Vegetative hyphal fusion is not essential for plant infection by Fusarium oxysporum

Vegetative hyphal fusion (VHF) is a ubiquitous phenomenon in filamentous fungi whose biological role is poorly understood. In Neurospora crassa, the mitogen-activated protein kinase (MAPK) Mak-2 and the WW domain protein So are required for efficient VHF. A MAPK orthologous to Mak-2, Fmk1, was previously shown to be essential for root penetration and pathogenicity of the vascular wilt fungus Fusarium oxysporum. Here we took a genetic approach to test two hypotheses, that (i) VHF and plant infection have signaling mechanisms in common and (ii) VHF is required for efficient plant infection. F. oxysporum mutants lacking either Fmk1 or Fso1, an orthologue of N. crassa So, were impaired in the fusion of vegetative hyphae and microconidial germ tubes. Δfmk1 Δfso1 double mutants exhibited a more severe fusion phenotype than either single mutant, indicating that the two components function in distinct pathways. Both Δfso1 and Δfmk1 strains were impaired in the formation of hyphal networks on the root surface, a process associated with extensive VHF. The Δfso1 mutants exhibited slightly reduced virulence in tomato fruit infection assays but, in contrast to Δfmk1 strains, were still able to perform functions associated with invasive growth, such as secretion of pectinolytic enzymes or penetration of cellophane sheets, and to infect tomato plants. Thus, although VHF per se is not essential for plant infection, both processes have some signaling components in common, suggesting an evolutionary relationship between the underlying cellular mechanisms.     

Chronic consumption of farmed salmon containing persistent organic pollutants causes insulin resistance and obesity in mice

Background

Dietary interventions are critical in the prevention of metabolic diseases. Yet, the effects of fatty fish consumption on type 2 diabetes remain unclear. The aim of this study was to investigate whether a diet containing farmed salmon prevents or contributes to insulin resistance in mice.

Methodology/Principal Findings

Adult male C57BL/6J mice were fed control diet (C), a very high-fat diet without or with farmed Atlantic salmon fillet (VHF and VHF/S, respectively), and Western diet without or with farmed Atlantic salmon fillet (WD and WD/S, respectively). Other mice were fed VHF containing farmed salmon fillet with reduced concentrations of persistent organic pollutants (VHF/S_-POPs). We assessed body weight gain, fat mass, insulin sensitivity, glucose tolerance, ex vivo muscle glucose uptake, performed histology and immunohistochemistry analysis, and investigated gene and protein expression. In comparison with animals fed VHF and WD, consumption of both VHF/S and WD/S exaggerated insulin resistance, visceral obesity, and glucose intolerance. In addition, the ability of insulin to stimulate Akt phosphorylation and muscle glucose uptake was impaired in mice fed farmed salmon. Relative to VHF/S-fed mice, animals fed VHF/S_-POPs had less body burdens of POPs, accumulated less visceral fat, and had reduced mRNA levels of TNFα as well as macrophage infiltration in adipose tissue. VHF/S_-POPs-fed mice further exhibited better insulin sensitivity and glucose tolerance than mice fed VHF/S.

Conclusions/Significance

Our data indicate that intake of farmed salmon fillet contributes to several metabolic disorders linked to type 2 diabetes and obesity, and suggest a role of POPs in these deleterious effects. Overall, these findings may participate to improve nutritional strategies for the prevention and therapy of insulin resistance.     

Treatment of Platelet Products with Riboflavin and UV Light: Effectiveness Against High Titer Bacterial Contamination

