The reaction of the mast cell population to the intratracheal administration of heparin and fucoidan]

Mast cell population of rat after single intratracheal injection of heparin and fukoidan was investigated with cytofluorometry and cytochemical methods. These polysaccharides have great differences in the molecular weight nevertheless they both were found to include in mast cells.     

Effect of non-fractionated and low-molecular heparin on the functional state of mast cells]

The state of the mast-cell population of rats treated with unfractionated and low-molecular weight heparins under stress conditions has been comparatively studied by the morphometrical assay. The stress was produced by 60 min immobilization followed by intravenous injection of unfractionated (UF) or low-molecular weight (LMW) heparin. The stress-induced heparin release from mast cells resulted in a 3.3-fold decrease of the index of saturation with heparin and in a significant increase of granulolysis and degranulation. The mast cell secretory status reached the preinjection level within 20 min in rats with UF heparin injected (15 unit/200 g). At the same time mast cells of rats with LMW heparin have no such ability. The data obtained indicate that LMW heparin in contrast to UF heparin cannot be accumulated (or accumulated very slowly) by mast cells. This fact as well as low affinity of LMW heparin to endothelium and blood platelets promote its preservation in blood for a long time.     

Heparin secretion by mast cells as an index of the status of the anticoagulant system

The status of the mast cell population was studied and compared after administration of trypsin or alpha-thrombin in similar molar concentrations. Morphometry disclosed a substantial shift of the mast cell population towards light, heparin-free cells within one minute after alpha-thrombin administration. The index of mast cell saturation with heparin dropped below 1. The maximal heparin secretion was observed at the 5th minute of experiment. The morphometric criteria of the mast cell population returned to basal level in 120 minutes. These data along with a significant increase in the level of complex heparin compounds and plasma thrombin time indicate heparin release as a result of the effector action of the anticoagulation system. No changes were observed in the activity of complex heparin compounds and in thrombin time after intravenous injection of trypsin. It is suggested that high heparin secretion by mast cells may serve as criterion of the active status of the anticoagulation system.     

Increased Sensitivity to Heparin Following Acute Myocardial Infarction

In vivo increased sensitivity to heparin has been demonstrated in patients following an acute myocardial infarction. An intravenous injection of 10,000 units of heparin was given to each of 18 patients with recent myocardial infarction in order to compare them with 17 patients who were not suffering from any acute illness. The changes in whole blood clotting time, recalcified plasma clotting time and prothrombin time were greater and more prolonged in the patients with recent myocardial infarction. Of the three tests, the one-stage prothrombin time provided the simplest and the most precise measurement of heparin sensitivity. The reason for this was not clear: it is possible that it is related to shock and congestive heart failure which were complications of the clinical course following myocardial infarction.     

Subcutaneous calcium heparin versus intravenous sodium heparin in treatment of established acute deep vein thrombosis of the legs: a multicentre prospective randomised trial.

One hundred patients with phlebographically proved acute deep vein thrombosis of the legs were prospectively randomised into two treatment groups to compare the safety and efficacy of subcutaneous calcium heparin versus intravenous sodium heparin administered by constant infusion pump. The dose of heparin was determined by daily measurement of the kaolin cephalin clotting time. Treatment was maintained for up to 14 days, after which phlebography was repeated. Of 49 patients who received subcutaneous calcium heparin, two showed an increase in thrombus size, while eight showed complete lysis. In the 47 patients who received intravenous sodium heparin thrombus increased in size in 13 while only one showed evidence of complete lysis. These differences were significant. There were no significant differences between the two groups in the incidence of serious complications, although almost half of those receiving intravenous heparin had some minor problem with the constant infusion pump and just over half of those receiving subcutaneous heparin had some bruising at the injection site. This study showed that subcutaneous calcium heparin was more effective in helping lyse existing thrombus and preventing its propagation than intravenous sodium heparin.     

