Platelet Subpopulations Research Articles

Activity and intracellular localization of senescence-associated β-galactosidase in aging Xenopus oocytes and eggs.

Senescence-associated β-galactosidase (SA-β-gal) serves as a marker of senescence in aging somatic cells. However, little is known about SA-β-gal dynamics in aging gamete cells. To address this issue, here we investigated activity and intracellular localization of SA-β-gal in freshly obtained and aging oocytes and eggs of the African clawed frog Xenopus laevis. Data base mining revealed the presence of several homologous β-galactosidase sequences in the annotated Xenopus genome. Some of them were predicted to contain an N-terminal signal peptide sequence, suggesting enzyme translocation to cellular organelles. Biochemical and microscopic analyses confirmed SA-β-gal localization in the particulate and cytosolic fractions of oocytes and eggs. SA-β-gal activity was found to reside predominantly within a fraction of dense cytoplasmic granules that were extensively stained with the lysosome-specific dye LysoTracker Green DND-26 and had an average size of 8.9 ± 5.6 μm. These features identify the SA-β-gal-containing granules as a subpopulation of yolk platelets, specialized late endosomes or lysosomes that accumulate and store processed protein in frog oocytes. Further analysis revealed an increase of SA-β-gal activity in Xenopus eggs, but not in oocytes, aged in vitro over 48 h. Our data suggest that endosomal acidification during egg aging may be responsible for this increase.

Flow Cytometry Assessment of Procoagulant Platelets Using a Dithiol-Reactive Probe.

Flow cytometry assessment of platelets using the combination of GSAO [4-(N-(S-glutathionylacetyl)amino)phenylarsonous acid], a dithiol-reactive probe, and P-selectin, a platelet activation marker, is a novel and powerful assay in the identification and quantification of the procoagulant subpopulation of platelets that has the capacity to support thrombin generation. In this chapter, we provide the flow cytometry protocols aimed at the study of procoagulant platelets under resting and agonist-stimulated conditions in whole blood and washed platelets of both human and murine (mouse) samples.

Necrotic but Not Apoptotic Platelets Are Functionally Procoagulant

There are numerous modes of cell death in mammalian cells. In platelets the primary identified modes are apoptosis and necrosis. Apoptosis is a form of programmed cell death, and in platelets it is considered to be important in their clearance. Besides pyknosis (cell shrinkage) and engulfment by resident phagocytes in vivo, a characteristic feature of apoptosis in nucleated cells is retraction of pseudopodia. However, in platelets, this does not occur. Instead, distinct from that which occurs during activation, lamelopodia and filopodia are not observed in apoptotic platelets suggesting that they have a limited potential to actively participate in coagulation. Necrosis, either regulated or pathologic, is characterized by cytoplasmic swelling (oncosis), swelling of cytoplasmic organelles, and, in later stages, rupture of the plasma membrane. Additionally, secondary necrosis can occur as a consequence of the activation of apoptotic pathways. Apoptosis is an ATP requiring process. In instances when an apoptotic signal was initiated and insufficient ATP is present for the completion of apoptosis, this ATP-depleted cell will undergo death with morphologic features of necrosis. Procoagulant platelets are those platelets that are capable of supporting the assembly of functional tenase and prothrombinase complexes. Due to the increased complexity of experiments demonstrating functional activity, the ability to support procoagulant activity has been presumed in subpopulations of platelets with variable degrees of phosphatidylserine exposure. Here we investigated the procoagulant function and the extent of PSer exposure in platelets undergoing different modes of cell death.Washed human platelets were stimulated with the BH-3 mimetic ABT-737 (100 nM) (apoptotic initiator) or convulxin (250 ng/mL, CVX) (initiator of regulated necrosis). The extent of PSer externalization was estimated by cytometric evaluation of annexin V (AnnV) binding, and functional activity was assessed using tissue factor-initiated thrombinoscopy. Upon stimulation two distinct subpopulations of PSer exposing platelets with “low” and “high” AnnV binding could be distinguished. In CVX-stimulated platelets only a single subpopulation (“high”) of PSer exposing platelets could be distinguished. But in platelets stimulated with the apoptotic initiator ABT-737 both “low” and “high” subpopulations of AnnV binding platelets could readily be discerned. To assess the procoagulant functionality of these various modes of cell death ABT-737 (generating ~38% “high” and ~46% “low” AnnV positive platelets) and CVX (~33% “high” AnnV) stimulated platelets were compared by thrombinoscopy. Relative to unstimulated platelets, inclusion of CVX-stimulated platelets within the reaction significantly shortened both the lag time (~5 to ~3 min (p<0.05)) and time to peak thrombin (~13 to ~8 min (p<0.05)). These shortened response times were accompanied by a ~2-fold increase in peak thrombin (p<0.05). In contrast to the potentiating effects of CVX-stimulation, ABT-737 stimulation had no effect on measures of thrombin; this despite having equivalent (when assessing only high) or even a greater percentage of AnnV binding (when assessing low and high).These results are the first to differentiate the mode of platelet cell death and its corresponding effects on PSer exposure and a functional measure of procoagulant function. Apoptotic and necrotic platelets are not only distinguished by their molecular mechanism of initiation, but also by their functional ability to support coagulation. Only platelets undergoing cell death by necrotic pattern were functionally procoagulant as measured by direct prothrombinase activity. Advancement in understanding the principles of cell death in procoagulant platelet formation might be beneficial for the determination and guidance of novel pharmacological strategy targeted for the treatment of procoagulant related hematopathologies. DisclosuresJobe:CSL: Consultancy; Shire: Consultancy; Octapharma: Consultancy.

