Publications by authors named "Donald F Brophy"

60 Publications

Dietary Bioactive Fatty Acids as Modulators of Immune Function: Implications on Human Health.

Nutrients 2019 Dec 5;11(12). Epub 2019 Dec 5.

Department of Pharmacotherapy and Outcomes Sciences, School of Pharmacy, Virginia Commonwealth University, Richmond, VA 23298, USA.

Diet is major modifiable risk factor for cardiovascular disease that can influence the immune status of the individual and contribute to persistent low-grade inflammation. In recent years, there has been an increased appreciation of the role of polyunsaturated fatty acids (PUFA) in improving immune function and reduction of systemic inflammation via the modulation of pattern recognition receptors (PRR) on immune cells. Extensive research on the use of bioactive lipids such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) and their metabolites have illustrated the importance of these pro-resolving lipid mediators in modulating signaling through PRRs. While their mechanism of action, bioavailability in the blood, and their efficacy for clinical use forms an active area of research, they are found widely administered as marine animal-based supplements like fish oil and krill oil to promote health. The focus of this review will be to discuss the effect of these bioactive fatty acids and their metabolites on immune cells and the resulting inflammatory response, with a brief discussion about modern methods for their analysis using mass spectrometry-based methods.
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http://dx.doi.org/10.3390/nu11122974DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6950193PMC
December 2019

Effect of Vitamin C Infusion on Organ Failure and Biomarkers of Inflammation and Vascular Injury in Patients With Sepsis and Severe Acute Respiratory Failure: The CITRIS-ALI Randomized Clinical Trial.

JAMA 2019 10;322(13):1261-1270

Virginia Commonwealth University, Richmond.

Importance: Experimental data suggest that intravenous vitamin C may attenuate inflammation and vascular injury associated with sepsis and acute respiratory distress syndrome (ARDS).

Objective: To determine the effect of intravenous vitamin C infusion on organ failure scores and biological markers of inflammation and vascular injury in patients with sepsis and ARDS.

Design, Setting, And Participants: The CITRIS-ALI trial was a randomized, double-blind, placebo-controlled, multicenter trial conducted in 7 medical intensive care units in the United States, enrolling patients (N = 167) with sepsis and ARDS present for less than 24 hours. The study was conducted from September 2014 to November 2017, and final follow-up was January 2018.

Interventions: Patients were randomly assigned to receive intravenous infusion of vitamin C (50 mg/kg in dextrose 5% in water, n = 84) or placebo (dextrose 5% in water only, n = 83) every 6 hours for 96 hours.

Main Outcomes And Measures: The primary outcomes were change in organ failure as assessed by a modified Sequential Organ Failure Assessment score (range, 0-20, with higher scores indicating more dysfunction) from baseline to 96 hours, and plasma biomarkers of inflammation (C-reactive protein levels) and vascular injury (thrombomodulin levels) measured at 0, 48, 96, and 168 hours.

Results: Among 167 randomized patients (mean [SD] age, 54.8 years [16.7]; 90 men [54%]), 103 (62%) completed the study to day 60. There were no significant differences between the vitamin C and placebo groups in the primary end points of change in mean modified Sequential Organ Failure Assessment score from baseline to 96 hours (from 9.8 to 6.8 in the vitamin C group [3 points] and from 10.3 to 6.8 in the placebo group [3.5 points]; difference, -0.10; 95% CI, -1.23 to 1.03; P = .86) or in C-reactive protein levels (54.1 vs 46.1 μg/mL; difference, 7.94 μg/mL; 95% CI, -8.2 to 24.11; P = .33) and thrombomodulin levels (14.5 vs 13.8 ng/mL; difference, 0.69 ng/mL; 95% CI, -2.8 to 4.2; P = .70) at 168 hours.

Conclusions And Relevance: In this preliminary study of patients with sepsis and ARDS, a 96-hour infusion of vitamin C compared with placebo did not significantly improve organ dysfunction scores or alter markers of inflammation and vascular injury. Further research is needed to evaluate the potential role of vitamin C for other outcomes in sepsis and ARDS.

Trial Registration: ClinicalTrials.gov Identifier: NCT02106975.
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http://dx.doi.org/10.1001/jama.2019.11825DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6777268PMC
October 2019

Enhanced potency of recombinant factor VIIa with increased affinity to activated platelets.

J Thromb Haemost 2020 01 14;18(1):104-113. Epub 2019 Oct 14.

Department of Pharmacotherapy and Outcomes Science, Virginia Commonwealth University, Richmond, VA, USA.

Background: Recombinant factor VIIa (rFVIIa) enhances thrombin generation in a platelet-dependent manner; however, rFVIIa binds activated platelets with relatively low affinity. Triggering receptor expressed on myeloid cells (TREM)-like transcript (TLT)-1 is expressed exclusively on activated platelets.

Objective: To enhance the potency of rFVIIa via binding TLT-1.

Methods: Recombinant FVIIa was conjugated to a TLT-1 binding Fab. In vitro potency of this platelet-targeted rFVIIa (PT-rFVIIa) was evaluated using factor X activation assays and by measuring viscoelastic changes in whole blood. In vivo potency was evaluated using a tail vein transection model in F8 mice expressing human TLT-1.

Results: PT-rFVIIa and rFVIIa had similar dissociation constant values for tissue factor binding and similar tissue factor-dependent factor X activation. However, PT-rFVIIa had increased catalytic efficiency on TLT-1-loaded vesicles and activated platelets. The in vitro potency in normal human blood with antibody-induced hemophilia A was dependent on assay conditions used; with maximally activated platelets, the half maximal effective concentration for clot time for PT-rFVIIa was 49-fold lower compared with rFVIIa. In the murine bleeding model, a 53-fold lower half maximal effective concentration was observed for blood loss for PT-rFVIIa, supporting the relevance of the assay conditions with maximally activated platelets. In vitro analysis of blood from subjects with hemophilia A confirmed the data obtained with normal blood.

Conclusions: Increasing the affinity of rFVIIa to activated platelets resulted in approximately 50-fold increased potency both in vitro and in the mouse model. The correlation of in vivo with in vitro data using maximally activated platelets supports that these assay conditions are relevant when evaluating platelet-targeted hemostatic concepts.
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http://dx.doi.org/10.1111/jth.14644DOI Listing
January 2020

A synthetic heparin mimetic that allosterically inhibits factor XIa and reduces thrombosis in vivo without enhanced risk of bleeding.

J Thromb Haemost 2019 12 10;17(12):2110-2122. Epub 2019 Sep 10.

Department of Medicinal Chemistry, Virginia Commonwealth University, Richmond, Virginia.

Background: Human factor XIa (FXIa) is an actively pursued target for development of safer anticoagulants. Our long-standing hypothesis has been that allosterism originating from heparin-binding site(s) on coagulation enzymes is a promising approach to yield safer agents.

