MECHANISM OF PLASMINOGEN ACTIVATION AT LOW TEMPERATURE IN PLASMA SAMPLES CONTAINING THERAPEUTIC CONCENTRATIONS OF t-PA

1987 ◽  
Author(s):  
D C Rijken ◽  
E Seifried ◽  
M M Barrett-Bergshoeff ◽  
C Kluft
Keyword(s):  
Time Course ◽  
Tissue Type ◽  
Plasminogen Activation ◽  
Plasma Samples ◽  
Rapid Reduction ◽  
Different Temperatures ◽  
Long Time ◽  
In Vitro Effects ◽  
C 50

It is known that plasminogen activation in blood samples taken during thrombolytic therapy with tissue-type plasminogen activator (t-PA) may continue during plasma handling, leading to artificially low fibrinogen (Fbg) and α2-antiplasmin (AP) values. Addition of D-Phe-Pro-Arg-CH2Cl or quenching antibodies against t-PA prevents this phenomenon, but these additions do not allow measurement of t-PA activity. The question of this study is, why the in vitro effects occur, even during freezing of the samples. Normal plasma was supplemented with various amounts of two-chain melanoma or recombinant t-PA and stored at -20°C, with and without a prior snap-freeze procedure. AP consumption (chromo-genic substrate assay) and Fbg degradation (Clauss method), measured in thawed samples, were most pronounced in the non snap-frozen samples. As it took a relatively long time before these samples were really frozen, the time course of the effects was studied at different temperatures. Plasma samples containing 1000 IU t-PA per ml were incubated at 37, 25,10, 0 and -8°C between 0 and 120 min. AP reduction was most rapid at 37°C (50% after 13 min), was less at 25°C (50% after 30 min), but did not further decrease at lower temperatures. The AP reduction at temperatures between 25 and -8°C corresponded to the effect of 40% t-PA activity at 37°C. The Fbg values gave a similar picture: the most rapid reduction occurred at 37°C, a slower reduction at 25°C, but no further reduction (even a small increase) was found from 25 to -8°C. The experiments were repeated in a purified system, consisting of t-PA, plasminogen, Fbg and AP. In contrast to the plasma system, AP reduction gradually decreased from 37 to 0°C. The apparent t-PA activity at 0°C was 6% of the activity at 37 °C.It is concluded that the in vitro effects in plasma samples containing t-PA can be, at least partially, explained by an abnormally strong plasminogen activation around 0°C. A normal temperature dependency in the purified system strongly suggests that unknown plasma factors enhance plasminogen activation at low temperatures.

Plasminogen Activation at Low Temperatures in Plasma Samples Gontaining Therapeutic Concentrations of Tissue-Type Plasm i nogen Activator or Other Thrombolytic Agents

1990 ◽  
Vol 64 (01) ◽  
pp. 047-052 ◽  
Author(s):  
D C Rijken ◽  
E Seifried ◽  
M M Barrett-Bergshoeffi ◽  
G Dooijewaard
Keyword(s):  
Thrombolytic Therapy ◽  
Low Temperatures ◽  
Incubation Temperature ◽  
Proteinase Inhibitors ◽  
Tissue Type ◽  
Plasminogen Activation ◽  
Plasma Samples ◽  
Blood Samples ◽  
Type Plasminogen Activator

SummaryIt is known that in vitro plasminogen activation in blood samples taken during thrombolytic therapy with tissue-type plasminogen activator (t-PA) may lead to artefactually low fibrinogen and α2-antiplasmin values. To mimic this phenomenon, pooled normal plasma was supplemented with 2.5 μg/ml t-PA and incubated at various temperatures. The rates of fibrinogen degradation and α2-antiplasmin consumption were most pronounced at 37° C, were less pronounced at 25° C, but surprisingly, did not further decrease at 10° C, 0° C or −8° C. In contrast, when plasma was supplemented with 10° IU/ml urokinase or 30 IU/ml streptokinase, the rates of fibrinogen degradation and α2-antiplasmin consumption gradually decreased with incubation temperature and were negligible at 10° C and lower temperatures. The rate of plasminogen activation also decreased gradually with temperature in mixtures of purified fibrinogen, plasminogeo, α2- antiplasmin and t-PA. These results imply that, in a plasma milieu, additional factors with a stimulatory activity are involved in t-PA-induced plasminogen activation at around 0° C. The abnormally high reaction rate at low temperatures explains in vitro plasminogen activation observed during the processing of t-PA-containing blood samples.In contrast to the activation of plasminogen by t-PA, the slow inhibition of t-PA (2.5 μg/ml) by proteinase inhibitors in plasma could be minimized to a negligible level by keeping the plasma samples at 0° C. This makes it possible to reliably monitor t-PA activity during thrombolytic therapy

