Abstract 98: Antiphospholipid Antibodies Induce Thrombosis by Activating Endothelial PP2A via ApoER2-Dab2-PSD95 Complex Formation

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Anastasia Sacharidou ◽  
Philip Shaul ◽  
Chieko Mineo
Keyword(s):  
Complex Formation ◽  
Antiphospholipid Antibodies ◽  
Thrombus Formation ◽  
Ldl Receptor ◽  
Surface Protein ◽  
Current Treatment ◽  
Wild Type ◽  
Mutant Proteins ◽  
Endothelial Cell Surface

In the antiphospholipid syndrome (APS), patients generate antiphospholipid antibodies (aPL) that promote thrombosis. We previous showed that aPL recognition of the endothelial cell surface protein β2-Glycoprotein I (β2GPI) causes β2GPI to interact with the LDL receptor family member ApoER2 and thereby antagonize endothelial NO synthase (eNOS). The decrease in bioavailable NO then leads to exaggerated thrombus formation. In the present work we sought to determine how aPL and the β2GPI-ApoER2 tandem antagonize eNOS. Initial experiments employed co-immunoprecipitation, RNAi-based gene silencing and adenoviral introduction of mutant proteins into human aortic endothelial cells. We discovered that in contrast to normal human IgG from healthy subjects (NHIgG), aPL invoke the formation of a complex between the cytoplasmic tail of ApoER2 and Dab-2 and PSD95, and that the formation of the complex is required for eNOS antagonism. We also found that ApoER2-Dab2-PSD95 complex formation in response to aPL potently activates the serine/threonine phosphatase PP2A, which in turn dephosphorylates eNOS-S1179, thereby extinguishing eNOS enzymatic activity. Furthermore, we found that upon aPL treatment, the PP2A catalytic and regulatory subunits are recruited to the ApoER2-Dab2-PSD95 complex. To test if these processes are operative in APS-related thrombosis in vivo, intravital microscopy of the mesenteric microcirculation was employed to evaluate thrombus formation in mice. In wild-type mice, aPL administration caused exaggerated thrombus formation compared to treatment with NHIgG. In contrast, ApoER2 did not promote thrombosis in knock-in ApoER2-EIG mice harboring a mutant receptor incapable of interacting with Dab-2. Moreover, in wild-type mice aPL treatment caused a dramatic increase in PP2A activity in the aorta, and administration of the PP2A inhibitor Endothall fully prevented thrombus formation induced by aPL. Having revealed the molecular underpinnings of the disorder, current treatment of APS with anticoagulation, which is often ineffective and fraught with complications, can potentially be replaced by new mechanism-based therapies targeting ApoER2 complex formation or PP2A.

Abstract 456: Antiphospholipid Antibodies Promote Platelet Activation and Thrombosis by Inhibition of Platelet eNOS

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Craig Morrell ◽  
AnneMarie Swaim ◽  
Tanika Martin ◽  
Guillermina Girardi ◽  
Jane E Salmon ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Antiphospholipid Antibodies ◽  
Thrombus Formation ◽  
Ldl Receptor ◽  
Wild Type ◽  
Human Igg ◽  
Increased Risk ◽  
Receptor Family

