Trauma patients have reduced ex vivo flow-dependent platelet hemostatic capacity in a microfluidic model of vessel injury.

Trauma is the leading cause of death in individuals up to 45 years of age. Alterations in platelet function are a critical component of trauma-induced coagulopathy (TIC), yet these changes and the potential resulting dysfunction is incompletely understood. The lack of clinical assays available to explore platelet function in this patient population has hindered detailed understanding of the role of platelets in TIC. The objective of this study was to assess trauma patient ex vivo flow-dependent platelet hemostatic capacity in a microfluidic model. We hypothesized that trauma patients would have flow-regime dependent alterations in platelet function. Blood was collected from trauma patients with level I activations (N = 34) within 60 min of hospital arrival, as well as healthy volunteer controls (N = 10). Samples were perfused through a microfluidic model of injury at venous and arterial shear rates, and a subset of experiments were performed after incubation with fluorescent anti-CD41 to quantify platelets. Complete blood counts were performed as well as plasma-based assays to quantify coagulation times, fibrinogen, and von Willebrand factor (VWF). Exploratory correlation analyses were employed to identify relationships with microfluidic hemostatic parameters. Trauma patients had increased microfluidic bleeding times compared to healthy controls. While trauma patient samples were able to deposit a substantial amount of clot in the model injury site, the platelet contribution to microfluidic hemostasis was attenuated. Trauma patients had largely normal hematology and plasma-based coagulation times, yet had elevated D-Dimer and VWF. Venous microfluidic bleeding time negatively correlated with VWF, D-Dimer, and mean platelet volume (MPV), while arterial microfluidic bleeding time positively correlated with oxygenation. Arterial clot growth rate negatively correlated with red cell count, and positively with mean corpuscular volume (MCV). We observed changes in clot composition in trauma patient samples reflected by significantly diminished platelet contribution, which resulted in reduced hemostatic function in a microfluidic model of vessel injury. We observed a reduction in platelet clot contribution under both venous and arterial flow ex vivo in trauma patient samples. While our population was heterogenous and had relatively mild injury severity, microfluidic hemostatic parameters correlated with different patient-specific data depending on the flow setting, indicating potentially differential mechanistic pathways contributing to platelet hemostatic capacity in the context of TIC. These data were generated with the goal of identifying key features of platelet dysfunction in bleeding trauma patients under conditions of flow and to determine if these features correlate with clinically available metrics, thus providing preliminary surrogate markers of physiological platelet dysfunction to be further studied across larger cohorts. Future studies will continue to explore those relationships and further define mechanisms of TIC and their relationship with patient outcomes.

Cold-stored platelet hemostatic capacity is maintained for three weeks of storage and associated with taurine metabolism.

BACKGROUND: Platelet (PLT) product transfusion is a life-saving therapy for actively bleeding patients. There is an urgent need to maintain PLT function and extend shelf life to improve outcomes in these patients. Cold-stored PLT (CS-PLT) maintain hemostatic potential better than room temperature-stored PLT (RT-PLT). However, whether function in long-term CS-PLT is maintained under physiological flow regimes and/or determined by cold-induced metabolic changes is unknown. OBJECTIVES: This study aimed to (i) compare the function of RT-PLT and CS-PLT under physiological flow conditions, (ii) determine whether CS-PLT maintain function after 3 weeks of storage, and (iii) identify metabolic pathways associated with the CS-PLT lesion. METHODS: We performed phenotypic and functional assessments of RT- and CS-PLT (22 °C and 4 °C storage, respectively; N = 10 unique donors) at storage days 0, 5, and/or 21 via metabolomics, flow cytometry, aggregation, thrombin generation, viscoelastic testing, and a microfluidic assay to measure primary hemostatic function. RESULTS: Day 21 4 °C PLT formed an occlusive thrombus under arterial shear at a similar rate to day 5 22 °C PLT. Day 21 4 °C PLTs had enhanced thrombin generation capacity compared with day 0 PLT and maintained functionality comparable to day RT-PLT across all assays performed. Key metrics from microfluidic assessment, flow cytometry, thrombin generation, and aggregation were associated with 4 °C storage, and metabolites involved in taurine and purine metabolism significantly correlated with these metrics. Taurine supplementation of PLT during storage improved hemostatic function under flow. CONCLUSION: CS-PLT stored for 3 weeks maintain hemostatic activity, and storage-induced phenotype and function are associated with taurine and purine metabolism.

