Functional Fibrinolysis Assays Reveal Different Mechanisms underlying Plasminogen Dysfunction in Ligneous Conjunctivitis

Abstract Background Ligneous conjunctivitis (LC) is a rare disorder associated with plasminogen deficiency characterized by chronic fibrin deposits in the eyelids. All patients with plasminogen deficiency do not develop LC, whose underlying mechanisms remain unknown. Objective We investigated whether fibrinolytic activity was correlated with phenotype and/or genotype in patients suffering from LC and their relatives. Methods Plasminogen activity/antigen levels and PLG mutations were determined in 10 patients with LC, 17 of their asymptomatic relatives, and 10 healthy individuals used as a control group. Plasma fibrinolytic activity was evaluated using three different assays: (1) tissue-plasminogen activator (t-PA) front lysis, (2) cell-based urokinase-dependent euglobulin clot lysis (ECLT) at the surface of corneal cells, and (3) urokinase-dependent plasminogen activation. Results Plasminogen activity varied from <10 to 40% in patients, 36 to 105% in relatives, and >80% in control healthy individuals. Homozygous K19E mutation was associated with normal antigenic plasminogen levels. In front-lysis experiments, all patients had a lower fibrinolysis rate as compared with their relatives and to control individuals. The cell-based ECLT and plasminogen activation assay demonstrated that urokinase-mediated fibrinolysis was not impaired in patients with homozygous K19E mutation compared with the other mutants. Conclusion We confirm that plasminogen levels fail to predict LC occurrence. In these conditions, t-PA clot lysis front is useful to predict clinical outcome in plasminogen deficiency. Moreover, we provide evidence that occurrence of LC overlaps quantitative and qualitative plasminogen deficiencies. The homozygous K19E mutation is associated with isolated impaired t-PA-mediated fibrinolysis compared with other mutants.

Download Full-text

Nanodroplet-mediated catheter-directed sonothrombolysis of retracted blood clots

Microsystems & Nanoengineering ◽

10.1038/s41378-020-00228-9 ◽

2021 ◽

Vol 7 (1) ◽

Author(s):

Leela Goel ◽

Huaiyu Wu ◽

Bohua Zhang ◽

Jinwook Kim ◽

Paul A. Dayton ◽

...

Keyword(s):

Drug Delivery ◽

Negative Pressure ◽

Boiling Point ◽

Clot Lysis ◽

Control Group ◽

Peak Negative Pressure ◽

Blood Clots ◽

Tissue Plasminogen ◽

First Time

AbstractOne major challenge in current microbubble (MB) and tissue plasminogen activator (tPA)-mediated sonothrombolysis techniques is effectively treating retracted blood clots, owing to the high density and low porosity of retracted clots. Nanodroplets (NDs) have the potential to enhance retracted clot lysis owing to their small size and ability to penetrate into retracted clots to enhance drug delivery. For the first time, we demonstrate that a sub-megahertz, forward-viewing intravascular (FVI) transducer can be used for ND-mediated sonothrombolysis, in vitro. In this study, we determined the minimum peak negative pressure to induce cavitation with low-boiling point phase change nanodroplets and clot lysis. We then compared nanodroplet mediated sonothrombolysis to MB and tPA mediate techniques. The clot lysis as a percent mass decrease in retracted clots was 9 ± 8%, 9 ± 5%, 16 ± 5%, 14 ± 9%, 17 ± 9%, 30 ± 8%, and 40 ± 9% for the control group, tPA alone, tPA + US, MB + US, MB + tPA + US, ND + US, and ND + tPA + US groups, respectively. In retracted blood clots, combined ND- and tPA-mediated sonothrombolysis was able to significantly enhance retracted clot lysis compared with traditional MB and tPA-mediated sonothrombolysis techniques. Combined nanodroplet with tPA-mediated sonothrombolysis may provide a feasible strategy for safely treating retracted clots.

