Phosphatidylserine: A comprehensive overview of synthesis, metabolism, and nutrition.

Phosphatidylserine (PtdS) is classified as a glycerophospholipid and a primary anionic phospholipid and is particularly abundant in the inner leaflet of the plasma membrane in neural tissues. It is synthesized from phosphatidylcholine or phosphatidylethanolamine by exchanging the base head group with serine, and this reaction is catalyzed by PtdS synthase-1 and PtdS synthase-2 located in the endoplasmic reticulum. PtdS exposure on the outside surface of the cell is essential for eliminating apoptotic cells and initiating the blood clotting cascade. It is also a precursor of phosphatidylethanolamine, produced by PtdS decarboxylase in bacteria, yeast, and mammalian cells. Furthermore, PtdS acts as a cofactor for several necessary enzymes that participate in signaling pathways. Beyond these functions, several studies indicate that PtdS plays a role in various cerebral functions, including activating membrane signaling pathways, neuroinflammation, neurotransmission, and synaptic refinement associated with the central nervous system (CNS). This review discusses the occurrence of PtdS in nature and biosynthesis via enzymes and genes in plants, yeast, prokaryotes, mammalian cells, and the brain, and enzymatic synthesis through phospholipase D (PLD). Furthermore, we discuss metabolism, its role in the CNS, the fortification of foods, and supplementation for improving some memory functions, the results of which remain unclear. PtdS can be a potentially beneficial addition to foods for kids, seniors, athletes, and others, especially with the rising consumer trend favoring functional foods over conventional pills and capsules. Clinical studies have shown that PtdS is safe and well tolerated by patients.

Whole-Cell Display of Phospholipase D in Escherichia coli for High-Efficiency Extracellular Phosphatidylserine Production.

Phospholipids are widely utilized in various industries, including food, medicine, and cosmetics, due to their unique chemical properties and healthcare benefits. Phospholipase D (PLD) plays a crucial role in the biotransformation of phospholipids. Here, we have constructed a super-folder green fluorescent protein (sfGFP)-based phospholipase D (PLD) expression and surface-display system in Escherichia coli, enabling the surface display of sfGFP-PLDr34 on the bacteria. The displayed sfGFP-PLDr34 showed maximum enzymatic activity at pH 5.0 and 45 °C. The optimum Ca2+ concentrations for the transphosphatidylation activity and hydrolysis activity are 100 mM and 10 mM, respectively. The use of displayed sfGFP-PLDr34 for the conversion of phosphatidylcholine (PC) and L-serine to phosphatidylserine (PS) showed that nearly all the PC was converted into PS at the optimum conditions. The displayed enzyme can be reused for up to three rounds while still producing detectable levels of PS. Thus, Escherichia coli/sfGFP-PLD shows potential for the feasible industrial-scale production of PS. Moreover, this system is particularly valuable for quickly screening higher-activity PLDs. The fluorescence of sfGFP can indicate the expression level of the fused PLD and changes that occur during reuse.

Phosphatidylserine synthesis controls oncogenic B cell receptor signaling in B cell lymphoma.

Cancer cells harness lipid metabolism to promote their own survival. We screened 47 cancer cell lines for survival dependency on phosphatidylserine (PS) synthesis using a PS synthase 1 (PTDSS1) inhibitor and found that B cell lymphoma is highly dependent on PS. Inhibition of PTDSS1 in B cell lymphoma cells caused a reduction of PS and phosphatidylethanolamine levels and an increase of phosphoinositide levels. The resulting imbalance of the membrane phospholipidome lowered the activation threshold for B cell receptor (BCR), a B cell-specific survival mechanism. BCR hyperactivation led to aberrant elevation of downstream Ca2+ signaling and subsequent apoptotic cell death. In a mouse xenograft model, PTDSS1 inhibition efficiently suppressed tumor growth and prolonged survival. Our findings suggest that PS synthesis may be a critical vulnerability of malignant B cell lymphomas that can be targeted pharmacologically.

Oncogenic KRAS is dependent upon an EFR3A-PI4KA signaling axis for potent tumorigenic activity.

