Plasma Coagulation on Microparticles Is Modulated by Water Activity. Relevance to Inflammation-Mediated Disruption of the Extracellular Matrix.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4048-4048
Author(s):  
Maria P. McGee ◽  
Lynne Li ◽  
Jorge Ollero
Keyword(s):  
Extracellular Matrix ◽  
Water Activity ◽  
Reaction Rates ◽  
Generation Rate ◽  
Limiting Factor ◽  
Plasma Extravasation ◽  
Factor X ◽  
In Vivo Models ◽  
Sorption Ability

Abstract The microenvironment of extravascular spaces influences the reactivities of extravasated plasma proteins. During inflammation, the structural and physicochemical characteristics of peri- and extra-vascular spaces can change widely depending on the severity of extracellular matrix disruption. Plasma extravasation after limited vascular injury results in transient changes in water activity (aw) and fibrin deposition. This work investigates whether changes in aw influence either pro- or anticoagulant pathways assembled on procoagulant microparticles. Based on conclusions from in vivo models indicating that factor Xa (fXa) concentration is the limiting factor in prothrombinase assembly we modeled the extravascular coagulation process in vitro, using plasma as the matrix; controlling aw as the independent variable; and measuring the fXa generation rate as the dependent variable. Initial rates of fXa generation were measured in human plasma (1/100 final dilution) containing procoagulant particles and 200 nM factor X. Water activity levels decreased from 0 to 0.8 atm relative to isosmolar plasma (~7 atm) with polyethylene glycol 8000 (~26 Å radius), while all reactant concentrations remained constant. Procoagulant particles were either recombinant tissue factor (TF) in PC/PS vesicles (at a mol ratio of 1/2666) or microvesicles collected from the monocytoid cell line THP-1 after 24-hour stimulation with endotoxin (1ug/ml). As the reaction environment’s aw progressively decreased, fXa generation rate first increased, and then decreased. Rate/aw profiles followed nearly symmetrical peaks with a maximum at ~0.2 atm and minimum at ~0.7 atm. Similar profiles were obtained with cell-derived procoagulant microvesicles. At each aw level, the fXa generation rate increased exponentially with TF concentration (25–100 pM). Activation and inhibition indices of ~1.5 and ~2.1, calculated as the ratio between the maximal and baseline and the maximal and minimal rates did not change significantly with TF concentration. Rates increased with added factor VIII (~2 units/ml) indicating fIX activation and a contribution of the intrinsic pathway protease to fXa generation. Furthermore, reaction rates changed with hirudin suggesting that thrombin-mediated regulatory loops also affect the rate profiles. Results in this simple model of extravascular coagulation indicate that aw modulates the rate of coagulation reactions stimulating or inhibiting them depending on whether the reaction environment’s aw is high or low. This conclusion in turn predicts that immediately after limited plasma extravasation coagulation rates accelerate with the decrease in aw as it tend to equilibrate with aw in the extravascular spaces. Continued water adsorption by an intact extracellular matrix would equilibrate aw and decrease coagulation rates. However, in a structurally damaged matrix with compromised water sorption ability, rates may remain longer at the higher level.

Structural similarity between the bone marrow extracellular matrix protein and neurokinin 1 could be the limiting factor in the hematopoietic effects of substance P

2002 ◽  
Vol 80 (5) ◽  
pp. 475-481 ◽  
Author(s):  
Pranela Rameshwar ◽  
Pedro Gascon ◽  
Persis S Bandari ◽  
Deval D Joshi ◽  
Annemarie Fernandes ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Extracellular Matrix ◽  
Substance P ◽  
Matrix Protein ◽  
Structural Similarity ◽  
Negative Regulator ◽  
Extracellular Matrix Protein ◽  
Limiting Factor ◽  
Neurokinin 1

