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.

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.

scholarly journals Natural heterogeneity of α2-antiplasmin: functional and clinical consequences

Blood ◽  
2016 ◽  
Vol 127 (5) ◽  
pp. 538-545 ◽  
Author(s):  
Shiraazkhan Abdul ◽  
Frank W. G. Leebeek ◽  
Dingeman C. Rijken ◽  
Shirley Uitte de Willige
Keyword(s):  
Fibrin Clot ◽  
Clot Lysis ◽  
Thrombotic Risk ◽  
Amino Terminal ◽  
Inhibitory Function ◽  
C Terminus ◽  
N Terminus ◽  
Clinical Consequences ◽  
Carboxyl Terminal ◽  
Plasmin Inhibitor

AbstractHuman α2-antiplasmin (α2AP, also called α2-plasmin inhibitor) is the main physiological inhibitor of the fibrinolytic enzyme plasmin. α2AP inhibits plasmin on the fibrin clot or in the circulation by forming plasmin-antiplasmin complexes. Severely reduced α2AP levels in hereditary α2AP deficiency may lead to bleeding symptoms, whereas increased α2AP levels have been associated with increased thrombotic risk. α2AP is a very heterogeneous protein. In the circulation, α2AP undergoes both amino terminal (N-terminal) and carboxyl terminal (C-terminal) proteolytic modifications that significantly modify its activities. About 70% of α2AP is cleaved at the N terminus by antiplasmin-cleaving enzyme (or soluble fibroblast activation protein), resulting in a 12-amino-acid residue shorter form. The glutamine residue that serves as a substrate for activated factor XIII becomes more efficient after removal of the N terminus, leading to faster crosslinking of α2AP to fibrin and consequently prolonged clot lysis. In approximately 35% of circulating α2AP, the C terminus is absent. This C terminus contains the binding site for plasmin(ogen), the key component necessary for the rapid and efficient inhibitory mechanism of α2AP. Without its C terminus, α2AP can no longer bind to the lysine binding sites of plasmin(ogen) and is only a kinetically slow plasmin inhibitor. Thus, proteolytic modifications of the N and C termini of α2AP constitute major regulatory mechanisms for the inhibitory function of the protein and may therefore have clinical consequences. This review presents recent findings regarding the main aspects of the natural heterogeneity of α2AP with particular focus on the functional and possible clinical implications.

Construction and functional evaluation of hirudin derivatives with low bleeding risk

2008 ◽  
Vol 99 (02) ◽  
pp. 324-330 ◽  
Author(s):  
Bin Yuan ◽  
Chunna Dong ◽  
Hongyang Yu ◽  
Lisheng Wang ◽  
Chuanling Zhang ◽  
...  
Keyword(s):  
Anticoagulant Activity ◽  
Vena Cava ◽  
Coagulation Factor ◽  
Bleeding Risk ◽  
Coagulation System ◽  
Bleeding Complications ◽  
Functional Evaluation ◽  
Thrombin Time ◽  
N Terminus

SummaryThe purpose of this study was to design and evaluate hirudin (HIR) derivatives with low bleeding risk. In these derivatives, the factor (F) XIa, FXa, and thrombin recognition peptides (EPR, GVYAR, and LGPR, respectively) were linked to the N-terminus of HIR. The intact derivatives have no anticoagulant activity because of the extension of the N-terminus of HIR. After cleavage by the corresponding coagulation factor that occurs on the activation of the coagulation system and in the presence of the thrombus, its activity is released. This limited the anticoagulant activity of these derivatives to the vicinity of the thrombus, and as a result, systemic bleeding complications were avoided. The definite antithrombotic effect and low bleeding parameters of these derivatives were investigated in rat carotid artery and inferior vena cava thrombosis models. In both models, the three derivatives showed significant antithrombotic effects, indicating that anticoagulant activity could be successfully released in vivo. Moreover, the bleeding parameters of these derivatives were lower than that of HIR as indicated by the values of activated partial thromboplastin time (APTT) and thrombin time (TT). To further assess the safety of these derivatives, bleeding time was measured in a mouse tail-cut model. Although the derivatives had obvious effects on bleeding at a dose of 6 mg/kg, the effect of these derivatives on bleeding was significantly weaker than that of HIR at a dose of 1.5 mg/kg. Thus, the benefit-to-risk profiles of the derivatives were superior to that of HIR.