The HRAS, NRAS, and KRAS genes are collectively mutated in a fifth of all human cancers. These mutations render RAS GTP-bound and active, constitutively binding effector proteins to promote signaling conducive to tumorigenic growth. To further elucidate how RAS oncoproteins signal, we mined RAS interactomes for potential vulnerabilities. Here we identify EFR3A, an adapter protein for the phosphatidylinositol kinase PI4KA, to preferentially bind oncogenic KRAS. Disrupting EFR3A or PI4KA reduces phosphatidylinositol-4-phosphate, phosphatidylserine, and KRAS levels at the plasma membrane, as well as oncogenic signaling and tumorigenesis, phenotypes rescued by tethering PI4KA to the plasma membrane. Finally, we show that a selective PI4KA inhibitor augments the antineoplastic activity of the KRASG12C inhibitor sotorasib, suggesting a clinical path to exploit this pathway. In sum, we have discovered a distinct KRAS signaling axis with actionable therapeutic potential for the treatment of KRAS-mutant cancers.

RRx-001 Increases Erythrocyte Preferential Adhesion to the Tumor Vasculature.

Red blood cells (RBCs) serve a variety of functions beyond mere oxygen transport both in health and pathology. Notably, RRx-001, a minimally toxic pleiotropic anticancer agent with macrophage activating and vascular normalization properties currently in Phase III trials, induces modification to RBCs which could promote vascular adhesion similar to sickle cells. This study assessed whether RBCs exposed to RRx-001 adhere to the tumor microvasculature and whether this adhesion alters tumor viability. We next investigated the biomechanics of RBC adhesion in the context of local inflammatory cytokines after treatment with RRx-001 as a potential mechanism for preferential tumor aggregation. Human HEP-G2 and HT-29 tumor cells were subcutaneously implanted into nu/nu mice and were infused with RRx-001-treated and Technetium-99m (99mTc)-labeled blood. RBC adhesion was quantified in an in vitro human umbilical vein endothelial cell (HUVEC) assay under both normoxic and hypoxic conditions with administration of either lipopolysaccharide (LPS) or Tumor necrosis alpha (TNFα) to mimic the known inflammation in the tumor microenvironment. One hour following administration of 99mTc labeled RBCs treated with 10 mg/kg RRx-001, we observed an approximate 2.0-fold and 1.5-fold increase in 99mTc-labeled RBCs compared to vehicle control in HEPG2 and HT-29 tumor models, respectively. Furthermore, we observed an approximate 40% and 36% decrease in HEP-G2 and HT-29 tumor weight, respectively, following treatment with RRx-001. To quantify RBC adhesive potential, we determined τ50, or the shear stress required for 50% disassociation of RBCs from HUVECs. After administration of TNF-α under normoxia, τ50 was determined to be 4.5 dynes/cm2 (95% CI: 4.3-4.7 dynes/cm2) for RBCs treated with 10 µM RRx-001, which was significantly different (p < 0.05) from τ50 in the absence of treatment. Under hypoxic conditions, the difference of τ50 with (4.8 dynes/cm2; 95% CI: 4.6-5.1 dynes/cm2) and without (2.6 dynes/cm2; 95% CI: 2.4-2.8 dynes/cm2) 10 µM RRx-001 treatment was exacerbated (p = 0.05). In conclusion, we demonstrated that RBCs treated with RRx-001 preferentially aggregate in HEP-G2 and HT-29 tumors, likely due to interactions between RRx-001 and cysteine residues within RBCs. Furthermore, RRx-001 treated RBCs demonstrated increased adhesive potential to endothelial cells upon introduction of TNF-α and hypoxia suggesting that RRx-001 may induce preferential adhesion in the tumor but not in other tissues with endothelial dysfunction due to conditions prevalent in older cancer patients such as heart disease or diabetic vasculopathy.

Phosphatidylserine biosynthesis pathways in lipid homeostasis: Toward resolution of the pending central issue for decades.

Enzymatic control of lipid homeostasis in the cell is a vital element in the complex organization of life. Phosphatidylserine (PS) is an essential anionic phospholipid of cell membranes, and conducts numerous roles for their structural and functional integrity. In mammalian cells, two distinct enzymes phosphatidylserine synthases-1 (PSS1) and -2 (PSS2) in the mitochondria-associated membrane (MAM) in the ER perform de novo synthesis of PS. It is based on base-exchange reactions of the preexisting dominant phospholipids phosphatidylcholine (PC) and phosphatidylethanolamine (PE). While PSS2 specifically catalyzes the reaction "PE â†’ PS," whether or not PSS1 is responsible for the same reaction along with the reaction "PC â†’ PS" remains unsettled despite its fundamental impact on the major stoichiometry. We propose here that a key but the only report that appeared to have put scientists on hold for decades in answering to this issue may be viewed consistently with other available research reports; PSS1 utilizes the two dominant phospholipid classes at a similar intrinsic rate. In this review, we discuss the issue in view of the current information for the enzyme machineries, membrane structure and dynamics, intracellular network of lipid transport, and PS synthesis in health and disease. Resolution of the pending issue is thus critical in advancing our understanding of roles of the essential anionic lipid in biology, health, and disease.

