L K Krishnan

Publications of L K Krishnan

• Adult stem cell homing and differentiation in vitro on composite fibrin matrix.

  Authors: P R Sreerekha, P Divya, L K Krishnan

  Cell proliferation. 09/2006; 39(4):301-12.

  Strategies to generate differentiated cells from haematopoetic progenitor cells will enhance potential use of adult stem cells for therapeutic transplantation or tissue engineering. TransplantationStrategies to generate differentiated cells from haematopoetic progenitor cells will enhance potential use of adult stem cells for therapeutic transplantation or tissue engineering. Transplantation of undifferentiated stem cells into recipient tissue hinges on the hypothesis of a milieu dependent differentiation and it has been suggested that a clot-equivalent scaffold is crucial for these circulating cells to anchor and multiply. Here a natural scaffold, fibrin along with fibronectin, gelatin and growth factors has been used to induce endothelial progenitor cells and smooth muscle progenitor cells to differentiate into endothelial cells and smooth muscle cells, respectively, from peripheral blood mononuclear cells. Characteristics of endothelial cells have been verified by the detection of mRNA for and immunostaining the cells for von Willebrand factor, uptake of acetylated low-density lipoproteins and measurement of released nitric oxide in the culture medium, as nitrite. The specific molecules that characterized smooth muscle cells were alpha smooth muscle actin and calponin, besides deposition of collagen type I and elastin, onto the culture matrix. The adhesive proteins used for the fabrication of endothelial progenitor cells matrix and smooth muscle progenitor cells matrix were the same, but specific differentiation was brought about by modulating the growth factor composition in the matrix and in the culture medium. Both endothelial and smooth muscle cells were consistently developed from 20 ml of human blood.

• Fibrin-mediated endothelial cell adhesion to vascular biomaterials resists shear stress due to flow.

  Authors: T R S Kumar, L K Krishnan

  Journal of materials science. Materials in medicine. 09/2002; 13(8):751-5.

  In vitro endothelial cell (EC) seeding onto biomaterials for blood-contacting applications can improve the blood compatibility of materials. Adhesive proteins adsorbed from serum that is supplementedIn vitro endothelial cell (EC) seeding onto biomaterials for blood-contacting applications can improve the blood compatibility of materials. Adhesive proteins adsorbed from serum that is supplemented with the culture medium intercede the initial cell adhesion and subsequent spreading on material surface during culture. Nevertheless, physical and chemical properties of vascular biomaterial surface fluctuate widely between materials resulting in dissimilarity in protein adsorption characteristics. Thus, a variation is expected in cell adhesion, growth and the ability of cell to resist shear stress when tissue engineering on to vascular biomaterials is attempted. This study was carried out with an objective to determine the significance of a matrix coating on cell adhesion and shear stress resistance when cells are cultured on materials such as polytetrafluoroethylene (PTFE, Teflon) and polyethyleneterephthalate (Dacron), ultra high molecular weight polyethylene (UHMWPE) and titanium (Ti), that are used for prosthetic devices. The study illustrates the distinction of EC attachment and proliferation between uncoated and matrix-coated surfaces. The cell attachment and proliferation on uncoated UHMWPE and titanium surfaces were not significantly different from matrix-coated surfaces. However, shear stress resistance of the cells grown on composite coated surfaces appeared superior compared to the cells grown on uncoated surface. On uncoated vascular graft materials, the cell adhesion was not supported by serum alone and proliferation was scanty as compared to matrix-coated surface. Therefore, coating of implant devices with a composite of adhesive proteins and growth factors can improve EC attachment and resistance of the cells to the forces of flow.

• Quantitation of platelet adhesion to Ti and DLC-coated Ti in vitro using (125)I-labeled platelets.

  Authors: L K Krishnan, N Varghese, C V Muraleedharan, G S Bhuvaneshwar, F Derangère, Y Sampeur, R Suryanarayanan

  Biomolecular engineering. 09/2002; 19(2-6):251-3.

  Measurement of platelet adhesion in vitro is a good indicator of its reactivity to implant devices in vivo. Platelets were labeled with I-125 without affecting its normal morphology and function andMeasurement of platelet adhesion in vitro is a good indicator of its reactivity to implant devices in vivo. Platelets were labeled with I-125 without affecting its normal morphology and function and the labeled platelets were suspended in platelet poor plasma and exposed to Ti and diamond like carbon-coated (DLC) Ti discs, under dynamic conditions, using a parallel plate flow chamber. The test materials were washed, dried, exposed to a phosphor screen and scanned to get the images. The number of platelets that adhered to Ti was found to be higher than those that adhered to DLC coated Ti sample, irrespective of the shear stress which was varied between 2 and 16 dynes/cm(2).

