Generation Of Factor XII-Dependent Plasminogen Activator Activity In Human Plasma Measured With A Fluorogenic Substrate

1981 ◽  
Author(s):  
G Dooijewaard ◽  
C Kluft
Keyword(s):  
Human Plasma ◽  
Plasminogen Activator ◽  
Minor Role ◽  
Factor Xii ◽  
Dextran Sulphate ◽  
Fluorogenic Substrate ◽  
A Value ◽  
Substrate Activation ◽  
Factor Xiia ◽  
A Minor

A rapid fluorometric assay for measurement of amidolytic activity in human plasma was developed, using the plasminogen activator sensitive synthetic substrate t-BOC-L-valyl--glycyl-L-arginine-β-naphthylamide. The plasma is diluted in a reaction cuvet containing 0.050 M Tris HC1 buffer (pH 8.0) and 150 μM substrate. Activation of plasminogen proactivator(s) is initiated at 37°C by the addition of 10 μg dextran sulphate (MW 500,000)/ml. The concentration of β-naphthyl- amide released is recorded fluorometrically as a function of time. The slope of this curve at any time t is proportional to the concentration of activator. Thus, in a single assay, the entire time-dependent profile of activation and subsequent inhibition is monitored; this provides 1. a value for an optimum plasminogen activator content in the plasma, and 2. the time it takes to reach the optimum. The plot of optimum activator content against μl of plasma added is linear for dilutions more than 100-fold, suggesting that under these conditions the optimum content approaches the content of proactivator(s) originally present.The activator content measured predominantly consists of contributions of a factor XII-dependent process since 1. without dextran sulphate or with plasmas deficient in factor XII or prekallikrein no activity could be generated, and 2. plots of optimum activator content against dextran sulphate concentration show sigmoidal-shaped saturation curves as found previously for the kallikrein generation in human plasma. Contributions of factor XIIa and kallikrein only partly account for the content measured and studies with plasmas deficient in factor XI point to a minor role for this factor, if any. Further identification of the activator (s) involved is in progress.

scholarly journals Human Prekallikrein (Plasminogen Proactivator): Purification, Characterization and Activation by Activated Factor XII

1977 ◽  
Author(s):  
Bonno N. Bouma ◽  
John H. Griffin
Keyword(s):  
Human Plasma ◽  
Plasminogen Activator ◽  
Clot Lysis ◽  
Factor Xii ◽  
Globulin Fraction ◽  
Factor Xi ◽  
Form Analysis ◽  
Plasminogen Activator Activity ◽  
Fibrin Plate ◽  
Factor Xiia

In order to resolve conflicting reports about the possible identity of prekallikrein and Factor XII-dependent plasminogen proactivator (FXII-PPA), the γ-globulin fractions of prekallikrein-deficient (Fletcher trait) and of normal plasma were assayed for FXII-PPA. Based on both fibrin plate and clot lysis tests, FXII-PPA in the γ-globulin fractions of prekallikrein-deficient plasmas from 2 unrelated patients was undetectable, i.e. <1% of the FXII-PPA in the normal γ-globulin fraction. However, PPA independent of FXII was detected in both the Fletcher and the normal γ-globulin fractions at 4% of the FXII-PPA present in the normal γ-globulin fraction.Human plasma prekallikrein was purified 2,000-fold (specific clotting activity 22 units/mg) and was greater than 95% homogeneous on SDS-gels. FXII-PPA was always copurified with prekallikrein and was totally separated from Factor XI. No Factor XII-dependent or Factor XII-independent plasminogen activator activity was detected in purified Factor XI preparations at 40 units/ml. Purified prekallikrein in its precursor form gave 2 protein bands on SDS-gels at 82,000 and 78,000 MW. Upon reduction, a single 85,000 MW band was observed. Kallikrein and plasminogen activator activity were generated upon incubation with purified human Factor XIIa (28,000 MW form). Analysis of this reaction mixture on SDS-gels without reduction showed 2 bands with apparently identical MW’s as the precursor protein bands, whereas reduction showed cleavage of both protein bands.These results suggest that prekallikrein is identical to FXII-PPA in normal human plasma and that activation of this zymogen by Factor XIIa involves limited proteolytic cleavage.

