Insulin-induced release of plasminogen activator from human blood platelets

1995 ◽  
Vol 268 (1) ◽  
pp. H117-H124
Author(s):  
N. N. Kahn ◽  
W. A. Bauman ◽  
A. K. Sinha
Keyword(s):  
Platelet Aggregation ◽  
Plasminogen Activator ◽  
Type I Diabetes ◽  
Platelet Rich Plasma ◽  
Blood Platelets ◽  
Tissue Type ◽  
Clot Lysis ◽  
Type I ◽  
Inhibition Of Platelet Aggregation ◽  
Lysis Time

Incubation of washed platelets in Tyrode buffer, pH 7.5, with insulin (200 microU/ml) and CaCl2 (1.2 mM) at 37 degrees C for 3 h resulted in a threefold increase of plasminogen activator activity in the supernatant over the basal level as determined by both the amidolytic assay and the proteolysis of alpha-casein through the formation of plasmin from plasminogen. This plasminogen activator showed no plasmin-like activity and was inhibited by anti-tissue plasminogen activator antibody as well as by type 1 plasminogen activator inhibitor. The substrate specificity and the inhibition of the enzymic activity by various inhibitors indicated that the platelet plasminogen activator (pPA) was related to tissue-type plasminogen activator of relative molecular weight 56,000. Fibrinolytic activity of pPA and its insulin-dependent release were demonstrated by the shortening of euglobulin lysis time and by the clot lysis time of platelet-rich plasma from normal and type I diabetes mellitus patients. Treatment of platelet membranes with insulin also increased the release of pPA. Increased levels of adenosine 3',5'-cyclic monophosphate (cAMP) in platelets by incubation with various agents completely inhibited the insulin-induced release of the activator. On the other hand, inhibition of platelet aggregation by aspirin had no effect on the release of pPA, indicating that the effect of cAMP was not due to the inhibition of platelet aggregation by the nucleotide.

Activated Protein C Increases Fibrin Clot Lysis by Neutralization of Plasminogen Activator Inhibitor No Evidence for a Cofactor Role of Protein S

1988 ◽  
Vol 60 (02) ◽  
pp. 328-333 ◽  
Author(s):  
N J de Fouw ◽  
Y F de Jong ◽  
F Haverkate ◽  
R M Bertina
Keyword(s):  
Plasminogen Activator ◽  
Protein C ◽  
Blood Platelets ◽  
Plasminogen Activator Inhibitor ◽  
Protein S ◽  
Tissue Type ◽  
Clot Lysis ◽  
Activator Inhibitor ◽  
Pai 1

summaryThe effect of purified human activated protein G (APC) on fibrinolysis was studied using a clot iysis system consisting of purified glu-plasminogen, tissue-type plasminogen activator, plasminogen activator inhibitor (released from endothelial cells or blood platelets), fibrinogen, 125T-fibrinogen and thrombin. All proteins were of human origin.In this system APC could increase fibrinolysis in a dose dependent way, without affecting fibrin formation or fibrin crosslinking. However, this profibrinolytic effect of APC could only be observed when plasminogen activator inhibitor (PAI-l) was present. The effect of APC was completely quenched by pretreatment of APC with anti-protein C IgG or di-isopropylfluorophosphate. Addition of the cofactors of APC:protein S, Ca2+-ions and phospholipid-alone or in combination did not enhance the profibrinolytic effect of APC. These observations indicate that human APC can accelerate in vitro clot lysis by the inactivation of PAI-1 activity. However, the neutralization of PAI-1 by APC is independent of the presence or absence of protein S, phospholipid and Ca2+-ions.

scholarly journals Plasminogen activator inhibitor 1: development of a radioimmunoassay and observations on its plasma concentration during venous occlusion and after platelet aggregation

Blood ◽  
1987 ◽  
Vol 70 (5) ◽  
pp. 1645-1653 ◽  
Author(s):  
EK Kruithof ◽  
G Nicolosa ◽  
F Bachmann
Keyword(s):  
Platelet Aggregation ◽  
Plasminogen Activator ◽  
Platelet Rich Plasma ◽  
Plasminogen Activator Inhibitor ◽  
Venous Occlusion ◽  
Tissue Type ◽  
Plasminogen Activator Inhibitor 1 ◽  
Bovine Endothelial Cell ◽  
Before And After ◽  
Pai 1

