THE PROTECTIVE EFFECT OF ACYLATI0N ON THE STABILITY OF EMINASE (APSAC) IN HUMAN PLASMA

1987 ◽  
Author(s):  
R Fears ◽  
H Ferres ◽  
R Standring
Keyword(s):  
Protective Effect ◽  
Human Plasma ◽  
Fibrinolytic Activity ◽  
Animal Studies ◽  
Active Form ◽  
Α2 Macroglobulin ◽  
The Stability ◽  
High Level ◽  
Activator Complex

Clinical and animal studies indicate that APSAC (anisoylated plasminogen.streptokinase activator complex, Eminase) circulates longer in the bloodstream in an active form than the other thrombolytics. In the present studies in vitro u/e have found that functional activity of APSAC is maintained in human plasma longer than that of SK.plasmin(ogen): the relative stability half-lives are similar to the plasma clearance haif-lives in patients. Some of the loss of activity of SK at early times can be attributed to neutralisation by inhibitors. Thus, the survival of fibrinolytically-active SK was promoted in plasma depleted in α2-antiplasmin (α2AP) and α2AP-SK.plasmin complexes (detected by immunoblotting) formed rapidly in normal plasma. Corresponding studies with α2 macroglobulin-depleted plasma suggested a slight, late influence on SK activity but the inhibitor complex has not been detected unequivocally. In addition, loss of SK activity can be attributed, in part, to. rapid degradation to low molecular products. The degradation of SK in APSAC was much slower. In other comparative studies, the stability of APSAC was found to be similar to the stability of prourokinase and much superior to that of SK which is similar to UK; t-PA is intermediate in stability.Maintenance of fibrinolytic activity vivo depends on the stability of the thrombolytic, its rate of clearance and mode of administration. The protective effect of acylation, demonstrated in these experiments, explains why the objective of maintaining a high level of fibrinolytic activity after intravenous bolus injection of APSAC is less compromised by opposing inactivation processes.

Protective Effect of Isothiocyanates from Cruciferous Vegetables on Breast Cancer: Epidemiological and Preclinical Perspectives

2020 ◽  
Vol 20 ◽  
Author(s):  
Suong N.T. Ngo ◽  
Desmond B. Williams
Keyword(s):  
Breast Cancer ◽  
Protective Effect ◽  
Animal Studies ◽  
Cancer Survival ◽  
Breast Cancer Survival ◽  
Human Studies ◽  
In Vitro Studies ◽  
Cochrane Library ◽  
Cruciferous Vegetables

Background: The effect of cruciferous vegetable intake on breast cancer survival is controversial at present. Glucosinolates are the naturally occurring constituents found across the cruciferous vegetables. Isothiocyanates are produced from the hydrolysis of glucosinolates and this reaction is catalysed by the plant-derived enzyme myrosinase. The main isothiocyanates (ITCs) from cruciferous vegetables are sulforaphane, benzyl ITC, and phenethyl ITC, which had been intensively investigated over the last decade for their antibreast cancer effects. Objective: The aim of this article is to systematically review the evidence from all types of studies, which examined the protective effect of cruciferous vegetables and/or their isothiocyanate constituents on breast cancer. Methods: A systematic review was conducted in Pubmed, EMBASE, and the Cochrane Library from inception to 27 April 2020. Peerreviewed studies of all types (in vitro studies, animal studies, and human studies) were selected. Results: The systematic literature search identified 16 human studies, 4 animal studies, and 65 in vitro studies. The effect of cruciferous vegetables and/or their ITCs intake on breast cancer survival was found to be controversial and varied greatly across human studies. Most of these trials were observational studies conducted in specific regions, mainly in the US and China. Substantial evidence from in vitro and animal studies was obtained, which strongly supported the protective effect of sulforaphane and other ITCs against breast cancer. Evidence from in vitro studies showed sulforaphane and other ITCs reduced cancer cell viability and proliferation via multiple mechanisms and pathways. Isothiocyanates inhibited cell cycle, angiogenesis and epithelial mesenchymal transition, as well as induced apoptosis and altered the expression of phase II carcinogen detoxifying enzymes. These are the essential pathways which promote the growth and metastasis of breast cancer. Noticeably, benzyl ITC showed a significant inhibitory effect on breast cancer stem cells, a new dimension of chemoresistance in breast cancer treatment. Sulforaphane and other ITCs displayed anti-breast cancer effects at variable range of concentrations and benzyl isothiocyanate appeared to have a relatively smallest inhibitory concentration IC50. The mechanisms underlying the cancer protective effect of sulforaphane and other ITCs have also been highlighted in this article. Conclusion: Current preclinical evidence strongly supports the role of sulforaphane and other ITCs as potential therapeutic agents for breast cancer, either as adjunct therapy or combined therapy with current anti-breast cancer drugs, with sulforaphane appeared to display the greatest potential.