Factor XII and Prekallikrein—Kallikrein—Kinin in the Cryoactivation of Human Plasma Prorenin

1984 ◽  
Vol 66 (5) ◽  
pp. 533-539 ◽  
Author(s):  
C. F. Brown ◽  
D. H. Osmond
Keyword(s):  
Plasma Renin Activity ◽  
Human Plasma ◽  
Plasma Renin ◽  
Determine Factor ◽  
Renin Activity ◽  
Factor Xii ◽  
Dextran Sulphate ◽  
Rat Uterus

1. Cryoactivation of human plasma ‘prorenin’ was followed for 24 h at −4°C. Chromogenic assays were used to determine factor XII (FXII), FXIIa, prekallikrein and kallikrein in relation to the observed cold-induced increase in plasma renin activity (PRA). Bradykinin activity was also determined using the rat uterus bioassay. 2. PRA increased rapidly and became significantly higher after just 6 h of cryoactivation, by which time prekallikrein had almost disappeared, while kallikrein and kinin levels increased. In contrast, FXII did not change notably, but some FXIIa was indeed formed. 3. The bacteriostat neomycin sulphate did not affect the course of cryoactivation, but did block the dextran sulphate- and kaolin-induced activation of prekallikrein and FXII respectively, and was therefore omitted. 4. Thus cryoactivation of prorenin is accompanied by, and may depend upon, the activation of FXII and prekallikrein, supporting other evidence in favour of this hypothesis.

Ethical perspectives on health technology assessment

2004 ◽  
Vol 20 (1) ◽  
pp. 71-76 ◽  
Author(s):  
Henk ten Have
Keyword(s):  
Health Technology Assessment ◽  
Technology Assessment ◽  
Medical Technology ◽  
Health Technology ◽  
Minor Role ◽  
Engineering Model ◽  
Ethical Aspects ◽  
A Value ◽  
Definition Of ◽  
A Minor

This study analyses why ethical aspects play a minor role in health technology assessment (HTA) studies, even when comprehensive approaches of technology assessment are advocated. Technology is often regarded as a value-neutral tool. At the same time, bioethics is dominated by an engineering model. Ethical contributions to evaluation of medical technology should go beyond issues of application in clinical practice and focus also on the definition of problems, the demarcation of technical and nontechnical issues, and the morally problematic implications of technologies.

The Contact-System Dependent Plasminogen Activator from Human Plasma: Identification and Characterization

1990 ◽  
Vol 64 (03) ◽  
pp. 390-397 ◽  
Author(s):  
D J Binnema ◽  
G Dooijewaard ◽  
J J L van Iersel ◽  
P N C Turion ◽  
C Kluft
Keyword(s):  
Human Plasma ◽  
Plasminogen Activator ◽  
Physiological Role ◽  
Contact System ◽  
Tissue Type ◽  
Antigenic Determinants ◽  
Dextran Sulphate ◽  
Single Chain ◽  
Native Form ◽  
Type Plasminogen Activator

SummaryApart from tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA), a third PA appears to occur in human plasma. Its activity is initiated when appropriate triggers of the contact system are added, and the activation depends on the presence of factor III and prekallikrein in plasma. The activity of this, so-called, contact-system dependent PA accounts for 30% of the PA activity in the dextran sulphate euglobulin fraction of plasma and was shown not to be an intrinsic property of one of the contact-system components, nor could it be inhibited by inhibitory antibodies against t-PA or u-PA. We have succeeded in identifying this third PA in dextran sulphate euglobulin fractions of human plasma. Its smallest unit (SDSPAGE) is an inactive 110 kDa single-chain polypeptide which upon activation of the contact system is converted to a cleaved, disulphide-bridged molecule with PA activity. The native form, presumably, is an oligomer, since the apparent M. on gelchromatography is 600,000. The IEP is 4.8, much lower than that of t-PA and u-PA. Although the active 110 kDa polypeptide cannot be inhibited by anti-u-PA, it yet comprises a 37 kDa piece with some u-PA related antigenic determinants. However, these determinants are in a latent or cryptic form, only detectable after denaturation by SDS. The 110 kDa polypeptide is evidently not a dimer of 55 kDa u-PA or a complex of u-PA with an inhibitor. It is probably a PA derived from a gene quite distinct from that of t-PA or u-PA, but sharing some homology with u-PA. The physiological role of this contact-system dependent PA remains to be established

