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.

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.

Demonstration of a Functionally Active tPA-Like Plasminogen Activator in Human Platelets

1994 ◽  
Vol 71 (04) ◽  
pp. 493-498 ◽  
Author(s):  
D L Wang ◽  
Y T Pan ◽  
J J Wang ◽  
C H Cheng ◽  
C Y Liu
Keyword(s):  
Plasminogen Activator ◽  
Fibrinolytic Activity ◽  
Protein Band ◽  
Human Platelets ◽  
Free Form ◽  
Plasminogen Activation ◽  
Single Chain ◽  
Western Blots ◽  
A Minor ◽  
Pai 1

SummaryThe mechanism of platelet-enhanced fibrinolysis is unclear. We therefore investigated the fibrinolytic activity of human platelets and demonstrated that they contain a tissue plasminogen activator (tPA)- like plasminogen activator, abbreviated as tPA-like-PA. This activator was detected by ELISA in platelet incubation medium and in platelet Triton extracts. Plasminogen activation assays showed that this tPA- like-PA could induce plasminogen activation to form plasmin. Western blots of Triton extracts incubated with anti-tPA antibody demonstrated a major 64-kD protein band, compared to a 70-kD band for standard single chain tPA, plus a minor 118-kD band corresponding to a complex of tPA-like-PA and plasminogen activator inhibitor (PAI-1). Western blots of Triton extracts incubated with anti-PAI-1 antibody produced an approximately similar high-molecular-weight (118 kD) protein band. Fibrin zymographic analysis of affinity-purified tPA-like- PA demonstrated a major and a minor fibrin lysis zone, which approximately corresponded to the tPA-like-PA and its complex with PAI-1 observed by Western blots. Immunogold labelling and electron microscopy demonstrated that platelet activator, either as the free form or co-localized with PAI-1, was present in granules and in channels of the open canalicular system. We conclude that platelets contain a functionally active tPA-like-PA, whose low fibrinolytic activity might be due to its readily forming a complex with PAI-1. This functionally active tPA-like-PA might contribute to the enhanced fibrinolytic activity of platelets observed in platelet-rich thrombi.

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.

scholarly journals A Refined Kinetic Analysis of Plasminogen Activation by Recombinant Bovine Tissue-Type Plasminogen Activator Indicates Two Interconvertible Activator Forms†

Biochemistry ◽  
1998 ◽  
Vol 37 (36) ◽  
pp. 12631-12639 ◽  
Author(s):  
Laust B. Johnsen ◽  
Peter Ravn ◽  
Lars Berglund ◽  
Torben E. Petersen ◽  
Lone K. Rasmussen ◽  
...  
Keyword(s):  
Kinetic Analysis ◽  
Plasminogen Activator ◽  
Tissue Type ◽  
Plasminogen Activation ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator ◽  
Bovine Tissue

Effect Of Neuraminidase On Plasmin Formation By Urokinase And The Vascular Plasminogen Activator

1981 ◽  
Author(s):  
M Geiger ◽  
B R Binder
Keyword(s):  
Sialic Acid ◽  
Kinetic Analysis ◽  
Plasminogen Activator ◽  
Human Urine ◽  
Test System ◽  
Time Dependent ◽  
Plasminogen Activation ◽  
Substrate Molecule ◽  
Neuraminidase Treatment ◽  
Enhancing Effect

It was the aim of the present study to determine whether sialic acid residues in the activator or substrate molecule play a role in the activation of plaminogen to plasmin by the vascular plasminogen activator (VPA) and urokinase (UK).Using a purified test system with UK (Mr=31.000, human urine), VPA (Mr=70.000, cadaver vessel eluates) as activators, Glu-plasminogen as substrate, and pretreatment with neuraminidase (C.perfringens,1.51U/mg) it could be shown by quantification of the formed plasmin with S-2251 as substrate that neuraminidase treatment enhances plasmin formation by UK as well as by VPA. Using Sepharose-bound neuraminidase (C.perfringens, 20-30U/g Sepharose) it could be shown that neuraminidase affects the substrate in a dose and time dependent fashion, while the activity of the activators seems to remain unchanged. Treatment of plasminogen with neuraminidase in a concentration of 2.5 mU per μmole plasminogen for 90 min. at 37°C increased the plasmin formation by VPA by about 50% which was the same as for UK.Kinetic analysis of plasmin formation from neuraminidase treated plasminogen by UK and VPA suggests increased binding of the substrate to the activators (unchanged Vmax and decreased KM). The enhancing effect of fibrin on plasmin formation by VPA seems to remain unaltered by neuraminidase treatment of the plasminogen.These data suggest that, as in the case of other protease substrates, removal of sialic acid residues results in increased cleavage of the substrate, i.e. enhanced plasminogen activation, regardless of the type of activator.

Binding Of Thrombin By Plasma Inhibitors

1981 ◽  
Author(s):  
J A Penner ◽  
H I Hassouna
Keyword(s):  
Experimental Approach ◽  
Specific Activity ◽  
Binding Capacity ◽  
Gel Filtration ◽  
Minor Role ◽  
Clotting System ◽  
Chloramine T ◽  
Presence And Absence ◽  
A Minor

Our study was undertaken with the purpose of clarifying the role of plasma inhibitors other than antithrombin III (ATIII) in binding thrombin in the presence and absence of heparin. In the clotting system, several antiproteinases are involved in neutralizing active factors. The capacity to destroy thrombin is attributed to ATIII whose concentration in plasma is ten times less than α1 antitrypsin (α1A) . If heparin is added to plasma, ATIII becomes an immediate inhibitor of thrombin. Several studies have indicated that a modest depression in ATIII activity leads to thrombosis, inferring that other plasma antiproteinases play a minor role. Experimental approach was as follows: Defibrinated platelet poor citrated human plasma was depleted of ATIII and α1 by means of monospecific insolubilized antibodies. Purified thrombin, labelled by Chloramine T incorporation of 125I in NaOH, had a specific activity of 1.3 × 105 CPM/μg. Total antiproteinase thrombin binding capacity of defibrinated plasma was estimated by incubating 4 × 10-33 μM thrombin with plasma (100 μl) for 2 hours and 24 hours in the absence of heparin and for 5 minutes in the presence of heparin (20 units). Free from bound *thrombin was separated by gel filtration on Sephacryl S200 and chromatograms monitored on gamma analyzer. Following the same protocol, ATIII deficient plasma was used to evaluate thrombin binding capacity not attributable to ATIII, and role of ATIII in the absence of α1A was assessed in α1A depleted plasma. As a result, ATIII binding capacity was found to be 62 μg *thrombin/100 μl of normal and α1A deficient plasma, in the presence and absence of heparin, eluting at a volume corresponding to 9 × 104 daltons. Binding capacity of other plasma inhibitors in ATIII deficient plasma amounted to 24 μg *thrombin in the absence of heparin and 7 μg in its presence. This confirms the role of ATIII as the major antithrombin, heparin cofactor. In its absence inactivation of thrombin is reduced by 61%.

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