Abstract 147: Plasma Factor XIII Binding to Cold-stored Platelets Results in Increased Fibrin Crosslinking and Clot Strength

Currently, platelets (PLTs) stored at room temperature (RT) for 5-7 days with gentle agitation are exclusively used for transfusion although FDA recently clarified that apheresis PLTs stored at 4°C for up to 72 hours may be used for treating active hemorrhage. We have demonstrated that cold (4C) storage of PLT is an attractive alternative to RT storage since it better preserves the PLT metabolic reserves, in vitro responses to agonists of activation, aggregation and physiologic inhibitors, as well as adhesion to thrombogenic surfaces. In this study, we tested the hypothesis that 4C-stored PLT will form clots with mechanical strength superior to those from RT-stored PLT due to higher hemostatic potential. From rheological measurements, we observed that the clots formed from 5 day 4C-stored PLTs are significantly stiffer (elastic modulus) and stronger (critical stress) than those formed from RT-stored PLT but comparable to fresh PLT (Fig. A). We also observed from ultrastructural microscopy that the fibrin fibers in clots from cold-stored PLT were thinner with more branch points than those from RT-stored PLTs, indicating the presence of increased crosslinks (Fig. B, C). Finally, molecular analysis revealed an increase FXIII transglutaminase activity due to the binding of plasma FXIII/fibrinogen to the surface of 4C-stored PLTs (Fig D).In conclusion, we have shown that cold-induced plasma FXIII binding to PLT surface result in increased fibrin crosslinking and enhanced clot strength. Our data, together with the benefit of reduced risk of late thrombosis due to their rapid clearance in vivo , underscores the consideration of 4C-stored PLT for acute response to hemorrhage. Figure 1 (A) RT-stored platelets form clots with less stiffness (n=5); (B) Representative SEM images of clots (n=3) (C) Clots from 4C-stored platelets have higher cross-linking density (n=5) (D) In FXIII-deficient plasma, 4C-stored platelets form clots with higher stiffness than fresh platelets (n=4)

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Synthesis and characterization of Chitosan-Catechol conjugates: Development and in vitro, in silico and in vivo evaluation of mucoadhesive pellets of lafutidine

Journal of Bioactive and Compatible Polymers ◽

10.1177/0883911521997849 ◽

2021 ◽

pp. 088391152199784

Author(s):

Loveleen Kaur ◽

Ajay Kumar Thakur ◽

Pradeep Kumar ◽

Inderbir Singh

Keyword(s):

Drug Release ◽

In Silico ◽

Ex Vivo ◽

Strong Interactions ◽

Dissolution Time ◽

Sem Images ◽

In Vivo Evaluation ◽

Release Studies

Present study was aimed to synthesize and characterize Chitosan-Catechol conjugates and to design and develop mucoadhesive pellets loaded with lafutidine. SEM images indicated the presence of fibrous structures responsible for enhanced mucoadhesive potential of Chitosan-Catechol conjugates. Thermodynamic stability and amorphous nature of conjugates was confirmed by DSC and XRD studies respectively. Rheological studies were used to evaluate polymer mucin interactions wherein strong interactions between Chitosan-Catechol conjugate and mucin was observed in comparison to pristine chitosan and mucin. The mucoadhesion potential of Chitosan-Catechol (Cht-C) versus Chitosan (Cht) was assessed in silico using molecular mechanics simulations and the results obtained were compared with the in vitro and ex vivo results. Cht-C/mucin demonstrated much higher energy stabilization (∆E ≈ −65 kcal/mol) as compared to Cht/mucin molecular complex. Lafutidine-loaded pellets were prepared from Chitosan (LPC) and Chitosan-Catechol conjugates (LPCC) and were evaluated for various physical properties viz. flow, circularity, roundness, friability, drug content, particle size and percent mucoadhesion. In vitro drug release studies on LPC and LPCC pellets were performed for computing t50%, t90% and mean dissolution time. The values of release exponent from Korsmeyer-Peppas model was reported to be 0.443 and 0.759 for LPC and LPCC pellets suggesting Fickian and non-Fickian mechanism representing drug release, respectively. In vivo results depicted significant controlled release and enhanced residence of the drug after being released from the chitosan-catechol coated pellets. Chitosan-Catechol conjugates were found to be a promising biooadhesive polymer for the development of various mucoadhesive formulations.