Abstract To study the effect of plasminogen activator inhibitors (PAI) on fibrinolysis it is essential to be able to specifically measure these proteins in plasma. To this end PAI-1 was purified from cortisol- stimulated HT 1080 fibrosarcoma cells and antisera raised in rabbits. The immunologic relationship of the purified inhibitor to PAI-1 in plasma and platelet extracts was established by immunoblotting and regular and reverse fibrin zymography. Furthermore, the purified product could be immunoprecipitated with antibodies to human or bovine endothelial cell-derived PAI-1. A radioimmunoassay was developed that measures both free and tissue-type plasminogen activator (t-PA)-bound PAI-1 in plasma and has an effective range of 8 to 250 ng/mL. PAI-1 antigen levels showed a twofold increase after 20 minutes of venous occlusion, partially due to hemoconcentration. Approximately one quarter of PAI-1 before and after venous occlusion is derived from platelets. After correction for hemoconcentration and the contribution of platelets to plasma PAI-1 levels, a still significant increase in PAI-1 levels was noted during venous occlusion, which suggests that the local vascular bed releases PAI-1. Concomitant with PAI-1, t-PA antigen levels increased eightfold and fibrinolytic activity 18-fold after 20 minutes of venous occlusion. PAI-1 and t-PA levels tend to augment with age: in a group of older healthy volunteers (mean age, 53 years) PAI-1 levels were twice and t-PA levels 1.7 times higher than those in a group with a mean age of 29 years. Determination of PAI-1 antigen levels before and after platelet aggregation demonstrated that 85% of PAI-1 in platelet-rich plasma is associated with platelets. The average amount of PAI-1 per platelet was 0.3 fg/platelet, ie, 4,000 molecules per platelet.

scholarly journals Plasminogen activator inhibitor 1: development of a radioimmunoassay and observations on its plasma concentration during venous occlusion and after platelet aggregation

Blood ◽  
1987 ◽  
Vol 70 (5) ◽  
pp. 1645-1653
Author(s):  
EK Kruithof ◽  
G Nicolosa ◽  
F Bachmann
Keyword(s):  
Platelet Aggregation ◽  
Plasminogen Activator ◽  
Platelet Rich Plasma ◽  
Plasminogen Activator Inhibitor ◽  
Venous Occlusion ◽  
Tissue Type ◽  
Plasminogen Activator Inhibitor 1 ◽  
Bovine Endothelial Cell ◽  
Before And After ◽  
Pai 1

To study the effect of plasminogen activator inhibitors (PAI) on fibrinolysis it is essential to be able to specifically measure these proteins in plasma. To this end PAI-1 was purified from cortisol- stimulated HT 1080 fibrosarcoma cells and antisera raised in rabbits. The immunologic relationship of the purified inhibitor to PAI-1 in plasma and platelet extracts was established by immunoblotting and regular and reverse fibrin zymography. Furthermore, the purified product could be immunoprecipitated with antibodies to human or bovine endothelial cell-derived PAI-1. A radioimmunoassay was developed that measures both free and tissue-type plasminogen activator (t-PA)-bound PAI-1 in plasma and has an effective range of 8 to 250 ng/mL. PAI-1 antigen levels showed a twofold increase after 20 minutes of venous occlusion, partially due to hemoconcentration. Approximately one quarter of PAI-1 before and after venous occlusion is derived from platelets. After correction for hemoconcentration and the contribution of platelets to plasma PAI-1 levels, a still significant increase in PAI-1 levels was noted during venous occlusion, which suggests that the local vascular bed releases PAI-1. Concomitant with PAI-1, t-PA antigen levels increased eightfold and fibrinolytic activity 18-fold after 20 minutes of venous occlusion. PAI-1 and t-PA levels tend to augment with age: in a group of older healthy volunteers (mean age, 53 years) PAI-1 levels were twice and t-PA levels 1.7 times higher than those in a group with a mean age of 29 years. Determination of PAI-1 antigen levels before and after platelet aggregation demonstrated that 85% of PAI-1 in platelet-rich plasma is associated with platelets. The average amount of PAI-1 per platelet was 0.3 fg/platelet, ie, 4,000 molecules per platelet.

