Trends and Sociodemographic Patterns in Keratoconus Management 2015-2020: An American Academy of Ophthalmology IRIS® Registry Analysis.

PURPOSE: Investigate trends in keratoconus (KCN) treatment patterns and diagnosis age from 2015 to 2020 and evaluate sociodemographic associations with the treatment approach. DESIGN: Retrospective cohort study. PARTICIPANTS: Patients with a new KCN diagnosis from 2015 to 2020 were identified in the Academy IRIS® Registry (Intelligent Research in Sight). METHODS: Associations between sociodemographic factors and treatment were evaluated using multivariable logistic regression. MAIN OUTCOME MEASURES: Outcomes included percentages and rates of each treatment (collagen crosslinking [CXL], keratoplasty, or no procedure) from 2015 to 2020, age at diagnosis during this period, and sociodemographic factors associated with treatment type. RESULTS: A total of 66 199 patients with a new diagnosis of KCN were identified. The percentage of patients undergoing CXL increased from 0.05% in 2015 to 29.5% in 2020 (P = 0.008). The average age (standard deviation) of KCN patients decreased from 44.1 (±16.9) years in 2015 to 39.2 (±16.9) years in 2020 (P < 0.001). In multivariable analyses comparing CXL versus no procedure and keratoplasty versus no procedure, patients undergoing CXL tended to be younger with the odds of having CXL decreasing with increasing age, for example, comparing CXL and no procedure patients, using ages 0-20 years as reference, the odds ratio (OR) (95% confidence interval [CI]) decreased from 0.62 (0.57-0.67; P < 0.0001) for patients aged 21-40 years to 0.03 (0.02-0.04; P < 0.0001) for patients aged > 60 years. Men were more likely than women to have CXL (OR, 1.31; 95% CI, 1.23-1.40; P < 0.0001) and keratoplasty (OR, 1.30; 95% CI, 1.19-1.42; P < 0.0001). Black patients were less likely than White patients to have CXL (OR, 0.70; 95% CI, 0.63-0.77; P < 0.0001) and more likely to have keratoplasty (OR, 2.24; 95% CI, 2.01-2.50; P < 0.0001). Likewise, Hispanic patients had higher odds of CXL (OR, 1.12; 95% CI, 1.00-1.24; P < 0.05) and keratoplasty (OR, 1.29; 95% CI, 1.12-1.50; P < 0.001) compared with non-Hispanic patients. Collagen crosslinking and keratoplasty also varied by region and insurance status. CONCLUSIONS: A significant increase in use of CXL was noted from 2015 to 2020. Sociodemographic differences in treatment among KCN patients may reflect differences in access, use, or care patterns, and future studies should aim to identify strategies to improve access for all patients. FINANCIAL DISCLOSURE(S): The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Thrombin spatial distribution determines protein C activation during hemostasis and thrombosis.

Rebalancing the hemostatic system by targeting endogenous anticoagulant pathways, like the protein C (PC) system, is being tested as a means of improving hemostasis in patients with hemophilia. Recent intravital studies of hemostasis demonstrated that, in some vascular contexts, thrombin activity is sequestered in the extravascular compartment. These findings raise important questions about the context-dependent contribution of activated PC (APC) to the hemostatic response, because PC activation occurs on the surface of endothelial cells. We used a combination of pharmacologic, genetic, imaging, and computational approaches to examine the relationships among thrombin spatial distribution, PC activation, and APC anticoagulant function. We found that inhibition of APC activity, in mice either harboring the factor V Leiden mutation or infused with an APC-blocking antibody, significantly enhanced fibrin formation and platelet activation in a microvascular injury model, consistent with the role of APC as an anticoagulant. In contrast, inhibition of APC activity had no effect on hemostasis after penetrating injury of the mouse jugular vein. Computational studies showed that differences in blood velocity, injury size, and vessel geometry determine the localization of thrombin generation and, consequently, the extent of PC activation. Computational predictions were tested in vivo and showed that when thrombin generation occurred intravascularly, without penetration of the vessel wall, inhibition of APC significantly increased fibrin formation in the jugular vein. Together, these studies show the importance of thrombin spatial distribution in determining PC activation during hemostasis and thrombosis.

