Effect of heparin on the production of matrix metalloproteinases and tissue inhibitors of metalloproteinases by human dermal fibroblasts

The influence of heparin and a heparin fragment devoid of anticoagulant activity on the production of matrix metalloproteinases and tissue inhibitors of metalloproteinases by human dermal fibroblasts was studied. Doses (0.1-400 microg/ml) responses were performed and data obtained were similar whatever heparin or fragment was used. The basal expression of collagenase by fibroblasts decreased quasi-linearly with increasing doses of heparins from 1 to 400 microg/ml. TIMP-1 levels were not affected by supplementing serum free culture medium with heparins. On the contrary, at low concentration, i.e. 1-10 microg/ml, heparins stimulated the secretion of both 72-kDa gelatinase (1.4-1.6-fold) and particularly TIMP-2 (>4-fold). At high doses, MMP-2 and TIMP-2 production by fibroblasts returned to basal levels. These results suggested that the local concentration of heparin released by mast cells could be instrumental in modulating fibroblast growth and proteolytic phenotype.     

Dual effect of heparin on cultured adult rat cardiomyocytes

Heparin has been widely reported to inhibit the growth of several cell types including neonatal rat cardiac myocyte (NRCM) but its effect on adult rat ventricular myocyte (ARVM) is unknown. To determine whether heparin is able to modulate ARVM protein synthesis capacity and if so which pathway is involved in this response, ARVM were cultured in presence or absence of 5% human serum and exposed to heparin (2-2,000 microg/ml) or its analogue xylan (0.5 and 50 microg/ml), and either the Ca(2+) chelator BAPTA/AM (10 microg/ml), or the calcineurin inhibitor FK506 (10 microg/ml), and heparinase I (0.1-10 U/ml) for 2 days. The protein synthesis (PS) was measured after 24 h incorporation of [14C]-Phenylalanine in ARVM. Independently of the serum presence, heparin and xylan altered PS in a bimodal dose-dependent manner. At high doses, heparin and xylan (2,000 and 50 microg/ml, respectively) either had no effect (without serum) or inhibited PS (with serum). In absence of serum, low doses of heparin or xylan (20 and 0.5 microg/ml, respectively) amplified the PS process in ARVM (2-fold, P < 0.05). FK506 inhibited the trophic response to 20 microg/ml heparin alone (-39%, P < 0.05). In presence of serum, the heparin induced-trophic effect, that was not significantly altered by FK506, was inhibited by BAPTA/AM (-32%, P < 0.05). Finally, heparinase I that increased PS in NRCM had no effect on ARVM growth. This study strongly suggests that heparin dose-dependently modulated PS in ARVM, this result being not observed in neonatal cells. Different mechanisms involving intracellular Ca(2+) play a role in the PS response of ARVM to low concentrations of heparin, the intracellular pathways depending on the presence of serum.     

Monocyte chemotaxis and activating factor production by keratinocytes in response to IFN-gamma

Monocytes accumulate in the epidermis and along the dermo-epidermal junction in several different inflammatory skin diseases. To determine whether human epidermal keratinocytes elaborate a specific chemotaxin responsible for the accumulation of monocytes at these anatomic sites, monocyte chemotactic activity in conditioned 16-h cultured keratinocyte supernatants were assayed using human peripheral blood monocytes as the target cell. Dilutional analysis revealed directed monocyte migration in IFN-gamma-treated (100 U/ml) keratinocyte supernatants (80% maximal FMLP response) which was 10-fold more than IFN-gamma itself or untreated keratinocyte activity alone. Gel filtration chromatography revealed that this activity eluted just ahead of a 12.5-kDa molecular mass marker. Blocking studies demonstrated that a rabbit polyclonal antibody to monocyte chemotaxis and activating factor (MCAF) inhibited all monocyte chemotaxis by greater than 80%. Keratinocytes were metabolically labeled with 35S-cysteine/methionine, and after 16 h incubation the supernatants immunoprecipitated with the same anti-MCAF antibody. MCAF was detected as a protein doublet of 12 and 9 kDa only in IFN-gamma-treated (100 U/ml) keratinocyte supernatants. Incubation with IFN-gamma and TNF-alpha (250 U/ml) in combination resulted in increased production of MCAF protein. By Northern blot analysis, MCAF mRNA was constitutively expressed in keratinocytes and upregulated only in the presence of IFN-gamma. TNF-alpha, IL-1 beta, transforming growth factor-beta and phorbol esters had no positive or negative influence on MCAF mRNA. These studies demonstrate that biologically active MCAF is elaborated by human epidermal keratinocytes upon activation by IFN-gamma, a cytokine also required for the induction of adherence between monocytes and keratinocytes. Keratinocyte-derived MCAF is likely to be important in the regulation of cutaneous monocyte trafficking and may also be responsible for the recruitment of Langerhans cells and dermal dendrocytes, which share many phenotypic features with monocytes/macrophages, to their anatomic locations in skin.     