• Development of viper-venom antibodies in chicken egg yolk and assay of their antigen binding capacity.

  Authors: C Maya Devi, M Vasantha Bai, L K Krishnan

  Toxicon : official journal of the International Society on Toxinology. 08/2002; 40(7):857-61.

  The therapeutic use of specific antibodies is invaluable in certain clinical conditions, such as administration of specific antivenom for snakebite envenomation. The production of antibodies andThe therapeutic use of specific antibodies is invaluable in certain clinical conditions, such as administration of specific antivenom for snakebite envenomation. The production of antibodies and their purification from mammalian blood has been found low yielding and laborious. Most antivenom is polyvalent whole serum or partially purified immunoglobulin. The side effects of anti-snake-venom therapy include serum sickness and can be reduced by using mono-specific antivenom in sufficiently pure form. We have attempted to standardize a simple method for producing avian antivenom in relatively pure form from eggs. The isolation is very simple and involves only two steps, namely, removal of lipids from the diluted egg yolk followed by gel filtration. Each egg produces 80-100mg of pure immunoglobulin, and specific antibodies are present for up to 100 days after immunization. Thus, large quantities of the Ig can be obtained in pure form using only small amounts of venom.Antigen binding was shown by Ouchterlony's double diffusion experiments and the avian antivenom neutralizes the thrombin-like activity of equivalent amounts of venom on human plasma. The LD(50) of the venom was approximately 3mg/kg body weight in mice and rats but when pre-incubated with equivalent amounts (by weight) of egg IgG injected subcutaneously, all the animals survived. In a similar experiment using a commercial horse IgG, 25% mortality is seen. These results indicate that the antivenom immunoglobulins purified from immunized chicken egg yolk is biologically active and the possibility of their therapeutic use will be investigated further.

• Endothelial cell growth factor (ECGF) enmeshed with fibrin matrix enhances proliferation of EC in vitro.

  Authors: T R Santhosh Kumar, L K Krishnan

  Biomaterials. 11/2001; 22(20):2769-76.

  The vascular biomaterials that are currently used for clinical implants have been considered as poor substrates for human endothelial cell adhesion and spreading. Therefore, thrombotic occlusion isThe vascular biomaterials that are currently used for clinical implants have been considered as poor substrates for human endothelial cell adhesion and spreading. Therefore, thrombotic occlusion is the predominant cause for the failure of small diameter vascular grafts made out of Dacron or Teflon. To reduce surface thrombogenicity of material surfaces used for vascular implants, in vitro seeding of endothelial cells using adhesive protein matrix is under evaluation in various laboratories. Evidences suggest that fibrin matrix is a suitable matrix for endothelial cell (EC) adhesion to the currently available vascular graft materials; however, poor proliferation of attached cells seems to be a major limitation. During this study we have also found that fibrin is a better matrix compared to gelatin to support cell attachment and spreading. However, the poor proliferation of initially attached human umbilical cord vein endothelial cell (HUVEC) necessitated modification of the matrix composition to get a monolayer within a limited period. Since fibrin can form a network of protein bundles, an effort is made to incorporate growth factors within the matrix. Endothelial cell growth factor (ECGF) isolated from bovine hypothalamus is immobilized on the surface with fibrin glue (FG) to promote proliferation of HUVEC. The results demonstrate that proteins with similar molecular weights as growth factors (GF) are retained within the matrix and released into the culture medium for 96 h, in quantities that would be sufficient to promote cell proliferation. When cells were seeded on the matrix composed with components of FG and ECGF, the HUVEC proliferated at a significantly higher rate compared to the cells on surfaces coated with gelatin or fibrin. The EC thus grown on the composite (FG + ECGF) resisted the shear stress as compared to the cells grown on gelatin. The HUVEC monolayer grown on the composite seems thromboresistant as adhesion and activation of platelets are negligible after platelet rich plasma is incubated with the monolayer for about 1 h with agitation. Therefore, the composite of fibrin and ECGF can be a suitable matrix for further evaluation of patients' autologous endothelial cell attachment and proliferation for clinical application.

• Effect of temperature on functional response of platelet concentrates stored in PVC bags.

  Authors: L K Krishnan, J Mathai

  The Indian journal of medical research. 04/1997; 105:117-24.