Open-Globe Injury Repairs in the American Academy of Ophthalmology IRIS® Registry 2014 - 2018: Incidence, Risk Factors, and Visual Outcomes.

PURPOSE: To estimate incidence and evaluate demographic risk factors and visual acuity (VA) outcomes of open-globe injuries requiring surgical repair in the IRIS® Registry (Intelligent Research in Sight). DESIGN: Retrospective cohort study. PARTICIPANTS: Patients with open-globe injury repairs (OGRs) were identified by Current Procedural Terminology codes (65275, 65280, 65285, 65286, 65235, 65260, and 65265) from 2014 through 2018 in the IRIS Registry. METHODS: Logistic regression models adjusting for age, sex, race, ethnicity, United States region, concurrent and subsequent surgeries, and baseline VA. MAIN OUTCOME MEASURES: Outcomes included annual and 5-year incidence rates per 100 000 people and factors associated with OGR, VA better than 20/40, and VA of 20/200 or worse at final follow-up (3-12 months after OGR). RESULTS: Thirteen thousand seven hundred sixty-six OGRs were identified; 5-year cumulative incidence was 28.0 per 100 000 patients. Open-globe repair was associated with age 21 to 40 years compared with younger than 21 years (odds ratio [OR], 1.6; 95% confidence interval [CI], 1.5-1.7]), male sex (OR, 2.8; 95% CI, 2.7-2.9), Black versus White race (OR, 1.3; 95% CI, 1.2-1.4), Hispanic versus non-Hispanic ethnicity (OR, 1.7; 95% CI, 1.6-1.8), and South (OR, 1.4; 95% CI, 1.3-1.5) and West (OR, 1.3; 95% CI, 1.2-1.4) versus Midwest regions and associated inversely with Asian versus White race (OR, 0.6; 95% CI, 0.6-0.7). Visual acuity outcomes, analyzed in a subset of 2966 patients with VA data available, showed vision impairment (VA < 20/40) at final follow-up was associated with VA of 20/200 or worse at presentation (20/200 better than 20/40; OR, 11.1; 95% CI, 8.0-15.7), older age (e.g., > 80 years vs. < 21 years; OR, 5.8; 95% CI, 3.2-10.7), and Black versus White race (OR, 1.8; 95% CI, 1.3-2.6). Risk factors were similar for VA of 20/200 or worse after OGR. Among the 1063 patients undergoing OGR with VA of 20/200 or worse at presentation, VA did not improve to better than 20/200 at follow-up in 35% of patients (1063/2996). CONCLUSIONS: Our findings bring to light racial disparities in risk of OGR and poor visual outcomes that warrant further exploration. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found after the references.

Interrelationships between structure and function during the hemostatic response to injury.

Extensive studies have detailed the molecular regulation of individual components of the hemostatic system, including platelets, coagulation factors, and regulatory proteins. Questions remain, however, about how these elements are integrated at the systems level within a rapidly changing physical environment. To answer some of these questions, we developed a puncture injury model in mouse jugular veins that combines high-resolution, multimodal imaging with functional readouts in vivo. The results reveal striking spatial regulation of platelet activation and fibrin formation that could not be inferred from studies performed ex vivo. As in the microcirculation, where previous studies have been performed, gradients of platelet activation are readily apparent, as is an asymmetrical distribution of fibrin deposition and thrombin activity. Both are oriented from the outer to the inner surface of the damaged vessel wall, with a greater extent of platelet activation and fibrin accumulation on the outside than the inside. Further, we show that the importance of P2Y12 signaling in establishing a competent hemostatic plug is related to the size of the injury, thus limiting its contribution to hemostasis to specific physiologic contexts. Taken together, these studies offer insights into the organization of hemostatic plugs, provide a detailed understanding of the adverse bleeding associated with a widely prescribed class of antiplatelet agents, and highlight differences between hemostasis and thrombosis that may suggest alternative therapeutic approaches.

