Dissecting Platelet's Role in Viral Infection: A Double-Edged Effector of the Immune System.

Platelets play a major role in the processes of primary hemostasis and pathological inflammation-induced thrombosis. In the mid-2000s, several studies expanded the role of these particular cells, placing them in the "immune continuum" and thus changing the understanding of their function in both innate and adaptive immune responses. Among the many receptors they express on their surface, platelets express Toll-Like Receptors (TLRs), key receptors in the inflammatory cell-cell reaction and in the interaction between innate and adaptive immunity. In response to an infectious stimulus, platelets will become differentially activated. Platelet activation is variable depending on whether platelets are activated by a hemostatic or pathogen stimulus. This review highlights the role that platelets play in platelet modulation count and adaptative immune response during viral infection.

The Pathophysiological Role of Platelet-Derived Extracellular Vesicles.

Platelets are very abundant in the blood, where they play a role in hemostasis, inflammation, and immunity. When activated, platelets undergo a conformational change that allows the release of numerous effector molecules as well as the production of extracellular vesicles, which are circulating submicron vesicles (10 to 1,000 nm in diameter) released into the extracellular space. Extracellular vesicles are formed by the budding of platelet and they carry some of its contents, including nucleic acids, surface proteins, and organelles. While platelets cannot cross tissue barriers, platelet-derived extracellular vesicles can enter the lymph, bone marrow, and synovial fluid. This allows the transfer of diverse contents carried by these platelet-derived vesicles to cell recipients and organs inaccessible to platelets where they can perform many functions. This review highlights the importance of these platelet-derived extracellular vesicles under different physiological and pathophysiological conditions.

University of Montreal's Clinician-Investigator Program: A 10-Year Descriptive Evaluation.

PURPOSE: Clinician-investigators have an important role in the development and implantation of new therapies and treatment modalities; however, there have been several reports highlighting a pending shortage in the clinician-investigators' workforce. In Canada, the Royal College has promoted the development of clinician-investigators programs (CIP) to facilitate the training of these individuals. There is currently a paucity of data regarding the outcomes of such programs. This study aims to identify the strengths and areas of improvement of the Montreal University CIP.  Methods: An internet-based 51-question survey was distributed to all the alumni from the University of Montreal CIP. Participation was voluntary and no incentives were provided. The response rate was 64%.  Results: Among respondents, 50% (n=16) had completed their clinical residency and all CIP requirements. The majority of these individuals (63%) had become independent investigators and had secured provincial and national funding. Satisfaction of the respondents was high regarding the overall program (85%), the research skills developed during the CIP (84%) and the financial support obtained during the program (72%). The satisfaction rate regarding career planning was lower (63%).  Conclusion: This survey demonstrates that, while indicators are favorable, some areas still require improvement. Several steps to improve the CIP have been identified; notably, the transition from the CIP to early independent career has been identified as critical in the development of clinician-investigators and steps have been taken to improve this progression.

Implication of Platelets in Immuno-Thrombosis and Thrombo-Inflammation.

In addition to their well-described hemostatic function, platelets are active participants in innate and adaptive immunity. Inflammation and immunity are closely related to changes in platelet reactions and enhanced platelet function in thrombo-inflammation, as well as in microbial and virus infections. A platelet's immune function is incompletely understood, but an important balance exists between its protective and pathogenic responses and its thrombotic and inflammatory functions. As the mediator of vascular homeostasis, platelets interact with neutrophils, bacteria and virus by expressing specific receptors and releasing granules, transferring RNA, and secreting mitochondria, which controls hemostasis and thrombosis, infection, and innate and adaptive immunity. This review focuses on the involvement of platelets during immuno-thrombosis and thrombo-inflammation.

The Role of the Proteasome in Platelet Function.

Platelets are megakaryocyte-derived acellular fragments prepped to maintain primary hemostasis and thrombosis by preserving vascular integrity. Although they lack nuclei, platelets harbor functional genomic mediators that bolster platelet activity in a signal-specific manner by performing limited de novo protein synthesis. Furthermore, despite their limited protein synthesis, platelets are equipped with multiple protein degradation mechanisms, such as the proteasome. In nucleated cells, the functions of the proteasome are well established and primarily include proteostasis among a myriad of other signaling processes. However, the role of proteasome-mediated protein degradation in platelets remains elusive. In this review article, we recapitulate the developing literature on the functions of the proteasome in platelets, discussing its emerging regulatory role in platelet viability and function and highlighting how its functional coupling with the transcription factor NF-κB constitutes a novel potential therapeutic target in atherothrombotic diseases.

