scholarly journals Platelet count reduction during in vitro membrane oxygenation affects platelet activation, neutrophil extracellular trap formation and clot stability, but does not prevent clotting

Perfusion ◽  
2021 ◽  
pp. 026765912198923
Author(s):  
Patrick Winnersbach ◽  
Jan Rossaint ◽  
Eva M. Buhl ◽  
Smriti Singh ◽  
Jonas Lölsberg ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Thrombus Formation ◽  
Clot Formation ◽  
Neutrophil Extracellular Trap ◽  
Bleeding Complications ◽  
In Vitro Test ◽  
Membrane Oxygenation ◽  
The Impact ◽  
Clot Stability

Introduction: Due to improved technology and increased application the mortality during extracorporeal membrane oxygenation (ECMO) is constantly declining. Nevertheless, complications including haemorrhage or thrombus formation remain frequent. Local mitigation of coagulation within an ECMO system to prevent thrombus formation on ECMO components and optimizing systemic anticoagulation is an approach to reduce clotting and bleeding complications at once. Foreign surfaces of ECMO systems, activate platelets (PLTs), which besides their major role in coagulation, can trigger the formation of neutrophil extracellular traps (NETs) contributing to robust thrombus formation. The impact of a reduced PLT count on PLT activation and NET formation is of paramount importance and worth investigating. Methods: In this study platelet poor (PLT–) and native (PLT+) heparinized human blood was circulated in two identical in vitro test circuits for ECMO devices for 6 hours. PLT reduction was achieved by a centrifugation protocol prior to the experiments. To achieve native coagulation characteristics within the test circuits, the initial heparin dose was antagonized by continuous protamine administration. Results: The PLT– group showed significantly lower platelet activation, basal NET formation and limited clot stability measured via thromboelastometry. Fluorescent and scanning electron microscope imaging showed differences in clot composition. Both groups showed equal clot formation within the circuit. Conclusions: This study demonstrated that the reduction of PLTs within an ECMO system is associated with limited PLT activation and NET formation, which reduces clot stability but is not sufficient to inhibit clot formation per se.

scholarly journals In vitro factor XIII supplementation increases clot firmness in Rotation Thromboelastometry (ROTEM®)

2010 ◽  
Vol 104 (08) ◽  
pp. 385-391 ◽  
Author(s):  
Lars Asmis ◽  
Burkhardt Seifert ◽  
Donat Spahn ◽  
Oliver Theusinger ◽  
Werner Baulig
Keyword(s):  
Factor Xiii ◽  
Clot Formation ◽  
Intensive Care Patients ◽  
Essential Parameter ◽  
Α Angle ◽  
The Impact ◽  
Clot Formation Time ◽  
Clot Stability ◽  
Maximum Clot Firmness

SummaryFactor XIII (F XIII) is an essential parameter for final clot stability. The purpose of this study was to determine the impact of the addition of factor (F)XIII on clot stability as assessed by Rotation Thromboelastometry (ROTEM®). In 90 intensive care patients ROTEM® measurements were performed after in vitro addition of F XIII 0.32 IU, 0.63 IU, 1.25 IU and compared to diluent controls (DC; aqua injectabile) resulting in approximate F XIII concentrations of 150, 300 and 600%. Baseline measurements without any additions were also performed. The following ROTEM® parameters were measured in FIBTEM and EXTEM tests: clotting time (CT), clot formation time (CFT), maximum clot firmness (MCF), maximum lysis (ML), maximum clot elasticity (MCE) and α-angle (αA). Additionally, laboratory values for FXIII, fibrinogen (FBG), platelets and haematocrit were contemporaneously determined. In the perioperative patient population mean FBG concentration was elevated at 5.2 g/l and mean FXIII concentration was low at 62%. The addition of FXIII led to a FBG concentration-dependent increase in MCF both in FIBTEM and EXTEM. Mean increases in MCF (FXIII vs. DC) of approximately 7 mm and 6 mm were observed in FIBTEM and EXTEM, respectively. F XIII addition also led to decreased CFT, increased αA, and reduced ML in FIBTEM and EXTEM. In vitro supplementation of FXIII to supraphysiologic levels increases maximum clot firmness, accelerates clot formation and increases clot stability in EXTEM and FIBTEM as assayed by ROTEM® in perioperative patients with high fibrinogen and low FXIII levels.