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Mobilization of Fibrinogen Receptor Sites on Human Platelets Stimulated with ADP is Prevented by Prostacyclin

10.1055/s-0039-1687387 ◽

1979 ◽

Author(s):

J. Hawiger ◽

S. Parkinson ◽

S. Timmons

Keyword(s):

Inhibitory Effect ◽

Human Platelets ◽

Affinity Constant ◽

Human Fibrinogen ◽

Fibrinogen Receptor ◽

Receptor Sites ◽

Plasma Factor ◽

Potent Inhibitory Effect

Fibrinogen is a plasma factor required for aggregation of human platelets by ADP. The mechanism of platelet-ADP-fibrinogen interaction was studied by measuring the equilibrium binding of 125I-fibrinogen to human platelets separated from plasma proteins. Binding of 125I-fibrinogen to platelets not stimulated with ADP was low and unaffected by an excess of unlabel led fibrinogen. However, when platelets were stimulated with 4μM of ADP, there was an eightfold increase In the number of available binding sites for human fibrinogen, with affinity constant of 1.9 x 109M-1. This striking increase in fibrinogen receptor sites on human platelets was specific for ADP as contrasted to ATP, AMP, and adenosine. Prostacyclin (Prostaglandin I2, PGI2), a novel prostaglandin produced by the blood vessel wall, completely blocked this ADP-induced increase in fibrinogen receptor sites on human platelets. The effect of PGI2 was prompt and concentration dependent, reaching maximum at 10-9M. 6-keto PGF2 a stable derivative ot PGI2, did not have such an effect. Thus movement of fibrinogen receptor sites on human platelet membrane stimulated with ADP is prevented by PGI2. This represents a new biologic property of this vascular hormone and contributes to better understanding of its potent inhibitory effect in vitro and in vivo on ADP-induced platelet aggregation requiring mobilization of fibrinogen receptor.

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Anti-leukemia properties of cadmium nanoparticles in the in vitro and in vivo condition: A chemobiological study

Archives of Medical Science ◽

10.5114/aoms/142465 ◽

2021 ◽

Author(s):

Yudi Miao ◽

Behnam Mahdavi ◽

Mohammad Zangeneh

Keyword(s):

Acute Myeloid Leukemia ◽

Cell Lines ◽

Aqueous Extract ◽

Myeloid Leukemia ◽

Antioxidant Properties ◽

Leukemia Cell ◽

Sem Images ◽

Acute Myeloid

IntroductionThe present study investigated the anti-acute myeloid leukemia effects of Ziziphora clinopodides Lam leaf aqueous extract conjugated cadmium nanoparticles.Material and methodsTo synthesize CdNPs, Z. clinopodides aqueous extract was mixed with Cd(NO3)2 .4H2O. The characterization of the biosynthesized cadmium nanoparticles was carried out using many various techniques such as UV-Vis. and FT-IR spectroscopy, XRD, FE-SEM, and EDS.ResultsThe uniform spherical morphology of NPs was proved by FE-SEM images with NPs the average size of 26.78cnm. For investigating the antioxidant properties of Cd(NO3)2, Z. clinopodides, CdNPs, and Daunorubicin, the DPPH test was used. The cadmium nanoparticles inhibited half of the DPPH molecules in a concentration of 196 µg/mL. To survey the cytotoxicity and anti-acute myeloid leukemia effects of Cd(NO3)2, Z. clinopodides, CdNPs, and Daunorubicin, MTT assay was used on the human acute myeloid leukemia cell lines i.e., Murine C1498, 32D-FLT3-ITD, and Human HL-60/vcr. The IC50 of the cadmium nanoparticles was 168, 205, and 210 µg/mL against Murine C1498, 32D-FLT3-ITD, and Human HL-60/vcr cell lines, respectively. In the part of in vivo study, DMBA was used for inducing acute myeloid leukemia in mice. CdNPs similar to daunorubicin ameliorated significantly (p≤0.01) the biochemical, inflammatory, RBC, WBC, platelet, stereological, histopathological, and cellular-molecular parameters compared to the other groups.ConclusionsAs mentioned, the cadmium nanoparticles had significant anti-acute myeloid leukemia effects. After approving the above results in the clinical trial studies, these cadmium nanoparticles can be used as a chemotherapeutic drug to treat acute myeloid leukemia in humans.