Dose-Dependent Von Willebrand Factor Inhibition by Aptamer BB-031 Correlates with Thrombolysis in a Microfluidic Model of Arterial Occlusion.

Von Willebrand Factor (VWF) plays a critical role in thrombus formation, stabilization, and propagation. Previous studies have demonstrated that targeted inhibition of VWF induces thrombolysis when administered in vivo in animal models of ischemic stroke. The study objective was to quantify dose-dependent inhibition of VWF-platelet function and its relationship with thrombolysis using BB-031, an aptamer that binds VWF and inhibits its function. VWF:Ac, VWF:RCo, T-TAS, and ristocetin-induced impedance aggregometry were used to assess BB-031-mediated inhibition of VWF. Reductions in original thrombus surface area and new deposition during administration of treatment were measured in a microfluidic model of arterial thrombolysis. Rotational thromboelastometry was used to assess changes in hemostasis. BB-031 induced maximal inhibition at the highest dose (3384 nM) in VWF:Ac, and demonstrated dose-dependent responses in all other assays. BB-031, but not vehicle, induced recanalization in the microfluidic model. Maximal lytic efficacy in the microfluidic model was seen at 1692 nM and not 3384 nM BB-031 when assessed by surface area. Minor changes in ROTEM parameters were seen at 3384 nM BB-031. Targeted VWF inhibition by BB-031 results in clinically measurable impairment of VWF function, and specifically VWF-GPIb function as measured by VWF:Ac. BB-031 also induced thrombolysis as measured in a microfluidic model of occlusion and reperfusion. Moderate correlation between inhibition and lysis was observed. Additional studies are required to further examine off-target effects of BB-031 at high doses, however, these are expected to be above the range of clinical targeted dosing.

Red cell glucose-6-phosphate dehydrogenase phenotypes in Iraq.

We attempted to characterize biochemically glucose-6-phosphate dehydrogenase (G6PD) variants in Iraqi individuals. Thus 758 healthy Iraqi males aged 18-60 years were randomly selected and 46 (6.1%) were G6PD deficient. Although the predominant non-deficient G6PD phenotype was G6PD B (92.6%), G6PD A+ was found in polymorphic frequency (1.3%). In the deficient group, 31 cases were fully characterized, including 17 cases with features consistent with G6PD Mediterranean variant, while 12 had other biochemical features and were labelled as non-Mediterranean variant. The remaining two deficient cases were characterized as G6PD A- variant. The presence of a significant number of non-Mediterranean variant was unexpected and may be related to the more heterogeneous background of the Iraqi people.

Pheromone binding proteins of Epiphyas postvittana (Lepidoptera: Tortricidae) are encoded at a single locus.

The light brown apple moth, Epiphyas postvittana (Tortricidae: Lepidoptera) uses a blend of (E)-11-tetradecenyl acetate and (E,E)-9,11-tetradecadienyl acetate as its sex pheromone. Odorant binding proteins, abundant in the antennae of male and female E. postvittana, were separated by native PAGE to reveal four major proteins with distinct mobilities. Microsequencing of their N-terminal residues showed that two were general odorant binding proteins (GOBPs) while two were pheromone binding proteins (PBPs). Full length cDNAs encoding these proteins were amplified using a combination of PCR and RACE-PCR. Sequence of the GOBPs revealed two genes (EposGOBP1, EposGOBP2), similar to orthologues in other species of Lepidoptera. Eleven cDNAs of the PBP gene were amplified, cloned and sequenced revealing two major phylogenetic clusters of PBP sequences differing by six amino acid substitutions. The position of the six amino acid differences on the protein was predicted by mapping onto the three-dimensional structure of PBP of Bombyx mori. All six substitutions were predicted to fall on the outside of the protein away from the inner pheromone binding pocket. One substitution does fall close to the putative dimerisation region of the protein (Ser63Thr). Expression of three of the cDNAs in a baculovirus expression system revealed that one class encodes an electrophoretically slow form (EposPBP1-12) while the other encodes a fast form (EposPBP1-2, EposPBP1-3). A native Western of these expressed proteins compared with antennal protein extracts demonstrated that PBP is also expressed in female antennae and that PBP may be present as a dimer as well as a monomer in E. postvittana. The fast and slow forms of EposPBP1 are allelic. Westerns on single antennal pair protein extracts and allele-specific PCR from genomic DNA both show a segregating pattern of inheritance in laboratory and wild populations. Radio labelled (E)-11-tetradecenyl acetate binds to both fast and slow PBP forms in gel assays. Taken together, the genetic and biochemical data do not support the hypothesis that these PBPs are specific for each component of the E. postvittana pheromone. However, duplication of this PBP locus in the future might allow such diversification to evolve, as observed in the other species.

