Morphological studies on the epiphyseal growth plate combined with biochemical and X-ray microprobe analyses

Summary The epiphyseal growth plate of the domestic pig was investigated topologically combining biochemical methods with electron microprobe microanalyses both correlated to histological controls. A lateral resolution of about 50 m was reached. Highest nuclease activity was found in the lower columnar cell zone, while alkaline phosphatase showed maximal activity in the hypertrophic area, connected with maximal values for extractable, organically bound phosphorus, and extractable Ca and Mg. Acid phosphatase activity reached maximal values in the zone of the lower primary spongiosa, while the extractable P_i had maximal values at the end of the zone of bone remodelling. Microprobe analyses have shown that the extracellular Ca content (per dry mass) remained relatively constant at 0.7% (about 58 mM/kg wet weight for 66% tissue fluid) in all zones of the plate increasing to 1% in the vicinity of the first foci of mineralization. The intracellular P content (per dry mass) was about 4.5%, the extracellular 0.1–0.2% (about 10–20 mM/kg wet weight) increasing also to about 1% in the vicinity of the first foci of mineralization. Thus the Ca x P product was much higher than the ion-product of 2 mM² which is necessary for an in vitro mineralization of connective tissue. The extracellular S content (per dry mass) as a probable indicator of sulfated proteoglycans was relatively constant at about 3.5% in the different zones but decreased to about 0.3% in the fully mineralized regions. This indicates a loss of sulfur containing substances with mineralization which is not so high since the concentrations per dry mass must be normalized to a unit volume of equal density of mass.     

Expression of tissue transglutaminase in skeletal tissues correlates with events of terminal differentiation of chondrocytes

Calcifying cartilages show a restricted expression of tissue transglutaminase. Immunostaining of newborn rat paw bones reveals expression only in the epiphyseal growth plate. Tissue transglutaminase appears first intracellularly in the proliferation/maturation zone and remains until calcification of the tissue in the lower hypertrophic zone. Externalization occurs before mineralization. Subsequently, the enzyme is present in the interterritorial matrix during provisional calcification and in the calcified cartilage cores of bone trabeculae. In trachea, mineralization occurring with maturation in the center of the cartilage is accompanied by expression of tissue transglutaminase at the border of the hydroxyapatite deposits. Transglutaminase activity also shows a restricted distribution in cartilage, similar to the one observed for tissue transglutaminase protein. Analysis of tissue homogenates showed that the enzyme is present in growth plate cartilage, but not in articular cartilage, and recognizes a limited set of substrate proteins. Osteonectin is coexpressed with tissue transglutaminase both in the growth plate and in calcifying tracheal cartilage and is a specific substrate for tissue transglutaminase in vitro. Tissue transglutaminase expression in skeletal tissues is strictly regulated, correlates with chondrocyte differentiation, precedes cartilage calcification, and could lead to cross-linking of the mineralizing matrix.     

Intracellular modulation of signaling pathways by annexin A6 regulates terminal differentiation of chondrocytes

Annexin A6 (AnxA6) is highly expressed in hypertrophic and terminally differentiated growth plate chondrocytes. Rib chondrocytes isolated from newborn AnxA6-/- mice showed delayed terminal differentiation as indicated by reduced terminal differentiation markers, including alkaline phosphatase, matrix metalloproteases-13, osteocalcin, and runx2, and reduced mineralization. Lack of AnxA6 in chondrocytes led to a decreased intracellular Ca(2+) concentration and protein kinase C α (PKCα) activity, ultimately resulting in reduced extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) activities. The 45 C-terminal amino acids of AnxA6 (AnxA6(1-627)) were responsible for the direct binding of AnxA6 to PKCα. Consequently, transfection of AnxA6-/- chondrocytes with full-length AnxA6 rescued the reduced expression of terminal differentiation markers, whereas transfection of AnxA6-/- chondrocytes with AnxA6(1-627) did not or only partially rescued the decreased mRNA levels of terminal differentiation markers. In addition, lack of AnxA6 in matrix vesicles, which initiate the mineralization process in growth plate cartilage, resulted in reduced alkaline phosphatase activity and Ca(2+) and inorganic phosphate (P(i)) content and the inability to form hydroxyapatite-like crystals in vitro. Histological analysis of femoral, tibial, and rib growth plates from newborn mice revealed that the hypertrophic zone of growth plates from newborn AnxA6-/- mice was reduced in size. In addition, reduced mineralization was evident in the hypertrophic zone of AnxA6-/- growth plate cartilage, although apoptosis was not altered compared with wild type growth plates. In conclusion, AnxA6 via its stimulatory actions on PKCα and its role in mediating Ca(2+) flux across membranes regulates terminal differentiation and mineralization events of chondrocytes.     