DIURNAL VARIATION OF COMPONENTS OF THE FIBRINOLYTIC SYSTEM

1987 ◽  
Author(s):  
V Grimaudo ◽  
E K O Kruithof ◽  
J Hauert ◽  
F Bachmann
Keyword(s):  
Plasminogen Activator ◽  
Fibrinolytic Activity ◽  
Fibrinolytic System ◽  
Tissue Type ◽  
Diurnal Fluctuation ◽  
Clot Lysis ◽  
Plasminogen Activator Inhibitor 1 ◽  
Entire Study ◽  
Lysis Time ◽  
Pai 1

To elucidate the causes of the well known diurnal variations of the fibrinolytic activity we have studied severed parameters of the fibrinolytic system in 8 healthy male volunteers during a period of 24 h. Blood sanples were taken at 8, 10 and 12 a.m., 4 and 8 p.m. and at 8 a.m. next morning. Hie following parameters were analyzed: euglobulin clot lysis time (ECLT), fibrinolytic activity of euglobulins on fibrin plates (FPA), antigen concentrations of tissue-type plasminogen activator (t-PA) by ELISA and of urokinase (u-PA) and plasminogen activator inhibitor-1 (PAI-1) by RIA, the overall PA-inhibitor activity by the indirect chranogenic substrate assay (Verheijen et al; 1984) and the electrophoretic-zymogra-phic analysis of the euglobulins fraction.Global fibrinolytic activity increased from 1.0 ±0.3 U/ml at 8 a.m. to 2.5 ±1.1 U/ml at 8 p.m. (p <0.002) as measured by ECLT (U=300/lysis time in minutes) and from 2.3 ±0.4 IU/ml to 4.7 ±1.1 IU/ml (p <0.001) as determined by FPA. In contrast, t-PA antigen decreased from 5.1 ±0.8 ng/ml at 8 a.m. to 2.9 ±1.5 ng/ml at 8 p.m. (p<0.01) and u-PA antigen from 8.1 ±1.2 ng/ml to 7.1 ±0.7 ng/ml (p<0.05). Most pronounced was the decrease of PAI-1 antigen from 23.5 ± 7.6 ng/ml in the morning to 8.8 ±2.6 ng/ml at 8 p.m. (p <0.001), while PAI activity decreased from 8.8 ±3.6 U/ml to 5.5 ±1.8 U/ml at 4 p.m. (p <0.002). During the entire study period the electro-phoretic-zymographic analysis did not reveal any free t-PA; the latter was present throughout in the form of the 110 kD t-PA/PAI-1 ccnplex. During the day the intensity of the 110 kD band decreased progressively.These findings confirm the physiological diurnal fluctuation of the fibrinolytic activity. All components of the fibrinolytic system are involved in this fluctuation and participate in the modulation of fibrinolytic activity.Our study demonstrates that the diurnal increase of fibrinolytic activity is due principally to a marked decline of the PAI-1 concentration during the day and not to an increase of PA antigen levels.

The Influence of Molsidomine and Its Active Metabolite SIN-1 on Fibrinolysis and Platelet Aggregation

1985 ◽  
Vol 54 (04) ◽  
pp. 746-749 ◽  
Author(s):  
M Basista ◽  
L Grodzińska ◽  
J święs
Keyword(s):  
Platelet Aggregation ◽  
Oral Administration ◽  
Active Metabolite ◽  
Ex Vivo ◽  
Blood Platelets ◽  
Clot Lysis ◽  
Lysis Time ◽  
Euglobulin Clot Lysis Time ◽  
Clot Lysis Time

SummaryMolsidomine and its active metabolite SIN-1 were examined in humans and animals for platelet suppressant and fibrinolytic activities.Following oral administration of molsidomine at doses of 6 or 15 mg/kg to rabbits, their blood platelets in PRP ex vivo required higher threshold concentrations of ADP, AA and thrombin to be aggregated. Unlike molsidomine, SIN-1 when infused (10 and 20 μg/kg i.v.) into anaesthetized cats caused a release of a substance disaggregating platelet clumps which had adhered to blood superfused collagen strip. The appearance of this unstable disaggregating substance was prevented by the pretreatment of cats with aspirin (50 mg/kg i.v.). It is suggested that SIN-1 may promote formation of a PGI2-like substance.In humans shortening of euglobulin clot lysis time was observed 60 min after a single ingestion of 2 mg of molsidomine. This fibrinolytic effect of molsidomine was not abolished by the pretreatment of patients with aspirin. Neither molsidomine nor SIN-1 activated fibrinolysis in preformed euglobulin clots in vitro.

