Effect Of Thrombin On Platelet Adherence To Cultured Vascular Cells: Modification By Dapa

1981 ◽  
Author(s):  
R L Czervionke ◽  
J C Hoak ◽  
D L Haycraft ◽  
G L Fry
Keyword(s):  
Endothelial Cells ◽  
Platelet Activation ◽  
Human Umbilical Cord ◽  
Vascular Cells ◽  
Aggregate Formation ◽  
Platelet Adherence ◽  
Vascular Origin ◽  
Fast Acting ◽  
Positive Controls

The role of thrombin in inducing adherence of platelets to cultured vascular cells is complex and may involve factors of both platelet and vascular origin. Dansylarginine N- (3-ethyl-l,5-pentanediyl)amide, DAPA, a fast-acting inhibitor of thrombin, was used in this study to evaluate platelet activation and endothelial PGI2 release induced by thrombin. Monolayers of endothelial cells and fibroblasts were cultured from human umbilical cord vessels. Empty culture dishes were used as a control. Some vascular cells were treated with aspirin (ASA) to inhibit PGI2 formation before they were incubated with 51Cr-platelets for platelet adherence studies. PGI2 was assayed by radioimmunoassay for 6-keto-PGF1α. In one study, endothelium was incubated with 0.3 U thrombin for 2 min. before 10 μM DAPA was added. Platelets were next mixed with this solution and adherence was determined. DAPA caused a decrease in thrombin-induced platelet adherence to normal endothelium from 8% to 2%. When ASA- treated endothelium was employed, DAPA decreased adherence from 59% to 2%. In another study, 51cr-platelets were first aggregated with 0.1 U thrombin, then 1.3 μM DAPA was added, and these aggregates were incubated with the monolayers. Similar experiments without DAPA served as positive controls. DAPA did not reverse the aggregate formation, but it did reduce platelet adherence to normal endothelium from 38% to 1%, and to ASA-treated endothelium from 68% to 11%. In contrast, DAPA had little effect with fibroblasts (81% to 71%), ASA-treated fibroblasts (82% to 67%), and the empty dish control (86% to 66%). These results suggest that thrombin-induced platelet adherence in this system involves more than just an effect upon platelets. In addition, they provide further evidence that the non-thrombogenic nature of endothelium persists despite the absence of PGI2.

Thrombin Augments Vascular Cell-dependent Migration of Human Mast Cells: Role of MGF

1997 ◽  
Vol 77 (03) ◽  
pp. 577-584 ◽  
Author(s):  
Mehrdad Baghestanian ◽  
Roland Hofbauer ◽  
Hans G Kress ◽  
Johann Wojta ◽  
Astrid Fabry ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Mast Cells ◽  
Umbilical Vein ◽  
Vascular Cells ◽  
Migratory Response ◽  
Thrombin Activation ◽  
Umbilical Vein Endothelial Cells ◽  
Primary Tissue ◽  
Local Expression

SummaryRecent data suggest that auricular thrombosis is associated with accumulation of mast cells (MC) in the upper endocardium (where usually no MC reside) and local expression of MGF (mast cell growth factor) (25). In this study, the role of vascular cells, thrombin-activation and MGF, in MC-migration was analyzed. For this purpose, cultured human auricular endocardial cells (HAUEC), umbilical vein endothelial cells (HUVEC) and uterine-(HUTMEC) and skin-derived (HSMEC) microvascular endothelial cells were exposed to thrombin or control medium, and the migration of primary tissue MC (lung, n = 6) and HMC-1 cells (human MC-line) against vascular cells (supernatants) measured. Supernatants (24 h) of unstimulated vascular cells (monolayers of endocardium or endothelium) as well as recombinant (rh) MGF induced a significant migratory response in HMC-1 (control: 3025 ± 344 cells [100 ± 11.4%] vs. MGF, 100 ng/ml: 8806 ± 1019 [291 ± 34%] vs. HAUEC: 9703 ± 1506 [320.8 ± 49.8%] vs. HUTMEC: 8950 ± 1857 [295.9 ± 61.4%] vs. HSMEC: 9965 ± 2018 [329.4 ± 66.7%] vs. HUVEC: 9487 ± 1402 [313.6 ± 46.4%], p <0.05) as well as in primary lung MC. Thrombin-activation (5 U/ml, 12 h) of vascular cells led to an augmentation of the directed migration of MC as well as to a hirudin-sensitive increase in MGF synthesis and release. Moreover, a blocking anti-MGF antibody was found to inhibit MC-migration induced by unstimulated or thrombin-activated vascular cells. Together, these data show that endocardial and other vascular cells can induce migration of human MC. This MC-chemotactic signal of the vasculature is associated with expression and release of MGF, augmentable by thrombin, and may play a role in the pathophysiology of (auricular) thrombosis.

