Role In Hemostasis Research Articles

Platelets, Protean Cells with All-Around Functions and Multifaceted Pharmacological Applications.

Platelets, traditionally known for their roles in hemostasis and coagulation, are the most prevalent blood component after erythrocytes (150,000-400,000 platelets/μL in healthy humans). However, only 10,000 platelets/μL are needed for vessel wall repair and wound healing. Increased knowledge of the platelet's role in hemostasis has led to many advances in understanding that they are crucial mediators in many other physiological processes, such as innate and adaptive immunity. Due to their multiple functions, platelet dysfunction is involved not only in thrombosis, mediating myocardial infarction, stroke, and venous thromboembolism, but also in several other disorders, such as tumors, autoimmune diseases, and neurodegenerative diseases. On the other hand, thanks to their multiple functions, nowadays platelets are therapeutic targets in different pathologies, in addition to atherothrombotic diseases; they can be used as an innovative drug delivery system, and their derivatives, such as platelet lysates and platelet extracellular vesicles (pEVs), can be useful in regenerative medicine and many other fields. The protean role of platelets, from the name of Proteus, a Greek mythological divinity who could take on different shapes or aspects, is precisely the focus of this review.

LL-37 Triggers Antimicrobial Activity in Human Platelets.

Platelets play a crucial role in hemostasis and the immune response, mainly by recognizing signals associated with vascular damage. However, it has recently been discovered that the antimicrobial peptide LL-37 activates platelets in functions related to thrombus formation and inflammation. Therefore, this work aims to evaluate the effect of LL-37 on the activation of antimicrobial functions of human platelets. Our results show that platelets treated with LL-37 increase the surface expression of receptors (Toll-like receptors (TLRs) 2 and -4, CD32, CD206, Dectin-1, CD35, LOX-1, CD41, CD62P, and αIIbβ3 integrins) for the recognition of microorganisms, and molecules related to antigen presentation to T lymphocytes (CD80, CD86, and HLA-ABC) secrete the antimicrobial molecules: bactericidal/permeability-increasing protein (BPI), azurocidin, human neutrophil peptide (HNP) -1, and myeloperoxidase. They also translate azurocidin, and have enhanced binding to Escherichia coli, Staphylococcus aureus, and Candida albicans. Furthermore, the supernatant of LL-37-treated platelets can inhibit E. coli growth, or platelets can employ their LL-37 to inhibit microbial growth. In conclusion, these findings demonstrate that LL-37 participates in the antimicrobial function of human platelets.

Platelet P2Y1 receptor exhibits constitutive G protein signaling and β-arrestin 2 recruitment

BackgroundPurinergic P2Y1 and P2Y12 receptors (P2Y1-R and P2Y12-R) are G protein-coupled receptors (GPCR) activated by adenosine diphosphate (ADP) to mediate platelet activation, thereby playing a pivotal role in hemostasis and thrombosis. While P2Y12-R is the major target of antiplatelet drugs, no P2Y1-R antagonist has yet been developed for clinical use. However, accumulating data suggest that P2Y1-R inhibition would ensure efficient platelet inhibition with minimal effects on bleeding. In this context, an accurate characterization of P2Y1-R antagonists constitutes an important preliminary step.ResultsHere, we investigated the pharmacology of P2Y1-R signaling through Gq and β-arrestin pathways in HEK293T cells and in mouse and human platelets using highly sensitive resonance energy transfer-based technologies (BRET/HTRF). We demonstrated that at basal state, in the absence of agonist ligand, P2Y1-R activates Gq protein signaling in HEK293T cells and in mouse and human platelets, indicating that P2Y1-R is constitutively active in physiological conditions. We showed that P2Y1-R also promotes constitutive recruitment of β-arrestin 2 in HEK293T cells. Moreover, the P2Y1-R antagonists MRS2179, MRS2279 and MRS2500 abolished the receptor dependent-constitutive activation, thus behaving as inverse agonists.ConclusionsThis study sheds new light on P2Y1-R pharmacology, highlighting for the first time the existence of a constitutively active P2Y1-R population in human platelets. Given the recent interest of P2Y12-R constitutive activity in patients with diabetes, this study suggests that modification of constitutive P2Y1-R signaling might be involved in pathological conditions, including bleeding syndrome or high susceptibility to thrombotic risk. Thus, targeting platelet P2Y1-R constitutive activation might be a promising and powerful strategy for future antiplatelet therapy.

