Absence Of Adenosine Diphosphate Research Articles

NanoGraphene Clot: A New Fibrinogen-Mimic Hemostatic Material.

Trauma is the leading cause of death for adults under the age of 44. Internal bleeding remains a significant challenge in medical emergencies, necessitating the development of effective hemostatic materials that could be administered by paramedics before a patient is in the hospital and treated by surgeons. In this study, we introduce a graphene oxide (GO)-based PEGylated synthetic hemostatic nanomaterial with an average size of 211 ± 83 nm designed to target internal bleeding by mimicking the role of fibrinogen. Functionalization of GO-g-PEG with peptides derived from the α-chain of fibrinogen, such as GRGDS, or the γ-chain of fibrinogen, such as HHLGGAKQAGDV:H12, was achieved with peptide loadings of 72 ± 6 and 68 ± 15 μM, respectively. In vitro studies with platelet-rich plasma (PRP) under confinement demonstrated aggregation enhancement of 39 and 24% for GO-g-PEG-GRGDS and GO-g-PEG-H12, respectively, compared to buffer, while adenosine diphosphate (ADP) alone induced a 5% aggregation. Compared to the same materials in the absence of ADP, GO-g-PEG-GRGDS achieved a 47% aggregation enhancement, while GO-g-PEG-H12 a 25% enhancement. This is particularly important for injectable hemostats and highlights the fact that our nanographene-based materials can only act as hemostats in the presence of agonists, reducing the possibility of unwanted clotting during circulation. Further studies on collagen-coated wells under dynamic flow revealed statistically significant augmentation of PRP fluorescence signal using GRGDS- or H12-coated GO-g-PEG compared to controls. Hemolysis studies showed <1% lysis of red blood cells (RBCs) at the highest PEGylated nanographene concentration. Finally, whole human blood coagulation studies reveal faster and more pronounced clotting using our nanohemostats vs PBS control from 3 min and below (blood is clotted with 10% CaCl2 within 4-5 min), with the biggest differences to be shown at 2 and 1 min. At 1 min, the clot weight was found to be ∼45% of that between 4 and 5 min, while no clot was formed in PBS-treated blood. Reduction of CaCl2 to 5 and 3%, or utilization of prostaglandin E1, an anticoagulant, still leads to clots but of smaller weight. The findings highlight the potential of our fibrinogen-mimic PEGylated nanographene as a promising non-hemolytic injectable scaffold for targeting internal bleeding, offering insights into its platelet aggregation capabilities under confinement and under dynamic flow as well as its pronounced coagulation abilities.

Structural basis for DEAH-helicase activation by G-patch proteins

RNA helicases of the DEAH/RHA family are involved in many essential cellular processes, such as splicing or ribosome biogenesis, where they remodel large RNA-protein complexes to facilitate transitions to the next intermediate. DEAH helicases couple adenosine triphosphate (ATP) hydrolysis to conformational changes of their catalytic core. This movement results in translocation along RNA, which is held in place by auxiliary C-terminal domains. The activity of DEAH proteins is strongly enhanced by the large and diverse class of G-patch activators. Despite their central roles in RNA metabolism, insight into the molecular basis of G-patch-mediated helicase activation is missing. Here, we have solved the structure of human helicase DHX15/Prp43, which has a dual role in splicing and ribosome assembly, in complex with the G-patch motif of the ribosome biogenesis factor NKRF. The G-patch motif binds in an extended conformation across the helicase surface. It tethers the catalytic core to the flexibly attached C-terminal domains, thereby fixing a conformation that is compatible with RNA binding. Structures in the presence or absence of adenosine diphosphate (ADP) suggest that motions of the catalytic core, which are required for ATP binding, are still permitted. Concomitantly, RNA affinity, helicase, and ATPase activity of DHX15 are increased when G-patch is bound. Mutations that detach one end of the tether but maintain overall binding severely impair this enhancement. Collectively, our data suggest that the G-patch motif acts like a flexible brace between dynamic portions of DHX15 that restricts excessive domain motions but maintains sufficient flexibility for catalysis.

Unique features of flight muscles mitochondria of honey bees (Apis mellifera L.).

