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Biomedical subjects

G H Rao

Publications and source records attributed to G H Rao.

At least 19 recordsLinked to original sources
PubMed2000

Use of plasma glow for surface-engineering biomolecules to enhance bloodcompatibility of Dacron and PTFE vascular prosthesis.

The search for a nonthrombogenic material having patency to be used for small diameter vascular graft applications continues to be a field of extensive investigation. The purpose of the present study was to examine whether surface modification of polytetra fluoroethylene (PTFE, Teflon) and polyethylene-terephthalate (Dacron) vascular grafts might extend graft biocompatibility without modifying the graft structure. A series of surface coatings were prepared by modifying the argon plasma-treated PTFE and Dacron grafts with collagen IV and laminin and subsequently immobilizing bioactive molecules like PGE1, heparin or phosphatidyl choline via the carbodiimide functionalities. Surface analysis by Fourier transform infrared spectroscopy-attenuated total reflectance revealed the presence of new functional groups on the modified graft surfaces. In vitro studies showed that fibrinogen adsorption and platelet adhesion on modified grafts were significantly reduced. This study proposes that surface grafting of matrix components (collagen-type IV and laminin) and subsequent immobilization of bioactive molecules (PGE1, heparin or phosphatidyl choline) changed the surface conditioning of vascular grafts and subsequently improved their biocompatibility. However, more detailed in vivo studies are needed to confirm these observations.

Biocompatible Materials↗
PubMed2000

Evaluation of heparin immobilized chitosan-PEG microbeads for charcoal encapsulation and endotoxin removal.

A technique is described to encapsulate activated charcoal for hemoperfusion to be used in an artificial liver support. Activated charcoal was encapsulated within chitosan-PEG matrix and subsequently surface modified with PGE1 or heparin (hep-AC-PEGCB) via the glutaraldehyde functionalities. This novel matrix was used as the supports for perfusion of endotoxin, under a flow rate of 30 ml/mt. Endotoxin adsorption was quantitatively measured by the method of Limulus Amebocyte lysate test. It seems, the hep-AC-PEGCB may be a good adsorbent system for the removal of toxic endotoxin, and the system may be useful for detoxification of blood. The hep-AC-PEGCB matrix had improved biocompatibility as demonstrated from their hemolytic potential and charcoal release. However, further studies are needed to determine their behaviour under clinical conditions.

Alprostadil↗
PubMed1999

Modifications in accessibility of membrane glycoproteins, binding of specific ligands and coagulation factor V during the activation of platelets in blood emerging from bleeding time wounds.

Dual color flow cytometric techniques were applied to micro-aliquots of whole blood obtained from bleeding time (BT) wounds. Modifications in platelet activation markers (P-selectin [CD62P]) and lysosomal related protein (UMPS [CD63]), presence of membrane glycoproteins (GPIb [CD42b], GPIIb-IIIa [CD41a], GDIV [CD36], binding of von Willebrand factor (vWF), fibrinogen (Fg) and factor V [FV]) were analyzed in normal donors (n = 10) and in a severe von Willebrand patient (type 3) von Willebrand disease [vWD]). Samples of blood (20 microl) were sequentially removed from BT wound edges for up to 5 min and fixed with paraformaldehyde. Antigens were detected using the corresponding tagged monoclonal antibodies (moAbs) and quantitative results were referred to those found on platelet samples obtained from venous blood obtained from the same individuals. A progressive increase in % of platelets positive for activation dependent antigens (CD62 from 7 +/- 2 to 48 +/- 19% and CD63 from 9 +/- 1 to 44 +/- 8%; initial vs. 4 min) was observed. Accessibility of GPIIb-IIIa epitopes on platelets from BT wounds remained slightly above levels observed in venous blood platelets, despite a progressive increase in the presence of platelets positive for Fgn. Binding of MoAb to GPIV increased at late stages of BT. A moderate decrease in the binding of a moAb to GPIb was observed on platelets obtained at late stages of the BT (14 +/- 9% and 20 +/- 6% at 4 and 5 min, respectively). This apparent decrease in GPIb epitopes paralleled an increased presence of platelets positive for vWF (26 +/- 12 and 38 +/- 15%). Binding of moAb to GPIb always remained above basal levels in platelets obtained from BTs performed in the patient with type 3 vWD. FV levels on platelets coming from the BT persisted at background levels in all the individuals and at all times studied.