scholarly journals Fibrinogenolysis by a neutrophil membrane protease generates an A α 1–21 fragment

1994 ◽  
Vol 298 (3) ◽  
pp. 689-695 ◽  
Author(s):  
S L Kelly ◽  
S A Adams ◽  
S C Robson ◽  
R E Kirsch ◽  
E G Shephard
Keyword(s):  
Amino Acids ◽  
Molecular Mass ◽  
Anticoagulant Activity ◽  
Acute Phase Reactant ◽  
Apparent Molecular Mass ◽  
Gamma Chain ◽  
Reactive Protein ◽  
C Terminus ◽  
N Terminus

The 600 kDa neutrophil membrane neutral protease, which had been shown to generate bioactive peptides from the acute-phase reactant C-reactive protein, has now been shown to have fibrinogenolytic activity that is distinct from fibrinogenolysis by plasmin and neutrophil lysosomal enzymes. This protease gradually reduces the apparent molecular mass of fibrinogen (340 kDa) to non-clottable products and generates terminal products with apparent molecular mass values of 270 kDa, 200 kDa, 100 kDa and less than 40 kDa through cleavage of all three of the constituent chains. Characteristics of fibrinogenolysis by this neutrophil protease are cleavage of the bond between amino acids valine and glutamic acid at positions 21 and 22 respectively from the N-terminus of the A alpha chain to release an A alpha 1-21 peptide, digestion of the B beta chain at positions within the C-terminus, and proteolysis of the bond between amino acids isoleucine and glycine at positions 394 and 395 respectively from the N-terminus of the gamma chain. This generates products that lack anticoagulant activity. The thrombin clotting time of the product with an apparent molecular mass of 330 kDa was prolonged, although clot formation was still observed. Loss of coagulability and inability to clot was found with further degradation of fibrinogen to an apparent molecular mass of 290 kDa. Activity of this neutrophil membrane protease in vivo could be important for the regulation of fibrin deposition at sites of inflammation, and may contribute to the reported plasma levels of the A alpha 1-21 peptide.

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.

scholarly journals Different expression levels of two KgmB-His fusion proteins

2005 ◽  
Vol 70 (12) ◽  
pp. 1401-1407 ◽  
Author(s):  
Sandra Markovic ◽  
Sandra Vojnovic ◽  
Milija Jovanovic ◽  
Branka Vasiljevic
Keyword(s):  
Recombinant Proteins ◽  
Fusion Proteins ◽  
Kanamycin Resistance ◽  
Expression Vectors ◽  
E Coli ◽  
C Terminus ◽  
N Terminus ◽  
Different Levels ◽  
Streptomyces Tenebrarius

The KgmB methylase from Streptomyces tenebrarius was expressed and purified using the QIAexpress System. Two expression vectors were made: pQEK-N, which places a (His)6 tag at the N-terminus, and pQEK-C, which places a (His)6 tag at the C-terminus of the recombinant KgmB protein. Kanamycin resistance of the E. coli cells containing either the pQEK-N or the pQEK-C recombinant plasmids confirmed the functionality of both KgmB-His fusion proteins in vivo. Interestingly, different levels of expression were observed between these two recombinant proteins. Namely, KgmB methylase with the (His)6 tag at the N-terminus showed a higher level of expression. Purification of the (His)6-tagged proteins using Ni-NTA affinity chromatography was performed under native conditions and the KgmB methylase with (His)6 tag at the N-terminus was purified to homogeneity >95 %. The recombinant KgmB protein was detected on a Western blot using anti-Sgm antibodies.

Cdc18p can block mitosis by two independent mechanisms

1998 ◽  
Vol 111 (20) ◽  
pp. 3101-3108 ◽  
Author(s):  
E. Greenwood ◽  
H. Nishitani ◽  
P. Nurse
Keyword(s):  
Dna Replication ◽  
S Phase ◽  
Checkpoint Control ◽  
Replication Checkpoint ◽  
Mitotic Kinase ◽  
C Terminus ◽  
N Terminus ◽  
Dna Replication Checkpoint ◽  
Checkpoint Genes