Contamination of platelet units by bacteria has long been acknowledged as a significant transfusion risk due to their post-donation storage conditions. Products are routinely stored at 22 °C on an agitating shaker, a condition that can promote bacterial growth. Although the total number of bacteria believed to be introduced into a platelet product is extremely low, these bacteria can multiply to a very high titer prior to transfusion, potentially resulting in serious adverse events. The aim of this study was to evaluate a riboflavin based pathogen reduction process against a panel of bacteria that have been identified as common contaminants of platelet products. This panel included the following organisms: S. epidermidis, S. aureus, S. mitis, S. pyogenes, S. marcescens, Y. enterocolitica, B. neotomae, B. cereus, E. coli, P. aeruginosa and K. pneumoniae. Each platelet unit was inoculated with a high bacterial load and samples were removed both before and after treatment. A colony forming assay, using an end point dilution scheme, was used to determine the pre-treatment and post-treatment bacterial titers. Log reduction was calculated by subtracting the post-treatment titer from the pre-treatment titer. The following log reductions were observed: S. epidermidis 4.7 log (99.998%), S. aureus 4.8 log (99.998%), S. mitis 3.7 log (99.98%), S. pyogenes 2.6 log (99.7%), S. marcescens 4.0 log (99.99%), Y. enterocolitica 3.3 log (99.95%), B. neotomae 5.4 log (99.9996%), B. cereus 2.6 log (99.7%), E. coli ≥5.4 log (99.9996%), P. aeruginosa 4.7 log (99.998%) and K. pneumoniae 2.8 log (99.8%). The results from this study suggest the process could help to lower the risk of severe adverse transfusion events associated with bacterial contamination.     

Plasma Components and Platelet Activation Are Essential for the Antimicrobial Properties of Autologous Platelet-Rich Plasma: An In Vitro Study

Autologous platelet concentrates are successfully adopted in a variety of medical fields to stimulate bone and soft tissue regeneration. The rationale for their use consists in the delivery of a wide range of platelet-derived bioactive molecules that promotes wound healing. In addition, antimicrobial properties of platelet concentrates have been pointed out. In this study, the effect of the platelet concentration, of the activation step and of the presence of plasmatic components on the antimicrobial activity of pure platelet-rich plasma was investigated against gram positive bacteria isolated from oral cavity. The antibacterial activity, evaluated as the minimum inhibitory concentration, was determined through the microdilution two-fold serial method. Results seem to suggest that the antimicrobial activity of platelet-rich plasma against Enterococcus faecalis, Streptococcus agalactiae, Streptococcus oralis and Staphylococcus aureus is sustained by a co-operation between plasma components and platelet-derived factors and that the activation of coagulation is a fundamental step. The findings of this study may have practical implications in the modality of application of platelet concentrates.     

Docking based design of diastereoisomeric MTCA as GPIIb/IIIa receptor inhibitor

In GPIIb/IIIa mediated arterial thrombosis platelet activation plays a central role. To discover platelet activation inhibitor the pharmacophores of GPIIb/IIIa receptor inhibitors and anti-thrombotic agents were analyzed. This led to the design of (1R,3S)- and (1S,3S)-1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acids as GPIIb/IIIa inhibitors. Comparing to (1S,3S)-isomer (1R,3S)-isomer had lower cdocker interaction energy. AFM image showed that the minimal effective concentration of (1S,3S)-isomer and (1R,3S)-isomer inhibiting platelet activation were 10^?5?M and 10^?6?M, respectively. In vivo 1?μmol/kg of oral (1S,3S)-isomer effectively inhibited the rats to form arterial thrombus and down regulated GPIIb/IIIa expression, but the activities were significantly lower than those of 1?μmol/kg of oral (1R,3S)-isomer. Both (1S,3S)-isomer and (1R,3S)-isomer can be safely used for structural modifications, but (1R,3S)-isomer should be superior to (1S,3S)-isomer.     

Vasopressin inhibits the adenylate cyclase activity of human platelet particulate fraction through V1-receptors

Arg⁸-vasopressin inhibited the adenylate cyclase activity of human platelet particulate fraction up to a maximum of 27% (IC₅₀ = 1.2 nM). This inhibition required the presence of 10 μM GTP and was optimal with 100 mM NaCl. Orn⁸-vasopressin had similar effects. 1-Deamino-Val⁴, D-Arg⁸-vasopressin did not by itself affect adenylate cyclase activity but competitively inhibited the action of Arg⁸-vasopressin (pA₂ = 7.74). Arg⁸-vasopressin did not inhibit adenylate cyclase in intact platelets but instead caused platelet aggregation, an effect that was also competitively inhibited by 1-deamino-Val⁴, D-Arg⁸-vasopressin (pA₂ = 7.82). Thus, platelets possess vasopressin receptors of the V₁ type that, under appropriate conditions, can mediate either inhibition of platelet adenylate cyclase or platelet aggregation.     