A role of mast cell glycosaminoglycans for the immunological expulsion of intestinal nematode, Strongyloides venezuelensis

We examined effects of mast cell glycosaminoglycans on the establishment of the intestinal nematode, Strongyloides venezuelensis, in the mouse small intestine. When intestinal mastocytosis occurred, surgically implanted adult worms could not invade and establish in the intestinal mucosa. In mast cell-deficient W/Wv mice, inhibition of adult worm invasion was not evident as compared with littermate +/+ control mice. Mucosal mastocytosis and inhibition of S. venezuelensis adult worm mucosal invasion was tightly correlated. To determine effector molecules for the invasion inhibition, adult worms were implanted with various sulfated carbohydrates including mast cell glycosaminoglycans. Among sulfated carbohydrates tested, chondroitin sulfate (ChS)-A, ChS-E, heparin, and dextran sulfate inhibited invasion of adult worms into intestinal mucosa in vivo. No significant inhibition was observed with ChS-C, desulfated chondroitin, and dextran. ChS-E, heparin, and dextran sulfate inhibited adhesion of S. venezuelensis adult worms to plastic surfaces in vitro. Furthermore, binding of intestinal epithelial cells to adhesion substances of S. venezuelensis, which have been implicated in mucosal invasion, was inhibited by ChS-E, heparin, and dextran sulfate. Because adult worms of S. venezuelensis were actively moving in the intestinal mucosa, probably exiting and reentering during infection, the possible expulsion mechanism for S. venezuelensis is inhibition by mast cell glycosaminoglycans of attachment and subsequent invasion of adult worms into intestinal epithelium.     

Growth factor-independent proliferation of normal human neonatal keratinocytes: production of autocrine- and paracrine-acting mitogenic factors

When normal human foreskin keratinocytes were cultured in the absence of polypeptide growth factors at densities above 5 x 10(3)/cells cm2, the cells proliferated continuously and the addition of IGF-I, EGF, TGF alpha, bFGF, or aFGF did not significantly alter growth rate. Heparin sulfate, TGF beta, or suramin inhibited keratinocyte growth factor-independent proliferation. The addition of EGF, TGF alpha, or aFGF reversed heparin-induced growth inhibition, while bFGF partially negated this effect. RIA of keratinocyte-derived conditioned medium (CM) indicated the presence of TGF alpha peptide at a concentration of approximately 235 pg/ml. In contrast, clonal growth of keratinocytes required the addition of growth factors to the basal medium. Keratinocyte-derived CM replaced EGF in stimulating keratinocyte clonal growth, and an anti-EGF receptor mAb inhibited CM-induced keratinocyte clonal growth. In addition to its effect on keratinocytes, keratinocyte-derived CM stimulated the incorporation of [3H]thymidine by quiescent cultures of human foreskin fibroblasts, mouse AKR-2B cells, and EGF-receptorless mouse NR6 cells. CM-stimulated [3H]thymidine incorporation into quiescent normal human fibroblasts was partially reduced in the presence of anti-EGF receptor mAb. Heparin sulfate partially inhibited CM-induced keratinocyte clonal growth and [3H]thymidine incorporation into quiescent AKR-2B cells. We hypothesize from these data that autocrine and paracrine-acting factors produced by keratinocytes mediated their effect through the activation of both EGF receptor-dependent and EGF receptor-independent mitogenic pathways and that some of these factors appear to be sensitive to inhibition by heparin.     

Effect of melanins from black yeast fungi on proliferation and differentiation of cultivated human keratinocytes and fibroblasts

The effects of melanin preparations from black yeast fungi (BYF) on the proliferation and differentiation of normal cultivated human skin keratinocytes and embryonic pulmonary fibroblasts have been investigated. Melanin preparations in the range of 5-0.1 microg/ml were optimally active, with a more pronounced effect on keratinocyte than on fibroblast proliferation. Of 17 dihydroxynaphthalene (DHN) natural melanin preparations and two commercial dihydroxyphenylalanine (DOPA) melanin preparations, only one preparation--DOPA melanin (of animal origin) significantly stimulated proliferation of keratinocytes at 5 microg/ml; four preparations (DHN melanin from BYF) significantly inhibited proliferation of these cells at 5 or 1 microg/ml. The remaining preparations had no significant effect. Similarly, of the 17 preparations of DHN melanin from BYF, one preparation significantly stimulated fibroblast proliferation, and four significantly inhibited proliferation at 5 microg/ml, one at all the concentrations, and three from 1 down to 0.1 microg/ml. These melanin preparations were also shown to affect the in vitro differentiation of keratinocytes.     