In Vitro Effects of Urokinase — Prevention by Different Inhibitors

1990 ◽  
Vol 64 (03) ◽  
pp. 402-406 ◽  
Author(s):  
M D Oethinger ◽  
E Seifried
Keyword(s):  
In Vitro Study ◽  
Thrombin Time ◽  
Plasma Samples ◽  
Temperature Dependent ◽  
Chloromethyl Ketone ◽  
Control Value ◽  
Dose Time ◽  
In Vitro Effects ◽  
Full Protection

SummaryThe present in vitro study investigated dose-, time- and temperature-dependent effects of two-chain urokinase plasminogen activato(u-PA, urokinase) on normal citrated plasma. When 10 μg/ml u-PA wereadded to pooled normal plasma and incubated for 30 min at an ambient temperature (25° C), α2-antiplas-min decreased to 8% of the control value. Incubation on ice yielded a decrease to 45% of control,whereas α2-antiplasmin was fully consumed at 37° C. Fibrinogen and plasminogen fell to 46% and 39%, respectively, after a 30 min incubation at 25° C. Thrombin time prolonged to 190% of control.Various inhibitors were studied with respect to their suitability and efficacy to prevent these in vitro effects. Aprotinin exhibited a good protective effect on fibrinogen at concentrations exceeding 500 KlU/ml plasma. Its use, however, was limited due to interferences with some haemostatic assays. We could demonstrate that L-Glutamyl-L-Glycyl-L-Arginyl chloromethyl ketone (GGACK) and a specific polyclonal anti-u-PA-antibody (anti-u-PA-IgG) effectively inhibited urokinase-induced plasmin generation without interfering with haemostatic assays. The anti-u-PA-antibody afforded full protection ofα2-antiplasmin at therapeutic levels of u-PA.It is concluded that u-PA in plasma samples from patients during thrombolytic therapy may induce in vitro effects which should be prevented by the use of a suitable inhibitor such as GGACK or specific anti-u-PA-antibody.

Comparison of Specific Antibody, D-Phe-Pro-Arg-CH2Cl and Aprotinin for Prevention of In Vitro Effects of Recombinant Tissue-Type Plasminogen Activator on Haemostasis Parameters

1987 ◽  
Vol 58 (03) ◽  
pp. 921-926 ◽  
Author(s):  
E Seifried ◽  
P Tanswell
Keyword(s):  
Specific Antibody ◽  
Plasma Concentrations ◽  
Fibrinolytic Therapy ◽  
Tissue Type ◽  
Control Value ◽  
Type Plasminogen Activator ◽  
In Vitro Effects ◽  
Fibrinolytic Parameters

SummaryIn vitro, concentration-dependent effects of rt-PA on a range of coagulation and fibrinolytic assays in thawed plasma samples were investigated. In absence of a fibrinolytic inhibitor, 2 μg rt-PA/ml blood (3.4 μg/ml plasma) caused prolongation of clotting time assays and decreases of plasminogen (to 44% of the control value), fibrinogen (to 27%), α2-antiplasmin (to 5%), FV (to 67%), FVIII (to 41%) and FXIII (to 16%).Of three inhibitors tested, a specific polyclonal anti-rt-PA antibody prevented interferences in all fibrinolytic and most clotting assays. D-Phe-Pro-Arg-CH2Cl (PPACK) enabled correct assays of fibrinogen and fibrinolytic parameters but interfered with coagulometric assays dependent on endogenous thrombin generation. Aprotinin was suitable only for a restricted range of both assay types.Most in vitro effects were observed only with rt-PA plasma concentrations in excess of therapeutic values. Nevertheless it is concluded that for clinical application, collection of blood samples on either specific antibody or PPACK is essential for a correct assessment of in vivo effects of rt-PA on the haemostatic system in patients undergoing fibrinolytic therapy.

