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.

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.

scholarly journals Sustained hypothermia accelerates microvascular thrombus formation in mice

2005 ◽  
Vol 289 (6) ◽  
pp. H2680-H2687 ◽  
Author(s):  
Nicole Lindenblatt ◽  
Michael D. Menger ◽  
Ernst Klar ◽  
Brigitte Vollmar
Keyword(s):  
Platelet Activation ◽  
Thrombus Formation ◽  
Specific Protein ◽  
Microvascular Thrombosis ◽  
Fibrinogen Binding ◽  
Increased Risk ◽  
Systemic Hypothermia ◽  
Risk For Thrombosis

Cold is supposed to be associated with alterations in blood coagulation and a pronounced risk for thrombosis. We studied the effect of clinically encountered systemic hypothermia on microvascular thrombosis in vivo and in vitro. Ferric chloride-induced microvascular thrombus formation was analyzed in cremaster muscle preparations from hypothermic mice. Additionally, flow cytometry and Western blot analysis was used to evaluate the effect of hypothermia on platelet activation. To test whether preceding hypothermia predisposes for enhanced thrombosis, experiments were repeated after hypothermia and rewarming to 37°C. Control animals revealed complete occlusion of arterioles and venules after 742 ± 150 and 824 ± 172 s, respectively. Systemic hypothermia of 34°C accelerated thrombus formation in arterioles and venules (279 ± 120 and 376 ± 121 s; P < 0.05 vs. 37°C). This was further pronounced after cooling to 31°C (163 ± 57 and 281 ± 71 s; P < 0.05 vs. 37°C). Magnitude of thrombin receptor activating peptide (TRAP)-induced platelet activation increased with decreasing temperatures, as shown by 1.8- and 3.0-fold increases in mean fluorescence after PAC-1 binding to glycoprotein (GP)IIb-IIIa and 1.6- and 2.9-fold increases of fibrinogen binding on incubation at 34°C and 31°C. Additionally, tyrosine-specific protein phosphorylation in platelets was increased at hypothermic temperatures. In rewarmed animals, kinetics of thrombus formation were comparable to those in normothermic controls. Concomitantly, spontaneous and TRAP-enhanced GPIIb-IIIa activation did not differ between rewarmed platelets and those maintained continuously at 37°C. Moderate systemic hypothermia accelerates microvascular thrombosis, which might be mediated by increased GPIIb-IIIa activation on platelets but does not cause predisposition with increased risk for microvascular thrombus formation after rewarming.

scholarly journals Increased tissue factor expression on circulating monocytes in chronic HIV infection: relationship to in vivo coagulation and immune activation

Blood ◽  
2010 ◽  
Vol 115 (2) ◽  
pp. 161-167 ◽  
Author(s):  
Nicholas T. Funderburg ◽  
Elizabeth Mayne ◽  
Scott F. Sieg ◽  
Robert Asaad ◽  
Wei Jiang ◽  
...  
Keyword(s):  
Hiv Infection ◽  
Tissue Factor ◽  
Immune Activation ◽  
Interleukin 6 ◽  
Plasma Levels ◽  
Thrombus Formation ◽  
Hiv Replication ◽  
Increased Risk

Abstract HIV infection is associated with an increased risk of thrombosis; and as antiretroviral therapy has increased the lifespan of HIV-infected patients, their risk for cardiovascular events is expected to increase. A large clinical study found recently that all-cause mortality for HIV+ patients was related to plasma levels of interleukin-6 and to D-dimer products of fibrinolysis. We provide evidence that this elevated risk for coagulation may be related to increased proportions of monocytes expressing cell surface tissue factor (TF, thromboplastin) in persons with HIV infection. Monocyte TF expression could be induced in vitro by lipopolysaccharide and flagellin, but not by interleukin-6. Monocyte expression of TF was correlated with HIV levels in plasma, with indices of immune activation, and with plasma levels of soluble CD14, a marker of in vivo lipopolysaccharide exposure. TF levels also correlated with plasma levels of D-dimers, reflective of in vivo clot formation and fibrinolysis. Thus, drivers of immune activation in HIV disease, such as HIV replication, and potentially, microbial translocation, may activate clotting cascades and contribute to thrombus formation and cardiovascular morbidities in HIV infection.