In the adult bone marrow (BM), immune cells are replenished through the process of definitive hematopoiesis, which is regulated by a complex process of cellular and humoral interactions. The latter include substance P (SP), a neurotransmitter that is produced by neural and nonneural cells. Neurokinin-1 (NK-1), the high-affinity SP receptor, shares structural similarity with fibronectin, a component of the BM extracellular matrix proteins. This study examines how such similarity could alter the effects of SP on the proliferation of the immature BM progenitors. In vitro studies show that 1 ng fibronectin/mL enhanced the stimulatory effect of SP on the proliferation of primitive BM progenitors. This finding was studied by computational studies: proteomics and three-dimensional molecular modeling. Use of surface-enhanced laser desorption/ionization ProteinChip® technology showed that despite the induction of neutral endopeptidase, exogenous fibronectin hindered the degradation of SP to SP(1–4). These findings support a protective role for fibronectin in the digestion of SP. Since SP(1–4) is a negative regulator of hematopoiesis, this report indicates that the structural similarity between fibronectin and NK-1 could be important for maintaining hematopoietic stimulation. These studies could be extrapolated to hematological disorders that are associated with SP–fibronectin complexes.Key words: substance P, fibronectin, stem cell, neuropeptide, bone marrow.

Neutrophil migration through in vitro interstitial matrix

1992 ◽  
Vol 50 (1) ◽  
pp. 894-895
Author(s):  
J. Roemer ◽  
S.R. Simon
Keyword(s):  
Extracellular Matrix ◽  
Smooth Muscle ◽  
Cell Migration ◽  
Smooth Muscle Cells ◽  
Inflammatory Cell ◽  
Neutrophil Migration ◽  
Culture Conditions ◽  
Aortic Smooth Muscle ◽  
Specific Culture

We are developing an in vitro interstitial extracellular matrix (ECM) system for study of inflammatory cell migration. Falcon brand Cyclopore membrane inserts of various pore sizes are used as a support substrate for production of ECM by R22 rat aortic smooth muscle cells. Under specific culture conditions these cells produce a highly insoluble matrix consisting of typical interstitial ECM components, i.e.: types I and III collagen, elastin, proteoglycans and fibronectin.

Cytological changes induced by platinum-thymine in sarcoma-180 cells: Electron microscopy study

1990 ◽  
Vol 48 (3) ◽  
pp. 290-291
Author(s):  
Gustav Ofosu
Keyword(s):  
Mammalian Cells ◽  
Antitumor Agent ◽  
Microscopy Study ◽  
Electron Microscopy Study ◽  
Limiting Factor ◽  
Sarcoma 180 ◽  
Electron Microscopic ◽  
Cytological Changes

Platinum-thymine has been found to be a potent antitumor agent, which is quite soluble in water, and lack nephrotoxicity as the dose-limiting factor. The drug has been shown to interact with DNA and inhibits DNA, RNA and protein synthesis in mammalian cells in vitro. This investigation was undertaken to elucidate the cytotoxic effects of piatinum-thymine on sarcoma-180 cells in vitro ultrastructurally, Sarcoma-180 tumor bearing mice were treated with intraperitoneal injection of platinum-thymine 40mg/kg. A concentration of 60μg/ml dose of platinum-thymine was used in in vitro experiments. Treatments were at varying time intervals of 3, 7 and 21 days for in vivo experiments, and 30, 60 and 120 min., 6, 12, and 24th in vitro. Controls were not treated with platinum-thymine.Electron microscopic analyses of the treated cells in vivo and in vitro showed drastic cytotoxic effect.

Development of an in vitro Model for the Study of the Response of Equine Tendon Fibroblasts to Injury and Medication

1997 ◽  
Vol 10 (01) ◽  
pp. 6-11 ◽  
Author(s):  
R. F. Rosenbusch ◽  
L. C. Booth ◽  
L. A. Dahlgren
Keyword(s):  
Electron Microscopy ◽  
Extracellular Matrix ◽  
Light Microscopy ◽  
Light And Electron Microscopy ◽  
Tendon Healing ◽  
Matrix Proteins ◽  
Isolated Cells ◽  
Superficial Digital Flexor Tendon ◽  
Vitro Model

