Binding Of Plasminogen And Vascular Plasminogen Activator To Fibrin And The Fibrin Alpha-Chain

1981 ◽  
Author(s):  
D A Lloyd ◽  
S A Cederholm-William ◽  
A A Sharp
Keyword(s):  
Plasminogen Activator ◽  
Binding Sites ◽  
Venous Occlusion ◽  
Plasminogen Activation ◽  
Catalytic Surface ◽  
Alpha Chain ◽  
Fibrin Monomer ◽  
Human Plasminogen ◽  
Early Degradation ◽  
Fibrin Structure

Investigation of the mechanism of plasminogen activation in the presence of fibrin has shown that fibrin acts as a regulatory and catalytic surface for both the reaction between activator and fibrin and between activator and plasminogen. Plasminogen (mol wt 84,000) and vascular activator form stable complexes with fibrin. We have carried out studies to determine the location of these binding sites in the fibrin structure.METHODS: Human plasminogen (mol wt 84,000) was iodinated with 125-I and vascular plasminogen activator was partially purified from venous occlusion plasma by chromatography on lysine-Sepharose. Sepharose coupled fibrinogen, fibrin monomer, fragments E and D and isolated fibrin alpha chain were prepared and the interactions between plasminogen and vascular activator studied.RESULTS: Plasminogen (mol wt 84,000), plasmin and vascular activator each showed affinity for Sepharosebound fibrin monomer and fibrin alpha chain. In addition the affinity of the vascular activator for fibrinogen and fragment E was increased by treatment with thrombin.CONCLUSION: The alpha chain of fibrin brings together plasminogen activator and plasminogen in such a way as to produce plasminogen activation resulting in the early degradation steps of fibrin.

Effect of Lipoprotein(a) and LDL on Plasminogen Binding to Extracellular Matrix and on Matrix-dependent Plasminogen Activation by Tissue Plasminogen Activator

1996 ◽  
Vol 75 (03) ◽  
pp. 497-502 ◽  
Author(s):  
Hadewijch L M Pekelharing ◽  
Henne A Kleinveld ◽  
Pieter F C.C.M Duif ◽  
Bonno N Bouma ◽  
Herman J M van Rijn
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Plasminogen Activator ◽  
Binding Sites ◽  
Umbilical Vein ◽  
Single Step ◽  
Plasminogen Activation ◽  
Structural Homology ◽  
Tissue Plasminogen ◽  
Umbilical Vein Endothelial Cells

SummaryLp(a) is an LDL-like lipoprotein plus an additional apolipoprotein apo(a). Based on the structural homology of apo(a) with plasminogen, it is hypothesized that Lp(a) interferes with fibrinolysis. Extracellular matrix (ECM) produced by human umbilical vein endothelial cells was used to study the effect of Lp(a) and LDL on plasminogen binding and activation. Both lipoproteins were isolated from the same plasma in a single step. Plasminogen bound to ECM via its lysine binding sites. Lp(a) as well as LDL were capable of competing with plasminogen binding. The degree of inhibition was dependent on the lipoprotein donor as well as the ECM donor. When Lp(a) and LDL obtained from one donor were compared, Lp(a) was always a much more potent competitor. The effect of both lipoproteins on plasminogen binding was reflected in their effect on plasminogen activation. It is speculated that Lp(a) interacts with ECM via its LDL-like lipoprotein moiety as well as via its apo(a) moiety.

scholarly journals The Plasminogen Activation System and the Regulation of Catecholaminergic Function

2012 ◽  
Vol 2012 ◽  
pp. 1-13 ◽  
Author(s):  
Hongdong Bai ◽  
Samir Nangia ◽  
Robert J. Parmer
Keyword(s):  
Plasminogen Activator ◽  
Binding Sites ◽  
Cell Function ◽  
Specific Binding ◽  
Neurosecretory Cells ◽  
Plasminogen Activation ◽  
Plasmin Activity ◽  
Plasminogen Activation System ◽  
Activation System ◽  
Catecholaminergic Cells

The local environment of neurosecretory cells contains the major components of the plasminogen activation system, including the plasminogen activators, tissue plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA), as well as binding sites for t-PA, the receptor for u-PA (uPAR), and also the plasminogen activator inhibitor, PAI-1. Furthermore, these cells express specific binding sites for plasminogen, which is available in the circulation and in interstitial fluid. Colocalization of plasminogen and its activators on cell surfaces provides a mechanism for promoting local plasminogen activation. Plasmin is retained on the cell surface where it is protected from its inhibitor,α2-antiplasmin. In neurosecretory cells, localized plasmin activity provides a mechanism for extracellular processing of secreted hormones. Neurotransmitter release from catecholaminergic cells is negatively regulated by cleavage products formed by plasmin-mediated proteolysis. Recently, we have identified a major plasminogen receptor, Plg-RKT. We have found that Plg-RKTis highly expressed in chromaffin cells of the adrenal medulla as well as in other catecholaminergic cells and tissues. Plg-RKT-dependent plasminogen activation plays a key role in regulating catecholaminergic neurosecretory cell function.