The DNA replication checkpoint is required to maintain the integrity of the genome, inhibiting mitosis until S phase has been successfully completed. The checkpoint preventing premature mitosis in Schizosaccharomyces pombe relies on phosphorylation of the tyrosine-15 residue on cdc2p to prevent its activation and hence mitosis. The cdc18 gene is essential for both generating the DNA replication checkpoint and the initiation of S phase, thus providing a key role for the overall control and coordination of the cell cycle. We show that the C terminus of the protein is capable of both initiating DNA replication and the checkpoint function of cdc18p. The C terminus of cdc18p acts upstream of the DNA replication checkpoint genes rad1, rad3, rad9, rad17, hus1 and cut5 and requires the wee1p/mik1p tyrosine kinases to block mitosis. The N terminus of cdc18p can also block mitosis but does so in the absence of the DNA replication checkpoint genes and the wee1p/mik1p kinases therefore acting downstream of these genes. Because the N terminus of cdc18p associates with cdc2p in vivo, we suggest that by binding the cdc2p/cdc13p mitotic kinase directly, it exerts an effect independently of the normal checkpoint control, probably in an unphysiological manner.

tPA Point Mutation at Autolysis Loop Enhances Resistance to PAI-1 Inhibition and Catalytic Activity

2018 ◽  
Vol 119 (01) ◽  
pp. 077-086 ◽  
Author(s):  
Shuangzhou Peng ◽  
Guangpu Xue ◽  
Shanli Chen ◽  
Zhuo Chen ◽  
Cai Yuan ◽  
...  
Keyword(s):  
Thrombolytic Therapy ◽  
Catalytic Efficiency ◽  
Bleeding Risk ◽  
Tissue Type ◽  
Clot Lysis ◽  
High Dose ◽  
Thrombolytic Activity ◽  
Serine Protease Domain ◽  
Pai 1

AbstractRecombinant tissue-type plasminogen activator (r-tPA) was approved by U.S. Food and Drug Administration as a thrombolytic drug. However, a high dose of r-tPA (up to 100 mg/person) is typically used in clinical applications. Such high dosage leads to severe side effects including haemorrhage and neurotoxicity, which can be fatal. To improve the proteolytic properties of tPA to enhance thrombolytic therapy, we designed a series of mutants in tPA serine protease domain (tPA-SPD) based on the crystal structure of tPA-SPD:plasminogen activators inhibitor-1 (PAI-1) complex that we determined recently. We found that the A146Y substitution in tPA-SPD(A146Y) enhanced resistance to PAI-1 inactivation by 30-fold compared with original tPA-SPD. Interestingly, the tPA-SPD(A146Y) variant showed fivefold higher activation for plasminogen compared with tPA-SPD. The variant also demonstrated thrombolytic activity stronger than tPA-SPD in a clot lysis assay. In vivo, we showed tPA-SPD(A146Y) possessed higher thrombolytic efficacy in a pulmonary embolism model compared with original tPA-SPD. Furthermore, a mouse tail bleeding assay showed that tPA-SPD(A146Y) did not increase bleeding risk compared with clinical drug r-tPA. Together, our findings reveal novel functions of A146Y variant, which not only increases the catalytic efficiency of the enzyme, but also enhances resistance to PAI-1 inhibition, and demonstrating that tPA-SPD (A146Y) variant is a much improved agent for thrombolytic therapy.

Pharmacological Characterization and Structure Activity Relationship of FXI Antisense Oligonucleotides in Cynomolgus Monkeys.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2101-2101
Author(s):  
Robert MacLeod ◽  
Jeff Crosby ◽  
Chenguang Zhao ◽  
Dacao Gao ◽  
Chris May ◽  
...  
Keyword(s):  
Antisense Oligonucleotides ◽  
Anticoagulant Activity ◽  
Bleeding Risk ◽  
Intrinsic Pathway ◽  
Antithrombotic Agents ◽  
Pharmacological Characterization ◽  
Protein Levels ◽  
Intravascular Thrombosis ◽  
Single Chemical

