Reverse fibrin autography: A method to detect and partially characterize protease inhibitors after sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Erickson, Laurence A.; Lawrence, Daniel A.; Loskutoff, David J.

doi:10.1016/0003-2697(84)90113-1

Cited by 172 publications

(74 citation statements)

References 22 publications

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“…CM collected from untreated cultures gave rise to a single lysis-resistant zone of Mr 50,000 (Fig. 3, lane 1), reflecting the presence of the antiactivator (22). Considerably less antiactivator activity was detected in CM prepared from APCtreated cells (Fig.…”

Section: Resultsmentioning

confidence: 97%

“…Thus, agents that alter the fibrinolytic activity of these cells may do so by changing PA, antiactivator, or both (24) tography (Fig. 3), a semi-quantitative technique in which the size of the lysis-resistant zone in the indicator film reflects the amount of inhibitor applied to the NaDodSO4 gel (22). CM collected from untreated cultures gave rise to a single lysis-resistant zone of Mr 50,000 (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The interaction of this urokinase with plasminogen caused the indicator film to lyse. The opaque lysis-resistant zones that develop in the otherwise clear indicator film result from the presence of inhibitors in the NaDodSO4/polyacrylamide gel (14,22).…”

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confidence: 99%

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Activated protein C stimulates the fibrinolytic activity of cultured endothelial cells and decreases antiactivator activity.

Sakata¹,

Curriden²,

Lawrence³

et al. 1985

Proc. Natl. Acad. Sci. U.S.A.

181

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The effects of bovine activated protein C (APC) on the fibrinolytic activity of cultured bovine aortic endothelial cells were investigated. Confluent monolayers were incubated with purified APC under various conditions and changes in total fibrinolytic activity and in the level of plasminogen activator and plasminogen activator inhibitor (antiactivator) were monitored. The addition of APC to the cells in the absence of other blood or plasma components led to a rapid, dose-dependent increase of fibrinolytic activity both in the media and in cellular extracts. For example, 3.4 jtg of APC per ml resulted in a 15-fold increase of fibrinolytic activity in the medium within 1 hour. The enhanced fibrinolytic activity reflected increases in both the urokinase-related and tissue-type plasminogen activators produced by these cells. Interestingly, treatment of-cells with APC also caused a rapid, dose-dependent decrease in antiactivator activity. Diisopropyl fluorophosphate-inactivated APC did not decrease antiactivator or increase plasminogen activator. Although a small but significant direct (i.e., cell-independent) effect of APC on both fibrinolytic activity and antiactivator activity could be demonstrated, the major portion of these changes appeared to be cellmediated. These observations indicate that the fibrinolytic potential of cultured endothelial cells is increased by APC and that the enzyme active site is essential for this change. Moreover, the results suggest that one of the primary mechanisms for this stimulation of endothelial cell fibrinolytic activity involves an APC-mediated decrease in antiactivator.

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Section: Resultsmentioning

confidence: 97%

Section: Resultsmentioning

confidence: 99%

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Activated protein C stimulates the fibrinolytic activity of cultured endothelial cells and decreases antiactivator activity.

Sakata¹,

Curriden²,

Lawrence³

et al. 1985

Proc. Natl. Acad. Sci. U.S.A.

181

View full text Add to dashboard Cite

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“…Triton X-100 for 2 x 4 5 min to remove SDS from the gel before being analyzed for PAI-1 activity. The reverse fibrin autography assay was performed according to Erickson et al (1984). Briefly, the gel containing the fractionated samples was laid onto a fibrin/agar indicator gel containing 2.4 mg/ml fibrinogen, 0.5 U/ml thrombin, 50 pg/ ml plasminogen and 0.01 U/ml uPA and incubated in a humid chamber at 37°C to allow autolysis.…”

Section: Preparation Of Ovarian Cellsmentioning

confidence: 99%

Transient and cell‐specific expression of tissue‐type plasminogen activator and plasminogen‐activator‐inhibitor type 1 results in controlled and directed proteolysis during gonadotropin‐induced ovulation