Honey bees Apis mellifera L. are one of the most studied insect species due to their economic importance. The interest in studying honey bees chiefly stems from the recent rapid decrease in their world population, which has become a problem of food security. Nevertheless, there are no systemic studies on the properties of the mitochondria of honey bee flight muscles. We conducted a research of the mitochondria of the flight muscles of A. mellifera L. The influence of various organic substrates on mitochondrial respiration in the presence or absence of adenosine diphosphate (ADP) was investigated. We demonstrated that pyruvate is the optimal substrate for the coupled respiration. A combination of pyruvate and glutamate is required for the maximal respiration rate. We also show that succinate oxidation does not support the oxidative phosphorylation and the generation of membrane potential. We also studied the production of reactive oxygen species by isolated mitochondria. The greatest production of H2 O2 (as a percentage of the rate of oxygen consumed) in the absence of ADP was observed during the respiration supported by α-glycerophosphate, malate, and a combination of malate with another NAD-linked substrate. We showed that honey bee flight muscle mitochondria are unable to uptake Ca2+ -ions. We also show that bee mitochondria are able to oxidize the respiration substrates effectively at the temperature of 50°С compared to Bombus terrestris mitochondria, which were more adapted to lower temperatures.

Mechanistic Study of Hemoglobin Induced Platelet Activation

Abstract 5262Nitric oxide (NO) has been previously shown to inhibit adenosine diphosphate (ADP)- and thrombin- mediated platelet activation. This inhibitory effect of NO is blocked by cell-free oxyhemoglobin (Villagra, Shiva et al. 2007). Cell-free hemoglobin (Hb) also appears to directly activate platelets even in the absence of ADP. However, the precise mechanisms for cell-free Hb-induced platelet activation have not been fully delineated. We hypothesized that oxyhemoglobin can promote platelet activation through one of three potential mechanisms: 1) direct apoprotein interaction with platelet receptors, 2) reactive oxygen species (ROS) production, and/or 3) scavenging of endogenous NO.Platelets were isolated from whole blood obtained from healthy volunteers (n=15) using sodium citrate blood collection tubes. Platelet activation was measured by examining immunofluorescent Pac-1 labeling by flow cytometry using activated glycoprotein IIb/IIIa as a measure of activation. In the presence of 10 uM concentration of NO donor mahmaNONOate, ADP-induced platelet activation was inhibited by 44%. Incubation of 100 uM cell-free oxyhemoglobin eliminated the inhibitory effect of NO. In addition, cell-free oxyhemoglobin (100 uM concentration) induced platelet activation by 17% above unstimulated controls even in the absence of ADP.Methemoglobin was used to test if direct Hb protein interaction with platelets was sufficient for platelet activation, independent of heme redox state. To produce methemoglobin, the ferrous iron in oxyhemoglobin was oxidized to its ferric form, thus methemoglobin is likely to preserve all direct protein interactions. However, unlike oxyhemoglobin, incubation of 100 uM methemoglobin did not induce platelet activation.Next, we tested whether reactive oxygen species production was responsible for hemoglobin-induced platelet activation. In the absence of hemoglobin, superoxide dismutase (SOD) (100 U/ml) and catalase (100 U/ml) reduced baseline platelet activation, verifying a ROS (Pignatelli, Pulcinelli et al. 1998). However, in the presence of oxyhemoglobin, SOD and catalase did not significantly abrogate the effect of Hb on platelet activation, suggesting that the ROS superoxide and hydrogen peroxide are not involved in hemoglobin-induced platelet activation.To determine if scavenging of endogenous NO was responsible for hemoglobin-induced platelet activation, platelets were incubated in the presence or absence of NO scavengers, carboxy-PTIO (5mM) and Fe-DTCS (3 mM), or nitric oxide synthase (NOS) inhibitor, LNMMA (100 uM). Results from these investigations suggest scavenging of endogenous NO was not responsible for hemoglobin-induced platelet activation. Overall our data suggest a complex mechanism for oxyhemoglobin-mediated platelet activation that is likely to involve more than a single contributing pathway.These data aid in elucidating the mechanism of platelet activation by cell-free hemoglobin and may provide novel insights to designing potential treatments to reduce the platelet activation underlying hypercoagulable states associated with a variety of pathological conditions including hemolytic anemias, stroke, and diabetes.This work was supported by NIH grants HL058091 and HL098032 Disclosures:Gladwin:Patents filed related to treating hemolysis.: Patents & Royalties. Kim-Shapiro:Patents filed related to treating hemolysis. Honororia from Sangart Inc.: Honoraria, Patents & Royalties.

