ◽  
Copper Chelator

Abstract Abstract 3049 Poster Board II-1025 Colocalization and assembly of blood coagulation factors in the presence of negatively charged phospholipids leads to a 1,000-fold increase in the rate of thrombin generation compared to the solution reaction. We have established prothrombin fragment 1, the region of prothrombin containing the γ-carboxy-glutamic acid domain, as a probe for anionic phospholipids including phosphatidylserine. Prothrombin fragment 1 binds with high affinity to phosphatidylserine-containing membranes in vitro and identifies phosphatidylserine exposure relevant for the site of assembly of coagulation complexes in vivo. In order to determine the effect of phosphatidylserine exposure on thrombus formation during the laser injury model in vivo, we treated mice orally with the Cu2+ chelator tetrathiomolybdate for one week prior to study. This treatment has been shown to suppress phosphatidylserine exposure in rats (PNAS, 100: 6700-05, 2003). After copper chelator treatment, normal partial thromboplastin times (39 sec vs 42 sec, p=0.5) and whole blood counts in treated versus untreated mice were similar, suggesting that copper chelation did not affect the function of coagulation factors or total blood cell counts. Annexin V and Prothrombin fragment 1 were also used to measure phosphatidylserine exposure after thrombin (1 U/ml) stimulation of washed platelets using flow cytometric analysis. Platelets from untreated mice exhibited 2-fold increase in binding of both Annexin V and Prothrombin fragment 1 after thrombin stimulation; these values are similar to those previously reported. In contrast, the platelets of treated mice did not expose phosphatidylserine upon thrombin stimulation. Treatment with copper chelator did not affect platelet degranulation, as determined by surface exposure of P-selectin in flow cytometry. In addition, total phospholipid content and the ratio of outer to inner membrane phospholipids was not affected by treatment with copper chelator, suggesting that any reduction in detection of phosphatidylserine was due to reduction in exposure on the cell surface in response to an appropriate stimulus rather than reduced biosynthesis. Fluorescently conjugated Prothrombin fragment 1 or fluorescently conjugated antibody directed against phosphatidylserine were used as probes to follow the kinetics of phosphatidylserine exposure after the laser injury of cremaster muscle arterioles of a living mouse using high speed fluorescence intravital microscopy. Endogenous platelets were labeled with a fluorescently conjugated Fab fragment of an anti-CD41 antibody and fibrin deposition was measured using a fluorescently conjugated antibody that recognizes fibrin but not fibrinogen. We observed a 42% reduction (median of 18 thrombi, p=0.02) in Prothrombin fragment 1 binding and a 60% reduction (median of 27 thrombi, p=0.0002) in anti-phosphatidylserine binding after laser injury compared to untreated animals (n=58 thrombi). The accumulation of platelets during thrombus formation was not affected by the treatment when compared to untreated mice (p=0.4). On the other hand, fibrin deposition was reduced by 64% in treated mice (median of 38 thrombi, p=0.001) when compared to untreated animals (39 of thrombi). These data suggest that suppression of phosphatidylserine exposure reduces assembly of coagulation complexes resulting in a suboptimal concentration of thrombin for full fibrin generation but sufficient thrombin to activate platelets to yield a normal platelet thrombus. This emphasizes the importance of the exposure of anionic phospholipids as the surface for the colocalization of the coagulation complexes in vivo. Disclosures No relevant conflicts of interest to declare.

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