Kinetics Of Plasminogen Activation By Tissue Plasminogen Activator. Role Of Fibrin

1981 ◽  
Author(s):  
M Hoylaerts ◽  
D C Rijken ◽  
H R Lijnen ◽  
D Collen
Keyword(s):  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Catalytic Efficiency ◽  
Reaction Scheme ◽  
Plasminogen Activation ◽  
Catalytic Rate Constant ◽  
Activation Rate ◽  
Tissue Plasminogen ◽  
Human Plasminogen ◽  
Straight Lines

The activation of human plasminogen (P) by two-chain tissue plasminogen activator (A) was studied in the presence of fibrin films (F) of increasing size and surface density. Initial rates of plasminogen activation (v) were determined as a function both of the plasminogen and fibrin concentration. The activation rate was strongly dependent on the presence of fibrin and plots of 1/v versus 1/ [p] or 1 /[F] yielded straight lines. The kinetic data were in agreement with the following reaction scheme.According to this model tissue plasminogen activator would bind to fibrin with a dissociation constant (KF of 0.2 µM and this complex fixes plasminogen with a Michaelis constant (Kp’) of 0.15 µM (Glu-plasminogen) or 0.02 µM (Lys-plasminogen) to form a ternary complex, converted to plasmin with a catalytic rate constant kcat = 0.05 s-1. This mechanism implies that both plasminogen and tissue plasminogen activator are concentrated on the fibrin surface through formation of a fibrin bridge. Activation of plasminogen in the absence of fibrin occurs with Km = 65 µM (Glu-plasminogen) or Km= 19 µM (Lys-plasminogen) and kcat = 0.05 s-1. Our data suggest that fibrin enhances the activation rate of plasminogen by tissue plasminogen activator by increasing the affinity of plasminogen for fibrin-bound tissue plasminogen activator and not by influencing the catalytic efficiency of the enzµMe. These data also support the hypothesis that fibrinolysis is both triggered by and directed towards fibrin.Generated plasmin was quantitated by measuring the rate of solubilization of 125I-labeled fibrin.

scholarly journals Competition between plasminogen and tissue plasminogen activator for cellular binding sites

Blood ◽  
1993 ◽  
Vol 82 (8) ◽  
pp. 2433-2441 ◽  
Author(s):  
J Felez ◽  
CJ Chanquia ◽  
P Fabregas ◽  
EF Plow ◽  
LA Miles
Keyword(s):  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Proteolytic Activity ◽  
Binding Sites ◽  
Cell Types ◽  
Receptor Expression ◽  
Plasminogen Activation ◽  
Single Chain ◽  
Cellular Receptors ◽  
Tissue Plasminogen

Cellular receptors for plasminogen and tissue plasminogen activator (t- PA) regulate plasminogen activation and cell-associated proteolytic activity. The characteristics of the interactions of both ligands with monocytes and monocytoid cell lines bear certain similarities, including affinity (kd approximately 1 mumol/L) capacity and susceptibility to carboxypeptidase treatment. Therefore, we have undertaken the present study to determine directly whether t-PA and plasminogen share common binding sites on cells. We found that recombinant human single-chain t-PA (rt-PA) could inhibit the binding of 125I-plasminogen to the cells and, conversely, plasminogen could inhibit 125I-rt-PA binding. This relationship was observed with 9 cell types, including both adherent cells and cells in suspension. In addition, under several conditions of cell treatment, plasminogen and t- PA receptor expression was modulated in parallel. Furthermore, molecules that have been implicated as candidate plasminogen receptors, gangliosides, and an alpha-enolase--related molecule, also interacted with t-PA. These results suggest that at least a component of the binding sites for plasminogen is shared with t-PA. Occupancy of these sites by either or both ligand(s) should result in arming the cells with the proteolytic activity of plasmin.

scholarly journals The resistance of fibrinogen and soluble fibrin monomer in blood to degradation by a potent plasminogen activator derived from cadaver limbs