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.

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.

Inhibitory effects of lycopene on in vitro platelet activation and in vivo prevention of thrombus formation

2005 ◽  
Vol 146 (4) ◽  
pp. 216-226 ◽  
Author(s):  
George Hsiao ◽  
Ying Wang ◽  
Nien-Hsuan Tzu ◽  
Tsorng-Hang Fong ◽  
Ming-Yi Shen ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Thrombus Formation ◽  
Inhibitory Effects ◽  
In Vitro Platelet Activation

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.

Critical Role for Cyclophilin D and the Mitochondrial Permeability Transition Pore (MPTP) in Platelet Activation.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1508-1508 ◽  
Author(s):  
Shawn M. Jobe ◽  
Katina M. Wilson ◽  
Lori Leo ◽  
Jeffery D. Molkentin ◽  
Steven R. Lentz ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Mitochondrial Permeability Transition ◽  
Critical Role ◽  
In Vitro Tests ◽  
Cyclophilin D ◽  
Wild Type ◽  
Glycoprotein Vi ◽  
Stimulation Of

Abstract Dual stimulation of platelets with thrombin and collagen results in the formation of a unique subpopulation of highly activated platelets. Characteristics of the highly activated platelet subpopulation includeincreased surface retention of procoagulant alpha granule proteins,high-level phosphatidylserine (PS) externalization, andmodulation of the fibrinogen receptor αIIbβ3 as evidenced by their decreased recognition by antibodies to activated αIIbβ3 such as PAC-1 and JON/A. Formation of the highly activated platelet subpopulation is closely correlated with a rapid loss of mitochondrial transmembrane potential (ΔΨm), a marker of MPTP formation. To test whether formation of the MPTP might regulate the development of the highly activated platelet subpopulation, platelet activation responses were examined in the presence of inhibitors and activators of MPTP formation. Cyclosporine, an inhibitor of MPTP formation, inhibited both PS externalization and αIIbβ3 modulation following dual stimulation with thrombin and the glycoprotein VI agonist convulxin (58 ± 4% vs. 9 ± 3%, p<0.01). Conversely, thrombin stimulation of platelets in the presence of H2O2 (100μM), an MPTP activator, increased PS externalization and αIIbβ3 modulation relative to platelets stimulated with thrombin alone (11 ± 3% vs. 48 ± 6%, p<0.05). Platelet activation responses were examined in cyclophilin D null (CypD −/−) mice, which have marked impairment of MPTP formation. Following dual agonist stimulation with thrombin and convulxin, both αIIbβ3 modulation and platelet PS externalization were significantly abrogated in CypD −/− platelets relative to wild type (7 ± 1% vs. 69 ± 1%, p<0.01). Alpha granule release, however, was unaffected in the absence of CypD. In vitro tests of platelet function similarly demonstrated that CypD −/− platelets had marked impairment of platelet prothrombinase activity relative to wild-type platelets after stimulation with thrombin and convulxin, but normal platelet aggregation responses. We then tested the hypothesis that CypD −/− mice would have an altered thrombotic response to arterial injury. Following photochemical injury of the carotid artery endothelium, a stable occlusive thrombus formed more rapidly in CypD −/− than in wild-type mice (16 ± 2 vs. 32 ± 7 min, p<0.05). Tail-bleeding time was unaffected. These results strongly implicate cyclophilin D and the MPTP as critical regulators of the subset of platelet activation responses occurring in the highly activated platelet subpopulation and suggest that activation of this novel platelet mitochondrial signaling pathway might play an important role in the regulation of the thrombotic response in vivo.

STIM1 Deficiency Results In Impaired Platelet Procoagulant Activity and Protection From Arterial Thrombosis

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 485-485
Author(s):  
Firdos Ahmad ◽  
Lucia Stefanini ◽  
Timothy Daniel Ouellette ◽  
Teshell K Greene ◽  
Stefan Feske ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Conflicts Of Interest ◽  
Thrombus Formation ◽  
Critical Role ◽  
Phosphatidyl Serine ◽  
Flow Chamber ◽  
Procoagulant Activity ◽  
Static Conditions

