P-selectin-targeting of the fibrin selective thrombolytic Desmodus rotundus salivary plasminogen activator α1

2004 ◽  
Vol 92 (11) ◽  
pp. 956-965 ◽  
Author(s):  
Ningzheng Dong ◽  
Valdeci Da Cunha ◽  
Andrej Citkowicz ◽  
Faye Wu ◽  
Jon Vincelette ◽  
...  
Keyword(s):  
Pulmonary Embolism ◽  
Fusion Protein ◽  
Plasminogen Activator ◽  
Therapeutic Potential ◽  
Bleeding Risk ◽  
Clot Lysis ◽  
Desmodus Rotundus ◽  
Dog Model

SummaryDuring thrombosis, P-selectin is expressed on the surface of activated endothelial cells and platelets. We hypothesized that targeting a plasminogen activator (PA) to P-selectin would enhance local thrombolysis and reduce bleeding risk. Previously, a urokinase (uPA)/anti-P-selectin antibody (HuSZ51) fusion protein was shown to increase fibrinolysis in a hamster pulmonary embolism model. To explore the therapeutic potential of this targeting strategy, we fused the fibrin-selective Desmodus rotundus salivary PA α1 (dsPAα1) to HuSZ51 and compared the fibrinolytic activity of P-selectin-targeted dsPAα1 (HuSZ51-dsPAα1) to unmodified dsPAα1 in vitro and in vivo. HuSZ51-dsPAα1 and dsPAα1 were expressed in CHO cells and purified to homogeneity by affinity chromatography. HuSZ51dsPAα1 bound to thrombin-activated human and dog platelets with comparable affinities to that of parental antibody SZ51. The fusion protein retained the catalytic activities of dsPAα1 in chromogenic and clot lysis assays, indicating that dsPAα1 is fully functional when fused to HuSZ51. Compared to dsPAα1, HuSZ51-dsPAα1 had similar thrombolytic efficacy in a rat pulmonary embolism model and anti-thrombotic potency in a dog model of femoral artery thrombosis. However, HuSZ51dsPAα1 was less effective in lysis of preexisting arterial thrombi in the dog model. The reduced arterial thrombolysis was not due to the pharmacokinetic properties of HuSZ51-dsPAα1 because antigen level and amidolytic activity were higher in plasma from HuSZ51-dsPAα1-treated groups than corresponding dsPAα1-treated groups. These data indicate that the thrombolytic efficacy of HuSZ51-dsPAα1 varied dependent on the physical composition of thrombi. The lack of stimulation by fibrin in arterial thrombi may contribute to the attenuated thrombolytic efficacy of HuSZ51-dsPAα1 in the dog model.

The Roles of α2-Antiplasmin and Plasminogen Activator Inhibitor 1 (PAI-1) in the Inhibition of Clot Lysis

1993 ◽  
Vol 70 (02) ◽  
pp. 301-306 ◽  
Author(s):  
Linda A Robbie ◽  
Nuala A Booth ◽  
Alison M Croll ◽  
Bruce Bennett
Keyword(s):  
Plasminogen Activator ◽  
Plasminogen Activator Inhibitor ◽  
Fibrin Clot ◽  
Clot Lysis ◽  
Plasminogen Activator Inhibitor 1 ◽  
Dependent Inhibition ◽  
Activator Inhibitor ◽  
Pai 1

SummaryThe relative importance of the two major inhibitors of fibrinolysis, α2-antiplasmin (α2-AP) and plasminogen activator inhibitor (PAI-1), were investigated using a simple microtitre plate system to study fibrin clot lysis in vitro. Cross-linked fibrin clots contained plasminogen and tissue plasminogen activator (t-PA) at concentrations close to physiological. Purified α2-AP and PAI-1 caused dose-dependent inhibition. All the inhibition due to normal plasma, either platelet-rich or poor, was neutralised only by antibodies to α2-AP. Isolated platelets, at a final concentration similar to that in blood, 2.5 × 108/ml, markedly inhibited clot lysis. This inhibition was neutralised only by antibodies to PAI-1. At the normal circulating ratio of plasma to platelets, α2-AP was the dominant inhibitor. When the platelet:plasma ratio was raised some 20-fold, platelet PAI-1 provided a significant contribution. High local concentrations of PAI-1 do occur in thrombi in vivo, indicating a role for PAI-1, complementary to that of α2-AP, in such situations.