Download Full-text

PHYSICAL ACTIVITY RELEASES TISSUE PLASMINOGEN ACTIVATOR FROM EXERCISING PARTS OF BODY

10.1055/s-0038-1643117 ◽

1987 ◽

Author(s):

I Keber ◽

K Potisk ◽

D Keber ◽

M Stegnar ◽

N Vene

Keyword(s):

Physical Activity ◽

Plasminogen Activator ◽

Tissue Plasminogen Activator ◽

Fibrinolytic Activity ◽

Venous Occlusion ◽

Clot Lysis ◽

Lysis Time ◽

Tissue Plasminogen ◽

Euglobulin Clot Lysis Time ◽

The Difference

To determine the origin of tissue plasminogen activator (t-PA) release during physical activity, we studied the separate and combined effects of venous occlusion and acute physical activity on t-PA release in arm and leg. In 15 healthy volunteers 20 min venous occlusions of arm and leg were performed simultaneously before physical activity ( maximal stress testing on treadmill)(occlusion I), immediately after physical activity and 45 min later (occlusion II). Blood samples were drawn from unoccluded arm before occlusion and after physical activity, and from occluded arm and leg after occlusion. Fibrinolytic activity was measured by euglobulin clot lysis time (ECLT) and t-PA activity assay. The amount of released t-PA during different stimuli (fibrinolytic potential) was calculated as the difference between post- and prestimulation fibrinolytic activity. Before physical activity there was a great increase in fibrinolytic activity due to t-PA in the occluded arm but no increase in the occluded leg. Physical activity itself caused a similar increase of systemic fibrinolytic activity as arm occlusion locally. After physical activity arm occlusion evoked equally good response than before it. Fibrinolytic activity during leg occlusion behaved differently: there was an increase in t-PA activity in the occluded leg which persisted one hour after physical activity, when systemic fibrinolytic activity already fell to initial level.These results demonstrated that walking and running triggered t-PA release from the leg vessels. Since leg occlusion was not a stimulus for t-PA release, it served only as a method to demonstrate the effect of physical activity.

Download Full-text

The pathogenesis of accelerated fibrinolysis in liver cirrhosis: a critical role for tissue plasminogen activator inhibitor

Blood ◽

10.1182/blood.v69.5.1315.1315 ◽

1987 ◽

Vol 69 (5) ◽

pp. 1315-1319 ◽

Cited By ~ 86

Author(s):

SL Hersch ◽

T Kunelis ◽

RB Jr Francis

Keyword(s):

Liver Cirrhosis ◽

Plasminogen Activator ◽

Tissue Plasminogen Activator ◽

Fibrinolytic Activity ◽

Blood Clot ◽

Critical Role ◽

Plasminogen Activator Inhibitor ◽

Clot Lysis ◽

Lysis Time ◽

Tissue Plasminogen

Abstract The pathogenesis of accelerated fibrinolysis in liver cirrhosis was investigated by comparing the results of specific assays for tissue plasminogen activator (tpa) antigen, tpa activity, tpa inhibitor, and alpha-2 plasmin inhibitor (a2PI) in 12 patients with cirrhosis and markedly accelerated fibrinolysis (dilute whole blood clot lysis time (DWBCLT) less than two hours), in nine patients with cirrhosis and moderately accelerated fibrinolysis (DWBCLT two to four hours), and in nine patients with cirrhosis and normal fibrinolysis (DWBCLT greater than four hours). Mean tpa antigen was markedly increased in all three groups, but no correlation was observed between overall fibrinolytic activity as measured by the DWBCLT and the level of tpa antigen. In contrast, there was a significant correlation between overall fibrinolytic activity and tpa activity and an equally significant correlation between fibrinolytic activity and decreased tpa inhibition. Mean a2PI activity was significantly lower than normal in groups 1 and 2 but was normal in group 3. The pathogenesis of accelerated fibrinolysis in liver cirrhosis thus appears to depend critically on the capacity of plasma inhibitors to inhibit increased circulating tpa antigen. Reduced a2PI also appears to play a role.