Efficient secretion expression of phospholipase D in Bacillus subtilis and its application in synthesis of phosphatidylserine by enzyme immobilization.

Transphosphatidylation catalyzed by phospholipase D has gained increasing attention for producing phosphatidylserine (PS), which can be used in functional food and medicine. In this study, we investigated the effects of six signal peptides on the secretion of PLD (PLDsa) from Streptomyces antibioticus TCCC 21059 in the food-grade GRAS bacterium Bacillus subtilis. It indicated that the optimal signal peptide DacB with an Ala-X-Ala sequence motif at the C-terminus showed the highest secretory expression ability, resulting in increased production of 2.84 U/mL PLDsa. Then PLDsa was immobilized on the epoxy-based carriers, and one of these carriers allowed PLDsa loading of up to 2.7 mg/g. The immobilized PLDsa was more stable over a wide range of pH value (4.5-7.5) and temperature (16 °C-60 °C) than free PLDsa. Subsequently, the synthesis of PS from soybean phosphatidylcholine (PC) was carried out in purely aqueous solution using immobilized PLDsa, leading to a high yield of 65%. The immobilized PLDsa catalyst maintained a relative PS production of 60% after 5 recycles. Notably, the use of toxic solvent was completely eliminated in the whole process, which would be more profitable for the application of PS.

Analysis of phospholipid synthesis in mitochondria.

Mitochondria and their associated membranes actively participate in biosynthesis, trafficking, and degradation of cellular phospholipids. Two crucial lipid biosynthetic activities of mitochondria include (i) the decarboxylation of phosphatidylserine to phosphatidylethanolamine and (ii) the de novo synthesis of cardiolipin. Here we describe protocols to measure these two activities, applying isotope-labeled or exogenous substrates in combination with thin-layer chromatography or mass spectrometry.

ES5 is involved in the regulation of phosphatidylserine synthesis and impacts on early senescence in rice (Oryza sativa L.).

Leaf senescence, which affects plant growth and yield in rice, is an ideal target for crop improvement and remarkable advances have been made to identify the mechanism underlying this process. We have characterized an early senile mutant es5 (early leaf senescence 5) in rice exhibiting leaf yellowing phenotype after the 4-leaf stage. This phenotype was confirmed by the higher accumulation of reactive oxygen species (ROS) and malondialdehyde (MDA), the disintegration of chloroplasts, reduction in chlorophyll content and photosynthetic rate and up-regulation of senescence-associated genes (SAGs) like Osh36, OsI57, and OsI85. Positional cloning revealed that the es5 phenotype is the result of one base substitution in ES5, encoding phosphatidylserine synthase (PSS) family protein, which is involved in the base-exchange type reaction to synthesize the minor membrane phospholipid phosphatidylserine. Functional complementation of ES5 in the es5 plants completely restored the wild-type phenotype. Ultra-high-performance liquid chromatography (UHPLC) analysis showed that es5 plants had increased levels of phosphatidylserine (PS) and decreased level of phosphatidylcholine (PC). These results provide evidence about the role of PS in rice leaf senescence.

Combining Cell Surface Display and DNA-Shuffling Technology for Directed Evolution of Streptomyces Phospholipase D and Synthesis of Phosphatidylserine.

Phospholipids have been widely used in food, medicine, cosmetics, and other fields because of their unique chemical structure and healthcare functions. Phospholipase D (PLD) is a key biocatalyst for the biotransformation of phospholipids. Here, an autodisplay expression system was constructed for rapid screening of mutants, and PLD variants were recombined using DNA shuffling technology and three beneficial mutations were obtained. The results of enzymatic performance and sequence information comparison indicated that C-terminal amino acids exerted a greater impact on the correct folding of PLDs, and N-terminal amino acids are more important for catalytic reaction. The best-performing recombinant enzyme in transphosphatidylation reactions was Recom-34, with a phosphatidylserine content accounting for 80.3% of total phospholipids and a 3.24-fold increased conversion rate compared to the parent enzyme. This study demonstrates great significance for screening ideal biocatalysts, facilitating soluble expression of inclusion body proteins, and identifying key amino acids.

Molecular and functional evidence of phosphatidylserine synthase in Vibrio parahaemolyticus.