Aspirin Reduces the Potentiating Effect of CD40L on Platelet Aggregation via Inhibition of Myosin Light Chain.

Background Antiplatelet therapy with aspirin (acetylsalicylic acid [ASA]) is less efficient in some coronary patients, which increases their risk of developing thrombosis. Elevated blood levels of thromboinflammatory mediators, like soluble CD40L (sCD40L), may explain such variabilities. We hypothesized that in the presence of elevated levels of sCD40L, the efficacy of ASA may vary and aimed to determine the effects of ASA on CD40L signaling and aggregation of platelets. Methods and Results The effects of ASA on CD40L-treated human platelets, in response to suboptimal concentrations of collagen or thrombin, were assessed at levels of aggregation, thromboxane A2 secretion, and phosphorylation of p38 mitogen-activated protein kinase, nuclear factor kappa B, transforming growth factor-ß-activated kinase 1, and myosin light chain. sCD40L significantly elevated thromboxane A2 secretion in platelets in response to suboptimal doses of collagen and thrombin, which was reversed by ASA. ASA did not inhibit the phosphorylation of p38 mitogen-activated protein kinase, nuclear factor kappa B, and transforming growth factor-ß-activated kinase 1, with sCD40L stimulation alone or with platelet agonists. sCD40L potentiated platelet aggregation, an effect completely reversed and partially reduced by ASA in response to a suboptimal dose of collagen and thrombin, respectively. The effects of ASA in sCD40L-treated platelets with collagen were related to inhibition of platelet shape change and myosin light chain phosphorylation. Conclusions ASA does not affect platelet sCD40L signaling but prevents its effect on thromboxane A2 secretion and platelet aggregation in response to collagen, via a mechanism implying inhibition of myosin light chain. Targeting the sCD40L axis in platelets may have a therapeutic potential in patients with elevated levels of sCD40L and who are nonresponsive or less responsive to ASA.

Role of NF-κB in Platelet Function.

Platelets are megakaryocyte-derived fragments lacking nuclei and prepped to maintain primary hemostasis by initiating blood clots on injured vascular endothelia. Pathologically, platelets undergo the same physiological processes of activation, secretion, and aggregation yet with such pronouncedness that they orchestrate and make headway the progression of atherothrombotic diseases not only through clot formation but also via forcing a pro-inflammatory state. Indeed, nuclear factor-κB (NF-κB) is largely implicated in atherosclerosis and its pathological complication in atherothrombotic diseases due to its transcriptional role in maintaining pro-survival and pro-inflammatory states in vascular and blood cells. On the other hand, we know little on the functions of platelet NF-κB, which seems to function in other non-genomic ways to modulate atherothrombosis. Therein, this review will resemble a rich portfolio for NF-κB in platelets, specifically showing its implications at the levels of platelet survival and function. We will also share the knowledge thus far on the effects of active ingredients on NF-κB in general, as an extrapolative method to highlight the potential therapeutic targeting of NF-κB in coronary diseases. Finally, we will unzip a new horizon on a possible extra-platelet role of platelet NF-κB, which will better expand our knowledge on the etiology and pathophysiology of atherothrombosis.

CD40L Priming of Platelets via NF-κB Activation is CD40- and TAK1-Dependent.

Background CD40 ligand (CD40L) is a thromboinflammatory molecule that predicts cardiovascular events. CD40L is a strong activator of nuclear factor kappa B (NF-κB) in platelets that primes and enhances platelet activation in response to thrombotic stimuli. In addition to its classical receptor CD40, CD40L binds αIIbß3, α5ß1, and αMß2 in various cell types. However, the function of the different CD40L receptors on platelets remains unexplored. The present study aims to identify the receptors of CD40L, involved in platelet NF-κB activation, their downstream signaling and their implication in platelet aggregation. Methods and Results We showed that platelets express CD40, αIIbß3, and α5ß1 and release CD40L in response to sCD40L stimulation. sCD40L alone dose-dependently induced platelet NF-κB activation; this effect was absent in CD40-/- mouse platelets and inhibited by the CD40 blockade, but was unaffected by the αIIbß3 or α5ß1 blockade in human platelets. sCD40L/CD40 axis activates transforming growth factor-ß-activated kinase 1 upstream of NF-κB. In functional studies, sCD40L alone did not affect platelet aggregation but potentiated the aggregation response in the presence of suboptimal doses of thrombin; this effect was abolished by CD40, transforming growth factor-ß-activated kinase 1, and NF-κB inhibitors. Conclusions CD40L primes platelets via signaling pathways involving CD40/transforming growth factor-ß-activated kinase 1/NF-κB, which predisposes platelets to enhanced activation and aggregation in response to thrombotic stimuli.