Objectives: To develop a synthetic heparin mimetic as an inhibitor of FXIa so as to reduce clot formation in vivo but not carry high bleeding risk.

Methods: We employed a gamut of methods involving synthetic chemistry, biophysical biochemistry, enzyme assays, blood and plasma coagulation assays, and in vivo thrombosis models in this work.

Results: Sulfated chiro-inositol (SCI), a non-saccharide mimetic of heparin, was synthesized in three steps in overall yields of >50%. SCI inhibited FXIa with potency of 280 nmol/L and preferentially engaged FXIa's heparin-binding site to conformationally alter its active site. SCI inhibition of FXIa could be rapidly reversed by common antidotes, such as protamine. SCI preferentially prolonged plasma clotting initiated with recalcification, rather than thromboplastin, alluding to its intrinsic pathway-based mechanism. Human blood thromboelastography indicated good ex vivo anticoagulation properties of SCI. Rat tail bleeding and maximum-dose-tolerated studies indicated that no major bleeding or toxicity concerns for SCI suggesting a potentially safer anticoagulation outcome. FeCl -induced arterial and thromboplastin-induced venous thrombosis model studies in the rat showed reduced thrombus formation by SCI at 250 μg/animal, which matched enoxaparin at 2500 μg/animal.

Conclusions: Overall, SCI is a highly promising, allosteric inhibitor of FXIa that induces potent anticoagulation in vivo. Further studies are necessary to assess SCI in animal models mimicking human clinical indications.
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http://dx.doi.org/10.1111/jth.14606DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6893084PMC
December 2019

Marstacimab, a tissue factor pathway inhibitor neutralizing antibody, improves coagulation parameters of ex vivo dosed haemophilic blood and plasmas.

Haemophilia 2019 Sep 23;25(5):797-806. Epub 2019 Jul 23.

Coagulation Advancement Laboratory, Department of Pharmacotherapy & Outcomes Science, Virginia Commonwealth University (VCU), Richmond, VA, USA.

Introduction: Tissue factor pathway inhibitor (TFPI) is an endogenous inhibitor of the extrinsic pathway that negatively regulates thrombin production during coagulation. Under haemophilic conditions, where the intrinsic coagulation pathway is impaired, inhibition of TFPI may improve clotting.

Aim: We investigated the ex vivo effects of a human TFPI neutralizing antibody, marstacimab (previously PF-06741086), in coagulation assays including rotational thromboelastometry (ROTEM), thrombin generation assay (TGA) and the dilute prothrombin time (dPT) assay, performed in haemophilic whole blood and plasmas. We compared the effects of marstacimab to the effects of recombinant coagulation factors and investigated the reproducibility of marstacimab in restoring haemostasis by comparing its effect in whole blood collected from the same study participants on differing days.

Methods: Citrated whole blood and plasmas obtained from haemophilia participants were supplemented ex vivo with vehicle, marstacimab, recombinant FVIII (rFVIII) or recombinant factor IX (rFIX) and analysed in ROTEM, TGA and the dPT assay using low tissue factor concentrations to trigger coagulation.

Results: Marstacimab induced pro-coagulant responses in ROTEM parameters including reduction in clotting times and increases in angle. Similarly, participant plasmas supplemented with marstacimab exhibited improvements in TGA parameters, including reduced lag times, increased peak thrombin concentrations and reductions in dPT clotting time. Concentrations of marstacimab tested showed activity comparable to addition of rFVIII or rFIX and were reproducible.

Conclusions: These studies show the ex vivo potency of marstacimab in restoring haemostasis in whole blood and plasmas from haemophilia participants and comparability to ex vivo reconstitution with recombination coagulation factors.
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http://dx.doi.org/10.1111/hae.13820DOI Listing
September 2019

Temporal map of the pig polytrauma plasma proteome with fluid resuscitation and intravenous vitamin C treatment.

J Thromb Haemost 2019 11 25;17(11):1827-1837. Epub 2019 Aug 25.

Department of Internal Medicine, Virginia Commonwealth University Medical Center, Richmond, Virginia.

Background: Fluid resuscitation plays a prominent role in stabilizing trauma patients with hemorrhagic shock yet there remains uncertainty with regard to optimal administration time, volume, and fluid composition (e.g., whole blood, component, colloids) leading to complications such as trauma-induced coagulopathies (TIC), acidosis, and poor oxygen transport. Synthetic fluids in combination with antioxidants (e.g., vitamin C) may resolve some of these problems.

Objectives: We applied quantitative mass spectrometry-based proteomics [liquid chromatography-mass spectrometry (LC-MS/MS)] to map the effects of fluid resuscitation and intravenous vitamin C (VitC) in a pig model of polytrauma (hemorrhagic shock, tissue injury, liver reperfusion, hypothermia, and comminuted bone fracture). The goal was to determine the effects of VitC on plasma protein expression, with respect to changes associated with coagulation and trauma-induced coagulopathy (TIC).

Methods: Longitudinal blood samples were drawn from nine male Sinclair pigs at baseline, 2 h post trauma, and 0.25, 2, and 4 h post fluid resuscitation with 500 mL hydroxyethyl starch. Pigs were treated intravenously (N = 3/treatment group) with saline, 50 mg VitC/kg (Lo-VitC), or 200 mg VitC/kg (Hi-VitC) during fluid resuscitation.

Results: A total of 436 plasma proteins were quantified of which 136 changed following trauma and resuscitation; 34 were associated with coagulation, complement cascade, and glycolysis. Unexpectedly, Lo-VitC and Hi-VitC treatments stabilized ADAMTS13 levels by ~4-fold (P = .056) relative to saline and enhanced ADAMTS13/von Willebrand factor (VWF) cleavage efficiency based on LC-MS/MS evidence for the semitryptic VWF cleavage product (VWF ).

Conclusions: This study provides the first comprehensive map of trauma-induced changes to the plasma proteome, especially with respect to proteins driving the development of TIC.
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http://dx.doi.org/10.1111/jth.14580DOI Listing
November 2019

Effects of a novel low volume resuscitation solutions on coagulation and platelet function.

PLoS One 2019 1;14(5):e0215386. Epub 2019 May 1.

Department of Surgery, Division of Acute Care Surgery, Virginia Commonwealth University School of Medicine, Richmond, VA, United States of America.

Background: Novel crystalloid solutions containing polyethylene glycol polymers (PEG-20k) produce dramatic resuscitation effects but dose-dependently produce a hypocoagulative state. The objective of this study was to examine possible mechanisms of this effect. Based on previous thromboelastography data, we hypothesize the effect is largely due to platelet interactions with the polymers.

Methods: Whole citrated blood from healthy volunteers was diluted ex-vivo 10% with crystalloids and tested for coagulation and platelet function. The specific tests included prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen and von Willebrand factor (vWf) activity, thrombin generation, thromboelastography with and without platelet mapping, platelet flow cytometry, and erythrocyte sedimentation rate.