Effects of epoxymethano analogues of prostaglandin endoperoxides on aggregation,on release of 5-hydroxytryptamine and on the metabolism of 3′,5′-cyclic AMP and cyclic GMP in human platelets

The effects on human platelets of two synthetic analogues of prostaglandin endoperoxides were examined in order to explore the relationship between aggregation and prostaglandin and cyclic nucleotide metabolism, and to help elucidate the role of the natural endoperoxide intermediates in regulating platelet function.Both analogues (Compound I, (15S)-hydroxy-9α,11α-(epoxymethano)-prosta-(5Z,13E)-dienoic acid, and Compound II, (15S)-hydroxy-11α,9α-(epoxymethano)-prosta-(5Z,13E)-dienoic acid) caused platelets to aggregate, an effect which could be inhibited by prostaglandin E₁ but not by indomethacin. Compound II produced primary, reversible aggregation at concentrations which did not induce release of 5-hydroxytryptamine. Production of thromboxane B₂ and malonyldialdehyde was monitored as an index of endogenous production of prostaglandin endoperoxides and thromboxane A₂ and were increased after incubation of human platelets with thrombin, collagen or arachidonic acid. However, neither malonydialdehyde nor thromboxane B₂ levels were significantly influenced by the endoperoxide analogues. Both analogues produced a small elevation of adenylate cyclase activity in platelet membranes and of cyclic AMP content in intact platelets, but neither had any modifying effect on the much greater stimulation of adenylate cyclase and cyclic AMP levels by prostaglandin E₁. Of all the aggregating agents tested, only arachidonic acid produced any significant increase in platelet cyclic GMP levels.These results suggest that the epoxymethano analogues of prostaglandin endoperoxides induce platelet aggregation independently of thromboxane biosynthesis and without inhibiting adenylate cyclase or lowerin platelet cyclic AMP levels. They therefore differ from better known aggregating agents such as ADP, epinephrine and collagen, which increase thromboxane A₂ production and reduce cyclic AMP levels, at least in platelets previously exposed to prostaglandin E₁.     

Correlation of DNA methylation patterns to the phenotypic features of Tibetan elite alpinists in extreme hypoxia

High altitude is an extreme environment that imposes hypoxic pressure on physiological processes, and natives living at high altitudes are more adaptive in certain physiological processes. So far, epigenetic modifications under extreme changes in hypoxic pressures are relatively less understood. Here, we recruit 32 Tibetan elite alpinists (TEAs), who have successfully mounted Everest (8848 m) at least five times. Blood samples and physiological phenotypes of TEAs and 32 matched non-alpinist Tibetan volunteers (non-TEAs) are collected for analysis. Genome-wide DNA methylation analysis identifies 23,202 differentially methylated CpGs (P_adj < 0.05, |β| > 0.1) between the two groups. Some differentially methylated CpGs are in hypoxia-related genes such as PPP1R13L, MAP3K7CL, SEPTI-9, and CUL2. In addition, Gene ontology enrichment analysis reveals several inflammation-related pathways. Phenotypic analysis indicates that 12 phenotypes are significantly different between the two groups. In particular, TEAs exhibit higher blood oxygen saturation levels and lower neutrophil count, platelet count, and heart rate. For DNA methylation association analysis, we find that two CpGs (cg16687447, cg06947206) upstream of PTEN were associated with platelet count. In conclusion, extreme hypoxia exposure leads to epigenetic modifications and phenotypic alterations of TEA, providing us clues for exploring the molecular mechanism underlying changes under extreme hypoxia conditions.     