A fusion protein with improved thrombolytic effect and low bleeding risk

2009 ◽  
Vol 102 (12) ◽  
pp. 1194-1203 ◽  
Author(s):  
Lisheng Wang ◽  
Qinglin Zhang ◽  
Yide Qin ◽  
Chutse Wu ◽  
Xiudong Wang ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Anticoagulant Activity ◽  
Bleeding Risk ◽  
Fibrin Clot ◽  
Clot Lysis ◽  
Recognition Sequence ◽  
Linker Peptide ◽  
C Terminus ◽  
N Terminus

SummaryTo resolve the therapeutic dilemma between efficacy of thrombolysis and bleeding risk associated with the use of a combination of thrombolytic and anticoagulant treatments, we created a fusion protein. Staphylokinase was fused to the N-terminus of hirudin using thrombin recognition sequence as linker peptide, resulting in a fusion protein STH.We hypothesised that STH would be cleaved by thrombin at the thrombus site, releasing staphylokinase and hirudin to perform bifunctionally, and attenuating bleeding risk. SDS-PAGE andWestern blot analyses indicated that the linker peptide could be specially recognised and cleaved by thrombin. Amidolytic and thromboelastogram assays showed that the N-terminus of hirudin in STH was blocked by staphylokinase and linker peptide, impeding hirudin’s anticoagulant activity. Once cleaved, STH displayed 35.7% of the anticoagulant activity of equimolar hirudin and exhibited anticoagulant effects in the fibrin clot lysis assay.Thrombin-binding and fibrin clot lysis assays showed that the C-terminus of hirudin retained its high affinity for thrombin. Moreover, STH showed improved thrombolytic effects and a lower bleeding risk in animals. Thus, STH may have the capacity to perform bifunctionally and release anticoagulant activity in a thrombus-targeted manner in vivo, which may reduce the bleeding risk that often accompanies high thrombolytic efficacy in the treatment of thromboembolic diseases.

scholarly journals Thrombolytic properties of Desmodus rotundus (vampire bat) salivary plasminogen activator in experimental pulmonary embolism in rats

Blood ◽  
1992 ◽  
Vol 79 (5) ◽  
pp. 1213-1217
Author(s):  
W Witt ◽  
B Baldus ◽  
P Bringmann ◽  
L Cashion ◽  
P Donner ◽  
...  
Keyword(s):  
Pulmonary Embolism ◽  
Plasminogen Activator ◽  
Tissue Type ◽  
Desmodus Rotundus ◽  
Type Plasminogen Activator ◽  
Blood Clots ◽  
Hemostatic Factors ◽  
The Difference ◽  
Vampire Bat