Platelet Phenotyping by Full Spectrum Flow Cytometry.

Platelets play key roles in hemostasis, immunity, and inflammation, and tests of platelet phenotype and function are useful in studies of disease biology and pathology. Full spectrum flow cytometry offers distinct advantages over standard tests and enables the sensitive and simultaneous detection of many biomarkers. A typical assay provides a wealth of information on platelet biology and allows the assessment of in vivo activation and in vitro reactivity, as well as the discovery of novel phenotypes. Here, we describe the analysis of platelets by full spectrum flow cytometry and discuss a range of controls and methods for interpreting results. © 2023 Wiley Periodicals LLC. Basic Protocol: Platelet phenotyping by full spectrum flow cytometry Support Protocol 1: Spectral unmixing Support Protocol 2: Data preprocessing.

Platelet Functional Testing Via High-Throughput Microtiter Plate-Based Assays.

Platelets play a critical role in hemostasis and thrombosis; therefore, in vitro assays that measure platelet reactivity are fundamental tools to gain insight into these physiologic processes, to diagnose platelet disorders, and to develop antithrombotic therapies. However, conventional platelet assays such as aggregometry, the clinical gold standard for assessing platelet function, are low throughput and require specialized equipment. Since platelets have a finite life span ex vivo, processes to miniaturize and multiplex assays allow a much broader overview of platelet function in significantly less time than conventional assays. Several groups have developed simplified, high-throughput approaches to quantify platelet activation with standard laboratory equipment to lower the barrier of entry to study platelet biology. This article describes a panel of optimized and validated high-throughput microplate assays to comprehensively assess platelet functionality, independently or in combination, to increase throughput and reduce costs. Specifically, following stimulation of platelets, a plate reader can be used to measure light transmission aggregation via absorbance; dense-granule secretion based on ATP-dependent luminescence generation; and cytosolic calcium levels with a cell-permeant, fluorescent Ca2+ -sensitive dye. Additionally, platelets are an easily accessible component of the blood that share signaling pathways with other cells, making them ideal for high-throughput drug screens. The highly adaptable and complementary assays presented in this article can be used to decipher the molecular mechanism underlying platelet activation or to identify novel inhibitors. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Microtiter plate-based light transmission aggregometry Basic Protocol 2: Measuring dense-granule secretion in high-throughput microplate assays Basic Protocol 3: Microtiter plate-based calcium mobilization Support Protocol: Platelet isolation and enumeration.

P478 Platelet-related indicators for predicting vedolizumab response in patients with Ulcerative Colitis