Factor XII-Dependent Fibrinolysis: A Double Function of Plasma Kallikrein and the Occurrence of a Previously Undescribed Factor XII- and Kallikrein-Dependent Plasminogen Proactivator

1979 ◽  
Vol 41 (04) ◽  
pp. 756-773 ◽  
Author(s):  
C Kluft ◽  
M M Trumpi-Kalshoven ◽  
A F H Jie ◽  
E C Veldhuyzen-Stolk
Keyword(s):  
Plasminogen Activator ◽  
Low Molecular Weight ◽  
Plasma Kallikrein ◽  
Factor Xii ◽  
Normal Plasma ◽  
Double Function ◽  
Plasma Fractions ◽  
A Minor ◽  
Molecular Weight Form ◽  
Minor Activity

SummaryFibrinolytic studies in euglobulin fractions of Fletcher trait plasma (deficient in prekallikrein) revealed reduced activities as compared to normal plasma. A quantitative assay for total plasminogen activator plus proactivator in plasma showed that the amount in Fletcher trait patients is about half of normal (normal = ± 100 blood activator units [BAU]/ ml). Plasma kallikrein partially purified in a high and low molecular weight form exerted plasminogen activator activity amounting to 10–15 BAU/ml plasma. So, the absence of kallikrein in the deficient plasma can not fully account for the reduction in activator activity. Additions of kallikrein preparations or normal plasma fractions resulted in additional activator activity in Fletcher trait plasma which was assessed at 30–40 BAU/ml. This activity was assumed to originate from a previously undescribed plasminogen proactivator whose activation is kallikrein- and factor XH-dependent.Fractionation experiments demonstrated the presence of two major activities and a minor activity caused by kallikrein in normal plasma.It is concluded that plasma kallikrein has two functions in the generation of factor XII- dependent fibrinolytic activity: one as a direct plasminogen activator and another as a factor in the activation of a major factor XH-dependent plasminogen proactivator.

Role of α2-Antiplasmin in Fibrin-Specific Clot Lysis with Single-Chain Urokinase-Type Plasminogen Activator in Human Plasma

1991 ◽  
Vol 65 (04) ◽  
pp. 394-398 ◽  
Author(s):  
P J Declerck ◽  
H R Lijnen ◽  
M Verstreken ◽  
D Collen
Keyword(s):  
Human Plasma ◽  
Plasminogen Activator ◽  
Clot Lysis ◽  
Minor Role ◽  
Single Chain ◽  
Plasma Clot ◽  
Normal Plasma ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator

SummaryThe role of plasma α2-antiplasmin (α2-AP) in the fibrinspecificity of clot lysis by recombinant single-chain urokinase-type plasminogen activator (rscu-PA) and in the conversion of rscu-PA to its two-chain derivative (rtcu-PA, urokinase) was investigated in an in vitro human plasma clot lysis system. Fifty % lysis in 2 h of a 0.1 ml 125l-fibrin labeled human plasma clot immersed in 0.5 ml normal human plasma was obtained with 1.4 ± 0.15 µg/ml rscu-PA (mean ± SD, n = 8). This was associated with degradation of 23 ± 7% of fibrinogen and generation of 0.20 ± 0.09 µg/ml rtcu-PA. In α2-AP-depleted plasrna 50% clot lysis in 2 h required 2-fold less rscu-PA which was associated with 3-fold more extensive fibrinogen degradation and 2-fold more rtcu-PA generation. Fifty % lysis in? h, of a 0.1 ml α2-AP-depleted plasma clot, subriersed in 0.5 ml normal plasma, was obtained with 0.80 ± 0.05 µg/ml rscu-PA (n = 3, p <0.001 vs normal clot) and was associated with 17 ± 6% fibrinogen breakdown (p : 0.22 vs normal clot) and 0.08 ± 0.02 µg/ml rtcu-PA generation (p < 0.05 vs normal clot). In α2-AP-depleted plasma the equipotent rscu-PA concentration was 4-fold lower than in normal plasma and was associated with 3-fold more fibrinogen degradation and a similar extent of rtcu-PA generationThus, α2-AP in plasma contributes significantly to the fibrinspecificity of rscu-PA, primarily via prevention of conversion in plasma of rscu-PA to rtcu-PA. Clot associated α2-AP increases the resistance of the clot to lysis with rscu-PA, but plays an only minor role in the fibrin-specificity of clot lysis in normal plasma.