Findings: Fibrinogen and vWF activities, PT, and aPTT were not affected by PEG-20k dilutions. Thrombin activity was mildly suppressed with PEG-20k (TTP- 20%). Platelet mapping demonstrated significantly greater % inhibition of both ADP and arachidonic acid-induced platelet aggregation with PEG-20k, but direct ADP-activated gpIIa/IIIb (PAC1) and P-selectin (CD62P) binding site expression was not altered. Mild dose-dependent suppression of TEG-MA was seen with PEG-20k using platelet poor plasma. Erythrocyte Sedimentation Rates (ESR) were dramatically accelerated after dilution with 10% PEG-20k, which was competitively blocked by smaller PEG polymers, suggesting nonspecific PEG-20k cell binding effects.

Conclusions: PEG-20k creates a mild hypocoagulative state in whole blood at concentrations ≥10%, which may be due to platelet-PEG interactions at the IIb/IIIa interface with lesser effects on fibrin polymerization. This interaction may cause a functional thrombasthenia induced by nonspecific platelet surface passivation by the PEG polymer.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0215386PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6493729PMC
January 2020

Platelets retain inducible alpha granule secretion by P-selectin expression but exhibit mechanical dysfunction during trauma-induced coagulopathy.

J Thromb Haemost 2019 05 18;17(5):771-781. Epub 2019 Mar 18.

Department of Emergency Medicine, University of Washington, Seattle, WA, USA.

Essentials Platelets in trauma-induced coagulopathy (TIC) are impaired, but the mechanism is not known. We performed comprehensive longitudinal platelet function testing in trauma patient samples. Platelets in TIC are widely impaired early after injury, but platelet activatability is intact. This suggests a mechanism of transient platelet cytoskeletal/integrin dysfunction during TIC. SUMMARY: Background Trauma-induced coagulopathy (TIC) is a common and deadly bleeding disorder. Platelet dysfunction is present during TIC, but its mechanisms remain unclear. Platelets are currently thought to become "exhausted," a state in which they have released their granule contents and can no longer aggregate or contract. Methods This prospective observational cohort study tested the hypothesis that platelet exhaustion is present during TIC and characterized the early time course of platelet dysfunction. Blood was collected from 95 adult trauma patients at a Level I trauma center at time of Emergency Department arrival and several time points over 72 h. Platelet activation state and function were characterized using CD62P (P-selectin) and PAC-1 surface membrane staining, platelet function analyzer (PFA-100), aggregometry, viscoelastic platelet mapping, and, to test for exhaustion, their ability to express CD62P after ex vivo adenosine diphosphate (ADP) agonism. Platelet function was compared between patients with and without TIC, defined by prothrombin time ≥18 s. Results Platelets in TIC showed no initial increase in their level of surface activation markers or impairment of their capacity to express CD62P in response to ADP stimulation. However, TIC platelets were impaired in nearly all functional assays, spanning adhesion, aggregation, and contraction. These effects largely remained after controlling for platelet count and fibrinogen concentration and resolved after 8 h. Conclusion The TIC platelets exhibit early impairment of adhesion, aggregation, and contraction with retained alpha granule secretion ability, suggesting a specific mechanism of cytoskeletal or integrin dysfunction that is not a result of more general platelet exhaustion.
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http://dx.doi.org/10.1111/jth.14414DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6494686PMC
May 2019

Use of global assays to monitor emicizumab prophylactic therapy in patients with haemophilia A with inhibitors.

Haemophilia 2019 03 12;25(2):e121-e123. Epub 2019 Feb 12.

Division of Hematology/Oncology, VCU School of Medicine, Richmond, Virginia.

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http://dx.doi.org/10.1111/hae.13689DOI Listing
March 2019

Thromboelastographic analysis of novel polyethylene glycol based low volume resuscitation solutions.

PLoS One 2018 15;13(11):e0207147. Epub 2018 Nov 15.

Department of Surgery, Division of Acute Care Surgery, Virginia Commonwealth University School of Medicine, Richmond, Virginia, United States of America.

Background: Low volume resuscitation (LVR) in shock prevents deleterious effects of crystalloid loading in pre-hospital settings. Polyethylene glycol 20,000 (PEG-20k) based LVR solutions are 20-fold more effective at maintaining perfusion and survival in shock compared to conventional crystalloids. The aim of this study was to determine coagulation and platelet function of whole blood treated with 10% PEG-20k.

Methods: Citrated blood from volunteers (n = 25) or early admission severely injured trauma patients (n = 9) were diluted 10% with various LVR solutions in a matched design with a paired volume control (saline), and studied using thromboelastography (TEG).

Findings: In healthy volunteers and patients, 10% PEG-20k significantly increased clot amplification time (k), decreased propagation (angle), maximal clot size and strength (MA), and the overall coagulation index (CI), but not clot initiation (R) or fibrinolysis (Ly30), relative to paired saline dilutional controls. Clinically, K, angle, and MA were just outside of the normal limits in volunteers but not in patients. No statistical differences existed between PEG-20k and Hextend (HES) in either patient population. In a dose response series using volunteer blood, all effects of 10% PEG-20k on TEG were reversed and normalized by lower concentrations (7.5% and 5%). Furthermore, 7.5% PEG-20k produced similar resuscitation effects as 10% PEG in rodent hemorrhagic shock models (n = 5).

Conclusions: In conclusion, PEG-20k based LVR solutions produced a dose-dependent minor hypocoagulative state, possibly associated with changes in clot propagation and platelet function, which can be reversed by dose reduction in concentration while providing superior LVR, microvascular rescue, and lactate clearance compared to saline or starch.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0207147PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6237338PMC
April 2019

A Unique Nonsaccharide Mimetic of Heparin Hexasaccharide Inhibits Colon Cancer Stem Cells via p38 MAP Kinase Activation.

Mol Cancer Ther 2019 01 18;18(1):51-61. Epub 2018 Oct 18.

Division of Hematology and Oncology, Department of Internal Medicine, Massey Cancer Center, Virginia Commonwealth University, Richmond, Virginia.