Abstract Background Ulcerative Colitis (UC) combine both inflammation and coagulation in their pathogenesis and clinical manifestations. Although platelets (PLT) are well known for their role in hemostasis, there are a rising number of studies supporting their considerable role as inflammatory amplifiers in chronic inflammatory conditions. However, whether PLT are related to the efficacy of biologic agents is unknown. This study aimed to investigate whether platelet ratio before and after induction therapy can be predictive biomarkers for the therapeutic outcomes of vedolizumab therapy in UC. Methods Patients with moderate-to-severe UC receiving vedolizumab in our hospital between November 2020 and November 2022 were retrospectively evaluated. Patients with concomitant corticosteroid treatment≥20 mg, immunomodulators, or other conditions that could modify the platelet count were excluded. The primary endpoint was clinical response at the end of the induction period (at 14 weeks). We analyzed the association between platelet count, neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), monocyte-to-lymphocyte ratio (MLR), C-reactive protein (CRP), fecal calprotectin, and the clinical efficacy of vedolizumab. The above indicators between the response and nonresponse groups were compared via a univariate analysis. The independent risk factors of nonresponse were identified via a multivariate analysis. Results Seventy-one patients with moderate-to-severe active UC started treatment with vedolizumab and 20 patients (28.2%) experienced loss of response (LOR) after induction therapy. The platelet count, albumin, CRP, NLR, PLR, MLR, and Particul Mayo score between the LOR and response groups significantly differed. The Platelet ratio before and after induction therapy cut-off value of 1.077 was predictive of LOR, using receiver operating characteristic analysis (sensitivity:78.0%, specificity:81.8%). A univariate analysis revealed a significant relationship between LOR and the platelet ratio (P＜0.05). Multivariate analysis indicated the platelet ratio as an independent prognostic factor for LOR (OR=11.219, 95% CI:3.316-37.959, P＜0.05). Conclusion Platelet ratio before and after induction therapy is a useful prognostic marker in patients with moderate-to-severe active UC treated with vedolizumab, and may contribute to appropriate use of vedolizumab LOR. A large sample from multiple centers is needed to validate the association between platelet-related measures and the rate of vedolizumab response.

Platelet count, temperature and pH value differentially affect hemostatic and immunomodulatory functions of platelets

Platelets are primarily recognized for their role in hemostasis, but also regulate immune responses by interacting with leukocytes. Their highly sensitive nature enables platelets to rapidly respond to micro-environmental changes, which is crucial under physiological condition but can jeopardize in vitro analyses. Thus, we tested how platelet count and changes in pH and temperatures, which are commonly experienced during inflammation and infection but also affected by ex vivo analyses, influence platelet-leukocyte interaction and immunomodulation.Reducing platelet count by up to 90 % slightly decreased platelet activation and platelet-leukocyte aggregate formation, but did not affect CD11b activation nor CD62L shedding of monocytes or neutrophils. Acidosis (pH 6.9) slightly elevated platelet degranulation and binding to innate leukocytes, though pH changes did not modulate leukocyte activation. While platelet responsiveness was higher at room temperature than at 37 °C, incubation temperature did not affect platelet-leukocyte aggregate formation. In contrast, platelet-mediated CD11b activation and CD62L expression increased with temperature.Our data thus demonstrate the importance of standardized protocols for sample preparation and assay procedure to obtain comparable data. Further, unspecific physiologic responses such as thrombocytopenia, acidosis or temperature changes may contribute to platelet dysfunction and altered platelet-mediated immunomodulation in inflammatory and infectious disease.

Abstract B036: Platelets promote in vitro osteosarcoma cell migration and in vivo metastatic lung seeding