The antiphospholipid syndrome (APS) is an autoimmune systemic disorder characterized by the persistent presence of antiphospholipid antibodies (aPL Ab) and increased risk of thrombosis, coronary artery disease and myocardial infarction. Although platelets are known direct targets of aPL Ab action, the molecular basis of aPL Ab actions on platelets remains unclear. Platelet endothelial NO synthase (eNOS) is a key regulator of platelet function, with NO causing blunted activation. We therefore determined whether aPL Ab modulate platelet eNOS. Normal human IgG (NH IgG) and human IgG containing polyclonal aPL Ab were obtained from healthy individuals and APS patients, respectively, and purified using protein G-Sepharose chromatography. Using both human and mouse platelets, we found that aPL Ab increased agonist-induced platelet activation whereas NH IgG did not. In contrast to the enhanced activation by aPL Ab in platelets from wild-type mice, aPL Ab had no effect on platelets isolated from eNOS null mice. Pre-treatment of platelets with aPL Ab also inhibited insulin-mediated eNOS stimulation as evidenced by diminished cGMP production and DAF2 fluorescence. Receptor associated protein (RAP), an antagonist of ligand binding to members of the LDL receptor family, blocked aPL Ab-induced increases in platelet activation. RAP also prevented aPL Ab-mediated antagonism of platelet eNOS, indicating that aPL Ab signal through the platelet ApoER2â ϵ™ (LRP8) to attenuate eNOS activity. Furthermore, using intravital microscopy of the mouse mesenteric circulation, we demonstrated that platelets from wild-type mice treated with aPL Ab have increased rolling on a stimulated endothelium and a decreased time to thrombus formation in vivo versus platelets treated with NH IgG. In contrast, aPL Ab did not alter the in vivo function of platelets from eNOS null mice. These cumulative in vitro and in vivo findings demonstrate that aPL Ab antagonism of platelet eNOS through LDL receptor family member binding underlies aPL Ab-mediated thrombosis.

Annexin A2 Deficient Mice Are Resistant to Pathogenic Effects of Antiphospholipid Antibodies in Vivo

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 421-421
Author(s):  
Zurina Romay-Penabad ◽  
Guadalupe Montiel-Manzano ◽  
Elizabeth Pappalardo ◽  
Katherine A. Hajjar ◽  
Tuya Shilagard ◽  
...  
Keyword(s):  
Antiphospholipid Antibodies ◽  
Cell Adhesion Molecule ◽  
Carotid Arteries ◽  
Thrombus Formation ◽  
Annexin A2 ◽  
Target Cells ◽  
Wild Type ◽  
Deficient Mice ◽  
Pathogenic Effects

Abstract Background: Thrombosis is an important cause of morbidity and mortality in Antiphospholipid Syndrome (APS) and in SLE patients with antiphospholipid antibodies (aPL). APL recognize β2 glycoprotein I (β2GPI)-bound to receptor (s) in endothelial cells (EC) and other target cells (i.e. platelets, monocytes) and trigger an intracellular signalling and a pro-coagulant and pro-inflammatory phenotype [i e.expression of tissue factor (TF), vascular cell adhesion molecule-1 (VCAM-1)] that lead to thrombosis. There is in vitro evidence that annexin A2 (A2), a receptor for tissue plasminogen activator (tPA) and plasminogen – and possibly other proteins such as toll-like receptors or the receptor for apolipoprotein E2′ - may be binding β2GPI on the membrane of target cells. Here, we examined the involvement of A2 in aPL-mediated pathogenic effects in vivo. We studied the effects of aPL Abs on thrombus formation, VCAM-1 expression in aortas of mice, and TF function in carotid artery homogenates in annexin A2 deficient (−/−) mice. Methods: A2 (−/−) mice and the corresponding wild-type (WT) mice, in groups of 10, were injected i.p. twice (0 and 48 hours later) with IgG from a patient with APS (IgG-APS) or with control IgG (IgG-NHS). Seventy-two hours after the first injection, several procedures were done in each mice: dynamics of thrombus formation (thrombus size), TF function in homogenates of carotid arteries, and c) VCAM-1 expression in the aortas using quantum dot nano crystals and two-photon excitation laser scanning microscopy. In addition, we examined the effect of an anti-A2 antibody on aPL-induced expression of intercellular cell-adhesion molecule (ICAM-1), E-selectin and TF acvitity on cultured endothelial cells (EC). Results: The titers of aCL and anti-β2GPI Abs in the sera of the mice at the time of surgery were medium-high positive in A2 (−/−) mice and in wild type mice injected with IgG-APS. Thrombus sizes were significantly larger in WT mice injected with IgG-APS when compared to similar type of mice treated with IgG-NHS (p=0.003). The size of thrombus in A2 (−/−) mice injected with IgG-APS was significantly smaller than mean thrombus size in WT mice injected with IgG-APS (p:0.0005). However, thrombus size in A2 (−/−) mice was larger in mice injected with IgG-APS when compared to same type of mice treated with control IgG-NHS (p=0.003), indicating a partial but significant abrogation of the thrombogenic effect. TF activity was significantly larger in WT mice treated with IgG-APS when compared to mice injected with IgG-NHS. Importantly, TF activity in carotid arteries homogenates of annexin A2 (−/−) mice injected with IgG-APS was significantly decreased (by 52%) when compared to wild type mice treated with IgG-APS. The expression of VCAM-1 in aorta of annexin A2 (−/−) ex vivo was also significantly reduced compared to LPS-treated mice (positive control) (p= 0.01). Interestingly, anti-A2 antibody significantly decreased aPL-induced expression of ICAM-1, E-sel and TF on cultured EC. Conclusions: Altogether these data indicate for the first time that A2 is involved in vivo pathogenic effects of aPL Abs. These findings may have important implications to devise new targeted and more specific therapeutic approaches to block the pathogenic effects of aPL Abs in patients with APS and SLE.