Apolipoprotein A-I Modulates Atherosclerosis Through Lymphatic Vessel-Dependent Mechanisms in Mice.

BACKGROUND: Subcutaneously injected lipid-free apoA-I (apolipoprotein A-I) reduces accumulation of lipid and immune cells within the aortic root of hypercholesterolemic mice without increasing high-density lipoprotein-cholesterol concentrations. Lymphatic vessels are now recognized as prerequisite players in the modulation of cholesterol removal from the artery wall in experimental conditions of plaque regression, and particular attention has been brought to the role of the collecting lymphatic vessels in early atherosclerosis-related lymphatic dysfunction. In the present study, we address whether and how preservation of collecting lymphatic function contributes to the protective effect of apoA-I. METHODS AND RESULTS: Atherosclerotic Ldlr-/- mice treated with low-dose lipid-free apoA-I showed enhanced lymphatic transport and abrogated collecting lymphatic vessel permeability in atherosclerotic Ldlr-/- mice when compared with albumin-control mice. Treatment of human lymphatic endothelial cells with apoA-I increased the adhesion of human platelets on lymphatic endothelial cells, in a bridge-like manner, a mechanism that could strengthen endothelial cell-cell junctions and limit atherosclerosis-associated collecting lymphatic vessel dysfunction. Experiments performed with blood platelets isolated from apoA-I-treated Ldlr-/- mice revealed that apoA-I decreased ex vivo platelet aggregation. This suggests that in vivo apoA-I treatment limits platelet thrombotic potential in blood while maintaining the platelet activity needed to sustain adequate lymphatic function. CONCLUSIONS: Altogether, we bring forward a new pleiotropic role for apoA-I in lymphatic function and unveil new potential therapeutic targets for the prevention and treatment of atherosclerosis.

Early outgrowth cells versus endothelial colony forming cells functions in platelet aggregation.

BACKGROUND: Endothelial progenitor cells (EPCs) have been implicated in neoangiogenesis, endothelial repair and cell-based therapies for cardiovascular diseases. We have previously shown that the recruitment of EPCs to sites of vascular lesions is facilitated by platelets where EPCs, in turn, modulate platelet function and thrombosis. However, EPCs encompass a heterogeneous population of progenitor cells that may exert different effects on platelet function. Recent evidence suggests the existence of two EPC subtypes: early outgrowth cells (EOCs) and endothelial colony-forming cells (ECFCs). We aimed at characterizing these two EPC subtypes and at identifying their role in platelet aggregation. METHODS: EOCs and ECFCs were generated from human peripheral blood mononuclear cells (PBMCs) seeded in conditioned media on fibronectin and collagen, respectively. The morphological, phenotypical and functional characteristics of EOCs and ECFCs were assessed by optical and confocal laser scanning microscopes, cell surface markers expression, and Matrigel tube formation. The impact of EOCs and ECFCs on platelet aggregation was monitored in collagen-induced optical aggregometry and compared with PBMCs and human umbilical vein endothelial cells (HUVECs). The levels of the anti-platelet agents' nitric oxide (NO) and prostacyclin (PGI2) released from cultured cells as well as the expression of their respective producing enzymes NO synthases (NOS) and cyclooxygenases (COX) were also assessed. RESULTS: We showed that EOCs display a monocytic-like phenotype whereas ECFCs have an endothelial-like phenotype. We demonstrated that both EOCs and ECFCs and their supernatants inhibited platelet aggregation; however ECFCs were more efficient than EOCs. This could be related to the release of significantly higher amounts of NO and PGI2 from ECFCs, in comparison to EOCs. Indeed, ECFCs, like HUVECs, constitutively express the endothelial (eNOS)-and inducible (iNOS)-NOS isoforms, and COX-1 and weakly express COX-2, whereas EOCs do not constitutively express these NO and PGI2 producing enzymes. CONCLUSION: The different morphological, phenotypic and more importantly the release of the anti-aggregating agents PGI2 and NO in each EPC subtype are implicated in their respective roles in platelet function and thus, may be linked to the increased efficiency of ECFCs in inhibiting platelet aggregation as compared to EOCs.