A Human Vascular Injury-on-a-Chip Model of Hemostasis.

Hemostasis is an innate protective mechanism that plays a central role in maintaining the homeostasis of the vascular system during vascular injury. Studying this essential physiological process is often challenged by the difficulty of modeling and probing the complex dynamics of hemostatic responses in the native context of human blood vessels. To address this major challenge, this paper describes a microengineering approach for in vitro modeling of hemostasis. This microphysiological model replicates the living endothelium, multilayered microarchitecture, and procoagulant activity of human blood vessels, and is also equipped with a microneedle that is actuated with spatial precision to simulate penetrating vascular injuries. The system recapitulates key features of the hemostatic response to acute vascular injury as observed in vivo, including i) thrombin-driven accumulation of platelets and fibrin, ii) formation of a platelet- and fibrin-rich hemostatic plug that halts blood loss, and iii) matrix deformation driven by platelet contraction for wound closure. Moreover, the potential use of this model for drug testing applications is demonstrated by evaluating the effects of anticoagulants and antiplatelet agents that are in current clinical use. The vascular injury-on-a-chip may serve as an enabling platform for preclinical investigation of hematological disorders and emerging therapeutic approaches against them.

Use of electron microscopy to study platelets and thrombi.

Electron microscopy has been a valuable tool for the study of platelet biology and thrombosis for more than 70 years. Early studies using conventional transmission and scanning electron microscopy (EM) provided a foundation for our initial understanding of platelet structure and how it changes upon platelet activation. EM approaches have since been utilized to study platelets and thrombi in the context of basic, translational and clinical research, and they are instrumental in the diagnosis of multiple platelet function disorders. In this brief review, we provide a sampling of the many contributions EM based studies have made to the field, including both historical highlights and contemporary applications. We will also discuss exciting new imaging modalities based on EM and their utility for the study of platelets, hemostasis and thrombosis into the future.

Detection of Sub-fM DNA with Target Recycling and Self-Assembly Amplification on Graphene Field-Effect Biosensors.

All-electronic DNA biosensors based on graphene field-effect transistors (GFETs) offer the prospect of simple and cost-effective diagnostics. For GFET sensors based on complementary probe DNA, the sensitivity is limited by the binding affinity of the target oligonucleotide, in the nM range for 20 mer targets. We report a ∼20 000× improvement in sensitivity through the use of engineered hairpin probe DNA that allows for target recycling and hybridization chain reaction. This enables detection of 21 mer target DNA at sub-fM concentration and provides superior specificity against single-base mismatched oligomers. The work is based on a scalable fabrication process for biosensor arrays that is suitable for multiplexed detection. This approach overcomes the binding-affinity-dependent sensitivity of nucleic acid biosensors and offers a pathway toward multiplexed and label-free nucleic acid testing with high accuracy and selectivity.

A systems approach to hemostasis: 1. The interdependence of thrombus architecture and agonist movements in the gaps between platelets.

Hemostatic thrombi develop a characteristic architecture in which a core of highly activated platelets is covered by a shell of less-activated platelets. Here we have used a systems biology approach to examine the interrelationship of this architecture with transport rates and agonist distribution in the gaps between platelets. Studies were performed in mice using probes for platelet accumulation, packing density, and activation plus recently developed transport and thrombin activity probes. The results show that intrathrombus transport within the core is much slower than within the shell. The region of slowest transport coincides with the region of greatest packing density and thrombin activity, and appears prior to full platelet activation. Deleting the contact-dependent signaling molecule, Sema4D, delays platelet activation, but not the emergence of the low transport region. Collectively, these results suggest a timeline in which initial platelet accumulation and the narrowing gaps between platelets create a region of reduced transport that facilitates local thrombin accumulation and greater platelet activation, whereas faster transport rates within the shell help to limit thrombin accumulation and growth of the core. Thus, from a systems perspective, platelet accumulation produces an altered microenvironment that shapes thrombus architecture, which in turn affects agonist distribution and subsequent thrombus growth.

