Stereochemistry- and concentration-dependent effects of phosphatidylserine enrichment on platelet function.

Platelets are small (1-2µm in diameter), circulating anuclear cell fragments with important roles in hemostasis and thrombosis that provide an excellent platform for studying the role of membrane components in cellular communication. Platelets use several forms of communication including exocytosis of three distinct granule populations, formation of bioactive lipid mediators, and shape change (allowing for adhesion). This work explores the role of stereochemistry and concentration of exogenous phosphatidylserine (PS) on platelet exocytosis and adhesion. PS, most commonly found in the phosphatidyl-l-serine (l-PS) form, is exposed on the outer leaflet of the cell membrane after the platelet is activated. Knowledge about the impact of exogenous phosphatidylserine on cell-to-cell communication is limited (particularly concentration and stereochemistry effects). This study found that platelets incubated in l-PS or phosphatidyl-d-serine (d-PS) are enriched to the same extent with their respective incubated PS. All levels of l-PS enrichment also showed an increase in platelet cholesterol, but only the 50µM d-PS incubation showed an increase in cholesterol. The uptake of d-PS induced the secretion of granules and manufactured platelet activating factor (PAF) in otherwise unstimulated platelets. The uptake of l-PS had a greater impact on platelet stimulation by decreasing both the amount of δ-granule secretion and the amount of PAF that was manufactured.

VAMP-7 links granule exocytosis to actin reorganization during platelet activation.

Platelet activation results in profound morphologic changes accompanied by release of granule contents. Recent evidence indicates that fusion of granules with the plasma membrane during activation provides auxiliary membrane to cover growing actin structures. Yet little is known about how membrane fusion is coupled with actin reorganization. Vesicle-associated membrane protein (VAMP)-7 is found on platelet vesicles and possesses an N-terminal longin domain capable of linking exocytosis to cytoskeletal remodeling. We have evaluated platelets from VAMP-7(-/-) mice to determine whether this VAMP isoform contributes to granule release and platelet spreading. VAMP-7(-/-) platelets demonstrated a partial defect in dense granule exocytosis and impaired aggregation. α Granule exocytosis from VAMP-7(-/-) platelets was diminished both in vitro and in vivo during thrombus formation. Consistent with a role of VAMP-7 in cytoskeletal remodeling, spreading on matrices was decreased in VAMP-7(-/-) platelets compared to wild-type controls. Immunoprecipitation of VAMP-7 revealed an association with VPS9-domain ankyrin repeat protein (VARP), an adaptor protein that interacts with both membrane-bound and cytoskeleton proteins and with Arp2/3. VAMP-7, VARP, and Arp2/3 localized to the platelet periphery during spreading. These studies demonstrate that VAMP-7 participates in both platelet granule secretion and spreading and suggest a mechanism whereby VAMP-7 links granule exocytosis with actin reorganization.

Platelet membrane variations and their effects on δ-granule secretion kinetics and aggregation spreading among different species.

Platelet exocytosis is regulated partially by the granular/cellular membrane lipids and proteins. Some platelets contain a membrane-bound tube, called an open canalicular system (OCS), which assists in granular release events and increases the membrane surface area for greater spreading. The OCS is not found in all species, and variations in membrane composition can cause changes in platelet secretion. Since platelet studies use various animal models, it is important to understand how platelets differ in both their composition and granular release to draw conclusions among various models. The relative phospholipid composition of the platelets with (mouse, rabbit) and without (cow) an OCS was quantified using UPLC-MS/MS. Cholesterol and protein composition was measured using an Amplex Red Assay and BCA Assay. TEM and dark field platelet images were gathered and analyzed with Image J. Granular release was monitored with single cell carbon fiber microelectrode amperometry. Cow platelets contained greater amounts of cholesterol and sphingomyelin. In addition, they yield greater serotonin release and longer δ granule secretion times. Finally, they showed greater spreading area with a greater range of spread. Platelets containing an OCS had more similarities in their membrane composition and secretion kinetics compared to cow platelets. However, cow platelets showed greater fusion pore stability which could be due to extra sphingomyelin and cholesterol, the primary components of lipid rafts. In addition, their greater stability may lead to many granules assisting in spreading. This study highlights fundamental membrane differences and their effects on platelet secretion.

Analytical characterization of the role of phospholipids in platelet adhesion and secretion.