  Haemostatic efficacy of platelet concentrates prepared and stored in locally available PVC triple bags was compared against a Japanese bag. In vitro functional parameters studied included shapeHaemostatic efficacy of platelet concentrates prepared and stored in locally available PVC triple bags was compared against a Japanese bag. In vitro functional parameters studied included shape change, aggregation and secretion in response to ADP. We have observed remarkable difference in the aggregatory response of platelets stored at slightly varying temperatures. The stimulatory responses of platelets stored with constant agitation at 70 strokes per min and 23 +/- 2 degrees C, deteriorated drastically by the time platelets were stored for 48 h. Both the rate and the extent of aggregation were affected showing no response to ADP at 72 h. However, when platelets were stored in a BOD incubator, thermostated at 22 +/- 0.5 degrees C, with continuous horizontal agitation at 70 strokes per min, 50 per cent functional response was retained till 72 h. We also demonstrated fragmentation of platelet membrane during storage. The membrane fragments collected by high speed centrifugation, expressed PF3 activity. Shedding of microvesicles indicates alterations at the membrane level that possibly cause functional lesion during storage. Our data suggest the significance of controlling the storage temperature steadily, to get maximum post transfusion efficacy.

• Morphological & ultrastructural changes of platelet concentrates stored in PVC bags.

  Authors: L K Krishnan, P V Sulochana, J Mathai, A John, R Sivakumar

  The Indian journal of medical research. 03/1997; 105:77-84.

  To assess the effect of storage bags on platelets, we studied the morphological and ultrastructural changes of samples drawn from platelet concentrates (PC) prepared and stored in triple, poly vinylTo assess the effect of storage bags on platelets, we studied the morphological and ultrastructural changes of samples drawn from platelet concentrates (PC) prepared and stored in triple, poly vinyl chloride (PVC) bags, manufactured in India. Using the scanning electron microscopy, we demonstrate formation of long pseudopods, and interaction through these to form aggregates. When platelets were stored at 23 +/- 2 degrees C, morphological changes were severe compared to the deleterious effects when kept at 22 +/- 0.5 degrees C. Ultrastructural analysis also showed that maintenance of discoid shape and prevention of granule secretion could be improved by storing the platelets at 22 +/- 0.5 degrees C. Significant degree of platelet fragmentation took place when the storage temperature was high. The morphology score done for platelets stored at both 22 +/- 0.5 degrees C and 23 +/- 2 degrees C showed that preservation of discoid shape was better with the former.

• Biochemical lesions of platelets stored as concentrates in PVC bags.

  Authors: L K Krishnan, J Mathai, P V Sulochana, J Jacob, R Sivakumar

  The Indian journal of medical research. 03/1997; 105:85-92.

  Acid-base status of platelet suspension during storage is a measure of the gas permeability of the bag material. To assess the efficacy of the bags available in our market to store platelets, weAcid-base status of platelet suspension during storage is a measure of the gas permeability of the bag material. To assess the efficacy of the bags available in our market to store platelets, we compared biochemical lesions of platelets stored in an Indian polyvinyl chloride (PVC) triple bag against a Japanese PVC bag standardized for 5 days platelet storage. Platelet concentrates prepared in both control and test PVC bags were stored for 72 h. Two ml samples were drawn 1 h after preparation, and then at 24 h intervals, for analysis. Our data show that the mean pH value in the test bags was maintained above 6.5. However, the CO2 tension was high and O2 tension was low. We also analyzed malondialdehyde (MDA) formation which is a measure of arachidonic acid metabolism, and seemed to be unaffected in stored platelets. Lactate dehydrogenase (LDH) was not released into the plasma excessively and hence significant platelet lysis was absent during storage. Hypotonic shock response (HSR) of platelets stored in both test and control bags was comparable, indicating the possibility of satisfactory post-transfusion recovery.

• Ajoene inhibition of platelet aggregation: possible mediation by a hemoprotein.

  Authors: M P Jamaluddin, L K Krishnan, A Thomas

  Biochemical and biophysical research communications. 06/1988; 153(1):479-86.

  Ajoene, an organosulfur compound derived from garlic, was found by spectral measurements, to interact, cooperatively, with a purified hemoprotein implicated, previously, in platelet activation. ItAjoene, an organosulfur compound derived from garlic, was found by spectral measurements, to interact, cooperatively, with a purified hemoprotein implicated, previously, in platelet activation. It modified the binding interactions of the protein with ligands, deemed to be physiologically relevant as effectors. The characteristics of the modifications were found to parallel those of ajoene induced modifications of agonist-induced aggregation kinetics of gel-filtered calf platelets.

• A rate equation for blood platelet aggregation.

  Authors: M P Jamaluddin, L K Krishnan

  Journal of theoretical biology. 12/1987; 129(2):257-61.

