Characterization of Cell-Associated Plasminogen Activation Catalyzed by Urokinase-Type Plasminogen Activator, but Independent of Urokinase Receptor (uPAR, CD87)

Blood ◽  
1999 ◽  
Vol 93 (11) ◽  
pp. 3839-3846 ◽  
Author(s):  
Colin Longstaff ◽  
R. Elizabeth Merton ◽  
Pere Fabregas ◽  
Jordi Felez
Keyword(s):  
Molecular Weight ◽  
Leukemic Cell ◽  
Plasminogen Activation ◽  
Single Chain ◽  
Amino Terminal ◽  
Leukemic Cell Lines ◽  
Upa Receptor ◽  
Type Plasminogen Activator

Abstract The 55-kD urokinase (uPA) receptor (uPAR, CD87) is capable of binding uPA and may be involved in regulating cell-associated plasminogen activation and pericellular proteolysis. While investigating the relationship between uPAR levels and plasmin generation, we found that uPA-catalyzed plasminogen activation is stimulated by cells which do not express uPAR. This uPAR-independent mechanism appears to be at least as effective in vitro as uPAR-dependent stimulation, such that stimulation on the order of 30-fold was observed, resulting from improvements in both apparent kcat and apparent Km. The mechanism depends on simultaneous binding of both uPA and plasminogen to the cell and requires the presence of the amino-terminal fragment (ATF), available in single chain and two chain high-molecular-weight uPA, but not low-molecular-weight uPA. Stimulation was observed in all leukemic cell lines investigated at similar optimum concentrations of 106to 107 cells/mL and may be more general. A mechanism is proposed whereby uPA can associate with binding sites on the cell surface of lower affinity, but higher capacity than uPAR, but these are sufficient to stimulate plasmin generation even at subphysiologic uPA concentrations. This mechanism is likely to operate under conditions commonly used for in vitro studies and may have some significance in vivo.

Characterization of Cell-Associated Plasminogen Activation Catalyzed by Urokinase-Type Plasminogen Activator, but Independent of Urokinase Receptor (uPAR, CD87)

Blood ◽  
1999 ◽  
Vol 93 (11) ◽  
pp. 3839-3846 ◽  
Author(s):  
Colin Longstaff ◽  
R. Elizabeth Merton ◽  
Pere Fabregas ◽  
Jordi Felez
Keyword(s):  
Molecular Weight ◽  
Leukemic Cell ◽  
Plasminogen Activation ◽  
Single Chain ◽  
Amino Terminal ◽  
Leukemic Cell Lines ◽  
Upa Receptor ◽  
Type Plasminogen Activator

The 55-kD urokinase (uPA) receptor (uPAR, CD87) is capable of binding uPA and may be involved in regulating cell-associated plasminogen activation and pericellular proteolysis. While investigating the relationship between uPAR levels and plasmin generation, we found that uPA-catalyzed plasminogen activation is stimulated by cells which do not express uPAR. This uPAR-independent mechanism appears to be at least as effective in vitro as uPAR-dependent stimulation, such that stimulation on the order of 30-fold was observed, resulting from improvements in both apparent kcat and apparent Km. The mechanism depends on simultaneous binding of both uPA and plasminogen to the cell and requires the presence of the amino-terminal fragment (ATF), available in single chain and two chain high-molecular-weight uPA, but not low-molecular-weight uPA. Stimulation was observed in all leukemic cell lines investigated at similar optimum concentrations of 106to 107 cells/mL and may be more general. A mechanism is proposed whereby uPA can associate with binding sites on the cell surface of lower affinity, but higher capacity than uPAR, but these are sufficient to stimulate plasmin generation even at subphysiologic uPA concentrations. This mechanism is likely to operate under conditions commonly used for in vitro studies and may have some significance in vivo.