scholarly journals Role of Endothelial Cells in Acute and Chronic Thrombosis

2019 ◽  
Vol 39 (02) ◽  
pp. 128-139 ◽  
Author(s):  
Magdalena L. Bochenek ◽  
Katrin Schäfer
Keyword(s):  
Endothelial Cells ◽  
Platelet Activation ◽  
Thrombus Formation ◽  
Blood Clot ◽  
Vascular Occlusion ◽  
Coagulation Factors ◽  
Activated Platelets ◽  
Endothelial Cell Response ◽  
Von Willebrand

AbstractHaemostasis encompasses a set of strictly regulated actions, such as vasoconstriction, platelet activation and blood coagulation. Endothelial cells play a crucial role in all of these processes and are an integral part of the vascular response to injury resulting in thrombus formation. Healthy endothelium expresses mediators to prevent platelet activation, including prostacyclin and nitric oxide, and to inhibit coagulation, such as thrombomodulin or RNase1. Upon activation, endothelial cells expose von Willebrand factor, integrins and other receptors to interact with activated platelets, erythrocytes and coagulation factors, respectively, resulting in blood clot formation. The endothelial cell response to cytokines and growth factors released from activated platelets and immune cells abundantly present in arterial and venous thrombi also plays an important role for thrombus resolution, whereas failure to completely resolve thrombi may initiate fibrotic remodelling and chronic vascular occlusion both in the arterial and venous tree. Therefore, endothelial cells are increasingly recognized as potential target to prevent thrombotic events and to accelerate thrombus resolution. Here, we discuss recent publications from our group in the context of other studies on the role of the endothelium during acute and chronic thrombotic events.

P-Selectin and Platelet Clearance

Blood ◽  
1998 ◽  
Vol 92 (11) ◽  
pp. 4446-4452 ◽  
Author(s):  
Gaëtan Berger ◽  
Daqing W. Hartwell ◽  
Denisa D. Wagner
Keyword(s):  
Flow Cytometry ◽  
Endothelial Cells ◽  
Platelet Activation ◽  
Soluble Form ◽  
Wild Type ◽  
Adhesion Receptor ◽  
Activated Platelets ◽  
Deficient Mice

P-selectin is an adhesion receptor for leukocytes expressed by activated platelets and endothelial cells. To assess a possible role of P-selectin in platelet clearance, we adapted an in vivo biotinylation technique in mice. Wild-type and P-selectin–deficient mice were infused with N-hydroxysuccinimido biotin. The survival of biotinylated platelets was followed by flow cytometry after labeling with fluorescent streptavidin. Both wild-type and P-selectin–deficient platelets presented identical life spans of about 4.7 days, suggesting that P-selectin does not play a role in platelet turnover. When biotinylated platelets were isolated, activated with thrombin, and reinjected into mice, the rate of platelet clearance was unchanged. In contrast, storage of platelets at 4°C caused a significant reduction in their life span in vivo but again no significant differences were observed between the two genotypes. The infused thrombin-activated platelets rapidly lost their surface P-selectin in circulation, and this loss was accompanied by the simultaneous appearance of a 100-kD P-selectin fragment in the plasma. This observation suggests that the platelet membrane P-selectin was shed by cleavage. In conclusion, this study shows that P-selectin, despite its binding to leukocytes, does not mediate platelet clearance. However, the generation of a soluble form of P-selectin on platelet activation may have biological implications in modulating leukocyte recruitment or thrombus growth.

scholarly journals Anti-inflammatory Role of Carotenoids in Endothelial Cells Derived from Umbilical Cord of Women Affected by Gestational Diabetes Mellitus

2019 ◽  
Vol 2019 ◽  
pp. 1-11 ◽  
Author(s):  
Mariangela Ucci ◽  
Pamela Di Tomo ◽  
Federica Tritschler ◽  
Vincenzo G. P. Cordone ◽  
Paola Lanuti ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Endothelial Cells ◽  
Gestational Diabetes ◽  
Umbilical Cord ◽  
Nuclear Translocation ◽  
Protective Action ◽  
Adhesion Assay ◽  
Human Umbilical Cord ◽  
Necrosis Factor Alpha