Increased Fibrinolytic Activity during Use of Oral Contraceptives Is Counteracted by an Enhanced Factor XI-independent down Regulation of Fibrinolysis

2000 ◽  
Vol 84 (07) ◽  
pp. 9-14 ◽  
Author(s):  
Saskia Middeldorp ◽  
Winnie Tekelenburg ◽  
Abraham van den Ende ◽  
Guido Tans ◽  
Martin Prins ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Oral Contraceptives ◽  
Fibrinolytic Activity ◽  
Tissue Type ◽  
Coagulation System ◽  
Clot Lysis ◽  
Factor Xi ◽  
Lysis Time ◽  
Pai 1 ◽  
Clot Lysis Time

SummaryThe effect of oral contraceptives (OC) on fibrinolytic parameters was investigated in a cycle-controlled cross-over study in which 28 non-OC using women were randomly prescribed either a representative of the so-called second (30 µg ethinylestradiol, 150 µg levonorgestrel) or third generation OC (30 µg ethinylestradiol, 150 µg desogestrel) and who switched OC after a two month wash out period. During the use of OC, the levels of tissue-type plasminogen activator (tPA) activity, plasminogen, plasmin-α2-antiplasmin complexes and D-dimer significantly increased (by 30 to 80%), while the levels of plasminogen activator inhibitor-1 (PAI-1) antigen, PAI-1 activity and tPA antigen significantly decreased (25 to 50%), suggesting an increase in endogenous fibrinolytic activity. These OC-induced changes were not different between the two contraceptive pills. TAFI (thrombin-activatable fibrinolysis inhibitor) levels increased on levonorgestrel, and even further increased on desogestrel. A clot lysis assay that probes both fibrinolytic activity and the efficacy of the coagulation system to generate thrombin necessary to down regulate fibrinolysis via TAFI showed no change of the clot lysis time during OC use. This finding suggests that the OC-induced increase in endogenous fibrinolytic activity is counteracted by an increased capacity of the coagulation system to down regulate fibrinolysis via TAFI. Indeed we observed that during OC use there was a significant increase of F1+2 generation during clot formation. When these assays were performed in the presence of an antibody against factor XI, we observed that the clot lysis time was significantly increased during OC use and that the increase in F1+2 generation during OC therapy was due to a factor XI-independent process, which was significantly higher on desogestrel than on levonorgestrel. These data indicate that the OC-induced inhibition of endogenous fibrinolysis takes place in a factor XI-independent way and is more pronounced on desogestrel than on levonorgestrel-containing OC.

Tissue-Type Plasminogen Activator (t-PA) and Dilute Blood Clot Lysis Time in Nephrotic Patients

1989 ◽  
Vol 61 (02) ◽  
pp. 270-274 ◽  
Author(s):  
Mircea P Cucuianu ◽  
Horea G Rus ◽  
Stefan Roman ◽  
Codruţa Mărcuşu ◽  
Constantin Spînu ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Blood Clot ◽  
Venous Occlusion ◽  
Plasma Fibrinogen ◽  
Tissue Type ◽  
Clot Lysis ◽  
Control Subjects ◽  
Lysis Time ◽  
Dilute Blood ◽  
Clot Lysis Time

SummaryWhen compared to normal weight normolipidemic control subjects, dilute blood clot lysis time was found to be obviously (p <0.001) prolonged in hypertriglyceridemic patients without proteinuria and slightly (p <0.05) accelerated in hyperlipidemic nephrotic patients in spite of their very high levels of plasma fibrinogen. As a result the ratio plasma fibrinogen (mg/dl) per clot lysis time (minutes) was 1.241 ± 0.08 (X ± SEM) in control subjects, 0.574 ± 0.07 in hypertriglyceridemic patients and 2.69 ± 0.172 in nephrotic patients. This finding suggesting that a larger amount of fibrin is rather readily dispersed from dilute blood clots of nephrotic patients was associated with higher levels of plasma t-PA: Ag (9.45 ng/ml ± 1.18 in nephrotic patients versus 5.8 ng/ml ± 1.23 in controls before venous occlusion and respectively 33.1 ng/ml ± 3.83 versus 20.3 ± 3.40 in controls after venous occlusion). Plasminogen activator activity of the euglobulins as assessed by the bovine fibrin-agarose plate was significantly higher in nephrotic patients only after venous occlusion. Plasma samples of nephrotic patients exerted a more potent inhibition of fibrinolysis in a urokinase activated system. This effect was, however, mainly due to the high levels of α2 macroglobulin in nephrotic plasma which apparently have little influence on dilute blood clot lysis time.