Abstract Osteosarcoma (OS) is the most common primary bone cancer worldwide where it mainly affects adolescents. Metastatic OS has a 20% survival rate and treatment options remain poor, thus understanding mechanisms that contribute to OS metastasis is critical for improved patient outcomes. Platelets, commonly recognized for their role in hemostasis, have been shown to promote metastasis in several cancer models. However, most previous studies investigating platelet contributions to metastasis have focused on carcinomas while their role in osteosarcoma remains poorly understood. Previous studies by our lab have shown that platelet interactions with breast cancer cells resulted in increased migration in vitro and increased lung metastases in vivo. Thus, we hypothesized that OS cell migration will be enhanced in the presence of platelets, and that platelet depletion in vivo will inhibit OS lung seeding. Cell suspensions of SaOs2 and MG63.3 cells in serum free media were fluorescently stained and seeded, in triplicates, in the top chamber of an 8um pore Fluoroblock migration assay wells. In the bottom chamber, media and either Tyrode’s buffer or 1.5x106 isolated human platelets were added. The presence of platelets resulted in a 3.5- and 4.6-fold increase of migrated MG63.3 and SaOs2 cells respectively. For in vivo analysis, cohorts of eight-week-old, male Nod Scid-Gamma (NSG), Nod Scid, and Nude mice were randomly assigned to either the control or platelet depletion group. Mice were depleted of platelets by administration of Anti-Platelet glycoprotein Ib alpha (GPIbα) antibody and, 5x105 GFP transduced MG63.3 cells were injected via tail vein. 24 hrs later, the mice were euthanized, and lungs were collected and microscopically imaged. Platelet depletion in NSG mice resulted in a 48% reduction in MG63.3 lung seeding compared to the isotype control recipient mice. This result was more dramatic in the less immunocompromised Nude and Nod Scid mice with a 61% and 87% reduction of MG63.3 lung seeding in the platelet depleted mice respectively. Finally, RNAseq and was performed on seven OS cell lines co-cultured with isolated mouse platelets, and alterations in gene expression were compared to control cells. Gene Enrichment Set Analysis (GSEA) of the data indicated that platelet co-incubation significantly upregulated pathways related to inflammatory responses, Tumor Necrosis Factor (TNF)-a signaling, and Interleukin (IL)-6 signaling. These results suggest that platelet secreted factors facilitate OS cell migration in vitro while an immune dependent mechanism is involved in the role of platelets in the in vivo lung seeding of OS cells. Citation Format: Olivia K. Travis, Ruishan Wang, Myriam Labelle. Platelets promote in vitro osteosarcoma cell migration and in vivo metastatic lung seeding [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr B036.

Toll-like Receptors and Thrombopoiesis.

Platelets are the second most abundant blood component after red blood cells and can participate in a variety of physiological and pathological functions. Beyond its traditional role in hemostasis and thrombosis, it also plays an indispensable role in inflammatory diseases. However, thrombocytopenia is a common hematologic problem in the clinic, and it presents a proportional relationship with the fatality of many diseases. Therefore, the prevention and treatment of thrombocytopenia is of great importance. The expression of Toll-like receptors (TLRs) is one of the most relevant characteristics of thrombopoiesis and the platelet inflammatory function. We know that the TLR family is found on the surface or inside almost all cells, where they perform many immune functions. Of those, TLR2 and TLR4 are the main stress-inducing members and play an integral role in inflammatory diseases and platelet production and function. Therefore, the aim of this review is to present and discuss the relationship between platelets, inflammation and the TLR family and extend recent research on the influence of the TLR2 and TLR4 pathways and the regulation of platelet production and function. Reviewing the interaction between TLRs and platelets in inflammation may be a research direction or program for the treatment of thrombocytopenia-related and inflammatory-related diseases.

Underlying mechanisms of thrombus formation/growth in atherothrombosis and deep vein thrombosis.

Thrombosis remains a leading cause of death worldwide despite technological advances in prevention, diagnosis, and treatment. The traditional view of arterial thrombus formation is that it is a platelet-dependent process, whereas that of venous thrombus formation is a coagulation-dependent process. Current pathological and basic studies on atherothrombosis and venous thrombosis have revealed the diverse participation of platelet and coagulation activation mechanisms in both thrombus initiation and growth processes during clinical thrombotic events. Atherosclerotic plaque cell-derived tissue factor contributes to fibrin formation and platelet aggregation. The degree of plaque disruption and a blood flow alteration promote atherothrombotic occlusion. While blood stasis/turbulent flow due to luminal stenosis itself initiates venous thrombus formation. The coagulation factor XI-driven propagation phase of blood coagulation plays a major role in venous thrombus growth, but a minor role in hemostasis. These lines of evidence indicate that atherothrombosis onset is affected by the thrombogenic potential of atherosclerotic plaques, the plaque disruption size, and an alteration in blood flow. Upon onset of venous thrombosis, enhancement of the propagation phase of blood coagulation under blood stasis and a hypercoagulable state contribute to large thrombus formation.

Determination of Antibody Activity by Platelet Aggregation.