Phosphatidylserine synthase (Pss) catalyzes phosphatidylserine synthesis, which is critical to synthesizing the component of cell membrane. However, few putative pss genes of bacteria have been studied. In this study, it was found that Vibrio parahaemolyticus, a common foodborne pathogen that causes human gastroenteritis, has a type I Pss with two HKD motifs and is a phospholipase D superfamily member. The transcriptional start site of pss was mapped through sequencing and was identified at -37 nucleotides upstream of the start codon. Pss mRNA was found to be expressed mainly during the exponential phase. In addition, the promoter was identified using a lux reporter assay and gel shift assay with an RNA polymerase. To analyze the catalytic activity, a soluble form of His6 -tagged recombinant Pss was overexpressed and purified from Escherichia coli. Using matrix-assisted laser desorption ionization-time of flight mass spectrometry, it was found that Pss can catalyze cytidine diphosphate diacylglycerol and L-serine to form phosphatidylserine. Since Pss is conserved in vibrios, the current study can promote understanding the biosynthesis of phospholipid in Vibrio bacteria that might cause vibriosis. This is the first report of molecular characterization of the pss gene and identification of Pss enzyme activity in V. parahaemolyticus using matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

Phosphatidylserine synthesis is essential for viability of the human fungal pathogen Cryptococcus neoformans.

Phospholipids are an integral part of the cellular membrane structure and can be produced by a de novo biosynthetic pathway and, alternatively, by the Kennedy pathway. Studies in several yeast species have shown that the phospholipid phosphatidylserine (PS) is synthesized from CDP-diacylglycerol and serine, a route that is different from its synthesis in mammalian cells, involving a base-exchange reaction from preexisting phospholipids. Fungal-specific PS synthesis has been shown to play an important role in fungal virulence and has been proposed as an attractive drug target. However, PS synthase, which catalyzes this reaction, has not been studied in the human fungal pathogen Cryptococcus neoformans Here, we identified and characterized the PS synthase homolog (Cn Cho1) in this fungus. Heterologous expression of Cn CHO1 in a Saccharomyces cerevisiae cho1Δ mutant rescued the mutant's growth defect in the absence of ethanolamine supplementation. Moreover, an Sc cho1Δ mutant expressing Cn CHO1 had PS synthase activity, confirming that the Cn CHO1 encodes PS synthase. We also found that PS synthase in C. neoformans is localized to the endoplasmic reticulum and that it is essential for mitochondrial function and cell viability. Of note, its deficiency could not be complemented by ethanolamine or choline supplementation for the synthesis of phosphatidylethanolamine (PE) or phosphatidylcholine (PC) via the Kennedy pathway. These findings improve our understanding of phospholipid synthesis in a pathogenic fungus and indicate that PS synthase may be a useful target for antifungal drugs.

Febrile Temperature Elevates the Expression of Phosphatidylserine on Plasmodium falciparum (FCR3CSA) Infected Red Blood Cell Surface Leading to Increased Cytoadhesion.

During the asexual intra-erythrocytic cycle, Plasmodium (P.) falciparum exports parasitic proteins to the surface of infected red blood cells (iRBCs) facilitating its cytoadhesion to various endothelial host receptors. This adhesive behavior is a critical contributor towards disease manifestation. However, little is known about the influence of recurring elevated temperature - a common symptom of the malaria infection - on the adhesive properties of iRBCs to endothelial receptors. To address this, we performed dual-micropipette step-pressure technique between P. falciparum (strain FCR3CSA) iRBCs and Chinese Hamster Ovary cells expressing Chondroitin sulfate A (CHO-CSA) after transient iRBCs incubation at febrile temperatures which revealed increase in adhesion parameters. Furthermore, flow cytometry analysis revealed an increase in phosphatidylserine (PS) expression on the iRBC surface following exposure to febrile temperature. The adhesion between iRBCs and CHO-CSA cells was remarkably reduced in presence of soluble Annexin V, indicating the mediation of PS on the adhesion events. Our results suggest that elevated PS recruitment on iRBC under thermally stressed conditions contributes to the increased adhesive behavior of iRBCs CSA-binding phenotype to CHO-CSA.

Historical perspective: phosphatidylserine and phosphatidylethanolamine from the 1800s to the present.