Assay of Fletcher Factor (Plasma Prekallikrein) Using an Artificial Clotting Reagent and a Modified Chromogenic Assay

1979 ◽  
Vol 42 (02) ◽  
pp. 538-547 ◽  
Author(s):  
Jean-Pierre Soulier ◽  
Danielle Gozin
Keyword(s):  
Human Plasma ◽  
Plasma Kallikrein ◽  
Factor Xii ◽  
Dextran Sulphate ◽  
Rabbit Plasma ◽  
Enzymatic Assays ◽  
Chromogenic Assay ◽  
Fletcher Factor

SummaryAn artificial clotting reagent lacking in Fletcher factor (plasma prekallikrein, PPK) was made by mixing human plasma, activated by 5 mg/ml of celite, then kept 16 hours at 37° to destroy most of the plasma kallikrein, plus rabbit plasma (which is devoid of XII and Fletcher activity).Chromogenic assay using a tripeptide substrate was also modified to exclude the interference of the endogenous contact factors. Celite eluate was used instead of kaolin or dextran sulphate for the activation.Using both these methods, it is possible to distinguish between Fletcher trait (PPK deficiency) and other contact factors such as factor XII and HMWK deficiencies, which do not activate with kaolin or dextran sulphate. These simple clotting and enzymatic assays give specific and well correlated results for PPK estimation.

scholarly journals The contact activation mechanism in human plasma: activation induced by dextran sulfate

Blood ◽  
1982 ◽  
Vol 59 (6) ◽  
pp. 1225-1233 ◽  
Author(s):  
F van der Graaf ◽  
FJ Keus ◽  
RA Vlooswijk ◽  
BN Bouma
Keyword(s):  
Human Plasma ◽  
Dextran Sulfate ◽  
Activation Mechanism ◽  
Factor Xii ◽  
Contact Activation ◽  
Prolonged Incubation ◽  
Normal Plasma ◽  
Essential Components ◽  
Factor Xiia ◽  
Kallikrein Activity

Abstract Incubation of normal human plasma with dextran sulfate for 7 min at 4 degrees C generates kallikrein amidolytic activity. No kallikrein activity is generated in factor XII or prekallikrein-deficient plasma and only small amounts (8%) in high molecular weight (HMW) kininogen- deficient plasma. Addition of specific antisera directed against prekallikrein or HMW kininogen to normal plasma blocked the generation of kallikrein activity by dextran sulfate. Thus, factor XII, prekallikrein, and HMW kininogen are essential components for optimal activation of prekallikrein. The role of limited proteolysis in the activation of prekallikrein induced by dextran sulfate was studied by adding 125I-prekallikrein to plasma. The generation of kallikrein activity paralleled the proteolytic cleavage of prekallikrein as judged on SDS gels in the presence of reducing agents. The same cleavage fragments were observed as obtained by activation of purified prekallikrein by beta-factor-XIIa. Addition of 131I-HMW kininogen and 125I-factor XII or 131I-HMW kininogen and 125I-prekallikrein to normal plasma followed by activation with dextran sulfate and analysis on SDS gels indicated that the observed cleavage of prekallikrein and HMW kininogen is fast compared to the observed cleavage of factor XII, which is much slower and less extensive. During the first minutes of incubation of normal plasma with dextran sulfate, mainly alpha-factor- XIIa is formed. During prolonged incubation, beta-factor-XIIa is also formed.