Platelet Contraction Force As a Biophysical Biomarker for Bleeding Risk in Patients with Immune Thrombocytopenia

Background: Immune Thrombocytopenia Purpura (ITP) is defined by a low platelet count in the absence of any known causes and affects over 4,000 US children and 8,000 adults each year (Terrell et al, 2010). Deciding when and how to treat these patients remains difficult as there is no biomarker or diagnostic test that identifies which patients will self-resolve and which are at risk for major bleeding and life-threatening intracranial hemorrhage (~10%). In addition, the medications used to treat ITP all have significant side effects, forcing clinicians to balance risks associated with bleeding and medication. As such, an ongoing debate in the field of clinical hematology centers around which patients require therapy (Flores & Buchanan, 2013)(Cooper 2017)(Gralnek 2008). Here, we show that a new quantitative measurement of platelet function, which is independent of known platelet biomarkers (Myers et al, 2017), has diagnostic potential in identifying bleeding risk in ITP patients. Specifically, using high-throughput platelet contraction cytometry (PCC) measurements of individual platelet forces (Myers et al, 2017), we found that low platelet forces strongly correlate with bleeding symptoms. Unlike existing tests of platelet function that use bulk measurements, our test operates at the single cell level and therefore does not depend on the platelet count, enabling a direct comparison of individual platelet function in health versus disease states. In the broad context of overall function, our findings of impaired force generation agree with previous research demonstrating that impaired platelet function, and not low platelet count, correlates with bleeding in ITP patients (Frelinger et al, 2015). In addition, as the first demonstration of a correlation between single cell force and disease, our novel approach may represent an entirely new class of “physics-based” diagnostics.Platelet Contraction Cytometry (PCC): Within the PCC, a single platelet attaches, spreads, and applies contractile force to a pair of fibrinogen microdots that are attached to a moveable, spring-like, surface. Since the applied platelet contractile force is directly proportional to the microdot displacement, the force is calculated from a single fluorescence image of the platelet. Using microfabrication technology, thousands of microdots are created on a single device to enable high-throughput measurements in tightly controlled mechanical, biochemical, and shear microenvironments (Fig 1). Here, gel-filtered platelets from patients are activated (1U/mL thrombin), plated on the device with a moderately stiff surface (75 kPa), and measured as described previously (Myers et al, 2017).Results: In a cohort of ITP patients (n = 27), we observed that patients with bleeding and/or bruising symptoms exhibited significantly lower average platelet contraction forces than asymptomatic patients regardless of platelet count. Using an average force cutoff value of 26nN, we found that low forces identified bleeding in ITP with 100% sensitivity and 89.4% specificity. From a mechanistic perspective, our preliminary data also suggests that mean platelet volume does not correlate with platelet force or function, although further studies on a single platelet level are needed to confirm this result. While this data indicates that patients with bleeding symptoms have platelets with low forces, it is unclear whether low forces correlate with bleeding or whether the patient always has had low platelet forces that therefore render them susceptible to bleeding. Our preliminary prospective data suggests that low platelet forces correlate with the bleeding symptoms themselves and return to higher values when bleeding symptoms cease.Importantly, as our approach enables “single-cell” examinations of platelet forces, we identified different platelet subpopulations unique to ITP. Healthy individuals have a distribution of contractile forces with a single peak, while ITP patients tend to have two prominent peaks, one with a lower contractile force and one with a higher one. Surprisingly, patients with bleeding symptoms only have a low force peak, suggesting that the lack of highly contractile platelets may be a biophysical biomarker for bleeding (Fig 2). Ongoing studies are focused on using this finding to further improve specificity as well as elucidating the mechanistic underpinnings of low platelet contraction in ITP. DisclosuresNo relevant conflicts of interest to declare.

