Biomedical subjects

Yahye Merhi

Publications and source records attributed to Yahye Merhi.

At least 19 recordsLinked to original sources

PubMed2006

CD40 ligand binds to alpha5beta1 integrin and triggers cell signaling.

It was originally thought that the critical role of the CD40 ligand (CD40L) in normal and inflammatory immune responses was mainly mediated through its interaction with the classic receptor, CD40. However, data from CD40L(-/-) and CD40(-/-) mice suggest that the CD40L-induced inflammatory immune response involves at least one other receptor. This hypothesis is supported by the fact that CD40L stabilizes arterial thrombi through an alphaIIbbeta3-dependent mechanism. Here we provide evidence that soluble CD40L (sCD40L) binds to cells of the undifferentiated human monocytic U937 cell line in a CD40- and alphaIIbbeta3-independent manner. Binding of sCD40L to U937 cells was inhibited by anti-CD40L monoclonal antibody 5C8, anti-alpha5beta1 monoclonal antibody P1D6, and soluble alpha5beta1. The direct binding of sCD40L to purified alpha5beta1 was confirmed in a solid phase binding assay. Binding of sCD40L to alpha5beta1 was modulated by the form of alpha5beta1 expressed on the cell surface as the activation of alpha5beta1 by Mn(2+) or dithiothreitol resulted in the loss of sCD40L binding. Moreover, sCD40L induced the translocation of alpha5beta1 to the Triton X-100-insoluble fraction of U937 cells, the rapid activation of the MAPK pathways ERK1/2, and interleukin-8 gene expression. The binding of sCD40L to CD40 on BJAB cells, an alpha5beta1-negative B cell line, and the resulting activation of ERK1/2 was not inhibited by soluble alpha5beta1, suggesting that sCD40L can bind concomitantly to both receptors. These results document the existence of novel CD40L-dependent pathways of physiological relevance for cells expressing multiple receptors (CD40, alpha5beta1, and alphaIIbbeta3) for CD40L.

Animals↗

PubMed2006

Essential role of protein kinase C delta in platelet signaling, alpha IIb beta 3 activation, and thromboxane A2 release.

The protein kinase C (PKC) family is an essential signaling mediator in platelet activation and aggregation. However, the relative importance of the major platelet PKC isoforms and their downstream effectors in platelet signaling and function remain unclear. Using isolated human platelets, we report that PKCdelta, but not PKCalpha or PKCbeta, is required for collagen-induced phospholipase C-dependent signaling, activation of alpha(IIb)beta(3), and platelet aggregation. Analysis of PKCdelta phosphorylation and translocation to the membrane following activation by both collagen and thrombin indicates that it is positively regulated by alpha(IIb)beta(3) outside-in signaling. Moreover, PKCdelta triggers activation of the mitogen-activated protein kinase-kinase (MEK)/extracellular-signal regulated kinase (ERK) and the p38 MAPK signaling. This leads to the subsequent release of thromboxane A(2), which is essential for collagen-induced but not thrombin-induced platelet activation and aggregation. This study adds new insight to the role of PKCs in platelet function, where PKCdelta signaling, via the MEK/ERK and p38 MAPK pathways, is required for the secretion of thromboxane A(2).

Blood Platelets↗

PubMed2006

Estradiol blocks the induction of CD40 and CD40L expression on endothelial cells and prevents neutrophil adhesion: an ERalpha-mediated pathway.

OBJECTIVE: Interferon gamma (IFN-gamma) was shown to induce CD40 and CD40L expression on endothelial cells (ECs) and consequently to promote neutrophil adhesion. The pro- and anti-inflammatory effects of estrogens are well recognized but their role on the regulation of CD40 and CD40L expression on ECs remains undefined. METHODS AND RESULTS: Treatment of porcine aortic endothelial cells (PAEC) with IFN-gamma for 24 h enhanced CD40 and CD40L expression by 97% and 78%, respectively. Pretreatment of PAEC with 17-beta-estradiol (17betaE) for 24 h prevented the latter expression of CD40/CD40L. Treatment of PAEC with antisense oligomers targeting ERalpha mRNA attenuated the ability of 17betaE to inhibit the IFN-gamma-induced CD40 and CD40L protein expression. The IFN-gamma activation pathway of CD40 is known to involve the phosphorylation of the Janus activated kinase (JAK) and the signal transducer and activator of transcription 1 (Stat1). 17betaE, acting via the estrogen receptor alpha (ERalpha), abrogated IFN-gamma-mediated effects on Stat1 but failed to inhibit Jak1 and Jak2 phosphorylation. Furthermore, 17betaE prevented neutrophil adhesion induced by IFN-gamma. CONCLUSION: In summary, 17betaE binding to ERalpha blocked IFN-gamma-induced Stat1 phosphorylation, CD40 and CD40L protein expression, and neutrophil adhesion onto ECs.

