Foxp1 regulation of neonatal vocalizations via cortical development.

The molecular mechanisms driving brain development at risk in autism spectrum disorders (ASDs) remain mostly unknown. Previous studies have implicated the transcription factor FOXP1 in both brain development and ASD pathophysiology. However, the specific molecular pathways both upstream of and downstream from FOXP1 are not fully understood. To elucidate the contribution of FOXP1-mediated signaling to brain development and, in particular, neocortical development, we generated forebrain-specific Foxp1 conditional knockout mice. We show that deletion of Foxp1 in the developing forebrain leads to impairments in neonatal vocalizations as well as neocortical cytoarchitectonic alterations via neuronal positioning and migration. Using a genomics approach, we identified the transcriptional networks regulated by Foxp1 in the developing neocortex and found that such networks are enriched for downstream targets involved in neurogenesis and neuronal migration. We also uncovered mechanistic insight into Foxp1 function by demonstrating that sumoylation of Foxp1 during embryonic brain development is necessary for mediating proper interactions between Foxp1 and the NuRD complex. Furthermore, we demonstrated that sumoylation of Foxp1 affects neuronal differentiation and migration in the developing neocortex. Together, these data provide critical mechanistic insights into the function of FOXP1 in the developing neocortex and may reveal molecular pathways at risk in ASD.

Dissecting the roles of dynamin and clathrin in platelet pinocytosis.

Platelets endocytose many molecules from their environment. However, this process of pinocytosis in platelets is poorly understood. Key endocytic regulators such as dynamin, clathrin, CDC42 and Arf6 are expressed in platelets but their roles in pinocytosis is not known. Stimulated platelets form two subpopulations of pro-aggregatory and procoagulant platelets. The effect of stimulation on pinocytosis is also poorly understood. In this study, washed human platelets were treated with a range of endocytosis inhibitors and stimulated using different activators. The rate of pinocytosis was assessed using pHrodo green, a pH-sensitive 10 kDa dextran. In unstimulated platelets, pHrodo fluorescence increased over time and accumulated as intracellular puncta indicating constituently active pinocytosis. Stimulated platelets (both pro-aggregatory and procoagulant) had an elevated pinocytosis rate compared to unstimulated platelets. Dynamin inhibition blocked pinocytosis in unstimulated, pro-aggregatory and procoagulant platelets indicating that most platelet pinocytosis is dynamin dependent. Although pinocytosis was clathrin-independent in unstimulated and procoagulant populations, clathrin partially contributed to pinocytosis in pro-aggregatory platelets.

Increasing the number and intensity of shock tube generated blast waves leads to earlier retinal ganglion cell dysfunction and regional cell death.

The purpose of this study was to examine the effect of a blast exposure generated from a shock tube on retinal ganglion cell (RGC) function and structure. Mice were exposed to one of three blast conditions using a shock tube; a single blast wave of 20 PSI, a single blast wave of 30 PSI, or three blast waves of 30 PSI given on three consecutive days with a one-day inter-blast interval. The structure and function of the retina were analyzed using the pattern electroretinogram (PERG), the optomotor reflex (OMR), and optical coherence tomography (OCT). The in vivo parameters were examined at baseline, and then again 1-week, 4-weeks, and 16-weeks following blast exposure. The number of surviving RGCs was quantified at the end of the study. Analysis of mice receiving a 20 PSI injury showed decreased PERG and OMR responses 16-weeks post blast, without evidence of changed retinal thickness or RGC death. Mice subjected to a 30 PSI injury showed decreased PERG responses 4 weeks and 16 weeks after injury, without changes in the retinal thickness or RGC density. Mice subjected to 30 PSI X 3 blast exposures had PERG deficits 1-week and 4-weeks post exposure. There was also significant change in retinal thickness 1-week and 16-weeks post blast exposure. Mice receiving 30 PSI X 3 blast injuries had regional loss of RGCs in the central retina, but not in the mid-peripheral or peripheral retina. Overall, this study has shown that increasing the number of blast exposures and the intensity leads to earlier functional loss of RGCs. We have also shown regional RGC loss only when using the highest blast intensity and number of blast injuries.