Analysis of procoagulant phosphatidylserine‐exposing platelets by imaging flow cytometry

BackgroundUpon platelet activation, a subpopulation of procoagulant platelets is formed, characterized by the exposure of the anionic aminophospholipid phosphatidylserine (PS) on the surface membrane. ObjectiveTo evaluate procoagulant PS‐exposing platelets by imaging flow cytometry. MethodsPlatelet ultrastructure was examined by transmission electron microscopy, and a comprehensive analysis of procoagulant platelets was performed using imaging flow cytometry; platelets were fluorescently labeled for the markers glycoprotein (GP)IX, activated integrin αIIbβ3, CD62P, and PS exposure. ResultsA subpopulation of platelets stimulated in suspension by the physiological agonists thrombin+collagen, and all platelets stimulated by the calcium ionophore A23187, had a distinct round morphology. These platelets were PS‐exposing, larger in size, had an increased circularity index, and had reduced internal complexity compared with non‐PS‐exposing platelets. They expressed CD62P and αIIbβ3 in an inactive conformation on the surface, and demonstrated depolarized inner mitochondrial membranes. For the first time, using imaging flow cytometry, a large proportion of PS‐exposing platelets possessing platelet‐associated extracellular vesicles (EVs) was observed, which demonstrated heterogeneous platelet marker expression that was different from free released EVs. ConclusionsInnovative imaging flow cytometry allowed detailed fluorescence‐based, quantitative morphometric analysis of PS‐exposing platelets; in becoming procoagulant, platelets undergo remarkable morphological changes, transforming into spherical “balloons,” almost devoid of their normal internal architecture. Almost all PS‐exposing platelets have associated EVs that are not detectable by traditional flow cytometry. While their functions have yet to be fully elucidated, the heterogeneity of platelet‐associated and released EVs suggests that they may contribute to different aspects of hemostasis and of thrombosis.

The potential contribution and role of a blood platelets in autoimmune thyroid diseases.

The blood platelets are multifunctional blood cells which are involved in the initiation of atheroma, endothelial dysfunction, and modulation of inflammatory and immune responses in the pathophysiology of many diseases. Because of their multifaceted pro‐inflammatory activity, platelets may be involved in the pathogenesis of autoimmune thyroid diseases (AITDs), such as Hashimoto's thyroiditis and Graves' disease. The aim of this study was to assess the level of activation and response ability of platelets in AITDs. We used the flow cytometry technique and kinetic measurement of aggregation to analyse platelet function immediately after blood collection and to demonstrate their activation in the circulation of patients with AITDs. We noted reorganization of platelet subpopulations (normal platelets, microparticles and aggregates) in AITDs, dependent on the degree of cell activation. We proved the elevated expression of the active form of integrin receptor GPIIb/IIIa, responsible for platelet aggregation, and in the kinetic test we confirmed the increased aggregation of platelets in different intracellular signal pathways (dependent on ADP, collagen, arachidonic acid). Our study demonstrates the high platelet activation level found in AITDs.