Platelets play an important role in hemostasis by forming clots and stopping bleeding. In immune thrombotic conditions, platelets and leukocytes are aberrantly activated by pathogenic antibodies resulting in platelet aggregates and NETosis, leading to thrombosis and thrombocytopenia. A simple assay that assesses platelet function and antibody activity is light transmission aggregometry. This assay can be used to determine antibody activity in patients with disorders such as heparin-induced thrombocytopenia (HIT) and vaccine-induced thrombotic thrombocytopenia (VITT). Briefly, for detection of pathogenic antibody, platelet-rich plasma (PRP) is treated with a specific agent (e.g., patient sera or purified patient antibodies) with constant stirring. Upon activation, platelets undergo a shape change and adhere to each other forming aggregates. This causes a reduction in opacity allowing more light to pass through PRP. Light transmission through the cuvette is proportional to the degree of platelet aggregation and is measured by the photocell over time. The advantage of this protocol is that it is a simple, reliable assay that can be applied to assess antibody activity in thrombotic conditions. Light transmission aggregometry does not require the use of radioactive reagents and is technically less demanding compared with 14C-serotonin release assay, another common assay for detecting antibody activity. Key features • This protocol can be used to assess platelet function and to detect platelet activating antibodies in diseases such as HIT and VITT. • Does not require radioactive reagents, requires an aggregometer; based on the light transmission aggregometry protocol, adapted for detection of VITT and other platelet-activating antibodies. • Two positive controls are required for reliable detection of antibodies in diseases such as HIT/VITT, namely a weak HIT/VITT antibody and a physiological agonist. • For detection of HIT/VITT antibodies, it is essential to use donors known to have platelets reactive to these antibodies to avoid false negative results.

Platelet-derived extracellular vesicles for drug delivery.

Platelet-derived extracellular vesicles (PEVs) are a subset of EVs that are released from platelets, which are small nuclear cell fragments that play a critical role in hemostasis and thrombosis. PEVs have been shown to have important roles in a variety of physiological and pathological processes, including inflammation, angiogenesis, and cancer. Recently, researchers, including our group have utilized PEVs as drug delivery platforms as PEVs could target inflammatory sites both passively and actively. This review summarizes the biological function of PEVs, introduces recent applications of PEVs in targeted drug delivery, and provides an outlook for the further development of utilizing PEVs for drug delivery.

Phenotypic Characterization of Acoustically Enriched Extracellular Vesicles from Pathogen-Activated Platelets

Extracellular vesicles (EVs) are derived from the membrane of platelets and released into the circulation upon activation or injury. Analogous to the parent cell, platelet-derived EVs play an important role in hemostasis and immune responses by transfer of bioactive cargo from the parent cells. Platelet activation and release of EVs increase in several pathological inflammatory diseases, such as sepsis. We have previously reported that the M1 protein released from the bacterial pathogen Streptococcus pyogenes directly mediates platelet activation. In this study, EVs were isolated from these pathogen-activated platelets using acoustic trapping, and their inflammation phenotype was characterized using quantitative mass spectrometry-based proteomics and cell-based models of inflammation. We determined that M1 protein mediated release of platelet-derived EVs that contained the M1 protein. The isolated EVs derived from pathogen-activated platelets contained a similar protein cargo to those from physiologically activated platelets (thrombin) and included platelet membrane proteins, granule proteins, cytoskeletal proteins, coagulation factors, and immune mediators. Immunomodulatory cargo, complement proteins, and IgG3 were significantly enriched in EVs isolated from M1 protein-stimulated platelets. Acoustically enriched EVs were functionally intact and exhibited pro-inflammatory effects on addition to blood, including platelet-neutrophil complex formation, neutrophil activation, and cytokine release. Collectively, our findings reveal novel aspects of pathogen-mediated platelet activation during invasive streptococcal infection.

Insights into Regulatory Factors in Megakaryocyte Development and Function: Basic Mechanisms and Potential Targets.

