The inflammatory action of CD40 ligand (CD154) expressed on activated human platelets is temporally limited by coexpressed CD40

Blood ◽  
2001 ◽  
Vol 98 (4) ◽  
pp. 1047-1054 ◽  
Author(s):  
Volker Henn ◽  
Sabine Steinbach ◽  
Kerstin Büchner ◽  
Peter Presek ◽  
Richard A. Kroczek
Keyword(s):  
Endothelial Cells ◽  
Inflammatory Reaction ◽  
Vascular System ◽  
Thrombus Formation ◽  
Constitutive Expression ◽  
Cd40 Ligand ◽  
Human Platelets ◽  
Soluble Form ◽  
Platelet Surface ◽  
Platelet Aggregate

Recently, we have demonstrated that human platelets carry preformed CD40 ligand (CD154) molecules, which rapidly appear on the platelet surface following stimulation by thrombin. Once on the surface, platelet CD154 induces an inflammatory reaction of CD40-bearing endothelial cells. This study shows that strong platelet agonists other than thrombin also lead to the expression of CD154 on the platelet surface. At the same time, several lines of evidence are presented that together indicate that thrombotic events in the vasculature are generally accompanied by activation of the inflammatory potential of platelet CD154. This study also reports the constitutive expression of CD40, the receptor for CD154, on platelets. The binding of CD154 to coexpressed CD40 in the platelet aggregate leads within minutes to hours to the cleavage of membrane-bound surface CD154 and the release of an 18-kd soluble form of the molecule. Soluble CD154 (sCD154), in contrast to transmembrane CD154, can no longer induce an inflammatory reaction of endothelial cells. These findings indicate that the interaction of platelet CD154 with CD40 on neighboring cells is temporally limited to prevent an uncontrolled inflammation at the site of thrombus formation. Thus, similar to the very tight regulation of the CD154-CD40 interaction in the immune system, an effective mechanism controls the inflammatory potential of platelet CD154 in the vascular system.

Regulated Shedding of sema4D from the Platelet Surface Produces a Bioactive Second Messenger in Thrombotic Disorders.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1641-1641
Author(s):  
Li Zhu ◽  
Wolfgang Bergmeier ◽  
Jie Wu ◽  
Hong Jiang ◽  
Nana Yeboah ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Platelet Activation ◽  
Transmembrane Domain ◽  
Thrombus Formation ◽  
Active Form ◽  
Extracellular Domain ◽  
Human Platelets ◽  
Biologically Active ◽  
Western Blots ◽  
Platelet Surface

Abstract Proteins that are expressed on the platelet surface can participate in contact-dependent signaling events which modulate thrombus formation or, after being shed from the platelet surface, serve as bioactive messengers that affect the function of nearby cells. Here we show for the first time that platelets express the class IV semaphorin known as sema4D or CD100, and that platelet activation causes the regulated shedding of the sema4D extracellular domain in a biologically-active form. Sema4D is a glycosylated 150 kDa disulfide-linked homodimer that has previously been implicated in interactions between T-cells and B-cells. Platelet activation by collagen, thrombin or PMA causes a transient increase in sema4D surface expression peaking at 15 min, followed by a complete loss of expression over 30–60 minutes. These events are accompanied by the release of the sema4D exodomain as a 130 kDa fragment, leaving a 25–30 kDa transmembrane and cytoplasmic domain fragment that is retained by the platelets. The cleavage event required to produce these fragments is inhibited by metalloprotease inhibitors and abolished in platelets from chimeric mice lacking the metalloprotease known as ADAM17 or TACE (TNF alpha converting enzyme). Incubation of recombinant sema4D with ADAM17 identified a single cleavage site just outside the predicted transmembrane domain. Western blots show that human platelets express ADAM17 in both its immature (zymogen) and mature (active) forms, and indicate that at least some of the ADAM17 is located on the platelet surface. ADAM17-dependent cleavage of sema4D does not require platelet aggregation, but the rate is accelerated when aggregation is allowed to occur and slowed when aggregation is prevented. Under both sets of conditions, cleavage of sema4D occurs to a greater extent and more rapidly than the ADAM17-dependent cleavage of GP Ib alpha, suggesting that there is a hierarchy of proteolytic events when platelets are activated. In terms of biological impact, the shedding of sema4D following platelet activation raises the possibility that the soluble extracellular domain of sema4D serves as a bioactive messenger. Two receptors for sema4D have been identified previously: CD72, which is present on lymphocytes where it regulates the activity of the tyrosine phosphatase, SHP-1, and plexin-B1, which is expressed in endothelial cells. Western blots suggest that both of these receptors are expressed on human platelets and show that SHP-1 is associated with CD72 in resting, but not activated platelets. Taken together, these results demonstrate that sema4D undergoes regulated ADAM17-dependent shedding when platelets are activated, and suggest that this results in the production of a bioactive form of the molecule that can affect responses in nearby platelets, lymphocytes and endothelial cells at sites of thrombosis.