Targeting of cancer stem cells (CSC) is expected to be a paradigm-shifting approach for the treatment of cancers. Cell surface proteoglycans bearing sulfated glycosaminoglycan (GAG) chains are known to play a critical role in the regulation of stem cell fate. Here, we show for the first time that G2.2, a sulfated nonsaccharide GAG mimetic (NSGM) of heparin hexasaccharide, selectively inhibits colonic CSCs G2.2-reduced CSCs (CD133/CXCR4, Dual hi) induced HT-29 and HCT 116 colon xenografts' growth in a dose-dependent fashion. G2.2 also significantly delayed the growth of colon xenograft further enriched in CSCs following oxaliplatin and 5-fluorouracil treatment compared with vehicle-treated xenograft controls. In fact, G2.2 robustly inhibited CSCs' abundance (measured by levels of CSC markers, e.g., CD133, DCMLK1, LGR5, and LRIG1) and self-renewal (quaternary spheroids) in colon cancer xenografts. Intriguingly, G2.2 selectively induced apoptosis in the Dual hi CSCs eluding to its CSC targeting effects. More importantly, G2.2 displayed none to minimal toxicity as observed through morphologic and biochemical studies of vital organ functions, blood coagulation profile, and analyses of normal intestinal (and bone marrow) progenitor cell growth. Through extensive , and mechanistic studies, we showed that G2.2's inhibition of CSC self-renewal was mediated through activation of p38α, uncovering important signaling that can be targeted to deplete CSCs selectively while minimizing host toxicity. Hence, G2.2 represents a first-in-class (NSGM) anticancer agent to reduce colorectal CSCs.
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http://dx.doi.org/10.1158/1535-7163.MCT-18-0104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6332501PMC
January 2019

Continuous-Flow Left Ventricular Assist Device-Related Gastrointestinal Bleeding.

Cardiol Clin 2018 Nov;36(4):519-529

Advanced Heart Failure and Transplant, Pauley Heart Center, Virginia Commonwealth University, 1200 East Broad Street, PO Box: 980204, Richmond, VA 23298-0036, USA. Electronic address:

Continuous-flow (CF) left ventricular assist devices (LVADs) are a safe and durable therapeutic option for patients with advanced heart failure as a bridge to transplant or as destination therapy. Despite the remarkable technological advances in device design and increasing familiarity with the physiologic effects of CF, major complications such as gastrointestinal bleeding (GIB) continue to cause significant morbidity. The causes underlying CF-LVAD-related GIB are multifactorial. Accordingly, management strategies for CF-LVAD-related GIB encompass a wide range of therapeutic modalities. This article reviews the epidemiology, pathophysiology, risk factors, and treatment options for the management of this complex complication.
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http://dx.doi.org/10.1016/j.ccl.2018.06.006DOI Listing
November 2018

Informing efficient pilot development of animal trauma models through quality improvement strategies.

Lab Anim 2019 Aug 9;53(4):394-404. Epub 2018 Oct 9.

3 Department of Internal Medicine, Virginia Commonwealth University Medical Center, Richmond, USA.

Poor quality data in preclinical trials can result from inconsistent and unstandardized experimental processes. Unpredictable pre-intervention variability generates unreliable data, biases outcomes and results in needless waste of animals and resources. We applied Define-Measure-Analyse-Improve-Control (DMAIC) quality improvement processes to pilot development of a swine model of trauma, haemorrhagic shock and coagulopathy. The goal was to reduce variability through protocol standardization and error reduction. Six male Sinclair swine were sequentially anesthetized, intubated, mechanically ventilated and instrumented, then subjected to multiple-hit injury, followed by fluid resuscitation monitoring and coagulation testing. Experimental tasks were defined and mapped. Performance measures were task performance times, subject stabilization time and number of task execution errors. Process improvement was assessed by reduced times and errors, and subject stability at target physiological values. Previously-overlooked performance errors and deficiencies were identified. 'Mistake-proofing' actions included personnel retraining, revisions of standard operating procedures and use of checklists. The quality improvement pilot trial produced a stable model with reduced protocol deviations. Data quality can be improved and animal waste minimized, if experimental planning incorporates strategies to ensure protocol adherence and reduced operator performance variation and errors. Properly designed pilot trials can be essential components of refinement and reduction strategies in animal-based research.
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http://dx.doi.org/10.1177/0023677218802999DOI Listing
August 2019

Evaluating the thrombin generation profiles of four different rFVIII products in FVIII-deficient plasma using FIXa and FXIa activation.

Haemophilia 2018 Sep 16;24(5):815-822. Epub 2018 Aug 16.

Novo Nordisk A/S, Bagsvaerd, Denmark.

Introduction: The thrombin generation assay (TGA) can be used to monitor factor replacement therapy in patients with haemophilia. The TGA assay is typically performed using tissue factor as the reaction activator; however, activating with FIXa or FXIa can enhance assay sensitivity when FVIII < 1%.

Aims: To evaluate the sensitivity of the TGA when FIXa (5 nmol/L) and FXIa (0.22 nmol/L) are used to activate the assay in platelet-poor plasma and to compare these data to the one-stage and chromogenic assays.

Methods: Plasma from 10 severe FVIII-deficient subjects was supplemented with FVIII (0%, 0.1%, 0.4%, 1.2%, 4%, 11% and 33%), using either Novo Eight , Advate , Eloctate , turoctocog alfa pegol or a control standard. The one-stage and chromogenic assays quantified the FVIII levels. The TGA assay was activated using either FIXa or FXIa.

Results: Both FIXa- and FXIa-activated TGA were sensitive across FVIII concentrations, with intra-assay coefficient of variation (CV) < 10%. The FXIa-activated assay had 25% CV at the lowest level of FVIII compared to 10% CV with FIXa activation. There were strong correlations between the FIXa- and FXIa-activated TGA tests (R  = 0.9912) and between the one-stage and chromogenic assays (R  = 0.9469). However, there were poor relationships between the TGA tests and one-stage and chromogenic assays.

Conclusions: Both FIXa- and FXIa activation results in similar TGA profiles across a FVIII range of 0.1%-33%; however, FIXa activation was more robust at the lowest levels of FVIII compared with FXIa activation.
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http://dx.doi.org/10.1111/hae.13597DOI Listing
September 2018

Interventional vitamin C: A strategy for attenuation of coagulopathy and inflammation in a swine multiple injuries model.

J Trauma Acute Care Surg 2018 07;85(1S Suppl 2):S57-S67

From the Department of Anesthesiology (P.S.R., B.D.S.), Department of Internal Medicine (B.J.F., J.M., C.S., P.M., M.E., E.F., A.A.F., R.N.), Department of Pharmacotherapy and Outcomes Science (E.J.M., D.F.B.), Virginia Commonwealth University, Richmond, Virginia; Department of Anesthesiology (P.S.R., B.D.S.), University of Florida, Gainesville, Florida; and Naval Medical Center Portsmouth (R.N.), Portsmouth, Virginia.

Background: Coagulopathy and inflammation induced by hemorrhagic shock and traumatic injury are associated with increased mortality and morbidity. Vitamin C (VitC) is an antioxidant with potential protective effects on the proinflammatory and procoagulant pathways. We hypothesized that high-dose VitC administered as a supplement to fluid resuscitation would attenuate inflammation, coagulation dysfunction, and end-organ tissue damage in a swine model of multiple injuries and hemorrhage.

