Structure-Function Relationships in Staphylokinase as Revealed by "Clustered Charge to Alanine" Mutagenesis

Silence, Karen; Hartmann, Manfred; Kh, Gührs; Gase, Ariane; Schlott, Bernhard; Collen, D.; Hr, Lijnen

doi:10.1074/jbc.270.45.27192

Cited by 45 publications

(44 citation statements)

References 25 publications

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“…When the SAK-plasmin complex is not fibrin-bound, it can be inhibited by the natural plasmin inhibitor, ␣ 2 -antiplasmin, present in plasma. In contrast, the fibrinbound plasminogen activator complex is much more resistant to ␣ 2 -antiplasmin-mediated inhibition (15). The result is a preferential plasminogen activation by SAK at the fibrin surface that contributes to the fibrin specificity of SAK in a plasma milieu.…”

mentioning

confidence: 72%

“…The high local concentration of clot-bound SAKM3-L-K1 can allow direct interaction of SAKM3-L-K1 with the adjacent clot-bound plasmin(ogen) to form functional plasminogen activators or to capture more plasmin(ogen) from plasma to the clot surface. Any plasminogen activated is likely to be clot bound and is less susceptible to ␣ 2 -antiplasminmediated inhibition (15).…”

Section: Plasma Clot Lysis Kinetics: Effects Of Sakm3-l-k1mentioning

confidence: 99%

“…In addition, it has some desirable features that are superior to tPA (11). Notably, SAK mediates the lysis of platelet-rich and retracted clots efficiently (12,13) and shows exceptional fibrin specificity (9,10,14,15). These properties can help minimize reocclusion and bleeding complications.…”

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confidence: 99%

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A Fast-acting, Modular-structured Staphylokinase Fusion with Kringle-1 from Human Plasminogen as the Fibrin-targeting Domain Offers Improved Clot Lysis Efficacy

Wong

2003

Journal of Biological Chemistry

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To develop a fast-acting clot dissolving agent, a clottargeting domain derived from the Kringle-1 domain in human plasminogen was fused to the C-terminal end of staphylokinase with a linker sequence in between. Production of this fusion protein in Bacillus subtilis and Pichia pastoris was examined. The Kringle domain in the fusion protein produced from B. subtilis was improperly folded because of its complicated disulfidebond profile, whereas the staphylokinase domain produced from P. pastoris was only partially active because of an N-linked glycosylation. A change of the glycosylation residue, Thr-30, to alanine resulted in a non-glycosylated biologically active fusion. The resulting mutein, designated SAKM3-L-K1, was overproduced in P. pastoris. Each domain in SAKM3-L-K1 was functional, and this fusion showed fibrin binding ability by binding directly to plasmin-digested clots. In vitro fibrin clot lysis in a static environment and plasma clot lysis in a flowcell system demonstrated that the engineered fusion outperformed the non-fused staphylokinase. The time required for 50% clot lysis was reduced by 20 to 500% under different conditions. Faster clot lysis can potentially reduce the degree of damage to occluded heart tissues.

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confidence: 72%

Section: Plasma Clot Lysis Kinetics: Effects Of Sakm3-l-k1mentioning

confidence: 99%

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A Fast-acting, Modular-structured Staphylokinase Fusion with Kringle-1 from Human Plasminogen as the Fibrin-targeting Domain Offers Improved Clot Lysis Efficacy

Wong

2003

Journal of Biological Chemistry

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“…Similarly, substitutions of Glu 46 , Lys 50 , Glu 65 and Asp 69 with alanine yielded mutants with impaired activation ability. 125 Szemraj and coworkers (2005) created a staphylokinase variant, SAK-RGD-K2-Hir by introducing the K2 domain of t-PA, the Arg-Gly-Asp tripeptide for preventing aggregation of platelets and the thrombin inhibitor hirudin to the C-terminus of staphylokinase. 126 Recently, another variant SAK-RGD-Hirulog has been developed with thrombin inhibition and antiplatelet aggregation abilities.…”

Section: (Iv) Monteplase (E6010)mentioning

confidence: 99%

The evolution of recombinant thrombolytics: Current status and future directions

Adivitiya

Khasa

2016

Bioengineered

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Cardiovascular disorders are on the rise worldwide due to alcohol abuse, obesity, hypertension, raised blood lipids, diabetes and age-related risks. The use of classical antiplatelet and anticoagulant therapies combined with surgical intervention helped to clear blood clots during the inceptive years. However, the discovery of streptokinase and urokinase ushered the way of using these enzymes as thrombolytic agents to degrade the fibrin network with an issue of systemic hemorrhage. The development of second generation plasminogen activators like anistreplase and tissue plasminogen activator partially controlled this problem. The third generation molecules, majorly t-PA variants, showed desirable properties of improved stability, safety and efficacy with enhanced fibrin specificity. Plasmin variants are produced as direct fibrinolytic agents as a futuristic approach with targeted delivery of these drugs using liposome technlogy. The novel molecules from microbial, plant and animal origin present the future of direct thrombolytics due to their safety and ease of administration.

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“…Unfortunately, owing to disorder, we could not observe the ®rst 15 residues of this structure. However, since several biochemical studies have shown that residues 11±15 of Sak are crucial for plasminogen activation (Silence et al, 1995;Gase et al, 1996), knowledge of the exact fold of these residues could be of particular importance to the understanding of the mechanism of complex formation between Sak and plasmin and the subsequent plasminogen activation. In order to obtain more accurate electron-density maps for this functionally important part of the N-terminal region in Sak, residues 1±10 were removed in an attempt to stabilize residues 11±15.…”

Section: Introductionmentioning

confidence: 99%

A fully functional deletion staphylokinase derivative crystallizes in two non-isomorphous monoclinic modifications

Rabijns

Bondt

Collen

et al. 1999

Acta Crystallogr D Biol Cryst

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Two non-isomorphous monoclinic types of diffraction-quality crystals of Á10Sak, a fully functional staphylokinase derivative lacking the ®rst ten amino acids, have been grown by the hanging-drop vapourdiffusion technique. Type I crystals grow from a solution containing Zn(OAc) 2 , Tris buffer pH 7.5 and PEG 8000, while type II crystals can be obtained from a solution containing MgCl 2 , Tris buffer pH 8.5 and PEG 4000. Both crystal types were suitable for data collection (to 2.4 and 2.6 A Ê resolution, respectively) and further structural investigation.

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Structure-Function Relationships in Staphylokinase as Revealed by "Clustered Charge to Alanine" Mutagenesis

Cited by 45 publications

References 25 publications

A Fast-acting, Modular-structured Staphylokinase Fusion with Kringle-1 from Human Plasminogen as the Fibrin-targeting Domain Offers Improved Clot Lysis Efficacy

A Fast-acting, Modular-structured Staphylokinase Fusion with Kringle-1 from Human Plasminogen as the Fibrin-targeting Domain Offers Improved Clot Lysis Efficacy

The evolution of recombinant thrombolytics: Current status and future directions

A fully functional deletion staphylokinase derivative crystallizes in two non-isomorphous monoclinic modifications

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