The cellular phospholipid membrane plays an important role in cell function and cell-cell communication, but its biocomplexity and dynamic nature presents a challenge for examining cellular uptake of phospholipids and the resultant effects on cell function. Platelets, small anuclear circulating cell bodies that influence a wide variety of physiological functions through their dynamic secretory and adhesion behavior, present an ideal platform for exploring the effects of exogenous phospholipids on membrane phospholipid content and cell function. In this work, a broad range of platelet functions are quantitatively assessed by leveraging a variety of analytical chemistry techniques, including ultraperformance liquid chromatography-tandem electrospray ionization mass spectrometry (UPLC-MS/MS), vasculature-mimicking microfluidic analysis, and single cell carbon-fiber microelectrode amperometry (CFMA). The relative enrichments of phosphatidylserine (PS) and phosphatidylethanolamine (PE) were characterized with UPLC-MS/MS, and the effects of the enrichment of these two phospholipids on both platelet secretory behavior and adhesion were examined. Results show that, in fact, both PS and PE influence platelet adhesion and secretion. PS was enriched dramatically and decreased platelet adhesion as well as secretion from δ-, α-, and lysosomal granules. PE enrichment was moderate and increased secretion from platelet lysosomes. These insights illuminate the critical connection between membrane phospholipid character and platelet behavior, and both the methods and results presented herein are likely translatable to other mammalian cell systems.

Dynamin-related protein-1 controls fusion pore dynamics during platelet granule exocytosis.

OBJECTIVE: Platelet granule exocytosis serves a central role in hemostasis and thrombosis. Recently, single-cell amperometry has shown that platelet membrane fusion during granule exocytosis results in the formation of a fusion pore that subsequently expands to enable the extrusion of granule contents. However, the molecular mechanisms that control platelet fusion pore expansion and collapse are not known. METHODS AND RESULTS: We identified dynamin-related protein-1 (Drp1) in platelets and found that an inhibitor of Drp1, mdivi-1, blocked exocytosis of both platelet dense and α-granules. We used single-cell amperometry to monitor serotonin release from individual dense granules and, thereby, measured the effect of Drp1 inhibition on fusion pore dynamics. Inhibition of Drp1 increased spike width and decreased prespike foot events, indicating that Drp1 influences fusion pore formation and expansion. Platelet-mediated thrombus formation in vivo after laser-induced injury of mouse cremaster arterioles was impaired after infusion of mdivi-1. CONCLUSIONS: These results demonstrate that inhibition of Drp1 disrupts platelet fusion pore dynamics and indicate that Drp1 can be targeted to control thrombus formation in vivo.

Advances in platelet granule biology.

PURPOSE OF REVIEW: This review will provide an overview of several recent advances in the field of platelet granule biology. RECENT FINDINGS: The past few years have witnessed a substantial evolution in our knowledge of platelet granules based on a number of discoveries and new experimental approaches. This article will cover recent studies in five areas. First, the vesicle trafficking pathways responsible for α-granule formation are beginning to be assembled as a result of the characterization of patients with α-granule deficiencies. Second, a revision of our understanding of which SNARE isoforms mediate platelet granule exocytosis has occurred following evaluation of patients with defects in platelet granule exocytosis and the generation of mice lacking specific SNAREs. Third, investigators have begun to establish how cargos are segregated among α-granules and determine whether or not different α-granule subpopulations exist in platelets. Fourth, an unanticipated role for α-granules in platelet spreading has been identified. Fifth, single-cell amperometry has revealed secretion kinetics with submillisecond temporal resolution enabling evaluation of the molecular control of the platelet fusion pore. SUMMARY: These new observations reveal a previously unappreciated complexity to platelet granule formation and exocytosis and challenge our earlier notions of how these granules are organized within platelets and contribute to the multitude of physiological activities in which platelets function.

Isotope-dilution UPLC-MS/MS determination of cell-secreted bioactive lipids.

Secreted bioactive lipids play critical roles in cell-to-cell communication and have been implicated in inflammatory immune responses such as anaphylaxis, vasodilation, and bronchoconstriction. Analysis of secreted bioactive lipids can be challenging due to their relatively short lifetimes and structural diversity. Herein, a method has been developed using ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) to quantify five cell-secreted, structurally and functionally diverse bioactive lipids (PGD2, LTC4, LTD4, LTE4, PAF) that play roles in inflammation. Sample analysis time is 5 min, and isotopically labeled internal standards are used for quantification. This method was applied to an immortal secretory cell line (RBL-2H3), a heterogeneous primary cell culture containing peritoneal mast cells, and murine platelets. In RBL cell supernatant samples, intrasample precisions ranged from 7.32-21.6%, averaging 17.0%, and spike recoveries in cell supernatant matrices ranged from 88.0-107%, averaging 97.0%. Calibration curves were linear from 10 ng mL(-1) to 250 ng mL(-1), and limits of detection ranged from 0.0348 ng mL(-1) to 0.803 ng mL(-1). This method was applied to the determination of lipid secretion from mast cells and platelets, demonstrating broad applicability for lipid measurement in primary culture biological systems.

CD73 Inhibits cGAS-STING and Cooperates with CD39 to Promote Pancreatic Cancer.