Antiphospholipid Antibodies Promote Leukocyte-Endothelial Cell Adhesion by Antagonizing Endothelial NO Synthase Via b2GPI and ApoER2.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3039-3039
Author(s):  
Cristina Tarango ◽  
Sangeetha Ramesh ◽  
Craig N. Morrell ◽  
Gail D. Thomas ◽  
Guillermina Girardi ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Antiphospholipid Antibodies ◽  
Ldl Receptor ◽  
Surface Protein ◽  
No Synthase ◽  
Cell Phenotype ◽  
No Donor ◽  
Endothelial No Synthase ◽  
Increased Risk

Abstract Abstract 3039 Poster Board II-1015 The antiphospholipid syndrome (APS) is an autoimmune disease characterized by the presence of antiphospholipid antibodies (aPL), with pathogenic aPL directed primarily against the cell surface protein β2 glycoprotein I (β2GPI), increased risk for thrombosis and cardiovascular events, and pregnancy morbidity. The endothelium is a critical direct target of aPL, which upregulate adhesion molecule expression and procoagulant activity. However, the molecular basis for aPL actions on endothelium is unknown. Nitric oxide (NO) generated by endothelial NO synthase (eNOS) has potent antiadhesive and antithrombotic properties. In this study we determined if aPL-induced alterations in endothelial cell phenotype are mediated by aPL actions on eNOS. Normal human IgG (NHIgG) and polyclonal aPL were obtained from healthy adults and APS patients, respectively. We found that aPL prevented acetylcholine (Ach)- or VEGF-mediated attenuation of monocyte adhesion to cultured endothelial cells and this was reversed by an NO donor, indicating a role for eNOS antagonism. In contrast, NHIgG did not affect adhesion. Whereas NHIgG did not alter eNOS activation, stimulation of eNOS by VEGF and other agonists was fully antagonized by aPL. In mice, NO-dependent, Ach-induced increases in carotid vascular conductance were unaffected by NHIgG but impaired by aPL, indicating that these processes are operative in vivo. Additional studies in culture showed that aPL attenuates eNOS activation by inhibiting Ser1179 phosphorylation, deprivation of β2GPI prevented aPL inhibition of eNOS, and monoclonal antibody against β2GPI mimicked the effects of aPL. Furthermore, receptor-associated protein (RAP) antagonism of LDL receptors, which are known to bind dimerized β2GPI, prevented aPL inhibition of eNOS in culture, and mice null for the LDL receptor apolipoprotein receptor 2 (apoER2) were protected from aPL inhibition of eNOS in vivo. Moreover, aPL-stimulated increases in leukocyte-endothelial adhesion were absent in both eNOS-/- and apoER2-/- mice. Thus, aPL-induced increases in leukocyte-endothelial adhesion are caused by eNOS antagonism, which is due to impaired S1179 phosphorylation mediated by β2GPI and apoER2. Novel therapies for APS can now be developed targeting these mechanisms. Disclosures Thomas: NIcox Research Institute: Research Funding. Thorpe:Peregrine Pharm.: Consultancy, sponsored research agreement.

scholarly journals Mutant tryptophan aporepressors with altered specificities of corepressor recognition.