Upregulation of CD40 ligand and enhanced monocyte-platelet aggregate formation are associated with worse clinical outcome after ischaemic stroke

2012 ◽  
Vol 107 (02) ◽  
pp. 346-355 ◽  
Author(s):  
Grzegorz Dworacki ◽  
Joanna Kufel-Grabowska ◽  
Cezary Watala ◽  
Wojciech Kozubski ◽  
Maria Lukasik
Keyword(s):  
Clinical Outcome ◽  
Predictive Marker ◽  
Cd40 Ligand ◽  
Surface Expression ◽  
Vascular Events ◽  
Platelet Surface ◽  
Increased Risk ◽  
Platelet Aggregate ◽  
Platelet Aggregates ◽  
Long Term Prognosis

SummaryThe white blood cell count and mean platelet volume determined shortly after the symptom onset are known as independent predictors for clinical outcome after stroke. In the present study we sought to evaluate the prognostic value of platelet-derived inflammatory biomarkers measured prospectively after an ischaemic event. Using five-colour flow cytometry, the platelet surface expression of CD40L, CD62P and subpopulations of leukocyte-platelet aggregates were assessed in 93 stroke patients on the first (V0), 10th (V1) and 90th (V2) day after stroke, and once in 65 disease controls. The clinical outcome was evaluated using the Scandinavian Stroke Scale (SSS) and modified Rankin Scale (mRS) at the same time points as blood sampling and 24 months after the event. Patients with either CD40L surface expression or the percentage of monocyte-platelet aggregates (M-plt) in the third tertile (T3) at V0 had a significantly lower score on the SSS at V1. Patients with the percentage M-plt at V0 higher than the median value of M-plt in controls were at increased risk of SSS < 40 at V1 (odds ratio: 2.6; 95% confidence interval [CI]: 1.4 – 8.7; p=0.006). Patients with the percentage of M-plt in T3 at V0 showed progressive decline in survival (hazard ratio [HR]: 1.6; 95% CI: 1.1–1.9; p=0.02) and a significantly higher number of recurrent vascular events (HR: 2.64; 95% CI: 1.3–3.2; p=0.02) when compared to the first tertile. In conclusion, increased levels of M-plt could be a predictive marker for both early outcome and long-term prognosis while increased CD40L was correlated with worse clinical outcome.The preliminary results of this study were presented in part during a poster session at the 62nd Annual Meeting of the American Academy of Neurology in Toronto, 7–17 April 2010.

Hydrogen sulphide reduces the activity of human endothelial cells

2020 ◽  
pp. 1-11
Author(s):  
Eberhard Grambow ◽  
Gina Klee ◽  
Wentao Xie ◽  
Clemens Schafmayer ◽  
Brigitte Vollmar
Keyword(s):  
Endothelial Cells ◽  
Thrombus Formation ◽  
Umbilical Vein ◽  
Protein S ◽  
Coagulation Factors ◽  
Human Platelets ◽  
Umbilical Vein Endothelial Cells ◽  
Von Willebrand ◽  
Biotin Switch

INTRODUCTION: The volatile endogenous mediator hydrogen sulfide (H2S) is known to impair thrombus formation by affecting the activity of human platelets. Beside platelets and coagulation factors the endothelium is crucial during thrombogenesis. OBJECTIVE: This study evaluates the effect of the H2S donor GYY4137 (GYY) on human umbilical vein endothelial cells (HUVECs) in vitro. METHODS: Flow cytometry of resting, stimulated or GYY-treated and subsequently stimulated HUVECs was performed to analyse the expression of E-selectin, ICAM-1 and VCAM-1. To study a potential reversibility of the GYY action, E-selectin expression was further assessed on HUVECs that were stimulated 24 h after GYY exposure. A WST-1 assay was performed to study toxic effects of the H2S donor. By using the biotin switch assay, protein S-sulfhydration of GYY-exposed HUVECs was assessed. Further on, the effects of GYY on HUVEC migration and von Willebrand factor (vWF) secretion were assessed. RESULTS: GYY treatment significantly reduced the expression of E-selectin and ICAM-1 but not of VCAM-1. When HUVECs were stimulated 24 h after GYY treatment, E-selectin expression was no longer affected. The WST-1 assay revealed no effects of GYY on endothelial cell viability. Furthermore, GYY impaired endothelial migration, reduced vWF secretion and increased protein S-sulfhydration. CONCLUSIONS: Summarizing, GYY dose dependently and reversibly reduces the activity of endothelial cells.