Methods: Male Sinclair swine (n = 24; mean body weight, 27 kg) were anesthetized, intubated, mechanically ventilated, and instrumented for physiologic monitoring. Following stabilization, swine were subjected to shock/traumatic injury (hypothermia, liver ischemia and reperfusion, comminuted femur fracture, hemorrhagic hypotension), resuscitated with 500 mL of hydroxyethyl starch, and randomized to receive either intravenous normal saline (NS), low-dose VitC (50 mg/kg; LO), or high-dose VitC (200 mg/kg; HI). Hemodynamics, blood chemistry, hematology, and coagulation function (ROTEM) were monitored to 4 hours postresuscitation. Histological and molecular analyses were obtained for liver, kidney, and lung.

Results: Compared with VitC animals, NS swine showed significant histological end-organ damage, elevated acute lung injury scores, and increased mRNA expression of tissue proinflammatory mediators (IL-1β, IL-8, TNFα), plasminogen activation inhibitor-1 and tissue factor. There were no statistically significant differences between treatment groups on mean arterial pressure or univariate measures of coagulation function; however, NS showed impaired multivariate clotting function at 4 hours.

Conclusion: Although correction of coagulation dysfunction was modest, intravenous high-dose VitC may mitigate the proinflammatory/procoagulant response that contributes to multiple organ failure following acute severe multiple injuries.

Level Of Evidence: Prospective randomized controlled blinded trial study, Preclinical (animal-based).
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http://dx.doi.org/10.1097/TA.0000000000001844DOI Listing
July 2018

Metabolomics and Precision Medicine in Trauma: The State of the Field.

Shock 2018 07;50(1):5-13

Department of Surgery, Division of Acute Care Surgical Services, School of Medicine, Virginia Commonwealth University, Richmond, Virginia.

Trauma is a major problem in the United States. Mortality from trauma is the number one cause of death under the age of 45 in the United States and is the third leading cause of death for all age groups. There are approximately 200,000 deaths per year due to trauma in the United States at a cost of over $671 billion in combined healthcare costs and lost productivity. Unsurprisingly, trauma accounts for approximately 30% of all life-years lost in the United States. Due to immense development of trauma systems, a large majority of trauma patients survive the injury, but then go on to die from complications arising from the injury. These complications are marked by early and significant metabolic changes accompanied by inflammatory responses that lead to progressive organ failure and, ultimately, death. Early resuscitative and surgical interventions followed by close monitoring to identify and rescue treatment failures are key to successful outcomes. Currently, the adequacy of resuscitation is measured using vital signs, noninvasive methods such as bedside echocardiography or stroke volume variation, and other laboratory endpoints of resuscitation, such as lactate and base deficit. However, these methods may be too crude to understand cellular and subcellular changes that may be occurring in trauma patients. Better diagnostic and therapeutic markers are needed to assess the adequacy of interventions and monitor responses at a cellular and subcellular level and inform clinical decision-making before complications are clinically apparent. The developing field of metabolomics holds great promise in the identification and application of biochemical markers toward the clinical decision-making process.
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http://dx.doi.org/10.1097/SHK.0000000000001093DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5995639PMC
July 2018

Multicenter Evaluation of Octreotide as Secondary Prophylaxis in Patients With Left Ventricular Assist Devices and Gastrointestinal Bleeding.

Circ Heart Fail 2017 Nov;10(11)

From The Pauley Heart Center (K.B.S., S.G., P.A.U., M.L.S.), Department of Pharmacotherapy and Outcomes Research (D.F.B.), and Division of Gastroenterology (G.B.S.), Virginia Commonwealth University, Richmond; Division of Cardiovascular Medicine, The Ohio State University, Columbus (S.E.); McGinnis Cardiovascular Institute, Allegheny General Hospital, Pittsburgh, PA (M.K.K.); Division of Cardiology, University of Chicago, IL (N.U.); Division of Cardiology, Columbia University, New York, NY (P.C.C.); and Abbott, Pleasanton, CA (J.C., D.J.F.).

Background: Gastrointestinal (GI) bleeding is one of the most common complications after continuous-flow left ventricular assist device implantation. More than one third of patients with incident bleed go on to develop recurrent GI bleeding. Octreotide, a somatostatin analog, is proposed to reduce the risk of recurrent GI bleeding in this population.

Methods And Results: This multicenter, retrospective analysis evaluated 51 continuous-flow left ventricular assist device patients who received secondary prophylaxis with octreotide after their index GI bleed from 2009 to 2015. All patients had a hospitalization for GI bleed and received octreotide after discharge. Patient demographics, medical and medication history, and clinical characteristics of patients who rebled after receiving octreotide were compared with non-rebleeders. These data were also compared with matched historical control patients previously enrolled in the HMII (HeartMate II) clinical trials, none of whom received octreotide, to provide a context for the bleeding rates. Twelve patients (24%) who received secondary octreotide prophylaxis developed another GI bleed, whereas 39 (76%) did not. There were similar intergroup demographics; however, significantly more bleeders had a previous GI bleeding history before left ventricular assist device placement (33% versus 5%; =0.02) and greater frequency of angiodysplasia confirmed during endoscopy (58% versus 23%; =0.03). Fewer patients in this study experienced a recurrent GI bleed compared with a matched historical control group that did not receive octreotide (24% versus 43%; =0.04).

Conclusions: Patients with continuous-flow left ventricular assist device receiving secondary prophylaxis with octreotide had a significantly lower GI bleed recurrence compared with historical controls not treated with octreotide. Additional prospective studies are needed to confirm these data.
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http://dx.doi.org/10.1161/CIRCHEARTFAILURE.117.004500DOI Listing
November 2017

Impact of high dose vitamin C on platelet function.

World J Crit Care Med 2017 Feb 4;6(1):37-47. Epub 2017 Feb 4.

Bassem M Mohammed, Department of Clinical Pharmacy, Faculty of Pharmacy, Cairo University, Cairo 11562, Egypt.

Aim: To examine the effect of high doses of vitamin C (VitC) on human platelets (PLTs).

Methods: Platelet concentrates collected for therapeutic or prophylactic transfusions were exposed to: (1) normal saline (control); (2) 0.3 mmol/L VitC (Lo VitC); or (3) 3 mmol/L VitC (Hi VitC, final concentrations) and stored appropriately. The VitC additive was preservative-free buffered ascorbic acid in water, pH 5.5 to 7.0, adjusted with sodium bicarbonate and sodium hydroxide. The doses of VitC used here correspond to plasma VitC levels reported in recently completed clinical trials. Prior to supplementation, a baseline sample was collected for analysis. PLTs were sampled again on days 2, 5 and 8 and assayed for changes in PLT function by: Thromboelastography (TEG), for changes in viscoelastic properties; aggregometry, for PLT aggregation and adenosine triphosphate (ATP) secretion in response to collagen or adenosine diphosphate (ADP); and flow cytometry, for changes in expression of CD-31, CD41a, CD62p and CD63. In addition, PLT intracellular VitC content was measured using a fluorimetric assay for ascorbic acid and PLT poor plasma was used for plasma coagulation tests [prothrombin time (PT), partial thrombplastin time (PTT), functional fibrinogen] and Lipidomics analysis (UPLC ESI-MS/MS).