The role of mast cells in vascular permeability disorders in rats subjected to immobilization stress]

Disturbances of vascular permeability were studied by the "vascular labeling" technique in the mesentry during the 24-hour immobilization of rats. Administration of dimebolin (an antihistaminic preparation) decreased the number of labeled vessels and labeling intensity. This effect was expressed in the presence of mast cells only and was accompanied by the mast cell degranulation. The authors suppose that the mast cells contain a substance preventing the disturbance of vascular permeability and released during degranulation. Such substance might be heparin. Experiments showed that small doses of heparin failed to produce such effect. These results allowed one to conclude that mast cells played a double role in the mechanisms of disturbance of vascular permeability during immobilization--the damaging (by the action of histamine and serotonine) and the protective (by the released heparin) action.     

The fibrinogenolytic activity of purified tryptase from human lung mast cells

The capacity of purified tryptase from human lung mast cells to metabolize human fibrinogen, fibrin, and plasminogen was evaluated. Tryptase (5 micrograms/ml) inactivated the thrombin-induced clotting activity of fibrinogen (100 micrograms/ml) with essentially similar t 1/2 values of 4.6 min in the absence of heparin and 5.8 min in the presence of heparin (20 micrograms/ml) that were not appreciably different than with lysine-Sepharose-purified plasmin (5 micrograms/ml). Fibrinogen treated with tryptase together with heparin lost all detectable clotting activity by 4 hr at 37 degrees C, whereas fibrinogen treated with tryptase alone resulted in destruction of only 80% of fibrinogen clotting equivalents after 16 hr. Tryptase alone was observed to cleave only the alpha-chains of fibrinogen by electrophoresis of tryptase-treated, denatured, and reduced fibrinogen in polyacrylamide gradient gels. Tryptase together with heparin cleaved first the alpha-chain and then the beta-chain, the latter cleavage corresponding to complete loss of fibrinogen clotting activity by 4 hr. No fibrinogen fragments with anticoagulant activity were generated by tryptase. In contrast, plasmin left no residual clotting activity after 4 hr of incubation and generated fibrinogen fragments with anticoagulant activity. Plasmin sequentially cleaved the alpha, beta, and gamma subunits of fibrinogen. Tryptase alone (6 micrograms/ml) or together with heparin (20 micrograms/ml) failed to activate plasminogen (0.6 mg/ml) after a 60-min incubation at 37 degrees C. Addition of urokinase to tryptase-treated or untreated plasminogen resulted in essentially identical plasmin activities (0.32 and 0.34 U/ml, respectively), indicating that tryptase neither activates nor destroys plasminogen. Tryptase (700 ng) also failed to substantially solubilize cross-linked fibrin (2.6 micrograms) or the corresponding amount of fibrinogen bound to plastic microtiter plates with or without heparin. The failure to solubilize fibrinogen and, possibly, fibrin is consistent with the observation that the apparent m.w. by SDS polyacrylamide gel electrophoresis of unreduced fibrinogen is not appreciably altered by prior treatment with tryptase, even though cleavage of alpha-and beta-chains is revealed after reduction. Fibrinogenolysis by tryptase complements other mast cell mediators with anticoagulant properties such as heparin and suggests a significant prevention of coagulation by activated mast cells.     

Cytofluorometric quantitation of 5-hydroxytryptamine and heparin in individual mast cell granules.

Cytofluorometric quantitation of 5-hydroxytryptamine (5-HT) and heparin in individual mast cell granules is described. The technique is based on micromanipulation of intact mast cells reacted with formaldehyde or stained with Berberine sulfate and the use of a cytofluorometer equipped with a sensitive peak detecting device. The quantities of 5-HT and heparin contained in mast cell granules which are of the order of 10(-16) and 10(-13) g, respectively were expressed as relative fluorescence guanta. The results of measurements on representative samples of mast cell granules indicate that all granules contain heparin as well as 5-HT, and that there are large variations in both 5-HT and heparin content within the granule populations of individual cells. A dose dependent increase in 5-HT content in both cells and individual mast cell granules occurred 24 hr after the injection of 10--50 mg L-5-hydroxytryptophan/kg intraperitoneally. There was no evidence for an increase in the heparin content of granules or cells, indicating that a new synthesis of granular macromolecules is not required for the 5-HT uptake. The results further suggest that 5-HT may be stored initially in a cytoplasmic extragranular pool and then taken up in the mast cell granules.     