CD40 ligand on activated platelets triggers an inflammatory reaction of endothelial cells

Nature ◽  
10.1038/35393 ◽  
1998 ◽  
Vol 391 (6667) ◽  
pp. 591-594 ◽  
Author(s):  
Volker Henn ◽  
Joseph R. Slupsky ◽  
Michael Gräfe ◽  
Ioannis Anagnostopoulos ◽  
Reinhold Förster ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Inflammatory Reaction ◽  
Cd40 Ligand ◽  
Activated Platelets

Anticoagulant and antithrombotic properties of platelet protease nexin-1

Blood ◽  
2010 ◽  
Vol 115 (1) ◽  
pp. 97-106 ◽  
Author(s):  
Yacine Boulaftali ◽  
Frédéric Adam ◽  
Laurence Venisse ◽  
Véronique Ollivier ◽  
Benjamin Richard ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Thrombus Formation ◽  
Human Platelets ◽  
Platelet Surface ◽  
Type Plasminogen Activator ◽  
Healthy Donors ◽  
Protease Nexin ◽  
Deficient Mice

AbstractProtease nexin–1 (PN-1) is a serpin that inhibits plasminogen activators, plasmin, and thrombin. PN-1 is barely detectable in plasma but is expressed by platelets. Here, we studied platelet PN-1 in resting and activated conditions and its function in thrombosis. Studies on human platelets from healthy donors and from patients with a Gray platelet syndrome demonstrate that PN-1 is present both at the platelet surface and in α-granules. The role of PN-1 was investigated in vitro using human platelets incubated with a blocking antibody and using platelets from PN-1–deficient mice. Both approaches indicate that platelet PN-1 is active on thrombin and urokinase-type plasminogen activator. Blockade and deficiency of platelet PN-1 result in accelerated and increased tissue factor-induced thrombin generation as indicated by calibrated automated thrombography. Moreover, platelets from PN-1–deficient mice respond to subthreshold doses of thrombin, as assessed by P-selectin expression and platelet aggregation. Thrombus formation, induced ex vivo by collagen in blood flow conditions and in vivo by FeCl3-induced injury, is significantly increased in PN-1–deficient mice, demonstrating the antithrombotic properties of platelet PN-1. Platelet PN-1 is thus a key player in the thrombotic process, whose negative regulatory role has been, up to now, markedly underestimated.

Human Platelets Contain and Release an Active ADAMTS-13-Like Metalloprotease.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3527-3527
Author(s):  
Li Liu ◽  
Huiwan Choi ◽  
Bernardo Aubrey ◽  
Angila Bergeron ◽  
Leticia Nolasco ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Platelet Activation ◽  
Fluid Shear Stress ◽  
Vascular Endothelial Cells ◽  
Severe Infection ◽  
Surface Expression ◽  
Human Platelets ◽  
Platelet Surface ◽  
The Difference ◽  
Adhesion Ligand

Abstract The adhesion ligand von Willebrand factor (VWF) is synthesized and stored in vascular endothelial cells and megakaryocytes/platelets. These cells release VWF in response to similar stimulations. However, a longstanding observation stated that, unlike endothelial cells, platelets do not release the ultra-large form (UL) of VWF upon activation. The lack of ULVWF release may be attributed to the difference in multimerization process in the storage granules or the intrinsic proteolysis of the hyperreactive ULVWF. To examine these possibilities, we analyzed the multimer compositions of VWF stored in and released from platelets. We found that ULVWF was detected by immunobloting of washed platelet lysates in the presence, but not in the absence of 5 mM EDTA. Similarly, when stimulated with 5 μM of thrombin receptor-activating peptide (TRAP), platelets released only VWF that was similar in sizes to plasma VWF, suggesting that platelet ULVWF may have been cleaved before or during its release. In support of this hypothesis, we found that platelets expressed ADAMTS-13-like molecule by western blot of platelet lysates and flow cytometry using two different ADAMTS-13 antibodies. The surface expression of ADAMTS-13-like molecule increased significantly upon platelet activation by TRAP and ADP. Consistent with these observations, the Triton-X-100 lysates of washed platelets and supernatant of washed platelets activated by TRAP contained VWF-cleaving activity, as demonstrated by the cleavage of ULVWF strings formed on histamine-stimulated endothelial cells under fluid shear stress. Our finding is consistent with a previous report, but further showed that this ADAMTS-13-like molecule exists not only in cytoplasma, but also on the surface of platelets. More importantly, the ADAMTS-13-like molecule increased its expression on platelet surface and was released upon platelet activation. The ADAMTS-13-like molecule either in platelets or released upon activation was active in cleaving ULVWF. This intrinsic mechanism for ULVWF proteolysis may be physiologically important. It may prevent the sudden release of hyper-reactive ULVWF from platelets and serve as the second pool of ADAMTS-13 to encounter the increase in ULVWF release from endothelial cells during conditions such as severe infection. The role of the platelet membrane-bound ADAMTS-13 in hemostasis remains to be further determined.