Download Full-text

The kinetics of plasminogen activation by thrombin-cleaved pro- urokinase and promotion of its activity by fibrin fragment E-2 and by tissue plasminogen activator

Blood ◽

10.1182/blood.v81.4.980.bloodjournal814980 ◽

1993 ◽

Vol 81 (4) ◽

pp. 980-987

Author(s):

JN Liu ◽

V Gurewich

Keyword(s):

Plasminogen Activator ◽

Tissue Plasminogen Activator ◽

Catalytic Efficiency ◽

Clot Lysis ◽

Plasminogen Activation ◽

Single Chain ◽

Carboxypeptidase B ◽

Thrombin Cleavage ◽

Tissue Plasminogen ◽

A Chain

Thrombin hydrolyzes the Arg156-Phe157 bond in pro-urokinase (pro-UK), two residues from the activation site, generating a two-chain form (thromb-UK) believed to have little activity and that is resistant to plasmin activation. The kinetic constants for thromb-UK against synthetic substrate (S2444) were found to be essentially identical to pro-UK. Against native plasminogen, thromb-UK had a lower Michaelis constant (KM) and a higher (2-fold) catalytic efficiency. However, this difference with pro-UK was nullified by carboxypeptidase B (CpB) treatment of thromb-UK to remove the C-terminal arginine on the A- chain. Plasminogen activation by thromb-UK was substantially promoted by fibrin fragment E-2 but not by other fibrin derivatives, a phenomenon previously observed with pro-UK. Similarly, clot lysis by thromb-UK was promoted by tissue plasminogen activator because their combined effect was synergistic. Fibrinogenolysis in plasma occurred at 80-fold the concentration of thromb-UK as pro-UK, reflecting the 90- fold greater plasmin resistance of thromb-UK. Addition of a CpB inhibitor to the plasma enhanced fibrinogenolysis by thromb-UK and pro- UK by approximately 16%, consistent with the promotion of both forms by certain C-terminal lysines. In conclusion, CpB-thromb-UK corresponds functionally to a plasmin resistant form of pro-UK, indicating that the catalytic site of the single-chain pro-UK is unaffected by thrombin cleavage. The effect of CpB indicates that the C-terminal Arg of thromb- UK slightly enhances its affinity for plasminogen. Thromb-UK has potential plasminogen-activating activity at surfaces where C-terminal lysines, functionally comparable to fragment E-2, are found.

Download Full-text

A Precise and Rapid Microtitre Plate Clot Lysis Assay: Methodology, Kinetic Modeling and Measurement of Catalytic Constants for Plasminogen Activation during Fibrinolysis

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647336 ◽

1990 ◽

Vol 64 (03) ◽

pp. 455-463 ◽

Cited By ~ 40

Author(s):

A J S Jones ◽

A M Meunier

Keyword(s):

Experimental Data ◽

Reference Method ◽

Clot Lysis ◽

Plasminogen Activation ◽

Microtitre Plate ◽

Plate Reader ◽

Tissue Plasminogen ◽

Reference Preparation ◽

Rapid Activation ◽

Kinetics Of

SummaryA rapid and precise turbidimetric clot lysis assay employing a microtitre plate reader and personal computer is described in detail. The use of such widely available instrumentation, the convenience and rapid throughput suggest the assay could be developed as a reference method with which to measure the potency of tissue plasminogen activator (t-PA) in conjunction with the WHO reference preparation. The method has been used to investigate molecular parameters involved in fibrinolysis. Aggregation status of the fibrin does not appear toinfluence the mechanism of plasminogen activation and clot lysis by plasmin. High ratios of plasminogen to fibrin resulted in a change in clot turbidity and in a change in the lysis profile of turbidity versus time. This is probably the result of plasminogen binding to fibrin and consequent restriction of the access of plasmin to its sites of cleavage in the fibrin. A simple model is proposed, and equations have been derived, for the kinetics of lysis which adequately describe the mechanism and which are confirmed by experimental data. This model results in estimates of the Km and kcat for the activation of plasminogen by t-PA during clot lysis of approximately 150 nM and0.1 s-1, respectively, in excellent agreement with published values The assay should therefore prove useful in quantitative evaluationsof the molecular phenomena occurring during fibrinolysis. The more rapid activation of lys-plasminogen than glu-plasminogen by t-PA was confirmed. However, evidence was obtained that the lys-form binds more tightly to fibrin by the same factor. This observation suggested that the appropriate substrate in the kinetic model is fibrin-bound plasminogen. When the data were re-analysed using published values ofthe affinities of the two forms, t-PA was found to activate both forms indistinguishably on the fibrin surface, consistent with suggestions that the conformation of glu-plasminogen changes to one more like that of lys-plasminogen upon binding to fibrin.