Oral Exposure to Phytomonas serpens Attenuates Thrombocytopenia and Leukopenia during Acute Infection with Trypanosoma cruzi

Mice infected with Trypanosoma cruzi, the agent of Chagas disease, rapidly develop anemia and thrombocytopenia. These effects are partially promoted by the parasite trans-sialidase (TS), which is shed in the blood and depletes sialic acid from the platelets, inducing accelerated platelet clearance and causing thrombocytopenia during the acute phase of disease. Here, we demonstrate that oral immunization of C57BL/6 mice with Phytomonas serpens, a phytoflagellate parasite that shares common antigens with T. cruzi but has no TS activity, reduces parasite burden and prevents thrombocytopenia and leukopenia. Immunization also reduces platelet loss after intraperitoneal injection of TS. In addition, passive transfer of immune sera raised in mice against P. serpens prevented platelet clearance. Thus, oral exposure to P. serpens attenuates the progression of thrombocytopenia induced by TS from T. cruzi. These findings are not only important for the understanding of the pathogenesis of T. cruzi infection but also for developing novel approaches of intervention in Chagas disease.     

First laboratory confirmation and sequencing of Zaire ebolavirus in Uganda following two independent introductions of cases from the 10th Ebola Outbreak in the Democratic Republic of the Congo,June 2019

Uganda established a domestic Viral Hemorrhagic Fever (VHF) testing capacity in 2010 in response to the increasing occurrence of filovirus outbreaks. In July 2018, the neighboring Democratic Republic of Congo (DRC) experienced its 10^th Ebola Virus Disease (EVD) outbreak and for the duration of the outbreak, the Ugandan Ministry of Health (MOH) initiated a national EVD preparedness stance. Almost one year later, on 10^th June 2019, three family members who had contracted EVD in the DRC crossed into Uganda to seek medical treatment.Samples were collected from all the suspected cases using internationally established biosafety protocols and submitted for VHF diagnostic testing at Uganda Virus Research Institute. All samples were initially tested by RT-PCR for ebolaviruses, marburgviruses, Rift Valley fever (RVF) virus and Crimean-Congo hemorrhagic fever (CCHF) virus. Four people were identified as being positive for Zaire ebolavirus, marking the first report of Zaire ebolavirus in Uganda. In-country Next Generation Sequencing (NGS) and phylogenetic analysis was performed for the first time in Uganda, confirming the outbreak as imported from DRC at two different time point from different clades. This rapid response by the MoH, UVRI and partners led to the control of the outbreak and prevention of secondary virus transmission.     

Requirements for different Ca2+ pools in the activation of rabbit platelets: I. Release reaction and protein phosphorylation

We examined the role of Ca²⁺, both extracellular and intracellular in origin, in the release reaction and protein phosphorylation in rabbit platelets stimulated with platelet activating factor (acetylglyceryl ether phosphorylcholine), thrombin, or ionophore A23187. In the presence of extracellular Ca²⁺, 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8), a putative antagonist of intracellular Ca²⁺ transport, blocked platelet activating factor-initiated serotonin release at a half-maximal inhibitor concentration of 40 μM, compared to 350 μM for thrombin-induced release and greater than 500 μM, for A23187-induced release. Platelet activating factor-induced phosphorylation of two platelet proteins of M_r=41 000 (P7P) and 20 000 (P9P) was inhibited by TMB-8, an effect which was additive to that caused by removing extracellular Ca²⁺. TMB-8 demonstrated only minor to non-existant inhibitory effects on phosphorylation in thrombin- or A23187-stimulated platelets. In contrast to P9P phosphorylation, phosphorylation of P7P caused by platelet activating factor was more dependent on a TMB-8 sensitive step than on the availability of extracellular Ca²⁺. Experiments with buffers containing fixed concentrations of free Ca²⁺ revealed that both processes (release and phosphorylation), when stimulated by platelet activating factor and thrombin, had the same threshold requirement (1–3 μM) for extracellular free Ca²⁺. These studies provide evidence that stimulation of rabbit platelets by platelet activating factor is more dependent on a TMB-8-sensitive intracellular Ca²⁺ source than is stimulation caused by thrombin. Furthermore, our data indicate that activation of different intracellular processes involved in platelet secretion (such as P7P and P9P phosphorylation) may require Ca²⁺ from different pools.     