Platelet-specific markers are associated with monocyte-platelet aggregate formation and thrombin generation potential in advanced atherosclerosis

2016 ◽  
Vol 115 (03) ◽  
pp. 615-621 ◽  
Author(s):  
Cihan Ay ◽  
Julia Riedl ◽  
Christoph W. Kopp ◽  
Beate Eichelberger ◽  
Renate Koppensteiner ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Thrombin Generation ◽  
Thrombus Formation ◽  
Cd40 Ligand ◽  
Aggregate Formation ◽  
Soluble Cd40 Ligand ◽  
Platelet Factor ◽  
Platelet Aggregate ◽  
Angioplasty And Stenting

SummaryPlatelet activation and thrombin generation are crucial steps in primary and secondary haemostasis. However, both also play major roles in intravascular thrombus formation and therefore in the development of adverse cardiovascular events. In the current study, we first sought to investigate the associations of the platelet biomarkers platelet factor (PF)-4, thrombospondin (TSP)-1, soluble CD40 ligand (sCD40L), and soluble P-selectin (sP-selectin) with each other and with monocyte-platelet aggregate (MPA) formation in 316 patients undergoing angioplasty and stenting. To better understand the interplay between platelet activation and thrombin generation, we subsequently investigated the associations of the platelet biomarkers with thrombin generation potential. The mostly platelet-specific markers PF-4, TSP-1 and sCD40L correlated strongly with each other (all p < 0.001), and the best correlation was observed between PF-4 and TSP-1 (r=0.91). In contrast, sP-selectin, which derives from platelets and endothelial cells, correlated rather poorly with TSP-1 (r=0.12, p=0.04), and did not correlate with PF-4 and sCD40L. While PF-4, TSP-1 and sP-selectin correlated significantly with in vivo MPA formation (all p < 0.001), no such association was found between sCD40L and MPA formation. PF-4, TSP-1 and sCD40L correlated strongly with peak thrombin generation (all p < 0.001) with the best correlation between PF-4 and peak thrombin generation (r=0.55), whereas sP-selectin did not correlate with peak thrombin generation. Likewise, PF-4, TSP-1 and sCD40L correlated significantly with the area under the thrombin generation curve (AUC; all p< 0.01), whereas sP-selectin did not correlate with the AUC. In conclusion, platelet-specific markers are associated with MPA formation and thrombin generation potential in patients with advanced atherosclerosis.

THE BINDING OF TISSUE PLASMINOGEN ACTIVATOR TO PLATELETS

1987 ◽  
Author(s):  
J Loscalzo ◽  
D E Vaughan
Keyword(s):  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Human Platelets ◽  
Tissue Type ◽  
Single Chain ◽  
Platelet Surface ◽  
Binding Domains ◽  
Tissue Plasminogen ◽  
Platelet Aggregate ◽  
A Site

Since the platelet surface has been shown to be a site for plasminogen conversion by tissue-type and other plasminogen activators, we examined the binding of tissue plasminogen activator (tPA) to human platelets. Resting, washed platelets were found to bind single chain, radioiodinated, recombinant tPA specifically and saturably with an apparent, estimated dissociation constant (KD) of 458 nM, binding approximately 570 molecules per platelet at saturation. Washed platelets activated with adenosine 5'-diphosphate in the presence of 0.1 mg/ml fibrinogen and 1 mM CaCl2 bound tPA with greater affinity, having an estimated apparent KD of 30.6 nM and binding approximately 29,000 molecules per platelet at saturation. Bound tPA could be completely displaced by an excess of unlabeled tPA. Interestingly, bound tPA could also be displaced from activated platelets with increasing concentrations of soluble fibrin with an estimated IC50 of 37.5 μg/ml of fibrin. In contrast, increasing concentrations of fibrinogen failed to reduce binding. These data show that tPA binds to the activated platelet surface by a mechanism that involves platelet-bound fibrinogen. In addition, these data suggest that on binding to the platelet surface, fibrinogen expresses domains that are similar to the tPA binding domains of fibrin. It is the presence of these domains within the platelet aggregate that likely supports tPA binding and facilitates plasminogen activation.