Download Full-text

The kinetics of plasminogen activation by thrombin-cleaved pro- urokinase and promotion of its activity by fibrin fragment E-2 and by tissue plasminogen activator

Blood ◽

10.1182/blood.v81.4.980.980 ◽

1993 ◽

Vol 81 (4) ◽

pp. 980-987 ◽

Cited By ~ 29

Author(s):

JN Liu ◽

V Gurewich

Keyword(s):

Plasminogen Activator ◽

Tissue Plasminogen Activator ◽

Catalytic Efficiency ◽

Clot Lysis ◽

Plasminogen Activation ◽

Single Chain ◽

Carboxypeptidase B ◽

Thrombin Cleavage ◽

Tissue Plasminogen ◽

A Chain

Abstract Thrombin hydrolyzes the Arg156-Phe157 bond in pro-urokinase (pro-UK), two residues from the activation site, generating a two-chain form (thromb-UK) believed to have little activity and that is resistant to plasmin activation. The kinetic constants for thromb-UK against synthetic substrate (S2444) were found to be essentially identical to pro-UK. Against native plasminogen, thromb-UK had a lower Michaelis constant (KM) and a higher (2-fold) catalytic efficiency. However, this difference with pro-UK was nullified by carboxypeptidase B (CpB) treatment of thromb-UK to remove the C-terminal arginine on the A- chain. Plasminogen activation by thromb-UK was substantially promoted by fibrin fragment E-2 but not by other fibrin derivatives, a phenomenon previously observed with pro-UK. Similarly, clot lysis by thromb-UK was promoted by tissue plasminogen activator because their combined effect was synergistic. Fibrinogenolysis in plasma occurred at 80-fold the concentration of thromb-UK as pro-UK, reflecting the 90- fold greater plasmin resistance of thromb-UK. Addition of a CpB inhibitor to the plasma enhanced fibrinogenolysis by thromb-UK and pro- UK by approximately 16%, consistent with the promotion of both forms by certain C-terminal lysines. In conclusion, CpB-thromb-UK corresponds functionally to a plasmin resistant form of pro-UK, indicating that the catalytic site of the single-chain pro-UK is unaffected by thrombin cleavage. The effect of CpB indicates that the C-terminal Arg of thromb- UK slightly enhances its affinity for plasminogen. Thromb-UK has potential plasminogen-activating activity at surfaces where C-terminal lysines, functionally comparable to fragment E-2, are found.

Download Full-text

Fibrinolysis-resistant carbonylated fibrin detected in thrombi attached to the vascular wall of abdominal aortic aneurysms

Scientific Reports ◽

10.1038/s41598-020-77582-1 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Yuko Suzuki ◽

Hiroki Tanaka ◽

Takahiro Horinouchi ◽

Hideto Sano ◽

Naoki Honkura ◽

...