Lipopolysaccharide stimulates monocyte adherence by effects on both the monocyte and the endothelial cell

The effects of the LPS moiety of endotoxin on monocyte adherence to an endothelial cell surface were investigated over times before the development of well described LPS-induced endothelial cell surface adhesive molecules. In an in vitro microtiter adherence assay, LPS in concentrations of 10 ng/ml to 10 micrograms/ml incubated for 20 to 60 min with human monocytes significantly stimulated monocyte adherence to human umbilical vein endothelial cell monolayers (HUVEC) and serum-coated plastic surfaces. The time course and concentration dependence of LPS-stimulated monocyte adherence to glutaraldehyde-fixed HUVEC did not differ significantly from that to unfixed HUVEC or serum-coated plastic surfaces. Pretreatment studies suggested that LPS acted on the monocyte within 25 min to stimulate adherence to untreated endothelial cells but required a minimum of 1.5 to 2 h to render the endothelial cell more adhesive for untreated monocytes. The potential role of TNF-alpha, IL-1 alpha, and IL-1 beta in this system was assessed by determining the ability of these cytokines (+/- cytokine antibodies) to increase monocyte adherence. TNF, but neither IL-1, stimulated early monocyte adherence (1 h). This TNF-stimulated monocyte adherence was abrogated by coincubation with anti-rTNF-alpha polyclonal antibody. However, the anti-rTNF antibody had no effect on LPS-induced monocyte adherence to endothelial cells or serum-coated plastic surfaces. An early action of LPS on the monocyte to induce adherence to endothelial cell surfaces may contribute to the initial localization of peripheral blood monocytes in tissues during endotoxemia. The later effects of LPS on the endothelial cell to stimulate monocyte adherence may then amplify these initial monocyte-endothelial cell interactions to prolong and intensify monocyte adherence prior to migration into tissues.     

Amphiregulin-dependent proliferation of cultured human keratinocytes: Autocrine growth,the effects of exogenous recombinant cytokine,and apparent requirement for heparin-like glycosaminoglycans

Amphiregulin, a member of the epidermal growth factor family with heparin binding affinity, functions as a natural regulator of keratinocyte growth. Autocrine signaling by amphiregulin and the effects of exogenous recombinant cytokine were studied in serum-free cultures of human neonatal keratinocytes. A metabolic inhibitor of proteoglycan sulfation was used to assess the role of cellular heparin-like glycosaminoglycans in amphiregulin-dependent growth. Keratinocytes plated at >10³ cells/cm² grew in an autocrine manner in the absence of exogenous epidermal growth factor or amphiregulin. Incubation of keratinocytes with an amphiregulin-blocking antibody indicated that ～70% of autocrine growth is mediated by endogenous amphiregulin. Proliferation potential in the presence of recombinant human amphiregulin was dose dependent and saturable and above ～1 ng/ml was comparable to that achieved with similar concentrations of epidermal growth factor. Sodium chlorate, which blocks glycosaminoglycan sulfation, reversibly inhibited epidermal growth factor-dependent proliferation by 42%, exogenous amphiregulin-dependent proliferation by 75%, and autocrine growth by 95%; concurrent incubation with 1-100 μg/ml heparin partially reversed this inhibition. Exogenous heparin in the absence of chlorate, however, nearly completely inhibited growth under autocrine conditions and in the presence of recombinant amphiregulin. Structure-function studies indicate that the polymerization level, high sulfate group density, and possibly iduronic acid content of heparin-like moieties correlate with their inhibitory activity. Collectively, these observations indicate that amphiregulin is the major autocrine factor for keratinocytes and demonstrate that exogenous amphiregulin is an effective growth promoting factor with molar potency similar to that of epidermal growth factor. Autocrine and paracrine signaling by amphiregulin may require cellular heparin-like glycosaminoglycans, presumably as matrix or membrane proteoglycans, whereas soluble glycosaminoglycans inhibit signaling, possibly by competing for cytokine binding. © 1994 wiley-Liss, Inc.     