Epigenetic Regulation of F2RL3 Associates With Myocardial Infarction and Platelet Function.

BACKGROUND: DNA hypomethylation at the F2RL3 (F2R like thrombin or trypsin receptor 3) locus has been associated with both smoking and atherosclerotic cardiovascular disease; whether these smoking-related associations form a pathway to disease is unknown. F2RL3 encodes protease-activated receptor 4, a potent thrombin receptor expressed on platelets. Given the role of thrombin in platelet activation and the role of thrombus formation in myocardial infarction, alterations to this biological pathway could be important for ischemic cardiovascular disease. METHODS: We conducted multiple independent experiments to assess whether DNA hypomethylation at F2RL3 in response to smoking is associated with risk of myocardial infarction via changes to platelet reactivity. Using cohort data (N=3205), we explored the relationship between smoking, DNA hypomethylation at F2RL3, and myocardial infarction. We compared platelet reactivity in individuals with low versus high DNA methylation at F2RL3 (N=41). We used an in vitro model to explore the biological response of F2RL3 to cigarette smoke extract. Finally, a series of reporter constructs were used to investigate how differential methylation could impact F2RL3 gene expression. RESULTS: Observationally, DNA methylation at F2RL3 mediated an estimated 34% of the smoking effect on increased risk of myocardial infarction. An association between methylation group (low/high) and platelet reactivity was observed in response to PAR4 (protease-activated receptor 4) stimulation. In cells, cigarette smoke extract exposure was associated with a 4.9% to 9.3% reduction in DNA methylation at F2RL3 and a corresponding 1.7-(95% CI, 1.2-2.4, P=0.04) fold increase in F2RL3 mRNA. Results from reporter assays suggest the exon 2 region of F2RL3 may help control gene expression. CONCLUSIONS: Smoking-induced epigenetic DNA hypomethylation at F2RL3 appears to increase PAR4 expression with potential downstream consequences for platelet reactivity. Combined evidence here not only identifies F2RL3 DNA methylation as a possible contributory pathway from smoking to cardiovascular disease risk but from any feature potentially influencing F2RL3 regulation in a similar manner.

Identification of diagnostic biomarkers used in the diagnosis of cardiovascular diseases and diabetes mellitus: A systematic review of quantitative studies.

AIMS: To perform a systematic review of studies that sought to identify diagnostic biomarkers for the diagnosis of cardiovascular diseases (CVDs) and diabetes mellitus (DM), which could be used in low- and middle-income countries (LMICs) where there is a lack of diagnostic equipment, treatments and training. MATERIALS AND METHODS: Papers were sourced from six databases: the British Nursing Index, Google Scholar, PubMed, Sage, Science Direct and Scopus. Articles published between January 2002 and January 2023 were systematically reviewed by three reviewers and appropriate search terms and inclusion/exclusion criteria were applied. RESULTS: A total of 18 studies were yielded, as well as 234 diagnostic biomarkers (74 for CVD and 160 for DM). Primary biomarkers for the diagnosis of CVDs included growth differentiation factor 15 and neurogenic locus notch homologue protein 1 (Notch1). For the diagnosis of DM, alpha-2-macroglobulin, C-peptides, isoleucine, glucose, tyrosine, linoleic acid and valine were frequently reported across the included studies. Advanced analytical techniques, such as liquid chromatography mass spectrometry, enzyme-linked immunosorbent assays and vibrational spectroscopy, were also repeatedly reported in the included studies and were utilized in combination with traditional and alternative matrices such as fingernails, hair and saliva. CONCLUSIONS: While advanced analytical techniques are expensive, laboratories in LMICs should carry out a cost-benefit analysis of their use. Alternatively, laboratories may want to explore emerging techniques such as infrared, Fourier transform-infrared and near-infrared spectroscopy, which allow sensitive noninvasive analysis.