scholarly journals Initiation and regulation of fibrinolysis in human plasma at the plasminogen activator level

Blood ◽  
1987 ◽  
Vol 69 (5) ◽  
pp. 1354-1362 ◽  
Author(s):  
TC Wun ◽  
A Capuano
Keyword(s):  
Human Plasma ◽  
Plasminogen Activator ◽  
Gel Electrophoresis ◽  
Fibrinolytic Activity ◽  
High Rate ◽  
Bovine Aortic Endothelial Cell ◽  
Hep G2 ◽  
Triton X ◽  
Sepharose 4B

The initiation and regulation of fibrinolysis has been studied by reconstitution of fibrinolytic activity in human plasma in vitro. Depletion of tissue plasminogen activator (tPA) antigen by immunoadsorption of human plasma with anti-tPA Ig Sepharose 4B leads to total loss of spontaneous fibrinolytic activity determined by lysis of a thrombin-induced clot. Addition of physiological concentrations of purified tPA to tPA-depleted plasma restores fibrinolytic activity as a function of the length of time between tPA addition and clotting. Addition of free tPA to tPA-depleted plasma followed by immediate clotting results in a high rate of fibrinolysis. In contrast, when free tPA is allowed to incubate in plasma for 10 to 60 minutes prior to clot formation, the fibrinolytic activity of tPA is gradually lost. The loss of tPA-induced fibrinolytic activity in unclotted plasma is accompanied by decreased partitioning of tPA antigen into fibrin after clotting and is kinetically correlated with the formation of a 100 kilodalton (kDa) tPA complex as demonstrated by SDS-gel electrophoresis and fibrin-agar zymography. These results suggest that free tPA is susceptible to complexation by the plasma inhibitor in the absence of a clot. Fibrin formation renders tPA relatively inaccessible to inhibition. The tPA antigen isolated from stored plasma consists mainly of 100 kDa activity in SDS-gel electrophoresis and zymography, indicating that the tPA complex is resistant to dissociation by SDS. Upon rezymography of the sliced gel, only a 60 kDa tPA activity is found, suggesting that the activity at 100 kDa is at least partly due to free tPA dissociated from the complex during the first zymography. Conversion of tPA complex to enzymatically active free tPA also occurs with brief SDS exposure followed by incubation in the presence of excess Triton X-100 or by hydroxylamine treatment. These results reconcile the apparent discrepancy of the 100 kDA inhibitor-tPA complex manifesting plasminogen activation activity during zymography. The plasma tPA- inhibitor complex is precipitated strongly by antisera against plasminogen activator inhibitors (PAIs) of human Hep G2 hepatoma and HT- 1080 fibrosarcoma cells and weakly by antiserum against bovine aortic endothelial cell PAI but not by antiserum against a placental PAI (PAI- 2) suggesting that the plasma inhibitor is immunologically related to Hep G2, HT-1080 and possibly endothedial cell PAIs. Based on the above findings, a simple model for the initiation and regulation of plasma fibrinolysis at the PA level has been formulated.

