Platelet‐derived CXCL12 (SDF‐1α): basic mechanisms and clinical implications

Platelets are a major source of CXCL12 (stromal cell‐derived factor ‐1α, SDF‐1α) and store CXCL12 as part of their α–granule secretome. Platelet activation enhances surface expression and release of CXCL12. Platelets and megakaryocytes express CXCR4, the major receptor for CXCL12, and interaction of CXCL12 with CXCR4 regulates megakaryopoiesis and the function of circulating platelets. Platelet‐derived CXCL12 also modulates paracrine mechanisms such as chemotaxis, adhesion, proliferation and differentiation of nucleated cells, including progenitor cells. Platelet‐derived CXCL12 enhances peripheral recruitment of progenitor cells to the sites of vascular and tissue injury both in vitro and in vivo and thereby promotes repair mechanisms. CXCL12 expression on platelets is elevated in patients with acute myocardial infarction, correlates with the number of circulating progenitor cells, is associated with preservation of myocardial function and is an independent predictor of clinical outcome. Administration of recombinant CXCL12 reduces infarct size following transient ischemia in mice. The present review summarizes the role of platelet‐derived CXCL12 in cardiovascular biology and its diagnostic and therapeutic implications.     

The endocannabinoid 2‐arachidonoylglycerol activates human platelets through non‐CB1/CB2 receptors

Summary. Background: The endocannabinoid 2‐arachidonoylglycerol (2‐AG) is an endogenous lipid that acts through the activation of G‐protein‐coupled cannabinoid receptors and plays essential roles in many physiological contexts. In the cardiovascular system 2‐AG is generated by both activated endothelial cells and platelets, and participates in the regulation of inflammation and thrombosis. Although human platelets actively metabolize endocannabinoids, 2‐AG also binds to platelet surface and leads to cell activation. Objective: To investigate the biological consequence of 2‐AG interactions with human platelets and to clarify the role of cannabinoid receptors. Methods: Gel‐filtered platelets were stimulated with 2‐AG in the presence or absence of various inhibitors. Platelet aggregation and secretion were measured in a lumiaggregometer. Calcium ion movements were measured in FURA‐2 loaded platelets. Thromboxane A₂ (TxA₂) generation was evaluated as Thromboxane B₂ accumulation with a commercial EIA assay. Results: 2‐AG induced platelet shape change, aggregation and secretion with a dose‐dependent mechanism that required engagement of platelet TxA₂ receptors. 2‐AG caused also cytosolic calcium increase; however, it was totally dependent on availability of TxA₂. Indeed 2‐AG was able to induce a robust generation of TxA₂ through the cyclooxygenase pathway. Treatment of platelets with inhibitors of monoacylglycerol lipase and fatty acid amide hydrolase did not affect the activation induced by 2‐AG. Moreover, neither CB₁ and CB₂ proteins nor CB₁/CB₂ mRNAs were detected in platelets. Conclusions: 2‐AG can be considered a new physiologic platelet agonist able to induce full platelet activation and aggregation with a non‐CB₁/CB₂ receptor‐mediated mechanism.     

Platelet‐derived transforming growth factor‐β1 promotes keratinocyte proliferation in cutaneous wound healing

Platelets are a recognised potent source of transforming growth factor‐β1 (TGFβ1), a cytokine known to promote wound healing and regeneration by stimulating dermal fibroblast proliferation and extracellular matrix deposition. Platelet lysate has been advocated as a novel personalised therapeutic to treat persistent wounds, although the precise platelet‐derived growth factors responsible for these beneficial effects have not been fully elucidated. The aim of this study was to investigate the specific role of platelet‐derived TGFβ1 in cutaneous wound healing. Using a transgenic mouse with a targeted deletion of TGFβ1 in megakaryocytes and platelets (TGFβ1^fl/fl.PF4‐Cre), we show for the first time that platelet‐derived TGFβ1 contributes to epidermal and dermal thickening and cellular turnover after excisional skin wounding. In vitro studies demonstrate that human dermal fibroblasts stimulated with platelet lysate containing high levels of platelet‐derived TGFβ1 did not exhibit enhanced collagen deposition or proliferation, suggesting that platelet‐derived TGFβ1 is not a key promoter of these wound healing processes. Interestingly, human keratinocytes displayed enhanced TGFβ1‐driven proliferation in response to platelet lysate, reminiscent of our in vivo findings. In summary, our novel findings define and emphasise an important role of platelet‐derived TGFβ1 in epidermal remodelling and regeneration processes during cutaneous wound healing.     

TC21/RRas2 regulates glycoprotein VI–FcRγ‐mediated platelet activation and thrombus stability

Essentials

• RAS proteins are expressed in platelets but their functions are largely uncharacterized.
• TC21/RRas2 is required for glycoprotein VI‐induced platelet responses and for thrombus stability in vivo.
• TC21 regulates platelet aggregation by control of α_IIbβ₃ integrin activation, via crosstalk with Rap1b.
• This is the first indication of functional importance of a proto‐oncogenic RAS protein in platelets.

Summary

Background

Many RAS family small GTPases are expressed in platelets, including RAC, RHOA, RAP, and HRAS/NRAS/RRAS1, but most of their signaling and cellular functions remain poorly understood. Like RRAS1, TC21/RRAS2 reverses HRAS‐induced suppression of integrin activation in CHO cells. However, a role for TC21 in platelets has not been explored.