Application of the Cellular Thermal Shift Assay (CETSA) to validate drug target engagement in platelets.

Small molecule drugs play a major role in the study of human platelets. Effective action of a drug requires it to bind to one or more targets within the platelet (target engagement). However, although in vitro assays with isolated proteins can be used to determine drug affinity to these targets, additional factors affect target engagement and its consequences in an intact platelet, including plasma membrane permeability, intracellular metabolism or compartmentalization, and level of target expression. Mechanistic interpretation of the effect of drugs on platelet activity requires comprehensive investigation of drug binding in the proper cellular context, i.e. in intact platelets. The Cellular Thermal Shift Assay (CETSA) is a valuable method to investigate target engagement within complex cellular environments. The assay is based on the principle that drug binding to a target protein increases that protein's thermal stability. In this technical report, we describe the application of CETSA to platelets. We highlight CETSA as a quick and informative technique for confirming the direct binding of drugs to platelet protein targets, providing a platform for understanding the mechanism of action of drugs in platelets, and which will be a valuable tool for investigating platelet signaling and function.

Platelets control blood clotting in health and disease. Small molecule drugs are often used to study human platelets. Here, describe how Cellular Thermal Shift Assay (CETSA) can be used in platelets to investigate the binding between these drugs and their targets inside platelets. This technique can be used to increase our understanding of how existing and future drugs work in platelets.

Characterization of orofacial features in sclerodermatous chronic graft-versus-host disease.

BACKGROUND: Chronic graft-versus-host disease (cGVHD) is a leading cause of morbidity and mortality following allogeneic hematopoietic cell transplantation (alloHCT). The sclerodermatous form of cGVHD can be particularly debilitating; however, orofacial sclerodermatous involvement remains poorly described. OBJECTIVE: To characterize orofacial features of sclerodermatous cGVHD in a single center cohort of patients who underwent alloHCT. STUDY DESIGN: Retrospective data were collected from electronic medical records and analyzed descriptively. RESULTS: There were 39 patients who received alloHCT between 1993 and 2017 and developed orofacial sclerodermatous cGVHD. Concomitant cutaneous sclerodermatous cGVHD was common (n = 20, 51%). Orofacial sclerodermatous cGVHD features included fibrous bands of the buccal mucosa (n = 23, 59%), limited mouth opening (n = 19, 54%), perioral fibrosis (n = 8, 21%), and focal gingival recession (n = 4, 10%). Oral mucosal fibrosis was observed at the site of active or resolved chronic lichenoid inflammation in 30 patients, with all but two also presenting with a history of ulcerations. Management included jaw stretching exercises (n = 10; 6 stable/improved), surgery (n = 3; 2 improved), and intralesional corticosteroid injections (n = 2; 2 improved). CONCLUSIONS: Orofacial involvement with sclerodermatous cGVHD can present with multiple manifestations including fibrous banding, limited mouth opening, perioral fibrosis, and focal gingival recession. Surgical and non-surgical management strategies may improve clinical function and reduce morbidity.

Pilot Collection and Evaluation of Head Kinematics in Stock Car Racing.

