NADPH Oxidases Are Required for Full Platelet Activation In Vitro and Thrombosis In Vivo but Dispensable for Plasma Coagulation and Hemostasis

Objective: Using 3KO (triple NOX [NADPH oxidase] knockout) mice (ie, NOX1 −/− /NOX2 −/− /NOX4 −/− ), we aimed to clarify the role of this family of enzymes in the regulation of platelets in vitro and hemostasis in vivo. Approach and Results: 3KO mice displayed significantly reduced platelet superoxide radical generation, which was associated with impaired platelet aggregation, adhesion, and thrombus formation in response to the key agonists collagen and thrombin. A comparison with single-gene knockouts suggested that the phenotype of 3KO platelets is the combination of the effects of the genetic deletion of NOX1 and NOX2, while NOX4 does not show any significant function in platelet regulation. 3KO platelets displayed significantly higher levels of cGMP—a negative platelet regulator that activates PKG (protein kinase G). The inhibition of PKG substantially but only partially rescued the defective phenotype of 3KO platelets, which are responsive to both collagen and thrombin in the presence of the PKG inhibitors KT5823 or Rp-8-pCPT-cGMPs, but not in the presence of the NOS (NO synthase) inhibitor L-NG-monomethyl arginine. In vivo, triple NOX deficiency protected against ferric chloride–driven carotid artery thrombosis and experimental pulmonary embolism, while hemostasis tested in a tail-tip transection assay was not affected. Procoagulatory activity of platelets (ie, phosphatidylserine surface exposure) and the coagulation cascade in platelet-free plasma were normal. Conclusions: This study indicates that inhibiting NOXs has strong antithrombotic effects partially caused by increased intracellular cGMP but spares hemostasis. NOXs are, therefore, pharmacotherapeutic targets to develop new antithrombotic drugs without bleeding side effects.

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Abstract 13598: The G-protein BetaGamma Subunits Regulate Platelet Function

Circulation ◽

10.1161/circ.142.suppl_3.13598 ◽

2020 ◽

Vol 142 (Suppl_3) ◽

Author(s):

Ahmed Alarabi ◽

Zubair Karim ◽

Victoria Hinojos ◽

Patricia A Lozano ◽

Keziah Hernandez ◽

...

Keyword(s):

G Protein ◽

Platelet Function ◽

Thrombus Formation ◽

Human Platelets ◽

Inhibitory Effects ◽

Clot Retraction ◽

Betagamma Subunits

Platelet activation involves tightly regulated processes to ensure a proper hemostasis response, but when unbalanced, can lead to pathological consequences such as thrombus formation. G-protein coupled receptors (GPCRs) regulate platelet function by interacting with and mediating the response to various physiological agonists. To this end, an essential mediator of GPCR signaling is the G protein Gαβγ heterotrimers, in which the βγ subunits are central players in downstream signaling pathways. While much is known regarding the role of the Gα subunit in platelet function, that of the βγ remains poorly understood. Therefore, we investigated the role of Gβγ subunits in platelet function using a Gβγ (small molecule) inhibitor, namely gallein. We observed that gallein inhibits platelet aggregation and secretion in response to agonist stimulation, in both mouse and human platelets. Furthermore, gallein also exerted inhibitory effects on integrin αIIbβ3 activation and clot retraction. Finally, gallein’s inhibitory effects manifested in vivo , as documented by its ability to modulate physiological hemostasis and delay thrombus formation. Taken together, our findings demonstrate, for the first time, that Gβγ directly regulates GPCR-dependent platelet function, in vitro and in vivo . Moreover, these data highlight Gβγ as a novel therapeutic target for managing thrombotic disorders.

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Study of Staphylococcus aureusPathogenic Genes by Transfer and Expression in the Less Virulent Organism Streptococcus gordonii

Infection and Immunity ◽

10.1128/iai.69.2.657-664.2001 ◽

2001 ◽

Vol 69 (2) ◽

pp. 657-664 ◽

Cited By ~ 32

Author(s):

P. Stutzmann Meier ◽

J. M. Entenza ◽

P. Vaudaux ◽

P. Francioli ◽

M. P. Glauser ◽

...