Spatial distribution of microbial biomass,activity, community structure,and the biodegradation of linear alkylbenzene sulfonate (LAS) and linear alcohol ethoxylate (LAE) in the subsurface

The vertical distribution of microbial biomass, activity, community structure and the mineralization of xenobiotic chemicals was examined in two soil profiles in northern Wisconsin. One profile was impacted by infiltrating wastewater from a laundromat, while the other served as a control. An unconfined aquifer was present 14 meters below the surface at both sites. Biomass and community structure were determined by acridine orange direct counts and measuring concentrations of phospholipid-derived fatty acids (PLFA). Microbial activity was estimated by measuring fluorescein diacetate (FDA) hydrolysis, thymidine incorporation into DNA, and mixed amino acid (MAA) mineralization. Mineralization kinetics of linear alkylbenzene sulfonate (LAS) and linear alcohol ethoxylate (LAE) were determined at each depth. Except for MAA mineralization rates, measures of microbial biomass and activity exhibited similar patterns with depth. PLFA concentration and rates of FDA hydrolysis and thymidine incorporation decreased 10–100 fold below 3 m and then exhibited little variation with depth. Fungal fatty acid markers were found at all depths and represented from 1 to 15% of the total PLFAs. The relative proportion of tuberculostearic acid (TBS), an actinomycete marker, declined with depth and was not detected in the saturated zone. The profile impacted by wastewater exhibited higher levels of PLFA but a lower proportion of TBS than the control profile. This profile also exhibited faster rates of FDA hydrolysis and amino acid mineralization at most depths. LAS was mineralized in the upper 2 m of the vadose zone and in the saturated zone of both profiles. Little or no LAS biodegradation occurred at depths between 2 and 14 m. LAE was mineralized at all depths in both profiles, and the mineralization rate exhibited a similar pattern with depth as biomass and activity measurements. In general, biomass and biodegradative activities were much lower in groundwater than in soil samples obtained from the same depth.     

Stereological studies on matrix vesicle distribution in the epiphyseal growth plate during healing of low phosphate, vitamin D deficiency rickets

A model of the healing phase of low phosphate, vitamin D deficiency was used to investigate the initial stages of mineralization. The matrix vesicle distribution between the zones of the growth plate was found to be bimodal with high volume densities in the resting and hypertrophic zones and low volume densities in the proliferative and calcifying zones. Healing of the rachitic lesion was associated with a decrease in matrix vesicle volume density in the calcifying zone, compared with the lower hypertrophic zone in florid rickets. The volume density differences were due to differences in the number of vesicles, as the variation in mean caliper diameter was rather small. The findings are compatible with the dynamic cell debris theory for matrix vesicle origin and distribution presented earlier, which favours the view that a major part of matrix vesicles are formed from cell debris. A role of matrix vesicles in the mineralization process is indicated by the finding of an association between mineralization and matrix vesicle degradation.     

Distribution of lipids associated with mineralization in the bovine epiphyseal growth plate

Calcium-acidic phospholipid-phosphate complexes, known to induce in vitro hydroxyapatite formation from metastable calcium phosphate sotutions, have been isolated from the morphologically defined zones of the bovine epiphyseal growth plate. The changes in zonal distribution of these complexes in epiphyseal cartilage correlate directly with other biochemical changes which occur prior to cartilage calcification. The concentration of calcium-acidic phospholipid-phosphate complexes increases going from the morphologically defined reserve zone to the proliferative zone, peaking in the hypertrophic zone, where mineralization is initiated, and decreasing in primary spongiosa and diaphyseal bone. Expressed as milligrams of calcium-phospholipid-phosphate complex per milligram hydroxyproline the concentration ranged from 19 (articular cartilage) to 535 (hypertrophic cell zone) decreasing to 43 (diaphyseal bone) with parallel changes being seen when the concentration was expressed per gram of demineralized dry tissue, per total lipid, per DNA, or, per 5′-AMPase activity.     

Calcification of rachitic cartilage to study matrix vesicle function.