The goal of this work was to collect on-track driver head kinematics using instrumented mouthpieces and characterize environmental exposure to accelerations and vibrations. Six NASCAR drivers were instrumented with custom-fit mouthpieces to collect head kinematic data. Devices were deployed at four tracks during practice and testing environments and configured to collect approximately 11 min of linear acceleration and rotational velocity data at 200 Hz. This continuous data collection, combined with film review, allowed extraction of complete laps of data. In addition to typical data processing methods, a moving-point average was calculated and subtracted from the overall signal for both linear acceleration and rotational velocity to determine the environmental component of head motion. The current analysis focuses on 42 full laps of data collected at four data collection events. The number of laps per track ranged from 2 to 23. Linear acceleration magnitudes for all 42 laps ranged from 2.46 to 7.48 g and rotational velocity ranged from 1.25 to 3.35 rad/s. After subtracting the moving average, linear acceleration ranged from 0.92 to 5.45 g and rotational velocity ranged from 0.57 to 2.05 rad/s. This study has established the feasibility of using an instrumented mouthpiece to measure head kinematics in NASCAR and presented a technique for isolating head motion due to cornering acceleration from those due to short-term perturbations experienced by the driver.

Platelet-Derived Extracellular Vesicles in Arterial Thrombosis.

Blood platelets are necessary for normal haemostasis but also form life-threatening arterial thrombi when atherosclerotic plaques rupture. Activated platelets release many extracellular vesicles during thrombosis. Phosphatidylserine-exposing microparticles promote coagulation. Small exosomes released during granule secretion deliver cargoes including microRNAs to cells throughout the cardiovascular system. Here, we discuss the mechanisms by which platelets release these extracellular vesicles, together with the possibility of inhibiting this release as an antithrombotic strategy.

P7C3-A20 treatment one year after TBI in mice repairs the blood-brain barrier, arrests chronic neurodegeneration, and restores cognition.

Chronic neurodegeneration in survivors of traumatic brain injury (TBI) is a major cause of morbidity, with no effective therapies to mitigate this progressive and debilitating form of nerve cell death. Here, we report that pharmacologic restoration of the blood-brain barrier (BBB), 12 mo after murine TBI, is associated with arrested axonal neurodegeneration and cognitive recovery, benefits that persisted for months after treatment cessation. Recovery was achieved by 30 d of once-daily administration of P7C3-A20, a compound that stabilizes cellular energy levels. Four months after P7C3-A20, electron microscopy revealed full repair of TBI-induced breaks in cortical and hippocampal BBB endothelium. Immunohistochemical staining identified additional benefits of P7C3-A20, including restoration of normal BBB endothelium length, increased brain capillary pericyte density, increased expression of BBB tight junction proteins, reduced brain infiltration of immunoglobulin, and attenuated neuroinflammation. These changes were accompanied by cessation of TBI-induced chronic axonal degeneration. Specificity for P7C3-A20 action on the endothelium was confirmed by protection of cultured human brain microvascular endothelial cells from hydrogen peroxide-induced cell death, as well as preservation of BBB integrity in mice after exposure to toxic levels of lipopolysaccharide. P7C3-A20 also protected mice from BBB degradation after acute TBI. Collectively, our results provide insights into the pathophysiologic mechanisms behind chronic neurodegeneration after TBI, along with a putative treatment strategy. Because TBI increases the risks of other forms of neurodegeneration involving BBB deterioration (e.g., Alzheimer's disease, Parkinson's disease, vascular dementia, chronic traumatic encephalopathy), P7C3-A20 may have widespread clinical utility in the setting of neurodegenerative conditions.

FoxP1 orchestration of ASD-relevant signaling pathways in the striatum.

Mutations in the transcription factor Forkhead box p1 (FOXP1) are causative for neurodevelopmental disorders such as autism. However, the function of FOXP1 within the brain remains largely uncharacterized. Here, we identify the gene expression program regulated by FoxP1 in both human neural cells and patient-relevant heterozygous Foxp1 mouse brains. We demonstrate a role for FoxP1 in the transcriptional regulation of autism-related pathways as well as genes involved in neuronal activity. We show that Foxp1 regulates the excitability of striatal medium spiny neurons and that reduction of Foxp1 correlates with defects in ultrasonic vocalizations. Finally, we demonstrate that FoxP1 has an evolutionarily conserved role in regulating pathways involved in striatal neuron identity through gene expression studies in human neural progenitors with altered FOXP1 levels. These data support an integral role for FoxP1 in regulating signaling pathways vulnerable in autism and the specific regulation of striatal pathways important for vocal communication.