Antibodies, Monoclonal↗
PubMed1999

Evaluation of modified alginate-chitosan-polyethylene glycol microcapsules for cell encapsulation.

A bioartificial pancreas, a medical device entrapping islets of Langerhans (islets) in an immunoisolative membrane, has been regarded as one of the most promising approaches to treat insulin-dependent diabetic patients. In this study, various modifications of alginate-chitosan microcapsules were made such as the inclusion of polyethylene glycol (PEG) and the use of crosslinkers such as carbodiimide (EDC) and glutaraldehyde (GA) in the core and onto the microcapsule membrane surface. A characterization of the modified microcapsules in terms of mechanical stability and albumin diffusion as well as their surface properties using SEM was performed. A mild GA treatment greatly enhanced the mechanical stability of the microcapsules, and this treatment did not affect the coating process of chitosan or PEG. The biological response to such microcapsules was evaluated by microencapsulation of red blood cells (RBC) and subsequent observation of their hemoglobin release. The encapsulated RBC in the PEG-GA coated microcapsules were found to be less hemolytic and had improved stability and biocompatibility. The results suggest the possibility of developing biological assist organs by microencapsulation of mammalian cells such as islets or liver cells in immunoisolative microcapsules in the near future.

Albumins↗
PubMed1999

Surface-immobilized biomolecules on albumin modified porcine pericardium for preventing thrombosis and calcification.

The search for a noncalcifying tissue material to be used for valve replacement application continues to be a field of extensive investigation. A series of porcine pericardial membranes was prepared by modifying the glutaraldehyde--treated tissues with albumin and subsequently immobilizing bioactive molecules like PGE1, PGI2 or heparin via the carbodiimide functionalities. The in vitro calcification and collagenase degradation of these modified tissues were studied as a function of exposure time. Furthermore, the biocompatibility aspects of such novel interfaces were established by platelet adhesion and fibrinogen adsorption. The results reported in this article propose that the treatment with antiplatelet agents such as albumin, heparin and prostaglandins (PGE1 or PGI2) change the surface conditioning of pericardial tissues, suggesting a possible role of deposited serum components in affecting mineralization process on bioprosthesis. Therefore, it is worthy to hypothesize that besides inhibiting the accumulation of calcium in the devitalized cells, the early formation of a conditioning layer on the bioprosthesis surface may affect salt precipitations, determining the propensity of the implant to calcify. More detailed studies are needed to understand the involvement of plasma proteins and cellular components of the recipient blood in tissue-associated calcification.

Albumins↗
PubMed1998

Changes in pericardial calcification due to antiplatelet agents: in vitro studies.

To develop tissue valves for prolonged use in the cardiovascular system, the complicated process of surface induced calcification must be better understood. Calcification was examined for 60 days on glutaraldehyde treated bovine pericardium (GABP) and enzyme extracted tissues fixed in glutaraldehyde (GATBP) incubated in metastable solutions of calcium phosphate, and the roles of aspirin and persantine in conjunction with vitamins C, B, or E, gentamycin (antibiotic), or pentothal sodium (anesthetic) in the medium were examined. Further, the diffusion of calcium across the GATBP was evaluated using a diffusion cell with 2 compartments. Pericardial calcification was also observed using scanning electron microscopy (SEM) techniques. It seems that the examined antiplatelet agents can modify the pericardial surfaces and subsequently their mineralization processes (GATBP, 31.7 micrograms/mg tissue; in the presence of 5 mg% vitamin C, 13.1 micrograms/mg tissue; in 1.5 mg% aspirin, 17.2 micrograms/mg tissue; and 1 mg% gentamycin, 14.8 micrograms/mg tissue) on exposure with the metastable calcium phosphate solution for 60 days. In addition, these agents may modify calcium transport and interfere with the adsorption at the surface, hence reducing calcium nodulation on GATBP. Scanning electron micrographs also revealed a reduction in calcium deposition on the pericardium due to these antiplatelet agents. It may be hypothesized that the influx of calcium on GATBP may be due to the cellular components or the involvement of plasma proteins like the fibrinogen molecule. The exact mechanism of these changes in the calcification of the pericardium are still unknown. From these in vitro findings, it appears that a combined vitamin therapy with low doses of aspirin may be beneficial for platelet suppression and thereby for prevention of thrombosis and calcification. However, more in vivo studies are needed to develop applications.

