Fusion proteins comprising annexin V and Kunitz protease inhibitors are highly potent thrombogenic site-directed anticoagulants

Chen, Hsiu‐Hui; Vicente, Cristina Pontes; He, Li; Tollefsen, Douglas M.; Wun, Tze-Chein

doi:10.1182/blood-2004-11-4435

Cited by 30 publications

(39 citation statements)

References 66 publications

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“…NP-7 contains a cluster of positively charged residues in the N-terminus and specifically binds to anionic phospholipids, preventing thrombin formation by the prothrombinase (Andersen et al, 2004). Finally, a bifunctional fusion protein containing Kunitz and annexin domains was shown recently to inhibit the initiation of blood coagulation (Chen et al, 2005).…”

Section: Group 1: Basic-tail Proteins (Btp)mentioning

confidence: 99%

The transcriptome of the salivary glands of the female western black-legged tick Ixodes pacificus (Acari: Ixodidae)

Francischetti

Pham

Mans

et al. 2005

Insect Biochemistry and Molecular Biology

147

156

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SummarySequencing of an Ixodes pacificus salivary gland cDNA library yielded 1068 sequences with an average undetermined nucleotide of 1.9% and an average length of 487 base pairs. Assembly of the expressed sequence tags yielded 557 contigs, 138 of which appear to code for secreted peptides or proteins based on translation of a putative signal peptide. Based on the BLASTX similarity of these contigs to 66 matches of Ixodes scapularis peptide sequences, only 58% sequence identity was found, indicating a rapid divergence of salivary proteins as observed previously for mosquito and triatomine bug salivary proteins. Here we report 106 mostly full-length sequences that clustered in 16 different families: Basic-tail proteins rich in lysine in the carboxy-terminal, Kunitz-containing proteins (monolaris, ixolaris and penthalaris families), proline-rich peptides, 5-kDa.-, 9.4 kDa.-, and 18.7 kDa.-proteins of unknown functions, in addition to metalloproteases (class PIII-like) similar to reprolysins. We also have found a family of disintegrins, named ixodegrins that display homology to variabilin, a GPIIb/IIIa antagonist from the tick Dermacentor variabilis. In addition, we describe peptides (here named ixostatins) that display remarkable similarities to the cysteine-rich domain of ADAMST-4 (aggrecanase). Many molecules were assigned in the lipocalin family (histaminebinding proteins); others appear to be involved in oxidant metabolism, and still others were similar to ixodid proteins such as the anticomplement ISAC. We also identified for the first time a neuropeptide-like protein (nlp-31) with GGY repeats that may have antimicrobial activity. In addition, 16 novel proteins without significant similarities to other tick proteins and 37 housekeeping proteins that may be useful for phylogenetic studies are described. Some of these proteins may be useful for studying vascular biology or the immune system, for vaccine development, or as immunoreagents to detect prior exposure to ticks. Electronic version of the manuscript can be found at http://www.ncbi.nlm.nih.gov/projects/omes/.

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Section: Group 1: Basic-tail Proteins (Btp)mentioning

confidence: 99%

The transcriptome of the salivary glands of the female western black-legged tick Ixodes pacificus (Acari: Ixodidae)

Francischetti

Pham

Mans

et al. 2005

Insect Biochemistry and Molecular Biology

147

156

View full text Add to dashboard Cite

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“…24,25 An alternative epitope for clot-targeting has been recently described: Phosphatidyl-L-serine/phosphatidylethanolamine, which is exposed on activated platelets and microparticles. 26 A 3-to 10-fold increase in anticoagulant potency could be achieved by this targeting strategy. 26 Another recent approach used P-selectin, which is expressed on activated platelets as well as on activated endothelial cells, as an epitope to target Desmodusrotundus salivary plasminogen activator alpha1.…”

Section: Discussionmentioning

confidence: 99%

Targeting Ligand-Induced Binding Sites on GPIIb/IIIa via Single-Chain Antibody Allows Effective Anticoagulation Without Bleeding Time Prolongation