Platelets are small, anucleate cellular fragments, which are produced by megakaryocytes, and play a key role in hemostasis and thrombus formation. The differentiation of megakaryocytes from hematopoietic stem cells in bone marrow and the development of megakaryocytes into platelets is a complex process. Various regulatory factorsin megakaryopoiesis including cytokines, growth factors, transcription factors, and gene expression, are all involved in the process of thrombocytopoiesis and play distinct roles in different stages of megakaryocytes development. In this review, we summarize the current state of knowledge ofmultiple regulatory factors including the TPO/Mpl signaling pathway, transcription factors, RasGTPases family, estrogen, and microRNAs. Altogether, we aimed to discuss more molecular mechanisms of megakaryocytes differentiation and maturation, and possess a better understanding of platelet formation.

Pharmacological Actions of 5-Hydroxyindolin-2 on Modulation of Platelet Functions and Thrombus Formation via Thromboxane A2 Inhibition and cAMP Production

Platelets play a very significant role in hemostasis while simultaneously posing a risk for the development of various cardiovascular diseases. Platelet-mediated issues can occur in blood vessels and trigger various medical problems. Therefore, controlling platelet function is important in the prevention of thrombosis. In this regard, we need to find compounds that provide potent antiplatelet activity with minimum side effects. Therefore, we examined the effect of 5-hydroxyindolin-2-one isolated from Protaetia brevitarsis larvae having antiplatelet properties and investigated different pathways that mediate the antiplatelet activity. We examined the effect of 5-hydroxyindolin-2-one (5-HI) on the regulation of phosphoproteins, thromboxane A2 generation, and integrin αIIbβ3 action. Our data showed that human platelet aggregation was inhibited by 5-HI (75, 100, 150, 200 μM) without cytotoxicity, and it suppressed intracellular Ca2+ concentration through the regulation of inositol 1, 4, 5-triphosphate receptor I (Ser1756) and extracellular signal-regulated kinase (ERK). Moreover, collagen-elevated thromboxane A2 production and αIIbβ3 action were inhibited by 5-HI through the regulation of cytosolic phospholipase A2 (cPLA2), mitogen-activated protein kinase p38 (p38MAPK), vasodilator-stimulated phosphoprotein (VASP), phosphoinositide 3-kinase (PI3K), and Akt (protein kinase B). Therefore, we suggested that 5-HI could be a potential substance for the prevention of thrombosis-mediated thrombosis.

Structure and dynamics of the von Willebrand Factor C6 domain

Von Willebrand disease (VWD) is a bleeding disorder with different levels of severity. VWD-associated mutations are located in the von Willebrand factor (VWF) gene, coding for the large multidomain plasma protein VWF with essential roles in hemostasis and thrombosis. On the one hand, a variety of mutations in the C-domains of VWF are associated with increased bleeding upon vascular injury. On the other hand, VWF gain-of-function (GOF) mutations in the C4 domain have recently been identified, which induce an increased risk of myocardial infarction. Mechanistic insights into how these mutations affect the molecular behavior of VWF are scarce and holistic approaches are challenging due to the multidomain and multimeric character of this large protein. Here, we determine the structure and dynamics of the C6 domain and the single nucleotide polymorphism (SNP) variant G2705R in C6 by combining nuclear magnetic resonance spectroscopy, molecular dynamics simulations and aggregometry. Our findings indicate that this mutation mostly destabilizes VWF by leading to a more pronounced hinging between both subdomains of C6. Hemostatic parameters of variant G2705R are close to normal under static conditions, but the missense mutation results in a gain-of-function under flow conditions, due to decreased VWF stem stability. Together with the fact that two C4 variants also exhibit GOF characteristics, our data underline the importance of the VWF stem region in VWF’s hemostatic activity and the risk of mutation-associated prothrombotic properties in VWF C-domain variants due to altered stem dynamics.