Blood ◽  
1975 ◽  
Vol 46 (4) ◽  
pp. 555-565
Author(s):  
V Gurewich ◽  
E Hyde ◽  
B Lipinski
Keyword(s):  
Plasminogen Activator ◽  
Solid Phase ◽  
Fibrin Clot ◽  
Reference Curve ◽  
Plasminogen Activation ◽  
Large Excess ◽  
Soluble Fibrin ◽  
Alternative Pathways ◽  
Fibrin Monomer ◽  
Fibrin Plate

The effect of a cadaver-derived vascular plasminogen activator (VA) on the degradation of fibrinogen, soluble fibrin monomer, and fibrin was studied and compared with the effect of equivalent fibrinolytic potencies of streptokinase (SK), urokinase (UK), and plasmin. The proteolytic activity of the three activators and plasmin was determined by a standard fibrin plate assay and was expressed in CTA units from a UK reference curve. Fibrinogen degradation was measured by clottable protein determinations and by an electrophoretic technique sensitive to small changes in the molecular weight of fibrinogen. When VA was incubated in plasma, no degradation of fibrinogen occurred, whereas rapid fibrinolysis took place after the plasma was clotted. By contrast, equivalent potencies of SK, UK, and plasmin caused extensive fibrinogenolysis. Since the plasmin added and that formed by the three activators had equivalent fibrinolytic activity, the failure of VA to induce fibrinogen degradation was attributed to antiactivators rather than antiplasmins. VA activity in plasma was consumed by clotting, whereas the antiactivator activity remained in the serum, suggesting dissociation of the VA-antiactivator complex on the fibrin clot. Fibrinogen and its soluble derivatives resisted degradation by VA in plasma because a solid phase appeared necessary for the complex to dissociate. The findings indicated that the degradation of fibrinogen or soluble fibrin in blood as a result of plasminogen activation by VA was unlikely to occur due to a large excess of antiactivator activity. Alternative pathways for their catabolism are discussed.

Regulation of plasminogen activation in isolated perfused rat kidney

1989 ◽  
Vol 256 (5) ◽  
pp. F787-F793
Author(s):  
W. Muellbacher ◽  
M. Maier ◽  
B. R. Binder
Keyword(s):  
Plasminogen Activator ◽  
Rat Kidney ◽  
Tissue Type ◽  
Plasminogen Activation ◽  
Isolated Perfused Rat Kidney ◽  
Plasminogen Activator Activity ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator ◽  
Perfused Rat Kidney ◽  
Human Plasminogen

To better understand the mechanism and regulation of plasminogen activation within the kidney, the release and excretion of plasminogen activator activities was studied in the isolated perfused rat kidney in the absence and presence of plasminogen substrate. In the absence of plasminogen, the kidneys released a constant amount of plasminogen activator activity into both the urine and the perfusate. On continuous infusion of purified human plasminogen into the perfusate, the release of plasminogen activator activity into the urine slightly increased, and plasmin generated could be detected in both urine and perfusate. With the use of specific antibodies against the tissue-type (t-PA) and the urokinase-type plasminogen activator (u-PA), respectively, the activity in the perfusate could be identified as t-PA, whereas the activity in the urine could be ascribed to u-PA. A bolus injection of either antibody into the plasminogen-supplemented perfusion medium completely inhibited plasminogen activator activity and generation of plasmin in the vascular or tubular compartment. Furthermore, intrarenal inhibition of t-PA activity by the specific antibody significantly increased the concentration of plasminogen in the perfusate, indicating decreased consumption. This effect was accompanied by increased excretion of u-PA into the urine, suggesting that the availability of intact plasminogen in the renal circulation directly or indirectly might participate in the regulation of u-PA excretion into the urine.

scholarly journals Binding of fibrin fragments to one-chain and two-chain tissue-type plasminogen activator

Blood ◽  
1992 ◽  
Vol 79 (9) ◽  
pp. 2313-2321 ◽  
Author(s):  
AA Hasan ◽  
WS Chang ◽  
AZ Budzynski
Keyword(s):  
Plasminogen Activator ◽  
Binding Sites ◽  
Solid Phase ◽  
Aminocaproic Acid ◽  
Tissue Type ◽  
Plasminogen Activation ◽  
Tissue Type Plasminogen Activator ◽  
Microtiter Plates ◽  
Type Plasminogen Activator ◽  
Epsilon Aminocaproic Acid