Effect of the medicinal leech salivary gland secretion on the state of rat subcutaneous mast cells

It is firstly showed that the medicinal leech salivary gland secretion (SGS) as a polycomponent system of proteins and low-molecular weight substances, activates rat subcutaneous mast cells in vitro prompting a decrease in the heparin saturation index and increasing some characteristic mast cells morphometric parameters. The same mast cell changes were detected by analysis of some specimens of subcutaneous cellular tissue in the point of skin injured by the leech bite. It is shown that these changes are saved during 3 days. The mechanical injury of rat skin does not effect the mast cells activation. Activation of mast cells by SGS is extended to the distant subcutaneous mast cells. It is expressed in sharp decreasing of heparin saturation index although not statistically positive. The secondary leeching on these distant points provokes reduction of mast cells activation and some decrease of post-leeching blood heparin content: 0.154 +/- 0.03 units/ml (n = 10) as compared with post-leeching blood heparin contents analysed from the wound after the primary leeching (0.160 +/- 0.03 units/ml, n = 10). Proceeding from these findings, participation of heparin secreted from activated mast cells in the support of post-leeching bleeding is suggested, the phenomenon which provides unloading of capillary pool by application of medicinal leeches for treatment many diseases.     

Immunologic mast cell-mediated responses and histamine release are attenuated by heparin.

Heparin has been shown to act as a competitive inhibitor of inositol 1,4,5-triphosphate (InsP3) receptors in various cell types. Because InsP3 is one of the second messengers involved in stimulus-secretion coupling in mast cells, it is possible that heparin may inhibit mast cell-mediated reactions. Therefore, in allergic sheep, we tested this hypothesis in two mast cell-mediated reactions induced by immunologic and nonimmunologic stimuli: immediate cutaneous reaction (ICR) and acute bronchoconstrictor response (ABR). In 12 sheep allergic to Ascaris suum antigen, the surface area of the skin wheal was determined 20 min after intradermal injection (0.05 ml) of increasing concentrations of specific antigen, compound 48/80, and histamine, without and after pretreatment with heparin (100, 300, or 1,000 U/kg i.v.). Antigen, compound 48/80, and histamine produced concentration-dependent increases in ICR. Heparin "partially" inhibited the ICR to antigen and compound 48/80 in a dose-dependent manner without modifying the ICR to histamine. The heparin preservative benzyl alcohol was ineffective. In 11 additional sheep, specific lung resistance was measured before and after inhalation challenges with antigen, compound 48/80, and histamine without and with aerosol heparin pretreatment (1,000 U/kg). Heparin blocked the antigen- and compound 48/80-induced bronchoconstriction without modifying the airway effects of histamine. In isolated human uterine mast cells, heparin inhibited the anti-immunoglobulin E- but not the calcium ionophore- (A23187) induced histamine release. These data suggest that heparin inhibits the ICR and ABR induced by stimuli that produce immunologic and nonimmunologic mast cell degranulation without attenuating the effects of histamine.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of an insulin load on rat mast cells]

The increase of heparin secretion by mast cells of kidney capsule and subcutaneous fat has been noted in rats after 30 min intravenous insulin administration in a dose 0.3 U/200 g (by this time the blood sugar concentration lowers by 40%). The index of mast cells saturation with heparin drops by 2.3 and 1.9 times correspondingly. After preliminary administration of protamine sulphate (2 mg/200 g that provokes in rats the status of temporary resistance to the hypoglycemic action of insulin the stimulatory effect of insulin on the function of mast cells does not occur.     