Diabetes is associated with vascular inflammation, endothelial dysfunction, and oxidative stress, promoting the development of cardiovascular diseases (CVD). Several studies showed that a carotenoid-rich diet is associated to a reduced cardiovascular risk in healthy and diabetic subjects, although the mechanisms of action are still unknown. Here, the potential role of β-carotene (BC) and lycopene (Lyc) in human endothelial cells isolated from human umbilical cord vein (HUVECs) of women with gestational diabetes (GD) and respective controls (C) has been investigated. Results showed that BC and Lyc reduced the tumor necrosis factor alpha- (TNF-α-) stimulated monocyte-endothelium interaction (adhesion assay), membrane exposure (flow cytometry), and total expression levels (Western blot) of VCAM-1 and ICAM-1 in both cell types. Moreover, the treatment with BC and Lyc reduced the TNF-α-induced nuclear translocation of NF-κB (image flow cytometry) by preserving bioavailability of nitric oxide (NO, flow cytometry, and cGMP EIA kit assay), a key vasoactive molecule. Notably, BC and Lyc pretreatment significantly reduced peroxynitrite levels (flow cytometry), contributing to the redox balance protection. These results suggest a new mechanism of action of carotenoids which exert vascular protective action in diabetic condition, thus reinforcing the importance of a carotenoid-rich diet in the prevention of diabetes cardiovascular complications.

Abstract 391: Platelet CD40L Affects Thrombus Formation via Phosphatidylinositol 3-kinase ß, But Not Via CD40 or IKKα

2015 ◽  
Vol 35 (suppl_1) ◽  
Author(s):  
Marijke J Kuijpers ◽  
Nadine J Mattheij ◽  
Lina Cipolla ◽  
Johanna P van Geffen ◽  
Toby Lawrence ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Thrombus Formation ◽  
Aggregate Formation ◽  
Phosphatidylinositol 3 Kinase ◽  
Akt Phosphorylation ◽  
Soluble Cd40l ◽  
Granule Secretion ◽  
Collagen Receptor

Objective: To investigate the roles and signaling pathways of CD40L and CD40 in platelet activation and thrombus formation under atherothrombotic conditions. Approach and Results: Mouse platelets lacking CD40L (Cd40lg -/- Apoe -/- ) showed diminished αIIbβ3 activation and α-granule secretion in response to collagen receptor (GPVI) stimulation, while CD40 deficient platelets (Cd40 -/- Apoe -/- ) showed increased responses. ADP- or thrombin-evoked activation was unaffected. In both Cd40lg -/- Apoe -/- and Cd40 -/- Apoe -/- mice, formation of multi-layered thrombi was decreased on both atherosclerotic plaque material and collagen, in comparison to controls. Addition of CD40L prior to perfusion over collagen or plaque material enhanced dense aggregate formation in Apoe -/- , Cd40lg -/- Apoe -/- and Cd40 -/- Apoe -/- blood. CD40L or low GPVI stimulation separately did not cause platelet aggregation. But when combined, aggregation was potentiated, even in the absence of CD40. This potentiation was antagonized by inhibiting PI3Kβ, as well as in platelets from Pik3cb R/R mice. CD40L enhanced Akt phosphorylation at low GPVI stimulation, which was again antagonized by PI3Kβ inhibition and absent in platelets from Pik3cb R/R mice. Finally, Chuk1 A/A Apoe -/- mice, deficient in IKKα, displayed no differences in platelet aggregation - with or without CD40L - nor in thrombus formation in whole blood, indicating that these effects are not mediated via IKKα/NFkB. Conclusions: Under atherothrombotic conditions, CD40L enforces collagen-dependent platelet activation, by supporting integrin αIIbβ3 activation, secretion and dense thrombus formation via PI3Kβ, but not IKKα. Since shedding of CD40L starts minutes after activation, these results point to a joint role of both platelet-bound and soluble CD40L in controlling the size of rapidly formed thrombi.

scholarly journals The Role of PPARs in the Endothelium: Implications for Cancer Therapy

PPAR Research ◽  
2008 ◽  
Vol 2008 ◽  
pp. 1-12 ◽  
Author(s):  
David Bishop-Bailey ◽  
Karen E. Swales
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Platelet Activation ◽  
Inflammatory Cell ◽  
Cell Layer ◽  
Cell Recruitment ◽  
Blood Vessel Formation ◽  
Endothelial Cell Layer ◽  
Inflammatory Cell Recruitment