Results: VitC supplementation significantly increased PLTs intracellular ascorbic acid levels from 1.2 mmol/L at baseline to 3.2 mmol/L (Lo VitC) and 15.7 mmol/L (Hi VitC, < 0.05). VitC supplementation did not significantly change PT and PTT values, or functional fibrinogen levels over the 8 d exposure period ( > 0.05). PLT function assayed by TEG, aggregometry and flow cytometry was not significantly altered by Lo or Hi VitC for up to 5 d. However, PLTs exposed to 3 mmol/L VitC for 8 d demonstrated significantly increased R and K times by TEG and a decrease in the α-angle ( < 0.05). There was also a fall of 20 mm in maximum amplitude associated with the Hi VitC compared to both baseline and day 8 saline controls. Platelet aggregation studies, showed uniform declines in collagen and ADP-induced platelet aggregations over the 8-d study period in all three groups ( > 0.05). Collagen and ADP-induced ATP secretion was also not different between the three groups ( > 0.05). Finally, VitC at the higher dose (3 mmol/L) also induced the release of several eicosanoids including thromboxane B and prostaglandin E, as well as products of arachidonic acid metabolism the lipoxygenases pathway such as 11-/12-/15-hydroxyicosatetraenoic acid ( < 0.05).

Conclusion: Alterations in PLT function by exposure to 3 mmol/L VitC for 8 d suggest that caution should be exerted with prolonged use of intravenous high dose VitC.
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http://dx.doi.org/10.5492/wjccm.v6.i1.37DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5295168PMC
February 2017

Unsupervised analysis of combined lipid and coagulation data reveals coagulopathy subtypes among dialysis patients.

J Lipid Res 2017 03 19;58(3):586-599. Epub 2016 Dec 19.

Department of Pharmacotherapy and Outcomes Science, School of Pharmacy, Virginia Commonwealth University (VCU), Richmond, VA

Hemodialysis (HD) and peritoneal dialysis (PD) are the primary means of managing end stage renal disease (ESRD). However, these treatment modalities are associated with the onset of coagulation abnormalities. Effective management of coagulation risk among these patients requires the identification of surrogate markers that provide an early indication of the coagulation abnormalities. The role of sphingolipids in the manifestation and prediction of coagulation abnormalities among dialysis patients have never been investigated. Herein, we report the first instance of an in depth investigation into the sphingolipid changes among ESRD patients undergoing HD and PD. The results reveal distinct differences in terms of perturbations to specific sphingolipid biosynthetic pathways that are highly dependent on the treatment modality. Our studies also demonstrated strong correlation between specific sphingolipids and coagulation parameters, such as HexCer(d18:1/26:0) and maximal amplitude (MA), SM(d18:1/24:1) and tissue factor pathway inhibitor, and sphingosine 1-phosphate d18:1 and FX (Spearman ρ of 0.93, 0.89, and -0.89, respectively). Furthermore, our study revealed the potential for using HexCer(d18:1/22:0), HexCer(d18:1/24:0), and HexCer(d18:1/26:0) (r = 0.71, 0.82, and 0.63, respectively) and coagulation parameter MA (r = 0.7) for successful diagnosis of differential coagulopathies among ESRD patients undergoing HD, providing an opportunity toward personalized disease management.
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http://dx.doi.org/10.1194/jlr.P068833DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5335589PMC
March 2017

Tolerability and Biological Effects of Long-Acting Octreotide in Patients With Continuous Flow Left Ventricular Assist Devices.

ASAIO J 2017 May/Jun;63(3):367-370

From the *Division of Pulmonary and Critical Care Medicine, Virginia Commonwealth University (VCU), Richmond, Virginia; †Division of Cardiology, Pauley Heart Center, VCU, Richmond, Virginia; ‡Department of Internal Medicine, VCU, Richmond, Virginia; §Johnson Center for Critical Care and Pulmonary Research, VCU, Richmond, Virginia; and ¶Department of Pharmacotherapy and Outcomes Science, VCU, Richmond, Virginia.

Patients with implanted continuous, nonpulsatile, left ventricular assist devices (LVADs) have increased the occurrence of gastrointestinal bleeding (GIB). Although the pathophysiology is multifactorial, there are few treatments beyond supportive care. Octreotide acetate is a somatostatin analog that reduces GIB in various patient populations. However, there are sparse case series that suggest octreotide acetate may reduce GIB in LVAD patients. This 10 patient, 28 week phase I study evaluated the safety and tolerability of octreotide acetate long-acting release (LAR) 20 mg depot injection every 4 weeks until week 16 after LVAD placement. Secondary aims were occurrence of GIB and measurement of vascular endothelial growth factor, fibrinogen, von Willebrand factor, and platelet aggregation across the study period. Ten patients were enrolled, and eight completed the study. The two study dropouts were not related to octreotide. None of the patients experienced side effects or safety concerns related to octreotide nor did GIB occur in the study population. Vascular endothelial growth factor levels were maintained in the reference range throughout the duration of the study. There did appear to be laboratory evidence of acquired von Willebrand syndrome, with mildly low platelet aggregation studies. In conclusion, octreotide acetate LAR 20 mg depot injection was safe and effective in this population.
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http://dx.doi.org/10.1097/MAT.0000000000000491DOI Listing
January 2018

Post-translational oxidative modification of fibrinogen is associated with coagulopathy after traumatic injury.

Free Radic Biol Med 2016 07 20;96:181-9. Epub 2016 Apr 20.

Bloodworks Northwest Research Institute, 1551 Eastlake Avenue E, Seattle, WA 98102, USA.

Victims of trauma often develop impaired blood clot formation (coagulopathy) that contributes to bleeding and mortality. Fibrin polymerization is one critical component of clot formation that can be impacted by post-translational oxidative modifications of fibrinogen after exposure to oxidants. In vitro evidence suggests that Aα-C domain methionine sulfoxide formation, in particular, can induce conformational changes that prevent lateral aggregation of fibrin protofibrils during polymerization. We used mass spectrometry of plasma from trauma patients to find that fibrinogen Aα-C domain methionine sulfoxide content was selectively-increased in patients with coagulopathy vs. those without coagulopathy. This evidence supports a novel linkage between oxidative stress, coagulopathy, and bleeding after injury.
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http://dx.doi.org/10.1016/j.freeradbiomed.2016.04.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4912420PMC
July 2016

Vitamin C promotes wound healing through novel pleiotropic mechanisms.

Int Wound J 2016 Aug 20;13(4):572-84. Epub 2015 Aug 20.

Division of Pulmonary Disease and Critical Care Medicine, Department of Internal Medicine, Virginia Commonwealth University, Richmond, VA, USA.

