Activity Regulation by Fibrinogen and Fibrin of Streptokinase from Streptococcus Pyogenes

<div><p>Streptokinase is a virulence factor of streptococci and acts as a plasminogen activator to generate the serine protease plasmin which promotes bacterial metastasis. Streptokinase isolated from group C streptococci has been used therapeutically as a thrombolytic agent for many years and its mechanism of action has been extensively studied. However, group A streptococci are associated with invasive and potentially fatal infections, but less detail is available on the mechanism of action of streptokinase from these bacteria. We have expressed recombinant streptokinase from a group C strain to investigate the therapeutic molecule (here termed rSK-H46A) and a molecule isolated from a cluster 2a strain from group A (rSK-M1GAS) which is known to produce the fibrinogen binding, M1 protein, and is associated with life-threatening disease. Detailed enzyme kinetic models have been prepared which show how fibrinogen-streptokinase-plasminogen complexes regulate plasmin generation, and also the effect of fibrin interactions. As is the case with rSK-H46A our data with rSK-M1GAS support a “trigger and bullet” mechanism requiring the initial formation of SK•plasminogen complexes which are replaced by more active SK•plasmin as plasmin becomes available. This model includes the important fibrinogen interactions that stimulate plasmin generation. In a fibrin matrix rSK-M1GAS has a 24 fold higher specific activity than the fibrin-specific thrombolytic agent, tissue plasminogen activator, and 15 fold higher specific activity than rSK-H46A. However, in vivo fibrin specificity would be undermined by fibrinogen stimulation. Given the observed importance of M1 surface receptors or released M1 protein to virulence of cluster 2a strain streptococci, studies on streptokinase activity regulation by fibrin and fibrinogen may provide additional routes to addressing bacterial invasion and infectious diseases.</p></div

The effect of known stimulators on Pgn activation by tPA, rSK-M1GAS and rSK-H46A.

<p>Bars show the degree of stimulation (rate with stimulator/rate without stimulator) for fixed concentrations of Pgn and tPA (blue), rSK-M1GAS (red) and rSK-H46A (black), using both glu- or lys-Pgn as substrate. Abbreviations are Fgn Ox, oxidised Fgn, CNBr, cyanogen bromide fragmented Fgn, and FDP-1 and FDP-2 are pooled samples from separate independent time courses of fibrin degradation products.</p

Model Parameters used in the model outlined in Fig 2 and simulated in Fig 3.

<p>Model Parameters used in the model outlined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170936#pone.0170936.g002" target="_blank">Fig 2</a> and simulated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170936#pone.0170936.g003" target="_blank">Fig 3</a>.</p

Comparison of Pgn activation data and simulated data for rSK-M1GAS over a range of Pgn and Fgn concentrations.

<p>Panels A (experimental data) and C (simulated data) show fitted surface plots of rate of Pm generation plotted against Fgn and Pgn concentrations as shown, for 1.6 nM rSK-M1GAS. Panels B and D present the same results as a surface and contour plots giving rates of Pm production in pM/s against Pgn concentration (0–1.6 μM) and Fgn concentration (log scale for 0–30 μM). Panel E is an overlay of surfaces for the data and simulation shown in panels A and B. Experimental data using rSK-M1GAS at 1.6 (closed circles) and 0.4 nM (open squares) over a range of Pgn concentrations were fitted to the Michaelis-Menten equation to determine k_cat and K_M values, and calculate k_cat/ K_M at each Fgn concentration and this is shown in panel F. The solid line is for the same values calculated from simulated data using the same ranges of Pgn and Fgn (the lines overlap for 2 hypothetical rSK-M1GAS concentrations of 1.6 and 0.4 nM. R scripts and data files are provided in Supporting Information.</p

Fibrin clot lysis by tPA, rSK-M1GAS and rSK-H46A over a range of Pgn concentrations.

<p>Fibrin clots were prepared using 3 mg/ml Fgn and incorporating Pgn from 0–1.6 μM. Rate of clot lysis was estimated from time to 50% clot lysis, as 1000x 1/time to 50% lysis in seconds. To get similar rates, activator concentrations used were 0.6 M tPA (blue circles), 0.3 nM rSK-H46A (black squares) and 0.02 nM rSK-GASM1 (red triangles). Detailed results from fitting to the Michaelis-Menten equation are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170936#pone.0170936.t003" target="_blank">Table 3</a>.</p

Inhibition of Pgn activation in a Fgn or fibrin environment by tranexamic acid (TA).

