The heparin target organ--the endothelium. Studies in a rat model.

Plasma levels of the antithrombotic drug heparin, as estimated by coagulation tests, are a poor indicator of antithrombotic effectiveness. The interaction of heparin with endothelium is a poorly studied but important factor in the clinical activity of heparin. This study describes the interaction of heparin with endothelium, following intragastric administration. The concentrations of heparin in endothelium and plasma were determined by gel electrophoresis following administration of heparin to rats by various routes. Heparin concentrations in endothelium versus plasma were approximately 100 times greater following intravenous or ex vivo administration and more than 1000 times greater when administered by intrapulmonary, subcutaneous, intraperitoneal and intragastric routes indicating that the route of administration affects the distribution of the drug. At 2.4 and 6 min after intravenous administration, 88 and 51% respectively of the administered dose was found associated with endothelium. Heparin was rapidly absorbed following intragastric administration and could be detected associated with endothelium at 2.4 min. At 6 min less than 1% of the administered dose was found in plasma, and 45% was associated with endothelium. These results show that endothelium is the main site of heparin distribution. Heparins could also be detected in cellular and pericellular fractions of cultured porcine aortic endothelial cells when 125I-heparin was added to medium. Bound radioactivity was released to medium from both cellular and pericellular fractions suggesting that heparin taken up by endothelium can be released. Intragastric administration of heparin and dextran sulphates significantly prevented thrombus formation in a rat model of thrombosis without significant changes in activated partial thromboplastin times.(ABSTRACT TRUNCATED AT 250 WORDS)

The internalization and release of heparins by cultured endothelial cells: the process is cell source, heparin source, time and concentration dependent.

Endothelial cells in vivo and in vitro take up heparin following administration. In in vitro systems, cellular and pericellular extracts of monolayers exposed to heparin, demonstrate internalization as well as cell surface attachment. To further investigate the characteristics of this exogenous heparin partitioning, we have exposed two types of endothelial cells to media containing cold and 125I labelled bovine lung, porcine mucosal or CY222 heparin. Heparin uptake, in pericellular and intracellular fractions of porcine cells, increased with concentration at 24 hrs exposure for all heparins. Only bovine heparin was preferentialy accumulated intracellularly. Cultured murine LE-II endothelial cells showed a greater accumulation in the intracellular fraction than porcine cells when exposed to porcine heparin. When bovine or CY222 heparin was administered for varying times, total heparin uptake increased with time. Heparin in the pericellular fraction was greater than intracellular at exposure durations from 5 mins to 6 hrs. At 16 hrs intracellular heparin markedly increased and far exceeded pericellular. The possibility of release of previously internalized heparin was studied by washing cultures previously exposed to heparin with heparin-free media. Heparin could be recovered up to 96 hours post exposure. A concurrent decrease in pericellular and cellular heparin was observed. These results show a differing distribution of heparin across endothelial cell membranes that is heparin source, concentration and time dependent. A previously unsuspected gradient-time mechanism is suggested. The degree of internalization differs for different endothelial cell sources. The protracted retention of heparin intracellularly by endothelial cells and subsequent release may be of therapeutic and physiological consequence.

The intracellular uptake and protracted release of exogenous heparins by cultured endothelial cells.

Heparins from bovine or porcine sources were fed in media for 48 hrs to cultured porcine aortic and human umbilical vein endothelial cells. Heparin was found in pericellular and cellular fractions after extraction by chemical methods and 125I radiolabelled heparins were recovered when radiolabelled heparin was included in the feed. Even after washing and media changes heparin was detected in media and cell fractions up to 6 days post feeding. Metachromatic vacuoles within cells were demonstrated histologically up to 7 days post feeding after staining with toluidine blue. This is the first report of protracted internalization of exogenous heparin by cultured endothelial cells with concurrent prolonged release of the heparin to the media. This clearly demonstrates that the endothelium plays an important role in the distribution and metabolism of heparin.

Heparin and sulfated mucopolysaccharides--a micro system for quantitative differentiation and determination.

Heparin (Hep), hyaluronic acid, chondroitins (sulfate) A, B, and C, and heparins (sulfate) A, B, C, and D were subjected to microelectrophoresis in barbital-agarose gel, fixed with cetylpyridinium chloride and stained with toluidine blue. The optical densities of the resulting bands were compared with optical densities obtained upon reaction with azure A in aqueous solution and with the carbazole reagent. A linear relation was obtained between optical density and concentration of purified sulfated mucopolysaccharide (SMP). Less than 1 microgram of Hep and 2 microgram of other SMPs are required for measurement by electrophoresis, while about 30 microgram of each is required with the carbazole reagent. The optical density of a mixture of SMPs was equal to the sum of the densities for the individual SMPs upon microelectrophoresis. It was demonstrated that the individual SMPs in mixtures were distinguishabed by reaction with specific enzymes and by changes in migration in agarose with barbital, phthalate, ethylenediamine, or propanediamine buffers, permitting ready demonstration and quantitation of various SMP species. Examples are shown of the application of the procedure to measure the total SMPs and individual SMPs in tissue extracts. The method is sensitive, reproducible, flexible, and measures quantities 1/30th of those measured colorimetrically, yet is relatively unaffected by protein, carbohydrate, or inorganic electrolytes.

Identification of acidic mucopolysaccharides by agarose gel electrophoresis.

A micro-method for the identification of most acidic mucopolysaccharides by agarose gel electrophoresis with three different buffer systems is described. In barbital buffer the mucopolysaccharides are fractionated from each other as a function of their net charge, whereas in a diamine buffer the fractionation is probably achieved according to the degree to which they are bound to the diamine. A combination of barbital and diaminopropane buffers in two-dimensional electrophoresis for the identification of mucopolysaccharides is also described.