Abstract Abstract 485 Platelet activation is a central event in thrombosis and hemostasis. We recently demonstrated that most aspects of platelet activation depend on synergistic signaling by two signaling modules: 1) Ca2+/CalDAG-GEFI/Rap1 and 2) PKC/P2Y12/Rap1. The intracellular Ca2+ concentration of platelets is regulated by Ca2+ release from the endoplasmic reticulum (ER) and store-operated calcium entry (SOCE) through the plasma membrane. Stromal interaction molecule 1 (STIM1) was recently identified as the ER Ca2+ sensor that couples Ca2+ store release to SOCE. In this study, we compared the activation response of platelets lacking STIM1−/− or CalDAG-GEFI−/−, both in vitro and in vivo. To specifically investigate Ca2+-dependent platelet activation, some of the experiments were performed in the presence of inhibitors to P2Y12. The murine Stim1 gene was deleted in the megakaryocyte/platelet lineage by breeding Stim flox/flox mice with PF4-Cre mice (STIM1fl/fl). STIM1fl/fl platelets showed markedly reduced SOCE in response to agonist stimulation. aIIbβ3 activation in STIM1fl/fl platelets was significantly reduced in the presence but not in the absence of the P2Y12 inhibitor, 2-MesAMP. In contrast, aIIbb3 activation was completely inhibited in 2-MesAMP-treated CalDAG-GEFI−/− platelets. Deficiency in STIM1, and to a lesser extent in CalDAG-GEFI, reduced phosphatidyl serine (PS) exposure in platelets stimulated under static conditions. PS exposure was completely abolished in both STIM1fl/fl and CalDAG-GEFI−/− platelets stimulated in the presence of 2-MesAMP. To test the ability of platelets to form thrombi under conditions of arterial shear stress, we performed flow chamber experiments with anticoagulated blood perfused over a collagen surface. Thrombus formation was abolished in CalDAG-GEFI−/− blood and WT blood treated with 2-MesAMP. In contrast, STIM1fl/fl platelets were indistinguishable from WT platelets in their ability to form thrombi. STIM1fl/fl platelets, however, were impaired in their ability to express PS when adhering to collagen under flow. Consistently, when subjected to a laser injury thrombosis model, STIM1fl/fl mice showed delayed and reduced fibrin generation, resulting in the formation of unstable thrombi. In conclusion, our studies indicate a critical role of STIM1 in SOCE and platelet procoagulant activity, but not in CalDAG-GEFI mediated activation of aIIbb3 integrin. Disclosures: No relevant conflicts of interest to declare.

Platelet JNK1 is involved in secretion and thrombus formation

Blood ◽  
2010 ◽  
Vol 115 (20) ◽  
pp. 4083-4092 ◽  
Author(s):  
Frédéric Adam ◽  
Alexandre Kauskot ◽  
Paquita Nurden ◽  
Eric Sulpice ◽  
Marc F. Hoylaerts ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Thrombus Formation ◽  
Blood Perfusion ◽  
Collagen Matrix ◽  
In Vivo Model ◽  
Wild Type ◽  
Platelet Secretion

Abstract The role of c-Jun NH2-terminal kinase 1 (JNK1) in hemostasis and thrombosis remains unclear. We show here, with JNK1-deficient (JNK1−/−) mice, that JNK1 plays an important role in platelet biology and thrombus formation. In tail-bleeding assays, JNK1−/− mice exhibited longer bleeding times than wild-type mice (396 ± 39 seconds vs 245 ± 32 seconds). We also carried out in vitro whole-blood perfusion assays on a collagen matrix under arterial shear conditions. Thrombus formation was significantly reduced for JNK1−/− platelets (51%). In an in vivo model of thrombosis induced by photochemical injury to cecum vessels, occlusion times were 4.3 times longer in JNK1−/− arterioles than in wild-type arterioles. Moreover, in vitro studies carried out in platelet aggregation conditions demonstrated that, at low doses of agonists, platelet secretion was impaired in JNK1−/− platelets, leading to altered integrin αIIbβ3 activation and reduced platelet aggregation, via a mechanism involving protein kinase C. JNK1 thus appears to be essential for platelet secretion in vitro, consistent with its role in thrombus growth in vivo. Finally, we showed that ERK2 and another isoform of JNK affect platelet aggregation through 2 pathways, one dependent and another independent of JNK1.

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