Crystal structure of glycogen synthase: homologous enzymes catalyze glycogen synthesis and degradation.

Glycogen and starch are the major readily accessible energy storage compounds in nearly all living organisms. Glycogen is a very large branched glucose homopolymer containing about 90% alpha-1,4-glucosidic linkages and 10% alpha-1,6 linkages. Its synthesis and degradation constitute central pathways in the metabolism of living cells regulating a global carbon/energy buffer compartment. Glycogen biosynthesis involves the action of several enzymes among which glycogen synthase catalyzes the synthesis of the alpha-1,4-glucose backbone. We now report the first crystal structure of glycogen synthase in the presence and absence of adenosine diphosphate. The overall fold and the active site architecture of the protein are remarkably similar to those of glycogen phosphorylase, indicating a common catalytic mechanism and comparable substrate-binding properties. In contrast to glycogen phosphorylase, glycogen synthase has a much wider catalytic cleft, which is predicted to undergo an important interdomain 'closure' movement during the catalytic cycle. The structures also provide useful hints to shed light on the allosteric regulation mechanisms of yeast/mammalian glycogen synthases.

Inhibition of Platelet Aggregation by Rat Trophoblasts

Objective: To determine the effect of rat trophoblast cell suspensions on adenosine diphosphate (ADP)-induced platelet aggregation. Materials and Methods: The trophoblasts were isolated from ectoplacental cones (a pre-placental tissue very rich in trophoblasts) developed in rat embryos on day 12 of pregnancy (normal period of gestation: 22–23 days). Platelet-rich plasma (PRP) was obtained from adult male rats. The trophoblasts were preincubated (37°C, 30 min), suspended in the medium, and then re-incubated with PRP for 3–5 min. Results: 5 × 10⁴ and 7 × 10⁴ trophoblasts inhibited ADP-induced platelet aggregation by 10 and 18%, respectively. When the trophoblast cell concentration was increased to 1– 2 × 10⁵ cells, a proaggregatory phenomenon was observed, even in the absence of ADP. However, there was no inhibition of aggregation or promotion of aggregation when fixed trophoblasts or live endometrial stromal cells were incubated with PRP. Conclusion: The results indicate that the aggregation inhibition response was cell specific and concentration dependent. A 68-kD protein was detected in the medium when it was conditioned with 5–7 × 10⁴ but not with 1–2 × 10⁵ trophoblasts. However, the inhibitory or stimulatory effect does not seem to be dependent on the presence of this protein.

KRDS–A Tetrapeptide Derived From Lactotransferrin–Inhibits Binding of Monoclonal Antibody Against Glycoprotein IIb-IIIa on ADP-Stimulated Platelets and Megakaryocytes

Short peptides isolated from fibrinogen and K-casein have been shown to inhibit platelet aggregation and fibrinogen binding to stimulated platelets. We studied the effects of synthetic peptides occurring in milk proteins (bovine K-casein, KNQDK, and human lactotransferrin, KRDS) and in fibrinogen (RGDS and L10) on subsequent binding of monoclonal antibodies (MoAb) against the glycoprotein (GP) IIb-IIIa complex (AP2 and P2) on adenosine diphosphate (ADP)-stimulated and unstimulated human platelets and megakaryocytes (MKs) by using an immunoperoxidase method to visualize antibody binding. Only KRDS (900 mumol/L) inhibited the binding of AP2 and P2 on ADP (5 mumol/L)-stimulated platelets, but not on unstimulated platelets. However, the binding of P2 was considerably more inhibited than that of AP2 as judged by immunoperoxidase intensity. Radiolabeled AP2 binding was inhibited by 30% with KRDS on ADP-stimulated platelets as compared with platelets incubated in the absence of ADP. KRDS did not inhibit the binding of MoAbs against GP IIIa (SZ 21), GP IIb (SZ 22), and GP Ib (SZ 2) on ADP-stimulated human platelets. Inhibition of P2 binding by KRDS was also observed in a section of MKs isolated from human bone marrow and stimulated by 15 or 20 micron ADP. A lower concentration of ADP (5 or 10 mumol/L) failed to produce any inhibition of binding. This indicates that MKs may not be equally responsive to agonists as platelets. Moreover, P2 binding inhibition was observed in a larger (P less than .001) percentage of mature MKs (29%) as compared with younger, maturing MKs (11%). The observations suggested that a functional ability possessed by platelets, namely, agonist-induced exposure of the site of interaction of KRDS, may occur at a late stage of MK development.