Animals↗

PubMed2006

Recombinant P-selectin glycoprotein-ligand-1 delays thrombin-induced platelet aggregation: a new role for P-selectin in early aggregation.

1. P-selectin is involved, with P-selectin glycoprotein (GP)-ligand-1 (PSGL-1), in platelet/leukocyte interactions during thrombo-inflammatory reactions; it also stabilizes platelet aggregates. Its antagonism accelerates thrombolysis and enhances the anti-aggregatory effects of GPIIb-IIIa inhibitors. This study was designed to investigate the mechanisms of P-selectin-mediated platelet aggregation. 2. In freshly isolated human platelets, P-selectin translocation after thrombin stimulation increased rapidly to 48, 72, and 86% positive platelets after 60, 120, and 300 s, respectively. Platelet aggregation at 60 s post-stimulation averaged 46.7 +/- 1.9% and its extent followed closely the kinetics of P-selectin translocation. 3. Pre-treatment of platelets with P-selectin antagonists, a recombinant PSGL-1 (rPSGL-Ig) or a blocking monoclonal antibody, significantly delayed platelet aggregation in a dose-dependent manner. At 100 microg ml(-1) of rPSGL-Ig, platelet aggregation was completely inhibited up to 60 s post-stimulation and increased thereafter to reach maximal aggregation at 5 min. The second phase of platelet aggregation, in the presence of rPSGL-Ig, was completely prevented by the addition of a GPIIb-IIIa antagonist (Reopro) at 60 s, whereas its addition in the absence of rPSGL-Ig was without any significant effect. 4. Combination of rPSGL-Ig with Reopro or with an inhibitor of Pi3K (LY294002), which reduces GPIIb-IIIa activation, showed to be more effective in inhibiting platelet aggregation, in comparison to the effects observed individually. 5. rPSGL-Ig blocks P-selectin, whereas Reopro and LY294002 block GPIIb-IIIa and its activation, respectively, without a major effect on the percentage of platelets expressing P-selectin. 6. In summary, platelet P-selectin participates with GPIIb-IIIa in the initiation of platelet aggregation. Its inhibition, with rPSGL-Ig, delays the aggregation process and increases the anti-aggregatory potency of Reopro. Thus, combination of P-selectin and GPIIb-IIIa antagonism may constitute a promising therapeutic option in the management of thrombotic disorders.

Blood Platelets↗

PubMed2005

Effect of sternotomy and extracorporeal circulation on pulmonary neutrophil kinetics in pigs.

Pulmonary margination of neutrophils may contribute to lung damage after extracorporeal circulation for cardiac surgery. We evaluated single-pass pulmonary neutrophil kinetics using the multiple indicator-dilution technique in control pigs (n = 10), after sternotomy alone (sterno, n = 10) or after 30 min of observation following a period of 90 min extracorporeal circulation (n = 7). Blood neutrophils increased in the control and sterno groups (p < 0.05) but remained unchanged in the extracorporeal circulation group. The transfer coefficient for neutrophil margination from the circulating to the lung-marginated pool (k(c-m)) and pulmonary neutrophil clearance (Cl(c-m)) were similar between the three groups. There was an inverse correlation between k(c-m) and the degree of lung tissue perfusion evaluated from the tracer-accessible extravascular lung water (r = -0.54, p < 0.01). There was no arterio-venous gradient of neutrophils in any of the groups, suggesting a dynamic equilibrium of the margination/demargination processes. We conclude that extracorporeal circulation does not significantly modify single pass pulmonary neutrophil kinetics 30 min after reperfusion. The rate of neutrophil margination to the tracer-accessible lung tissue suggests that lung tissue de-recruitment is associated with increased neutrophil margination.

Animals↗

PubMed2005

Biodegradable membrane-covered stent from chitosan-based polymers.