Red cell glucose-6-phosphate dehydrogenase phenotypes in Iraq

We attempted to characterize biochemically glucose-6-phosphate dehydrogenase [G6PD] variants in Iraqi individuals. Thus 758 healthy Iraqi males aged 18-60 years were randomly selected and 46 [6.1%] were G6PD deficient. Although the predominant non-deficient G6PD phenotype was G6PD B [92.6%], G6PD A+ was found in polymorphic frequency [1.3%]. In the deficient group, 31 cases were fully characterized, including 17 cases with features consistent with G6PD Mediterranean variant, while 12 had other biochemical features and were labelled as non-Mediterranean variant. The remaining two deficient cases were characterized as G6PD A- variant. The presence of a significant number of non-Mediterranean variant was unexpected and may be related to the more heterogeneous background of the Iraqi people

Vesicular storage of adenosine triphosphate in the guinea-pig cochlear lateral wall and concentrations of ATP in the endolymph during sound exposure and hypoxia.

Previous studies have revealed putative vesicular stores of adenosine triphosphate (ATP) in the marginal cells of the cochlear stria vascularis which may serve as a source of ATP for purinergic signalling. This study aimed to provide further evidence of ATP storage in the cochlea and to see whether ATP levels in the endolymph are affected by noise and hypoxia. Tissues from the lateral wall and organ of Corti of the guinea-pig cochlea were fractionated to obtain vesicular (VF) and mitochondrial (MF) fractions. Free and total ATP were then measured by the luciferase-luciferin reaction from which membrane-bound vesicular ATP was calculated. In the lateral wall, the VF contained 2.02+/-0.04 nmol ATP/mg protein (n = 5), significantly greater (p < 0.001; paired Student's t-test) than the concentration of ATP in the MF (0.36+/-0.05). In the organ of Corti, the VF contained 0.69+/-0.08 nmol ATP/mg protein (n = 4), significantly smaller than the amount in the VF of the lateral wall tissues (p < 0.001; non-paired Student's t-test). Small amounts of fumarase. an enzyme of the mitochondrial matrix, in the VF, excluded the possibility of mitochondrial ATP contamination. To investigate the effect of hypoxia and noise on the ATP concentrations in the endolymph, fluid samples were collected from the first (basal) cochlear turn of anaesthetized guinea-pigs. As a result of hypoxia (15 min, 13% F1O2), ATP concentrations (nM, mean +/- SEM) increased from 6.2+/-2.3 to 9.3+/-4.5 (n = 4), but the difference was not statistically significant. As a result of noise (15 min, 10 kHz, 110 dB SPL. broad band), the ATP levels increased significantly from 7.4+/-1.2 to 16.0+/-1.8 (p = 0.01; Student's t-test: n = 4). This study has demonstrated the presence of a vesicular store of ATP in the stria vascularis of the cochlea and described an increase in the ATP levels in the endolymph during noise exposure. The findings suggest that ATP is actively secreted from the vesicular store under conditions of metabolic stress. The presence of ATP under basal conditions supports a role for ATP in the sound transduction process during normal function.

Alkaline phosphatase of cancerous larynx tissue in comparison with the placental enzyme. Biochemical and histochemical studies.

Tissue specimens of squamous cell carcinoma of the larynx from twenty patients were processed for histological and histopathological characterization. A histochemical study of alkaline phosphatase (ALP) was carried out using the simultaneous azo coupling method, and biochemical studies were performed using disodium phenylphosphate as substrate. Full-term, normal placentae were used for comparison. The specific activity of ALP from cancerous laryngeal tissue was 8.9 mKAU/mg protein compared with 154.7 mKAU/mg protein in the placenta. The ALP was localized histochemically in tumor cells (tumor-specific), blood vessels (vascular) and fibrous tissue (interstitial). The tumor-specific phosphatase was sensitive to inhibition by L-phenylalanine, L-leucine and to a lesser degree by L-tryptophan and levamisole. Placental ALP, on the other hand, was completely inhibited by levamisole, more resistant to leucine and more sensitive to phenylalanine and tryptophan. Biochemical estimation of ALP in cancerous laryngeal tissue combined with inhibition studies revealed that the tumor-specific activity of ALP constitutes 15% of the total ALP activity while the major isoenzyme was the vascular ALP, and around one-third of ALP activity was attributed to the interstitial enzyme. The characterization and localization of these isoenzymes are described and compared with that of the placenta. The significance and implications of the above findings are presented.