Sulforaphane Switches Cyclin E Isoform Expression and Blocks Leukemic Cell Cycling.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4405-4405
Author(s):  
Laura M. Gorham ◽  
Gigi Frye ◽  
Michelle Miranda ◽  
Richard A. Steinman
Keyword(s):  
Molecular Weight ◽  
Cyclin E ◽  
Leukemic Cell ◽  
Full Length ◽  
Leukemic Cells ◽  
Pharmacokinetic Studies ◽  
Leukemic Cell Lines ◽  
Myeloid Leukemic Cell

Abstract Sulforaphane (SFN), an isothiocyanate derived from cruciferous vegetables, has been shown to inhibit the growth of prostate cancer cells in vitro and in vivo. We were interested in exploring potential antileukemic effects of SFN. The viability of multiple myeloid leukemic cell lines was decreased by 25uM SFN. Pharmacokinetic studies reported in rats suggest that this serum concentration can be achieved through oral dosing. Lower SFN concentrations (1–5 uM) inhibited leukemic cell growth without affecting cell viability. Synchronized HL-60 cells exposed to 25uM SFN were blocked at the G1/S phase transition. Kinetic analysis of cell cycle proteins demonstrated that the G1/S block arose from downmodulation of cyclins D3 and E rather than upregulation of cdk-inhibitors. Interestingly, we found that HL-60 cells expressed a low molecular weight (LMW, 36 kD) variant of cyclin E rather than (50 kD) full-length cyclin E. Treatment with SFN for as little as 2 hours caused a decrease in expression of the LMW cyclin E and induced the expression of a higher molecular weight (~50 kD) cyclin E isoform. Because LMW cyclin E has been associated with increased cdk2 activity and p27 resistance compared to full-length cyclin E, we postulate that SFN-mediated cyclin E isoform-switching contributed to growth inhibition of these leukemic cells. The signaling pathway through which SFN altered cyclin E expression appeared to be distinct from MEK/ERK and JNK pathways that have been implicated in the apoptotic effects of SFN. Given that cyclin E overexpression and, particularly, LMW cyclin E expression are correlated with poor prognosis in multiple cancers, the mechanism through which SFN decreases LMW cyclin E expression in these leukemic cells could have therapeutic significance.

Interaction of Plasminogen Activators and Plasminogen with Heparin: Effect of Ionic Strength

1993 ◽  
Vol 70 (05) ◽  
pp. 867-872 ◽  
Author(s):  
Dingeman C Rijken ◽  
Gerard A W de Munk ◽  
Annie F H Jie
Keyword(s):  
Molecular Weight ◽  
Ionic Strength ◽  
Plasminogen Activator ◽  
Plasminogen Activators ◽  
Fibrinolytic System ◽  
Tissue Type ◽  
Single Chain ◽  
Physiological Conditions ◽  
Amino Terminal ◽  
Type Plasminogen Activator

SummaryIn order to define the possible effects of heparin on the fibrinolytic system under physiological conditions, we studied the interactions of this drug with plasminogen and its activators at various ionic strengths. As reported in recent literature, heparin stimulated the activation of Lys-plasminogen by high molecular weight (HMW) and low molecular weight (LMW) two-chain urokinase-type plasminogen activator (u-PA) and two-chain tissue-type plasminogen activator (t-PA) 10- to 17-fold. Our results showed, however, that this stimulation only occurred at low ionic strength and was negligible at a physiological salt concentration. Direct binding studies were performed using heparin-agarose column chromatography. The interaction between heparin and Lys-plasminogen appeared to be salt sensitive, which explains at least in part why heparin did not stimulate plasminogen activation at 0.15 M NaCl. The binding of u-PA and t-PA to heparinagarose was less salt sensitive. Results were consistent with heparin binding sites on both LMW u-PA and the amino-terminal part of HMW u-PA. Single-chain t-PA bound more avidly than two-chain t-PA. The interactions between heparin and plasminogen activators can occur under physiological conditions and may modulate the fibrinolytic system.