scholarly journals Repeated social defeat exaggerates fibrin-rich clot formation in FeCl3-induced arterial thrombosis mouse model by enhancing NETs formation via modulation of neutrophil functional properties

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
T Sugimoto ◽  
H Yamada ◽  
H Kubota ◽  
D Miyawaki ◽  
M Saburi ◽  
...  
Keyword(s):  
Functional Properties ◽  
Arterial Thrombosis ◽  
Social Defeat ◽  
Arterial Injury ◽  
Physical Contact ◽  
Clot Formation ◽  
Positive Staining ◽  
Double Positive ◽  
The Impact

Abstract Background and objective Depression is an independent risk factor of cardiovascular disease (CVD). We have recently shown that repeated social defeat (RSD) precipitates depressive-like behaviors in apoE−/− mice and exaggerates atherosclerosis development by enhancing neutrophil extracellular traps (NETs) formation. Here, we investigated the impact of RSD on arterial thrombosis. Methods and results Eight-week-old male WT mice were exposed to RSD by housing with a larger CD-1 mouse in a shared home cage. They were subjected to vigorous physical contact daily for 10 consecutive days. Control mice were housed in the same gage without physical contact. After social interaction test to confirm depressive-like behaviors, defeated mice (19 of 31) and control mice (12 of 14) were underwent arterial injury at 10 wks of age. A filter paper saturated with 10% FeCl3 was applied on the adventitial surface of left carotid artery for 3 min and analyzed 3 hrs later. The volume of thrombi was comparable between the two groups. However, fibrinogen/fibrin-positive areas in immunofluorescent images significantly increased in defeated mice (27.8% vs. 48.8%, p<0.01). The number of Ly-6G-positive cells in thrombi was markedly higher in defeated mice (144/mm2 vs. 878/mm2, p<0.05). Further, Ly-6G-positive cells were almost accumulated at the inner surface of injured artery, which were co-localized with neutrophil elastase, Cit-H3, and CD41-positive staining. Treatment with DNase I completely diminished the exaggerated fibrin-rich clot formation in defeated mice to an extent similar to that in control mice (25.7% vs. 22.3%, p = ns), without affecting the volume of thrombi and accumulation of Ly-6G-positive cells. Given that platelet aggregations induced by ADP or collagen were comparable between the two groups, neutrophil functional properties primarily contribute to the exaggerated fibrin-rich clot formation in defeated mice. We then examined neutrophil subset and vulnerability to NETs formation. At 3 hrs after FeCl3 application, the numbers of immature neutrophils (Ly6Glo/+CXCR2-) were comparable between the two groups in both bone marrow (BM) and peripheral blood (PB). In contrast, the number of PB mature neutrophils (Ly6G+CXCR2+) was markedly higher in defeated mice than control mice (580±68 /μl vs. 1265±114, p<0.01). We next examined in vitro NETs formation upon PMA in BM mature neutrophils by FACS and nucleic acid staining. The percentage of double-positive cells (Cit-H3, MPO) was significantly higher in defeated mice (7.5% vs. 10.2%, p<0.05), as well as SYTOX green-positive cells expelling DNA fibers (8.1% vs. 11.8%, p<0.05). Conclusions Our findings demonstrate for the first time that repeated social defeat enhances fibrin-rich clot formation after arterial injury by enhancing NETs formation via modulation of neutrophil functional properties, suggesting that NETosis could be a new therapeutic target in depression-related CVD development. Funding Acknowledgement Type of funding source: None

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

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.