Heat-stable alkaline phosphatase. A putative tumor marker of head and neck squamous cell carcinoma.

Serum total alkaline phosphatase (AP) activity and heat-stable AP (HSAP) were investigated in patients with uncontrolled squamous cell carcinoma of the head and neck before and after treatment. No significant differences in AP activity were seen between normal subjects and cancer patients. However, the HSAP fraction of the total AP activity was significantly elevated prior to treatment and the level declined and remained low during successful treatment, while it increased with tumor progression or recurrences. Heat-stable AP was found to be a useful tumor marker of potential usefulness in the management of patients with squamous cell carcinoma of the head and neck.

Concomitant natural infection with L. donovani and L. major: a case report from Iraq.

This is a case report of kala-azar with cutaneous leishmaniasis. Upon admission, the patient had fever, hepatosplenomegaly and an ulcer on her cheek. The patient responded to Pentostam. Isoenzyme studies of parasite isolates from the bone marrow and from the cutaneous lesion revealed that these were L. donovani and L. major, respectively. This is the first report in Iraq of a proven concomitant infection with two species of leishmania parasites.

Axenic cultivation of amastigotes of Leishmania donovani and Leishmania major and their infectivity.

Two clones of promastigotes, one of Leishmania donovani and one of L. major, and an uncloned stock of L. major were axenically transformed to heat-shock amastigotes, at 35 and 37 degrees C, respectively. Of the four different culture media tested, a relatively cheap, liquid medium, RBLM, was found to be the best, both for the transformation of the promastigotes and the serial, axenic cultivation of the amastigotes. In an experiment of 30 days duration, serial cultivation, in an atmosphere with 5% CO2, was possible by subculturing every three days. There were significant differences in virulence in BALB/c mice between axenically-cultured amastigotes and promastigotes, both in terms of the weights, lengths and parasite burdens of the spleens of mice infected intraperitoneally (ip) with L. donovani or L. major and of the appearance, type and size of the cutaneous lesions which developed in mice given L. major by intradermal inoculation.

Solubilization and kinetic characterization of mitochondrial adenosine triphosphatase from Leishmania donovani promastigotes.

Oligomycin-sensitive particulate ATPase (MB ATPase) from L. donovani promastigotes was solubilized by chloroform treatment. Polyacrylamide gel electrophoresis revealed several protein bands, with the major one possessing ATPase activity. The solubilized enzyme had Mg2+-ATPase and Ca2+-ATPase but no K+-dependent alkaline phosphatase activity. The Mg2+-ATPase activity was stimulated by monovalent cations and was not sensitive to oligomycin. Hence it is referred to as F1 ATPase. It had optimum activity at pH 7.6 and 30 degrees C. The Arrhenius plot for MB ATPase was biphasic with activation energies (Ea) of 16.2 and 3.4 kcal mol-1, while F1 ATPase exhibited a linear plot with Ea = 10.1 kcal mol-1. Lineweaver-Burk plots were biphasic with Km values of 0.17 and 1.25 mM for MB ATPase and 0.18 and 1.33 mM for F1 ATPase. The enzyme could be preserved at -15 degrees C in Tris-SO2-(4)-EDTA-ATP-glycerol (t1/2 = 20 days).

Isolation and identification of coenzyme Q from Leishmania donovani.

Crystalline coenzyme Q (CoQ) was obtained from Leishmania donovani promastigotes by extraction of the unsaponifiable material with hexane, chromatography on a Florisil column and crystallization from ethanol. Crystalline CoQ in ethanol revealed an ultraviolet-peak, at 275 nm, which disappeared upon reduction with NaBH4. Polarographic and mass fragmentographic measurements were characteristic of CoQ homologs. Reversed-phase thin-layer chromatography revealed that it is CoQ9.