KRDS--a tetrapeptide derived from lactotransferrin--inhibits binding of monoclonal antibody against glycoprotein IIb-IIIa on ADP-stimulated platelets and megakaryocytes

Short peptides isolated from fibrinogen and K-casein have been shown to inhibit platelet aggregation and fibrinogen binding to stimulated platelets. We studied the effects of synthetic peptides occurring in milk proteins (bovine K-casein, KNQDK, and human lactotransferrin, KRDS) and in fibrinogen (RGDS and L10) on subsequent binding of monoclonal antibodies (MoAb) against the glycoprotein (GP) IIb-IIIa complex (AP2 and P2) on adenosine diphosphate (ADP)-stimulated and unstimulated human platelets and megakaryocytes (MKs) by using an immunoperoxidase method to visualize antibody binding. Only KRDS (900 mumol/L) inhibited the binding of AP2 and P2 on ADP (5 mumol/L)- stimulated platelets, but not on unstimulated platelets. However, the binding of P2 was considerably more inhibited than that of AP2 as judged by immunoperoxidase intensity. Radiolabeled AP2 binding was inhibited by 30% with KRDS on ADP-stimulated platelets as compared with platelets incubated in the absence of ADP. KRDS did not inhibit the binding of MoAbs against GP IIIa (SZ 21), GP IIb (SZ 22), and GP Ib (SZ 2) on ADP-stimulated human platelets. Inhibition of P2 binding by KRDS was also observed in a section of MKs isolated from human bone marrow and stimulated by 15 or 20 micron ADP. A lower concentration of ADP (5 or 10 mumol/L) failed to produce any inhibition of binding. This indicates that MKs may not be equally responsive to agonists as platelets. Moreover, P2 binding inhibition was observed in a larger (P less than .001) percentage of mature MKs (29%) as compared with younger, maturing MKs (11%). The observations suggested that a functional ability possessed by platelets, namely, agonist-induced exposure of the site of interaction of KRDS, may occur at a late stage of MK development.

Single-headed binding of a spin-labeled-HMM-ADP complex to F-actin. Saturation transfer electron paramagnetic resonance and sedimentation studies

The interaction of actin and spin-labeled heavy meromyosin (MSL-HMM) was studied in the presence and absence of adenosine diphosphate or 5'-adenyl-yl-imidodiphosphate (AMPPNP) to determine the contributions of single and double-headed binding. The extent of single-headed binding to actin was deduced from a comparison of the fraction of immobilized heads (fi) with the fraction of bound molecules (fs) determined by saturation-transfer EPR (ST-EPR) and sedimentation, respectively. The ST-EPR measurements depend on the reduced motion of the spin label rigidly bound to the HMM heads upon the interaction of the latter with actin. During titration of acto-MSL-HMM with nucleotide, we measured changes in fi and fs brought about by dissociation of MSL-HMM from actin. On titration with ADP, fs changed very little, remaining above 0.8, while fi decreased to approximately 0.5 at 10mM ADP, a result consistent with extensive single-headed binding of MSL-HMM to actin. On titration with AMPPNP, single-headed binding was not detected; viz., fi and fs decreased in parallel. It was not necessary to postulate a nucleotide induced state of the bound heads, differing in motional properties from that of rigor heads, to account for the results.

Alteration of oxidative phosphorylation in uremia.

The effect of ultrafiltrate of uremic serum upon the oxygen consumption of rat liver mitochondria was studied. Oxygen consumption in the absence of adenosine diphosphate (State 4) was increased by the addition of ultrafiltered serum of uremic patients, causing decrease of P/O ratio and respiratory control ratio, indicating the uncoupler action of uremic serum. Such uncoupler action was noted when succinate was used as the substrate, and also when α-ketoglutarate was used as the nicotinamide adenine dinucleotide-linked substrate. The mitochondrial respiration in the presence of adenosine diphosphate (State 3) was decreased, suggesting the inhibitory action of uremic serum on energy transfer. Oxygen consumption after 20 minute incubation with uremic serum showed the decrease in either state, leaving ground for the presence of inhibitory action against electron transport. Similar results were obtained in liver mitochondria of rats in acute renal insufficiency caused by bilateral nephrectomy. Based on these facts, the nature of metabolic inhibitors in uremic serum was discussed.

