Pneumolysin induces platelet destruction, not platelet activation, which can be prevented by immunoglobulin preparations in vitro

Abstract Community-acquired pneumonia by primary or superinfections with Streptococcus pneumoniae can lead to acute respiratory distress requiring mechanical ventilation. The pore-forming toxin pneumolysin alters the alveolar-capillary barrier and causes extravasation of protein-rich fluid into the interstitial pulmonary tissue, which impairs gas exchange. Platelets usually prevent endothelial leakage in inflamed pulmonary tissue by sealing inflammation-induced endothelial gaps. We not only confirm that S pneumoniae induces CD62P expression in platelets, but we also show that, in the presence of pneumolysin, CD62P expression is not associated with platelet activation. Pneumolysin induces pores in the platelet membrane, which allow anti-CD62P antibodies to stain the intracellular CD62P without platelet activation. Pneumolysin treatment also results in calcium efflux, increase in light transmission by platelet lysis (not aggregation), loss of platelet thrombus formation in the flow chamber, and loss of pore-sealing capacity of platelets in the Boyden chamber. Specific anti-pneumolysin monoclonal and polyclonal antibodies inhibit these effects of pneumolysin on platelets as do polyvalent human immunoglobulins. In a post hoc analysis of the prospective randomized phase 2 CIGMA trial, we show that administration of a polyvalent immunoglobulin preparation was associated with a nominally higher platelet count and nominally improved survival in patients with severe S pneumoniae–related community-acquired pneumonia. Although, due to the low number of patients, no definitive conclusion can be made, our findings provide a rationale for investigation of pharmacologic immunoglobulin preparations to target pneumolysin by polyvalent immunoglobulin preparations in severe community-acquired pneumococcal pneumonia, to counteract the risk of these patients becoming ventilation dependent. This trial was registered at www.clinicaltrials.gov as #NCT01420744.

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Layer-by-layer coating of stainless steel plates mediated by surface priming treatment to improve antithrombogenic properties

Biomedical Science and Engineering ◽

10.4081/bse.2016.22 ◽

2016 ◽

Author(s):

Irene Carmagnola ◽

Tiziana Nardo ◽

Francesca Boccafoschi ◽

Valeria Chiono

Keyword(s):

Stainless Steel ◽

Surface Modification ◽

Platelet Activation ◽

Colorimetric Assay ◽

Thrombus Formation ◽

Human Platelets ◽

Steel Plates ◽

Blue Dye ◽

Layer By Layer

The stainless steel (SS) stents have been used in clinics since 1994. However, typical drawbacks are restenosis and thrombus formation due to limited endothelialisation and hemocompatibility. Surface modification is a smart strategy to enhance antithrombogenicity by promoting endothelialisation. In this work, the layer-by-layer (LbL) technique was applied for coating SS model substrates, after surface priming by functionalisation with 3-aminopropyl triethoxysilane (APTES). A LbL coating made of 14 layers of poly(styrene sulfonate)/poly(diallyldimethylammonium chloride) and heparin as last layer was deposited. FTIR-ATR analysis and contact angle measurements showed that LbL was an effective method to prepare nanostructured coatings. XPS analysis and colorimetric assay employing 1,9-dimethylmethylene blue dye to detect -COOH groups confirmed the successful polyelectrolyte deposition on the coated samples. Preliminary in vitro cell tests, using whole blood and human platelets, were performed to evaluate how surface modification affects platelet activation. Results showed that SS and SS-APTES surfaces induced platelet activation, as indicated by platelet spreading and filopodia formation. After surface modification by LbL coating, the platelets assumed a round shape and no fibrin nets were detected. Data demonstrated that LbL coating is a promising technique to fabricate antithrombogenic surface.

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Characterization of GPVI- or GPVI-CD39-Coated Nanoparticles and Their Impact on In Vitro Thrombus Formation

International Journal of Molecular Sciences ◽

10.3390/ijms23010011 ◽

2021 ◽

Vol 23 (1) ◽

pp. 11

Author(s):

Jeremy A. Nestele ◽

Anne-Katrin Rohlfing ◽

Valerie Dicenta ◽

Alexander Bild ◽

Daniela Eißler ◽

...