Endothelial progenitor cells inhibit platelet function in a P-selectin-dependent manner.

BACKGROUND: The role of endothelial progenitor cells (EPCs) in vascular repair is related to their recruitment at the sites of injury and their interaction with different components of the circulatory system. We have previously shown that EPCs bind and inhibit platelet function and impair thrombus formation via prostacyclin secretion, but the role of EPC binding to platelet P-selectin in this process has not been fully characterized. In the present study, we assessed the impact of EPCs on thrombus formation and we addressed the implication of P-selectin in this process. METHODS: EPCs were generated from human peripheral blood mononuclear cells cultured on fibronectin in conditioned media. The impact of EPCs on platelet aggregation and thrombus formation was investigated in P-selectin deficient (P-sel(-/-)) mice and their wild-type (WT) counterparts. RESULTS: EPCs significantly and dose-dependently impaired collagen-induced whole blood platelet aggregation in WT mice, whereas no effects were observed in P-sel(-/-) mice. Moreover, in a ferric chloride-induced arterial thrombosis model, infusion of EPCs significantly reduced thrombus formation in WT, but not in P-sel(-/-) mice. Furthermore, the relative mass of thrombi generated in EPC-treated P-sel(-/-) mice were significantly larger than those in EPC-treated WT mice, and the number of EPCs recruited within the thrombi and along the arterial wall was reduced in P-sel(-/-) mice as compared to WT mice. CONCLUSION: This study shows that EPCs impair platelet aggregation and reduce thrombus formation via a cellular mechanism involving binding to platelet P-selectin. These findings add new insights into the role of EPC-platelet interactions in the regulation of thrombotic events during vascular repair.

Development of a Polyester Coating Combining Antithrombogenic and Cell Adhesive Properties: Influence of Sequence and Surface Density of Adhesion Peptides.

Biofunctionalization strategies have been developed to improve small-diameter vascular grafts. However, a fully successful coating featuring antithrombogenic properties while allowing for endothelialization has not been achieved yet. In this report, we explored the combination of low-fouling polyethylene glycol (PEG) and adhesion peptides, namely, RGD, YIGSR, and REDV, grafted on top of polyvinylamine (PVAm)-coated polyester. The peptides were grafted over a wide range of density (ca. 20-2000 pmol/cm(2)) on top of a dense PEG underlayer. The coating performances were assessed through HUVEC adhesion, platelet attachment, and protein adsorption, which were all drastically diminished on PEG-coated samples. RGD exhibited the expected high adhesive properties, toward both HUVEC and platelets. REDV had no effect neither on platelet attachment, as expected, nor on HUVEC adhesion, in contrast with previous reports. YIGSR was the most promising sequence even though its combination with other agents should be explored to further decrease thrombogenicity for vascular graft applications.

Interaction of CD154 with different receptors and its role in bidirectional signals.

In addition to its classical receptor, CD40, it is now well established that CD154 also binds αIIbß3, α5ß1, and αMß2 integrins. Although these integrins are all members of the same family, they bind CD154 differently. The current investigation aims to analyze the interaction of CD154 with α5ß1 and αMß2 and investigate its role in bidirectional signals in various human cell lines. Results obtained herein indicate that the CD154 residues involved in the interaction with α5ß1 are N151 and Q166, whereas those involved in αMß2 binding are common to residues required for CD40, namely Y145 and R203. Soluble CD40/CD154 or αMß2/CD154 complexes do not interfere with the binding of CD154 to α5ß1-positive cells, but inhibit the binding of CD154 to CD40- or αMß2-positive cells, respectively. Ligation of CD154 on CD154-positive cells with soluble CD40, αIIbß3, α5ß1, or αMß2 stimulates intracellular signaling, including MAPK phosphorylation. Given that CD154 exists as a trimer, our data strongly suggest that CD154 may bind concomitantly to two receptors of the same or different family, and biologically activate cells expressing both receptors. The characterization of CD154/receptor interactions helps the identification of new therapeutic targets for the prevention and/or treatment of CD154-associated autoimmune and inflammatory diseases.