Mass Cytometry Reveals Distinct Platelet Subtypes in Healthy Subjects and Novel Alterations in Surface Glycoproteins in Glanzmann Thrombasthenia

Mass cytometry (MC) uses mass spectrometry to simultaneously detect multiple metal-conjugated antibodies on single cells, thereby enabling the detailed study of cellular function. Here, for the first time, we applied MC to the analysis of platelets. We developed a panel of 14 platelet-specific metal-tagged antibodies (targeting cluster of differentiation [CD] 9, CD29, CD31, CD36, CD41, CD42a, CD42b, CD61, CD62P, CD63, CD107a, CD154, glycoprotein [GP] VI and activated integrin αIIbβ3) and compared this panel with two fluorescence flow cytometry (FFC) panels (CD41, CD42b, and CD61; or CD42b, CD62P, and activated integrin αIIbβ3) in the evaluation of activation-dependent changes in glycoprotein expression on healthy subject and Glanzmann thrombasthenia (GT) platelets. High-dimensional analysis of surface markers detected by MC identified previously unappreciated subpopulations of platelets in healthy donors. As expected, MC and FFC revealed that GT platelets had significantly reduced CD41, CD61, and activated integrin αIIbβ3 surface expression. MC also revealed that surface expression of CD9, CD42a and CD63 were elevated, CD31, CD154 and GPVI were reduced and CD29, CD36, CD42b, CD62P and CD107a were similar on GT platelets compared to healthy donor platelets. In summary, MC revealed distinct platelet subtypes in healthy subjects and novel alterations in surface glycoproteins on GT platelets.

Tumor-Educated Platelets as a Noninvasive Biomarker Source for Cancer Detection and Progression Monitoring.

Liquid biopsies represent a potential revolution in cancer diagnostics as a noninvasive method for detecting and monitoring diseases, complementary to or even replacing current tissue biopsy approaches. Several blood-based biosources and biomolecules, such as cell-free DNA and RNA, proteins, circulating tumor cells, and extracellular vesicles, have been explored for molecular test development. We recently discovered the potential of tumor-educated blood platelets (TEP) as a noninvasive biomarker trove for RNA biomarker panels. TEPs are involved in the progression and spread of several solid tumors, and spliced TEP RNA surrogate signatures can provide specific information on the presence, location, and molecular characteristics of cancers. So far, TEP samples from patients with different tumor types, including lung, brain, and breast cancers, have been tested, and it has been shown that TEPs from patients with cancer are distinct from those with inflammatory and other noncancerous diseases. It remains to be investigated how platelets are "educated," which mechanisms cause intraplatelet RNA splicing, and whether the relative contribution of specific platelet subpopulations changes in patients with cancer. Ultimately, TEP RNA may complement currently used biosources and biomolecules employed for liquid biopsy diagnosis, potentially enhancing the detection of cancer in an early stage and facilitating noninvasive disease monitoring. Cancer Res; 78(13); 3407-12. ©2018 AACR.

Identification of eNOS‐based Megakaryocyte Subpopulations and Their Pharmacological Characterization by IFNγ and IL‐10