A systems approach to hemostasis: 2. Computational analysis of molecular transport in the thrombus microenvironment.

Hemostatic thrombi formed after a penetrating injury have a heterogeneous architecture in which a core of highly activated, densely packed platelets is covered by a shell of less-activated, loosely packed platelets. In the first manuscript in this series, we show that regional differences in intrathrombus protein transport rates emerge early in the hemostatic response and are preserved as the thrombus develops. Here, we use a theoretical approach to investigate this process and its impact on agonist distribution. The results suggest that hindered diffusion, rather than convection, is the dominant mechanism responsible for molecular movement within the thrombus. The analysis also suggests that the thrombus core, as compared with the shell, provides an environment for retaining soluble agonists such as thrombin, affecting the extent of platelet activation by establishing agonist-specific concentration gradients radiating from the site of injury. This analysis accounts for the observed weaker activation and relative instability of platelets in the shell and predicts that a failure to form a tightly packed thrombus core will limit thrombin accumulation, a prediction tested by analysis of data from mice with a defect in clot retraction.

A systems approach to hemostasis: 3. Thrombus consolidation regulates intrathrombus solute transport and local thrombin activity.

Hemostatic thrombi formed after a penetrating injury have a distinctive structure in which a core of highly activated, closely packed platelets is covered by a shell of less-activated, loosely packed platelets. We have shown that differences in intrathrombus molecular transport emerge in parallel with regional differences in platelet packing density and predicted that these differences affect thrombus growth and stability. Here we test that prediction in a mouse vascular injury model. The studies use a novel method for measuring thrombus contraction in vivo and a previously characterized mouse line with a defect in integrin αIIbß3 outside-in signaling that affects clot retraction ex vivo. The results show that the mutant mice have a defect in thrombus consolidation following vascular injury, resulting in an increase in intrathrombus transport rates and, as predicted by computational modeling, a decrease in thrombin activity and platelet activation in the thrombus core. Collectively, these data (1) demonstrate that in addition to the activation state of individual platelets, the physical properties of the accumulated mass of adherent platelets is critical in determining intrathrombus agonist distribution and platelet activation and (2) define a novel role for integrin signaling in the regulation of intrathrombus transport rates and localization of thrombin activity.

Investigating clot-flow interactions by integrating intravital imaging with in silico modeling for analysis of flow, transport, and hemodynamic forces.

As a blood clot forms, grows, deforms, and embolizes following a vascular injury, local clot-flow interactions lead to a highly dynamic flow environment. The local flow influences transport of biochemical species relevant for clotting, and determines the forces on the clot that in turn lead to clot deformation and embolization. Despite this central role, quantitative characterization of this dynamic clot-flow interaction and flow environment in the clot neighborhood remains a major challenge. Here, we propose an approach that integrates dynamic intravital imaging with computer geometric modeling and computational flow and transport modeling to develop a unified in silico framework to quantify the dynamic clot-flow interactions. We outline the development of the methodology referred to as Intravital Integrated In Silico Modeling or IVISim, and then demonstrate the method on a sample set of simulations comprising clot formation following laser injury in two mouse cremaster arteriole injury model data: one wild-type mouse case, and one diYF knockout mouse case. Simulation predictions are verified against experimental observations of transport of caged fluorescent Albumin (cAlb) in both models. Through these simulations, we illustrate how the IVISim methodology can provide insights into hemostatic processes, the role of flow and clot-flow interactions, and enable further investigations comparing and contrasting different biological model scenarios and parameter variations.

Disparities in Retinal Vein Occlusion Presentation and Initiation of Anti-VEGF Therapy: An Academy IRIS® Registry Analysis.