  A kinetic scheme for agonist-induced aggregation of blood platelets was formulated in terms of the kinetics of agonist interaction with the nonaggregable, discoid platelets, formation of aggregableA kinetic scheme for agonist-induced aggregation of blood platelets was formulated in terms of the kinetics of agonist interaction with the nonaggregable, discoid platelets, formation of aggregable forms by shape-change reactions, and interactions among shape-changed forms. Taking into account the relative magnitudes of the rate constants of the different steps and assuming aggregation to be by hydrophobic forces, an equation similar in form to the Michaelis-Menten equation was derived to characterize aggregation kinetics. The kinetic formulation could account for several empirical observations and may be used to interpret kinetic effects of antiplatelet drugs more informatively than at present.

• A spectrophotometric method for following initial rate kinetics of blood platelet aggregation.

  Authors: M P Jamaluddin, L K Krishnan

  Journal of biochemical and biophysical methods. 08/1987; 14(4):191-200.

  A double-beam recording spectrophotometer was used to assay platelet aggregation. Agonist-induced turbidity changes, at 540 nm, in dilute suspensions of platelets (1 ml, 6-8 X 10(7) platelets) wereA double-beam recording spectrophotometer was used to assay platelet aggregation. Agonist-induced turbidity changes, at 540 nm, in dilute suspensions of platelets (1 ml, 6-8 X 10(7) platelets) were recorded differentially against a reference cuvette, also containing platelets, as a function of time. The curves obtained showed downward pen deflections (decrease of turbidity) abolished by preincubation with the aggregation inhibitor, citrate. The turbidity decrease occurred simultaneously with microscopically determined single platelet recruitment into aggregates and its initial slope (r0) was a linear function of platelet concentration upto approximately 1.5 X 10(8) per ml. At a fixed platelet concentration the r0 values of ADP-induced aggregation of calf platelet-rich plasma varied as a hyperbolic function of ADP concentration at both 32 degrees C and 37 degrees C. The kinetic data at 37 degrees C were comparable to those, in the literature, obtained by following single platelet recruitment into aggregates. The increase in turbidity due to ADP-induced shape-change (6 +/- 1%, mean +/- S.E.M., n = 7) measured in the present study was substantially greater than that (3%) measured in the aggregometer.

• Conformational responses of an arachidonate- and U46619-binding haemoprotein in relation to platelet activation.

  Authors: L K Krishnan, M P Jamaluddin

  FEBS letters. 03/1987; 212(2):213-9.

  Antibodies were raised, in rabbits, against an arachidonate- and U46619-binding protein purified from calf platelets. Spectral measurements and immunodiffusion experiments were employed to followAntibodies were raised, in rabbits, against an arachidonate- and U46619-binding protein purified from calf platelets. Spectral measurements and immunodiffusion experiments were employed to follow conformational responses of the protein in relation to platelet activation. Upon treatment with the platelet agonists, arachidonate and PGH2, as well as the common haem ligands, imidazole and CN-, the purified protein had its Soret band red-shifted, with hypochromicity, but the protein saturated with the agonists, not with the haem ligands, showed altered antigenic properties in immunodiffusion experiments. In an analogous manner activation of gel-filtered calf platelets with high concentrations of ADP and A23187, as well as by cold, had Soret bands of extracts of sonicated platelets red-shifted, with hypochromicity; concomitantly, antigenically different conformations of the protein appeared in Triton X-100 extracts of the activated platelets. A protein immunologically related to the platelet protein was detected in Triton X-100 extracts of calf neutrophils. It is suggested that conformational changes of the protein induced by arachidonate or prostaglandin endoperoxides or H2O2 formed in different compartments during platelet activation by different stimuli may be a biochemical mechanism of stimulus-response coupling and that similar mechanisms might operate in other cell types.

• A molecular mechanism for the biphasic effect of exogenous arachidonate on platelets.

  Authors: M P Jamaluddin, L K Krishnan

  FEBS letters. 05/1985; 183(2):287-92.

  A haemoprotein (Mr approximately 40 000) made up of two apparently identical subunits, and having Soret maximum at 405 nm, but displaying it mostly at about 410 nm or often at 415 nm in crudeA haemoprotein (Mr approximately 40 000) made up of two apparently identical subunits, and having Soret maximum at 405 nm, but displaying it mostly at about 410 nm or often at 415 nm in crude extracts owing to different bound small molecules, found in platelets of several species, has been purified from calf platelets. The purified protein could bind the stable PGH2 analogue, U-46619, without co-operativity (h = 1.0 and half-maximal saturation concentration, S0.5 = 10 microM) and arachidonate with co-operativity that increased with arachidonate concentration (h increasing from approximately 2 to approximately 4). S0.5 of arachidonate was 1.5 microM. Arachidonate binding to the protein was accompanied by its oligomerization.