SummaryEquine tendon fibroblasts were isolated from explants of superficial digital flexor tendon, subcultured and maintained in monolayers. The cells were characterized by light microscopy, electron microscopy and radiolabel studies for proteoglycan production. Two predominant cell morphologies were identified. The cells dedifferentiated toward a more spindle shape with repeated subcultures. Equine tendon fibroblasts were successfully cryopreserved and subsequently subcultured. The ability to produce proteoglycan was preserved.The isolated cells were identified as fibroblasts, based on their characteristic shape by light microscopy and ultrastructure and the active production of extracellular matrix proteins. Abundant rough endoplasmic reticulum and the production of extracellular matrix products demonstrated active protein production and export. Proteoglycans were measurable via liquid scintillation counting in both the cell-associated fraction and free in the supernatant. This model is currently being utilized to study the effects of polysulfated glycosaminoglycan on tendon healing. Future uses include studying the effects of other pharmaceuticals, such as hyaluronic acid, on tendon healing.A model was developed for in vitro investigations into tendon healing. Fibroblasts were isolated from equine superficial digital flexor tendons and maintained in monolayer culture. The tenocytes were characterized via light and electron microscopy. Proteoglycan production was measured, using radio-label techniques. The fibroblasts were cryopreserved and subsequently subcultured. The cells maintained their capacity for proteoglycan production, following repeated subculturing and cryopreservation.

The Anticoagulant Properties of a Modified Form of Protein S

1988 ◽  
Vol 60 (02) ◽  
pp. 298-304 ◽  
Author(s):  
C A Mitchell ◽  
S M Kelemen ◽  
H H Salem
Keyword(s):  
Monoclonal Antibody ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Factor Xa ◽  
Protein S ◽  
Human Neutrophil Elastase ◽  
Factor X ◽  
Sds Page

SummaryProtein S (PS) is a vitamin K-dependent anticoagulant that acts as a cofactor to activated protein C (APC). To date PS has not been shown to possess anticoagulant activity in the absence of APC.In this study, we have developed monoclonal antibody to protein S and used to purify the protein to homogeneity from plasma. Affinity purified protein S (PSM), although identical to the conventionally purified protein as judged by SDS-PAGE, had significant anticoagulant activity in the absence of APC when measured in a factor Xa recalcification time. Using SDS-PAGE we have demonstrated that prothrombin cleavage by factor X awas inhibited in the presence of PSM. Kinetic analysis of the reaction revealed that PSM competitively inhibited factor X amediated cleavage of prothrombin. PS preincubated with the monoclonal antibody, acquired similar anticoagulant properties. These results suggest that the interaction of the monoclonal antibody with PS results in an alteration in the protein exposing sites that mediate the observed anticoagulant effect. Support that the protein was altered was derived from the observation that PSM was eight fold more sensitive to cleavage by thrombin and human neutrophil elastase than conventionally purified protein S.These observations suggest that PS can be modified in vitro to a protein with APC-independent anticoagulant activity and raise the possibility that a similar alteration could occur in vivo through the binding protein S to a cellular or plasma protein.

Correlation between Thromboplastin Sensitivity and In Vitro F1 + 2 Generation Rate

1995 ◽  
Vol 73 (03) ◽  
pp. 547-547
Author(s):  
Guglielmo Mariani ◽  
Gualtiero Palareti ◽  
Giuliana Guazzaloca ◽  
Alberto Abate ◽  
Gian Domenico Di Nucci ◽  
...  
Keyword(s):  
Generation Rate

Perturbation of Platelet Adhesion to Endothelial Cells by Plasminogen Activation In Vitro

1997 ◽  
Vol 78 (02) ◽  
pp. 934-938 ◽  
Author(s):  
Hsiun-ing Chen ◽  
Yueh-I Wu ◽  
Yu-Lun Hsieh ◽  
Guey-Yueh Shi ◽  
Meei-Jyh Jiang ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Platelet Adhesion ◽  
Treatment Time ◽  
Shape Change ◽  
Umbilical Vein ◽  
Tissue Type ◽  
Apical Surface ◽  
Plasminogen Activation

SummaryTo investigate whether the endothelium-platelet interactions may be altered by plasminogen activation, cultured human umbilical vein endothelial cells (ECs) were treated with tissue-type plasminogen activator (t-PA) in the presence of plasminogen, and platelet adhesion to ECs was subsequently measured by using a tapered flow chamber. Our results demonstrated that platelets adhered more readily to t-PA treated EC monolayer than to the control monolayer at all shear stress levels tested. This phenomenon was treatment time-dependent and dose-dependent, and it could be blocked by adding plasmin inhibitors, such as e-amino caproic acid and aprotinin. Adherent platelets on t-PA treated EC monolayer underwent more severe shape change than those on the control monolayer. While the extracellular matrix directly treated with t-PA attracted less platelets than the control matrix did, platelet adhesion to the matrix that was produced by t-PA-treated ECs was unaltered. These data suggest that t-PA treatment on ECs compromised antiplatelet-adhesion capability on their apical surface without altering the reactivity of their extracellular matrix towards platelets.