Absence of Activation in Vitro of Renin in Rat Plasma

1982 ◽  
Vol 62 (4) ◽  
pp. 435-437 ◽  
Author(s):  
M. H. De Keijzer ◽  
A. P. Provoost ◽  
F. H. M. Derkx
Keyword(s):  
Human Plasma ◽  
Active Form ◽  
Plasma Renin ◽  
Rat Plasma ◽  
Plasma Renin Concentration ◽  
Inactive Form ◽  
Inactive Renin

1. Rat plasma was subjected at 4°C to various treatments known to convert inactive renin into its active form in human plasma. 2. No statistical differences in plasma renin concentration were found when the levels after the various treatments were compared with that of untreated rat plasma. 3. It is concluded that, in contrast to human plasma, no inactive form of renin is present in rat plasma.

Plasminogen: an enigmatic zymogen

Blood ◽  
2021 ◽  
Author(s):  
Charithani B Keragala ◽  
Robert L Medcalf
Keyword(s):  
Animal Studies ◽  
Wound Repair ◽  
Active Form ◽  
Cell Behaviour ◽  
Surface Receptors ◽  
Enzymatic Cascades ◽  
Inflammatory Processes ◽  
Over 40 Years

Plasminogen is an abundant plasma protein that exists in various zymogenic forms. Plasmin, the proteolytically active form of plasminogen, is known for its essential role in fibrinolysis. The therapeutic targeting of the fibrinolytic system to date has been for two purposes: to promote plasmin generation for thromboembolic conditions, or to stop plasmin to reduce bleeding. However, both plasmin and plasminogen serve other important functions, some of which are unrelated to fibrin removal. Indeed, for over 40 years, the anti-fibrinolytic agent, tranexamic acid, has been administered for its serendipitously discovered skin whitening properties. Plasmin also plays an important role in the removal of misfolded/aggregated proteins and can trigger other enzymatic cascades including complement. In addition, plasminogen, via binding to one of its dozen cell-surface receptors, can modulate cell behaviour and further influence immune and inflammatory processes. Plasminogen administration itself has been reported to improve thrombolysis and to accelerate wound repair. While many of these more recent findings have been derived from in vitro or animal studies, the use of anti-fibrinolytics to reduce bleeding in humans has revealed additional clinically relevant consequences, particularly in relation to reducing infection risk that is independent of its haemostatic effects. The finding that many viruses harness the host plasminogen to aid infectivity has suggested that anti-fibrinolytic agents may have anti-viral benefits. Here we review the broadening role of the plasminogen activating system in physiology and pathophysiology and how manipulation of this system may be harnessed for benefits unrelated to its conventional application in thrombosis and haemostasis.

DETECTION OF INACTIVATED α2-MACROGLOBULIN (α2M) IN HUMAN PLASMA USING MONOCLONAL ANTIBODIES

1987 ◽  
Author(s):  
J Abbink ◽  
J Nuijens ◽  
C Huijbregts ◽  
E Hack
Keyword(s):  
Monoclonal Antibodies ◽  
Longitudinal Studies ◽  
Human Plasma ◽  
Dextran Sulfate ◽  
Optimal Conditions ◽  
Α2 Macroglobulin ◽  
In Vitro Activation

Monoclonal antibodies (mAbs) were raised against human a2M. Five mAbs that bound to α2M in ELISA were further analyzed by a radioimmunoassay (RIA) for their reaction with three types of α2M: native α2M, chemically inactivated α2M (iα2M) (methylamine treated), and proteolytically iα2M. One mAb reacted with all forms of α2M, while four mAbs bound both forms of ia2M but not native α2M. One of these latter mAbs (Ml) was used to develop a RIA (the Ml-assay) for the detection of iα2M in plasma: Ml coupled to Sepharose is incubated with the plasma to be tested, and bound iα2M is detected by a subsequent incubation with polyclonal 125I-anti-α2M antibodies. As little as 5 ng of iα2M can be detected with this assay in the presence of an excess of native α2M. This assay was then applied to measure inactivation of α2M in vitro and in vivo. In vitro activation of the contact system in plasma by dextran sulfate results in the inactivation of ca 10% of α2M. When blood from normal donors was collected under optimal conditions, about 0.5% of the total α2M content appeared to be iα2M. Longitudinal studies in patients (a.o. with septicaemie, during cardiopulmunary bypass) revealed that increased levels of iα2M occurred sporadically. The Ml-assay appears to be useful to monitor the role of α2M in human diseases.