Utilizing orthodontic appliances to retain and disguise a pediatric maxillary obturator throughout growth.

Unique challenges are encountered when providing an obturator for an adolescent patient. Challenges include the need to modify the obturator throughout growth, engaging the mixed dentition or partially erupted teeth with minimal undercuts, the psychosocial challenges of an actively maturing patient, and the possible need for coincidental orthodontic therapy. This clinical report describes the ongoing rehabilitation of a patient who presented at the age of 10 with a biopsy-confirmed palatal mucoepidermoid carcinoma. Incorporating orthodontic brackets for retention of innovative adjustable obturator clasps allowed for the favorable function of the obturator prosthesis and the ability to alter the prosthesis over time.

Accuracy of Digital Impressions at Varying Implant Depths: An In Vitro Study.

PURPOSE: Implants placed at variable depths may vary the amount of visible scannable surface of a scan body. Intraoral scanner technology uses advanced optical principles to record the surface of the scan body to accurately capture the implant position. The purpose of this study is to investigate the effect implant placement depth has on the accuracy of digital implant impressions using an intraoral scanner. MATERIALS AND METHODS: A partially edentulous gypsum master model was fabricated to allow the positioning of a single implant analog at different depths. Four groups were created based on the planned implant depths of 7, 6, 3, and 0 mm and corresponding visibility of the scan body at 2, 3, 6, and 9 mm. The model was digitized with a laboratory scanner for the reference scan and with an intraoral scanner to generate 15 test scans per group, with a total of 60 scans. The test scans were superimposed onto the reference scan using the best fit algorithm to analyze and measure the positional (dXYZ) and angular deviation (d⍬) of the scan body using three-dimensional metrology software. Statistical analysis was performed using a one-way ANOVA and pairwise comparison was done with a Tukey-Kramer HSD test (α = 0.05). RESULTS: The one-way ANOVA of the groups for the dXYZ and dθ parameters was statistically significant (F3,56 = 11.45, p < 0.001, F3,56 = 24.04, p < 0.001). Group D (9 mm) showed the least positional deviation at 38.41 µm (95% CI 30.26; 46.56) and the least angular deviation of 0.17° (95% CI 0.12; 0.21). Group A (2 mm) showed the greatest positional deviation of 77.17 µm (95% CI 65.23; 89.11) and greatest angular deviation of 0.84° (95% CI 0.65; 1.03). The positional and angular deviation increased with increased implant depth. CONCLUSIONS: The accuracy of digital impressions is influenced by the implant depth and the amount of visibility of the scan body. The trueness and precision are highest when the implant is placed at 0 mm depth with complete visibility of the scan body and decreases with subgingival implant placement.

Immune responses in mice after blast-mediated traumatic brain injury TBI autonomously contribute to retinal ganglion cell dysfunction and death.

PURPOSE: The purpose of this study was to examine the role of the immune system and its influence on chronic retinal ganglion cell (RGC) dysfunction following blast-mediated traumatic brain injury (bTBI). METHODS: C57BL/6J and B6.129S7-Rag1tm1Mom/J (Rag-/-) mice were exposed to one blast injury of 140 kPa. A separate cohort of C57BL/6J mice was exposed to sham-blast. Four weeks following bTBI mice were euthanized, and splenocytes were collected. Adoptive transfer (AT) of splenocytes into naïve C57BL/6J recipient mice was accomplished via tail vein injection. Three groups of mice were analyzed: those receiving AT of splenocytes from C57BL/6J mice exposed to blast (AT-TBI), those receiving AT of splenocytes from C57BL/6J mice exposed to sham (AT-Sham), and those receiving AT of splenocytes from Rag-/- mice exposed to blast (AT-Rag-/-). The visual function of recipient mice was analyzed with the pattern electroretinogram (PERG), and the optomotor response (OMR). The structure of the retina was evaluated using optical coherence tomography (OCT), and histologically using BRN3A-antibody staining. RESULTS: Analysis of the PERG showed a decreased amplitude two months post-AT that persisted for the duration of the study in AT-TBI mice. We also observed a significant decrease in the retinal thickness of AT-TBI mice two months post-AT compared to sham, but not at four or six months post-AT. The OMR response was significantly decreased in AT-TBI mice 5- and 6-months post-AT. BRN3A staining showed a loss of RGCs in AT-TBI and AT-Rag-/- mice. CONCLUSION: These results suggest that the immune system contributes to chronic RGC dysfunction following bTBI.