Genetics ◽  
1991 ◽  
Vol 128 (1) ◽  
pp. 29-35
Author(s):  
D N Arvidson ◽  
M Shapiro ◽  
P Youderian
Keyword(s):  
Dna Binding ◽  
Binding Pocket ◽  
Side Chain ◽  
Wild Type ◽  
Mutant Proteins ◽  
Naturally Occurring ◽  
Repressor Complex ◽  
Genes Encoding ◽  
Active Repressor

Abstract The Escherichia coli trpR gene encodes tryptophan aporepressor, which binds the corepressor ligand, L-tryptophan, to form an active repressor complex. The side chain of residue valine 58 of Trp aporepressor sits at the bottom of the corepressor (L-tryptophan) binding pocket. Mutant trpR genes encoding changes of Val58 to the other 19 naturally occurring amino acids were made. Each of the mutant proteins requires a higher intracellular concentration of tryptophan for activation of DNA binding than wild-type aporepressor. Whereas wild-type aporepressor is activated better by 5-methyltryptophan (5-MT) than by tryptophan, Ile58 and other mutant aporepressors prefer tryptophan to 5-MT as corepressor, and Ala58 and Gly58 prefer 5-MT much more strongly than wild-type aporepressor in vivo. These mutant aporepressors are the first examples of DNA-binding proteins with altered specificities of cofactor recognition.

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 Intragenic suppression among CDC34 (UBC3) mutations defines a class of ubiquitin-conjugating catalytic domains.

1995 ◽  
Vol 15 (10) ◽  
pp. 5635-5644 ◽  
Author(s):  
Y Liu ◽  
N Mathias ◽  
C N Steussy ◽  
M G Goebl
Keyword(s):  
Temperature Sensitive ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Cloning And Sequencing ◽  
Mutant Proteins ◽  
Catalytic Domains ◽  
Conserved Regions ◽  
E2 Enzymes ◽  
Key Residues

Ubiquitin-conjugating (E2) enzymes contain several regions within their catalytic domains that are highly conserved. However, within some of these conserved regions are several residues that may be used to define different classes of catalytic domains for the E2 enzymes. One class can be defined by the Ubc1 protein, which contains K-65, D-90, and D-120, while the corresponding positions within the Cdc34 (Ubc3) protein, which defines a second class of enzymes, contain S-73, S-97, and S-139, respectively. The presence of these differences within otherwise highly conserved regions of this family suggests that these residues may be critical for the specificity of Cdc34 function or regulation. Therefore, we have constructed a series of cdc34 alleles encoding mutant proteins in which these serine residues have been changed to other amino acid residues, including alanine and aspartic acid. In vivo complementation studies showed that S-97, which lies near the active site C-95, is essential for Cdc34 function. The addition of a second mutation in CDC34, which now encoded both the S97D and S73K changes, restored partial function to the Cdc34 enzyme. Moreover, the deletion of residues 103 to 114 within Cdc34, which are not present in the Ubc1-like E2s, allowed the S73K/S97D mutant to function as efficiently as wild-type Cdc34 protein. Finally, the cloning and sequencing of the temperature-sensitive alleles of CDC34 indicated that A-62 is also unique to the Cdc34 class of E2 enzymes and that mutations at this position can be detrimental to Cdc34 function. Our results suggest that several key residues within conserved regions of the E2 enzyme family genetically interact with each other and define a class of E2 catalytic domains.