PurposeRecently, our laboratory identified in human blood two platelet subpopulations based on the presence or absence of a functional endothelial nitric oxide synthase (eNOS)‐signalling pathway and the ability to produce nitric oxide (NO). We have found that eNOS‐negative (eNOSneg) platelets, although less abundant are more reactive than eNOS‐positive (eNOSpos) platelets and initiate aggregate/thrombus formation, while eNOS‐positive platelets through their ability to generate NO limit aggregate size. As platelets derive from bone marrow megakaryocytes, we hypothesized that eNOSneg and eNOSpos subpopulations of megakaryocytes exist and give rise to their respective eNOS‐based platelet subpopulations. Additionally, we hypothesized that interferon‐γ (IFNγ) and interleukin‐10 (IL‐10), which are known to counter‐regulate eNOS expression and megakaryocyte differentiation, would determine in a concentration manner the ratio of eNOSneg to eNOSpos megakaryocytes.MethodsThe human megakaryoblastic cell line (Meg‐01) and megakaryocytes isolated from eNOS‐GFP transgenic mice were studied. RT‐PCR and DNA sequencing were performed to validate eNOS presence within Meg‐01. Flow cytometry was used to identify eNOSneg and eNOSpos megakaryocytes, and NO production was measured using DAF‐FM, a cell‐permeable fluorescent probe. Further, Meg‐01 cells were treated for 48 hours with IFNγ (0 – 100 ng/ml) and IL‐10 (0 – 100 ng/ml), and eNOS as well as inducible NOS (iNOS) were measured by flow cytometry and immunoblot, respectively.ResultsThe presence of eNOS was confirmed in Meg‐01 and eNOS‐GFP transgenic mice. Similar to human platelets, the majority of Meg‐01 cells were eNOSpos (91.5%±1.2 vs. 8.5%±1.2 eNOSneg, P&lt;0.05) and NO‐producing (92.0%±3.34 vs. 8.0%±6.68 non‐producing, P &lt; 0.05). Conversely, similar to mouse platelets, the majority of mouse megakaryocytes were eNOSneg (95.8%±0.7 vs. 4.2%±0.7 eNOSpos, P &lt; 0.05). The proinflammatory cytokine IFNγ decreased the percent eNOSpos Meg‐01 in a concentration‐dependent manner and also increased the level of iNOS within Meg‐01. IL‐10 in a concentration‐dependent manner (10–100ng/ml) reversed IFNγ‐induced iNOS expression, but not the decrease in percent eNOSpos Meg‐01. The changes in eNOS expression within Meg‐01 were further confirmed by qPCR within the total Meg‐01 population and demonstrated that IFNγ (10 ng/ml) caused a 6.4 fold decrease of eNOS mRNA but only 2.2 fold decrease in the presence IL‐10 (100ng/ml).ConclusionseNOS‐based megakaryocyte subpopulations exist within the Meg‐01 cell line and in eNOS‐GFP transgenic mice. Similar to previously shown for platelets, major species differences exist in eNOSpos to eNOSneg ratios between human and mouse megakaryocytes. IFNγ decreases eNOS expression and percent eNOSpos Meg‐01; while increasing iNOS expression. However, high concentrations of IL‐10 attenuated this effect. Our data show that pro‐inflammatory IFN‐γ may promote growth/differentiation of megakaryocytes with decreased levels/absence of eNOS, which may lead to formation of higher numbers of more reactive eNOSneg platelets potentially leading to higher risk of a pro‐thrombotic state. Further experiments are required to confirm whether eNOSpos and eNOSneg megakaryocytes give rise to their respective platelet subtypes.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Platelets Drive Thrombus Propagation in a Hematocrit and Glycoprotein VI-Dependent Manner in an In Vitro Venous Thrombosis Model.

The objective of this study was to measure the role of platelets and red blood cells on thrombus propagation in an in vitro model of venous valvular stasis. A microfluidic model with dimensional similarity to human venous valves consists of a sinus distal to a sudden expansion, where for sufficiently high Reynolds numbers, 2 countercurrent vortices arise because of flow separation. The primary vortex is defined by the points of flow separation and reattachment. A secondary vortex forms in the deepest recess of the valve pocket characterized by low shear rates. An initial fibrin gel formed within the secondary vortex of a tissue factor-coated valve sinus. Platelets accumulated at the interface of the fibrin gel and the primary vortex. Red blood cells at physiological hematocrits were necessary to provide an adequate flux of platelets to support thrombus growth out of the valve sinus. A subpopulation of platelets that adhered to fibrin expose phosphatidylserine. Platelet-dependent thrombus growth was attenuated by inhibition of glycoprotein VI with a blocking Fab fragment or D-dimer. A 3-step process regulated by hemodynamics was necessary for robust thrombus propagation: First, immobilized tissue factor initiates coagulation and fibrin deposition within a low flow niche defined by a secondary vortex in the pocket of a model venous valve. Second, a primary vortex delivers platelets to the fibrin interface in a red blood cell-dependent manner. Third, platelets adhere to fibrin, activate through glycoprotein VI, express phosphatidylserine, and subsequently promote thrombus growth beyond the valve sinus and into the bulk flow.

Toward the Relevance of Platelet Subpopulations for Transfusion Medicine.

Circulating platelets consist of subpopulations with different age, maturation state and size. In this review, we address the association between platelet size and platelet function and summarize the current knowledge on platelet subpopulations including reticulated platelets, procoagulant platelets and platelets exposing signals to mediate their clearance. Thereby, we emphasize the impact of platelet turnover as an important condition for platelet production in vivo. Understanding of the features that characterize platelet subpopulations is very relevant for the methods of platelet concentrate production, which may enrich or deplete particular platelet subpopulations. Moreover, the concept of platelet size being associated with platelet function may be attractive for transfusion medicine as it holds the perspective to separate platelet subpopulations with specific functional capabilities.