Q94 is not a selective modulator of proteinase-activated receptor 1 (PAR1) in platelets.

Thrombin is a potent platelet activator, acting through proteinase-activated receptors -1 and -4 (PAR1 and PAR4). Of these, PAR-1 is activated more rapidly and by lower thrombin concentrations. Consequently, PAR-1 has been extensively investigated as a target for anti-platelet drugs to prevent myocardial infarction. Q94 has been reported to act as an allosteric modulator of PAR1, potently and selectively inhibiting PAR1-Gαq coupling in multiple cell lines, but its effects on human platelet activation have not been previously studied. Platelet Ca2+ signaling, integrin αIIbß3 activation and α-granule secretion were monitored following stimulation by a PAR1-activating peptide (PAR1-AP). Although Q94 inhibited these responses, its potency was low compared to other PAR1 antagonists. In addition, αIIbß3 activation and α-granule secretion in response to other platelet activators were also inhibited with similar potency. Finally, in endothelial cells, Q94 did not inhibit PAR1-dependent Ca2+ signaling. Our data suggest that Q94 may have PAR1-independent off-target effects in platelets, precluding its use as a selective PAR1 allosteric modulator.

All-Terrain Vehicle-Related Emergency Department Visits: Interaction of Sex and Age, NEISS, 2019.

BACKGROUND: Epidemiologic patterns of all-terrain vehicle (ATV)-related emergency department (ED) visits by male and female individuals may vary at different ages. To our knowledge, this has not been researched previously. OBJECTIVE: The purpose of this study was to determine the interaction of sex and age differences in their association with ATV-related ED visits. METHODS: Data from the 2019 National Electronic Injury Surveillance System were extracted for ATV-related ED visits, including sex, age, race, location of crash, injured body part, and whether alcohol was involved. Descriptive statistics and logistic regression analyses were conducted. We modeled sex in separate multivariable models, adjusting for the same independent variables. RESULTS: There were an estimated 95,995 (unweighted n = 1999) ATV-related ED visits. There was a significant age-by-sex interaction in the association between ATV-related ED visits vs. other ED injuries, indicating that the effect of age on ATV-related ED visits differed by sex and vice versa. Overall, male individuals were 1.7 times as likely to have an ATV-related ED visit as female individuals. In the stratified analysis for female individuals, odds were substantially greater for girls younger than 18 years (adjusted odds ratio [AOR] 2.33; 95% confidence interval [CI] 1.61-3.69) and women aged 18-35 years (AOR 4.76; 95% CI 3.48-6.51) compared with woman older than 35 years. For men, odds were significant for ages 18-35 years (AOR 2.21; 95% CI 1.72-2.85) compared with men older than 35 years. CONCLUSIONS: As newer ATVs become more powerful and faster, there is a need to know who is at greatest risk for ATV-related ED visits to develop policies and safety measures.

Cortical Foxp2 Supports Behavioral Flexibility and Developmental Dopamine D1 Receptor Expression.

