Fibrin-targeted recombinant hirudin inhibits fibrin deposition on experimental clots more efficiently than recombinant hirudin.

Bode, Christoph; Hudelmayer, M; Mehwald, P; Bauer, Silvia; Freitag, Mathias; Hodenberg, Eberhard von; Kübler, W.; Haber, Edgar; Runge, M S

doi:10.1161/01.cir.90.4.1956

Cited by 40 publications

(14 citation statements)

References 30 publications

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“…10,11 Furthermore, a chemical conjugate of the fibrinspecific mAb 59D8 and the direct thrombin inhibitor hirudin inhibited fibrin deposition on experimental clots 12 and demonstrated an increase in antithrombotic potency in baboons. 13 To increase the yield and activity of antibody-targeted hirudin and to further improve the risk/benefit ratio of anticoagulation, we have developed a recombinant fusion molecule consisting of an antifibrin single-chain antibody and hirudin.…”

Section: Discussionmentioning

confidence: 98%

“…12 Clots were initiated by the addition of CaCl 2 (16.6 mmol/L) and 2.5 vol% of Actin 7 FS-activated PTT reagent (Dade International) to anticoagulated blood (citric acid, 11 mmol/L) from healthy volunteers. Whole blood was immediately drawn into a silicone tubing (4-mm inner diameter), and clots were allowed to form at 37°C for 1 hour.…”

Section: Whole Blood Clot Assaymentioning

confidence: 99%

“…9 Coupling of mAb 59D8 to plasminogen activators resulted in enhanced thrombolytic potency and specificity in vitro and in vivo. 10,11 A chemical conjugate between hirudin and 59D8 effectively inhibited fibrin deposition on experimental clots 12 and demonstrated potent antithrombotic activity in nonhuman primates. 13 Nevertheless, chemical coupling of hirudin to mAbs has several limitations, the major ones being low yield and loss of hirudin activity.…”

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confidence: 99%

“…13 Nevertheless, chemical coupling of hirudin to mAbs has several limitations, the major ones being low yield and loss of hirudin activity. 12 We tried to bypass these limitations by the use of recombinant technology.…”

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confidence: 99%

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Construction and Functional Evaluation of a Single-Chain Antibody Fusion Protein With Fibrin Targeting and Thrombin Inhibition After Activation by Factor Xa

et al. 2000

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Background-Recombinant technology was used to produce a new anticoagulant that is preferentially localized and active at the site of the clot. Methods and Results-The variable regions of the heavy and light chains of a fibrin-specific antibody were amplified by polymerase chain reaction (PCR) with hybridoma cDNA. To obtain a functional single-chain antibody (scFv), a linker region consisting of (Gly 4 Ser) 3 was introduced by overlap PCR. After the scFv clones were ligated with DNA encoding the pIII protein of the M13 phage, high-affinity clones were selected by 10 rounds of panning on the B␤15-22 peptide of fibrin (␤-peptide). Hirudin was genetically fused to the C-terminus of the variable region of the light chain. To release the functionally essential N-terminus of hirudin at the site of a blood clot, a factor Xa recognition site was introduced between scFv 59D8 and hirudin. The fusion protein was characterized by its size on SDS-PAGE (36 kDa), by Western blotting, by its cleavage into a 29-kDa (single chain alone) and 7-kDa (hirudin) fragment, by its binding to ␤-peptide, and by thrombin inhibition after Xa cleavage. Finally, the fusion protein inhibited appositional growth of whole blood clots in vitro more efficiently than native hirudin. Conclusions-A fusion protein was constructed that binds to a fibrin-specific epitope and inhibits thrombin after its activation by factor Xa. This recombinant anticoagulant effectively inhibits appositional clot growth in vitro. Its efficient and fast production at low cost should facilitate a large-scale evaluation to determine whether an effective localized antithrombin activity can be achieved without systemic bleeding complications.

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Section: Discussionmentioning

confidence: 98%

Section: Whole Blood Clot Assaymentioning

confidence: 99%

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confidence: 99%

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Construction and Functional Evaluation of a Single-Chain Antibody Fusion Protein With Fibrin Targeting and Thrombin Inhibition After Activation by Factor Xa

et al. 2000

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“…Fibrin-containing blood clots have been extensively used as a target for site-specific delivery of imaging agents and anticlotting agents to thrombi (8)(9)(10). Delivering anticoagulants into vessels where clotting is taking place has been shown to be effective at reducing the formation and expansion of clots, and it also decreases the risk of systemic side effects (11,12).…”

Section: Ardiovascular Disease Affects 1 In 3 People In the Unitedmentioning

confidence: 99%

Targeting atherosclerosis by using modular, multifunctional micelles

Peters

Kastantin

Kotamraju

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

257

252

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Subtle clotting that occurs on the luminal surface of atherosclerotic plaques presents a novel target for nanoparticle-based diagnostics and therapeutics. We have developed modular multifunctional micelles that contain a targeting element, a fluorophore, and, when desired, a drug component in the same particle. Targeting atherosclerotic plaques in ApoE-null mice fed a high-fat diet was accomplished with the pentapeptide cysteine-arginine-glutamic acid-lysine-alanine, which binds to clotted plasma proteins. The fluorescent micelles bind to the entire surface of the plaque, and notably, concentrate at the shoulders of the plaque, a location that is prone to rupture. We also show that the targeted micelles deliver an increased concentration of the anticoagulant drug hirulog to the plaque compared with untargeted micelles.cysteine-arginine-glutamic acid-lysine-alanine ͉ hirulog ͉ plaque ͉ imaging ͉ nanoparicles C ardiovascular disease affects 1 in 3 people in the United States during their lifetime, and accounts for nearly a third of the deaths that occur each year (1). Atherosclerosis is one of the leading causes of cardiovascular disease, and it results in raised plaques in the arterial wall that can occlude the vascular lumen and block blood flow through the vessel. Recently, it has become clear that not all plaques are the same. Those susceptible to rupture, fissuring, and subsequent thrombosis are most frequently the cause of acute coronary syndromes and death (2).Rupture of an atherosclerotic plaque exposes collagen and other plaque components to the bloodstream. This rupture initiates hemostasis in the blood vessel and leads to activation of thrombin and a thrombus to form at the site of rupture. Elevated levels of activated thrombin bound to the vessel wall have been observed up to 72 h after vascular injury (3). These elevated thrombin levels not only induce clot formation but also have been implicated in the progression of atherosclerosis by causing smooth muscle cells to bind circulating low density lipoprotein (4). Subtle clotting in plaques is also indicated by deposition of fibrin(ogen) both inside and on the surface of atherosclerotic plaques, which has been well documented since the 1940s (5-7).Fibrin-containing blood clots have been extensively used as a target for site-specific delivery of imaging agents and anticlotting agents to thrombi (8-10). Delivering anticoagulants into vessels where clotting is taking place has been shown to be effective at reducing the formation and expansion of clots, and it also decreases the risk of systemic side effects (11,12). Antibodies and peptides that bind to molecular markers specifically expressed on atherosclerotic plaques have shown promise for plaque imaging in vivo (13-16), but clotting on the plaque has not been used as a target. We reasoned that the fibrin deposited on plaques could serve as a target for delivering diagnostic and therapeutic compounds to plaques.We chose the clot-binding peptide cysteine-arginine-glutamic acid-lysine-alanine (CREKA) to te...

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