ON THE MECHANISM OF THE IN VIVO SYNERGISM BETWEEN TISSUE-TYPE PLASMINOGEN ACTIVATOR (t-PA) AND SINGLE CHAIN UROKINASE-TYPE PLASMINOGEN ACTIVATOR (scu-PA)

1987 ◽  
Author(s):  
J M Stassen ◽  
D Collen
Keyword(s):  
Plasminogen Activator ◽  
Rabbit Model ◽  
Sequential Therapy ◽  
Tissue Type ◽  
Single Chain ◽  
Molar Ratios ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator

t-PA and scu-PA, in molar ratios between 1:4 and 4:1 do not act synergically in vitro (Thromb. Haemost. 56,35,1986) but display marked synergism in a rabbit model (Circulation 74, 838, 1986) and in man (Am. Heart J. 112, 1083, 1986). To investigate the mechanism of in vivo synergism in the rabbit model (J. Clin. Invest. 71, 368, 1983), t-PA and scu-PA were infused 1) simultaneously over 4 hrs, 2) t-PA over 1 hr, then 15 min later scu-PA over 2 hrs and 3) scu-PA over 1 hr, then 15 min later t-PA over 2 hrs.Significant synergism on thrombolysis is observed when t-PA and scu-PA are infused simultaneously or when t-PA is followed by scu-PA but not when scu-PA is followed by t-PA. These results suggest that low dose t-PA induces some plasminogen activation, sufficient to partially degrade fibrin, exposing COOH-terminal lysines with high affinity for plasminogen (Eur. J. Biochem. 140, 513, 1984). scu-PA might then activate surface-bound Glu-pla-minogen more efficiently.Sequential therapy with t-PA (or any other agent which "predigests" the thrombus), followed by scu-PA might constitute an alternative to simultaneous infusion of synergistic thrombolytic agents.

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 Akacid Medical Formulation Induces Apoptosis in Myeloid and Lymphatic Leukemic Cell Lines In Vitro and In Vivo

PLoS ONE ◽  
2015 ◽  
Vol 10 (2) ◽  
pp. e0117806 ◽  
Author(s):  
Hannes Neuwirt ◽  
Elisabeth Wabnig ◽  
Clemens Feistritzer ◽  
Iris E. Eder ◽  
Christina Salvador ◽  
...  
Keyword(s):  
Cell Lines ◽  
Leukemic Cell ◽  
Leukemic Cell Lines

EVIDENCE FOR SYNERGISM BETWEEN TISSUE-TYPE PLASMINOGEN ACTIVATOR AND SINGLE CHAIN UROKINASE ON IN VITRO PLASMA CLOT LYSIS

1987 ◽  
Author(s):  
R S Rappaport ◽  
M R Blume ◽  
R L Vogel ◽  
M H Levner ◽  
P P Hung
Keyword(s):  
Plasminogen Activator ◽  
Tissue Type ◽  
Clot Lysis ◽  
Single Chain ◽  
Plasma Clot ◽  
Tissue Type Plasminogen Activator ◽  
Molar Ratios ◽  
Type Plasminogen Activator