The growth and metastasis of cancers intimately involve the vasculature and in particular the endothelial cell layer. Tumours require new blood vessel formation via angiogenesis to support growth. In addition, inflammation, coagulation, and platelet activation are common signals in the growth and metastasis of tumour cells. The endothelium plays a central role in the homeostatic control of inflammatory cell recruitment, regulating platelet activation and coagulation pathways. PPAR, -/, and - are all expressed in endothelial cells. This review will discuss the roles of PPARs in endothelial cells in relation to angiogenesis, inflammation, coagulation, and platelet control pathways. In particular, we will discuss the recent evidence that supports the hypothesis that PPAR and PPAR are antiangiogenic receptors, while PPAR/ is proangiogenic.

Steroid hormones and the stroma-vascular cells of the adipose tissue

2013 ◽  
Vol 15 (1) ◽  
Author(s):  
Fanny Volat ◽  
Anne Bouloumié
Keyword(s):  
Endothelial Cells ◽  
Adipose Tissue ◽  
Progenitor Cells ◽  
Steroid Hormones ◽  
Immune Cells ◽  
Potential Role ◽  
Vascular Cells ◽  
Stroma Vascular Fraction ◽  
Corticosteroid Hormones

AbstractThe stroma-vascular fraction (SVF) of adipose tissue (AT) is a heterogeneous cell fraction composed of progenitor cells, endothelial cells, and immune cells. SVF plays a key role in AT homeostasis and growth as well as in obesity-associated pathologies. The SVF cell composition and phenotype are distinct according to AT location and adiposity. Such discrepancies influence AT function and are involved in obesity-associated disorders such as chronic inflammation. Investigations performed in recent years in rodents and humans provided evidence that the stroma-vascular cells contribute to the conversion of steroid hormones in AT and are also steroid targets. This review describes the link between steroids and SVF depending on gender, adiposity, and AT location and highlights the potential role of sex and corticosteroid hormones in adipogenesis, angiogenesis, and their contributions in AT inflammation.

P-Selectin and Platelet Clearance

Blood ◽  
1998 ◽  
Vol 92 (11) ◽  
pp. 4446-4452 ◽  
Author(s):  
Gaëtan Berger ◽  
Daqing W. Hartwell ◽  
Denisa D. Wagner
Keyword(s):  
Flow Cytometry ◽  
Endothelial Cells ◽  
Platelet Activation ◽  
Soluble Form ◽  
Wild Type ◽  
Adhesion Receptor ◽  
Activated Platelets ◽  
Deficient Mice

Abstract P-selectin is an adhesion receptor for leukocytes expressed by activated platelets and endothelial cells. To assess a possible role of P-selectin in platelet clearance, we adapted an in vivo biotinylation technique in mice. Wild-type and P-selectin–deficient mice were infused with N-hydroxysuccinimido biotin. The survival of biotinylated platelets was followed by flow cytometry after labeling with fluorescent streptavidin. Both wild-type and P-selectin–deficient platelets presented identical life spans of about 4.7 days, suggesting that P-selectin does not play a role in platelet turnover. When biotinylated platelets were isolated, activated with thrombin, and reinjected into mice, the rate of platelet clearance was unchanged. In contrast, storage of platelets at 4°C caused a significant reduction in their life span in vivo but again no significant differences were observed between the two genotypes. The infused thrombin-activated platelets rapidly lost their surface P-selectin in circulation, and this loss was accompanied by the simultaneous appearance of a 100-kD P-selectin fragment in the plasma. This observation suggests that the platelet membrane P-selectin was shed by cleavage. In conclusion, this study shows that P-selectin, despite its binding to leukocytes, does not mediate platelet clearance. However, the generation of a soluble form of P-selectin on platelet activation may have biological implications in modulating leukocyte recruitment or thrombus growth.