Perturbation of Platelet Adhesion to Endothelial Cells by Plasminogen Activation In Vitro

1997 ◽  
Vol 78 (02) ◽  
pp. 934-938 ◽  
Author(s):  
Hsiun-ing Chen ◽  
Yueh-I Wu ◽  
Yu-Lun Hsieh ◽  
Guey-Yueh Shi ◽  
Meei-Jyh Jiang ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Platelet Adhesion ◽  
Treatment Time ◽  
Shape Change ◽  
Umbilical Vein ◽  
Tissue Type ◽  
Apical Surface ◽  
Plasminogen Activation

SummaryTo investigate whether the endothelium-platelet interactions may be altered by plasminogen activation, cultured human umbilical vein endothelial cells (ECs) were treated with tissue-type plasminogen activator (t-PA) in the presence of plasminogen, and platelet adhesion to ECs was subsequently measured by using a tapered flow chamber. Our results demonstrated that platelets adhered more readily to t-PA treated EC monolayer than to the control monolayer at all shear stress levels tested. This phenomenon was treatment time-dependent and dose-dependent, and it could be blocked by adding plasmin inhibitors, such as e-amino caproic acid and aprotinin. Adherent platelets on t-PA treated EC monolayer underwent more severe shape change than those on the control monolayer. While the extracellular matrix directly treated with t-PA attracted less platelets than the control matrix did, platelet adhesion to the matrix that was produced by t-PA-treated ECs was unaltered. These data suggest that t-PA treatment on ECs compromised antiplatelet-adhesion capability on their apical surface without altering the reactivity of their extracellular matrix towards platelets.

Assay of Functional Plasminogen in Rat Plasma Applicable to Experimental Studies of Thrombolysis

2000 ◽  
Vol 84 (08) ◽  
pp. 299-306 ◽  
Author(s):  
Kristian Bangert ◽  
Sixtus Thorsen
Keyword(s):  
Plasminogen Activator ◽  
Experimental Studies ◽  
Fold Increase ◽  
Rat Plasma ◽  
Tissue Type ◽  
Plasminogen Activation ◽  
Type Plasminogen Activator ◽  
Plasmin Inhibitor

SummaryAn improved sensitive, specific, precise and accurate assay of plasminogen in rat plasma was developed. It is performed in 96-well microtiter plates and can be completed within one hour. The assay is based on activation of plasminogen by human urokinase-type plasminogen activator (uPA) and simultaneous measurement of generated plasmin with the specific plasmin substrate H-D-Val-Phe-Lys-4-nitroanilide (S-2390), using purified native rat plasminogen for calibration. The concentration of S-2390 in the final reaction mixture during the whole reaction period is much greater than the K m value (≈20 µM) for rat plasmin-cleavage of S-2390 ensuring that hydrolysis of substrate follows zero order kinetics and that the substrate produces a 20-35 fold decrease in rate of inhibition of plasmin by its target inhibitors in plasma. Analogous to the human system the target plasma inhibitors of rat plasmin are shown to be plasmin inhibitor and α-macroglobulins. Tranexamic acid (0.8 mM) is incorporated in the reaction mixture resulting in a 19-fold increase in the rate of plasminogen activation and presumably an about 50-fold decrease in the rate of inhibition of generated plasmin by plasmin inhibitor. The assay is suitable for accurate measurement of plasminogen in samples obtained from animals containing pharmacological concentrations of uPA or tissue-type plasminogen activator (tPA) in their plasma when in vitro plasminogen activation is blocked at pH 5 by collecting blood in acidic anticoagulant. Judged from in vitro experiments formation of catalytic active plasmin-α-macroglobulin complexes during massive activation of plasminogen in vivo does not interfere with the assay.

scholarly journals Muscle fatigue examined at different temperatures in experiments on intact mammalian (rat) muscle fibers

2009 ◽  
Vol 106 (2) ◽  
pp. 378-384 ◽  
Author(s):  
H. Roots ◽  
G. Ball ◽  
J. Talbot-Ponsonby ◽  
M. King ◽  
K. McBeath ◽  
...  
Keyword(s):  
Muscle Fatigue ◽  
Power Output ◽  
Force Depression ◽  
Output Force ◽  
Different Temperatures ◽  
Force Velocity ◽  
C 30 ◽  
C 50 ◽  
Shortening Contractions