Objectives

To determine TC21 expression in platelets, TC21 activation in response to platelet agonists, and roles of TC21 in platelet function in in vitro and in vivo thrombosis.

Results

We demonstrate that TC21 is expressed in human and murine platelets, and is activated in response to agonists for the glycoprotein (GP) VI–FcRγ immunoreceptor tyrosine‐based activation motif (ITAM)‐containing collagen receptor, in an Src‐dependent manner. GPVI‐induced platelet aggregation, integrin α_IIbβ₃ activation, and α‐granule and dense granule secretion, as well as phosphorylation of Syk, phospholipase Cγ2, AKT, and extracellular signal‐regulated kinase, were inhibited in TC21‐deficient platelets ex vivo. In contrast, these responses were normal in TC21‐deficient platelets following stimulation with P2Y, protease‐activated receptor 4 and C‐type lectin receptor 2 receptor agonists, indicating that the function of TC21 in platelets is GPVI–FcRγ‐ITAM‐specific. TC21 was required for GPVI‐induced activation of Rap1b. TC21‐deficient mice did not show a significant delay in injury‐induced thrombosis as compared with wild‐type controls; however, thrombi were unstable. Hemostatic responses showed similar effects.

Conclusions

TC21 is essential for GPVI–FcRγ‐mediated platelet activation and for thrombus stability in vivo via control of Rap1b and integrins.

     

Flavin monooxygenase 3, the host hepatic enzyme in the metaorganismal trimethylamine N‐oxide‐generating pathway,modulates platelet responsiveness and thrombosis risk

Essentials

• Microbe‐dependent production of trimethylamine N‐oxide (TMAO) contributes to thrombosis risk.
• The impact of host flavin monooxygenase 3 (FMO3) modulation on platelet function is unknown.
• Genetic manipulation of FMO3 in mice alters systemic TMAO levels and thrombosis potential.
• Genetic manipulation of FMO3 is associated with alteration of gut microbial community structure.

Summary

Background

Gut microbes play a critical role in the production of trimethylamine N‐oxide (TMAO), an atherogenic metabolite that impacts platelet responsiveness and thrombosis potential. Involving both microbe and host enzymatic machinery, TMAO generation utilizes a metaorganismal pathway, beginning with ingestion of trimethylamine (TMA)‐containing dietary nutrients such as choline, phosphatidylcholine and carnitine, which are abundant in a Western diet. Gut microbial TMA lyases use these nutrients as substrates to produce TMA, which upon delivery to the liver via the portal circulation, is converted into TMAO by host hepatic flavin monooxygenases (FMOs). Gut microbial production of TMA is rate limiting in the metaorganismal TMAO pathway because hepatic FMO activity is typically in excess.

Objectives

FMO3 is the major FMO responsible for host generation of TMAO; however, a role for FMO3 in altering platelet responsiveness and thrombosis potential in vivo has not yet been explored.

Methods

The impact of FMO3 suppression (antisense oligonucleotide‐targeting) and overexpression (as transgene) on plasma TMAO levels, platelet responsiveness and thrombosis potential was examined using a murine FeCl₃‐induced carotid artery injury model. Cecal microbial composition was examined using 16S analyses.

Results

Modulation of FMO3 directly impacts systemic TMAO levels, platelet responsiveness and rate of thrombus formation in vivo. Microbial composition analyses reveal taxa whose proportions are associated with both plasma TMAO levels and in vivo thrombosis potential.

Conclusions

The present studies demonstrate that host hepatic FMO3, the terminal step in the metaorganismal TMAO pathway, participates in diet‐dependent and gut microbiota‐dependent changes in both platelet responsiveness and thrombosis potential in vivo .

     

Magnetic labeling of pancreatic β‐cells modulates the glucose‐ and insulin‐induced phosphorylation of ERK1/2 and AKT

This study was undertaken to investigate the effect of a magnetic resonance imaging (MRI) contrast agent, superparamagnetic iron oxide nanoparticle (SPIO), on signal transduction by glucose and insulin in pancreatic β‐cells. INS‐1 cells were labeled in culture medium containing clinically approved SPIO for 24 h. Labeled and unlabeled cells were stimulated with glucose (25 mM) or insulin (0.1–1 µM) for 12 h. The phosphorylation of extracellular signal‐regulated kinase1/2 (ERK1/2) and protein kinase B (AKT) and intracellular insulin protein levels were assessed by Western blotting. After labeling with increasing amounts of SPIO, cytotoxicity was not observed, yet the intracellular iron concentration increased in a dose‐dependent manner. SPIO labeling (200 µg Fe ml^?1) induced a significant increase in ERK1/2 and AKT phosphorylation (labeled vs unlabeled, p < 0.05), but significantly reduced the glucose‐stimulated phosphorylation of ERK1/2 and AKT and insulin‐stimulated phosphorylation of AKT (labeled vs unlabeled, p < 0.05). The level of intracellular insulin protein was found to be lower in labeled cells than unlabeled cells (labeled vs unlabeled, p < 0.05). This study demonstrates that SPIO labeling alters some fundamental functional variables, at least in INS‐1 cells, through modulation of the glucose‐ or insulin‐induced activation of ERK1/2 and AKT, which leads to insulin biosynthesis. Copyright © 2012 John Wiley & Sons, Ltd.