Keyword(s):

Single Gene ◽

Individual Factors ◽

Gene Products ◽

Streptococcus Gordonii ◽

Pathogenic Role ◽

Gain Of Function ◽

Function Strategy

ABSTRACT Because Staphylococcus aureus strains contain multiple virulence factors, studying their pathogenic role by single-gene inactivation generated equivocal results. To circumvent this problem, we have expressed specific S. aureus genes in the less virulent organism Streptococcus gordonii and tested the recombinants for a gain of function both in vitro and in vivo. Clumping factor A (ClfA) and coagulase were investigated. Both gene products were expressed functionally and with similar kinetics during growth by streptococci and staphylococci. ClfA-positive S. gordoniiwas more adherent to platelet-fibrin clots mimicking cardiac vegetations in vitro and more infective in rats with experimental endocarditis (P < 0.05). Moreover, deletingclfA from clfA-positive streptococcal transformants restored both the low in vitro adherence and the low in vivo infectivity of the parent. Coagulase-positive transformants, on the other hand, were neither more adherent nor more infective than the parent. Furthermore, coagulase did not increase the pathogenicity ofclfA-positive streptococci when both clfA andcoa genes were simultaneously expressed in an artificial minioperon in streptococci. These results definitively attribute a role for ClfA, but not coagulase, in S. aureus endovascular infections. This gain-of-function strategy might help solve the role of individual factors in the complex the S. aureus-host relationship.

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IκB kinase 2 is not essential for platelet activation

Blood Advances ◽

10.1182/bloodadvances.2019001044 ◽

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.

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Nitric oxide enhancement of erythropoietin production in the isolated perfused rat kidney

AJP Cell Physiology ◽

10.1152/ajpcell.1995.269.4.c917 ◽

1995 ◽

Vol 269 (4) ◽

pp. C917-C922 ◽

Cited By ~ 10

Author(s):

K. Yoshioka ◽

J. W. Fisher

Keyword(s):

Nitric Oxide ◽

Rat Kidney ◽

Mrna Levels ◽

Isolated Perfused Rat Kidney ◽

Intracellular Cgmp ◽

Perfused Rat Kidney ◽

Arterial Po2

We have previously reported that nitric oxide (NO) and guanosine 3',5'-cyclic monophosphate (cGMP) may be involved in the regulation of erythropoietin (Epo) production in response to hypoxia both in vivo and in vitro (20). In the present studies, we have used the isolated perfused rat kidney to assess the role of NO in oxygen sensing and Epo production. When arterial PO2 was reduced from 100 mmHg (normoxemic) to 30 mmHg (hypoxemic) in the perfusate of this system, perfusate levels of Epo were significantly increased. This hypoxia-induced increase in Epo production was significantly decreased by the addition of NG-nitro-L-arginine methyl ester (L-NAME; 1 mM) to the perfusates. Hypoxemic perfusion also produced a significant increase, and L-NAME significantly inhibited this increase, in intracellular cGMP levels in the kidney when compared with normoxemic perfused kidneys. Quantitative reverse transcription-polymerase chain reaction also revealed that hypoxemic perfusion produced significant increases in Epo mRNA levels in the kidney, which was blocked by L-NAME. Our findings further support an important role for the NO/cGMP system in hypoxic regulation of Epo production.

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Impact of Antithrombin Deficiency on Efficacies of DU-176b, a Novel Orally Active Direct Factor Xa Inhibitor, and Antithrombin Dependent Anticoagulants, Fondaparinux and Heparin.

Blood ◽

10.1182/blood.v106.11.1874.1874 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1874-1874 ◽

Cited By ~ 1

Author(s):

Toshio Fukuda ◽

Yuko Honda ◽

Chikako Matsumoto ◽

Nobutoshi Sugiyama ◽

Tadashi Matsushita ◽

...

Keyword(s):