Platelet subpopulations remain despite strong dual agonist stimulation and can be characterised using a novel six-colour flow cytometry protocol

It is recognised that platelets respond differently to activation, where a subpopulation of platelets adopt a procoagulant phenotype while others are aggregatory. However, it has not been thoroughly tested whether these subpopulations will remain in maximally activated samples, or if they are merely a result of different platelet sensitivities to agonist activation. Here platelets were activated with gradually increasing concentrations of thrombin and/or the GPVI agonist cross-linked collagen-related peptide (CRP-XL). Platelet activation was investigated using a novel six-colour flow cytometry protocol evaluating exposure of phosphatidylserine, active conformation of the fibrinogen receptor αIIbβ3, α-granule and lysosomal release (P-selectin and LAMP-1 exposure), mitochondrial membrane integrity and platelet fragmentation. Upon activation by CRP-XL or thrombin+CRP-XL, platelets formed three differently sized subpopulations. Normal-sized platelets showed high exposure of aggregatory active αIIbβ3 and intact mitochondria, while the smaller platelets and platelet fragments showed high exposure of procoagulant phosphatidylserine. The distribution of platelets between the differently sized subpopulations remained stable despite high agonist concentrations. All three were still present after 30 and 60 min of activation, showing that all platelets will not have the same characteristics even after maximal stimulation. This suggests that platelet subpopulations with distinct activation patterns exist within the total platelet population.

ВЛИЯНИЕ ГЕЛИОГЕОФИЗИЧЕСКИХ ФАКТОРОВ НА СВЕРТЫВАЮЩУЮ СИСТЕМУ КРОВИ БЕРЕМЕННЫХ С ПЛАЦЕНТАРНЫМИ НАРУШЕНИЯМИ

Geomagnetic storms are one of the natural abiotic risk factors for human health. The response of the organism to their impact depends on the individual adaptive abilities, which were formed during the evolution, but can change in the conditions of pathological processes. Evaluation of the effect of a weak magnetic storm (G4v (22 < Ar < 39 nTl) on hemorheological parameters of blood of pregnant women with placental insufficiency compared to healthy pregnant women showed an increase in platelet count (p = 0.05), fibrinogen content (p = 0.02), thrombocyte (p = 0.03), erythrocyte sedimentation rate (p = 0.004). At the same time, a decrease in the number of red blood cells (p = 0.04), hemoglobin concentration (p = 0.001), hematocrit value (p = 0.0009) was registered. We found a strong direct correlation between subpopulations of platelets, which are characterized by different functional activity. Heterogeneity of platelets, their multidirectional correlations with indicators of blood coagulation system of healthy pregnant women can be considered as a response to the change in the disturbance of the earth's magnetic field. For pregnant women with placental disorders, the magnetic storm was associated with increased activity of the blood coagulation system and a significant increase in its components in the blood of patients. Despite the fact that changes in the blood coagulation system occur from the beginning of pregnancy and are often adaptive, in pregnant women with placental disorders, the additional impact of a weak magnetic storm can be a risk of hemostasis disorders and the development of platelet thrombosis, timely diagnosis and therapy of which is important.

Differential eNOS-signalling by platelet subpopulations regulates adhesion and aggregation.

In addition to maintaining haemostasis, circulating blood platelets are the cellular culprits that form occlusive thrombi in arteries and veins. Compared to blood leucocytes, which exist as functionally distinct subtypes, platelets are considered to be relatively simple cell fragments that form vascular system plugs without a differentially regulated cellular response. Hence, investigation into platelet subpopulations with distinct functional roles in haemostasis/thrombosis has been limited. In our present study, we investigated whether functionally distinct platelet subpopulations exist based on their ability to generate and respond to nitric oxide (NO), an endogenous platelet inhibitor. Utilizing highly sensitive and selective flow cytometry protocols, we demonstrate that human platelet subpopulations exist based on the presence and absence of endothelial nitric oxide synthase (eNOS). Platelets lacking eNOS (approximately 20% of total platelets) fail to produce NO and have a down-regulated soluble guanylate cyclase-protein kinase G (sGC-PKG)-signalling pathway. In flow chamber and aggregation experiments eNOS-negative platelets primarily initiate adhesion to collagen, more readily activate integrin αIIbβ3 and secrete matrix metalloproteinase-2, and form larger aggregates than their eNOS-positive counterparts. Conversely, platelets having an intact eNOS-sGC-PKG-signalling pathway (approximately 80% of total platelets) form the bulk of an aggregate via increased thromboxane synthesis and ultimately limit its size via NO generation. These findings reveal previously unrecognized characteristics and complexity of platelets and their regulation of adhesion/aggregation. The identification of platelet subpopulations also has potentially important consequences to human health and disease as impaired platelet NO-signalling has been identified in patients with coronary artery disease.