The ectonucleotidases CD39 and CD73 catalyze extracellular ATP to immunosuppressive adenosine, and as such, represent potential cancer targets. We investigated biological impacts of CD39 and CD73 in pancreatic ductal adenocarcinoma (PDAC) by studying clinical samples and experimental mouse tumors. Stromal CD39 and tumoral CD73 expression significantly associated with worse survival in human PDAC samples and abolished the favorable prognostic impact associated with the presence of tumor-infiltrating CD8+ T cells. In mouse transplanted KPC tumors, both CD39 and CD73 on myeloid cells, as well as CD73 on tumor cells, promoted polarization of infiltrating myeloid cells towards an M2-like phenotype, which enhanced tumor growth. CD39 on tumor-specific CD8+ T cells and pancreatic stellate cells also suppressed IFNγ production by T cells. Although therapeutic inhibition of CD39 or CD73 alone significantly delayed tumor growth in vivo, targeting of both ectonucleotidases exhibited markedly superior antitumor activity. CD73 expression on human and mouse PDAC tumor cells also protected against DNA damage induced by gemcitabine and irradiation. Accordingly, large-scale pharmacogenomic analyses of human PDAC cell lines revealed significant associations between CD73 expression and gemcitabine chemoresistance. Strikingly, increased DNA damage in CD73-deficient tumor cells associated with activation of the cGAS-STING pathway. Moreover, cGAS expression in mouse KPC tumor cells was required for antitumor activity of the CD73 inhibitor AB680 in vivo. Our study, thus, illuminates molecular mechanisms whereby CD73 and CD39 seemingly cooperate to promote PDAC progression.

SRF617 Is a Potent Inhibitor of CD39 with Immunomodulatory and Antitumor Properties.

CD39 (ENTPD1) is a key enzyme responsible for degradation of extracellular ATP and is upregulated in the tumor microenvironment (TME). Extracellular ATP accumulates in the TME from tissue damage and immunogenic cell death, potentially initiating proinflammatory responses that are reduced by the enzymatic activity of CD39. Degradation of ATP by CD39 and other ectonucleotidases (e.g., CD73) results in extracellular adenosine accumulation, constituting an important mechanism for tumor immune escape, angiogenesis induction, and metastasis. Thus, inhibiting CD39 enzymatic activity can inhibit tumor growth by converting a suppressive TME to a proinflammatory environment. SRF617 is an investigational, anti-CD39, fully human IgG4 Ab that binds to human CD39 with nanomolar affinity and potently inhibits its ATPase activity. In vitro functional assays using primary human immune cells demonstrate that inhibiting CD39 enhances T-cell proliferation, dendritic cell maturation/activation, and release of IL-1ß and IL-18 from macrophages. In vivo, SRF617 has significant single-agent antitumor activity in human cell line-derived xenograft models that express CD39. Pharmacodynamic studies demonstrate that target engagement of CD39 by SRF617 in the TME inhibits ATPase activity, inducing proinflammatory mechanistic changes in tumor-infiltrating leukocytes. Syngeneic tumor studies using human CD39 knock-in mice show that SRF617 can modulate CD39 levels on immune cells in vivo and can penetrate the TME of an orthotopic tumor, leading to increased CD8+ T-cell infiltration. Targeting CD39 is an attractive approach for treating cancer, and, as such, the properties of SRF617 make it an excellent drug development candidate.

Electroanalytical eavesdropping on single cell communication.

This article reviews measurement of single cell exocytosis with microelectrodes, covering history, basic instrumentation, cell types investigated, and fundamental insight gained.

Cholesterol effects on vesicle pools in chromaffin cells revealed by carbon-fiber microelectrode amperometry.

Cell-cell communication is often achieved via granular exocytosis, as in neurons during synaptic transmission or neuroendocrine cells during blood hormone control. Owing to its critical role in membrane properties and SNARE function, cholesterol is expected to play an important role in the highly conserved process of exocytosis. In this work, membrane cholesterol concentration is systematically varied in primary culture mouse chromaffin cells, and the change in secretion behavior of distinct vesicle pools as well as pool recovery following stimulation is measured using carbon-fiber microelectrode amperometry. Amperometric traces obtained from activation of the younger readily releasable and slowly releasable pool (RRP/SRP) vesicles at depleted cholesterol levels showed fewer sustained fusion pore features (6.1 ± 1.1% of spikes compared with 11.2 ± 1.0% for control), revealing that cholesterol content influences fusion pore formation and stability during exocytosis. Moreover, subsequent stimulation of RRP/SRP vesicles showed that cellular cholesterol level influences both the quantal recovery and kinetics of the later release events. Finally, diverging effects of cholesterol on RRP and the older reserve pool vesicle release suggest two different mechanisms for the release of these two vesicular pools.

Bioanalytical tools for single-cell study of exocytosis.