Membrane-covered devices could help treat disease of the vasculature such as aneurysm, rupture, and fistulas. They are also investigated to reduce embolic complication associated with revascularization of saphenous vein graft. The aim of this study is to design a clinically applicable biodegradable membrane-covered stent based on the natural polysaccharide chitosan, which has been developed. The mechanical properties of the membrane is optimized through blending with polyethylene oxide (70:30% Wt CH:PEO). The membrane was able to sustain the mechanical deformation of the supporting self-expandable metallic stents during its deployment. The membrane was demonstrated to resist physiological transmural pressure (burst pressure resistance >500 mm Hg) and presented a high-water permeation resistance (1 mL/cm(2) min(-1) at 120 mmHg). The CH-PEO membrane showed a good hemocompatibility in an ex vivo assay. Heparin and hyaluronan surface complexation with the membrane further reduced platelet adhesion by 50.1 and 63% (p = 0.05). The ability of the membrane-covered devices to be used as a drug reservoir was investigated using the nitric oxide donor sodium nitroprusside (SNP). SNP-loaded membranes displayed significantly reduced platelet adhesion.

Biodegradation, Environmental↗

PubMed2005

Therapeutic potential of nanoparticulate systems for macrophage targeting.

The use of non-viral nanoparticulate systems for the delivery of therapeutic agents is receiving considerable attention for medical and pharmaceutical applications. This increasing interest results from the fact that these systems can be designed to meet specific physicochemical requirements, and they display low toxic and immunogenic effects. Among potential cellular targets by drug-loaded nanoparticles, macrophages are considered because they play a central role in inflammation and they act as reservoirs for microorganisms that are involved with deadly infectious diseases. The most common and potent drugs used in macrophage-mediated diseases treatment often induce unwanted side effects, when applied as a free form, due to the necessity of high doses to induce a satisfactory effect. This could result in their systemic spreading, a lack of bioavailability at the desired sites, and a short half-life. Therefore, the use of drug-loaded nanoparticles represents a good alternative to avoid, or at least decrease, side effects and increase efficacy. In this manuscript, we present an overview of the usefulness of nanoparticles for macrophage-mediated therapies in particular. We discuss, though not exhaustively, the potential of therapeutic agent-loaded nanoparticles for some macrophage-mediated diseases. We also underline the most important parameters that affect the interaction mechanisms of the macrophages and the physicochemical aspects of the particulate systems that may influence their performance in macrophage-targeted therapies.

Anti-Inflammatory Agents↗

PubMed2005

Neutrophil P-selectin-glycoprotein-ligand-1 binding to platelet P-selectin enhances metalloproteinase 2 secretion and platelet-neutrophil aggregation.

Platelets and neutrophils constitute a high source of metalloproteinases (MMPs), and their interactions via P-selectin and P-selectin-glycoprotein-ligand-1 (PSGL-1) are involved in thrombosis, vascular remodelling, and restenosis. We investigated the impact of these interactions on platelet MMP-2 secretion and function in platelet and neutrophil aggregation. The secretion of MMP-2 from human platelets was significantly increased three-fold after thrombin activation, and enhanced two-fold in the presence of neutrophils. Neutrophil supernatant had no effect on platelet MMP-2 secretion. While no MMP-2 was detected in the supernatant of neutrophils, a high amount of MMP-9 was released by neutrophils, and remained unchanged upon thrombin activation or in the presence of platelets. Platelet P-selectin, which increased significantly after activation, triggered platelet binding to neutrophils that was completely inhibited by P-selectin or PSGL-1 antagonists, and was reduced by 50% with a GPIIb/ IIIa antagonist. P-selectin or PSGL-1 antagonism abolished the enhanced secretion of platelet MMP-2 in the presence of neutrophils and reduced platelet-neutrophil aggregation. Platelet activation and binding to neutrophils enhance the secretion of platelet MMP-2 via an adhesive interaction between P-selectin and PSGL-1, which contribute to increase platelet-neutrophil aggregation.

Antibodies↗

PubMed2004

Angiopoietins can directly activate endothelial cells and neutrophils to promote proinflammatory responses.

Angiopoietin-1 (Ang1) and -2 (Ang2) are endothelial growth factors that bind to the tyrosine kinase receptor Tie2 and contribute to orchestrate blood vessel formation during angiogenesis. Ang1 mediates vessel maturation and integrity by the recruitment of pericytes. In contrast, Ang2 is classically considered as a Tie2 antagonist, counteracting the stabilizing action of Ang1. Inflammation exists in a mutually dependent association with angiogenesis and we have therefore studied the capacity of angiopoietins to modulate proinflammatory activities, namely P-selectin translocation and neutrophil adhesion onto endothelial cells. We observed that both Ang1 and Ang2 increased these biologic activities. Furthermore, combination of Ang1/Ang2 induced an additive effect on neutrophil adhesion but not on P-selectin translocation. In an attempt to clarify this phenomenon, we found that angiopoietins can directly activate neutrophils through Tie2 signaling as well as modulate platelet-activating factor (PAF) synthesis and beta(2) integrin functional up-regulation. Together, our data demonstrate that angiopoietins could promote acute recruitment of leukocytes, which might contribute to facilitate vascular remodeling and angiogenesis.