To explore whether fibrin fragments have binding affinity for the tissue-type plasminogen activator (t-PA) molecule, the interactions were studied of (DD)E complex and fragments DD, E1, and E3 with one- chain and two-chain t-PA. For this purpose, a solid-phase binding assay was developed using microtiter plates with nitrocellulose filters. It was found that (DD)E complex and fragments DD and E3 retained the t-PA binding function of the parent fibrin molecule, thus demonstrating that t-PA binds to both the D and E domains of fibrin. Unexpectedly, fragment E1 did not bind t-PA. Fibrin fragments had different binding properties for one-chain and two-chain t-PA. (DD)E complex had the highest and fragment E3 the lowest affinity for one-chain t-PA, both binding curves being consistent with one class of binding sites. However, binding of the fragments with two-chain t-PA was distinguished by more than one class of binding sites, with fragment E3 having the highest affinity for this form of the activator. epsilon-Aminocaproic acid, even at 50 mmol/L concentration, had only minimal effect on binding of (DD)E complex or fragment DD to either one-chain or two- chain t-PA. The potentiating effect of fibrin fragments on plasminogen activation by t-PA was measured by a chromogenic substrate assay. Fragment DD was the most effective stimulator of plasminogen activation by t-PA. In conclusion, (DD)E complex and fragment DD retained most of the regulatory functions of fibrin, which included t-PA binding and t- PA-mediated acceleration of plasminogen activation to plasmin.

A colorimetric assay for releasable plasminogen activator.

1982 ◽  
Vol 28 (5) ◽  
pp. 1125-1128 ◽  
Author(s):  
E E Campbell ◽  
M A Shifman ◽  
J G Lewis ◽  
J J Pasqua ◽  
S V Pizzo
Keyword(s):  
Plasminogen Activator ◽  
Colorimetric Assay ◽  
Systolic Pressure ◽  
Venous Occlusion ◽  
Clin Pathol ◽  
Before And After ◽  
Report Data ◽  
Human Plasminogen ◽  
Form Blood Vessel ◽  
Radioactive Substrate

Abstract We describe an equilibrium assay for measuring release of plasminogen activator form blood-vessel walls and report data from 125 individuals free of overt thromboembolic disease. Excess human plasminogen is added to the euglobulin fraction of plasma obtained before and after venous occlusion at mean systolic pressure. To measure plasmin generation in these samples, we used the chromogenic plasmin substrate D-Val-Leu-Lys-p-nitroanilide, which liberates p-nitroaniline upon cleavage. Releasable plasminogen activator in 24 subjects was determined by this colorimetric assay and by the radiocasein assay previously reported by this laboratory (Am. J. Clin. Pathol. 76,403-409, 1981), and the results were compared. The correlation coefficient was 0.97. The colorimetric assay offers several advantages over the radiocasein assay: shorter incubation (6 vs 16 h) and no preparation or quantification of a radioactive substrate and its cleavage products.

scholarly journals Binding of fibrin fragments to one-chain and two-chain tissue-type plasminogen activator

Blood ◽  
1992 ◽  
Vol 79 (9) ◽  
pp. 2313-2321 ◽  
Author(s):  
AA Hasan ◽  
WS Chang ◽  
AZ Budzynski
Keyword(s):  
Plasminogen Activator ◽  
Binding Sites ◽  
Solid Phase ◽  
Aminocaproic Acid ◽  
Tissue Type ◽  
Plasminogen Activation ◽  
Tissue Type Plasminogen Activator ◽  
Microtiter Plates ◽  
Type Plasminogen Activator ◽  
Epsilon Aminocaproic Acid

Abstract To explore whether fibrin fragments have binding affinity for the tissue-type plasminogen activator (t-PA) molecule, the interactions were studied of (DD)E complex and fragments DD, E1, and E3 with one- chain and two-chain t-PA. For this purpose, a solid-phase binding assay was developed using microtiter plates with nitrocellulose filters. It was found that (DD)E complex and fragments DD and E3 retained the t-PA binding function of the parent fibrin molecule, thus demonstrating that t-PA binds to both the D and E domains of fibrin. Unexpectedly, fragment E1 did not bind t-PA. Fibrin fragments had different binding properties for one-chain and two-chain t-PA. (DD)E complex had the highest and fragment E3 the lowest affinity for one-chain t-PA, both binding curves being consistent with one class of binding sites. However, binding of the fragments with two-chain t-PA was distinguished by more than one class of binding sites, with fragment E3 having the highest affinity for this form of the activator. epsilon-Aminocaproic acid, even at 50 mmol/L concentration, had only minimal effect on binding of (DD)E complex or fragment DD to either one-chain or two- chain t-PA. The potentiating effect of fibrin fragments on plasminogen activation by t-PA was measured by a chromogenic substrate assay. Fragment DD was the most effective stimulator of plasminogen activation by t-PA. In conclusion, (DD)E complex and fragment DD retained most of the regulatory functions of fibrin, which included t-PA binding and t- PA-mediated acceleration of plasminogen activation to plasmin.

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