Genetic studies have associated FOXP2 variation with speech and language disorders and other neurodevelopmental disorders (NDDs) involving pathology of the cortex. In this brain region, FoxP2 is expressed from development into adulthood, but little is known about its downstream molecular and behavioral functions. Here, we characterized cortex-specific Foxp2 conditional knockout mice and found a major deficit in reversal learning, a form of behavioral flexibility. In contrast, they showed normal activity levels, anxiety, and vocalizations, save for a slight decrease in neonatal call loudness. These behavioral phenotypes were accompanied by decreased cortical dopamine D1 receptor (D1R) expression at neonatal and adult stages, while general cortical development remained unaffected. Finally, using single-cell transcriptomics, we identified at least five excitatory and three inhibitory D1R-expressing cell types in neonatal frontal cortex, and we found changes in D1R cell type composition and gene expression upon cortical Foxp2 deletion. Strikingly, these alterations included non-cell-autonomous changes in upper layer neurons and interneurons. Together, these data support a role for Foxp2 in the development of dopamine-modulated cortical circuits and behaviors relevant to NDDs.

Comparison of putative BH3 mimetics AT-101, HA14-1, sabutoclax and TW-37 with ABT-737 in platelets.

Platelet lifespan is regulated by intrinsic apoptosis. Platelet apoptosis can be triggered by BH3 mimetics that inhibit the pro-survival Bcl-2 family protein, Bcl-xL. Here, we investigated several small molecules that are reported to act as BH3 mimetics and compared their effects to the well-established BH3 mimetic, ABT-737. Platelet phosphatidylserine (PS) exposure was determined by flow cytometry. Changes in cytosolic Ca2+ signaling were detected using Cal-520. Plasma membrane integrity was determined by calcein leakage. The roles of caspases and calpain in these processes were determined using Q-VD-OPh and calpeptin, respectively. As previously reported, ABT-737 triggered PS exposure in a caspase-dependent manner and calcein loss in a caspase and calpain-dependent manner. In contrast, AT-101 and sabutoclax triggered PS exposure independently of caspases. HA14-1 also triggered PS exposure in a caspase-independent but calpain-dependent manner. There were also significant differences in the pattern and protease-dependency of cytosolic Ca2+ signaling in response to these drugs compared to ABT-737. Since there are clear differences between the action of ABT-737 and the other putative BH3 mimetics investigated here, AT-101, HA14-1 and sabutoclax cannot be considered as acting as BH3 mimetics in platelets. Furthermore, the platelet death caused by these drugs is likely to be distinct from apoptosis.

High extracellular phosphate increases platelet polyphosphate content.

Platelet-derived extracellular polyphosphate (PolyP) is a major mediator of thrombosis. PolyP is a linear chain of inorganic phosphate (Pi) and is stored in platelet dense granules. Pi enters cells from the extracellular fluid through phosphate transporters and may be stored as PolyP. Here we show that high extracellular Pi concentration in vitro increases platelet PolyP content, in a manner dependent on phosphate transporters, IP6K and V-type ATPases. The increased PolyP also enhanced PolyP-dependent coagulation in platelet-rich plasma. These data suggest a mechanistic link between hyperphosphatemia, PolyP and enhanced coagulation, which may be important in pathologies such as chronic kidney disease.

Cytosolic and mitochondrial Ca2+ signaling in procoagulant platelets.

SummaryPlatelets are the major cellular contributor to arterial thrombosis. However, activated platelets form two distinct subpopulations during thrombosis. Pro-aggregatory platelets aggregate to form the main body of the thrombus. In contrast, procoagulant platelets expose phosphatidylserine on their outer surface and promote thrombin generation. This apparently all-or-nothing segregation into subpopulations indicates that, during activation, platelets commit to becoming procoagulant or pro-aggregatory. Although the signaling pathways that control this commitment are not understood, distinct cytosolic and mitochondrial Ca2+ signals in different subpopulations are likely to be central. In this review, we discuss how these Ca2+ signals control procoagulant platelet formation and whether this process can be targeted pharmacologically to prevent arterial thrombosis.