OBJECTIVE: Investigate disparities in retinal vein occlusion (RVO) presentation and initiation of anti-VEGF treatment. DESIGN: Retrospective cohort study. SUBJECTS: Patients in the American Academy of Ophthalmology IRIS® (Intelligent Research in Sight) Registry database (2015-2021) with branch or central RVO and macular edema (ME). METHODS: The association of demographic characteristics and presenting visual acuity (VA) with anti-VEGF treatment initiation were quantified using multivariable logistic regression. MAIN OUTCOME MEASURES: Treatment with ≥ 1 anti-VEGF injection within 12 months after RVO diagnosis. RESULTS: A total of 304 558 eligible patients with RVO and ME were identified. Age at presentation varied by race, ethnicity, sex, and RVO type (all P values < 0.001). Within the first year after RVO presentation, 192 602 (63.2%) patients received ≥ 1 anti-VEGF injection. In a multivariable regression model adjusting for relevant covariates, female (vs. male) patients had lower odds of receiving injections (odds ratio [OR], 0.95; 95% confidence interval [CI], 0.93-0.96; P < 0.0001) as did Black/African American (vs. White) patients (OR, 0.90; 95% CI, 0.88-0.92; P < 0.0001) and Asian (vs. White) patients (OR, 0.95; 95% CI, 0.91-0.99; P = 0.02), whereas older patients (vs. patients aged < 51 years) had higher odds (61-70 years: OR, 1.20; 95% CI, 1.16-1.24; 71-80 years: OR, 1.20; 95% CI, 1.16-1.24; > 80 years: OR, 1.15; 95% CI, 1.11-1.18; all P values < 0.0001). Hispanic (vs. non-Hispanic) patients had a small increased odds of treatment initiation (OR, 1.08; 95% CI, 1.04-1.11; P < 0.0001). Results were similar in the subset of 226 143 patients with VA data. In this subset, patients with presenting VA < 20/40 to 20/200 were most frequently treated in the first year after diagnosis (∼ 70%) and patients with light perception/no light perception (LP-NLP) vision or VA of 20/20 or better were treated least frequently (36.9% and 41.9%, respectively). CONCLUSIONS: In this large national clinical registry, 37% of RVO patients with ME had no anti-VEGF treatment documented in the first year after diagnosis. Black/African American, Asian, and female patients and patients with VA of LP-NLP were least likely to receive treatment. Awareness of this undertreatment and these disparities highlight the need for initiatives to ensure all RVO patients receive timely anti-VEGF injections for optimized visual outcomes. FINANCIAL DISCLOSURES: Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Increased platelet storage time is associated with mitochondrial dysfunction and impaired platelet function.

BACKGROUND: Hemorrhagic shock is a leading cause of death following severe trauma, and platelet transfusions are frequently necessary to achieve hemostasis. Platelets, however, require special storage conditions, and storage time has been associated with loss of platelet quality. We hypothesized that standard storage conditions have a deleterious effect on platelet mitochondrial function and platelet activation. MATERIALS AND METHODS: Platelet donations were collected from healthy donors (n = 5) and stored in gas-permeable collection bags according to American Association of Blood Bank recommendations. Platelet units were sampled from day of collection (day 0) until day 7. High-resolution respirometry was used to assess baseline mitochondrial respiration, maximal oxygen utilization, and individual mitochondrial complex-dependent respiration. Fluorescence-activated cell sorting was performed to analyze mitochondrial content, mitochondrial reactive oxygen species, the expression of P-selectin (both before and after challenge with thrombin receptor-activating peptide), and apoptosis. Data were analyzed using analysis of variance and Pearson correlation (P < 0.05 significant). RESULTS: Mitochondrial respiration decreased significantly in platelets stored longer than 2 d (P < 0.05). Platelets also demonstrated a persistent decrease in response to stimulation with thrombin receptor-activating peptide by the third day of storage (P < 0.05) as well as an increase in mitochondrial reactive oxygen species and apoptosis (P < 0.05). Mitochondrial respiration significantly correlated with platelet capacity to activate (r = 0.8, P < 0.05). CONCLUSIONS: Platelet mitochondrial respiratory function and activation response decrease significantly in platelets stored for 3 d or more. Because platelet transfusions almost universally occur between the third and fifth day of storage, our findings may have significant clinical importance and warrant further in vivo analysis.