C1-Inactivator as a Determining Factor in Contact Activation of Fibrinolysis

1975 ◽  
Author(s):  
C. Kluft
Keyword(s):  
Fibrinolytic Activity ◽  
Slow Rate ◽  
Contact Activation ◽  
Activation Rate ◽  
Activity Generation ◽  
High Level ◽  
Fletcher Factor ◽  
Determining Factor

The rate of contact activation of fibrinolysis is considered to reflect the activation rate of proactivator and Hageman factor. This study was undertaken to determine the role of Cl-inactivator in this process.Contact activation of fibrinolysis was performed according to Ogston et al. (1969), J. Clin. Invest. 48, 1786-1801. The rate of activity generation was measured in plasma with various levels of Cl-inactivator and appeared to be dependent on that level; i.e., a high level of Cl-inactivator corresponds with a slow rate of activity generation.It has recently been demonstrated that the fibrinolytic activity of euglobulin fractions is strongly inhibited by Cl-inactivator also present in this fraction. The activity generation of contact activation is found to be accompanied by a gradual decrease in functional Cl-inactivator in the euglobulin fraction. The fibrinolytic activity is set free by this disappearance of inhibition.It is concluded that the rate of contact activation of fibrinolysis must be interpreted in terms of the inactivation of Cl-inactivator rather than of the activation of proenzymes. All enzymes capable of inactivating Cl-inactivator can contribute to the process of contact activation of fibrinolysis. This mechanism might account for the observed defects in fibrinolysis in vitro in Fletcher Factor deficient patients.

In Vitro Effects of the Acylated StreptokinasePlasminogen Activator Complex BRL 33 575 Incubated with Normal Human Plasma

1984 ◽  
Vol 51 (03) ◽  
pp. 403-405 ◽  
Author(s):  
B Lämmle ◽  
G Noll ◽  
T H Tran ◽  
A Lohri ◽  
F Duckert
Keyword(s):  
Human Plasma ◽  
Clinical Studies ◽  
Systemic Effects ◽  
In Vitro Experiments ◽  
Normal Human Plasma ◽  
Normal Human ◽  
Repeated Doses ◽  
In Vitro Effects ◽  
Activator Complex

SummaryThrombolysis with acylated streptokinase-plasminogen complexes is aimed to achieve fibrinolysis without systemic fibrinogenolysis. The p-aminobenzoyl-streptokinase-(Lys)-plasminogen-complex (BRL 33 575) should be particularly useful due to its slow deacylation rate. Unexpectedly, repeated doses of 10 mg of BRL 33 575 (corresponding to 310'000 streptokinase equivalent units) induced systemic effects in patients though less than streptokinase alone. In vitro incubation of normal human plasma with BRL 33 575 at concentrations used in patients resulted in nearly complete consumption of α2-antiplasmin and plasminogen and significant fibrinogenolysis within 3 hr. This demonstrates that - despite of slow deacylation of BRL 33 575 - the small amounts of activator generated are highly efficacious in activating plasma plasminogen under conditions in which no physiological clearance of the free activator takes place. Simulating the calculated activator release from BRL 33 575 by infusing equivalent amounts of streptokinase into plasma resulted in less pronounced effects. This is probably explained by anti-streptokinase antibodies which will neutralize the initially infused streptokinase but will be bound by BRL 33 575.Our in vitro experiments indicate that further clinical studies should be done with lower doses of BRL 33 575 or prolonged dosage intervals.