Anesthetics, Intravenous↗
PubMed1998

Platelet adhesion and spreading on protein-coated surfaces: variations in behavior in washed cells, PRP, and whole blood.

Platelet attachment and spreading were monitored on glass and various protein coated glass, under shear with washed platelets, platelet rich plasma (PRP) and whole blood, using fluorescence Optimas imaging system and software. Results showed that the platelet adhesion and spreading were sensitive to the nature of precoated proteins and the type of medium used for introducing platelet suspension for the study. In general, the cell adhesion and spreading were higher with fibrinogen (Fg), fibronectin (Fn), von Willebrand Factor (vWF), and collagen precoated surfaces. In the presence of albumin on the surface, however, platelets could not attach and spread fully when using washed cells. But, the surface attachment and spreading of the cells were higher on albumin substrates on exposure to PRP or whole blood. This may be due to the replacement of precoated albumin by other plasma proteins, like Fg to facilitate the platelet-surface attachment. The composition of this layer determines the extent of platelet activation and the adhesive strength between platelets and polymer surface. These results indicate that multiple adhesion receptors can mediate platelet adhesion and spread to matrix proteins immobilized on surfaces. Further, these studies combined with some of our earlier observations and suggestions propose the need for developing in vitro tests that resemble in vivo conditions.

Adsorption↗
PubMed1998

Microtubule coils versus the surface membrane cytoskeleton in maintenance and restoration of platelet discoid shape.

The discoid form of blood platelets is important to their function in hemostasis. Recent studies have suggested that the spectrin-rich surface membrane cytoskeleton and the cytoplasmic, actin-rich cytoskeleton are responsible for discoid shape, shape change, and recovery after activation or chilling. Earlier studies had suggested that circumferential coils of microtubules supported the disc shape of resting platelets and that their repositioning or reassembly restored disc shape after exposure to low temperature. The present study has used the chilling-rewarming model, together with microtubule stabilizing (taxol) and disassembling (vincristine) agents to retest the relative importance of the surface membrane cytoskeleton and circumferential microtubules in platelet discoid shape and its restoration. Washed platelet samples were rested at 37 degrees C and chilled to 4 degrees C; chilled and rewarmed to 37 degrees C for 60 minutes; or chilled, rewarmed, and exposed to the same cycle in the presence or absence of vincristine or taxol and fixed for study by disseminated interference phase contrast microscopy and electron microscopy. Rhodamine-phalloidin and flow cytometry were used to measure changes in actin filament assembly. Chilling caused loss of disc shape, pseudopod extension, disassembly of microtubule coils, and assembly of new actin filaments. Rewarming resulted in restoration of disc shape, pseudopod retraction, disassembly of new actin filaments, and reassembly of circumferential microtubule coils. Vincristine converted discoid platelets to rounded cells that extended pseudopods when chilled and retracted them when rewarmed, leaving spheres that could undergo the same sequence of changes when chilled and rewarmed again. Taxol prevented cold-induced disassembly of microtubules and limited pseudopod formation. Rewarming caused retraction of pseudopods on taxol-treated, discoid cells. Cytochalasin B, an agent that blocks new actin filament assembly, alone or in combination with taxol, inhibited the cold-induced shape change but not dilation of the open canalicular system. Rewarming eliminated open canalicular system dilation and restored lentiform appearance. The results indicate that microtubule coils are the major structural elements responsible for disc shape and its restoration after submaximal stimulation or rewarming of chilled platelets.

Actins↗
PubMed1997

Influence of ionized calcium on thrombin-induced down regulation of GPIb/IX receptors on human platelets.

The influence of ionized calcium on the down-regulation of GPIb/IX receptors on human platelets was evaluated by flow cytometry using monoclonal antibodies. Addition of EDTA alone to a washed platelet suspension did not cause decreased monoclonal antibody binding to the cells. However, introduction of thrombin to the washed platelets containing EDTA resulted in a marked decrease in binding of monoclonal antibodies to the GPIb/IX receptors. If calcium at 1-3 mmol/L was added to buffered platelets instead of EDTA before thrombin, down-regulation was prevented or significantly reduced. Restoring calcium to EDTA platelets after the thrombin-stimulated down-regulation had been in progress for 1-3 minutes caused reversal of decreased antibody binding by GPIb/IX to near resting levels. Results demonstrate that extracellular calcium is a major factor regulating thrombin-induced down-regulation.