Inner Mitochondrial Membrane Disruption Links Apoptotic and Agonist-Initiated Phosphatidylserine Externalization in Platelets.

Phosphatidylserine exposure mediates platelet procoagulant function and regulates platelet life span. Apoptotic, necrotic, and integrin-mediated mechanisms have been implicated as intracellular determinants of platelet phosphatidylserine exposure. Here, we investigate (1) the role of mitochondrial events in platelet phosphatidylserine exposure initiated by these distinct stimuli and (2) the cellular interactions of the procoagulant platelet in vitro and in vivo. Key mitochondrial events were examined, including cytochrome c release and inner mitochondrial membrane (IMM) disruption. In both ABT-737 (apoptotic) and agonist (necrotic)-treated platelets, phosphatidylserine externalization was temporally correlated with IMM disruption. Agonist stimulation resulted in rapid cyclophilin D-dependent IMM disruption that coincided with phosphatidylserine exposure. ABT-737 treatment caused rapid cytochrome c release, eventually followed by caspase-dependent IMM disruption that again closely coincided with phosphatidylserine exposure. A nonmitochondrial and integrin-mediated mechanism has been implicated in the formation of a novel phosphatidylserine-externalizing platelet subpopulation. Using image cytometry, this subpopulation is demonstrated to be the result of the interaction of an aggregatory platelet and a procoagulant platelet rather than indicative of a novel intracellular mechanism regulating platelet phosphatidylserine externalization. Using electron microscopy, similar interactions between aggregatory and procoagulant platelets are demonstrated in vitro and in vivo within a mesenteric vein hemostatic thrombus. Platelet phosphatidylserine externalization is closely associated with the mitochondrial event of IMM disruption identifying a common pathway in phosphatidylserine-externalizing platelets. The limited interaction of procoagulant platelets and integrin-active aggregatory platelets identifies a potential mechanism for procoagulant platelet retention within the hemostatic thrombus.

Platelet-Mediated Modulation of Fibrinolysis.

Platelets are crucial to the hemostatic response. Their role in coagulation is well documented and they have been considered for some time to promote resistance of thrombi to fibrinolysis. Platelets confer resistance to lysis by promoting clot retraction of the immediate fibrin network and through release of plasminogen activator inhibitor-1 from their α-granules. However, recent developments in the field indicate that the role of platelets in fibrinolysis is much more diverse. Indeed, novel studies suggest that platelets form different subpopulations upon activation that play varied roles in regulating hemostasis. Likewise the developments in our understanding of thrombus formation, architecture, and changes in fibrin deposition and composition suggest that these different subpopulations of platelets may populate distinct areas within thrombi and potentially dictate the local hemostatic balance in these areas. This review will discuss the diverse roles of platelets in fibrinolysis and highlight the recent developments in the field and the contribution of both the intracellular pool of modulators as well as the membrane surface in regulating these processes.

The role of RNA uptake in platelet heterogeneity.

The role of platelets in regulating vascular homeostasis has expanded beyond mediation of haemostasis and thrombosis. The discovery of platelet RNA and the presence of subpopulations of platelets containing varying amounts of RNA suggest a role for platelet transcripts in vascular function. As the RNA in anucleated platelets is biologically functional and may transfer to other vascular cells, we hypothesised that platelet RNA diminishes over the lifespan of the platelet with diminishing platelet size due to horizontal cellular transfer. The purpose of this study is to determine if platelet RNA variance is the result of horizontal cellular transfer between platelets and other vascular cells. Utilising platelet sorting and RNA sequencing, we found that smaller platelets contained a more diverse set of transcripts than larger platelets. Further investigation using fluorescence imaging, gene expression analyses and in vitro and in vivo modelling revealed that platelets take up RNA from other vascular cells in a complex manner, revealing a dynamic role for platelets in modulating vascular homeostasis through bidirectional RNA transfer. The resultant RNA profile heterogeneity suggests unique functional roles for platelets dependent on size and complexity. This study expands our basic understanding of platelet function and heterogeneity and is the first to evaluate endogenous vascular RNA uptake and its relation to platelet processes. Our findings describe a novel endogenous phenomenon that can help elucidate the platelet's role in these non-thrombotic and haemostatic fields, as well as present potential for diagnostic and therapeutic development.