Sympathetic Ophthalmia in Patients with Enucleation or Evisceration: Pathology Laboratory and IRIS® Registry Experience.

Introduction: Sympathetic ophthalmia (SO) is a rare bilateral granulomatous panuveitis that can follow surgical or nonsurgical ocular trauma in one eye. Because its diagnosis requires clinical-pathologic correlation, the true incidence of SO is unknown, and there is a need to understand the recent trends in risk factors and frequency of this condition. Methods: Pathology records of all enucleated or eviscerated (ENEV) eyes at three pathology laboratories were reviewed. Data collected included patient demographics, procedure indication, pathology diagnosis, and clinical history of trauma and uveitis. IRIS® Registry (Intelligent Research in Sight) was searched for all patients with SO, acquired absence of eye (AAE), and/or ENEV. Data obtained included patient demographics, ocular procedures, and preoperative diagnoses within 30 days of AAE/ENEV. Results: In the pathology laboratory setting, the incidence of SO over a 36-year period in patients who underwent ENEV was 0.2% (20/9,092); the 5-year incidence ranged from 0.0 to 0.3%. Among the 20 eyes with SO, the inciting event was surgical trauma in 50% (10/20), nonsurgical trauma in 45% (9/20), and missing/undetermined in 5% (1/20). SO was suspected preoperatively in 7/20 (35%) patients. Clinical concern for SO and ruptured globe were indications for ENEV in 50/9,092 (0.5%) and 872/9,092 (10%) patients, respectively. In the IRIS Registry, 0.7% (199/27,830) of patients with AAE/ENEV had diagnosis of SO. The frequency of SO between 2015 and 2020 was 0.01% (7,371/62,318,249); of these 7,371 cases, 199 (3%) had AAE/ENEV. In 25,975 patients with available data, injury and SO were listed as diagnoses less than 30 days prior to AAE/ENEV in 909 (4%) and 63 (0.2%) cases, respectively. Conclusion: The frequency of SO in recent decades has been low. Most cases of SO are not managed with eye removal. In histopathology-confirmed SO, surgical trauma is as frequent as nonsurgical trauma as an inciting etiology of disease.

Incidence and Risk Factors for Retinal Detachment and Retinal Tear after Cataract Surgery: IRIS® Registry (Intelligent Research in Sight) Analysis.

Objective: To report the incidence of and evaluate demographic, ocular comorbidities, and intraoperative factors for rhegmatogenous retinal detachment (RRD) and retinal tear (RT) after cataract surgery in the American Academy of Ophthalmology IRIS® Registry (Intelligent Research in Sight). Design: Retrospective cohort study. Participants: Patients aged ≥ 40 years who underwent cataract surgery between 2014 and 2017. Methods: Multivariable logistic regression was used to evaluate demographic, comorbidity, and intraoperative factors associated with RRD and RT after cataract surgery. Main Outcome Measures: Incidence and risk factors for RRD or RT within 1 year of cataract surgery. Results: Of the 3 177 195 eyes of 1 983 712 patients included, 6690 (0.21%) developed RRD and 5489 (0.17%) developed RT without RRD within 1 year after cataract surgery. Multivariable logistic regression odds ratios (ORs) showed increased risk of RRD and RT, respectively, among men (OR 3.15; 95% confidence interval [CI], 2.99-3.32; P < 0.001 and 1.79; 95% CI, 1.70-1.89; P < 0.001), and younger ages compared with patients aged > 70, peaking at age 40 to 50 for RRD (8.61; 95% CI, 7.74-9.58; P < 0.001) and age 50 to 60 for RT (2.74; 95% CI, 2.52-2.98; P < 0.001). Increased odds of RRD were observed for procedure eyes with lattice degeneration (LD) (10.53; 95% CI, 9.82-11.28; P < 0.001), hypermature cataract (1.61; 95% CI, 1.06-2.45; P = 0.03), complex cataract surgery (1.52; 95% CI, 1.4-1.66; P < 0.001), posterior vitreous detachment (PVD) (1.24; 95% CI, 1.15-1.34; P < 0.001), and high myopia (1.2; 95% CI, 1.14-1.27; P < 0.001). Lattice degeneration conferred the highest odds of RT (43.86; 95% CI, 41.39-46.49; P < 0.001). Conclusion: In the IRIS Registry, RRD occurs in approximately 1 in 500 cataract surgeries in patients aged > 40 years within 1 year of surgery. The presence of LD conferred the highest odds for RRD and RT after surgery. Additional risk factors for RRD included male gender, younger age, hypermature cataract, PVD, and high myopia. These data may be useful during the informed consent process for cataract surgery and help identify patients at a higher risk of retinal complications. Financial Disclosures: The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Models of electrical activity: calibration and prediction testing on the same cell.

