From snake venom’s disintegrins and C-type lectins to anti-platelet drugs

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. Snake venoms are attractive natural sources for drug discovery and development, with a number of substances either in clinical use or in research and development. These drugs were developed based on RGD-containing snake venom disintegrins, which efficiently antagonize fibrinogen activation of αIIbβ3 integrin (glycoprotein GP IIb/IIIa). Typical examples of anti-platelet drugs found in clinics are Integrilin (Eptifibatide), a heptapeptide derived from Barbourin, a protein found in the venom of the American Southeastern pygmy rattlesnake and Aggrastat (Tirofiban), a small molecule based on the structure of Echistatin, and a protein found in the venom of the saw-scaled viper. Using a similar drug discovery approach, linear and cyclic peptides containing the sequence K(R)TS derived from VP12, a C-type lectin protein found in the venom of Israeli viper venom, were used as a template to synthesize Vipegitide, a novel peptidomimetic antagonist of α2β1 integrin, with anti-platelet activity. This review focus on drug discovery of these anti-platelet agents, their indications for clinical use in acute coronary syndromes and percutaneous coronary intervention based on several clinical trials, as well as their adverse effects

Biocompatibility studies of fluorescent diamond particles-(Nv)~800nm (part v): In vitro kinetics and in vivo localization in rat liver following long-term exposure

© 2019 Gerstenhaber et al. Background: We recently reported on long-term comprehensive biocompatibility and biodistribution study of fluorescent nanodiamond particles (NV)-Z-average 800nm (FNDP- (NV)) in rats. FNDP-(NV) primary deposition was found in the liver, yet liver function tests remained normal. Purpose: The present study aimed to gain preliminary insights on discrete localization of FNDP-(NV) in liver cells of the hepatic lobule unit and venous micro-vasculature. Kinetics of FDNP-(NV) uptake into liver cells surrogates in culture was conducted along with cell cytokinesis as markers of cells' viability. Methods: Preserved liver specimens from a pilot consisting of two animals which were stained for cytoskeletal elements (fluorescein-isothiocyanate-phalloidin) were examined for distribution of FNDP-(NV) by fluorescent microscopy (FM) and Confocal-FM (CFM) using near infra-red fluorescence (NIR). Hepatocellular carcinoma cells (HepG-2) and human umbilical vein endothelial cells (HUVEC) were cultured with FNDP-(NV) and assayed for particle uptake and location using spectrophotometric technology and microscopy. Results: HepG-2 and HUVEC displayed rapid (<30 mins) onset and concentration-dependent FNDP-(NV) internalization and formation of peri-nuclear corona. FM/CFM of liver sections revealed FNDP-(NV) presence throughout the hepatic lobules structures marked by spatial distribution, venous microvascular spaces and parenchyma and non-parenchyma cells. Conclusion: The robust presence of FNDP-(NV) throughout the hepatic lobules including those internalized within parenchyma cells and agglomerates in the liver venous microcirculation were not associated with macro or micro histopathological signs nor vascular lesions. Cells cultures indicated normal cytokinesis in cells containing FNDP-(NV) agglomerates. Liver parenchyma cells and the liver microcirculation remain agnostic to presence of FNDP-(NV) in the sinusoids or internalized in the hepatic cells

Pilot study on biocompatibility of fluorescent nanodiamond-(NV)-Z~800 particles in rats: Safety, pharmacokinetics, and bio-distribution (Part III)

© 2018 Barone et al. Introduction: We hereby report on studies aimed to characterize safety, pharmacokinetics, and bio-distribution of fluorescent nanodiamond particles (NV)-Z~800 (FNDP-(NV)) administered to rats by intravenous infusion in a single high dose. Methods: Broad scale biological variables were monitored following acute (90 minutes) and subacute (5 or 14 days) exposure to FNDP-(NV). Primary endpoints included morbidity and mortality, while secondary endpoints focused on hematology and clinical biochemistry biomarkers. Particle distribution (liver, spleen, lung, heart, and kidney) was assessed by whole organ near infrared imaging using an in vivo imaging system. This was validated by the quantification of particles extracted from the same organs and visualized by fluorescent and scanning electron microscopy. FNDP-(NV)-treated rats showed no change in morbidity or mortality and preserved normal motor and sensory function, as assessed by six different tests. Results: Blood cell counts and plasma biochemistry remained normal. The particles were principally distributed in the liver and spleen. The liver particle load accounted for 51%, 24%, and 18% at 90 minutes, 5 days, and 14 days, respectively. A pilot study of particle clearance from blood indicated 50% clearance 33 minutes following the end of particle infusion. Conclusion: We concluded that systemic exposure of rats to a single high dose of FDNP-(NV)-Z~800 (60 mg/kg) appeared to be safe and well tolerated over at least 2 weeks. These data suggest that FNDP-(NV) should proceed to preclinical development in the near future