Keyword(s):

Abdominal Aortic Aneurysms ◽

Vascular Wall ◽

Fibrin Clot ◽

Hexanoic Acid ◽

Aortic Aneurysms ◽

Clot Lysis ◽

Plasminogen Activation ◽

Long Period ◽

Abdominal Aortic ◽

Tissue Plasminogen

AbstractIn this study, we investigated how carbonylation of fibrinogen by acrolein modified its indispensable function to enhance fibrinolysis after being converted to fibrin and contributed to generating a fibrinolysis-resistant fibrin clot. Acrolein-treated fibrinogen was subjected to tissue plasminogen activator-induced fibrinolysis assay and the effect of lysine residue carbonylation in fibrinogen on fibrinolysis was analyzed. The acrolein-treated fibrinogen-derived fibrin clot appeared more resistant to fibrinolysis and the N-acetyl 3-formyl-3,4-dehydropiperidino (FDP)-Lysine levels in the lysed solution were positively correlated with the duration of clot lysis. The lysine analog 6-amino hexanoic acid (6AHA), which mimics the C-terminal lysine of fibrin, was carbonylated and its enhancing effect on Glu1-plasminogen activation was evaluated. After incubation with acrolein, 6AHA was converted to N-acetyl FDP-6AHA, losing its ability to enhance Glu1-plasminogen activation. These results suggest that fibrinogen carbonylation by acrolein to generate N-acetyl FDP-Lysine resulted in the generation of fibrinolysis-resistant fibrin by attenuating the C-terminal lysine-dependent activation of the Glu1-plasminogen. In abdominal aortic aneurysms, fibrin(ogen) containing the acrolein adduct N-acetyl FDP-Lysine was detected in the vascular wall-attached thrombi. These results suggest that this mechanism is likely involved in the modification of fibrinolysis-resistant thrombi and to their persistence for a long period.

Download Full-text

The pathogenesis of accelerated fibrinolysis in liver cirrhosis: a critical role for tissue plasminogen activator inhibitor

Blood ◽

10.1182/blood.v69.5.1315.bloodjournal6951315 ◽

1987 ◽

Vol 69 (5) ◽

pp. 1315-1319

Author(s):

SL Hersch ◽

T Kunelis ◽

RB Jr Francis

Keyword(s):

Liver Cirrhosis ◽

Plasminogen Activator ◽

Tissue Plasminogen Activator ◽

Fibrinolytic Activity ◽

Blood Clot ◽

Critical Role ◽

Plasminogen Activator Inhibitor ◽

Clot Lysis ◽

Lysis Time ◽

Tissue Plasminogen

The pathogenesis of accelerated fibrinolysis in liver cirrhosis was investigated by comparing the results of specific assays for tissue plasminogen activator (tpa) antigen, tpa activity, tpa inhibitor, and alpha-2 plasmin inhibitor (a2PI) in 12 patients with cirrhosis and markedly accelerated fibrinolysis (dilute whole blood clot lysis time (DWBCLT) less than two hours), in nine patients with cirrhosis and moderately accelerated fibrinolysis (DWBCLT two to four hours), and in nine patients with cirrhosis and normal fibrinolysis (DWBCLT greater than four hours). Mean tpa antigen was markedly increased in all three groups, but no correlation was observed between overall fibrinolytic activity as measured by the DWBCLT and the level of tpa antigen. In contrast, there was a significant correlation between overall fibrinolytic activity and tpa activity and an equally significant correlation between fibrinolytic activity and decreased tpa inhibition. Mean a2PI activity was significantly lower than normal in groups 1 and 2 but was normal in group 3. The pathogenesis of accelerated fibrinolysis in liver cirrhosis thus appears to depend critically on the capacity of plasma inhibitors to inhibit increased circulating tpa antigen. Reduced a2PI also appears to play a role.

Download Full-text

Tissue Plasminogen Activator in Human Megakaryocytes and Platelets: Immunocytochemical Localization, Immunoblotting and Zymographic Analysis

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647528 ◽

1988 ◽

Vol 59 (03) ◽

pp. 529-534 ◽

Cited By ~ 12

Author(s):

C Jeanneau ◽

Y Sultan

Keyword(s):