Growth plate cartilage from rachitic rats was studied to assess the role of extra-cellular matrix vesicles in the reinstitution of calcification during healing. The concentration and distribution of matrix vesicles was found to be normal in rachitic growth plate, and although the rachitic cartilage matrix was largely uncalcified, an occasional vesicle did contain internal mineral. Matrix vesicles served as initial loci for mineralization when healing was brought about either by in vivo injection of phosphate or in vitro incubation of growth plates in a metastable calcifying solution. During in vitro calcification a distinct line of mineralization developed in the upper growth plate which was shown by electron microscopy to reflect mineralization by the vesicles. The appearance of this vesicle-associated calcification line was inhibited by preheating or repeated freezing and thawing, and by 30 minutes preincubation in deoxycholate, ethane-1-hydroxy-1,1-diphosphonate, or beryllium sulfate. Our results suggest that vesicle calcification is dependent on the structural and enzymatic integrity of the vesicle membrane. Enzymes that may well play a role in vesicle calcification are phosphatases (e. g., alkaline phosphatase, pyrophosphatase and ATPase), which are known to be concentrated in vesicle membranes.     

Role of the progressive ankylosis gene (ank) in cartilage mineralization

Mineralization of growth plate cartilage is a critical event during endochondral bone formation, which allows replacement of cartilage by bone. Ankylosis protein (Ank), which transports intracellular inorganic pyrophosphate (PP(i)) to the extracellular milieu, is expressed by hypertrophic and, especially highly, by terminally differentiated mineralizing growth plate chondrocytes. Blocking Ank transport activity or ank expression in terminally differentiated mineralizing growth plate chondrocytes led to increases of intra- and extracellular PP(i) concentrations, decreases of alkaline phosphatase (APase) expression and activity, and inhibition of mineralization, whereas treatment of these cells with the APase inhibitor levamisole led to an increase of extracellular PP(i) concentration and inhibition of mineralization. Ank-overexpressing hypertrophic nonmineralizing growth plate chondrocytes showed decreased intra- and extracellular PP(i) levels; increased mineralization-related gene expression of APase, type I collagen, and osteocalcin; increased APase activity; and mineralization. Treatment of Ank-expressing growth plate chondrocytes with a phosphate transport blocker (phosphonoformic acid [PFA]) inhibited uptake of inorganic phosphate (P(i)) and gene expression of the type III Na(+)/P(i) cotransporters Pit-1 and Pit-2. Furthermore, PFA or levamisole treatment of Ank-overexpressing hypertrophic chondrocytes inhibited APase expression and activity and subsequent mineralization. In conclusion, increased Ank activity results in elevated intracellular PP(i) transport to the extracellular milieu, initial hydrolysis of PP(i) to P(i), P(i)-mediated upregulation of APase gene expression and activity, further hydrolysis and removal of the mineralization inhibitor PP(i), and subsequent mineralization.     

Collagen/annexin V interactions regulate chondrocyte mineralization

Physiological mineralization in growth plate cartilage is highly regulated and restricted to terminally differentiated chondrocytes. Because mineralization occurs in the extracellular matrix, we asked whether major extracellular matrix components (collagens) of growth plate cartilage are directly involved in regulating the mineralization process. Our findings show that types II and X collagen interacted with cell surface-expressed annexin V. These interactions led to a stimulation of annexin V-mediated Ca(2+) influx resulting in an increased intracellular Ca(2+) concentration, [Ca(2+)](i), and ultimately increased alkaline phosphatase activity and mineralization of growth plate chondrocytes. Consequently, stimulation of these interactions (ascorbate to stimulate collagen synthesis, culturing cells on type II collagen-coated dishes, or overexpression of full-length annexin V) resulted in increase of [Ca(2+)](i), alkaline phosphatase activity, and mineralization of growth plate chondrocytes, whereas inhibition of these interactions (3,4-dehydro-l-proline to inhibit collagen secretion, K-201, a specific annexin channel blocker, overexpression of N terminus-deleted mutant annexin V that does not bind to type II collagen and shows reduced Ca(2+) channel activities) decreased [Ca(2+)](i), alkaline phosphatase activity, and mineralization. In conclusion, the interactions between collagen and annexin V regulate mineralization of growth plate cartilage. Because annexin V is up-regulated during pathological mineralization events of articular cartilage, it is possible that these interactions also regulate pathological mineralization.     

Histochemical localization of calcium in growth plate mitochondria and matrix vesicles.