Role of fibrinolysis and α2-macroglobulin in growth of primary adenocarcinoma and rectal polyps.

2017 ◽  
Vol 35 (15_suppl) ◽  
pp. e15093-e15093
Author(s):  
Evgeniy N. Kolesnikov ◽  
Elena Alekseevna Nikipelova ◽  
Elena Mikhaylovna Frantsiyants ◽  
Larisa Kozlova ◽  
Irina V. Kaplieva ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Growth Factors ◽  
Protective Effect ◽  
Tumor Angiogenesis ◽  
Active Form ◽  
Primary Adenocarcinoma ◽  
Α2 Macroglobulin ◽  
Rectal Polyps ◽  
Resection Line

e15093 Background: An important role in tumor angiogenesis induction is played by hydrolytic systems, in particular plasmin one which provides degradation of the extracellular matrix, activates growth factors and metalloproteinases. Our purpose was to analyze the tissue fibrinolytic system and α2-macroglobulin (α2M) in primary adenocarcinoma (PA) and polyps (P) of the rectum (R). Methods: Tissues of tumors (РА, G2, T2-3N0M0, 42-73 years) and their perifocal zone (PZ) in R (n = 24) and Р (n = 27) were studied by ELISA; the result was calculated per 1 mg of tissue. Results: Resection line (RL) tissues contained levels of tРА antigen and its active form (tРА-Ag and tРА-act) 6.7 and 33.8 times higher than prourokinase and urokinase levels (uPA-Ag and uPA-act). RL tissues in P did not differ significantly from R tissues at PA resection. uPA-act in PA was higher than in RL by 3.2 times, uPA-Ag - 8 times higher, tРА-act - 2.3 times lower; tPA-Ag was similar to RL tissue values. Plasmin-α2-antiplasmin complex (PAP) in PA was 1.4 times higher than in RL. Plasminogen (PG) in PA was 1.5 times lower than in RL (p < 0.01). Activity of α2M in PA and RL did not differ. PAP and α2М in PA PZ were similar to RL, and PG did not differ from PA. Other indices were between tumor and RL levels. P tissue did not show changes in uPA; tPA-act activity was 1.3 times lower than in RL, and tPA-Ag 1.3 times higher (p < 0.01). PAP in tissues of 88.2% P was 1.5 times higher than in RL and 1.7 times lower than in PA. PG and α2М were similar in P and RL. PAP in PZ of P was 2.3 times higher than in RL and 1.5 times higher than in PA PZ. PG in P PZ was 1.4 times lower than in RL (p < 0.01). α2М activity in P PZ was higher than in RL and P by 5.4 times on average. uPA in P PZ did not differ from RL; tРА-Ag and tРА-act were higher than in RL by 1.5 and 1.9 times and exceeded P by 2.1 and 1.5 times (p < 0.01). Prevalence of uPA, activation of PG in PA and its PZ indicated degradation of the extracellular matrix compared with the corresponding P tissues. Conclusions: The role of R tumor PZ as a "metabolic" tumor field in neoplasm progression was confirmed. Activation of fibrinolysis in PA and its PZ with a deficiency of inhibitors stimulates the migration and proliferation of cells. Increasing tРА, РАР and α2М in PZ of polyps has a protective effect.

Factors affecting the stability of tPA and PAI-1 during storage and handling of human plasma for in vitro studies

1990 ◽  
Vol 1 (5) ◽  
pp. 393-400 ◽  
Author(s):  
F. Hamon ◽  
C. Masson-Lunven ◽  
B. Boutiere ◽  
C. Boyer-Neumann ◽  
M. J. Larri??u ◽  
...  
Keyword(s):  
Human Plasma ◽  
In Vitro Studies ◽  
Factors Affecting ◽  
The Stability ◽  
Pai 1
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