Thrombus Formation ◽

Factor Xa ◽

In Vitro Study ◽

Antithrombotic Effects ◽

At Deficiency ◽

Fxa Inhibitor ◽

Orally Active ◽

Relative Potencies

Abstract Antithrombin (AT) is a major physiological inhibitor of coagulation factors, primarily inhibiting thrombin and factor Xa (FXa). Binding of heparin and its related pentasaccharides, fondaparinux, to AT dramatically accelerates inhibition of thrombin and FXa. Entire AT-dependency of heparins may result in decreased anticoagulant effects in patients with inherited or acquired AT deficiencies. Objectives: We have developed an orally active direct (i.e. AT-independent) FXa inhibitor, DU-176b. The objectives of this study were to examine the anticoagulant and antithrombotic effects of DU-176b, fondaparinux, and heparin in heterozygous AT deficient (AT+/−) mice (Refs 1, 2), and to determine the impact of AT deficiency on the efficacies of these anticoagulants. Methods: [In vitro study] Plasma obtained from wild type (AT+/+, C57BL/6J) and AT+/− mice were subjected to measurement of levels of AT antigen and activity. The anticoagulant effects on prothrombin time (PT) and activated partial thromboplastin time (APTT) was measured and the drug concentrations were calculated required to double the clotting time (CT2). [In vivo study] Male AT+/+ and AT+/− mice were fasted over night. Thrombosis was induced in the inferior vena cava by applying filter paper (1 x 5 mm) presoaked in 15% FeCl3 for 10 min. Thrombus was removed 60 min after FeCl3 treatment and its protein content was assessed by Bradford method. DU-176b was orally administered 60 min before, fondaparinux was given s.c. 30 min before, and heparin was injected into the jugular vein 3 min before thrombus induction. Relative potencies of antithrombotic effects in AT+/− mice to those in AT+/+ mice were analyzed by parallel line assay. Results: [In vitro study] Plasma levels of AT antigen and activity in AT+/− mice were deceased to 40% compared with AT+/+ plasma. PT-CT2 of DU-176b was 0.72 μM in AT+/+ plasma and 0.74 μM in AT+/− plasma, respectively, indicating that anticoagulant activity of the direct FXa inhibitor was not affected by heterozygous AT deficiency. APTT-CT2 of fondaparinux and heparin in AT+/+ plasma was 3.8 μM and 14 mU/mL, respectively, whereas APTT-CT2 in AT+/− plasma was 9.2 μM and 20 mU/mL, respectively. Therefore, anticoagulant activities of such AT-dependent inhibitors were attenuated in AT+/− plasma. [In vivo study] All three anticoagulants inhibited venous thrombus formation of AT+/+ mice in dose-dependent manners. In AT+/− mice, the antithrombotic effects of fondaparinux and heparin were less potent than those in AT+/+ mice. In contrast, DU-176b prevented thrombus formation equipotently in both mice. Relative potencies of DU-176b, fondaparinux and heparin were 0.84, 0.40, and 0.70, respectively. Conclusion: DU-176b exerts a comparable antithrombotic effect even in individuals with low plasma AT antigens and activities. Thus, DU-176b may be prioritized over AT-dependent agents for use at the fixed dose in patients with lower plasma AT concentrations.

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Platelet JNK1 is involved in secretion and thrombus formation

Blood ◽

10.1182/blood-2009-07-233932 ◽

2010 ◽

Vol 115 (20) ◽

pp. 4083-4092 ◽

Cited By ~ 72

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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Tissue factor–positive neutrophils bind to injured endothelial wall and initiate thrombus formation

Blood ◽

10.1182/blood-2012-06-437772 ◽

2012 ◽

Vol 120 (10) ◽

pp. 2133-2143 ◽

Cited By ~ 143

Author(s):

Roxane Darbousset ◽

Grace M. Thomas ◽

Soraya Mezouar ◽

Corinne Frère ◽

Rénaté Bonier ◽

...

Keyword(s):

Endothelial Cells ◽

Tissue Factor ◽

Blood Coagulation ◽

Thrombus Formation ◽

Coagulation Cascade ◽

Factor Xii ◽

Long Time ◽

Platelet Accumulation

AbstractFor a long time, blood coagulation and innate immunity have been viewed as interrelated responses. Recently, the presence of leukocytes at the sites of vessel injury has been described. Here we analyzed interaction of neutrophils, monocytes, and platelets in thrombus formation after a laser-induced injury in vivo. Neutrophils immediately adhered to injured vessels, preceding platelets, by binding to the activated endothelium via leukocyte function antigen-1–ICAM-1 interactions. Monocytes rolled on a thrombus 3 to 5 minutes postinjury. The kinetics of thrombus formation and fibrin generation were drastically reduced in low tissue factor (TF) mice whereas the absence of factor XII had no effect. In vitro, TF was detected in neutrophils. In vivo, the inhibition of neutrophil binding to the vessel wall reduced the presence of TF and diminished the generation of fibrin and platelet accumulation. Injection of wild-type neutrophils into low TF mice partially restored the activation of the blood coagulation cascade and accumulation of platelets. Our results show that the interaction of neutrophils with endothelial cells is a critical step preceding platelet accumulation for initiating arterial thrombosis in injured vessels. Targeting neutrophils interacting with endothelial cells may constitute an efficient strategy to reduce thrombosis.