K-pyroantimonate is an anion that forms an electron-dense precipitate with cellular cations that is readily visualized at the ultrastructural level. The staining process is made relatively specific for calcium by comparing pyroantimonate treated sections to sections pretreated with ethylene glycol-bis-N,N'-tetraacetic acid, a chelating agent that removes calcium but not other cellular cations. By these means, it is shown that the antimonate-calcium complex is located predominantly in mitochondria and cell membranes throughout most of the growth plate. In the degenerating zone, however, there is a gradual loss of stain complex from the mitochondria and cell membranes and a concomitant accumulation of the stain complex by matrix vesicles. The latter are the initial site of mineralization in the growth plate as detected by these means. Thus, this study suggests that intracellular calcium plays a significant role in matrix calcification.     

Induction of alkaline phosphatase in primary cultures of epiphyseal growth plate chondrocytes by a serum-derived factor

Alkaline phosphatase (AP) activity in epiphyseal growth plate cartilage increases markedly during differentiation of the chondrocytes, and reaches high levels in the zone of hypertrophy where vascular penetration and provisional mineralization begin. A proteinaceous factor has been discovered in serum that stimulates the expression of AP in chicken growth plate chondrocytes when these cells are grown in serum-free media. Sera from a variety of vertebrate species (goat, fetal bovine, horse, human, and chicken) all contained detectable levels of the inducing activity. The chondrocyte AP-induction factor (CAP-IF) from fetal bovine serum was precipitated with ammonium sulfate between 33% and 50% saturation, and purified by dye-ligand affinity chromatography. The active fraction, which eluted from an Affi-Gel Blue column between 0.10 and 0.15 M NaCl, was further resolved on a QMA anion exchange column. The most active and almost homogeneous fraction contained primarily a 64.5 kDa protein; about 3 micrograms/ml medium induced 50% of the maximal level of AP induction. CAP-IF is stable to heat (100 degrees C for 3 min) and dithiothreitol (50 mM) treatment, and is only mildly inactivated by 2 h treatment with trypsin. CAP-IF caused no significant effect on cell division as measured by 3H-thymidine uptake. Time-course studies revealed that at least 18-24 h exposure of the chondrocytes to CAP-IF is required to produce major increases in AP activity. Longer exposure time generally further increases the response. Cycloheximide almost completely blocked the increase in AP activity, indicating that de novo protein synthesis is required for induction.     

Annexin-mediated Ca2+ influx regulates growth plate chondrocyte maturation and apoptosis

Maturation of epiphyseal growth plate chondrocytes plays an important role in endochondral bone formation. Previously, we demonstrated that retinoic acid (RA) treatment stimulated annexin-mediated Ca(2+) influx into growth plate chondrocytes leading to a significant increase in cytosolic Ca(2+), whereas K-201, a specific annexin Ca(2+) channel blocker, inhibited this increase markedly. The present study addressed the hypothesis that annexin-mediated Ca(2+) influx into growth plate chondrocytes is a major regulator of terminal differentiation, mineralization, and apoptosis of these cells. We found that K-201 significantly reduced up-regulation of expression of terminal differentiation marker genes, such as cbfa1, alkaline phosphatase (APase), osteocalcin, and type I collagen in RA-treated cultures. Furthermore, K-201 inhibited up-regulation of annexin II, V, and VI gene expression in these cells. RA-treated chondrocytes released mineralization-competent matrix vesicles, which contained significantly higher amounts of annexins II, V, and VI as well as APase activity than vesicles isolated from untreated or RA/K-201-treated cultures. Consistently, only RA-treated cultures showed significant mineralization. RA treatment stimulated the whole sequence of terminal differentiation events, including apoptosis as the final event. After a 6-day treatment gene expression of bcl-2, an anti-apoptotic protein, was down-regulated, whereas caspase-3 activity and the percentage of TUNEL-positive cells were significantly increased in RA-treated cultures compared with untreated cultures. Interestingly, the cytosolic calcium chelator BAPTA-AM and K-201 protected RA-treated chondrocytes from undergoing apoptotic changes, as indicated by higher bcl-2 gene expression, reduced caspase-3 activity, and the percentage of TUNEL-positive cells. In conclusion, annexin-mediated Ca(2+) influx into growth plate chondrocytes is a positive regulator of terminal differentiation, mineralization, and apoptosis events in growth plate chondrocytes.     