Regulated exocytosis is a fundamental biological process used to deliver chemical messengers for cell-cell communication via membrane fusion and content secretion. A plethora of cell types employ this chemical-based communication to achieve crucial functions in many biological systems. Neurons in the brain and platelets in the circulatory system are representative examples utilizing exocytosis for neurotransmission and blood clotting. Single-cell studies of regulated exocytosis in the past several decades have greatly expanded our knowledge of this critical process, from vesicle/granule transport and docking at the early stages of exocytosis to membrane fusion and to eventual chemical messenger secretion. Herein, four main approaches that have been widely used to study single-cell exocytosis will be highlighted, including total internal reflection fluorescence microscopy, capillary electrophoresis, single-cell mass spectrometry, and microelectrochemistry. These techniques are arranged in the order following the route of a vesicle/granule destined for secretion. Within each section, the basic principles and experimental strategies are reviewed and representative examples are given revealing critical spatial, temporal, and chemical information of a secretory vesicle/granule at different stages of its lifetime. Lastly, an analytical chemist's perspective on potential future developments in this exciting field is discussed.

Preclinical Antitumor Activity and Biodistribution of a Novel Anti-GCC Antibody-Drug Conjugate in Patient-derived Xenografts.

Guanylyl cyclase C (GCC) is a unique therapeutic target with expression restricted to the apical side of epithelial cell tight junctions thought to be only accessible by intravenously administered agents on malignant tissues where GCC expression is aberrant. In this study, we sought to evaluate the therapeutic potential of a second-generation investigational antibody-dug conjugate (ADC), TAK-164, comprised of a human anti-GCC mAb conjugated via a peptide linker to the highly cytotoxic DNA alkylator, DGN549. The in vitro binding, payload release, and in vitro activity of TAK-164 was characterized motivating in vivo evaluation. The efficacy of TAK-164 and the relationship to exposure, pharmacodynamic marker activation, and biodistribution was evaluated in xenograft models and primary human tumor xenograft (PHTX) models. We demonstrate TAK-164 selectively binds to, is internalized by, and has potent cytotoxic effects against GCC-expressing cells in vitro A single intravenous administration of TAK-164 (0.76 mg/kg) resulted in significant growth rate inhibition in PHTX models of metastatic colorectal cancer. Furthermore, imaging studies characterized TAK-164 uptake and activity and showed positive relationships between GCC expression and tumor uptake which correlated with antitumor activity. Collectively, our data suggest that TAK-164 is highly active in multiple GCC-positive tumors including those refractory to TAK-264, a GCC-targeted auristatin ADC. A strong relationship between uptake of 89Zr-labeled TAK-164, levels of GCC expression and, most notably, response to TAK-164 therapy in GCC-expressing xenografts and PHTX models. These data supported the clinical development of TAK-164 as part of a first-in-human clinical trial (NCT03449030).

Fluorous polymeric membranes for ionophore-based ion-selective potentiometry: how inert is Teflon AF?

Fluorous media are the least polar and polarizable condensed phases known. Their use as membrane materials considerably increases the selectivity and robustness of ion-selective electrodes (ISEs). In this research, a fluorous amorphous perfluoropolymer was used for the first time as a matrix for an ISE membrane. Electrodes for pH measurements with membranes composed of poly[4,5-difluoro-2,2-bis(trifluoromethyl)-1,3-dioxole]-co-poly(tetrafluoroethylene) (87% dioxole monomer content; known as Teflon AF2400) as polymer matrix, a linear perfluorooligoether as plasticizer, sodium tetrakis[3,5-bis(perfluorohexyl)phenyl]borate providing for ionic sites, and bis[(perfluorooctyl)propyl]-2,2,2-trifluoroethylamine as H+ ionophore were investigated. All electrodes had excellent potentiometric selectivities, showed Nernstian responses to H+ over a wide pH range, exhibited enhanced mechanical stability, and maintained their selectivity over at least 4 weeks. For membranes of low ionophore concentration, the polymer affected the sensor selectivity noticeably at polymer concentrations exceeding 15%. Also, the membrane resistance increased quite strongly at high polymer concentrations, which cannot be explained by the Mackie-Meares obstruction model. The selectivities and resistances depend on the polymer concentration because of a functional group associated with Teflon AF2400, with a concentration of one functional group per 854 monomer units of the polymer. In the fluorous environment of these membranes, this functional group binds to Na+, K+, Ca2+, and the unprotonated ionophore with binding constants of 10(3.5), 10(1.8), 10(6.8), and 10(4.4) M(-1), respectively. Potentiometric and spectroscopic evidence indicates that these functional groups are COOH groups formed by the hydrolysis of carboxylic acid fluoride (COF) groups originally present in Teflon AF2400. The use of higher ionophore concentrations removes the undesirable effect of these COOH groups almost completely. Alternatively, the C(=O)F groups can be eliminated chemically, or they can be used to readily introduce new functionalities.