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A Novel Type V CRISPR System with Potential for Genome Editing in the Liver

Blood ◽

10.1182/blood-2021-154505 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1862-1862

Author(s):

Gregory J. Cost ◽

Morayma Temoche-Diaz ◽

Janet Mei ◽

Cristina N. Butterfield ◽

Christopher T. Brown ◽

...

Keyword(s):

Amino Acid ◽

Genome Editing ◽

Mammalian Cells ◽

Conflicts Of Interest ◽

Attractive Alternative ◽

Vitro Assay ◽

Crispr System ◽

Type V

Abstract RNA guided CRISPR genome editing systems can make specific changes to the genomes of mammalian cells and have the potential to treat a range of diseases including those that can be addressed by editing hepatocytes. Attempts to edit the liver in vivo have relied almost exclusively on the Cas9 nucleases derived from the bacteria S treptococcus pyogenes or Staphylococcus aureus to which humans are commonly exposed. Pre-existing immunity to both these proteins has been reported in humans which raises concerns about their in vivo application. In silico analysis of a large metagenomics database followed by testing in mammalian cells in culture identified MG29-1, a novel CRISPR system which is a member of the Type V family but exhibits only 41 % amino acid identity to Francisella tularensis Cas12a/cpf1. MG29-1 is a 1280 amino acid RNA programmable nuclease that utilizes a single guide RNA comprised of a 22 nucleotide (nt) constant region and a 20 to 25 nt spacer, recognizes the PAM KTTN (predicted frequency 1 in 16 bp) and generates staggered cuts. MG29-1 was derived from a sample taken from a hydrothermal vent and it is therefore unlikely that humans will have developed pre-existing immunity to this protein. A screen for sgRNA targeting serum albumin in the mouse liver cell line Hepa1-6 identified 6 guides that generated more than 80% INDELS. The MG29-1 system was optimized for in vivo delivery by screening chemical modifications to the guide that improve stability in mammalian cell lysates while retaining or improving editing activity. Two lead guide chemistries were evaluated in mice using MG29-1 mRNA and sgRNA packaged in lipid nanoparticles (LNP). Three days after a single IV administration on-target editing was evaluated in the liver by Sanger sequencing. The sgRNA that was the most stable in the in vitro assay generated INDELS that ranged from 20 to 25% while a sgRNA with lower in vitro stability failed to generate detectable INDELs. The short sgRNA and small protein size compared to spCas9 makes MG29-1 an attractive alternative to spCas9 for in vivo editing applications. Evaluation of the potential of MG29-1 to perform gene knockouts and gene additions via non-homologous end joining is ongoing. Disclosures No relevant conflicts of interest to declare.

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Anatomic and Biochemical Correlates of the Dopamine Transporter Ligand 11C-PE2I in Normal and Parkinsonian Primates: Comparison with 6-[18F]Fluoro-L-Dopa

Journal of Cerebral Blood Flow & Metabolism ◽

10.1097/00004647-200107000-00003 ◽

2001 ◽

Vol 21 (7) ◽

pp. 782-792 ◽

Cited By ~ 39

Author(s):

Thomas Poyot ◽

Françoise Condé ◽

Marie-Claude Grégoire ◽

Vincent Frouin ◽

Christine Coulon ◽

...

