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.

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 Plasminogen activators in dextran sulfate-activated euglobulin fractions: a molecular analysis of factor XII- and prekallikrein- dependent fibrinolysis

Blood ◽  
1989 ◽  
Vol 73 (4) ◽  
pp. 994-999
Author(s):  
J Hauert ◽  
G Nicoloso ◽  
WD Schleuning ◽  
F Bachmann ◽  
M Schapira
Keyword(s):  
Plasminogen Activator ◽  
Fibrinolytic Activity ◽  
Dextran Sulfate ◽  
Plasminogen Activators ◽  
Contact System ◽  
Tissue Type ◽  
Plasma Kallikrein ◽  
Factor Xii ◽  
Single Chain ◽  
Type Plasminogen Activator

To elucidate the mechanism by which activation of the contact system of blood coagulation leads to expression of fibrinolytic activity, we have determined the molecular characteristics of the plasminogen activators present in dextran sulfate-treated euglobulin fractions by electrophoretic-zymographic analysis and specific immunoadsorption. In addition to free and protease inhibitor-bound tissue-type plasminogen activator (t-PA), dextran sulfate precipitates of euglobulins contained the complex formed between plasma kallikrein and C1-inhibitor, an indicator of prekallikrein activation. These precipitates also contained substantial fibrinolytic activity related to urinary-type plasminogen activator (u-PA). Autoradiographic analysis was then used to evaluate the cleavage of 125I-single-chain u-PA (prourokinase) in dextran sulfate euglobulins as well as after exposure to kallikrein or beta-factor XIIa. This analysis supported the conclusion that plasma kallikrein-mediated cleavage and activation of single-chain u-PA is the mechanism operative for the development of lytic activity in euglobulin precipitates following activation of the contact system.

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.

Plasminogen Activator (PA) Activity Of Urokinase (UK) , The Vascular Plasminogen Activator (VPA) , Plasma Kallikrein (PK) , And Hageman Factor (HF) In The Presence And Absence Of Fibrin

1981 ◽  
Author(s):  
B R Binder ◽  
R Beckmann ◽  
M Jörg
Keyword(s):  
Kinetic Analysis ◽  
Plasminogen Activator ◽  
Active Sites ◽  
Minor Role ◽  
Plasma Kallikrein ◽  
Plasminogen Activation ◽  
Apparent Homogeneity ◽  
Presence And Absence ◽  
A Minor ◽  
Substrate Concentrations

It could be shown previously by us and others that the PA activity of purified VPA is increased in the presence of fibrin, while the activity of UK remains unchanged. However, there had been some discussion on the mechanisms involved and the relative potency of the different PAs in man. In the present study kinetic analysis of plasminogen activation with different PAs was performed in the presence and absence of fibrin.Human UK (urine, Mr=56.000) , VPA (cadaver vessel eluates) , PK (HF-fragments activated) were purified to apparent homogeneity, and active sites were determined by 3H-DFP incorporation. Plasminogen activation was evaluated by incubation of different amounts of PAs with Glu-plasminogen at different substrate concentrations with and without fibrin (fibrinogen- Sepharose treated with thrombin) at 37°C for 3 to 120 minutes; the plasmin generated was measured by a synthetic para- nitroanilide substrate.Under these conditions VPA exhibited an apparent KM≃0.5 μM and a kcat≃0.15 s-1. In the presence of fibrin KM decreased significantely (<<0.01 μM) while kcat remained unchanged; untreated fibrinogen-Sepharose had no effect. UK, on the other hand, showed an apparent KM≃1 μM and a kcat≃0.5 s-1 which were both unaffected by fibrin. PK cleaved plasminogen with an apparent KM≃0.2 μM and a kcat≃0.004 s-1 regardless of the presence of fibrin. Preliminary data using intact HF together with kaolin and phospholipids revealed an apparent KM for plasminogen activation of that mixture of 3.5 μM and a kcat≃0.07 s-1 which were again unaffected by fibrin.From these data it can be concluded that in the absence of fibrin UK is a more effective PA than the others, while in the presence of fibrin VPA is by fare the most effective one. From kinetic analysis PK and HF should play only a minor role in plasminogen activation.

scholarly journals Plasminogen activators in dextran sulfate-activated euglobulin fractions: a molecular analysis of factor XII- and prekallikrein- dependent fibrinolysis

Blood ◽  
1989 ◽  
Vol 73 (4) ◽  
pp. 994-999 ◽  
Author(s):  
J Hauert ◽  
G Nicoloso ◽  
WD Schleuning ◽  
F Bachmann ◽  
M Schapira
Keyword(s):  
Plasminogen Activator ◽  
Fibrinolytic Activity ◽  
Dextran Sulfate ◽  
Plasminogen Activators ◽  
Contact System ◽  
Tissue Type ◽  
Plasma Kallikrein ◽  
Factor Xii ◽  
Single Chain ◽  
Type Plasminogen Activator