rDSPA alpha 1 (recombinant Desmodus salivary plasminogen activator alpha 1) is a recombinant protein corresponding to a natural plasminogen activator from the vampire bat Desmodus rotundus. The thrombolytic properties of rDSPA alpha 1 and tissue-type plasminogen activator (t-PA) were compared in a rat model of pulmonary embolism. Whole blood clots, produced in vitro and labeled with 125I-fibrinogen, were embolized into the lungs of anesthetized rats. Thrombolysis was calculated from the difference between initial clot radioactivity and that remaining in the lungs at 60 minutes. Blood was sampled for gamma counting, measurement of hemostatic factors, and plasminogen activator antigen levels. Thrombolysis at 3, 10, 30, and 100 nmol/kg intravenously (10% bolus, 90% over 60 minutes) amounted to 30% +/- 2%, 51% +/- 4%, 85% +/- 4%, 98% +/- 0% for rDSPA alpha 1 and 30% +/- 3%, 41% +/- 3%, 57% +/- 6%, 93% +/- 2% for t-PA (controls: 29% +/- 2%; mean +/- SEM, n greater than or equal to 6). t-PA at 100 nmol/kg significantly decreased fibrinogen, plasminogen, and alpha 2- antiplasmin levels by 33% +/- 7%, 38% +/- 8%, and 61% +/- 9%, whereas rDSPA alpha 1 at 100 nmol/kg only lowered alpha 2-antiplasmin significantly (by 29% +/- 6%). Compared with t-PA, rDSPA alpha 1 is the more potent and more clot selective (fibrin specific) thrombolytic agent. These results suggest that rDSPA alpha 1 may be safer and more efficacious than currently used thrombolytics.

scholarly journals Inhibition of clot lysis and decreased binding of tissue-type plasminogen activator as a consequence of clot retraction

Blood ◽  
1992 ◽  
Vol 79 (6) ◽  
pp. 1420-1427 ◽  
Author(s):  
S Kunitada ◽  
GA FitzGerald ◽  
DJ Fitzgerald
Keyword(s):  
Plasminogen Activator ◽  
Tissue Type ◽  
Clot Lysis ◽  
Plasminogen Activator Inhibitor 1 ◽  
Clot Retraction ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator ◽  
Pai 1

Tissue-type plasminogen activator (t-PA) is less active in vivo and in vitro against clots that are enriched in platelets, even at therapeutic concentrations. The release of radioactivity from 125I-fibrin-labeled clots was decreased by 47% 6 hours after the addition of t-PA 400 U/mL when formed in platelet-rich versus platelet-poor plasma. This difference was not due to the release of plasminogen activator inhibitor-1 (PAI-1) by platelets. Thus, the fibrinolytic activity of t- PA in the supernatant was similar in the two preparations and fibrin autography demonstrated only a minor degree of t-PA-PAI-1 complex formation. Furthermore, a similar platelet-dependent reduction in clot lysis was seen with a t-PA mutant resistant to inhibition by PAI-1. The reduction in t-PA activity correlated with a decrease in t-PA binding to platelet-enriched clot (60% +/- 3% v platelet-poor clot, n = 5). This reduction in binding was also shown using t-PA treated with the chloromethylketone, D-Phe-Pro-Arg-CH2Cl (PPACK) (36% +/- 13%, n = 3), and with S478A, a mutant t-PA in which the active site serine at position 478 has been substituted by alanine (43% +/- 6%, n = 3). In contrast, fixed platelets and platelet supernatants had no effect on the binding or lytic activity of t-PA. Pretreatment with cytochalasin D 1 mumol/L, which inhibits clot retraction, also abolished the platelet- induced inhibition of lysis and t-PA binding by platelets. These data suggest that platelets inhibit clot lysis at therapeutic concentrations of t-PA as a consequence of clot retraction and decreased access of fibrinolytic proteins.

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 Lack of interference by heparin with thrombolysis or binding of tissue- type plasminogen activator to thrombi

Blood ◽  
1988 ◽  
Vol 71 (5) ◽  
pp. 1347-1352 ◽  
Author(s):  
ET Fry ◽  
BE Sobel
Keyword(s):  
Plasminogen Activator ◽  
Plasma Concentrations ◽  
Concomitant Administration ◽  
Tissue Type ◽  
Clot Lysis ◽  
Chloromethyl Ketone ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator

Abstract Coronary thrombolysis with t-PA is generally implemented with concomitant administration of heparin. However, results of studies in vitro suggest that heparin competes with fibrin for binding of tissue- type plasminogen activator (t-PA), augments activation of free plasminogen, decreases fibrin specificity, and impairs thrombolysis. To define the biological implications of these observations, we characterized effects of therapeutic concentrations of heparin on the binding of t-PA to thrombi formed in whole blood, effects of heparin on activation of plasminogen by t-PA in plasma, and effects of heparin on thrombolysis induced by t-PA in a clot lysis system designed to simulate conditions in vivo. The amount of t-PA bound to thrombi was not affected by heparin (0, 0.5, 1.0, and 5.0 U/mL). When t-PA activity was selectively and irreversibly inhibited by D-Phe-Pro-Arg- chloromethyl ketone (PPACK) the amount of t-PA-PPACK bound was similarly unaffected by heparin. Thrombolysis measured by 125I- fibrin(ogen) release and by reduction of mass of thrombi were not altered by heparin. Heparin did not affect plasminogen consumption induced by t-PA. Plasma concentrations of alpha-2-antiplasmin after exposure of blood to t-PA were less depressed with increasing concentrations of heparin. Thus, heparin in therapeutic concentrations does not interfere with binding of t-PA to thrombi, augment activation of free plasminogen, or inhibit thrombolysis. Accordingly, it appears likely that concomitant administration of heparin will not impair thrombolysis with t-PA implemented clinically.

scholarly journals In Vitro Simulation of Extremely Activated Thrombolysis

2008 ◽  
Vol 14 (2) ◽  
pp. 238-240
Author(s):  
Thomas W. Stief
Keyword(s):  
Pulmonary Embolism ◽  
Plasminogen Activator ◽  
Microtiter Plate ◽  
Massive Pulmonary Embolism ◽  
Tissue Type ◽  
Clot Lysis ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator ◽  
Life Threatening

A life-threatening thrombus in massive pulmonary embolism has to be eliminated within minutes. Extremely activated plasmatic fibrinolysis destroys such thrombi in time: 50 µL plasma clots were incubated with urokinase or tissue-type plasminogen activator and 50 µL pooled normal plasma. The microtiter plate clot lysis assay was performed. The time point at which 50% of the clot has been lysed is 4 minutes for 8333 IU/mL urokinase or an equimolar concentration of tissue-type plasminogen activator (52498 IU/mL = 105 µg/mL). The effective dose 50% at 5 minutes lysis time is about 800 nM (4320 IU/mL) urokinase or (27220 IU/mL = 54 µg/mL) tissue-type plasminogen activator. Addition of plasminogen to the plasmatic clot supernatant improves thrombolysis if 65 IU/mL of urokinase acts for 10 minutes. The risk for severe intracranial hemorrhage in massive thrombolysis might be much lower than the lethality of a massive pulmonary embolism. Extremely activated plasmatic thrombolysis could be clinically indicated.

scholarly journals Inhibition of clot lysis and decreased binding of tissue-type plasminogen activator as a consequence of clot retraction

Blood ◽  
1992 ◽  
Vol 79 (6) ◽  
pp. 1420-1427 ◽  
Author(s):  
S Kunitada ◽  
GA FitzGerald ◽  
DJ Fitzgerald
Keyword(s):  
Plasminogen Activator ◽  
Tissue Type ◽  
Clot Lysis ◽  
Plasminogen Activator Inhibitor 1 ◽  
Clot Retraction ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator ◽  
Pai 1