Keyword(s):

Gold Standard ◽

Dopaminergic Neurons ◽

Emission Tomography ◽

Attractive Alternative ◽

Input Rate ◽

Nigrostriatal Pathway ◽

Positron Emission ◽

Neuroprotective Strategies

Positron emission tomography (PET) coupled to 6-[18F]Fluoro-L-Dopa (18F-Dopa) remains the gold standard for assessing dysfunctionality concerning the dopaminergic nigrostriatal pathway in Parkinson's disease and related disorders. The use of ligands of the dopamine transporters (DAT) is an attractive alternative target; consequently, the current aim was to validate one of them, 11C-PE2I, using a multiinjection modeling approach allowing accurate quantitation of DAT densities in the striatum. Experiments were performed in three controls, three MPTP-treated (parkinsonian) baboons, and one reserpine-treated baboon. 11C-PE2I B′max values obtained with this approach were compared with 18F-Dopa input rate constant values (Ki), in vitro Bmax binding of 125I-PE2I, and the number of dopaminergic neurons in the substantia nigra estimated postmortem by stereology. In the caudate nucleus and putamen, control values for 11C-PE2I B'max were 673 and 658 pmol/mL, respectively, whereas it was strongly reduced in the MPTP-treated (B′max = 26 and 36 pmol/mL) and reserpine-treated animals (B′max = 338 and 483 pmol/mL). In vivo11C-PE2I B′max values correlated with 18F-Dopa Ki values and in vitro125I-PE2I Bmax values in the striatum and with the number of nigral dopaminergic neurons. Altogether, these data support the use of 11C-PE2I for monitoring striatal dopaminergic disorders and the effect of potential neuroprotective strategies.

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Oxygen pressure-dependent control of carbonic anhydrase synthesis in chick embryonic erythrocytes

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.1991.261.5.r1188 ◽

1991 ◽

Vol 261 (5) ◽

pp. R1188-R1196 ◽

Cited By ~ 5

Author(s):

D. Million ◽

P. Zillner ◽

R. Baumann

Keyword(s):

Carbonic Anhydrase ◽

Pertussis Toxin ◽

Second Messengers ◽

Fold Increase ◽

Rapid Onset ◽

Inhibitor Molecule ◽

Activation Of Transcription ◽

Plasma Factor

During chick embryonic development carbonic anhydrase (CA) expression of erythrocytes is kept at a very low level until the last week of incubation (i.e., up to day 14). We have previously obtained evidence that hypoxia is the physiological stimulus for rapid onset of CA synthesis before hatching. Looking for putative signals we have carried out in vitro incubations of embryonic erythrocytes, screening a large number of hormones and second messengers, which were all ineffective, with the exception of the A1 agonist N6-phenylisopropyladenosine (adenosine had no effect). However, incubation with embryonic plasma (10%) from embryos greater than 6 days caused a 10-fold increase of the CA activity during 24 h. This increase was not observed when the incubation was carried out with the addition of actinomycin D, cycloheximide, aluminum fluoride, pertussis toxin, or heat-inactivated plasma. Mammalian plasma had no effect on CA activity. Filtration experiments show that the molecular mass of the factor is less than 2,000 Da. We conclude that embryonic plasma contains a heat-labile factor which stimulates CA synthesis via activation of transcription and whose receptor is coupled to a pertussis toxin-sensitive G protein. In vivo the action of the plasma factor is suppressed as long as blood Po2 is high, suggesting the presence of an inhibitor molecule whose synthesis is controlled by the Po2.

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Baculoviruses as Vectors for Gene Therapy against Human Prostate Cancer

Journal of Biomedicine and Biotechnology ◽

10.1155/s1110724303209049 ◽

2003 ◽

Vol 2003 (2) ◽

pp. 79-91 ◽

Cited By ~ 20

Author(s):

Lindsay J. Stanbridge ◽

Vincent Dussupt ◽

Norman J. Maitland

Keyword(s):

Prostate Cancer ◽

Gene Therapy ◽

Viral Entry ◽

Mammalian Cells ◽

Primary Tumour ◽

Genetic Material ◽

Cell Types ◽

Attractive Alternative

Current curative strategies for prostate cancer are restricted to the primary tumour, and the effect of treatments to control metastatic disease is not sustained. Therefore, the application of gene therapy to prostate cancer is an attractive alternative. Baculoviruses are highly restricted insect viruses, which can enter, but not replicate in mammalian cells. Baculoviruses can incorporate large amounts of extra genetic material, and will express transgenes in mammalian cells when under the control of a mammalian or strong viral promoter. Successful gene delivery has been achieved both in vitro and in vivo and into both dividing and nondividing cells, which is important since prostate cancers divide relatively slowly. In addition, the envelope protein gp64 is sufficiently mutable to allow targeted transduction of particular cell types. In this review, the advantages of using baculoviruses for prostate cancer gene therapy are explored, and the mechanisms of viral entry and transgene expression are described.