ADAM15 is an adhesion receptor for platelet GPIIb-IIIa and induces platelet activation

2005 ◽  
Vol 94 (09) ◽  
pp. 555-561 ◽  
Author(s):  
Harald Langer ◽  
Andreas E. May ◽  
Andreas Bültmann ◽  
Meinrad Gawaz
Keyword(s):  
Endothelial Cells ◽  
Platelet Activation ◽  
Platelet Adhesion ◽  
Thrombus Formation ◽  
Maximum Level ◽  
High Shear ◽  
Adhesion Receptor ◽  
Aminoacid Sequence ◽  
Novel Target

SummaryCell adhesion and proteolytic matrix degradation are central processes in atherosclerosis. Being a member of the family of ADAMs (“a disintegrin and metalloproteinase”), metargidin (ADAM15) combines a metalloproteinase domain and an RGD aminoacid sequence. We studied the potential role of ADAM15 as an adhesion receptor on endothelial cells and interactions between platelets and ADAM15 with respect to platelet adhesion, activation and thrombus formation. ADAM15 was found to be expressed on cultured endothelial cells (HUVEC). Platelet adhesion to immobilized recombinantADAM15 was effectively enhanced under both static and high shear rate conditions reaching the maximum level of adhesion to fibrinogen. Consistently, platelet adhesion onto ADAM15 overexpressing endothelial cells was significantly increased. Adhesion to ADAM15 was reduced by blockade of GPIIb-IIIa using neutralizing anti-αIIbβ3 mAbs (7E3, 2G12), but not by anti-α v β3 (LM609). Soluble ADAM15 binds to activated but not to resting GPIIb-IIIa. Moreover, platelets adherent to ADAM15 additionally attracted platelets under high shear rates indicating an initial role of platelet- ADAM15 interactions for thrombus formation. Furthermore, incubation of platelets with solubleADAM15 showed a dose-dependent increase in secretion of CD62P and CD40L. ADAM15 is expressed on endothelial cells and can serve as an adhesion receptor for platelets via GPIIb-IIIa binding. Platelet adhesion to ADAM15 leads to platelet activation, secretion and promotes thrombus formation. Thus, ADAM15 may represent a novel target for antithrombotic strategies in cardiovascular pathologies.

scholarly journals HIV Antivirals Affect Endothelial Activation and Endothelial-Platelet Crosstalk

2020 ◽  
Vol 127 (11) ◽  
pp. 1365-1380
Author(s):  
Akif A. Khawaja ◽  
Kirk A. Taylor ◽  
Andrew O. Lovell ◽  
Mark Nelson ◽  
Brian Gazzard ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Antiretroviral Therapy ◽  
Endothelial Function ◽  
Platelet Activation ◽  
Tenofovir Disoproxil Fumarate ◽  
People Living With Hiv ◽  
Human Umbilical Cord ◽  
Increased Risk ◽  
Tenofovir Alafenamide ◽  
Living With Hiv

Rationale: People living with HIV on effective antiretroviral therapy are at increased risk of cardiovascular complications, possibly due to off-target drug effects. Some studies have associated antiretroviral therapy with increased risk of myocardial infarction and endothelial dysfunction, but a link between endothelial function and antiretrovirals has not been established. Objective: To determine the effects of antiretrovirals in common clinical use upon in vitro endothelial function to better understand cardiovascular risk in people living with HIV. Methods and Results: Human umbilical cord vein endothelial cells or human coronary artery endothelial cells were pretreated with the antiretrovirals abacavir sulphate (ABC), tenofovir disoproxil fumarate, or tenofovir alafenamide. Expression of adhesion molecules, ectonucleotidases (CD39 and CD73), tissue factor (TF), endothelial-derived microparticle (EMP) numbers and phenotype, and platelet activation were evaluated by flow cytometry. TF and ectonucleotidase activities were measured using colourimetric plate-based assays. ABC-treated endothelial cells had higher levels of ICAM (intercellular adhesion molecule)-1 and TF expression following TNF (tumor necrosis factor)-α stimulation. In contrast, tenofovir disoproxil fumarate and tenofovir alafenamide treatment gave rise to greater populations of CD39 + CD73 + cells. These cell surface differences were also observed within EMP repertoires. ABC-treated cells and EMP had greater TF activity, while tenofovir disoproxil fumarate- and tenofovir alafenamide-treated cells and EMP displayed higher ectonucleotidase activity. Finally, EMP isolated from ABC-treated cells enhanced collagen-evoked platelet integrin activation and α-granule release. Conclusions: We report differential effects of antiretrovirals used in the treatment of HIV upon endothelial function. ABC treatment led to an inflammatory, prothrombotic endothelial phenotype that promoted platelet activation. In contrast, tenofovir disoproxil fumarate and tenofovir alafenamide conferred potentially cardioprotective properties associated with ectonucleotidase activity. These observations establish a link between antiretrovirals and specific functional effects that provide insight into cardiovascular disease in people living with HIV.

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