Long-term biocompatibility of fluorescent diamonds-(NV)-Z~800 nm in rats: Survival, morbidity, histopathology, particle distribution and excretion studies (part IV)

© 2019 Barone et al. Background: Thromboembolic events are a major cause of heart attacks and strokes. However, diagnosis of the location of high risk vascular clots is hampered by lack of proper technologies for their detection. We recently reported on bio-engineered fluorescent diamond-(NV)-Z~800nm (FNDP-(NV)) conjugated with bitistatin (Bit) and proven its ability to identify iatrogenic blood clots in the rat carotid artery in vivo by Near Infra-Red (NIR) monitored by In Vivo Imaging System (IVIS). Purpose: The objective of the present research was to assess the in vivo biocompatibility of FNDP-(NV)-Z~800nm infused intravenously to rats. Multiple biological variables were assessed along this 12 week study commissioned in anticipation of regulatory requirements for a long-term safety assessment. Methods: Rats were infused under anesthesia with aforementioned dose of the FNDP-(NV), while equal number of animals served as control (vehicle treated). Over the 12 week observation period rats were tested for thriving, motor, sensory and cognitive functions. At the termination of study, blood samples were obtained under anesthesia for comprehensive hematology and biochemical assays. Furthermore, 6 whole organs (liver, spleen, brain, heart, lung and kidney) were collected and examined ex vivo for FNDP-NV) via NIR monitored by IVIS and histochemical inspection. Results: All animals survived, thrived (no change in body and organ growth). Neuro-behavioral functions remain intact. Hematology and biochemistry (including liver and kidney functions) were normal. Preferential FNDP-(NV) distribution identified the liver as the main long-term repository. Certified pathology reports indicated no outstanding of finding in all organs. Conclusion: The present study suggests outstanding biocompatibility of FNDP-(NV)-Z~800nm after long-term exposure in the rat

The Human Polyoma JC Virus Agnoprotein Acts as a Viroporin

Virus infections can result in a range of cellular injuries and commonly this involves both the plasma and intracellular membranes, resulting in enhanced permeability. Viroporins are a group of proteins that interact with plasma membranes modifying permeability and can promote the release of viral particles. While these proteins are not essential for virus replication, their activity certainly promotes virus growth. Progressive multifocal leukoencephalopathy (PML) is a fatal demyelinating disease resulting from lytic infection of oligodendrocytes by the polyomavirus JC virus (JCV). The genome of JCV encodes six major proteins including a small auxiliary protein known as agnoprotein. Studies on other polyomavirus agnoproteins have suggested that the protein may contribute to viral propagation at various stages in the replication cycle, including transcription, translation, processing of late viral proteins, assembly of virions, and viral propagation. Previous studies from our and other laboratories have indicated that JCV agnoprotein plays an important, although as yet incompletely understood role in the propagation of JCV. Here, we demonstrate that agnoprotein possesses properties commonly associated with viroporins. Our findings demonstrate that: (i) A deletion mutant of agnoprotein is defective in virion release and viral propagation; (ii) Agnoprotein localizes to the ER early in infection, but is also found at the plasma membrane late in infection; (iii) Agnoprotein is an integral membrane protein and forms homo-oligomers; (iv) Agnoprotein enhances permeability of cells to the translation inhibitor hygromycin B; (v) Agnoprotein induces the influx of extracellular Ca2+; (vi) The basic residues at amino acid positions 8 and 9 of agnoprotein key are determinants of the viroporin activity. The viroporin-like properties of agnoprotein result in increased membrane permeability and alterations in intracellular Ca2+ homeostasis leading to membrane dysfunction and enhancement of virus release

Digalactosyl-diacylglycerol-deficiency lowers the thermal stability of thylakoid membranes