Keyword(s):

Platelet Aggregation ◽

Antithrombotic Therapy ◽

Light Transmission ◽

Thrombus Formation ◽

Bleeding Risk ◽

Significant Inhibition ◽

Site Specific ◽

Glycoprotein Vi ◽

Coated Nanoparticles

Traditional antithrombotic agents commonly share a therapy-limiting side effect, as they increase the overall systemic bleeding risk. A novel approach for targeted antithrombotic therapy is nanoparticles. In other therapeutic fields, nanoparticles have enabled site-specific delivery with low levels of toxicity and side effects. Here, we paired nanotechnology with an established dimeric glycoprotein VI-Fc (GPVI-Fc) and a GPVI-CD39 fusion protein, thereby combining site-specific delivery and new antithrombotic drugs. Poly(lactic-co-glycolic acid) (PLGA) nanoparticles, NP-BSA, NP-GPVI and NP-GPVI-CD39 were characterized through electron microscopy, atomic force measurements and flow cytometry. Light transmission aggregometry enabled analysis of platelet aggregation. Thrombus formation was observed through flow chamber experiments. NP-GPVI and NP-GPVI-CD39 displayed a characteristic surface coating pattern. Fluorescence properties were identical amongst all samples. NP-GPVI and NP-GPVI-CD39 significantly impaired platelet aggregation. Thrombus formation was significantly impaired by NP-GPVI and was particularly impaired by NP-GPVI-CD39. The receptor-coated nanoparticles NP-GPVI and the bifunctional molecule NP-GPVI-CD39 demonstrated significant inhibition of in vitro thrombus formation. Consequently, the nanoparticle-mediated antithrombotic effect of GPVI-Fc, as well as GPVI-CD39, and an additive impact of CD39 was confirmed. In conclusion, NP-GPVI and NP-GPVI-CD39 may serve as a promising foundation for a novel therapeutic approach regarding targeted antithrombotic therapy.

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Polyvalent immunoglobulin preparations inhibit pneumolysin-induced platelet destruction

Thrombosis and Haemostasis ◽

10.1055/a-1723-1880 ◽

2021 ◽

Author(s):

Friederike Wiebe ◽

Stefan Handtke ◽

Jan Wesche ◽

Annabel Schnarre ◽

Raghavendra Palankar ◽

...

Keyword(s):

Respiratory Distress ◽

Platelet Function ◽

Calcium Release ◽

Intravenous Immunoglobulins ◽

Flow Chamber ◽

Platelet Destruction ◽

Immunoglobulin Preparations ◽

Immunoglobulin Preparation ◽

Polyvalent Immunoglobulin

Platelets play an important role in the development and progression of respiratory distress. Functional platelets are known to seal inflammatory endothelial gaps and loss of platelet function has been shown to result in loss of integrity of pulmonary vessels. This leads to fluid accumulation in the pulmonary interstitium, eventually resulting in respiratory distress. Streptococcus pneumoniae is one of the major pathogens causing community-acquired pneumonia. Previously, we have shown that its major toxin pneumolysin forms pores in platelet membranes and renders them non-functional. In vitro, this process was inhibited by polyvalent intravenous immunoglobulins (IVIG). In this study, we compared the efficacy of a standard intravenous immunoglobulin preparation (IVIG, 98% IgG; Privigen, CSL Behring, USA) and an IgM/IgA-enriched immunoglobulin preparation (21% IgA, 23% IgM, 56% IgG; trimodulin, Biotest AG, Germany) to inhibit pneumolysin-induced platelet destruction. Platelet destruction and functionality were assessed by flow cytometry, intracellular calcium release, aggregometry, platelet viability, transwell, and flow chamber assays. Overall, both immunoglobulin preparations efficiently inhibited pneumolysin-induced platelet destruction. The capacity to antagonize pneumolysin mainly depended on the final IgG content. As both polyvalent immunoglobulin preparations efficiently prevent pneumolysin-induced platelet destruction and maintain platelet function in vitro, they represent promising candidates for clinical studies on supportive treatment of pneumococcal pneumonia to reduce progression of respiratory distress.

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Supplemental Fibrinogen Restores Platelet Inhibitor-Induced Reduction in Thrombus Formation without Altering Platelet Function: An In Vitro Study

Thrombosis and Haemostasis ◽

10.1055/s-0040-1715445 ◽

2020 ◽

Vol 120 (11) ◽

pp. 1548-1556

Author(s):

Thomas Bärnthaler ◽

Elisabeth Mahla ◽

Gabor G. Toth ◽

Rufina Schuligoi ◽

Florian Prüller ◽

...

Keyword(s):

Antiplatelet Therapy ◽

Platelet Activation ◽

Platelet Function ◽

Dual Antiplatelet Therapy ◽

Thrombus Formation ◽

In Vitro Study ◽

Coronary Intervention ◽

Calcium Flux ◽

Major Surgery

Abstract Background For patients treated with dual antiplatelet therapy, standardized drug-specific 3-to-7 day cessation is recommended prior to major surgery to reach sufficient platelet function recovery. Here we investigated the hypothesis that supplemental fibrinogen might mitigate the inhibitory effects of antiplatelet therapy. Methods and Results To this end blood from healthy donors was treated in vitro with platelet inhibitors, and in vitro thrombus formation and platelet activation were assessed. Ticagrelor, acetylsalicylic acid, the combination of both, and tirofiban all markedly attenuated the formation of adherent thrombi, when whole blood was perfused through collagen-coated microchannels at physiological shear rates. Addition of fibrinogen restored in vitro thrombus formation in the presence of antiplatelet drugs and heparin. However, platelet activation, as investigated in assays of P-selectin expression and calcium flux, was not altered by fibrinogen supplementation. Most importantly, fibrinogen was able to restore in vitro thrombogenesis in patients on maintenance dual antiplatelet therapy after percutaneous coronary intervention. Conclusion Thus, our in vitro data support the notion that supplementation of fibrinogen influences the perioperative hemostasis in patients undergoing surgery during antiplatelet therapy by promoting thrombogenesis without significantly interfering with platelet activation.