Chondroitin sulfate coatings display low platelet but high endothelial cell adhesive properties favorable for vascular implants.

This study highlights the advantages of chondroitin sulfate (CS) as a sublayer combining selective low-fouling properties, low-platelet adhesion and pro-adhesive properties on endothelial cells, making CS promising for vascular graft applications. These properties were evaluated by comparing CS with well-known low-fouling coatings such as poly(ethylene glycol) (PEG) and carboxymethylated dextran (CMD), which were covalently grafted on primary amine-rich plasma polymerized (LP) films. Protein adsorption studies by quartz crystal microbalance with dissipation monitoring (QCM-D) and fluorescence measurements showed that CS is as effective as PEG in reducing fibrinogen adsorption (~90% reduction). CS also largely reduced adsorption of bovine serum albumin (BSA) as well as fetal bovine serum (FBS) but to a lower extent than PEG and CMD surfaces (72% vs 85% for BSA and 66% vs 89% for FBS). Whole blood perfusion assays indicated that, while LP surfaces were highly reactive with platelets, PEG, CMD, and CS grafted surfaces drastically decreased platelet adhesion and activation to levels significantly lower than polyethylene terephthalate (PET) surfaces. Finally, while human umbilical vein endothelial cell (HUVEC) adhesion and growth were found to be very limited on PEG and CMD, they were significantly increased on CS compared to that on bare PET and reached similar values as those for tissue culture polystyrene positive controls. Interestingly, HUVEC retention during perfusion with blood was found to be excellent on CS but poor on PET. Overall, our results suggest that the CS surface has the advantage of promoting HUVEC growth and resistance to flow-induced shear stress while preventing fibrinogen and platelet attachment. Such a nonthrombogenic but endothelial-cell adhesive surface is thus promising to limit vascular graft occlusion.

Soluble CD40 ligand stimulates the pro-angiogenic function of peripheral blood angiogenic outgrowth cells via increased release of matrix metalloproteinase-9.

The role of endothelial progenitor cells in vascular repair is related to their incorporation at sites of vascular lesions, differentiation into endothelial cells, and release of various angiogenic factors specifically by a subset of early outgrowth endothelial progenitor cells (EOCs). It has been shown that patients suffering from cardiovascular disease exhibit increased levels of circulating and soluble CD40 ligand (sCD40L), which may influence the function of EOCs. We have previously shown that the inflammatory receptor CD40 is expressed on EOCs and its ligation with sCD40L impairs the anti-platelet function of EOCs. In the present study, we aimed at investigating the effect of sCD40L on the function of EOCs in endothelial repair. Human peripheral blood mononuclear cell-derived EOCs express CD40 and its adaptor proteins, the tumor necrosis factor receptor-associated factors; TRAF1, TRAF2 and TRAF3. Stimulation of EOCs with sCD40L increased the expression of TRAF1, binding of TRAF2 to CD40 and phosphorylation of p38 mitogen activated protein kinase (MAPK). In an in vitro wound healing assay, stimulation of EOCs with sCD40L increased the release of matrix metalloproteinase 9 (MMP-9) in a concentration-dependent manner and significantly enhanced the angiogenic potential of cultured human umbilical vein endothelial cells (HUVECs). Inhibition of p38 MAPK reversed sCD40L-induced MMP-9 release by EOCs, whereas inhibition of MMP-9 reversed their pro-angiogenic effect on HUVECs. This study reveals the existence of a CD40L/CD40/TRAF axis in EOCs and shows that sCD40L increases the pro-angiogenic function of EOCs on cultured HUVECs by inducing a significant increase in MMP-9 release via, at least, the p38 MAPK signaling pathway.

A versatile star PEG grafting method for the generation of nonfouling and nonthrombogenic surfaces.