Involvement of keratinocyte growth factor (KGF)-KGF receptor signaling in developmental estrogenization syndrome of mouse vagina

Exposure of mice to estrogen or keratinocyte growth factor (KGF) in vivo during the neonatal period results in estrogen-independent persistent proliferation and cornification of the vaginal epithelium when the animals become adults. Here, whether and how KGF-signaling is involved in the effects of estrogen on the neonatal mouse vagina were studied with an in vitro method. Newborn mouse vaginae were cultured for 3 days in serum-free medium containing various combinations of estradiol-17 (E₂), KGF, anti-KGF antibody, KGFR inhibitory peptide and heparin, and then transplanted into ovariectomized host mice for 35 days. The vaginae cultured with 5 g/ml E₂ or 5 g/ml KGF had a cornified thick epithelium, while the epithelium of the vehicle-treated controls stayed thin. The E₂ effect was blocked by concurrent treatment with anti-KGF antibody or KGFR inhibitory peptide. KGF treatment alone at doses less than 500 ng/ml did not induce permanent vaginal changes but such changes did occur in vaginae treated with heparin plus as little as 10 ng/ml KGF. On the other hand, heparin inhibited the permanent vaginal changes induced by estrogen. These results suggest that irreversible vaginal changes are induced by the direct action of KGF on the developing vagina and that the developmental estrogenization syndrome of mouse vagina is caused by intensification of endogenous KGF/KGFR signaling by exogenous estrogen.This work was supported by Grants-in-Aid for Scientific Research on Priority Areas (A) and for Encouragement of Young Scientists from the Ministry of Education Science, Sports and Culture, Japan to M.M.     

In vitro effects of propolis on Giardia duodenalis trophozoites

In order to improve the current chemotherapy of Giardia infection, potential antigiardial agents have been screened, including natural products. Propolis, a resinous hive product collected by bees, has attracted attention as a useful and popular substance with several therapeutic activities. The present study was carried out aiming to evaluate the in vitro effects of an ethanolic extract of propolis on the growth and adherence of Giardia duodenalis trophozoites. Propolis inhibited the growth of trophozoites and the level of inhibition varied according to the extract concentration and incubation times. The highest reduction of parasite growth was observed in cultures exposed to 125, 250 and 500 microg/ml of propolis, in all incubation periods (24, 48, 72 and 96 h). Growth reduction by 50% was observed in 125 microg/ml propolis-treated cultures, while the concentrations of 250 and 500 microg/ml were able to inhibit growth by more than 60%. Propolis also inhibited parasite adherence and all assayed propolis concentrations promoted the detachment of trophozoites. Light microscope observations revealed changes of the pear-shaped aspect of the cell and reduction of flagellar beating frequency in the great part of the trophozoites. Our results hold the perspective for the utilization of propolis as an antigiardial agent.     

Dried plum polyphenols attenuate the detrimental effects of TNF-alpha on osteoblast function coincident with up-regulation of Runx2, Osterix and IGF-I

Previous studies have demonstrated that dried plums which contain high amounts of polyphenols can restore bone mass and structure, and significantly increase indices of bone formation. The purpose of this study was to determine how dried plum polyphenols influence osteoblast activity and mineralized nodule formation under normal and inflammatory conditions. MC3T3-E1 cells were plated and pretreated with dried plum polyphenols (0, 2.5, 5, 10 and 20 microg/ml) and 24 h later stimulated with TNF-alpha (0 or 1.0 ng/ml). The 5, 10 and 20 microg/ml doses of polyphenols significantly increased intracellular ALP activity under normal conditions at 7 and 14 days, and restored the TNF-alpha-induced suppression of intracellular ALP activity by 14 days (P<.001). Polyphenols also increased mineralized nodule formation under normal and inflammatory conditions. In the absence of TNF-alpha, 5 microg/ml of polyphenols significantly up-regulated the growth factor, IGF-I, compared to controls, and the 5 and 10 microg/ml doses increased the expression of lysyl oxidase involved in collagen crosslinking. TNF-alpha decreased the expression of Runx2, Osterix and IGF-I, and polyphenols restored their mRNA levels to that of the controls. Although TNF-alpha failed to alter lysyl oxidase at 18 h, the polyphenols up-regulated its expression (P<.05) in the presence of TNF-alpha. As expected, TNF-alpha up-regulated RANKL mRNA and polyphenols suppressed RANKL expression without altering OPG. Based on these findings, we conclude that dried plum polyphenols enhance osteoblast activity and function by up-regulating Runx2, Osterix and IGF-I and increasing lysyl oxidase expression, and at the same time attenuate osteoclastogenesis signaling.     