Decrease of platelet aggregation and spreading via inhibition of the cAMP phosphodiesterase by trapidil

Trapidil (N,N-diethyl-5-methyl[l,2,4]triazolo[l,5-α]pyrimidine-7-amine) inhibits platelet spreading and aggregation induced by arachidonic acid (AA), a stable analogue of prostaglandin (PG) endoperoxides (U46619), ADP, and low concentrations of thrombin, but not by A23187 and high concentrations of thrombin. Trapidil does not affect platelet adenylate cyclase but inhibits the cAMP PDE by approx. 50%. PDE inhibition proceeds via a competitive mechanism (K_i = 0.52 mM) and is not mediated by calmodulin inhibition. Trapidil does not change the platelet basal cAMP level but potentiates an increase of cAMP induced by the stable prostacyclin analogue (6β-PGI_i). These results suggest that trapidil antiplatelet effects may be due to the inhibition of platelet PDE.     

Requirement for different Ca2+ pools in the activation of rabbit platelets: II. Phospholipase activity

The effects of extracellular Ca²⁺ concentration and the putative antagonist of intracellular Ca²⁺ movement, 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) on platelet phospholipase activity and thromboxane B₂ synthesis were examined in rabbit platelets stimulated by platelet activating factor, thrombin and ionophore A23187. TMB-8 markedly inhibited the platelet activating factor-induced decrease in [¹⁴C]arachidonate content in platelet phsophatidylacholine and phosphatidylinositol, while showing minimal effects on thrombin-induced phospholipase activation. A23187 stimulation of these processes was inhibited to an intermediated degree by TMB-8. In contrast, extracellular Ca²⁺ removal inhibited phospholipase activity to a similar degree with all three stimuli. Moreover, the threshold concentration of extracelullar Ca²⁺ for phospholiphase activation, as measured by thromboxane B₂ synthesis, was similar for platelet activating factor- and thrombin-stimulated platelets. The data provide evidence that, while platelet activating factor and thrombin may, to some extent, have similar requirements for extracellular Ca²⁺, they utilize a TMB-8 sensitive step to different degrees during activation of platelet phospholipase.     

N49 phospholipase A2, a unique subgroup of snake venom group II phospholipase A2

A novel phospholipase A₂ (PLA₂) with Asn at its site 49 was purified from the snake venom of Protobothrops mucrosquamatus by using SP-Sephadex C25, Superdex 75, Heparin-Sepharose (FF) and HPLC reverse-phage C₁₈ chromatography and designated as TM-N49. It showed a molecular mass of 13.875 kDa on MALDI-TOF. TM-N49 does not possess enzymatic, hemolytic and hemorrhagic activities. It fails to induce platelet aggregation by itself, and does not inhibit the platelet aggregation induced by ADP. However, it exhibits potent myotoxic activity causing inflammatory cell infiltration, severe myoedema, myonecrosis and myolysis in the gastrocnemius muscles of BALB/c mice. Phylogenetic analysis found that that TM-N49 combined with two phospholipase A₂s from Trimeresurus stejnegeri, TsR6 and CTs-R6 cluster into one group. Structural and functional analysis indicated that these phospholipase A₂s are distinct from the other subgroups (D49 PLA₂, S49 PLA₂ and K49 PLA₂) and represent a unique subgroup of snake venom group II PLA₂, named N49 PLA₂ subgroup.     