Polyethylene glycol (PEG) grafting has a great potential to create nonfouling and nonthrombogenic surfaces, but present techniques lack versatility and stability. The present work aimed to develop a versatile PEG grafting method applicable to most biomaterial surfaces, by taking advantage of novel primary amine-rich plasma-polymerized coatings. Star-shaped PEG covalent binding was studied using static contact angle, X-ray photoelectron spectroscopy (XPS), and quartz crystal microbalance with dissipation monitoring (QCM-D). Fluorescence and QCM-D both confirmed strong reduction of protein adsorption when compared to plasma-polymerized coatings and pristine poly(ethyleneterephthalate) (PET). Moreover, almost no platelet adhesion was observed after 15 min perfusion in whole blood. Altogether, our results suggest that primary amine-rich plasma-polymerized coatings offer a promising stable and versatile method for PEG grafting in order to create nonfouling and nonthrombogenic surfaces and micropatterns.

CD154: the atherosclerotic risk factor in rheumatoid arthritis?

Atherosclerosis, now regarded as a chronic inflammatory disease of the arterial wall, and its clinical manifestations have increasingly been associated with rheumatoid arthritis (RA), supporting the notion that autoimmune diseases and vascular disorders share common etiological features. Indeed, evidence pertaining to this matter indicates that inflammation and its multiple components are the driving force behind the pathogenesis of these disorders. Interestingly, CD154 and its receptors have emerged as major players in the development of RA and atherosclerosis, which raises the possibility that this axis may represent an important biological link between both complications. Indeed, CD154 signaling elicits critical inflammatory responses that are common to the pathogenesis of both diseases. Here, we provide an overview of the traditional and disease-related interrelations between RA and vascular abnormalities, while focusing on CD154 as a potential mediator in the development of atherosclerotic events in RA patients.

Involvement of nuclear factor κB in platelet CD40 signaling.

CD40 ligand (CD40L) is a thrombo-inflammatory molecule that predicts cardiovascular events. Platelets constitute the major source of soluble CD40L (sCD40L), which has been shown to potentiate platelet activation and aggregation, in a CD40-dependent manner, via p38 mitogen activated protein kinase (MAPK) and Rac1 signaling. In many cells, the CD40L/CD40 dyad also induces activation of nuclear factor kappa B (NF-κB). Given that platelets contain NF-κB, we hypothesized that it may be involved in platelet CD40 signaling and function. In human platelets, sCD40L induces association of CD40 with its adaptor protein the tumor necrosis factor receptor associated factor 2 and triggers phosphorylation of IκBα, which are abolished by CD40L blockade. Inhibition of IκBα phosphorylation reverses sCD40L-induced IκBα phosphorylation without affecting p38 MAPK phosphorylation. On the other hand, inhibition of p38 MAPK phosphorylation has no effect on IκBα phosphorylation, indicating a divergence in the signaling pathway originating from CD40 upon its ligation. In functional studies, inhibition of IκBα phosphorylation reverses sCD40L-induced platelet activation and potentiation of platelet aggregation in response to a sub-threshold concentration of collagen. This study demonstrates that the sCD40L/CD40 axis triggers NF-κB activation in platelets. This signaling pathway plays a critical role in platelet activation and aggregation upon sCD40L stimulation and may represent an important target against thrombo-inflammatory disorders.

Functional interaction of CD154 protein with α5ß1 integrin is totally independent from its binding to αIIbß3 integrin and CD40 molecules.

In addition to its classical CD40 receptor, CD154 also binds to αIIbß3, α5ß1, and αMß2 integrins. Binding of CD154 to these receptors seems to play a key role in the pathogenic processes of chronic inflammation. This investigation was aimed at analyzing the functional interaction of CD154 with CD40, αIIbß3, and α5ß1 receptors. We found that the binding affinity of CD154 for αIIbß3 is ∼4-fold higher than for α5ß1. We also describe the generation of sCD154 mutants that lost their ability to bind CD40 or αIIbß3 and show that CD154 residues involved in its binding to CD40 or αIIbß3 are distinct from those implicated in its interaction to α5ß1, suggesting that sCD154 may bind simultaneously to different receptors. Indeed, sCD154 can bind simultaneously to CD40 and α5ß1 and biologically activate human monocytic U937 cells expressing both receptors. The simultaneous engagement of CD40 and α5ß1 activates the mitogen-activated protein kinases, p38, and extracellular signal-related kinases 1/2 and synergizes in the release of inflammatory mediators MMP-2 and -9, suggesting a cross-talk between these receptors.