Vitamin C (VitC) or ascorbic acid (AscA), a cofactor for collagen synthesis and a primary antioxidant, is rapidly consumed post-wounding. Parenteral VitC administration suppresses pro-inflammatory responses while promoting anti-inflammatory and pro-resolution effects in human/murine sepsis. We hypothesised that VitC could promote wound healing by altering the inflammatory, proliferative and remodelling phases of wound healing. Mice unable to synthesise VitC (Gulo(-/-) ) were used in this study. VitC was provided in the water (sufficient), withheld from another group (deficient) and supplemented by daily intra-peritoneal infusion (200 mg/kg, deficient + AscA) in a third group. Full thickness excisional wounds (6 mm) were created and tissue collected on days 7 and 14 for histology, quantitative polymerase chain reaction (qPCR) and Western blotting. Human neonatal dermal fibroblasts (HnDFs) were used to assess effects of In conclusion, VitC favorably on proliferation. Histological analysis showed improved wound matrix deposition and organisation in sufficient and deficient +AscA mice. Wounds from VitC sufficient and deficient + AscA mice had reduced expression of pro-inflammatory mediators and higher expression of wound healing mediators. Supplementation of HnDF with AscA induced the expression of self-renewal genes and promoted fibroblast proliferation. VitC favourably impacts the spatiotemporal expression of transcripts associated with early resolution of inflammation and tissue remodelling.
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http://dx.doi.org/10.1111/iwj.12484DOI Listing
August 2016

Laboratory assessment of warfarin reversal with global coagulation tests versus international normalized ratio in patients with intracranial bleeding.

Blood Coagul Fibrinolysis 2015 Jun;26(4):443-7

aDepartment of Pharmacotherapy and Translational Research, University of Florida College of Pharmacy, Gainesville, Florida bCoagulation Advancement Laboratory, Department of Pharmacotherapy and Outcomes Science, Virginia Commonwealth University, Richmond, Virginia, USA cFaculty of Pharmacy, Department of Clinical Pharmacy, Cairo University, Cairo, Egypt dNeuroscience Intensive Care Unit, Virginia Commonwealth University Medical Center, Richmond, Virginia, USA.

We assess the in-vivo relationship between international normalized ratio (INR) and global coagulation tests in patients with life-threatening bleeding who received prothrombin complex concentrate (PCC) for warfarin reversal. This was a prospective pilot study in adult patients with intracranial bleeding related to anticoagulation with warfarin. Thromboelastography (TEG), thrombin generation parameters and INR were assessed at baseline, 30  min, 2 and 24  h after PCC. Changes in laboratory parameters and relationship between INR and global coagulation tests were assessed over time. Eight patients mean [standard deviation (SD)] age 72 (16) were included and received mean (SD) dose of PCC 24 (5) units/kg. Four patients died during the study, all with INR values more than 1.5 thirty minutes after PCC. Mean (SD) INR was 3.0 (1.3) and decreased significantly to 1.8 (0.48) thirty minutes after PCC (P < 0.01). Baseline endogenous thrombin potential and thrombin peak were 890  nmol/min and 123  nmol and increased significantly to 1943  nmol/min (P < 0.01) and 301  nmol (P < 0.01) 30  min after PCC administration. Reaction (R)-time decreased significantly (P = 0.02), and maximum amplitude and overall coagulation index (CI) significantly increased during treatment (P < 0.01, respectively). Thrombin generation and TEG values corrected after PCC administration; however, INR did not fully correct. Patients that died tended to be older with prolonged INR values across the study period. INR and TEG values correlated well with thrombin generation before administration of PCC, but this relationship was lost afterward.
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http://dx.doi.org/10.1097/MBC.0000000000000270DOI Listing
June 2015

Clot Formation Is Associated With Fibrinogen and Platelet Forces in a Cohort of Severely Injured Emergency Department Trauma Patients.

Shock 2015 Aug;44 Suppl 1:39-44

*Division of Emergency Medicine, University of Washington, and Puget Sound Blood Center Research Institute, Seattle, WA; †Coagulation Advancement Laboratory, Department of Pharmacotherapy & Outcomes Science, Virginia Commonwealth University, Richmond, VA; ‡Department of Clinical Pharmacy, Faculty of Pharmacy, Cairo University, Cairo, Egypt; §Pharmacotherapy & Outcomes Science and Department of Neurosurgery, Virginia Commonwealth University, Richmond, VA; ‖Department of Anesthesiology, Virginia Commonwealth University, Richmond, VA; ¶US Army Medical Research and Materiel Command, Fort Detrick, MD; #Michigan Center for Integrative Research in Critical Care, University of Michigan, Ann Arbor, Michigan.

Introduction: Anticoagulation, fibrinogen consumption, fibrinolytic activation, and platelet dysfunction all interact to produce different clot formation responses after trauma. However, the relative contributions of these coagulation components to overall clot formation remain poorly defined. We examined for sources of heterogeneity in clot formation responses after trauma.

Methods: Blood was sampled in the emergency department from patients meeting trauma team activation criteria at an urban trauma center. Plasma prothrombin time of 18 s or longer was used to define traumatic coagulopathy. Mean kaolin-activated thrombelastography (TEG) parameters were calculated and tested for heterogeneity using analysis of means. Discriminant analysis and forward stepwise variable selection with linear regression were used to determine if prothrombin time, fibrinogen, platelet contractile force (PCF), and D-dimer concentration, representing key mechanistic components of coagulopathy, each contribute to heterogeneous TEG responses after trauma.

Results: Of 95 subjects, 16% met criteria for coagulopathy. Coagulopathic subjects were more severely injured with greater shock and received more blood products in the first 8 h compared with noncoagulopathic subjects. Mean (SD) TEG maximal amplitude (MA) was significantly decreased in the coagulopathic group (57.5 [SD, 4.7] mm vs. 62.7 [SD, 4.7], t test P < 0.001). The MA also exceeded the ANOM predicted upper decision limit for the noncoagulopathic group and the lower decision limit for the coagulopathic group at α = 0.05, suggesting significant heterogeneity from the overall cohort mean. Fibrinogen and PCF best discriminated TEG MA using discriminant analysis. Fibrinogen, PCF, and D-dimer were primary covariates for TEG MA using regression analysis.

Conclusions: Heterogeneity in TEG-based clot formation in emergency department trauma patients was linked to changes in MA. Individual parameters representing fibrin polymerization, PCFs, and fibrinolysis were primarily associated with TEG MA after trauma and should be the focus of early hemostatic therapies.
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http://dx.doi.org/10.1097/SHK.0000000000000342DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4504845PMC
August 2015

Intact stable isotope labeled plasma proteins from the SILAC-labeled HepG2 secretome.

Proteomics 2015 Sep 14;15(18):3104-15. Epub 2015 Feb 14.

Department of Pharmaceutics, Virginia Commonwealth University, Richmond, VA, USA.