Bioprotective Role of Platelet-Derived Microvesicles in Hypothermia: Insights from the Differential Characteristics of Peripheral and Splenic Platelets

While platelets play an important role in hemostasis and blood coagulation, they also contribute to tissue repair. Most platelets are present in peripheral blood, but some are stored in the spleen. Since tissue environments of peripheral blood vessels and the spleen are quite distinctive, the properties of the platelets present in each may also differ. However, no studies have addressed this difference.We have reported that accidental hypothermia activates splenic platelets, but not peripheral blood, whose biological significance is unknown. Accidental hypothermia is an involuntary drop in core body temperature to less than 35°C due to exposure to low environmental temperatures. Shivering, an involuntary small contraction of skeletal muscles throughout the body, is thought to be an automatic defense system against cold, in which skeletal muscles contract to increase heat production. Shivering occurs in muscles throughout the body, so when muscles are damaged by it, the tissue must be repaired by a mechanism that covers the entire body.Platelets, when activated, release microvesicles that encapsulate many bioactive substances. These microvesicles are called platelet-derived microvesicles (PDMV). It is generally believed that when cells take up extracellular vesicles, they are affected by the action of the encapsulated substances. Therefore, we hypothesized that PDMV released by platelet activation in the spleen at hypothermia may be involved in the regeneration of skeletal muscle throughout the body. C57Bl/6 mice were placed in an environment of -20°C and the rectal temperature was lowered to 15°C to model hypothermia. Platelets, PDMV, and skeletal muscle tissue were collected from these mice for analysis.Transcriptomic changes between splenic and peripheral platelets were greater in hypothermic mice than in normal mice. Pathway analysis of these Transcriptomic changes showed alterations related to Wnt signaling, cytoskeletal remodeling, and cell adhesion. Electron microscopy and real-time RT-PCR analysis revealed that platelets activated in the spleen by hypothermia internalize the transcripts involved in tissue repair, such as Wnt3a and Ctnnb1, into PDMV and release them into plasma. Plasma microvesicles from hypothermic mice promoted wound healing in the mouse myoblast cell line C2C12. Skeletal muscles of hypothermic mice were damaged but recovered within 24 hours after rewarming. However, splenectomy delayed recovery from skeletal muscle injury even after the mice were rewarmed.These results indicate that PDMV released from activated platelets in the spleen plays an important role in the repair of skeletal muscle damaged by shivering of hypothermia, suggesting a novel function of the spleen not previously known.

Mouse Model for Platelet Aggregation using Flow Cytometry

Abstract Platelets play crucial roles in hemostasis, bleeding, and thrombosis. Within the United States, an estimated 7,000 platelet units are transfused daily. The gold standard to measure platelet survival in humans is to determine post-transfusion recovery after an autologous transfusion of radiolabeled platelet units, however, the ability to detect platelets circulating after transfusion does not provide information on how well these platelets function in hemostasis. Clinically, platelet unit function is routinely measured using aggregometry, which requires large volumes of platelet concentrates and normal platelet counts. While the clinical demand for platelets continues to increase with advances in medical care, there remains a lack of in vitro measures to properly assess platelet function in animal models. Here we outline an in vitro method for characterizing platelet function using flow cytometry to assess platelet activation and aggregation simultaneously in a mouse model. Using two commercially available transgenic mouse lines, one with platelets expressing red fluorescent protein (RFP) and the other with platelets expressing green fluorescent protein (GFP), whole blood is collected from each transgenic mouse line by aseptic cardiac puncture, and leukocyte-reduced platelet rich plasma (LRPRP) is isolated after two consecutive centrifuge spins and leukocyte reduction. RFP- and GFP-labeled platelets can be visually separated by flow cytometry. Platelets are activated after incubation with 4 mM GPRP peptide followed by exposure to 0.5U/mL high activity bovine thrombin for 2 minutes. Platelets are then stained with anti-CD41a, labelling all platelets, and anti-CD62P, labelling activated platelets; these platelet populations can be analyzed by flow cytometry. When RFP- and GFP-LRPRP are combined prior to activation with thrombin, RFP- and GFP-labeled platelet aggregation can be measured as the number of cells doubly expressing RFP and GFP by flow cytometry. Thrombin stimulation activated 93.31% of the total number of RFP- and GFP-platelets. Of the activated, CD62P-positive platelets, 11.03% expressed both RFP and GFP, indicating that these platelets were aggregated. Of the inactivated, CD62P-negative platelets, 0.81% expressed both RFP and GFP. These results suggest that thrombin exposure activates platelets and causes platelet aggregation, while <1% of inactivated platelets aggregate. Additionally, platelet aggregation is easily measured by flow without differentially labeling the two separate platelet populations. These data indicate that our model is a viable measure of platelet function in vitro. Our model of platelet activation and aggregation provides a baseline measure of in vitro platelet function in RFP and GFP mice. This in vitro method of assessing platelet function can be applied to various experimental populations and used to quantify the effects of experimental conditions on platelet function in mice. Using this model of platelet function, factors influencing platelet storage can be characterized and ultimately used to improve platelet transfusion therapies.