Partially desulfated heparin modulates the interaction between anti-protamine/heparin antibodies and platelets

2016 ◽  
Vol 115 (02) ◽  
pp. 324-332 ◽  
Author(s):  
Rabie Jouni ◽  
Heike Zöllner ◽  
Ahmad Khadour ◽  
Jan Wesche ◽  
Anne Grotevendt ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Platelet Factor ◽  
Standard Drug ◽  
Platelet Surface ◽  
Minimal Impact ◽  
Platelet Survival ◽  
Heparin Complexes ◽  
The Impact

SummaryProtamine (PRT) is the standard drug to neutralise heparin. PRT/heparin complexes induce an immune response similar to that observed in heparin-induced thrombocytopenia (HIT). Partially desulfated heparin (ODSH) was shown to interfere with anti-platelet factor 4/heparin antibodies (Abs), which are responsible for HIT. In this study, we analyse the impact of ODSH on the interaction between anti-PRT/heparin Abs and platelets. The ability of ODSH to prevent anti-PRT/heparin Ab-induced platelet destruction in vivo was investigated using the NOD/ SCID mouse model. ODSH improved platelet survival in the presence of PRT, heparin and anti-PRT/heparin Abs (median platelet survival after 300 minutes (min) with 20 μg/ml ODSH: 75 %, range 70–81 % vs without ODSH: 49%, range 44–59%, p=0.006). Furthermore, when ODSH was applied 60 min after Ab injection platelet survival was improved (median platelet survival after 300 min with ODSH: 83 %, range 77–93 % vs without ODSH: 59 %, range 29–61 %, p=0.02). In in vitro experiments ODSH inhibited platelet activation at concentrations > 16 μg/mL (p< 0.001), as well as PRT/heparin complex binding to platelets (mean fluorescence intensity [MFI] without ODSH: 85 ± 14 vs with ODSH: 15 ± 0.6, p=0.013). ODSH also displaced pre-bound complexes from the platelet surface (MFI without ODSH: 324 ± 43 vs with 32 μg/ml ODSH: 53 ± 9, p< 0.001). While interfering with platelet activation by anti-PRT/heparin Abs, up to a concentration of 16 μg/ml, ODSH had only minimal impact on neutralisation of heparin by PRT. In conclusion, our study shows that ODSH is able to inhibit platelet activation and destruction suggesting a potential clinical use to reduce anti-PRT/heparin Ab-mediated adverse effects.

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

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.

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.

Platelet Signaling in Primary Haemostasis and Arterial Thrombus Formation: Part 1

2018 ◽  
Vol 38 (04) ◽  
pp. 203-210 ◽  
Author(s):  
Rüdiger Scharf
Keyword(s):  
Platelet Activation ◽  
Thrombus Formation ◽  
Vascular Lesions ◽  
Arterial Thrombus ◽  
Platelet Signaling ◽  
Transduction Mechanisms ◽  
Granule Secretion ◽  
Von Willebrand ◽  
The Impact

AbstractPlatelets react immediately in response to traumatic vascular injury by adhesion, activation, aggregation and subsequent haemostatic plug formation. While this reaction pattern is essential for haemostasis, platelet responses can also cause occlusive thrombi in diseased arteries, leading to myocardial infarction or stroke. Initially, flowing platelets are captured from the circulation to vascular lesions. This step is mediated by glycoprotein (GP) Ib-IX-V interacting with immobilized von Willebrand factor (VWF) on exposed subendothelial components. Tethered platelets can now bind to collagen through GPVI and integrin α2β1. Outside-in signals from the adhesion receptors act synergistically with inside-out signals from soluble stimuli and induce platelet activation. These mediators operate through G protein–coupled receptors and reinforce adhesion and activation. Typical manifestations of activated platelets include calcium mobilization, procoagulant activity, cytoskeletal reorganization, granule secretion and aggregation. This requires activation of integrin αIIbβ3 with shifting into a high-affinity state and is indispensable to bind soluble fibrinogen, VWF and fibronectin. The multiple interactions and the impact of thrombin result in firm adhesion and recruitment of circulating platelets into growing aggregates. A fibrin meshwork supports stabilization of haemostatic thrombi and prevents detachment by the flowing blood. This two-part review provides an overview of platelet activation and signal transduction mechanisms with a focus on αIIbβ3-mediated outside-in signaling in integrin variants. In the first part, a three-stage model of platelet recruitment and activation in vivo is presented. Along with that, platelet responses upon exposure to thrombogenic surfaces followed by platelet-to-platelet interactions and formation of haemostatic thrombi are discussed. Moreover, several determinants involved in pathological thrombosis will be reviewed.

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.

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.

Sign in / Sign up

Export Citation Format

Share Document