In experiments on small bundles of intact fibers from a rat fast muscle, in vitro, we examined the decline in force in repeated tetanic contractions; the aim was to characterize the effect of shortening and of temperature on the initial phase of muscle fatigue. Short tetanic contractions were elicited at a control repetition rate of 1/60 s, and fatigue was induced by raising the rate to 1/5 s for 2–3 min, both in isometric mode (no shortening) and in shortening mode, in which each tetanic contraction included a ramp shortening at a standard velocity. In experiments at 20°C ( n = 12), the force decline during a fatigue run was 25% in the isometric mode but was significantly higher (35%) in the shortening mode. In experiments at different temperatures (10–30°C, n = 11), the tetanic frequency and duration were adjusted as appropriate, and for shortening mode, the velocity was adjusted for maximum power output. In isometric mode, fatigue of force was significantly less at 30°C (∼20%) than at 10°C (∼30%); the power output (force × velocity) was >10× higher at 30°C than at 10°C, and power decline during a fatigue run was less at 30°C (∼20–30%) than at 10°C (∼50%). The finding that the extent of fatigue is increased with shortening contractions and is lower at higher temperatures is consistent with the view that force depression by inorganic phosphate, which accumulates within fibers during activity, may be a primary cause of initial muscle fatigue.

scholarly journals Dependence of plasmin-mediated degradation of platelet adhesive receptors on temperature and Ca2+

Blood ◽  
1990 ◽  
Vol 76 (8) ◽  
pp. 1546-1557
Author(s):  
KJ Winters ◽  
PR Eisenberg ◽  
AS Jaffe ◽  
SA Santoro
Keyword(s):  
Platelet Activation ◽  
Direct Consequence ◽  
Tissue Type ◽  
Plasminogen Activation ◽  
Membrane Glycoproteins ◽  
Type Plasminogen Activator ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Marked Dependence

The effects of activation of plasminogen by streptokinase and tissue- type-plasminogen activator on platelet activation and the membrane glycoproteins (GPs) that mediate platelet adhesion and aggregation are not yet fully defined. To clarify effects on platelets during activation of plasminogen in vitro, we used monoclonal antibodies (MoAbs), flow cytometry, and platelets surface-labeled with 125I to characterize changes in receptors for fibrinogen (GPIIb-IIIa), von Willebrand factor (GPIb), and collagen (GPIa-IIa). Activation of plasminogen in plasma with pharmacologic concentrations of plasminogen activators did not degrade GPIIb-IIIa or GPIb, and caused only a modest decrease in GPIa. In washed platelets GPIIb-IIIa was extensively degraded by plasmin at 37 degrees C in the absence of exogenous Ca2+, conditions that destabilize the IIb-IIIa complex. Degradation of GPIb in washed platelets displayed a similar although less-marked dependence on temperature and the absence of Ca2+. The binding of activation- specific MoAbs did not increase during activation of plasminogen in plasma. We conclude that during pharmacologic fibrinolysis, reported inhibition of platelet function in plasma is not due to degradation of platelet-adhesive receptors. In addition, platelet activation observed during thrombolytic therapy does not appear to be a direct consequence of plasminogen activation.

scholarly journals A monoclonal antibody preventing binding of tissue-type plasminogen activator to fibrin: useful to monitor fibrinogen breakdown during t-PA infusion

Blood ◽  
1986 ◽  
Vol 67 (5) ◽  
pp. 1482-1487 ◽  
Author(s):  
P Holvoet ◽  
HR Lijnen ◽  
D Collen
Keyword(s):  
Plasminogen Activator ◽  
Tissue Type ◽  
Clot Lysis ◽  
Plasminogen Activation ◽  
Plasma Clot ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator ◽  
Additional Support