This article provides a historical account of the discovery, chemistry, and biochemistry of two ubiquitous phosphoglycerolipids, phosphatidylserine (PS) and phosphatidylethanolamine (PE), including the ether lipids. In addition, the article describes the biosynthetic pathways for these phospholipids and how these pathways were elucidated. Several unique functions of PS and PE in mammalian cells in addition to their ability to define physical properties of membranes are discussed. For example, the translocation of PS from the inner to the outer leaflet of the plasma membrane of cells occurs during apoptosis and during some other specific physiological processes, and this translocation is responsible for profound life-or-death events. Moreover, mitochondrial function is severely impaired when the PE content of mitochondria is reduced below a threshold level. The discovery and implications of the existence of membrane contact sites between the endoplasmic reticulum and mitochondria and their relevance for PS and PE metabolism, as well as for mitochondrial function, are also discussed. Many of the recent advances in these fields are due to the use of isotope labeling for tracing biochemical pathways. In addition, techniques for disruption of specific genes in mice are now widely used and have provided major breakthroughs in understanding the roles and metabolism of PS and PE in vivo.

Mining of a phospholipase D and its application in enzymatic preparation of phosphatidylserine.

Phosphatidylserine (PS) is useful as the additive in industries for memory improvement, mood enhancement and drug delivery. Conventionally, PS was extracted from soybeans, vegetable oils, egg yolk, and biomass; however, their low availability and high extraction cost were limiting factors. Phospholipase D (PLD) is a promising tool for enzymatic synthesis of PS due to its transphosphatidylation activity. In this contribution, a new and uncharacterized PLD was first obtained from GenBank database via genome mining strategy. The open reading frame consisted of 1614 bp and potentially encoded a protein of 538-amino-acid with a theoretical molecular mass of 60 kDa. The gene was successfully cloned and expressed in Escherichia coli. Its enzymatic properties were experimentally characterized. The temperature and pH optima of PLD were determined to be 60°C and 7.5, respectively. Its hydrolytic activity was improved by addition of Ca2+ at 5 mM as compared with the control. The enzyme displayed suitable transphosphatidylation activity and PS could be synthesized with L-serine and soybean lecithin as substrates under the catalysis of PLD. This PLD enzyme might be a potential candidate for industrial applications in PS production. To the best of our knowledge, this is the first report on genome mining of PLDs from GenBank database.

Phosphatidylserine synthesis at membrane contact sites promotes its transport out of the ER.

Close contacts between organelles, often called membrane contact sites (MCSs), are regions where lipids are exchanged between organelles. Here, we identify a novel mechanism by which cells promote phospholipid exchange at MCSs. Previous studies have shown that phosphatidylserine (PS) synthase activity is highly enriched in portions of the endoplasmic reticulum (ER) in contact with mitochondria. The objective of this study was to determine whether this enrichment promotes PS transport out of the ER. We found that PS transport to mitochondria was more efficient when PS synthase was fused to a protein in the ER at ER-mitochondria contacts than when it was fused to a protein in all portions of the ER. Inefficient PS transport to mitochondria was corrected by increasing tethering between these organelles. PS transport to endosomes was similarly enhanced by PS production in regions of the ER in contact with endosomes. Together, these findings indicate that PS production at MCSs promotes PS transport out of the ER and suggest that phospholipid production at MCSs may be a general mechanism of channeling lipids to specific cellular compartments.

Properties of donated red blood cell components from patients with hereditary hemochromatosis.

BACKGROUND: Red blood cells (RBCs) contain large amounts of iron, and periodic therapeutic phlebotomy is thus the main treatment for hereditary hemochromatosis (HH). However, the donation of therapeutic phlebotomy products from asymptomatic patients for transfusion purposes remains controversial. In this study, we compared the quality of RBCs obtained from HH patients with those of non-HH RBCs, within the allowed 42-day storage period. STUDY DESIGN AND METHODS: RBCs were obtained from HH patient donors and random regular blood donors by whole blood collection. RBCs were stored for up to 42 days, according to national regulations and standard blood bank conditions in France. The following variables were assessed: hematologic and biochemical results, RBC membrane and soluble inflammatory markers, and the proinflammatory potential of HH RBC supernatant toward endothelial cells in an in vitro model. RESULTS: There were no major differences between the two groups in terms of biophysical, biochemical, or soluble immunomodulatory factors. However, we observed small but significant differences in changes in RBC membrane proteins during storage, including increased phosphatidylserine expression and decreased hemolysis in HH compared with normal RBCs. However, there were no differences in terms of bioactivity of soluble immunomodulatory factors in the RBC supernatant during storage between HH and control donors, as determined by their effects on endothelial cells in vitro. CONCLUSIONS: These in vitro studies suggest that RBCs from HH patients appear, while exhibiting subtle differences, to be suitable for transfusion purposes according to currently accepted criteria.