Oxidative phosphorylation by muscle mitochondria of dysstrophic mice.

Oxidative phosphorylation was studied in mitochondria isolated from the skeletal muscle of control and dystrophic mice of the Jackson Laboratory strain 129/Re, aged 32–104 days. The isolation procedure included a preliminary incubation of the muscle minced in a medium containing a proteinase (Nagarse) followed by gentle homogenization and differential centrifugation. Polarographic estimations in the presence and absence of adenosine diphosphate (ADP) indicated that the rate of oxygen uptake, ADP/0 ratio, respiratory control ratio, and phosphorylation rate were not significantly different in the mitochondria isolated from control and dystrophic mice. Bovine serum albumin increased the ADP/0 and respiratory control ratios, but the values for the control and dystrophic preparations again did not differ significantly in the presence of albumin.

Automated Method for the Determination of Serum Creatine Kinase Activity

Abstract An automated (AutoAnalyzer) method for the colorimetric determination of creatine kinase activity in serum is described. This method includes reactivation of creatine kinase with cysteine, incubation of the active enzyme with creatine phosphate and adenosine diphosphate at 37.5°, and subsequent inactivation of enzyme and binding of cysteine by phenylmercuric borate. The enzymatically produced creatine is dialyzed against a solution of diacetyl and reacted with α-naphthol in an alkaline solution. The absorbance of the colored end product is measured at 550 mµ. Individual blanks are determined in the absence of adenosine diphosphate. Comparison of results obtained by this method and a manual procedure shows satisfactory agreement.

P:O and ADP:O Ratios and Quantum Yields in Chloroplasts, with the Use of Chloranil as Electron Acceptor

Abstract It has been shown that, when chloranil is used as electron acceptor in photophosphorylation, phosphorylation has an absolute requirement for molecular oxygen. Although oxygen consumption does proceed in the absence of adenosine diphosphate, the rate of oxygen consumption is doubled in the presence of ADP. The observed P:O and ADP:O ratios at high light intensities are over 3 when chloranil is the acceptor. Under these conditions, the maximum quantum yields at low light intensity for phosphorylation were 0.6, and the quantum yields for electrons (with the use of oxygen as the acceptor) were 1.0. Arguments and data supporting the thesis that chloranil acts only as a noncyclic electron acceptor are given. These high P:O ratios are observed only at low concentrations (approximately 10-8 m) of the oxidized acceptor.

Correlation of Light-induced Absorbance Changes with Photophosphorylation in Rhodospirillum rubrum Extracts

Abstract The relationship between light-induced absorbance changes and photophosphorylation has been studied in extracts of the photosynthetic bacterium Rhodospirillum rubrum purified by partition in dextran-methyl cellulose mixtures. In these preparations infrared light induces absorbance changes in the visible region of the spectrum which are modified by the addition of reductants required for photophosphorylation. The kinetics of photophosphorylation has been compared with light-induced absorbance changes, by measuring ATP formation in flashing light. With submaximal flash duration, the ultimate yield of ATP per flash parallels the absorbance change induced during the flash. The light-off absorbance change is characterized by a rapid phase and slower subsequent phase, or phases. The dark stage of photophosphorylation coincides with the rapid phase of the light-off absorbance change (the first 0.1 sec). The effects of potent inhibitors of photophosphorylation upon absorbance change kinetics are limited to the rapid phase of the light-off reaction. This phase is almost eliminated by antimycin A and other inhibitors of similar characteristics. It is significantly slowed by phenylmercuric acetate and by oligomycin. The carbonyl cyanide phenylhydrazones stimulate the rapid phase of the absorbance change, but only in the absence of adenosine diphosphate or in the presence of oligomycin. The rapid phase of the light-off reaction is also stimulated by adenosine diphosphate. These results suggest that the rapid phase of the light-off absorbance change may represent electron transport associated with phosphorylation.