The plasma proteome remains an attractive biospecimen for MS-based biomarker discovery studies. The success of these efforts relies on the continued development of quantitative MS-based proteomics approaches. Herein we report the use of the SILAC-labeled HepG2 secretome as a source for stable isotope labeled plasma proteins for quantitative LC-MS/MS measurements. The HepG2 liver cancer cell line secretes the major plasma proteins including serum albumin, apolipoproteins, protease inhibitors, coagulation factors, and transporters that represent some of the most abundant proteins in plasma. The SILAC-labeled HepG2 secretome was collected, spiked into human plasma (1:1 total protein), and then processed for LC-MS/MS analysis. A total of 62 and 56 plasma proteins were quantified (heavy:light (H/L) peptide pairs) from undepleted and depleted (serum albumin and IgG), respectively, with log2 H/L = ± 6. Major plasma proteins quantified included albumin, apolipoproteins (e.g., APOA1, APOA2, APOA4, APOB, APOC3, APOE, APOH, and APOM), protease inhibitors (e.g., A2M and SERPINs), coagulation factors (e.g., Factor V, Factor X, fibrinogen), and transport proteins (e.g., TTR). The average log2 H/L values for shared plasma proteins in both undepleted and depleted plasma samples were 0.43 and 0.44, respectively. This work further expands the SILAC strategy into MS-based biomarker discovery of clinical biospecimens.
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http://dx.doi.org/10.1002/pmic.201400369DOI Listing
September 2015

Resolution of sterile inflammation: role for vitamin C.

Mediators Inflamm 2014 9;2014:173403. Epub 2014 Sep 9.

Division of Pulmonary Disease and Critical Care Medicine, Department of Internal Medicine, Virginia Commonwealth University, P.O. Box 980050, Richmond, VA 23298-0050, USA.

Introduction: Macrophage reprogramming is vital for resolution of acute inflammation. Parenteral vitamin C (VitC) attenuates proinflammatory states in murine and human sepsis. However information about the mechanism by which VitC regulates resolution of inflammation is limited.

Methods: To examine whether physiological levels of VitC modulate resolution of inflammation, we used transgenic mice lacking L-gulono-γ-lactone oxidase. VitC sufficient/deficient mice were subjected to a thioglycollate-elicited peritonitis model of sterile inflammation. Some VitC deficient mice received daily parenteral VitC (200 mg/kg) for 3 or 5 days following thioglycollate infusion. Peritoneal macrophages harvested on day 3 or day 5 were examined for intracellular VitC levels, pro- and anti-inflammatory protein and lipid mediators, mitochondrial function, and response to lipopolysaccharide (LPS). The THP-1 cell line was used to determine the modulatory activities of VitC in activated human macrophages.

Results: VitC deficiency significantly delayed resolution of inflammation and generated an exaggerated proinflammatory response to in vitro LPS stimulation. VitC sufficiency and in vivo VitC supplementation restored macrophage phenotype and function in VitC deficient mice. VitC loading of THP-1 macrophages attenuated LPS-induced proinflammatory responses.

Conclusion: VitC sufficiency favorably modulates macrophage function. In vivo or in vitro VitC supplementation restores macrophage phenotype and function leading to timely resolution of inflammation.
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http://dx.doi.org/10.1155/2014/173403DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4175383PMC
June 2015

Targeting the GPIbα binding site of thrombin to simultaneously induce dual anticoagulant and antiplatelet effects.

J Med Chem 2014 Apr 25;57(7):3030-9. Epub 2014 Mar 25.

Department of Medicinal Chemistry and Institute for Structural Biology and Drug Discovery, Virginia Commonwealth University , Richmond, Virginia 23219, United States.

Exosite 2 of human thrombin contributes to two opposing pathways, the anticoagulant pathway and the platelet aggregation pathway. We reasoned that an exosite 2 directed allosteric thrombin inhibitor should simultaneously induce anticoagulant and antiplatelet effects. To assess this, we synthesized SbO4L based on the sulfated tyrosine-containing sequence of GPIbα. SbO4L was synthesized in three simple steps in high yield and found to be a highly selective, direct inhibitor of thrombin. Michelis-Menten kinetic studies indicated a noncompetitive mechanism of inhibition. Competitive inhibition studies suggested ideal competition with heparin and glycoprotein Ibα, as predicted. Studies with site-directed mutants of thrombin indicated that SbO4L binds to Arg233, Lys235, and Lys236 of exosite 2. SbO4L prevented thrombin-mediated platelet activation and aggregation as expected on the basis of competition with GPIbα. SbO4L presents a novel paradigm of simultaneous dual anticoagulant and antiplatelet effects achieved through the GPIbα binding site of thrombin.
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http://dx.doi.org/10.1021/jm4020026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4203406PMC
April 2014

Phase I safety trial of intravenous ascorbic acid in patients with severe sepsis.

J Transl Med 2014 Jan 31;12:32. Epub 2014 Jan 31.

Division of Pulmonary Disease and Critical Care Medicine, Department of Internal Medicine, School of Medicine, Virginia Commonwealth University, PO Box 980050, Richmond, VA 23298-0050, USA.

Background: Parenterally administered ascorbic acid modulates sepsis-induced inflammation and coagulation in experimental animal models. The objective of this randomized, double-blind, placebo-controlled, phase I trial was to determine the safety of intravenously infused ascorbic acid in patients with severe sepsis.

Methods: Twenty-four patients with severe sepsis in the medical intensive care unit were randomized 1:1:1 to receive intravenous infusions every six hours for four days of ascorbic acid: Lo-AscA (50 mg/kg/24 h, n = 8), or Hi-AscA (200 mg/kg/24 h, n = 8), or Placebo (5% dextrose/water, n = 8). The primary end points were ascorbic acid safety and tolerability, assessed as treatment-related adverse-event frequency and severity. Patients were monitored for worsened arterial hypotension, tachycardia, hypernatremia, and nausea or vomiting. In addition Sequential Organ Failure Assessment (SOFA) scores and plasma levels of ascorbic acid, C-reactive protein, procalcitonin, and thrombomodulin were monitored.

Results: Mean plasma ascorbic acid levels at entry for the entire cohort were 17.9 ± 2.4 μM (normal range 50-70 μM). Ascorbic acid infusion rapidly and significantly increased plasma ascorbic acid levels. No adverse safety events were observed in ascorbic acid-infused patients. Patients receiving ascorbic acid exhibited prompt reductions in SOFA scores while placebo patients exhibited no such reduction. Ascorbic acid significantly reduced the proinflammatory biomarkers C-reactive protein and procalcitonin. Unlike placebo patients, thrombomodulin in ascorbic acid infused patients exhibited no significant rise, suggesting attenuation of vascular endothelial injury.

Conclusions: Intravenous ascorbic acid infusion was safe and well tolerated in this study and may positively impact the extent of multiple organ failure and biomarkers of inflammation and endothelial injury.

Trial Registration: ClinicalTrials.gov identifier NCT01434121.
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http://dx.doi.org/10.1186/1479-5876-12-32DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3937164PMC
January 2014