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Mice expressing a mutant desmosomal cadherin exhibit abnormalities in desmosomes, proliferation, and epidermal differentiation.

The Journal of Cell Biology ◽

10.1083/jcb.133.6.1367 ◽

1996 ◽

Vol 133 (6) ◽

pp. 1367-1382 ◽

Cited By ~ 104

Author(s):

E Allen ◽

Q C Yu ◽

E Fuchs

Keyword(s):

Granular Layer ◽

Transgenic Animals ◽

Pemphigus Vulgaris ◽

Epidermal Differentiation ◽

Wild Type Counterpart ◽

Tail Tip ◽

Cadherin Superfamily

Desmogleins are members of the cadherin superfamily which form the core of desmosomes. In vitro studies indicate that the cytoplasmic domain of desmogleins associates with plakoglobin; however, little is known about the role of this domain in desmosome recognition or assembly in vivo, or about the possible relation of desmoglein mutations to epidermal differentiation and disease. To address these questions we used transgenic mouse technology to produce an NH2-terminally truncated desmoglein (Pemphigus Vulgaris Antigen or Dsg3) in cells known to express its wild-type counterpart. Within 2 d, newborn transgenic animals displayed swelling of their paws, flakiness on their back, and blackening of the tail tip. When analyzed histologically and ultrastructurally, widening of intercellular spaces and disruption of desmosomes were especially striking in the paws and tail. Desmosomes were reduced dramatically in number and were smaller and often peculiar in structure. Immunofluorescence and immunoelectron microscopy revealed no major abnormalities in localization of hemidesmosomal components, but desmosomal components organized aberrantly, resulting in a loss of ultrastructure within the plaque. In regions where desmosome loss was prevalent but where some adhesive structures persisted, the epidermis was thickened, with a marked increase in spinous and stratum corneum layers, variability in granular layer thickness, and parakeratosis in some regions. Intriguingly, a dramatic increase in cell proliferation was also observed concomitant with biochemical changes, including alterations in integrin expression, known to be associated with hyperproliferation. An inflammatory response was also detected in some skin regions. Collectively, these findings demonstrate that a mutation in a desmoglein can perturb epidermal cell-cell adhesion, triggering a cascade of changes in the skin.

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The distal carboxyl-terminal domains of ADAMTS13 are required for regulation of in vivo thrombus formation

Blood ◽

10.1182/blood-2008-07-169359 ◽

2009 ◽

Vol 113 (21) ◽

pp. 5323-5329 ◽

Cited By ~ 58

Author(s):

Fumiaki Banno ◽

Anil K. Chauhan ◽

Koichi Kokame ◽

Jin Yang ◽

Shigeki Miyata ◽

...

Keyword(s):

Shear Rate ◽

Genetic Background ◽

Thrombus Formation ◽

Short Form ◽

Von Willebrand ◽

Willebrand Factor ◽

Terminal Domains

Abstract ADAMTS13 is a multidomain protease that limits platelet thrombogenesis through the cleavage of von Willebrand factor (VWF). We previously identified 2 types of mouse Adamts13 gene: the 129/Sv-strain Adamts13 gene encodes the long-form ADAMTS13 having the same domains as human ADAMTS13, whereas the C57BL/6-strain Adamts13 gene encodes the short-form ADAMTS13 lacking the distal C-terminal domains. To assess the physiologic significance of the distal C-terminal domains of ADAMTS13, we generated and analyzed 129/Sv-genetic background congenic mice (Adamts13S/S) that carry the short-form ADAMTS13. Similar to wild-type 129/Sv mice (Adamts13L/L), Adamts13S/S did not have ultralarge VWF multimers in plasma, in contrast to 129/Sv-genetic background ADAMTS13-deficient mice (Adamts13−/−). However, in vitro thrombogenesis under flow at a shear rate of 5000 s−1 was accelerated in Adamts13S/S compared with Adamts13L/L. Both in vivo thrombus formation in ferric chloride–injured arterioles and thrombocytopenia induced by collagen plus epinephrine challenge were more dramatic in Adamts13S/S than in Adamts13L/L but less than in Adamts13−/−. These results suggested that the C-terminally truncated ADAMTS13 exhibited decreased activity in the cleavage of VWF under high shear rate. Role of the C-terminal domains may become increasingly important under prothrombotic conditions.

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

Blood ◽

10.1182/blood-2009-05-222273 ◽

2009 ◽

Vol 114 (16) ◽

pp. 3464-3472 ◽

Cited By ~ 130

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.

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