We investigated the effects of digalactosyl-diacylglycerol (DGDG) on the organization and thermal stability of thylakoid membranes, using wild-type Arabidopsis thaliana and the DGDG-deficient mutant, dgd1. Circular-dichroism measurements reveal that DGDG-deficiency hampers the formation of the chirally organized macrodomains containing the main chlorophyll a/b light-harvesting complexes. The mutation also brings about changes in the overall chlorophyll fluorescence lifetimes, measured in whole leaves as well as in isolated thylakoids. As shown by time-resolved measurements, using the lipophylic fluorescence probe Merocyanine 540 (MC540), the altered lipid composition affects the packing of lipids in the thylakoid membranes but, as revealed by flash-induced electrochromic absorbance changes, the membranes retain their ability for energization. Thermal stability measurements revealed more significant differences. The disassembly of the chiral macrodomains around 55°C, the thermal destabilization of photosystem I complex at 61°C as detected by green gel electrophoresis, as well as the sharp drop in the overall chlorophyll fluorescence lifetime above 45°C (values for the wild type—WT) occur at 4–7°C lower temperatures in dgd1. Similar differences are revealed in the temperature dependence of the lipid packing and the membrane permeability: at elevated temperatures MC540 appears to be extruded from the dgd1 membrane bilayer around 35°C, whereas in WT, it remains lipid-bound up to 45°C and dgd1 and WT membranes become leaky around 35 and 45°C, respectively. It is concluded that DGDG plays important roles in the overall organization of thylakoid membranes especially at elevated temperatures

Fibulin-5, an integrin-binding matricellular protein: its function in development and disease

Interactions between the extracellular matrix (ECM) and cells are critical in embryonic development, tissue homeostasis, physiological remodeling, and tumorigenesis. Matricellular proteins, a group of ECM components, mediate cell-ECM interactions. One such molecule, Fibulin-5 is a 66-kDa glycoprotein secreted by various cell types, including vascular smooth muscle cells (SMCs), fibroblasts, and endothelial cells. Fibulin-5 contributes to the formation of elastic fibers by binding to structural components including tropoelastin and fibrillin-1, and to cross-linking enzymes, aiding elastic fiber assembly. Mice deficient in the fibulin-5 gene (Fbln5) exhibit systemic elastic fiber defects with manifestations of loose skin, tortuous aorta, emphysematous lung and genital prolapse. Although Fbln5 expression is down-regulated after birth, following the completion of elastic fiber formation, expression is reactivated upon tissue injury, affecting diverse cellular functions independent of its elastogenic function. Fibulin-5 contains an evolutionally conserved arginine-glycine-aspartic acid (RGD) motif in the N-terminal region, which mediates binding to a subset of integrins, including α5β1, αvβ3, and αvβ5. Fibulin-5 enhances substrate attachment of endothelial cells, while inhibiting migration and proliferation in a cell type- and context-dependent manner. The antagonistic function of fibulin-5 in angiogenesis has been demonstrated in vitro and in vivo; fibulin-5 may block angiogenesis by inducing the anti-angiogenic molecule thrompospondin-1, by antagonizing VEGF165-mediated signaling, and/or by antagonizing fibronectin-mediated signaling through directly binding and blocking the α5β1 fibronectin receptor. The overall effect of fibulin-5 on tumor growth depends on the balance between the inhibitory property of fibulin-5 on angiogenesis and the direct effect of fibulin-5 on proliferation and migration of tumor cells. However, the effect of tumor-derived versus host microenvironment-derived fibulin-5 remains to be evaluated

Bitistatin-functionalized fluorescent nanodiamond particles specifically bind to purified human platelet integrin receptor α<inf>iib</inf>β<inf>3</inf> and activated platelets

© 2017 Marcinkiewicz et al. Thromboembolic events (TEE) underwrite key causes of death in developed countries. While advanced imaging technologies such as computed tomography scans serve to diagnose blood clots during acute cardiovascular events, no such technology is available in routine primary care for TEE risk assessment. Here, we describe an imaging platform technology based on bioengineered fluorescent nanodiamond particles (F-NDPs) functionalized with bitistatin (Bit), a disintegrin that specifically binds to the αIIbβ3 integrin, platelet fibrinogen receptor (PFR) on activated platelets. Covalent linkage of purified Bit to F-NDP was concentration-dependent and saturable, as validated by enzyme-linked immunosorbent assay using specific anti-Bit antibodies. F-NDP–Bit interacted with purified PFR, either in immobilized or soluble form. Lotrafiban, a nonpeptide, αIIbβ3 receptor antagonist, specifically blocked F-NDP–Bit–PFR complex formation. Moreover, F-NDP–Bit specifically binds to activated platelets incorporated into a clot generated by thrombin-activated rat platelet-rich plasma (PRP). Our results suggest that engineered F-NDP–Bit particles could serve as noninvasive, “real-time” optical diagnostics for clots present in blood vessels