Structural and histochemical study of the effect of 1,25-dihydroxyvitamin D3 on long-bone growth center in suckling mice

The in vivo effects of 1 alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3] on cellular structure and response, matrix metachromasia and mineralization have been studied in the epiphysis and growth plate of humeri in normal neonatal mice. A relatively low dose of the metabolite, 50 ng/kg body weight, significantly increased the overall size of humeral growth plate, the zone of proliferating cells and that of hypertrophic chondrocytes. The response of the tissue to the metabolite changed with the increase in dose administered so that it was only the zone of proliferation that still showed increases in size in comparison to untreated controls. 1,25 (OH)2D3 led to an increase in the metachromatic reaction of the cartilage matrix in the chondroblastic zone, and to marked increase in matrix mineralization in the hypertrophic zone. Qualitative changes were also noted in the structure of chondroblasts and hypertrophic chondrocytes. 1,25(OH)2D3 affected the osteoblastic and osteocytic populations of cells along the metaphysis and diaphysis of the humerus. High doses of 1,25(OH)2D3 brought about distinct atrophic changes in the above cells. Chondrocytes in the epiphysis were found to synthesize type I collagen and fibronectin. These findings indicate that excessive doses of 1,25(OH)2D3 in an intact growing animal affect the normal differentiative pathway of prechondroblasts and thereby affect long bone development.     

Rapid activity-dependent sprouting of optic fibers into a local area denervated by application of beta-bungarotoxin in goldfish tectum

The retinotectal projection is known to be capable of extensive long-term expansion of connections, but it is not known how fast such changes can occur or what triggers sprouting of terminals. We studied sprouting of optic fibers into an area denervated by local microinjection of beta-bungarotoxin (β-BTX), a specific presynaptic neurotoxin with phospholipase A₂ activity that destroys nerve terminals at the neuromuscular junction. After injection of 0.1 pmol of β-BTX, the optic terminals fired spontaneously with decreasing amplitude and became silent within 1 to 2 h. Outside the injection zone, the retinotectal map was normal, so the silent zone was associated with a scotoma in the visual field. Horseradish peroxidase (HRP) staining of the entire optic nerve showed a denervated region at the injection site with beaded, degenerating fibers at its edge. Between 3 and 9 days later, optic units were recorded within the injection zone whose receptive fields lay just outside the scotoma in the visual field, indicating that intact surrounding terminals had sprouted into the area. These sprouts made functional connections, as indicated by field potential recordings and current source-density analysis. At this time, HRP staining also demonstrated retinal innervation within the injection zone. By 12 days, normal maps with no scotoma were recorded and HRP staining was normal at the injection site, indicating that the β-BTX-damaged fibers had regenerated to reclaim their tectal sites. The results show that the retinotectal projection of goldfish is very dynamic, since intact optic fibers can sprout into adjacent vacant postsynaptic territory within 2 to 3 days, much faster than previously reported. In a final experiment, we showed that this sprouting is activity-dependent, since it could be prevented by blocking retinal activity with intraocular tetrodotoxin (TTX) during the first 2 days postinjection, even though TTX block of activity does not block regeneration in this system. One possible mechanism for this rapidly triggered sprouting is that arachidonic acid liberated by β-BTX acts as a sprouting factor to attract surrounding healthy fibers into the denervated region but requires activity at the terminals to be effective. © 1996 John Wiley & Sons, Inc.     

Mineralization Capacity of Bacteria and Fungi from the Rhizosphere-Rhizoplane of a Semiarid Grassland

A radiotracer glucose mineralization assay was used with streptomycin and actidione to monitor the relative seasonal contributions of bacteria and fungi to mineralization processes in soils derived from the rhizosphere-rhizoplane zone of plants from a shortgrass prairie ecosystem. Bacteria played a major role in glucose mineralization in both the rhizosphere and rhizoplane. These results indicate that the bacteria may play a greater role in glucose mineralization processes in the rhizosphere and rhizoplane zones of a semiarid grassland than would be assumed, based on available biomass estimates. This technique appears to be valuable for determining bacterial versus fungal contributions to glucose mineralization in the rhizosphere and rhizoplane and may be useful for measuring the decomposition of other more complex substances in this zone of intense microbial activity.     