Abstract One (MA-1C8) of 36 monoclonal antibodies obtained by fusion of P3X63- Ag8–6.5.3 myeloma cells with spleen cells of mice immunized with purified human tissue-type plasminogen activator (t-PA) blocked the activity of t-PA on fibrin plates but not on chromogenic substrates. MA- 1C8 at a concentration of 200 micrograms/mL inhibited plasma clot lysis and binding of t-PA to the clot. MA-1C8 had no influence on the activation of plasminogen by t-PA, which obeys Michaelis-Menten kinetics with Km = 105 mumol/L and kcat = 0.05 s-1; however, it abolished the influence of CNBr-digested fibrinogen on Km. These findings confirm that the stimulatory effect of fibrin on the activation of plasminogen by t-PA is mediated by binding of t-PA to fibrin and provide additional support for the kinetic model. Addition of t-PA to pooled fresh human plasma to a concentration of 5 micrograms/mL resulted in extensive fibrinogen breakdown after incubation for one hour at 37 degrees C or during storage at -20 degrees C for one day. In both instances, fibrinogen degradation was completely prevented by addition of MA-1C8 to a concentration of 200 micrograms/mL of plasma. MA-1C8 also effectively prevented in vitro fibrinogen degradation and in vitro plasminogen activation in plasma samples obtained during infusion of recombinant t-PA in patients with thromboembolic disease. Thus, MA-1C8 is a useful tool for discriminating between in vivo and in vitro fibrinolysis during thrombolytic therapy with t-PA.

IN VIVO MUSCLE LENGTH CHANGES IN BUMBLEBEES AND THE IN VITRO EFFECTS ON WORK AND POWER

1993 ◽  
Vol 183 (1) ◽  
pp. 101-113 ◽  
Author(s):  
K. M. Gilmour ◽  
C. P. Ellington
Keyword(s):  
High Speed ◽  
Time Course ◽  
Second Harmonic ◽  
Muscle Length ◽  
Muscle Fibres ◽  
Bombus Lucorum ◽  
Length Changes ◽  
In Vitro Effects

The amplitude and time course of muscle length changes were examined in vivo in tethered, flying bumblebees Bombus lucorum. A ‘window’ was cut in the dorsal cuticle and aluminium particles were placed on the exposed dorsal longitudinal muscle fibres. Muscle oscillations were recorded using high-speed video and a high-magnification lens. The amplitude of muscle length changes was 1.9 % (s.d.=0.5 %, N=7), corresponding to the commonly quoted strain of 1–3 % for asynchronous muscle. Higher harmonics, particularly the second, were found in the muscle oscillations and in the wing movements. The second harmonic for wing movements was damped in comparison to that for muscle length changes, probably as a result of compliance in the thoracic linkage. Inclusion of the second harmonic in the driving signal for in vitro experiments on glycerinated fibres generally resulted in a decrease in the work and power, but a substantial increase was found for some fibres.

Effect of external Ca2+ concentration on stretch-augmented natriuretic peptide secretion by rat atria

1991 ◽  
Vol 260 (4) ◽  
pp. C756-C762 ◽  
Author(s):  
E. Page ◽  
J. Upshaw-Earley ◽  
G. E. Goings ◽  
D. A. Hanck
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Natriuretic Peptide ◽  
Time Course ◽  
Rate Coefficient ◽  
Time Dependent ◽  
Dependent Manner ◽  
Dependent Inactivation ◽  
Long Time ◽  
Peptide Secretion

An in vitro noncontracting rat atrial preparation stretched at 37 degrees C by a distending pressure of 5.1 mmHg was used to examine effects of external Ca2+ concentration ([Ca2+]out, 0.05-3.0 mM) on secretion of immunoreactive atrial natriuretic peptide (ANP) in presence of saxitoxin (STX) and in presence or absence of ryanodine. Under these conditions, the time course of the amount (y) of ANP secreted per milligram dry atrium during 44 min could be approximated by a rate coefficient (k) according to the relation y = s[1 - e(-kt)], where s is the maximal amount secreted after a long time (t). Although k, the rate coefficient for stretch-augmented secretion, increased significantly as [Ca2+]out was raised, secretion inactivated progressively in a time- and [Ca2+]out-dependent manner. This time-dependent decrease was not prevented by ryanodine. We conclude that a component of ANP secreted by quiescent atria in vitro is positively modulated by [Ca2+]out and does not require ryanodine-sensitive Ca2+ release from sarcoplasmic reticulum. The [Ca2+]out-sensitive processes underlying time-dependent inactivation of secretion remain undetermined.

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