Blood Platelets↗
PubMed1996

Aggregated-disaggregated, refractory platelets retain sensitivity to ristocetin.

The present investigation has used an aggregation-disaggregation-reaggregation model to determine if exposure to potent aggregating agents causing a refractory state in deaggregated platelets results in down-regulation and clearance of GPIb/IX, the receptor for von willebrand factor. Thrombin, the thrombin receptor activating peptide (TRAP) and the thromboxane A2 mimic, U46619, caused irreversible aggregation and secretion when stirred on an aggregometer with plateletrich plasma (PRP). Addition of prostaglandin E1 (PGE1) after the plateau of maximum aggregation was reached caused rapid dissociation of platelet aggregates. Dissociated platelets were refractory to a second exposure to the primary stimulus or to other aggregating agents. Exposure of the refractory cells to epinephrine before the primary agent restored sensitivity resulting in a second wave of irreversible aggregation. Deaggregated, refractory platelets, however, retained their sensitivity to ristocetin. Amounts of the antibiotic causing agglutination of resting PRP also caused agglutination of disaggregated, refractory platelets. Addition of epinephrine to samples of refractory platelets less sensitive to low concentrations of the antibiotic restored their capacity to undergo irreversible ristocetin-induced agglutination. Analysis of the frequency of gold particles associated with monoclonal antibodies directed against GPIb/IX on control platelets and disaggregated, refractory platelets showed no significant difference in the number of receptors. The findings indicate that significant numbers of GPIb/IX receptors remain on platelet surfaces following exposure to potent aggregating agents.

Adult↗
PubMed1996

Human platelet activation is inhibited by the occupancy of glycoprotein IIb/IIIa receptor.

We studied the effect of glycoprotein GPIIb/IIIa (integrin alpha IIb beta 3) receptor occupancy by adenosine 5',1-thiotriphosphate (ATP alpha S), a competitive inhibitor of the ADP receptor, by fibrinogen, and by peptides containing the RGD (Arg-Gly-Asp) sequence as RGDW (Arg-Gly-Asp-Trp), RGDS (Arg-Gly-Asp-Ser), or the negative control RGGW (Arg-Gly-Gly-Trp) on human platelet physiological functions: aggregation, ATP secretion, and [Ca2+]in. As the presence of a nucleotide binding site on GPIIb alpha has been demonstrated in platelets [N. J. Greco, N. Yamamoto, B. W. Jackson, N. N. Tandon, M. Moos, and G. A. Jamieson (1991) J. Biol. Chem. 266, 13627-13633], we studied the effect of ATP alpha S, which specifically binds to this site, on platelet activation. We observed that ATP alpha S inhibited aggregation by thrombin, ADP, PMA, and ionophore A23187. Moreover, ATP alpha S dose dependently inhibited ATP secretion by ionophore A23187 and Ca2+ transients by thrombin and vasopressin in both the presence and absence of external Ca2+. Fibrinogen, although induced by a potentiation of platelet aggregation, inhibited ATP secretion and [Ca2+]in elevation induced by low thrombin concentrations or by vasopressin, interfering with both Ca2+ entry and Ca2+ release by the intracellular stores. RGD peptides, which specifically bind to GPIIb/IIIa, inhibited aggregation, secretion, and Ca2+ transients by thrombin, whereas the negative control RGGW did not exert any effect. We conclude that the occupancy of the GPIIb/IIIa receptor binding sites modulates platelet function by giving an inhibitory outside-in signal in platelets, particularly effective in platelets stimulated with low agonist doses. We suggest that ATP alpha S, fibrinogen, or RGD compounds, by interacting with GPIIb/IIIa receptor, prime some intracellular negative feedback mechanisms, which prevent further activation of circulating platelets by low-intensity stimuli and intravascular aggregation.

Adenosine Triphosphate↗
PubMed1996

Platelet adhesion to novel phospholipid materials: modified phosphatidylcholine covalently immobilized to silica, polypropylene, and PTFE materials.