Angiopoietin-1↗

PubMed2004

VEGF-mediated endothelial P-selectin translocation: role of VEGF receptors and endogenous PAF synthesis.

The acute increase in vascular permeability produced by vascular endothelial growth factor (VEGF-A(165)) requires activation of endothelial Flk-1 receptors (VEGFR-2) and stimulation of platelet-activating factor (PAF) synthesis. Like PAF, VEGF-A(165) promotes translocation of P-selectin to the endothelial cell (EC) surface. However, the mechanisms involved remain unknown. By treating human umbilical vein endothelial cells (HUVECs) with VEGF analogs, we show that activation of VEGFR-1 or VEGFR-2 or both induced a rapid and transient translocation of endothelial P-selectin and neutrophil adhesion to activated ECs. The effects mediated by VEGF-A(165) and VEGF-A(121) (VEGFR-1/VEGFR-2 agonists) were blocked by a selective VEGFR-2 inhibitor, SU1498. VEGF-A(165) was twice as potent as VEGF-A(121), which can be explained by the binding capacity of VEGF-A(165) to its coreceptor neuropilin-1 (NRP-1). Indeed, treatment with NRP-1 antagonist (GST-Ex7) reduced the effect of VEGF-A(165) to the levels observed upon stimulation with VEGF-A(121). Finally, the use of selective PAF receptor antagonists reduced VEGF-A(165)-mediated P-selectin translocation. Together, these data show that maximal P-selectin translocation and subsequent neutrophil adhesion was mediated by VEGF-A(165) on the activation of VEGFR-2/NRP-1 complex and required PAF synthesis.

Cell Adhesion↗

PubMed2004

Radionuclides-hyaluronan-conjugate thromboresistant coatings to prevent in-stent restenosis.

Catheter-based brachytherapy is one of the most effective modalities to inhibit hyperplasia following revascularization procedures. Radioactive stents have failed, however, to prevent clinical hyperplasia due to excessive late lumen loss on the edge of the devices. Numerous strategies have been proposed to circumvent the drawbacks of irradiation therapies, such as the use of more appropriate radionuclides or the "hot-end" stents approach. This paper describes versatile radioactive devices obtained by coating plasma functionalized surfaces-stents or catheters-with a hyaluronan (HA)-diethylenetriamine pentaacetic acid (DTPA) conjugate (HA-DTPA) complexed with a gamma or beta radionuclide. Yttrium and indium were used as radionuclide models, due to their suitability for endovascular radiotherapy. X-ray photoelectron microscopy and time-of-flight secondary ions mass spectrometry analyses confirmed the successful immobilization of the HA-DTPA conjugate on both the metallic (NiTi) and polymeric (Teflon) plasma functionalized surfaces. HA-DTPA-coated surfaces were significantly more hydrophilic than bare surfaces (39.5 degrees vs. 67 degrees on NiTi substrate and 29 degrees vs. 128 degrees on Teflon substrate). Therapeutic doses of yttrium and indium were easily loaded onto the surfaces and remained stable over 2 weeks with a radionuclide loss of about 6%. The HA-DTPA-coated Teflon surfaces presented significantly less fibrinogen adsorption than uncoated materials in an in vitro flow model. This approach, which combines the hemocompatibility of HA-coated surfaces and the anti-proliferative effects of an appropriate radiotherapy, constitutes a promising methodology to alleviate the restenosis induced by existing devices.

Adsorption↗

PubMed2004

Prevention of in-stent restenosis via reduction of thrombo-inflammatory reactions with recombinant P-selectin glycoprotein ligand-1.