Biochemical and histological study of the ossification in the early developing pedicle of the fallow deer (Dama dama)

To date, no histochemical data exist concerning the process of ossification of developing pedicles in deer. Four different zones of the growing pedicle (subcutaneous tissue; fibrous layer of the periosteum; cambial layer of the periosteum; women bone of the primary spongiosa) were analysed in direct correlation to their histological appearance. The level of extractable specific alkaline phosphatase in the preosseous zones of the pedicle was 4-fold higher than levels in the epiphyseal growth plate previously reported. These results reflect that rapid bone formation takes place in the growing pedicle. Highest buffer-extractable alkaline phosphatase activity was found in the cambial layer directly in front of the mineralization area of the pedicle-bone, connected with maximal values for organically bound phosphate and inorganic phosphate. Moreover, the values for buffer-extractable alkaline phosphatase, organically bound phosphate and inorganic phosphate decreased with increasing mineralization in the zone of the primary spongiosa. The present histological and biochemical findings on the process of ossification in the pedicle show similarities to typical endochondral ossification. The process of pedicle growth may serve as a new and important system for chondrogenic and osteogenic studies, including a better understanding of antler development.     

The identification of matrix Gla protein in cartilage

The vitamin K-dependent bone protein matrix gamma-carboxyglutamic acid (Gla) protein (MGP) has been identified by radioimmunoassay in the guanidine extract of rat cartilage. MGP was present in all cartilages tested at levels comparable to the MGP level in bone. Western blot analysis indicated that the molecular weight of cartilage MGP is the same as bone MGP, and Northern blot analysis revealed that MGP mRNA from cartilage is the same size as the MGP mRNA from bone. The structurally related vitamin K-dependent protein bone Gla protein could not be detected in cartilage by radioimmunoassay or by Northern blot analysis. The discovery that MGP is synthesized by growth plate cartilage could provide an explanation for the excessive growth plate mineralization disorder seen in rats treated with the vitamin K antagonist warfarin and the punctate mineralization of the growth plate seen in infants whose mothers received warfarin in the first trimester of pregnancy (the fetal warfarin syndrome). Both disorders appear to be caused by the inactivation of a vitamin K-dependent mineralization inhibitor in cartilage, an inhibitor which we suggest is MGP.     

Bioremediation Assessment of Hydrocarbon-Contaminated Soils from the High Arctic

The bioremediation potential of hydrocarbon-contaminated soils from the most northerly inhabited station in the world, Canadian Forces Station - Alert, was assessed. Microbial enumeration, by both viable plate counts and direct counts, combined with molecular analysis (polymerase chain reaction and colony hybridization) for hydrocarbon catabolic genes (alkB, ndoB, xylE), demonstrated the presence of significant numbers of cold-adapted hydrocarbon-degrading microorganisms. The degradative activity of these populations was assessed by mineralization of ¹⁴Clabeled hexadecane (C16) at 5°C in untreated and treated soils. Although very low rates of C16 mineralization were observed in the untreated soils, nutrient supplementation with a fertilizer markedly increased C16 mineralization. Highly active cold-adapted hydrocarbon-degrading consortia were prepared from soil slurries, and their degradative potentials were monitored by biomass measurements and mineralization activity. Bio augmentation of the contaminated soils with consortia containing the greatest percentages of degradative bacteria resulted in the shortest C16 mineralization acclimation period. However, treatment with the consortia plus fertilizer did not appreciably increase C16 mineralization or reduce total petroleum hydrocarbon concentrations to a greater extent than did the fertilizer treatment alone. These results indicate that the soils possessed sufficient numbers of cold-adapted degradative bacteria, and that fertilizer application alone was sufficient to obtain elevated levels of degradative activity at low ambient summer temperatures.     

A rapid and strong increase of plasminogen activator induced by experimental anaphylaxis in rabbits.

Anaphylactic shock was induced in rabbits by injecting bovine serum albumin (BSA) as an antigen. Measurements of the enzyme activities in the fibrinolytic system confirmed that a rapid and strong increase of plasminogen activator (PA) was induced during anaphylaxis. The euglobulin fibrinolytic activity (EFA) as estimated by the plasminogen-rich fibrin plate method rose significantly, peaking at 15 min after the BSA injection (when the arterial pressure was minimum). However, EFA was not detected by the plasminogen-poor fibrin plate method. The tissue-type PA (t-PA) activity using the natural substrate plasminogen increased significantly with a peak at 15 min. The amidolytic activity also simultaneously increased significantly using the t-PA substrate, H-D-Ile-Pro-Arg-pNA. The plasminogen activator inhibitor (PAI) activity remained at baseline levels until 30 min, but rose fourfold at 90 min. The main plasma fibrinolytic enzyme which increased in anaphylaxis was proved by zymography to be t-PA with a molecular weight (MW) of 69,000.