Plasminogen Activator ◽

Tissue Plasminogen Activator ◽

Fibrinolytic Activity ◽

Blood Platelets ◽

Fibrinolytic System ◽

Plasminogen Activation ◽

Sds Page ◽

Tissue Plasminogen ◽

Zymographic Analysis

SummaryTwo approaches were used to identify and characterize the presence of tissue plasminogen activator (t-PA) in megakaryocytes and platelets. We investigated the fibrinolytic activity of human megakaryocytes (MK) and platelets. The presence of t-PA antigen in megakaryocytes and platelets was demonstrated using immunocytochemical techniques and polyclonal or monoclonal antibodies specific for t-PA. When cells were applied to fibrin plates, lysis zones developed around isolated human megakaryocytes, whereas no fibrinolytic activity appeared when either intact washed platelets or platelet lysate were deposited. After SDS-PAGE of platelet and MK extracts (Triton X-100) immunoblotting and peroxidase staining identified t-PA antigen in several bands. Zymographic analysis of SDS-PAGE carried out on fibrin film overlays identified one or two zones corresponding to free or complexed t-PA. These results indicate that t-PA is present in platelets as well as in the precursor cells, however, in platelets, t-PA may not be immediately available for plasminogen activation and fibrin degradation. From our findings and from previous work of others, it appears that platelets may either activate or inhibit the fibrinolytic system. Therefore the conditions of plasminogen activation by platelet t-PA and plasmin inhibition by platelet α2-antiplasmin or other inhibitors have to be precised before the role of platelets in clot dissolution is understood.The physiological role of platelets in fibrinolysis and clot dissolution remains unclear. In 1953, the antifibrinolytic activity of blood platelets was demonstrated (1) and in the early 1960’s a fibrinolytic activity, increasing with platelet concentration in the experimental system, was shown (2, 3). In 1979, it was demonstrated that metabolically active platelets were necessary for platelets to play a role in the fibrinolytic system (4). More recently it was established by Plow and Collen (5) that the specific plasmin inhibitor, α2-antiplasmin is a constituent of platelet α-granules.In the present study, we investigated the fibrinolytic components and activity of human megakaryocytes and platelets, using zymographic and immunochemical techniques. We report here our observations that human megakaryocytes and platelets contain tissue plasminogen (t-PA) which possesses fibrinolytic activity.

Download Full-text

Plasma Fibronectin Enhances Fibrinolytic System In Vitro

Thrombosis and Haemostasis ◽

10.1055/s-0038-1660088 ◽

1985 ◽

Vol 54 (03) ◽

pp. 639-644 ◽

Cited By ~ 10

Author(s):

Nisan Gilboa ◽

John E Kaplan

Keyword(s):

Fibrinolytic System ◽

Clot Lysis ◽

Plasminogen Activation ◽

Plasma Fibronectin ◽

Amidolytic Activity ◽

Lysis Time ◽

Tissue Plasminogen ◽

Clot Lysis Time

SummaryThe effects of plasma fibronectin on the fibrinolytic system were studied in vitro. Fibronectin caused a time and concentration-dependent increase (up to 99% with 330 ug/ml) in the amidolytic activity of tissue plasminogen activator (TPA) but not of urokinase. In the presence of fibronectin the Km of the amidolytic activity of TPA decreased without a change in Vmax. It also caused a concentration-dependent increase in lys-plas-minogen activation by TPA (up to 825% with 375 ug/ml) and by urokinase (up to 400% with 250 ug/ml), as well as in the amidolytic activity of plasmin (up to 55% with 300 ug/ml). Fibronectin did not enhance the activation of glu-plasminogen. In the presence of fibronectin the Km of lys-plasminogen activation decreased without a change in Vmax. In purified systems fibronectin significantly shortened the clot lysis time (CLT) by up to 28% and 30% in TPA- and plasmin-activated lysis, respectively. The presence of Ca2+ did not change fibronectin’s effect on CLT. Clots of non-fibronectin-depleted plasma were lysed up to about twice as fast as the clots of fibronectin-depleted plasma. In conclusion, physiologic concentrations of fibronectin enhanced the fibrinolytic system in vitro. Further studies will be required to elucidate the mechanisms involved and to document whether fibronectin has a similar effect in vivo.

Download Full-text