Mathematical models are increasingly important in biology, and testability is becoming a critical issue. One limitation is that one model simulation tests a parameter set representing one instance of the biological counterpart, whereas biological systems are heterogeneous in their properties and behavior, and a model often is fitted to represent an ideal average. This is also true for models of a cell's electrical activity; even within a narrowly defined population there can be considerable variation in electrophysiological phenotype. Here, we describe a computational experimental approach for parameterizing a model of the electrical activity of a cell in real time. We combine the inexpensive parallel computational power of a programmable graphics processing unit with the flexibility of the dynamic clamp method. The approach involves 1), recording a cell's electrical activity, 2), parameterizing a model to the recording, 3), generating predictions, and 4), testing the predictions on the same cell used for the calibration. We demonstrate the experimental feasibility of our approach using a cell line (GH4C1). These cells are electrically active, and they display tonic spiking or bursting. We use our approach to predict parameter changes that can convert one pattern to the other.

Fast-activating voltage- and calcium-dependent potassium (BK) conductance promotes bursting in pituitary cells: a dynamic clamp study.

The electrical activity pattern of endocrine pituitary cells regulates their basal secretion level. Rat somatotrophs and lactotrophs exhibit spontaneous bursting and have high basal levels of hormone secretion, while gonadotrophs exhibit spontaneous spiking and have low basal hormone secretion. It has been proposed that the difference in electrical activity between bursting somatotrophs and spiking gonadotrophs is due to the presence of large conductance potassium (BK) channels on somatotrophs but not on gonadotrophs. This is one example where the role of an ion channel type may be clearly established. We demonstrate here that BK channels indeed promote bursting activity in pituitary cells. Blocking BK channels in bursting lacto-somatotroph GH4C1 cells changes their firing activity to spiking, while further adding an artificial BK conductance via dynamic clamp restores bursting. Importantly, this burst-promoting effect requires a relatively fast BK activation/deactivation, as predicted by computational models. We also show that adding a fast-activating BK conductance to spiking gonadotrophs converts the activity of these cells to bursting. Together, our results suggest that differences in BK channel expression may underlie the differences in electrical activity and basal hormone secretion levels among pituitary cell types and that the rapid rate of BK channel activation is key to its role in burst promotion.

GRK2 regulates ADP signaling in platelets via P2Y1 and P2Y12.