Lymphocytosis produced by heparin and other sulphated polysaccharides in mice and rats

Lymphocytosis has been produced in mice and rats using heparin and other sulphated polysaccharides. Two hours after heparin (50 mg/kg ip) the concentration of lymphocytes in mouse blood increased threefold; it fell to control levels after 9 hr. The height of the lymphocytosis was related to the dose of heparin injected. After intravenous heparin in rats there was a comparable lymphocytosis maximal 1 hr after injection. In mice other negatively charged sulphated polysaccharides also caused lymphocytoses, which were greater and occurred later with increase in molecular weight of the substance injected. Results in rats were similar. No lymphocytosis followed the injection of negatively charged phosphated dextrans, positively charged DEAE dextran, or neutral dextran. There was no correlation between the effect of these substances on lymphocytes and their effect on coagulation of blood, hepatic phagocytosis, or the immune response to sheep red blood cells.     

Heparin and mast cells in the dynamics of experimental extrahepatic cholestasis]

The content of heparin in the blood and mast cell count in rat tissues were studied during different periods of experimental extrahepatic cholestasis induced in rats by ligation of the common bile duct. During cholestasis the blood heparin level proved to increase and the mast cell count fell on the 3rd day, increased on the 7th day and fell again on the 14th day. Correlation between the degranulated and nondegranulated forms of mast cells altered in favour of the latter. The fluctuation of mast cell counts and increase of degranulated forms is considered to be the best result of mast cell irritation produced by bile acids and pigments which accumulated in the organism.     

Heparin augments osteoclast resorption-stimulating activity in serum

Increased numbers of mast cells are commonly seen at sites of increased bone resorption and in osteoporosis. Long-term administration of heparin, a major component of mast cell granules, causes osteoporosis. We therefore tested the effect of heparin on bone resorption by osteoclasts disaggregated from neonatal rat long bones. We found that, in the absence of serum, heparin was without effect on osteoclast function. However, in the presence of newborn calf serum, rat serum, or bovine platelet-poor plasma-derived serum, heparin, in the range 25-100 micrograms/ml, induced an increase in osteoclastic bone resorption. Heparin appeared to act through binding and enhancement of an osteoclast resorption-stimulating activity (ORSA) present in serum. A number of known factors that show an affinity for heparin, including transforming growth factor-beta, platelet-derived growth factors, insulin-like growth factors I or II, acidic or basic fibroblast growth factors, fibronectin, or laminin, could not substitute for ORSA, suggesting that the activity may represent a novel heparin-binding factor. The ability of glycosaminoglycans (GAGs) and related molecules to enhance resorption was dependent on the degree of sulfation and on their size: The high molecular weight GAG heparan sulfate and polysaccharides fucoidan or dextran sulfate showed a similar effect, while low molecular weight heparin, chondroitin-2-sulfate, chondroitin-4-sulfate, and chondroitin-6-sulfate were without effect. We propose that mast cells or heparin therapy increases bone resorption through augmentation of the activity of a factor involved in the local and systemic regulation of osteoclastic bone resorption.     

Effect of exogenous heparin on secretory status of rat mast cells under immobilization stress]

The significant increase of heparin release from mast cells was observed in rats under stress conditions induced by 60 min immobilization. The index of its saturation with heparin became 4 times lower. The highest secretory activity of mast cells was observed during the first 30 min of immobilization. It was shown that at that time the heparin release from mast cells occurred by granulolysis (merocrine type of secretion). In the rats received heparin (15 or 150 u/200 g) during the first 15 min of immobilization the mast cells released heparin with the same intensity as in a 4 control animals. But then in rats with high heparin blood concentration the heparin release from mast cells ceased and mast cells began to accumulate heparin from blood. By the 30th min of immobilization the heparin content in the mast cells has become normal.     