Microscopic Description of Platelet Aggregates Induced by Escherichia coli Strains.

In addition to their role in haemostasis, platelets are also involved in the inflammatory and antimicrobial process. Interactions between pathogens and platelets, mediated by receptors can lead to platelet activation, which may be responsible for a granular secretion process or even aggregation, depending on the bacterial species. Granular secretion releases peptides with bactericidal activity as well as aggregating factors. To our knowledge, these interactions have been poorly studied for Escherichia coli (E. coli). Few studies have characterised the cellular organization of platelet-E. coli aggregates. The objective of our study was to investigate the structure of platelet aggregates induced by different E. coli strains as well as the ultrastructure of platelet-E. coli mixtures using a scanning and transmission electron microscopy (SEM and TEM) approach. Our results show that the appearance of platelet aggregates is mainly dependent on the strain used. SEM images illustrate the platelet activation and aggregation and their colocalisation with bacteria. Some E. coli strains induce platelet activation and aggregation, and the bacteria are trapped in the platelet magma. However, some strains do not induce significant platelet activation and are found in close proximity to the platelets. The structure of the E. coli strains might explain the results obtained.

Inhibition of platelet aggregation by activation of platelet intermediate conductance Ca2+‐activated potassium channels

BackgroundWithin the vasculature platelets and endothelial cells play crucial roles in hemostasis and thrombosis. Platelets, like endothelial cells, possess intermediate conductance Ca2+‐activated K+ (IKCa) channels and generate nitric oxide (NO). Although NO limits platelet aggregation, the role of IKCa channels in platelet function and NO generation has not yet been explored. ObjectivesWe investigated whether IKCa channel activation inhibits platelet aggregation, and per endothelial cells, enhances platelet NO production. MethodsPlatelets were isolated from human volunteers. Aggregometry, confocal microscopy, and a novel flow chamber model, the Quartz Crystal Microbalance (QCM) were used to assess platelet function. Flow cytometry was used to measure platelet NO production, calcium signaling, membrane potential, integrin αIIb/β3 activation, granule release, and procoagulant platelet formation. ResultsPlatelet IKCa channel activation with SKA‐31 inhibited aggregation in a concentration‐dependent manner, an effect reversed by the selective IKCa channel blocker TRAM‐34. The QCM model along with confocal microscopy demonstrated that SKA‐31 inhibited platelet aggregation under flow conditions. Surprisingly, IKCa activation by SKA‐31 inhibited platelet NO generation, but this could be explained by a concomitant reduction in platelet calcium signaling. IKCa activation by SKA‐31 also inhibited dense and alpha‐granule secretion and integrin αIIb/β3 activation, but maintained platelet phosphatidylserine surface exposure as a measure of procoagulant response. ConclusionsPlatelet IKCa channel activation inhibits aggregation by reducing calcium‐signaling and granule secretion, but not by enhancing platelet NO generation. IKCa channels may be novel targets for the development of antiplatelet drugs that limit atherothrombosis, but not coagulation.