Stoll

Bassler

Hagemeyer

et al. 2007

ATVB

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Objective-Therapeutic anticoagulation is widely used, but limitations in efficacy and bleeding complications cause an ongoing search for new agents. However, with new agents developed it seems to be an inherent problem that increased efficiency is accompanied by an increase in bleeding complications. We investigate whether targeting of anticoagulants to activated platelets provides a means to overcome this association of potency and bleeding. Methods and Results-Ligand-induced binding sites (LIBS) on fibrinogen/fibrin-binding GPIIb/IIIa represent an abundant clot-specific target. We cloned an anti-LIBS single-chain antibody (scFv anti-LIBS ) and genetically fused it with a potent, direct factor Xa (fXa) inhibitor, tick anticoagulant peptide (TAP). Specific antibody binding of fusion molecule scFv anti-LIBS -TAP was proven in flow cytometry; anti-fXa activity was demonstrated in chromogenic assays. In vivo anticoagulative efficiency was determined by Doppler-flow in a ferric chloride-induced carotid artery thrombosis model in mice. ScFv anti-LIBS -TAP prolonged occlusion time comparable to enoxaparine, recombinant TAP, and nontargeted mutant-scFv-TAP. ScFv anti-LIBS -TAP revealed antithrombotic effects at low doses at which the nontargeted mutant-scFv-TAP failed. In contrast to the other anticoagulants tested, bleeding times were not prolonged by scFv anti-LIBS -TAP. Key Words: GPIIb/IIIa Ⅲ anticoagulation Ⅲ single-chain Ⅲ fXa Ⅲ thrombosis T herapeutic anticoagulation is used extensively in many areas of medicine. Despite the overall benefits achieved, the currently used therapeutic anticoagulants are also a major source of mortality and morbidity, caused by limitations in efficacy and even more so by bleeding complications. 1 In an effort to overcome these problems, a plethora of new agents have been developed. 2,3 However, it seems that more efficient therapeutic anticoagulation is inevitably associated with an increase in bleeding complications. Targeting of anticoagulants to the clot may represent a means to break this association. The success of this targeting is dependent on the abundance and specificity of the epitope chosen as the target. We have previously demonstrated that fibrin, which satisfies both requirements, can be used successfully for clot targeting. 4 -7 In the present study, we investigated whether activated platelets can be used as an alternative and potentially more efficient clot-target. Platelets are highly abundant in particular in thrombi within the arterial system, as with atherosclerosis-induced thrombi, eg, in myocardial infarction. Activated platelets are highly specific for clots and are not typically found in the circulation. Thus, both requirements for efficient clot-targeting, abundance and specificity, are highly satisfied. Besides these favorable properties, the use of activated platelets as epitopes for clot-targeting may have additional advantages compared with fibrin, because platelet activation may precede fibrin formation. 8 One of the most abundantly expressed molecul...

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“…5,7,8) Following injury to the endothelium and vessel wall, platelets adhere rapidly to the sub-endothelial matrix, then become activated and form the primary hemostatic plug. 9,10) In the meanwhile, phospholipids such as phosphatidylserine (PS) are rapidly exposed on the membrane surfaces of activated platelets. [11][12][13][14] The activated platelets provide anionic surfaces for the assembly and catalysis of intrinsic tenase (VIIIa/IXa), prothrombinase (Va/Xa), and XIa complexes, leading to explosive generation of thrombin and consolidation of the fibrin-platelet plug.…”

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confidence: 99%

“…[26][27][28][29] Many efforts have been made to construct ANXV based chimeras for evaluation of their targeting effect at a potential thrombus. 6,10,30,31) Staphylokinase (SAK) is one of the promising blood clot dissolving agents. 1,6,[32][33][34] Although the thrombolytic potency of SAK has been found to be comparable to that of tPA, clinical trials have indicated that SAK has a superior fibrin specificity.…”

mentioning

confidence: 99%

“…[15][16][17] Thus, rapid exposure of PS on the cell surfaces of platelets is the key thrombogenic stimulus initiating and propagating the clotting cascade. 9,10,17) Annexins (ANXs) are a family of calcium-dependent phospholipid binding proteins. 18,19) Structurally, ANXs are characterized by a highly -helical and tightly packed protein core domain of four to eight conserved repeats plus the N-terminal region, which is variable both in length and in sequence.…”

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confidence: 99%

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Staphylokinase-Annexin XI Chimera Exhibited Efficientin VitroThrombolytic Activities

Chiou¹,

Woon²,

Cheng³

et al. 2007

Bioscience, Biotechnology, and Biochemistry

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Fusion proteins comprising annexin V and Kunitz protease inhibitors are highly potent thrombogenic site-directed anticoagulants

Cited by 30 publications

References 66 publications

The transcriptome of the salivary glands of the female western black-legged tick Ixodes pacificus (Acari: Ixodidae)

The transcriptome of the salivary glands of the female western black-legged tick Ixodes pacificus (Acari: Ixodidae)

Targeting Ligand-Induced Binding Sites on GPIIb/IIIa via Single-Chain Antibody Allows Effective Anticoagulation Without Bleeding Time Prolongation

Staphylokinase-Annexin XI Chimera Exhibited Efficientin VitroThrombolytic Activities

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