Abstract To elucidate the mechanism by which activation of the contact system of blood coagulation leads to expression of fibrinolytic activity, we have determined the molecular characteristics of the plasminogen activators present in dextran sulfate-treated euglobulin fractions by electrophoretic-zymographic analysis and specific immunoadsorption. In addition to free and protease inhibitor-bound tissue-type plasminogen activator (t-PA), dextran sulfate precipitates of euglobulins contained the complex formed between plasma kallikrein and C1-inhibitor, an indicator of prekallikrein activation. These precipitates also contained substantial fibrinolytic activity related to urinary-type plasminogen activator (u-PA). Autoradiographic analysis was then used to evaluate the cleavage of 125I-single-chain u-PA (prourokinase) in dextran sulfate euglobulins as well as after exposure to kallikrein or beta-factor XIIa. This analysis supported the conclusion that plasma kallikrein-mediated cleavage and activation of single-chain u-PA is the mechanism operative for the development of lytic activity in euglobulin precipitates following activation of the contact system.

Cold Promoted Activation of Factor VII

1972 ◽  
Vol 28 (02) ◽  
pp. 169-181 ◽  
Author(s):  
H Gjønnæss
Keyword(s):  
Low Temperature ◽  
Oral Contraceptives ◽  
Fold Increase ◽  
Factor Vii ◽  
Plasma Kallikrein ◽  
Factor Xii ◽  
Overnight Incubation ◽  
Esterolytic Activity

SummaryThe activating principle (CPA) of the factor VII activation seen in plasmas of women taking oral contraceptives after overnight incubation of the plasmas at 0° C was investigated. The reaction was dependent on low temperature, and factor XII was indispensable. Concomitant with the activation of factor VII a 10–30 fold increase in TAME esterolytic activity was observed together with a near 100 per cent drop in plasma kininogen concentration. The results indicated that the activation of factor VII is linked to activation of the kallikrein system, and that the activator may be plasma kallikrein.

Properties of Chimeric (Tissue-Type/Urokinase-Type) Plasminogen Activators Obtained by Fusion at the Plasmin Cleavage Site

1993 ◽  
Vol 69 (05) ◽  
pp. 466-472 ◽  
Author(s):  
M Colucci ◽  
L G Cavallo ◽  
G Agnelli ◽  
A Mele ◽  
R Bürgi ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Plasminogen Activator ◽  
Cleavage Site ◽  
Plasminogen Activators ◽  
Tissue Type ◽  
Low Molecular Weight ◽  
Single Chain ◽  
Type Plasminogen Activator ◽  
Kringle 2 ◽  
Fibrin Monomers

SummaryTwo hybrid plasminogen activators (K2tu-PA and FK2tu-PA), linking the kringle 2 domain or the finger plus the kringle 2 domains of tissue-type plasminogen activator (t-PA) to the catalytic domain of single-chain urokinase-type plasminogen activator (scu-PA) were studied. At variance with similar constructs previously reported, they were obtained by fusion of the t-PA and scu-PA derived portions at their plasmin cleavage site (between Arg275 of t-PA and Ile159 of scu-PA), thus eliminating from scu-PA the two peptide bonds (Glu143-Leu144 and Arg156-Phe157) that lead to low molecular weight scu-PA and to thrombin-inactivated tcu-PA. The specific activities of K2tu-PA and FK2tu-PA, as measured by fibrin plate were 2.5 × 106 and 1.0 × 106 t-PA equivalent units/mg, respectively. Activation of plasminogen by hybrid PAs was stimulated by both CNBr-digested fibrinogen (40- and 80-fold) and Des-A-fibrin monomers (6- and 12-fold). The relatively weak stimulation of chimeric PAs by minimally degraded fibrin monomers was consistent with their reduced fibrin binding capacity. Like scu-PA, the chimeric PAs, in the single-chain form, were insensitive to inhibition, as they retained full activity after prolonged incubation in plasma and did not interact with SDS-reactivated recombinant PAI-1. The concentration producing 50% lysis of blood clots in 3 h was 0.5 μg/ml for K2tu-PA and 1 μg/ml for FK2tu-PA, as compared to 0.5 μg/ml and >2 μg/ml for t-PA and scu-PA, respectively. Plasminogen and α2-antiplasmin consumption induced by the hybrid PAs in clot-free plasma was comparable to (K2tu-PA) or lower than (FK2tu-PA) that induced by either t-PA or scu-PA. When exposed to plasmin, the hybrids were completely converted into two-chain molecules with full enzymatic activity. At variance with u-PA, however, the two-chain recombinant activators still required fibrin for full expression of activity. These data indicate that the products of such “artificial” fusion behave like true chimeras without loss of biological activity. The insensitivity to thrombin inactivation and to the proteolytic cleavage leading to low molecular weight scu-PA might confer enhanced stability to the molecules, especially at thrombus level. Moreover, if the thrombolytic activity observed in vitro is maintained in vivo, the prolonged half life of these hybrids should result in higher plasma levels of activator and thus in more extensive and rapid lysis.

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