The binding of leukocyte P-selectin glycoprotein ligand-1 (PSGL-1) to platelet P-selectin is central to post-angioplasty restenosis. Although intracoronary stents limit the mechanical component of restenosis, they cause marked thrombo-inflammation and neointimal proliferation leading to greater late luminal loss. We sought to demonstrate that P-selectin antagonism, using recombinant PSGL-1 (rPSGL-Ig), is effective in reducing platelet-leukocyte reactions and in-stent restenosis in double-injured porcine coronary arteries. Two weeks after initial injury by angioplasty to the coronary arteries, stents were implanted at the injury-induced lesion site, 15 min after an i.v. bolus administration of a vehicle or rPSGL-Ig (1 mg/kg). Four weeks later, adhesion of (51)Cr-platelets and (111)In-neutrophils and histomorphometric analyses were performed. In-stent residual lumen was almost 3 fold larger in rPSGL-Ig-treated arteries (3.1 +/- 0.4 mm(2)) as compared to control (1.1 +/- 0.2 mm(2)), which correspond to 64% vascular stenosis in control with no change in rPSGL-Ig animals. For a similar injury score, in-stent neointima was significantly reduced by 30 to 40% in the rPSGL-Ig group and quantitative coronary angiography showed a significant 35% reduction in late lumen loss. These effects of rPSGL-Ig were associated with a respective 70% and 53% reduction in platelet and neutrophil adhesion. In conclusion, pretreatment with rPSGL-Ig reduces thrombo-inflammatory responses, neointimal proliferation, and in-stent restenosis. P-selectin antagonism offers a promising therapy to improve clinical outcomes of coronary stenting.

Angioplasty↗

PubMed2003

In vitro cytotoxicity evaluation of a 50.8% NiTi single crystal.

To our knowledge, the biocompatibility of nickel-titanium (NiTi) single crystals has not been reported. Yet certain orientations of single crystals present several advantages over the polycrystalline form in terms of maximal strain, fatigue resistance, and temperature range of superelasticity. Therefore we tested the in vitro biocompatibility of 50.8% NiTi single crystals in the orientation <001> after four different heat treatments in a helium atmosphere followed by mechanical polishing. The study was performed on the material extracts after immersion of the specimens in cell culture medium (DMEM) for 7 days at 37 degrees C. Cytotoxicity studies were performed on L-929 mouse fibroblasts using the MTT assay. J-774 macrophages were used to assess the potential inflammatory effect of the extracts by IL1-beta and TNF-alpha dosages (sandwich ELISA method). Exposure of L-929 to material extracts did not affect cell viability. In addition, IL1-beta and TNF-alpha secretion was not stimulated after incubation with NiTi extracts compared to the negative controls. These results were predictable since atomic absorption spectroscopy did not detect nickel ions in the extracts with a resolution of 1 ppm. Within the limits of in vitro testing, our results demonstrate that the TiNi(50.8%) single crystals do not trigger a cytotoxic reaction.

Alloys↗

PubMed2003

Nanocoatings onto arteries via layer-by-layer deposition: toward the in vivo repair of damaged blood vessels.

The deposition of polysaccharide-based self-assembled nanocoatings onto damaged arteries is described as a means not only to protect a damaged artery against thrombogenesis, but also to control the healing processes by incorporating biologically active components within the multilayer. As shown by confocal microscopy, the polysaccharide multilayer was retained on the artery in physiological condition and prevented platelet adhesion. Diffusion of the polysaccharides within the artery was also observed and may be used to efficiently target the vascular wall. The NO-precursor l-arginine was used a drug model and incorporated within the self-assembled layers.

Animals↗

PubMed2003

Atrial ischemia promotes atrial fibrillation in dogs.

BACKGROUND: Coronary artery disease is a significant risk factor for atrial fibrillation (AF), but the basis for this association is incompletely understood. The present study evaluated the hypothesis that atrial ischemia can create a substrate for AF maintenance. METHODS AND RESULTS: Atrial ischemia was induced by occlusion of an atrial arterial branch that did not provide blood flow to the ventricles. Atrial-arterial occlusion increased the duration of AF induced by burst pacing from 57+/-32 seconds (control) to 803+/-214 seconds (P<0.001) after 0.5 hour of occlusion and to 887+/-209 seconds (P<0.001) after 3 hours of occlusion. Prolonged AF (>20 minutes) was induced in 0 of 16 dogs (0%) under control conditions, 7 of 16 (44%, P<0.01) at 0.5 to 3 hours, and 5 of 13 (38%, P<0.01) 3 to 5 hours after occlusion. Atrial conduction was slowed substantially within the ischemic zone: eg, conduction delay was 8+/-1 ms at a cycle length of 200 ms, control, versus 22+/-5 ms (P<0.01) after 0.5 hours and 27+/-5 ms (P<0.001) after 3 hours of ischemia. Refractoriness was initially unaffected but was prolonged 5 hours after occlusion. Phase-delay analysis and high-density mapping confirmed severe conduction slowing in the ischemic zone. Histological examination confirmed the location of ischemic regions and revealed extensive ischemia-induced necrosis at sites of conduction delay. CONCLUSIONS: Experimental atrial ischemia creates a substrate for AF maintenance, apparently by causing local conduction slowing that promotes reentry. These results suggest that atrial ischemia may significantly promote AF, and may be relevant to AF mechanisms in association with coronary artery disease.