Abstract Tissue-type plasminogen activator (t-PA) is less active in vivo and in vitro against clots that are enriched in platelets, even at therapeutic concentrations. The release of radioactivity from 125I-fibrin-labeled clots was decreased by 47% 6 hours after the addition of t-PA 400 U/mL when formed in platelet-rich versus platelet-poor plasma. This difference was not due to the release of plasminogen activator inhibitor-1 (PAI-1) by platelets. Thus, the fibrinolytic activity of t- PA in the supernatant was similar in the two preparations and fibrin autography demonstrated only a minor degree of t-PA-PAI-1 complex formation. Furthermore, a similar platelet-dependent reduction in clot lysis was seen with a t-PA mutant resistant to inhibition by PAI-1. The reduction in t-PA activity correlated with a decrease in t-PA binding to platelet-enriched clot (60% +/- 3% v platelet-poor clot, n = 5). This reduction in binding was also shown using t-PA treated with the chloromethylketone, D-Phe-Pro-Arg-CH2Cl (PPACK) (36% +/- 13%, n = 3), and with S478A, a mutant t-PA in which the active site serine at position 478 has been substituted by alanine (43% +/- 6%, n = 3). In contrast, fixed platelets and platelet supernatants had no effect on the binding or lytic activity of t-PA. Pretreatment with cytochalasin D 1 mumol/L, which inhibits clot retraction, also abolished the platelet- induced inhibition of lysis and t-PA binding by platelets. These data suggest that platelets inhibit clot lysis at therapeutic concentrations of t-PA as a consequence of clot retraction and decreased access of fibrinolytic proteins.

scholarly journals Efficacy of antiangiogenic targeted toxins against glioblastoma multiforme

2006 ◽  
Vol 20 (4) ◽  
pp. E23 ◽  
Author(s):  
Walter A. Hall ◽  
Daniel A. Vallera
Keyword(s):  
Endothelial Cells ◽  
Glioblastoma Multiforme ◽  
Fusion Protein ◽  
Plasminogen Activator ◽  
Systemic Exposure ◽  
Treatment Modalities ◽  
Target Tissue ◽  
Targeted Toxins

Object Because the prognosis for patients with glioblastoma multiforme (GBM) remains poor, investigators have focused on developing new and more effective treatment modalities. Targeted toxins represent a new class of compounds composed of a potent protein toxin and a carrier ligand that will recognize cell surface antigens located on target tissue. A recombinant fusion protein was created that contains the translocation and catalytic portions of diphtheria toxin that are responsible for cell entry and killing, respectively, fused to the noninternalizing aminoterminal fragment portion of human plasminogen activator. This diptheria toxin–uPA fusion protein (DTAT) has the advantage over other fusion proteins of targeting malignant glioma cells and the endothelial cells of the neovasculature that express the urokinase-type plasminogen activator receptor (uPAR). Another protein, DTAT13, was synthesized to target uPAR on the neovasculature and the uPAR and interleukin-13 receptor-expressing GBM cells. The authors describe the in vitro and in vivo efficacy of DTAT and DTAT13 against GBM. Methods The in vitro cytotoxicity of DTAT and DTAT13 was measured using cell proliferation assays. In vivo studies were performed in which DTAT, DTAT13, or a control protein was injected directly into GBM flank tumors in athymic nude mice. Tumor volume was assessed over time and analyzed using the Student t-test. The systemic organ effects of DTAT and DTAT13 were examined functionally and histologically in tumor-free C57BL/6 mice. In vitro, DTAT and DTAT13 were found to be highly potent and selective against U118MG, U87MG, and U373MG GBM cell lines and human umbilical vein endothelial cells. In vivo, DTAT and DTAT13 both caused a statistically significant (p < 0.05) regression of U87MG GBM flank tumors when administered every other day at 10 μg/day for five doses. No tumor regression was seen in control animals. Both DTAT and DTAT13 had little effect on histological findings in the liver, kidney, spleen, and lungs. Serum analysis did not demonstrate an effect on blood urea nitrogen levels, but liver alanine aminotransferase levels rose to statistically significant (p = 0.046) but not life-threatening levels. Also, DTAT13 was less toxic than DTAT in studies of mortality rates. Conclusions Both DTAT and DTAT13 might have potential for clinical application against GBM because of their ability to target both the tumor cells and neovasculature simultaneously with an absence of serious systemic side effects. The discovery that DTAT13 was less toxic than DTAT indicated that the bispecific fusion protein might target a broader subset of antigenetically diverse patients with tumors while reducing the systemic exposure to toxin that would be necessary if two agents were administered separately.

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