Desmopressin (DDAVP) Enhances Platelet Adhesion to the Extracellular Matrix of Cultured Human Endothelial Cells through Increased Expression of Tissue Factor

1997 ◽  
Vol 77 (05) ◽  
pp. 0975-0980 ◽  
Author(s):  
Angel Gálvez ◽  
Goretti Gómez-Ortiz ◽  
Maribel Díaz-Ricart ◽  
Ginés Escolar ◽  
Rogelio González-Sarmiento ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Tissue Factor ◽  
Platelet Adhesion ◽  
Umbilical Vein ◽  
Procoagulant Activity ◽  
Experimental Conditions ◽  
Chromogenic Assay

SummaryThe effect of desmopressin (DDAVP) on thrombogenicity, expression of tissue factor and procoagulant activity (PCA) of extracellular matrix (ECM) generated by human umbilical vein endothelial cells cultures (HUVEC), was studied under different experimental conditions. HUVEC were incubated with DDAVP (1, 5 and 30 ng/ml) and then detached from their ECM. The reactivity towards platelets of this ECM was tested in a perfusion system. Coverslips covered with DD A VP-treated ECMs were inserted in a parallel-plate chamber and exposed to normal blood anticoagulated with low molecular weight heparin (Fragmin®, 20 U/ml). Perfusions were run for 5 min at a shear rate of 800 s1. Deposition of platelets on ECMs was significantly increased with respect to control ECMs when DDAVP was used at 5 and 30 ng/ml (p <0.05 and p <0.01 respectively). The increase in platelet deposition was prevented by incubation of ECMs with an antibody against human tissue factor prior to perfusion. Immunofluorescence studies positively detected tissue factor antigen on DDAVP derived ECMs. A chromogenic assay performed under standardized conditions revealed a statistically significant increase in the procoagulant activity of the ECMs produced by ECs incubated with 30 ng/ml DDAVP (p <0.01 vs. control samples). Northern blot analysis revealed increased levels of tissue factor mRNA in extracts from ECs exposed to DDAVP. Our data indicate that DDAVP in vitro enhances platelet adhesion to the ECMs through increased expression of tissue factor. A similar increase in the expression of tissue factor might contribute to the in vivo hemostatic effect of DDAVP.

RAPID IN VITRO PROPAGATION OF WESTERN GHATS CULTIVAR - COLOCASIA ESCULENTA FOR THE HIGH FREQUENCY OF PLANTLETS

2019 ◽  
Vol 7 (2) ◽  
Author(s):  
Jyothi R ◽  
Srinivasa Murthy K M ◽  
Hossein . ◽  
Veena .
Keyword(s):  
Tissue Culture ◽  
Wide Distribution ◽  
Colocasia Esculenta ◽  
Limiting Factor ◽  
Promising Alternative ◽  
Rapid Multiplication ◽  
Medical Plant ◽  
Total Fat ◽  
Planting Materials

Colocasia esculenta is commonly known as Taro, it is referred to as cocoyam in Nigeria. They are cherished for their rich taste, nutritional and medicinal properties. Every 100 g of taro corms possess 112 Kcal, 26.46 g carbohydrate, 1.50 g protein, 0.20 g total fat and 4.1g fiber (USDA National Nutrient Data Base). Besides its nutritional value, taro is used as a medical plant and provides bioactive compounds used as an anti-cancer drugs. Traditionally, cocoyams are vegetative propagated from tuber fragments, a practice that encourages pathogen distribution. Colocasia esculenta is a widely distributed food crop in the humid tropics and subtropics. Despite of its wide distribution, Taro plants are commonly infected with DsMV and other pathogens. This virus induces conspicuous mosaic, malformation, dwarfing or feathering on leaves in taro. As the results of infection, it reduces the quality and yield of taro production greatly. This virus is thus considered as a major limiting factor in the production of taro. Here plays the importance of  tissue culture plays a major role in producing the disease resistant plants round the year with high quality. For rapid multiplication and production of quality planting materials, tissue culture technology offers promising alternative compared to the traditional production methods. KEYWORDS: Colocasia esculenta, Virus, Pathogens, Conventional propagation, Micropropagation, Yield, Rapid multiplication, Quality

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