Heparin modulates the growth and adherence and augments the growth-inhibitory action of TNF-alpha on cultured human keratinocytes

Previous works suggest the involvement of mast cells in the epithelialization of chronic wounds. Since heparin is a major mediator stored in the secretory granules of mast cells, the purpose of this work was to elucidate the function of heparin in epithelialization using in vitro culture models. For this, low- and high-calcium media in monolayer and epithelium cultures of keratinocytes were used. Also, an assay based on keratinocyte adherence onto plastic surface was used as well. Heparin (0.02-200 microg/ml) inhibited keratinocyte growth in a non-cytotoxic and dose-dependent manner in low- and high-calcium media, Keratinocyte-SFM and DMEM, in the absence of growth factors and serum. Also, heparin inhibited the growth of keratinocyte epithelium in the presence of 10% fetal calf serum and DMEM. Instead, in the presence of Keratinocyte-SFM and growth factors, heparin at 2 microg/ml inhibited the growth by 18% but at higher heparin concentrations the inhibition was reversed to baseline. TNF-alpha is another preformed mediator in mast cell granules and it inhibited keratinocyte growth in monolayer and epithelium cultures. Interestingly, heparin at 2-20 microg/ml augmented or even potentiated this growth-inhibitory effect of TNF-alpha. The association of TNF-alpha with heparin was shown by demonstrating that TNF-alpha bound tightly to heparin-Sepharose chromatographic material. However, heparin could not augment TNF-alpha-induced cell cycle arrest at G0/G1 phase or intercellular adhesion molecule-1 expression in keratinocytes. In the cell adherence assay, heparin at 2 microg/ml inhibited significantly by 12-13% or 33% the adherence of keratinocytes onto the plastic surface coated with fibronectin or collagen, respectively, but this inhibition was reversed back to baseline at 20 or 200 microg/ml heparin. Also, heparin affected the cell membrane rather than the protein coat on the plastic surface. In conclusion, heparin not only inhibits or modulates keratinocyte growth and adherence but it also binds and potentiates the growth-inhibitory function of TNF-alpha.     

Changes in numbers and heparin content of peritoneal fluid mast cells of growing rats measured by flow cytofluorometry

A cytofluorometric method, based on berberine staining of mast cell heparin, was used for flow cytofluorometric counting and heparin quantitation of mast cells in crude peritoneal suspensions of growing rats. The automatic flow cytofluorometric counting of mast cells correlated well with hemocytometer cell counts. The mean mast cell heparin content obtained by flow cytofluorometry showed good agreement with such obtained by cytofluorometry of microscopically identified mast cells. The number of peritoneal mast cells and the mean mast cell heparin content was found to increase as the animals grew older. The results of the microscope fluorometric measurements suggested that the heparin content was normally distributed within mast cell populations of both young and old rats. However, the heparin distributions obtained by flow cytofluorometry were often positively skewed but did not fulfill the condition of the log-normal distribution.     

Decreased sensitivity to the hypoglycemic action of insulin in animals with a lowered blood heparin concentration

It has been shown that in animals with reduced heparin blood concentration the hypoglycemic insulin action was considerably low. This was established for rats with alloxan diabetes and ageing rats with depressed anticoagulation system and atherosclerosis enhanced by prolonged atherogenic diet. With insulin injection (0.2-0.3 U/200 g), blood sugar concentration in such animals was 2-2.5 times lower than in normal ones. The compensation of endogenous heparin deficiency by an intravenous injection of heparin normalizes the reaction of animals to exogenous insulin.     

Mast cell population in rats with experimental atherosclerosis

Mast cell population was studied in rats with experimental atherosclerosis. It has been established that animals kept for 8 months on atherogenic diet revealed marked changes in mast cell population. Predominance of light cells and cell defects were noted. Heparin saturation index was reduced (0.35), as compared to the control (3.9). Stimulation of anticoagulation system by DIP-alpha-thrombin in such animals revealed no heparin in the blood. Mast cell subpopulation was characterized by light cell predominance and low heparin saturation index. The nature of cell degradation remained unchanged. The data obtained indicate the defects in mast cell pool in animals with experimental atherosclerosis.