scholarly journals The In Vivo Association of BiP with Newly Synthesized Proteins Is Dependent on the Rate and Stability of Folding and Not Simply on the Presence of Sequences That Can Bind to BiP

1999 ◽  
Vol 144 (1) ◽  
pp. 21-30 ◽  
Author(s):  
Rachel Hellman ◽  
Marc Vanhove ◽  
Annabelle Lejeune ◽  
Fred J. Stevens ◽  
Linda M. Hendershot
Keyword(s):  
Secretory Pathway ◽  
Variable Region ◽  
Wild Type ◽  
Mutant Proteins ◽  
Protein Subunits ◽  
Hsp70 Family ◽  
Nascent Chain ◽  
Kinetic Traps

Immunoglobulin heavy chain-binding protein (BiP) is a member of the hsp70 family of chaperones and one of the most abundant proteins in the ER lumen. It is known to interact transiently with many nascent proteins as they enter the ER and more stably with protein subunits produced in stoichiometric excess or with mutant proteins. However, there also exists a large number of secretory pathway proteins that do not apparently interact with BiP. To begin to understand what controls the likelihood that a nascent protein entering the ER will associate with BiP, we have examined the in vivo folding of a murine λI immunoglobulin (Ig) light chain (LC). This LC is composed of two Ig domains that can fold independent of the other and that each possess multiple potential BiP-binding sequences. To detect BiP binding to the LC during folding, we used BiP ATPase mutants, which bind irreversibly to proteins, as “kinetic traps.” Although both the wild-type and mutant BiP clearly associated with the unoxidized variable region domain, we were unable to detect binding of either BiP protein to the constant region domain. A combination of in vivo and in vitro folding studies revealed that the constant domain folds rapidly and stably even in the absence of an intradomain disulfide bond. Thus, the simple presence of a BiP-binding site on a nascent chain does not ensure that BiP will bind and play a role in its folding. Instead, it appears that the rate and stability of protein folding determines whether or not a particular site is recognized, with BiP preferentially binding to proteins that fold slowly or somewhat unstably.

Macrovascular thrombosis is driven by tissue factor derived primarily from the blood vessel wall

Blood ◽  
2005 ◽  
Vol 105 (1) ◽  
pp. 192-198 ◽  
Author(s):  
Sharlene M. Day ◽  
Jennifer L. Reeve ◽  
Brian Pedersen ◽  
Diana M Farris ◽  
Daniel D. Myers ◽  
...  
Keyword(s):  
Blood Vessel ◽  
Tissue Factor ◽  
Vessel Wall ◽  
Thrombus Formation ◽  
Vena Cava ◽  
Vascular Wall ◽  
Blood Vessel Wall ◽  
Wild Type

Abstract Leukocytes and leukocyte-derived microparticles contain low levels of tissue factor (TF) and incorporate into forming thrombi. Although this circulating pool of TF has been proposed to play a key role in thrombosis, its functional significance relative to that of vascular wall TF is poorly defined. We tested the hypothesis that leukocyte-derived TF contributes to thrombus formation in vivo. Compared to wild-type mice, mice with severe TF deficiency (ie, TF–/–, hTF-Tg+, or “low-TF”) demonstrated markedly impaired thrombus formation after carotid artery injury or inferior vena cava ligation. A bone marrow transplantation strategy was used to modulate levels of leukocyte-derived TF. Transplantation of low-TF marrow into wild-type mice did not suppress arterial or venous thrombus formation. Similarly, transplantation of wild-type marrow into low-TF mice did not accelerate thrombosis. In vitro analyses revealed that TF activity in the blood was very low and was markedly exceeded by that present in the vessel wall. Therefore, our results suggest that thrombus formation in the arterial and venous macrovasculature is driven primarily by TF derived from the blood vessel wall as opposed to leukocytes.

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

Blood ◽  
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.

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.

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