Regulation of aerobic fermentation in Leishmania donovani promastigotes by NADP+-dependent malic enzyme.

NADP+-dependent malic enzyme (decarboxylating) was extracted from Leishmania donovani promastigotes with Triton X-100. The enzyme was specific for NADP+ and did not decarboxylate oxaloacetate (OA). The substrate activity relationship was hyperbolic for both L-malate and NADP+, and Km values were calculated as 0.18 and 0.12 mM, respectively. The enzyme exhibited a broad pH optimum of 7.5-8.0. Pyruvate, NADPH and OA inhibited the reaction in a competitive manner with apparent Ki values of 0.2, 0.04 and 0.04 mM, respectively, while oxalate inhibition was of the mixed type. The kinetic results obtained indicate that malic enzyme is involved in the regulation of carbon flow towards aerobic fermentation, complete oxidation of dicarboxylic acids or biosynthetic purposes.

Numerical taxonomy of some Old World Leishmania species.

Iraqi Leishmania stocks originating from human cutaneous and visceral infections, others from the sandfly Sergentomyia baghdadis, and one visceral stock from a dog, were compared among themselves and with Old World reference stocks, by electrophoresis of 11 enzymes. 79 unit characters emerged. Paired comparison technique and complete-linkage cluster analysis revealed heterogeneity within the L. donovani complex. The Iraqi L. donovani stocks appear to be more closely related to an Ethiopian reference L. donovani than to an L. donovani infantum. Two Indian visceral stocks were linked to the L. donovani complex and the L. tropica complex by similarity indices of the same range. The results show a marginally closer association between L. tropica and L. donovani complexes than between L. tropica and L. major.

Purification and properties of cytochrome c555 from Leishmania donovani.

A hemoprotein present in minute quantities in Leishmania donovani promastigotes was isolated, purified and spectrally characterized as cytochrome c555. Physicochemical characterization revealed electrophoretic mobility of 1.6 X 10(-5) cm2 V-1 s-1, an isoelectric point (pI) of 9.9, a relative molecular weight (Mr) of 11.9 kDa and half-wave potential (Ep) of -1.058 V. Values obtained were compared with those of horse heart cytochrome c subjected to the same analyses. Heme c555 was also prepared and spectrally characterized. The results indicated that leishmanial cytochrome c555 is similar but not identical to its crithidial counterpart.

Affinity separation and partial characterization of serologically active Leishmania donovani antigens.

Leishmania donovani-soluble antigens capable of antibody production in rabbits were separated from the total antigenic make-up of the parasite by adsorption on to anti-Leishmania immunoglobulins coupled to CNBr-activated Sepharose 4B (ALIG). Immune rabbit sera were produced either by intravenous inoculation of living promastigotes or by intradermal and subcutaneous inoculation of soluble antigens. Approximately four times as much soluble antigen was bound to ALIG produced by i.v. than i.d. and s.c. inoculation although the percentage recovery was less. The antigenic fractions recovered were subjected to immunoelectrophoresis, isoelectric point and molecular weight determination. The results of affinity chromatography indicate high antigen-antibody interaction affinity, the recovery of at least 28 antigens (with 0.15 M NaCl, pH 11.0) and complete loss of serological activity (determined by immunoelectrophoresis) on treatment with 8 M urea.

Comparative diagnostic study of kala azar.

Sera from 58 children with hepatosplenomegaly were tested for kala azar by gel diffusion immunoelectrophoresis, countercurrent immunoelectrophoresis and micro-ELISA. The results are compared with those from bone marrow cultures: 40 cases were diagnosed as kala azar by the four immunological techniques and Leishmania parasites were grown in 37 of 40 bone marrow cultures. The techniques used are discussed in terms of sensitivity, specificity and simplicity. The micro-ELISA technique was the most sensitive and convenient to carry out and is strongly recommended for use in routine diagnosis and epidermiological surveys.

Isoenzyme characterization of Leishmania species from Iraq.

Leishmaniasis in Iraq takes both the visceral and cutaneous forms. The causative organisms are identified according to the electrophoretic variation of the enzymes MDH, GPI, 6PGD, PGM and IDH. The 37 visceral stocks investigated fall into two groups which differ only with respect to GPI. The six cutaneous stocks were divided into three groups. Group 3 represents Leishmania major, while groups 4 and 5 refer to L. tropica showing intraspecific variation with regard to 6PGD.