Experimental Acute Hepatic Encephalopathy: Comparison of the Electroencephalographic Changes in the Liverless and in the Eviscerated Rat

1974 ◽  
Vol 47 (6) ◽  
pp. 599-608 ◽  
Author(s):  
Francine Degos ◽  
J.-D. Degos ◽  
Denise Bourdiau ◽  
Michèle Peignoux ◽  
Danièle Prandi ◽  
...  
Keyword(s):  
Hepatic Encephalopathy ◽  
Vena Cava ◽  
Portacaval Shunt ◽  
Ammonia Concentration ◽  
Minor Role ◽  
Plasma Ammonia ◽  
A Value ◽  
Slow Sleep ◽  
The Mean ◽  
A Minor

1. The present work was carried out to compare the electroencephalographic changes in liverless rats and eviscerated rats and to determine whether substances released from the intestine, in particular ammonia, play a major part in the mechanism of hepatic encephalopathy. 2. The animals were prepared according to a three-stage procedure: ligation of inferior vena cava; 3 weeks later, end-to-side portacaval shunt; 2 days later, removal of the liver (liverless rats) or removal of the liver, spleen, stomach, intestine and pancreas (eviscerated rats). 3. In liverless rats, the electroencephalographic changes began 4–8 h after hepatectomy with a predominance of ‘slow’ sleep pattern followed by increasing changes, which consisted successively of (a) alteration of, then disappearance of, spindles of high-voltage waves; (b) predominance of slow waves; (c) depression in voltage and finally flat tracing. The mean duration of survival was 18.4 h. Mean plasma ammonia concentration 15 h after hepatectomy was 353 μmol/l. 4. In eviscerated rats, the electroencephalographic changes were similar. The mean duration of survival was 21.3 h, which is not statistically different from that of liverless rats. Mean plasma ammonia concentration 15 h after evisceration was 148 μmol/l, a value significantly lower than that of liverless rats. 5. These results suggest that ammonia, and substances released from the intestine in general, play no part or at most a minor role in the mechanism of hepatic encephalopathy of the liverless rat.

An Analysis of the Activators of Single-Chain Urokinase-Type Plasminogen Activator (scu-PA) in the Dextran Sulphate Euglobulin Fraction of Normal Plasma and of Plasmas Deficient in Factor XII and Prekallikrein

1991 ◽  
Vol 65 (02) ◽  
pp. 144-148 ◽  
Author(s):  
D J Binnema ◽  
G Dooijewaard ◽  
P N C Turion
Keyword(s):  
Plasminogen Activator ◽  
Gel Chromatography ◽  
Factor Xii ◽  
Dextran Sulphate ◽  
Contact Activation ◽  
Single Chain ◽  
Activity Peak ◽  
Normal Plasma ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator

SummaryAn analysis was made of the various possible activators of single-chain urokinase-type plasminogen activator (scu-PA) in the dextran sulphate euglobulin fraction (DEF) of human plasma. scu-PA activators were detected in an assay system in which the substrate scu-PA, in physiological concentration (50 pM), was immuno-immobilized. After activation of the immobilized scu-PA for a certain period of time the activity of the generated amount of immuno-immobilized two-chain u-PA was determined with plasminogen and the chromogenic substrate S-2251. The scu-PA activator activity (scuPA-AA) in the DEF of plasmas deficient in factor XII or prekallikrein was about half of that in the DEF of normal plasma. Separation of scuPA-AA in the DEF by gel chromatography showed to major peaks, one eluting with an apparent Mr of 500,000 and the other around Mr 100,000. The former peak, which coincided with the activity peak of the kallikrein-kininogen complex, was absent in the DEF of plasma depleted of prekallikrein and therefore was identified as kalli-krein. The latter peak was still present in the depleted plasma and most likely represents plasmin, because its scuPA-AA coincided with the activity peak of plasmin and could be fully inhibited by antibodies raised against human plasminogen. It is concluded that plasmin and the contact-activation factor kallikrein each contribute for about 50% to the scuPA-AA in the DEF. Compared on a molar basis, however, plasmin was found to be almost 1,000 times more effective than kallikrein, and we conclude, therefore, that in vivo plasmin is the primary activator of scu-PA and the role of the contact system is of secondary importance.

Sign in / Sign up

Export Citation Format

Share Document