Nerve growth factor modifies the expression of inflammatory cytokines by mast cells via a prostanoid-dependent mechanism

Nerve growth factor (NGF) is well recognized to have a number of potent effects on mast cells, including increasing mast cell numbers in vivo and inducing mast cell degranulation in vitro. More recently, NGF has been demonstrated to induce PGD2 production by mast cells through the induction of mast cell cyclooxygenase expression. We have observed that NGF at doses as low as 10 ng/ml will induce IL-6 production and inhibit TNF-alpha release from rat peritoneal mast cells in the presence of lysophosphatidylserine as a cofactor. NGF synergizes with LPS treatment of peritoneal mast cells (PMC) for the induction of IL-6. Examination of the mechanism of this phenomenon has revealed that NGF can induce both rat PMC and mouse bone marrow-derived cultured mast cells to produce substantial levels of PGE2. This response is maximal at later time points 18-24 h after NGF activation. The ability of NGF to induce PGE2 is not dependent on mast cell degranulation. Other stimuli capable of inducing IL-6, such as LPS, do not induce production of this prostanoid. Inhibition of cyclooxygenase activity by PMC using either flurbiprofen or indomethacin inhibited both the NGF-induced PGE2 synthesis and the NGF-induced alterations in TNF-alpha and IL-6 production. These results suggest a role for mast cell-derived prostanoids in the regulation of local inflammatory responses and neuronal degeneration after tissue injury involving induction of NGF production.     

Testosterone and estradiol differentially regulate TSH-induced thyrocyte proliferation in immature and adult rats

Though sex steroids are found to influence thyroid pathogenesis in human and in animals, their role in normal thyroid growth and thyrocyte proliferation is not yet understood fully. The present study is addressed to know the effect of testosterone and estradiol on the basal and TSH-induced thyrocyte proliferation in immature and adult rats in vitro. The male and female Wistar rats were gonadectomized (GDX) and one group of GDX rats were supplemented with either testosterone or estradiol. After the experimental period, the rats were sacrificed by decapitation and thyroid glands were removed, washed in Hank's Balanced Salt Solution (HBSS), pH 7.4 and digested with the enzyme mixture containing 0.08% collagenase and 0.12% dispase in HBSS. The isolated follicles were washed thrice with Dulbecco's modified Eagle's medium (DMEM) containing 0.5% fetal bovine serum (FBS), and were cultured in Falcon's tissue culture flasks containing 5 ml DMEM with FBS (5%) transferrin (5 microg/ml), hydrocortisone (10(-8) M), somatostatin (10 microg/ml), insulin (10 microg/ml) and glycyl-L-histidyl-L-lysine acetate (10 microg/ml). The cells (2.5 x 10(4)) were exposed to various exponential doses of TSH or testosterone (6.25-800 ng/ml) or estradiol (6.25-800 pg/ml). It is suggested from the present study that both TSH and sex steroids enhance thyrocyte proliferation. The mitogenic effect of TSH is greater than that of sex steroids. Sex steroids modulate TSH-induced cell proliferation in a gender-specific manner.     

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)     

Mast cell-mediated allergic response is suppressed by Sophorae flos: inhibition of SRC-family kinase

Complementary and alternative medicines are considered as a promising direction for the development of anti-allergic therapies in oriental countries. We screened approximately 100 oriental herbal medicines for anti-allergic activity. Sophorae flos exhibited the most potent effect on degranulation in antigen-stimulated mast cells. We further investigated the effect of Sophorae flos on the IgE-mediated allergic response in vivo and its mechanism of action in mast cells. Sophorae flos exhibited a significant inhibitory effect on degranulation in antigen-stimulated mast cells with IC(50) values of approximately 31.6 microg/mL (RBL-2H3 mast cells) and approximately 47.8 microg/mL (bone marrow-derived mast cells). Sophorae flos also suppressed the expression and secretion of TNF-alpha and IL-4 in the cells and IgE-mediated passive cutaneous anaphylaxis (PCA) in mice. Sophorae flos inhibited the activating phosphorylation of Syk and LAT in mast cells. Further downstream, activating phosphorylation of Akt and the prototypic MAP kinases, namely, p38, ERK1/2, and JNK, were also inhibited. These results suggest that Sophorae flos inhibits the Src family kinase-dependent signaling cascades in mast cells and may thus exert anti-allergic activity.     

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.