Animals↗

PubMed2003

Kinetic analysis of pulmonary neutrophil retention in vivo using the multiple-indicator-dilution technique.

Multiple-indicator-dilution experiments were performed in the lungs of 13 anesthetized dogs by simultaneous bolus injection of 111In-labeled neutrophils, 51Cr-labeled red blood cells, and Evans blue-labeled albumin. Concomitant counts of unlabeled neutrophils were similar in pulmonary artery and aortic blood samples, demonstrating a dynamic balance across the lungs in the physiological state. Outflow profiles of labeled neutrophils were analyzed on the basis of a recirculatory pharmacokinetic model of labeled albumin. The outflow profiles of the recovered neutrophils were composed of a throughput component of circulating neutrophils and a component of reversibly marginated neutrophils. They were interpreted by a model incorporating neutrophil margination (transfer coefficient = 0.195 +/- 0.081 s-1), rapid demargination (0.054 +/- 0.027 s-1), and transfer to a slow marginated pool (0.023 +/- 0.018 s-1). It will be interesting to apply the analysis in future studies aimed at determining whether it could be a useful research tool to investigate the interactions between the pulmonary endothelium and neutrophils in physiological and diseased states.

Albumins↗

PubMed2003

Chronic platelet and neutrophil adhesion: a causal role for neointimal hyperplasia in in-stent restenosis.

PURPOSE: To investigate the relationship between platelets and neutrophils and the progression of neointimal hyperplasia after angioplasty versus stenting of porcine coronary arteries. METHODS: Balloon angioplasty or implantation of a balloon-expandable stent was randomly performed in the left and right coronary arteries of 16 Yorkshire swine; the animals were euthanized 1 hour (n=6), 24 hours (n=4), or 1 month (n=6) after the interventions. The adhesion of chromium 51-labeled platelets and indium 111-labeled neutrophils was quantified (per cm2), and histological and morphometric analyses were performed. RESULTS: The acute adhesion of platelets and neutrophils observed on the injured segments 1 hour after the interventions was similar between the treated groups. However, at 24 hours, stenting was associated with 2-fold more platelets and 3-fold more neutrophils (p<0.05) than was dilation, and stented arteries remained more thrombogenic at 1 month (p<0.05). Neointimal formation was more intense after stent implantation (3.80+/-0.77 mm, p<0.05) than after dilation (0.81+/-0.21 mm), and it correlated positively with the adhesion of platelets (r=0.81, p<0.002) and neutrophils (r=0.69, p<0.01). CONCLUSIONS: These results indicate that stent implantation is associated with a more intense acute and chronic, low-grade inflammatory response than is dilation. It appears that the chronic inflammatory response is, at least in part, platelet- and neutrophil-driven and contributes to the progression of neointimal proliferation after stenting.

Angioplasty, Balloon↗

PubMed2003

Differential regulation of P-selectin expression by protein kinase A and protein kinase G in thrombin-stimulated human platelets.

P-selectin is rapidly translocated from platelet alpha-granules following activation. Intracellular cyclic AMP (cAMP) is a potent inhibitory pathway that results in global downregulation of platelet activation. While cAMP-dependent protein kinase (PKA) has long been considered as the main mediator of cAMP-dependent effects, no study has yet evaluated its effect on P-selectin expression in human platelets. Pretreatment of thrombin-stimulated platelets with forskolin resulted in a concentration- dependent inhibition of P-selectin expression that correlated with adenylyl cyclase activity. Inhibition of PKA with H-89 reversed cAMP-induced inhibition of P-selectin while cGMP-dependent protein kinase (PKG) inhibition with KT5823 significantly potentiated cAMP-dependent inhibition of P-selectin. Similar results were also observed in a platelet/neutrophil binding assay. In conclusion, cAMP-induced inhibition of P-selectin expression is, in large part, mediated through activation of PKA. PKG appears to be solicited for P-selectin expression when cAMP levels are elevated which suggest a cAMP/PKG-dependent pathway of platelet activation.

Adenylyl Cyclases↗