Topographic cues of a novel bilayered scaffold modulate dental pulp stem cells differentiation by regulating YAP signalling through cytoskeleton adjustments

© 2019 The Authors. Cell Proliferation Published by John Wiley & Sons Ltd. Objectives: Topographic cues can modulate morphology and differentiation of mesenchymal stem cells. This study aimed to determine how topographic cues of a novel bilayered poly (lactic-co-glycolic acid) (PLGA) scaffold affect osteogenic/odontogenic differentiation of dental pulp stem cells (DPSCs). Methods: The surface morphology of the scaffolds was visualized by scanning electron microscope, and the surface roughness was measured by profilometry. DPSCs were cultured on each side of the scaffolds. Cell morphology, expression of Yes-associated protein (YAP) and osteogenic/odontogenic differentiation were analysed by immunohistochemistry, real-time polymerase chain reaction, and Alizarin Red S staining. In addition, cytochalasin D (CytoD), an F-actin disruptor, was used to examine the effects of F-actin on intracellular YAP localisation. Verteporfin, a YAP transcriptional inhibitor, was used to explore the effects of YAP signalling on osteogenic/odontogenic differentiation of DPSCs. Results: The closed side of our scaffold showed smaller pores and less roughness than the open side. On the closed side, DPSCs exhibited enhanced F-actin stress fibre alignment, larger spreading area, more elongated appearance, predominant nuclear YAP localization and spontaneous osteogenic differentiation. Inhibition of F-actin alignments was correlated with nuclear YAP exclusion of DPSCs. Verteporfin restricted YAP localisation to the cytoplasm, down-regulated expression of early osteogenic/odontogenic markers and inhibited mineralization of DPSCs cultures. Conclusions: The surface topographic cues changed F-actin alignment and morphology of DPSCs, which in turn regulated YAP signalling to control osteogenic/odontogenic differentiation

Bitistatin-functionalized fluorescent nanodiamond particles specifically bind to purified human platelet integrin receptor &alpha;IIb&beta;3 and activated platelets

Cezary Marcinkiewicz,1,2 Jonathan A Gerstenhaber,1 Mark Sternberg,2 Peter I Lelkes,1 Giora Feuerstein1,2 1Department of Bioengineering, College of Engineering, Temple University, Philadelphia, 2Debina Diagnostic, Inc., Newton Square, PA, USA Abstract: Thromboembolic events (TEE) underwrite key causes of death in developed countries. While advanced imaging technologies such as computed tomography scans serve to diagnose blood clots during acute cardiovascular events, no such technology is available in routine primary care for TEE risk assessment. Here, we describe an imaging platform technology based on bioengineered fluorescent nanodiamond particles (F-NDPs) functionalized with bitistatin (Bit), a disintegrin that specifically binds to the &alpha;IIb&beta;3 integrin, platelet fibrinogen receptor (PFR) on activated platelets. Covalent linkage of purified Bit to F-NDP was concentration-dependent and saturable, as validated by enzyme-linked immunosorbent assay using specific anti-Bit antibodies. F-NDP&ndash;Bit interacted with purified PFR, either in immobilized or soluble form. Lotrafiban, a nonpeptide, &alpha;IIb&beta;3 receptor antagonist, specifically blocked F-NDP&ndash;Bit&ndash;PFR complex formation. Moreover, F-NDP&ndash;Bit specifically binds to activated platelets incorporated into a clot generated by thrombin-activated rat platelet-rich plasma (PRP). Our results suggest that engineered F-NDP&ndash;Bit particles could serve as noninvasive, &ldquo;real-time&rdquo; optical diagnostics for clots present in blood vessels. Keywords: carbon nanoparticles, blood clots, imaging, platelet fibrinogen receptor, fluorescence, disintegrin, thromboembolic complications, thrombosi