Based on the premise of achieving blood compatibility through mimicking the chemical constitutents of the biologically insert surface of the unactivated platelet membrane, a process was developed that entails the covalent grafting of modified phosphatidylcholine molecules to materials including silica, polypropylene, and polytetrafluoroethylene (PTFE) polymer films. These materials were characterized using x-ray photoelectron spectroscopy (XPS) and contactangle measurements. The phosphatidylcholine-containing materials (PC materials) were used as substrates in the plateletadhesion assays and were subjected to enzymatic degradation evaluation. Phosphatidylcholine-grafted silica materials do not support platelet adhesion. In addition the number of adherent platelets correlate with the amount of grafted phospholipid present, as indicated by the phosphorus/ carbon ratio obtained by XPS analysis. Platelet adhesion to phosphatidylcholine-grafted polypropylene and PTFE was inhibited 80% and 90%, respectively, when compared with platelet adhesion to unmodified polypropylene and PTFE.

Biocompatible Materials↗
PubMed1996

Physiology and function of platelets from patients with Alzheimer's disease.

The discovery that intact Alzheimer amyloid precursor protein is present in platelet granules, has created a great interest in the biochemistry, physiology and function of platelets of patients with Alzheimer disease (AD). In this study we monitored various biochemical and physiological parameters, such as serotonin and adenine nucleotide levels, membrane fluidity, agonist-mediated release of arachidonic acid, thromboxane formation, calcium mobilization, as well as irreversible aggregation and secretion of granule contents. Platelets of patients with AD responded poorly when stirred with weak or potent agonists on a platelet aggregometer. Although capable of agonist-mediated calcium mobilization and synthesis of thromboxanes, the aggregation response of platelets of patients with AD to thrombin and archidonate was considerably compromised. In view of the normal biochemistry and signal transduction capabilities, the compromised response of these cells to potent agonists like thrombin suggested an extrinsic defect. The present study has shown that a plasmatic factor is at least in part responsible for the functional abnormalities of AD platelets.

Adenine Nucleotides↗
PubMed1995

Retention of glycoprotein Ib/IX receptors on external surfaces of thrombin-activated platelets in suspension.

The present study has evaluated the hypothesis stating that glycoprotein (GP) Ib/IX, the receptor for von Willebrand factor (vWF), is downregulated and cleared from exposed surfaces to channels of the open canalicular system (OCS) on platelets activated by thrombin in suspension. Cryosections of resting and thrombin-activated platelets fixed at intervals of 1 to 30 minutes after stimulation by thrombin and stained with antiglycocalicin antibody and protein A gold showed no decrease in the density of GPIb/IX receptors on the platelet surface or increase on linings of the OCS at any interval after stimulation by thrombin. Thin sections of platelets exposed to thrombin in suspension followed by settling onto a plastic chamber for intervals of 1 to 30 minutes revealed retention of GPIb/IX receptors on exposed surfaces detected by vWF, anti-vWF, and protein A gold throughout the 30-minute period of study. Results of this investigation indicate that GPIb/IX receptors remain on the surface of platelets activated by thrombin in suspension, are not cleared to the OCS, and retain the ability to bind vWF for at least 30 minutes.

Blood Platelets↗
PubMed1995

Influx of extracellular calcium and agonist-coupling appear essential for the activation of thromboxane A2-dependent phospholipase A2 in human platelets.

The present study demonstrates the existence of a unique mechanism for arachidonic acid (AA)-specific phospholipase A2 (PLA2) activation, which requires both sustained elevation of cytosolic Ca2+ coupled to the influx of extracellular Ca2+ and agonist interaction in platelets. The activation of PLA2 in platelets exposed to thapsigargin was abolished by the inhibition of cyclooxygenase (COX), thus suggesting a requirement of endogenously produced COX metabolite(s) for the activation of this enzyme. A thromboxane A2 (TXA2) analog, U46619, restored the activation of this AA-specific PLA2 activation supporting the requirements of COX metabolite(s) especially TXA2. Our subsequent studies demonstrated that both the effects of TXA2, and U46619 could be mimicked by collagen. Neither the transient cytosolic Ca2+ rise nor the agonists such as U46619 or collagen alone were sufficient to prime the activation of this PLA2 in the absence of thapsigargin. Since collagen behaves very similarly to TXA2, we suggest that this PLA2, is not only responsive to TXA2, but also to other agonists such as collagen, as shown in this study. We suggest that the activation of this distinct TXA2- and collagen-sensitive PLA2 involves two steps: (a) sustained elevation of cytosolic Ca2+ coupled to the influx of extracellular Ca2+; and (b) interaction with agonists such as TXA2 and collagen.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