Integration of VEK‐30 peptide enhances fibrinolytic properties of staphylokinase

Abstract

Staphylokinase (SAK), a 136 amino acid bacterial protein with profibrinolytic properties, has emerged as an important thrombolytic agent because of its fibrin specificity and reduced inhibition by α‐2 antiplasmin. In an attempt to enhance the clot dissolution ability of SAK, a 30 amino acid peptide (VEK‐30) derived from a plasminogen (Pg) binding protein (PAM), was fused at the C‐terminal end of SAK with a RGD (Arg–Gly–Asp) linker. The chimeric protein, SAKVEK, was expressed in E. coli and purified as a soluble protein. Pg activation by equimolar complexes of SAKVEK and SAK with plasmin revealed that the fusion of VEK‐30 peptide has significantly enhanced the catalytic activity of SAK. The kinetic constant, kcat/Km, of SAKVEK for the substrate Pg appeared 2.7 times higher than that of SAK and the time required for the fibrin and platelet rich clot lysis was shortened by 30% and 50%, respectively. The binary activator complex of SAKVEK with plasmin gets inhibited by α2‐ antiplasmin but remains protected in the presence of fibrin, very similar to SAK. Thus, the present study suggests that SAKVEK is more potent and effective as a thrombolytic agent due to its higher catalytic activity for Pg activation in a fibrin‐specific manner and its ability to clear platelet‐rich plasma clot faster than SAK.

Extended Mechanism and Rate-Limiting Step of the Plasminogen Activator Staphylokinase Revealed by Global Kinetic Analysis

“…We applied the same methodology of conventional initial rate analysis to our kinetic data (Figure 2a,b,d,e; Supplementary Table S1) and compared the obtained values with those from the literature. 19,24,25,28,[34][35][36][37][38][39] Values similar to the reported range (Table 1) were obtained for both the turnover number kcat (0.011 ± 0.001 s -1 ) and Michaelis constant Km (21 ± 1 µM). Such results confirmed the consistency of our kinetic data with those previously reported.…”

“…We validated our kinetic data by first applying a conventional data analysis approach and obtained comparable values of underestimated steady-state kinetic parameters as those reported previously. 19,24,25,28,[34][35][36][37][38][39] It should be noted that some studies have reported values of kcat approximately 10-fold higher and out of the range of other consistent results. 22,23,26,27,43 However, those data were collected at elevated temperatures where exponentially higher rates are expected.…”

“…Although many prior reports have focused on the kinetic characterization of SAK and its modified variants, 19,[22][23][24][25][26][27][28][34][35][36][37][38][39]43 we were unable to find a single study reporting the analysis of SAK kinetics by a combination of steady-state and binding affinity datasets. Some studies conducted both types of experiments 19,23,26,40 but did not combine the data to obtain global estimates of kinetic parameters.…”

“…19,20 Even though there have been several attempts to improve the SAK thrombolytic biocatalytic potential by engineering, none of the generated proteins exhibited a significant increase of effectivity. 15,[21][22][23][24][25][26][27][28] This was mainly due to the complex SAK molecular mechanism, which makes it difficult to properly identify the real limiting step, which is critical to be known for rational protein engineering. 29,30 The major challenge is that the generated Plm "product" can also act as a "partner" and form more of the SAK.Plm complex, continuously changing equilibria without reaching a real steadystate phase.…”

Whole-Genome Analysis Reveals That Bacteriophages Promote Environmental Adaptation of Staphylococcus aureus via Gene Exchange, Acquisition, and Loss

“…Furthermore, sciderin is an important protein associated with host defense that interferes with the activation of the human complement system [40]. Additionally, staphylokinase is a fibrinolytic agent that plays an important role in dissolving blood clots on fibrin surfaces [41]. β-Haemolysin acts as a hemolytic in sheep, contributes to biofilm formation in rabbit endocarditis models, and enhances the ability of S. aureus to colonize murine skin [42].…”