Abstract Abstract 2101 Poster Board II-78 The absence of abnormal bleeding associated with congenital deficiencies of the intrinsic coagulation pathway factor, FXI, suggests that this pathway is not important for normal blood coagulation in vivo. However, recent work in mice and higher species demonstrates that the intrinsic pathway is an important contributor to pathologic intravascular thrombosis, suggesting that targeting this pathway may yield effective antithrombotic agents with high safety. We have previously demonstrated that second generation antisense oligonucleotides (ASOs) targeting the intrinsic pathway member FXI were potent antithrombotic agents and that this activity was achieved in several mouse models of thrombosis without any increase in bleeding risk (Zhang et al. ASH 2008). Here we characterize the pharmacological activity, chemical SAR and safety profile of FXI antisense oligonucleotides administered subcutaneously in Cynomologus monkeys. Our first monkey study was designed to address the PK/PD kinetics of FXI ASOs from a single chemical series (20 mers, 5-10-5 MOE Gapmers). Two dose escalation regimes were evaluated (1) 5mg/kg (3wks), 10mg/kg (3 wks) followed by 25 mg/kg (6wks) and (2) 5mg/kg (3wks) followed by 10mg/kg (9 wks). The subsequent study was designed to evaluate chemical SAR around the active ASOs identified in the study 1. FXI antisense oligonucleotides FXI-AS1, FXI-AS2 demonstrated dose and time dependent pharmacologic activity, including, decreased FXI mRNA in liver (up to 90%), decreased FXI protein levels and FXI activity measured in plasma, with a maximal inhibition of >80% observed at 25mg/kg on both schedules, prolonged activated partial thromboplastin times (aPTT), maximal aPTT ratio of 2.O, but no change in prothrombin time (PT) as expected. Similar to previous studies in mouse the anticoagulant activity observed from FXI depletion in monkeys was not associated with increased bleeding risk as assessed in a skin bleeding time test. Additionally, FXI ASOs had no deleterious effects on organ weights, platelets, or on measures of liver and kidney function. The second study confirmed the effects of FXI-AS1 and FXI-AS2 and demonstrated that shorter ASOs with decreased gap size were equally potent. These results further support the development of FXI ASOs as human therapeutics for the treatment of coagulation related disorders with the potential for improved safety profiles. Disclosures: MacLeod: Isis Pharmaceuticals: Employment. Crosby:Isis Pharmaceuticals, Inc.: Employment. Zhao:Isis Pharmaceuticals, Inc.: Employment. Gao:Isis Pharmaceuticals, Inc.: Employment. May:Isis Pharmaceuticals, Inc.: Employment. Zhang:Isis Pharmaceuticals, Inc.: Consultancy. Lowenberg:Isis Pharmaceuticals, Inc.: Consultancy. Levi:Isis Pharmaceuticals, Inc.: Consultancy. Monia:Isis Pharmaceuticals, Inc.: Employment.

Synergistic Effects of Bifunctional Antibodies Against beta3 Integrin on Dissolution of Platelet Thrombus,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3350-3350
Author(s):  
Wei Zhang ◽  
Suying Dang ◽  
Thomas Wisniewski
Keyword(s):  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Synergistic Effects ◽  
Fibrin Clot ◽  
Clot Lysis ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Platelet Thrombus ◽  
Platelet Aggregates

Abstract Abstract 3350 HIV-ITP patients have a unique Ab against platelet GPIIIa49-66 which induces oxidative platelet fragmentation in the absence of complement (Cell 106: 551, 2001; JCI 113: 973, 2004). Using a phage display single-chain antibody (scFv) library, we developed a novel human monoclonal scFv Ab against GPIIIa49-66 (named A11), which act similarly to the parental Ab (JBC 283: 3224, 2008). We then produced a bifunctional GPIIIa49-66 agent (named SLK), that targets newly deposited fibrin strands within and surrounding the platelet thrombus and has reduced effects on non-activated circulating platelets (Blood 116: 2336, 2010). In this study, we produced another bifunctional GPIIIa49-66 agent (named APAC), which homes to activated platelets. Like SLK, APAC destroys platelet aggregates ex vivo in an identical fashion with ∼85% destruction of platelet aggregates at 2 hrs. Platelet aggregate dissolution with a combination of SLK and APAC was ∼2 fold greater than either agent alone at 0.025 μM. Platelet-rich clot lysis experiments demonstrated the time required for 50% platelet-rich fibrin clot lysis (T50%) by APAC (95±6.1 min) was significantly longer than that by APAC+SLK (65±7.6 min) at a final concentration of 0.025 μM (APAC+SLK vs APAC, p<0.01). In comparison with APAC alone, the T50% of APAC+SLK was shortened by 1.56, 1.67 and 2.1 fold at the concentrations of 0.025, 0.5 and 0.1μM, respectively. Thus these low concentrations of a combination of both agents are likely to be more effective and less toxic when used therapeutically in vivo. Disclosures: No relevant conflicts of interest to declare.

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