Polymorphisms in Canine Platelet Glycoproteins Identify Potential Platelet Antigens

Human alloimmune thrombocytopenic conditions caused by exposure to a platelet-specific alloantigen include neonatal alloimmune thrombocytopenia, posttransfusion purpura, and platelet transfusion refractoriness. More than 30 platelet-specific alloantigens have been defined in the human platelet antigen (HPA) system; however, there is no previous information on canine platelet-specific alloantigens. Using the HPA system as a model, we evaluated the canine ITGB3, ITGA2B, and GP1BB genes encoding GPIIIa (β₃), GPIIb (α_IIb), and GPIbβ, respectively, which account for 21 of 27 HPA, to determine whether amino acid polymorphisms are present in the orthologous canine genes. A secondary objective was to perform a pilot study to assess possible association between specific alleles of these proteins and a diagnosis of idiopathic thrombocytopenic purpura (ITP) in dogs. By using genomic DNA from dogs of various breeds with and without ITP, sequencing of PCR products encompassing all coding regions and exon–intron boundaries for these 3 genes revealed 4 single-nucleotide polymorphisms in ITGA2B resulting in amino acid polymorphisms in the canine genome, 3 previously reported and 1 newly identified (Gly[GGG]/Arg[AGG] at amino acid position 576 of ITGA2B. Of 16 possible ITGA2B protein alleles resulting from unique combinations of the 4 polymorphic amino acids, 5 different protein isoforms were present in homozygous dogs and explain all of the genotype combinations in heterozygous dogs. There was no amino acid polymorphism or protein isoform that was specific for a particular breed or for the diagnosis of ITP.Abbreviations: HPA, human platelet antigen; ITP, idiopathic thrombocytopenic purpura; PTR, platelet transfusion refractoriness; SNP, single-nucleotide polymorphismHuman alloimmune thrombocytopenic conditions caused by exposure to a platelet-specific alloantigen, through pregnancy or transfusion, include neonatal alloimmune thrombocytopenia, posttransfusion purpura, and platelet transfusion refractoriness (PTR). More than 30 platelet-specific alloantigens have been defined in the human platelet antigen (HPA) system, with 12 alloantigens grouped into 6 biallelic systems (HPA1 through HPA5 and HPA15) in which alloantibodies against both the common (designated ‘a’) and rare (designated ‘b’) alleles have been identified.^5,12 For the remaining 21 platelet alloantigens (HPA6bw through HPA14bw and HPA16bw through HPA27bw), only antibodies against the rare allele (designated ‘bw’) have been detected. Although HPA traditionally were defined by using immune sera, the molecular basis of these antigens has now been characterized.^5,12 The HPA reside in platelet membrane glycoproteins, the most common being GPIIIa, which accounts for 14 HPA. In all but one (HPA14bw), the platelet alloantigens are defined by a single amino-acid substitution caused by a single-nucleotide polymorphism (SNP) in the gene encoding the relevant membrane glycoprotein.^5,12 The Platelet Nomenclature Committee, which includes members of both the International Society of Blood Transfusion and the International Society of Thrombosis and Haemostasis, has set guidelines for defining new platelet antigens, which include: 1) determining the genetic basis of the alloantigen by genomic DNA sequence analysis, and 2) demonstrating an association between the genetic mutation and the reactivity of alloantibodies with the allelic forms of the protein.¹² For patients suspected of having alloimmune thrombocytopenia, well-defined platelet genotyping methods and serologic assays for the detection of platelet antibodies are available to guide case management.In contrast to the well-characterized HPA system and wealth of information on alloimmune thrombocytopenic conditions in humans, there is no information on canine platelet-specific alloantigens in the literature. However, early serologic studies using antiHPA1a alloantiserum resulted in direct immunoprecipitation of a 90-kDa protein from canine platelets, suggesting that the antigenic determinant for HPA1a has been conserved between humans and dogs.¹⁰ Neonatal alloimmune thrombocytopenia has not yet been documented in dogs, and there is a single case report of suspected posttransfusion purpura in a dog with hemophilia A, in which severe thrombocytopenia was noted 1 to 2 wk after transfusion (cryoprecipitate, fresh whole blood), with an increased amount of platelet surface-associated IgG and rapid resolution (less than 1 wk) of the thrombocytopenia.³⁰ PTR, however, has been well documented in dogs for more than 25 y, because this species has been used as an experimental model to evaluate a variety of methods of preventing platelet alloimmunization.^21-23 The occurrence of platelet alloimmunization in dogs receiving platelets from dog leukocyte antigen-identical littermates indicates that nondog leukocyte antigen immunizing platelet antigens, perhaps platelet-specific antigens, are responsible for the development of PTR.²¹Using the HPA system as a model, our main objective in this study was to evaluate the canine ITGB3, ITGA2B, and GP1BB genes encoding the GPIIIa (β₃), GPIIb (α_IIb), and GPIbβ, respectively, proteins that account for 21 of 27 HPA, to determine whether there are amino acid polymorphisms that could define canine platelet-specific alloantigens. A secondary objective was to perform a pilot study to assess the possible association between canine platelet antigen protein alleles or single amino acid substitutions and primary immune-mediated thrombocytopenia, also referred to as idiopathic thrombocytopenic purpura (ITP), in dogs. The identification of a canine platelet antigen system would improve our understanding of the molecular basis of alloimmune thrombocytopenic conditions in dogs and help guide effective platelet transfusion support of these critical patients.     