The critical role of G protein-coupled receptor kinase 2 (GRK2) in regulating cardiac function has been well documented for >3 decades. Targeting GRK2 has therefore been extensively studied as a novel approach to treating cardiovascular disease. However, little is known about its role in hemostasis and thrombosis. We provide here the first evidence that GRK2 limits platelet activation and regulates the hemostatic response to injury. Deletion of GRK2 in mouse platelets causes increased platelet accumulation after laser-induced injury in the cremaster muscle arterioles, shortens tail bleeding time, and enhances thrombosis in adenosine 5'-diphosphate (ADP)-induced pulmonary thromboembolism and in FeCl3-induced carotid injury. GRK2-/- platelets have increased integrin activation, P-selectin exposure, and platelet aggregation in response to ADP stimulation. Furthermore, GRK2-/- platelets retain the ability to aggregate in response to ADP restimulation, indicating that GRK2 contributes to ADP receptor desensitization. Underlying these changes in GRK2-/- platelets is an increase in Ca2+ mobilization, RAS-related protein 1 activation, and Akt phosphorylation stimulated by ADP, as well as an attenuated rise of cyclic adenosine monophosphate levels in response to ADP in the presence of prostaglandin I2. P2Y12 antagonist treatment eliminates the phenotypic difference in platelet accumulation between wild-type and GRK2-/- mice at the site of injury. Pharmacologic inhibition of GRK2 activity in human platelets increases platelet activation in response to ADP. Finally, we show that GRK2 binds to endogenous Gßγ subunits during platelet activation. Collectively, these results show that GRK2 regulates ADP signaling via P2Y1 and P2Y12, interacts with Gßγ, and functions as a signaling hub in platelets for modulating the hemostatic response to injury.

The contribution of TFPIα to the hemostatic response to injury in mice.

BACKGROUND: Tissue factor pathway inhibitor (TFPI) is an essential regulator of coagulation, limiting thrombin generation and preventing thrombosis. In humans and mice, TFPIα is the sole isoform present in platelets. OBJECTIVE: Here, we asked whether TFPIα, because of its release from platelets at sites of injury, has a unique role in limiting the hemostatic response. METHODS: TFPIα-mutant (TfpiΔα/Δα ) mice were generated by introducing a stop codon in the C-terminus. Platelet accumulation, platelet activation, and fibrin accumulation were measured following penetrating injuries in the jugular vein and cremaster muscle arterioles, and imaged by fluorescence and scanning electron microscopy. Time to bleeding cessation was recorded in the jugular vein studies. RESULTS: TfpiΔα/Δα mice were viable and fertile. Plasma TFPI levels were normal in the TfpiΔα/Δα mice, no TFPI protein or activity was present in their platelets and thrombin-antithrombin complex levels were indistinguishable from Tfpi+/+ littermates. There was a small, but statistically significant reduction in the time to bleeding cessation following jugular vein puncture injury in the TfpiΔα/Δα mice, but no measurable changes in platelet or fibrin accumulation or in hemostatic plug architecture following injury of the micro- or macrovasculature. CONCLUSION: Loss of TFPIα expression does not produce a global prothrombotic state in mice. Platelet TFPIα is expected to be released or displayed in a focal manner at the site of injury, potentially accumulating to high concentrations in the narrow gaps between platelets. If so, the data from the vascular injury models studied here indicate this is not essential for a normal hemostatic response in mice.

Modeling how reproductive ecology can drive protein diversification and result in linkage disequilibrium between sperm and egg proteins.

Gamete-recognition proteins determine whether sperm and eggs are compatible at fertilization, and they often evolve rapidly. The source of selection driving the evolution of these proteins is still debated. It has been suggested that sexual conflict can result in proliferation of genetic variation and possibly linkage disequilibrium between sperm and egg proteins. Empirical evidence suggests that both male and female reproductive success can be predicted by their sperm ligand genotype, but why female success can be predicted by a protein expressed only in males is unknown. Here we use mathematical modeling to investigate the interaction between reproductive behavior and sperm availability on the evolution of sperm ligands and egg receptors. We consider haploid and diploid expression in gametes in two possible ecological scenarios, monogamous spawning and competitive spawning. Reproductive behavior plays an important role in determining possible outcomes resulting from sexual conflict. Sperm limitation selects for common genotypes regardless of mating behavior. Under conditions of sperm abundance, competitive spawning provides conditions for the persistence of allelic variation and gametic disequilibrium. With monogamous spawning, such conditions are more restrictive.