Aqueous-Solid System for Highly Efficient and Environmentally Friendly Transphosphatidylation Catalyzed by Phospholipase D To Produce Phosphatidylserine.

The purely aqueous system of phospholipase D (PLD)-mediated transphosphatidylation using pre-existing carriers for the adsorption of phosphatidylcholine (PC) to act as an "artificial interface" was introduced to replace the liquid-liquid system. Toxic organic solvents are avoided during the reaction, and the free enzyme can be simply reused by centrifugation. Special attention has been paid to the effect of the pore diameter and surface area of silica gel 60H covered with PC molecules on the yield of phosphatidylserine (PS). Results indicated that the highest PS yield of 99.5% was achieved. Moreover, 73.6% of the yield of PS was obtained after being used for six batches. This is the first description of the remarkably high reusability of free enzymes for enzymatic synthesis of PS as well. The excellent results make the aqueous-solid system more promising candidates for the industrial production of PS.

n-3 Polyunsaturated Fatty Acids Reduce Neonatal Hypoxic/Ischemic Brain Injury by Promoting Phosphatidylserine Formation and Akt Signaling.

BACKGROUND AND PURPOSE: Omega-3 polyunsaturated fatty acids (n-3 PUFAs) attenuate neonatal hypoxic/ischemic (H/I) brain damage, but the underlying mechanisms are not fully understood. This study tested the hypothesis that n-3 PUFAs enhance Akt-dependent prosurvival signaling by promoting the biosynthesis of phosphatidylserine in neuronal cell membranes. METHODS: Dietary n-3 PUFA supplementation was initiated on the second day of pregnancy in dams. H/I was induced in 7-day-old rat pups by ipsilateral common carotid artery occlusion followed by hypoxia (8% oxygen for 2.5 hours). Neurological outcomes, brain tissue loss, cell death, and the activation of signaling events were assessed after H/I. The effects of n-3 PUFAs (docosahexaenoic acid and eicosapentaenoic acid) on oxygen-glucose deprivation-induced cell death and the underlying mechanism of protection were also examined in primary cortical neuron cultures. RESULTS: n-3 PUFAs reduced brain tissue loss at 7 days after H/I and improved neurological outcomes, whereas inhibition of PI3K/Akt signaling by LY294002 partially abrogated this neuroprotective effect. Docosahexaenoic acid/eicosapentaenoic acid also prevented ischemic neuronal death through the Akt prosurvival pathway in vitro. Furthermore, docosahexaenoic acid/eicosapentaenoic acid increased the production of phosphatidylserine, the major membrane-bound phospholipids, after ischemia both in vitro and in vivo. A reduction in membrane phosphatidylserine by shRNA-mediated knockdown of phosphatidylserine synthetase-1 attenuated Akt activation and neuronal survival after docosahexaenoic acid/eicosapentaenoic acid treatment in the oxygen-glucose deprivation model. CONCLUSIONS: n-3 PUFAs robustly protect against H/I-induced brain damage in neonates by activating Akt prosurvival pathway in compromised neurons. In addition, n-3 PUFAs promote the formation of membrane phosphatidylserine, thereby promoting Akt activity and improving cellular survival.

Collagen can selectively trigger a platelet secretory phenotype via glycoprotein VI.

Platelets are not only central actors of hemostasis and thrombosis but also of other processes including inflammation, angiogenesis, and tissue regeneration. Accumulating evidence indicates that these "non classical" functions of platelets do not necessarily rely on their well-known ability to form thrombi upon activation. This suggests the existence of non-thrombotic alternative states of platelets activation. We investigated this possibility through dose-response analysis of thrombin- and collagen-induced changes in platelet phenotype, with regards to morphological and functional markers of platelet activation including shape change, aggregation, P-selectin and phosphatidylserine surface expression, integrin activation, and release of soluble factors. We show that collagen at low dose (0.25 µg/mL) selectively triggers a platelet secretory phenotype characterized by the release of dense- and alpha granule-derived soluble factors without causing any of the other major platelet changes that usually accompany thrombus formation. Using a blocking antibody to glycoprotein VI (GPVI), we further show that this response is mediated by GPVI. Taken together, our results show that platelet activation goes beyond the mechanisms leading to platelet aggregation and also includes alternative platelet phenotypes that might contribute to their thrombus-independent functions.