Studies on the Release of a Plasminogen Activator Inhibitor by Human Platelets

1986 ◽  
Vol 55 (02) ◽  
pp. 201-205 ◽  
Author(s):  
E K O Kruithof ◽  
C Tran-Thang ◽  
F Bachmann
Keyword(s):  
Platelet Aggregation ◽  
Plasminogen Activator ◽  
Platelet Rich Plasma ◽  
Plasminogen Activator Inhibitor ◽  
Human Platelets ◽  
Tissue Type ◽  
Blood Collection ◽  
Platelet Poor Plasma ◽  
Bovine Endothelial Cell ◽  
Activator Inhibitor

SummaryThe relative contribution of platelets to plasminogen activator inhibitor (PA-inhibitor) activity in blood was investigated. From the difference in PA-inhibitor levels in platelet-poor plasmas of 12 donors (3 ± 1 U/ml, mean ±95% confidence limits) and in the corresponding platelet-rich plasmas after induction of platelet aggregation by collagen, ADP or epinephrine (7 ± 1 U/ml), it may be concluded that a greater amount of PA-inhibitor in blood is associated with platelets than with plasma. In collagen-stimulated platelets maximal release of PA-inhibitor and of beta-thromboglobulin (β-TG) was attained within fifteen seconds, whereas in ADP-stimulated platelets the release of both factors was slower. In platelet-poor plasma no correlation was found between the level of PA-inhibitor and that of P-TG. Thus, the PA-inhibitor found in plasma is not derived from platelets that had been stimulated after blood collection. The rate of complex formation and the Mr of the principal complexes of radioiodinated tissue-type plasminogen activator (t-PA) or urokinase (UK), in platelet-poor plasma, in platelet-rich plasma after platelet aggregation or in an extract of washed platelets was the same. Moreover, complexes of UK or t-PA with plasmatic PA-inhibitor or with the PA-inhibitor(s) from platelets bound to immobilized antibodies against bovine endothelial cell-derived PA-inhibitor. These results show that the PA-inhibitors in plasma and in platelets are very similar or identical.

Differential Role of Components of the Fibrinolytic System in the Formation and Removal of Thrombus Induced by Endothelial Injury

1999 ◽  
Vol 81 (04) ◽  
pp. 601-604 ◽  
Author(s):  
Hiroyuki Matsuno ◽  
Osamu Kozawa ◽  
Masayuki Niwa ◽  
Shigeru Ueshima ◽  
Osamu Matsuo ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Thrombus Formation ◽  
Endothelial Injury ◽  
Fibrinolytic System ◽  
Tissue Type ◽  
Clot Lysis ◽  
Wild Type ◽  
Type Plasminogen Activator ◽  
Pai 1

SummaryThe role of fibrinolytic system components in thrombus formation and removal in vivo was investigated in groups of six mice deficient in urokinase-type plasminogen activator (u-PA), tissue-type plasminogen activator (t-PA), or plasminogen activator inhibitor-1 (PAI-1) (u-PA-/-, t-PA-/- or PAI-1-/-, respectively) or of their wild type controls (u-PA+/+, t-PA+/+ or PAI-1+/+). Thrombus was induced in the murine carotid artery by endothelial injury using the photochemical reaction between rose bengal and green light (540 nm). Blood flow was continuously monitored for 90 min on day 0 and for 20 min on days 1, 2 and 3. The times to occlusion after the initiation of endothelial injury in u-PA+/+, t-PA+/+ or PAI-1+/+ mice were 9.4 ± 1.3, 9.8 ± 1.1 or 9.7 ± 1.6 min, respectively. u-PA-/- and t-PA-/- mice were indistinguishable from controls, whereas that of PAI-1-/- mice were significantly prolonged (18.4 ± 3.7 min). Occlusion persisted for the initial 90 min observation period in 10 of 18 wild type mice and was followed by cyclic reflow and reocclusion in the remaining 8 mice. At day 1, persistent occlusion was observed in 1 wild type mouse, 8 mice had cyclic reflow and reocclusion and 9 mice had persistent reflow. At day 2, all injured arteries had persistent reflow. Persistent occlusion for 90 min on day 0 was observed in 3 u-PA-/-, in all t-PA-/- mice at day 1 and in 2 of the t-PA-/-mice at day 2 (p <0.01 versus wild type mice). Persistent patency was observed in all PAI-1-/- mice at day 1 and in 5 of the 6 u-PA-/- mice at day 2 (both p <0.05 versus wild type mice). In conclusion, t-PA increases the rate of clot lysis after endothelial injury, PAI-1 reduces the time to occlusion and delays clot lysis, whereas u-PA has little effect on thrombus formation and spontaneous lysis.

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