Probing the interaction of a glycoprotein IIb/IIIa receptor antagonist with bound platelets using electrochemical impedance

A label-free assay is described to monitor the interaction of abciximab, a glycoprotein IIb/IIIa receptor antagonist (ReoPro), with platelets bound to a fibrinogen-functionalised electrode surface. Firstly, fibrinogen is deposited in a defined pattern onto a gold electrode using microcontact printing, and then platelets from whole blood are captured on the patterned surface. Patterning influences the spreading of platelets, which is strikingly different to that observed on homogeneously coated surfaces. The drug–platelet interaction has been investigated using AC impedance on uniform and patterned fibrinogen-modified surfaces. The results demonstrate that patterned fibrinogen surfaces can provide deep insights into the interaction of abciximab with different platelet sub-populations. The key advantages of this approach are that it is rapid, label free and does not require pre-processing of patient blood samples.

Platelet subpopulation bearing leukocyte specific antigen and tissue factor.

Platelets bearing leukocyte antigen CD45 were identified in the blood of patients with myocardial infarction (MI) and healthy donors by flow cytofluorimetry. Part of these platelets contained tissue factor (TF)-primary initiator of blood clotting. The number of CD45+ and CD45+/TF+ platelets in MI patients at the first day was comparable with their level in healthy donors, but was increased at 8-12 days after MI onset. At that time in some patients the amount of CD45+ and CD45+/TF+ platelets reached 5-6 and 2-3% of their total number. It is assumed that CD45+/TF+ platelets could be formed as a result of platelet interaction with leukocytes or leukocyte produced membrane microparticles.

Dynamics of calcium spiking, mitochondrial collapse and phosphatidylserine exposure in platelet subpopulations during activation

Essentials The sequence and logic of events leading to platelet procoagulant activity are poorly understood. Confocal time-lapse microscopy was used to investigate activation of single adherent platelets. Platelet transition to the procoagulant state followed cytosolic calcium oscillations. Mitochondria did not collapse simultaneously and membrane potential loss could be reversible. Background Activated platelets form two subpopulations, one of which is able to efficiently aggregate, and another that externalizes phosphatidylserine (PS) and thus accelerates membrane-dependent reactions of blood coagulation. The latter, procoagulant subpopulation is characterized by a high cytosolic calcium level and the loss of inner mitochondrial membrane potential, and there are conflicting opinions on their roles in its formation. Methods We used confocal microscopy to investigate the dynamics of subpopulation formation by imaging single, fibrinogen-bound platelets with individual mitochondria in them upon loading with calcium-sensitive and mitochondrial potential-sensitive dyes. Stimulation was performed with thrombin or the protease-activated receptor (PAR) 1 agonist SFLLRN. Stochastic simulations with a computational systems biology model of PAR1 calcium signaling were employed for analysis. Results Platelet activation resulted in a series of cytosolic calcium spikes and mitochondrial calcium uptake in all platelets. The frequency of spikes decreased with time for SFLLRN stimulation, but remained high for a long period of time for thrombin. In some platelets, uptake of calcium by mitochondria led to the mitochondrial permeability transition pore opening and inner mitochondrial membrane potential loss, which could be either reversible or irreversible. The latter resulted in an increase in the cytosolic calcium level and PS exposure. These platelets had higher cytosolic calcium levels before activation, and their mitochondria collapsed not simultaneously but one after another. Conclusions These results support a model of procoagulant subpopulation development following a series of stochastic cytosolic calcium spikes that are accumulated by mitochondria, leading to a collapse, and suggest important roles of individual platelet reactivity and signal exchange between different mitochondria of a platelet.