There is mounting evidence from animal models and the clinic that combination thrombolytic therapy with tissue-type plasminogen activator (tPA) and single chain urokinase (scuPA) is synergistic. Yet, efforts to demonstrate synergism between these two plasminogen activators in vitro have met with discordant results. Collen et al (Thromb. Haemostasis, 56:35, 1986) reported an absence of synergism between these two agents on clot lysis in an in vitro plasma milieu when they were evaluated at molar ratios of 1:4 (tPA:scuPA and vice versa). Gurewich and Pannell (Thromb. Res., 44:217, 1986), however, reported a synergistic effect on fibrin-specific clot lysis in vitro when the agents were combined in concentrations exceeding molar ratios of 1:4 (tPA:scuPA). Here, we present evidence that synergism between tPA and scuPA may be demonstrated in vitro provided that the molar ratio of tPA to scuPA exceeds 1:4 and that the concentration of clot bound or unbound tPA is minimized. In order to achieve this experimental condition, the standard in vitro plasma clot lysis assay was modified. Human plasma clots were incubated first for a short time in plasma containing varying amounts of tPA. After incubation, the clots were washed thoroughly and reimmersed in plasma alone or in plasma containing varying amounts of scuPA or tPA. Under these conditions, lysis proceeded at a greater rate and to a greater extent when tPA clots were immersed in plasma containing an appropriate amount of scuPA than when they were immersed in plasma alone or in plasma containing appropriate amounts of tPA. Lysis of untreated clots or clots exposed first to scuPA and then to plasma containing varying amounts of scuPA proceeded far less efficiently with a characteristic lag. The enhanced lysis produced by tPA and scuPA obeyed the classical definition of synergy: the same biological effect can be obtained with two drugs together at algebraic fractional combinations of less than 1 (Berenbaum, M.C., Clin. Exp. Immunol., 28:1-18, 1977). Thus, conditions that more closely mimic the in vivo situation resulting from a bolus injection of tPA followed by infusion with scuPA, may provide a system for duplication of in vivo synergism in. vi tro and investigation of the mechanism thereof.

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.

scholarly journals The mechanism of plasminogen activation and fibrin dissolution by single chain urokinase-type plasminogen activator in a plasma milieu in vitro

Blood ◽  
1989 ◽  
Vol 73 (7) ◽  
pp. 1864-1872 ◽  
Author(s):  
HR Lijnen ◽  
B Van Hoef ◽  
F De Cock ◽  
D Collen
Keyword(s):  
Plasminogen Activator ◽  
Conformational Transition ◽  
Resistant Mutant ◽  
Fibrin Clot ◽  
Plasminogen Activation ◽  
Single Chain ◽  
Plasma Clot ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator

Abstract The relative contribution of several mechanisms to plasminogen activation and fibrin dissolution by urokinase-type plasminogen activator (u-PA) in vitro was quantitated. The activation of plasminogen by recombinant single chain u-PA (rscu-PA), by its two chain derivative (rtcu-PA) and by a plasmin-resistant mutant, rscu-PA- Glu158, obeys Michaelis-Menten kinetics with catalytic efficiencies of 0.00064, 0.046, and 0.00005 L/mumol.s for native plasminogen (Glu- plasminogen) and of 0.0061, 1.21, and 0.0004 L/mumol.s for partially degraded plasminogen (Lys-plasminogen). In a purified system consisting of a fibrin clot submerged in a plasminogen solution, the equi- effective doses (50% lysis in one hour) for rscu-PA, rtcu-PA, and rscu- PA-Glu158 were 16, 6.5, and 32,000 ng/mL for Glu-plasminogen and two- to fourfold lower for Lys-plasminogen. In a plasma milieu, 50% lysis in two hours was obtained for a plasma clot with 2.1 micrograms/mL rscu- PA, 0.5 micrograms/mL rtcu-PA, and greater than 200 micrograms/mL rscu- PA-Glu158 and for a purified fibrin clot with 1.3 micrograms/mL rscu-PA and 0.27 microgram/mL rtcu-PA. After predigestion of a purified fibrin clot with plasmin, the apparent potency of rscu-PA and rtcu-PA increased by 40% and 20%, respectively. In conclusion, rscu-PA has an intrinsic plasminogen activating potential that is only about 1% of that of rtcu-PA and that is 13 times higher than that of rscu-PA- Glu158. Conformational transition of Glu-plasminogen to Lys-plasminogen enhances its sensitivity to activation by all u-PA moieties ten- to 20- fold. Predigestion of fibrin clots with associated increased binding of plasminogen results in a minor apparent increase of the fibrinolytic potency of rscu-PA and rtcu-PA. The relative fibrinolytic potency of rtcu-PA is two to three orders of magnitude higher than that of rscu-PA- Glu158 but only two- to five-fold higher than that of rscu-PA, both in purified systems and in a plasma milieu. These results indicate that conversion of rscu-PA to rtcu-PA constitutes the primary mechanism of fibrin dissolution.

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