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

Journal of Laboratory and Clinical Medicine ◽

10.1016/j.lab.2005.03.018 ◽

2005 ◽

Vol 146 (4) ◽

pp. 216-226 ◽

Cited By ~ 27

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

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

Blood ◽

10.1182/blood.v104.11.3526.3526 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3526-3526 ◽

Cited By ~ 1

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.

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

Blood ◽

10.1182/blood.v116.21.485.485 ◽

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.

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Gβ1 a Component Of The Heterotrimeric G Protein Is a New Protein Phosphatase 1c Interacting Protein That Regulates Platelet Activation

Blood ◽

10.1182/blood.v122.21.3508.3508 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3508-3508

Author(s):

Subhashree Pradhan ◽

Tanvir Khatlani ◽

Satya P. Kunapuli ◽

K. Vinod Vijayan

Keyword(s):

Signal Transduction ◽

G Protein ◽

Platelet Activation ◽

Protein Interactions ◽

Protein Phosphatase ◽

Conflicts Of Interest ◽

Thrombus Formation ◽

Adaptor Protein ◽

Gpcr Signaling

Abstract Platelet activation at the site of injury is tied to signal transduction events that are mediated by protein kinases and phosphatases. Reversible tyrosine, serine/threonine (Ser/Thr) phosphorylation-dependent assembly and/or disassembly of effector (cytoskeletal, signaling and adaptor) protein complexes propagate signaling downstream of G protein coupled receptors (GPCRs). Compared to kinases, the contribution of Ser/Thr phosphatases and its effectors in GPCR signaling studies is not well explored. Our previous studies had revealed that the catalytic subunit of protein phosphatase 1γ (PP1cγ) support GPCR signaling and thrombus formation. Since cell signaling networks are dependent on protein-protein interactions, we sought to identify the potential effectors of PP1cγ. We employed yeast two-hybrid interaction studies with the full length PP1cγ fused to GAL4 activating domain as bait and screened human bone marrow library. A novel interaction of PP1cγ with a protein called Gβ1 (GNB1) was identified. Gβ1 is a component of the heterotrimeric G proteins like the Gα and couple to GPCR. However, unlike Gα subunits, Gβ1 is unexplored in platelets. Co-immunoprecipitation (co-IP) studies validated PP1cγ-Gβ1 interaction in 293 cells expressing PP1cγ-HA and Gβ1-FLAG. Importantly, Gβ1 interacted with all the PP1c isoforms, suggesting that Gβ1 could target all PP1c isoforms to the GPCR complex. Purified PP1c bound to recombinant Gβ1-GST protein but not to GST protein, indicating that the in vitro interaction of PP1c with Gβ1 was direct and independent of Gα and Gγ subunits. A small molecule inhibitor of G protein βγ, gallein decreased thrombin-induced human platelet aggregation and adhesion to immobilized fibrinogen. There is a paucity of Gβ1-/- platelets because Gβ1-/- mice die within 2 days of birth due to microencephaly. siRNA mediated depletion of Gβ1 in murine megakaryocytes reduced PAR4-activating peptide induced soluble fibrinogen binding to αIIbβ3. These studies suggest a functional role for Gβ1 in GPCR signaling. PP1c co-immunoprecipitated with Gβ1 in resting platelets and agonist (thrombin and ADP) treatment under non-stirring conditions induced dissociation of PP1c from Gβ1. These studies indicate that Gβ1-PP1c complex in platelets is responsive to agonist. Furthermore, PP1c and Gβ1 associated with P2Y12 receptor in resting but not agonist activated platelets in a co-IP assay, suggesting a role for this complex in G protein signaling. Finally, agonist induced dissociation of PP1c from Gβ1 correlated with the association of PP1c with the downstream GPCR effector phospholipase C β3 (PLCβ3) with a concomitant dephosphorylation of PLCβ3 at Ser1105. Since previous studies have revealed that PLCβ3 activity is inhibited by Ser1105 phosphorylation, our observation suggest that agonist-induced association of PP1c with PLCβ3 facilitates dephosphorylation and activation of PLCβ3. These studies highlight a coupling of GPCR signaling with the phosphatase driven signal transduction during platelet activation. Disclosures: No relevant conflicts of interest to declare.

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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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