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Motility of bile canaliculi in the living animal: implications for bile flow.

The Journal of Cell Biology ◽

10.1083/jcb.113.5.1069 ◽

1991 ◽

Vol 113 (5) ◽

pp. 1069-1080 ◽

Cited By ~ 93

Author(s):

N Watanabe ◽

N Tsukada ◽

C R Smith ◽

M J Phillips

Keyword(s):

Bile Flow ◽

Cytochalasin B ◽

Time Lapse ◽

Sodium Fluorescein ◽

Branch Points ◽

Bile Canaliculi ◽

Enhanced Fluorescence ◽

Microscopic Techniques

Modern fluorescence microscopic techniques were used to image the bile canalicular system in the intact rat liver, in vivo. By combining the use of sodium fluorescein secretion into bile, with digitally enhanced fluorescence microscopy and time-lapse video, it was possible to capture and record the canalicular motility events that accompany the secretion of bile in life. Active bile canalicular contractions were found predominantly in zone 1 (periportal) hepatocytes of the liver. The contractile movements were repetitive, forceful, and appeared unidirectional moving bile in a direction towards the portal bile ducts. Contractions were not seen in the network of canaliculi on the surface of the liver. Cytochalasin B administration resulted in reduced canalicular motility, progressive dilation of zone 1 canaliculi, and impairment of bile flow. Canalicular dilations invariably involved the branch points of the canalicular network. The findings add substantively to previous in vitro studies using couplets, and suggest that canalicular contractions contribute physiologically to bile flow in the liver.

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An Optimized 3D Coculture Assay for Preclinical Testing of Pro- and Antiangiogenic Drugs

SLAS DISCOVERY Advancing Life Sciences ◽

10.1177/2472555216686529 ◽

2017 ◽

Vol 22 (5) ◽

pp. 602-613 ◽

Cited By ~ 1

Author(s):

Daniela Unterleuthner ◽

Nina Kramer ◽

Karoline Pudelko ◽

Alexandra Burian ◽

Markus Hengstschläger ◽

...

Keyword(s):

Basal Membrane ◽

Cell Interaction ◽

Resistance Mechanisms ◽

Culture Conditions ◽

Angiogenic Factors ◽

Branch Points ◽

Intrinsic Resistance ◽

Antiangiogenic Drugs

Angiogenesis is a promising target for anticancer therapies, but also for treating other diseases with pathologic vessel development. Targeting the vascular endothelial growth factor (VEGF) pathway did not proof as effective as expected due to emerging intrinsic resistance mechanisms, as well as stromal contributions leading to drug insensitivity. Therefore, alternative strategies affecting the interaction of endothelial cells (ECs) with other stromal cells seem to be more promising. Human preclinical in vitro angiogenesis models successfully recapitulating these interactions are rare, and two-dimensional (2D) cell cultures cannot mimic tissue architecture in vivo. Consequently, models combining three-dimensionality with heterotypic cell interaction seem to be better suited. Here, we report on an improved human fibroblast–EC coculture assay mimicking sprouting angiogenesis from EC-covered microbeads resembling existing endothelial structures. Culture conditions were optimized to assess pro- and antiangiogenic compounds. Important characteristics of angiogenesis, that is, the number of sprouts and branch points, sprout length protrusion, and overall vessel structure areas, were quantified. Notably, the endothelial sprouts display lumen formation and basal membrane establishment. In this model, angiogenesis can be inhibited by genetic interference of pro-angiogenic factors expressed in the fibroblasts. Moreover, bona fide antiangiogenic drugs decreased, whereas pro-angiogenic factors increased vessel formation in 24-well and 96-well settings, demonstrating the applicability for screening approaches.

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