scholarly journals Activation and homologous desensitization of human endothelial cells by CD40 ligand, tumor necrosis factor, and interleukin 1.

1996 ◽  
Vol 184 (1) ◽  
pp. 173-182 ◽  
Author(s):  
K Karmann ◽  
W Min ◽  
W C Fanslow ◽  
J S Pober
Keyword(s):  
Endothelial Cells ◽  
Tumor Necrosis Factor ◽  
Adhesion Molecule ◽  
Tumor Necrosis ◽  
Interleukin 1 ◽  
Cd40 Ligand ◽  
Intercellular Adhesion Molecule ◽  
Soluble Form ◽  
Homologous Desensitization ◽  
Necrosis Factor

We have reported previously that activation of human umbilical vein endothelial cells (HUVECs) through CD40, using a recombinant soluble form of trimerized CD40 ligand, leads to induction of E-selectin, vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1). Here, we compare the effects of CD40 ligand with those of tumor necrosis factor (TNF) and interleukin 1 (IL-1). All three ligands induce transient increases in E-selectin (peak 4 h) and VCAM-1 (peak 8-24 h), as well as sustained increases in ICAM-1 (plateau 24 h). Quantitatively, TNF is more potent than IL-1, which is much more potent than CD40 ligand. The same hierarchy is observed for transcriptional activation of an E-selectin promoter reporter gene construct in transiently transfected HUVECs. TNF and CD40 ligand each induced activation of the transcription factors NF-kappa B, IRF-1, and ATF-2/c-Jun, measured by electrophoretic mobility shift assays, but this response appeared quantitatively similar. All three agents transiently (peak 30 min) activated Jun NH2-terminal kinase (JNK), which has been implicated in transcription of E-selectin through its actions on ATF-2/c-Jun. Activation of JNK again showed a hierarchy of potency (TNF &gt; IL-1 &gt; CD40 ligand), although the time course of induction was similar for all three agents. After 44 h of pretreatment, TNF, IL-1, and CD40 ligand each display homologous desensitization for reinduction of surface expression of E-selectin. A similar pattern of homologous desensitization for reactivation of JNK was observed. We conclude that TNF, IL-1, and CD40 ligand all activate similar responses in ECs, and that homologous desensitization of JNK may explain the inability of individual cytokines to reinduce E-selectin expression.

Increased soluble and platelet-associated CD40 ligand in essential thrombocythemia and reactive thrombocytosis

Blood ◽  
2002 ◽  
Vol 99 (7) ◽  
pp. 2612-2614 ◽  
Author(s):  
Jean-François Viallard ◽  
Anne Solanilla ◽  
Bruno Gauthier ◽  
Cécile Contin ◽  
Julie Déchanet ◽  
...  
Keyword(s):  
Platelet Count ◽  
Essential Thrombocythemia ◽  
Healthy Subjects ◽  
Cd40 Ligand ◽  
Soluble Form ◽  
Platelet Surface ◽  
Reactive Thrombocytosis ◽  
Potential Marker ◽  
High Platelet Count ◽  
Tight Correlation

CD40 ligand (CD40L) is expressed on activated CD4+ T lymphocytes and at the activated platelet surface. A circulating soluble form of CD40L (sCD40L) is generated by the way of a proteolytic cleavage. We measured sCD40L in the plasma of either healthy subjects; patients with inflammatory disorders and low, normal, or high platelet count (reactive thrombocytosis); or patients with essential thrombocythemia (ET). A tight correlation was found between the platelet count and plasma sCD40L. ET patients had the highest levels of sCD40L. Platelet-associated CD40L was increased in ET and reactive thrombocytosis, conditions associated with increased platelet regeneration. Platelet-associated CD40L was released upon platelet activation. In conclusion, platelets appear as a reservoir of CD40L that may be a major contributor to circulating sCD40L. Platelet-associated CD40L may be a potential marker of platelet regeneration.

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