Engineering an Endothelialized Vascular Graft: A Rational Approach to Study Design in a Non-Human Primate Model

After many years of research, small diameter, synthetic vascular grafts still lack the necessary biologic integration to perform ideally in clinical settings. Endothelialization of vascular grafts has the potential to improve synthetic graft function, and endothelial outgrowth cells (EOCs) are a promising autologous cell source. Yet no work has established the link between endothelial cell functions and outcomes of implanted endothelialized grafts. This work utilized steady flow, oscillatory flow, and tumor necrosis factor stimulation to alter EOC phenotype and enable the formulation of a model to predict endothelialized graft performance. To accomplish this, EOC in vitro expression of coagulation and inflammatory markers was quantified. In parallel, in non-human primate (baboon) models, the platelet and fibrinogen accumulation on endothelialized grafts were quantified in an ex vivo shunt, or the tissue ingrowth on implanted grafts were characterized after 1mth. Oscillatory flow stimulation of EOCs increased in vitro coagulation markers and ex vivo platelet accumulation. Steady flow preconditioning did not affect platelet accumulation or intimal hyperplasia relative to static samples. To determine whether in vitro markers predict implant performance, a linear regression model of the in vitro data was fit to platelet accumulation data—correlating the markers with the thromboprotective performance of the EOCs. The model was tested against implant intimal hyperplasia data and found to correlate strongly with the parallel in vitro analyses. This research defines the effects of flow preconditioning on EOC regulation of coagulation in clinical vascular grafts through parallel in vitro, ex vivo, and in vivo analyses, and contributes to the translatability of in vitro tests to in vivo clinical graft performance.     

Long-lived enzymatic metabolites of thromboxane B2 in the human circulation

Thromboxane A₂, a potent vasoconstrictor and platelet agonist, is an evanescent cyclooxygenase product of arachidonic acid. Assessment of thromboxane biosynthesis commonly relies upon analysis of the stable but biologically inactive hydration product, thromboxane B₂. However, measurement of this compound in plasma is readily confounded by platelet activation ex vivo. We have identified 11-dehydro-thromboxane B₂, 11-dehydro-13,14-dihydro-15-keto-thromboxane B₂, and 2,3-dinor-thromboxane B₂ as enzymatic products of infused thromboxane B₂ in the human circulation. Biosynthesis of deuterated standards permitted the development of quantitative analyses for these compounds, employing capillary gas chromatography-negative ion chemical ionization-mass spectrometry. We thus established that the postinfusion half-lives of 11-dehydrothromboxane B₂ and the keto-dihydro metabolite approximated 1 hour, while that of the dinor metabolite ranged from 15 to 17 min. Combined analysis of short- and long-lived enzymatic metabolites of thromboxane B₂ promises to bypass the problem of ex vivo platelet activation and enhance the likelihood of relating a discreet clinical event to an alteration in the biosynthesis of thromboxane A₂ in the human circulation.