Peng

Hsueh

1993

European Journal of Biochemistry

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Proteolytic activity generated by the plasminogen-activator system (PA system) is associated with many biological processes. However, it is not known how the proteolytic activity is regulated in vivo in order to obtain directed proteolysis while, at the same time, protecting unrestrained tissue destruction. Using gonadotropin-induced ovulation as a model, we have studied how two components of the PA system, tissue-type plasminogen activator (tPA) and plasminogen-activator-inhibitor type 1 (PAI-l), are regulated temporally and spatially by gonadotropins, leading to the initiation and termination of a well-directed proteolytic process. In-situ hybridization and in-situ zymography were used to analyze the expression of tPA and PAI-1 mRNA and PA-activity in specific ovarian cell types. Both tPA and PAI-1 were found to be regulated and to have a distinct expression pattern in different ovarian compartments. tPA was expressed in both granulosa and thecal-interstitial cells ; the highest levels of tPA mRNA were found in the granulosa cells of preovulatory follicles, just prior to ovulation. Consistent with a role for luteinizing hormonekhorionic gonadotropin (LWCG) in triggering ovulation, the cells and follicles that actively expressed tPA also contained high levels of LH-receptor mRNA while cumulus cells that contain undetectable amounts of tPA mRNA were devoid of LH-receptor expression. The highest levels of PAI-1 mRNA were found about 6 h before ovulation and mainly in the thecal-interstitial cells and ovarian stroma tissue which encapsulate the follicle. Preovulatory follicles, protruding onto the surface of the ovary with less surrounding stroma tissue, expressed less PAI-1 compared to small non-ovulatory follicles embedded in inner part of the ovary. In-situ zymography also revealed that the PA activity was colocalized to the surface of the ovary just prior to ovulation. Our studies suggest that a proteolytic activity provided by tPA and modulated by PAI-1 is responsible for a controlled and directed proteolysis leading to rupture of selected follicles during ovulation. Extracellular proteolysis, mediated by the plasminogenactivator (PA) system, involving the conversion of plasminogen into active plasmin, has been associated with many physiological and pathological processes. These processes include ovulation, mammary involution, embryo implantation, angiogenesis and tumor invasion (for review and references see Dan0 et al., 1985;Saksela and Rifkin 1988;Vassalli et al., 1991). All these processes involve proteolytic degradation of the extracellular matrix components, which must be conducted with appropriate specificity in order to prevent unrestrained tissue destruction.Many in-vitro studies have shown that the regulation of the PA system is complex and occurs at multiple levels. The Correspondence to T. Ny, Dept of Cell and Molecular BiologyFax: t 4 6 90 11 1420. Abbreviations. PA, plasminogen activator; uPA, urokinase-type plasminogen activator; tPA, tissue-type plasminogen activator; PAI-1, plasminogen-activa...

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“…The fractions were then assayed for both PAI-1 antigen and PA1 activity, and highly enriched fractions were pooled (5 ml or 7.5 ml) for further analysis. [32], immunodiffusion analysis according to Ouchterlony [33] and immunoblotting with MA-7F5 according to Towbin et al [34].…”

Section: Purification Of Pai-i From Conditioned Mediamentioning

confidence: 99%

Purification and characterization of natural and recombinant human plasminogen activator inhibitor‐1 (PAI‐1)

Alessi

Declerck

Mol

et al. 1988

European Journal of Biochemistry

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Human plasminogen activator inhibitor-I (PAI-1) was purified from the conditioned medium of endotoxinstimulated umbilical vein endothelial cell cultures by combinations of zinc-chelate-Sepharose chromatography, gel filtration on Sephacryl S-300 and immunoadsorption on an insolubilized murine monoclonal antibody (MA-7D4). The final product was obtained with a recovery of approximately 20% from conditioned medium containing about 3 pg/ml PAI-1. The yield of PAI-1 was 15-100 pg/umbilical cord, depending on the culture and harvest conditions. SDS gel electrophoresis revealed a main band with M , = 46000 both under reducing and non-reducing conditions. On gel filtration on Sephacryl S-300, however, the material was separated in two fractions, one eluting at the void volume, which contains active PAI-1, and one with M , = 46000 containing inactive material that could be reactivated with 12 M urea. SDS gel electrophoresis of the isolated high-M, fraction revealed several bands including a main 46 000-M, component, which reacted with anti-(PAI-1) antibodies on immunoblotting and neutralized tissue-type plasminogen activator (t-PA). The active high-M, fraction and the reactivated low-M, fraction of PAI-1 inhibited t-PA very rapidly with an apparent second-order rate constant of (1.5 -4) x lo7 M-' SC1.

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Reverse fibrin autography: A method to detect and partially characterize protease inhibitors after sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Cited by 172 publications

References 22 publications

Activated protein C stimulates the fibrinolytic activity of cultured endothelial cells and decreases antiactivator activity.

Activated protein C stimulates the fibrinolytic activity of cultured endothelial cells and decreases antiactivator activity.

Transient and cell‐specific expression of tissue‐type plasminogen activator and plasminogen‐activator‐inhibitor type 1 results in controlled and directed proteolysis during gonadotropin‐induced ovulation

Purification and characterization of natural and recombinant human plasminogen activator inhibitor‐1 (PAI‐1)

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