<p>Data are presented as the % activity remaining relative to activation with no TA where rSK-M1GAS (red symbols) or rSK-H46A (black symbols) is activator. Open symbols and dashed lines are for data in the presence of Fgn and solid symbols and lines are in fibrin. Curve fitting to a 4 parameter model suggests a significant difference between IC₅₀for rSK-M1GAS in the presence of Fgn (14.5 μM) and fibrin (133 μM). Inhibition of Pgn activation by rSK-H46A was inhibited at higher TA and there was no significant difference with Fgn or fibrin.</p

Kinetic parameters for clot lysis by tPA, rSK-M1GAS and rSK-H46A from data shown in Fig 5.

<p>Kinetic parameters for clot lysis by tPA, rSK-M1GAS and rSK-H46A from data shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170936#pone.0170936.g005" target="_blank">Fig 5</a>.</p

Scheme outlining the pathway of activation of Pgn by rSK-M1GAS and stimulation by Fgn.

<p>The scheme follows a trigger and bullet mechanism where an initial activator complex of SK•Pgn (BG) is replaced by SK•Pm (BE) as Pm (E) is generated due to the higher affinity binding of E to SK. Fgn (F) associates weakly with Pgn (G), while formation of active Michaelis complexes, GFBG and GFBE have improved dissociation constants. An improved rate of Pgn activation is achieved by GFBE relative to GFBG due to lower K_M, while the k_cat for formation of Pm is unchanged. Derivation of the constants shown is detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170936#pone.0170936.t002" target="_blank">Table 2</a>.</p

Fractal Kinetic Behavior of Plasmin on the Surface of Fibrin Meshwork

Intravascular fibrin clots are resolved by plasmin acting at the interface of gel-phase substrate and fluid-borne enzyme. The classic Michaelis–Menten kinetic scheme cannot describe satisfactorily this heterogeneous-phase proteolysis because it assumes homogeneous well-mixed conditions. A more suitable model for these spatial constraints, known as fractal kinetics, includes a time-dependence of the Michaelis coefficient <i>K</i>_<i>m</i>^<i>F</i> <i>=</i> <i>K</i>_<i>m</i>0^<i>F</i>(1 + <i>t</i>)^<i>h</i>, where <i>h</i> is a fractal exponent of time, <i>t</i>. The aim of the present study was to build up and experimentally validate a mathematical model for surface-acting plasmin that can contribute to a better understanding of the factors that influence fibrinolytic rates. The kinetic model was fitted to turbidimetric data for fibrinolysis under various conditions. The model predicted <i>K</i>_<i>m</i>0^<i>F</i>= 1.98 μM and <i>h</i> = 0.25 for fibrin composed of thin fibers and <i>K</i>_<i>m</i>0^<i>F</i>= 5.01 μM and <i>h</i> = 0.16 for thick fibers in line with a slower macroscale lytic rate (due to a stronger clustering trend reflected in the <i>h</i> value) despite faster cleavage of individual thin fibers (seen as lower <i>K</i>_<i>m</i>0^<i>F</i>). ε-Aminocaproic acid at 1 mM or 8 U/mL carboxypeptidase-B eliminated the time-dependence of <i>K</i>_<i>m</i>^<i>F</i> and increased the lysis rate suggesting a role of C-terminal lysines in the progressive clustering of plasmin. This fractal kinetic concept gained structural support from imaging techniques. Atomic force microscopy revealed significant changes in plasmin distribution on a patterned fibrinogen surface in line with the time-dependent clustering of fluorescent plasminogen in confocal laser microscopy. These data from complementary approaches support a mechanism for loss of plasmin activity resulting from C-terminal lysine-dependent redistribution of enzyme molecules on the fibrin surface

Glycated albumin modulates the contact system with implications for the kallikrein-kinin and intrinsic coagulation systems

Background: Human serum albumin (HSA) is the most abundant plasma protein and is sensitive to glycation in vivo. The chronic hyperglycemic conditions in patients with diabetes mellitus (DM) induce a nonenzymatic Maillard reaction that denatures plasma proteins and forms advanced glycation end products (AGEs). HSA-AGE is a prevalent misfolded protein in patients with DM and is associated with factor XII activation and downstream proinflammatory kallikrein-kinin system activity without any associated procoagulant activity of the intrinsic pathway. Objectives: This study aimed to determine the relevance of HSA-AGE toward diabetic pathophysiology. Methods: The plasma obtained from patients with DM and euglycemic volunteers was probed for activation of FXII, prekallikrein (PK), and cleaved high-molecular-weight kininogen by immunoblotting. Constitutive plasma kallikrein activity was determined via chromogenic assay. Activation and kinetic modulation of FXII, PK, FXI, FIX, and FX via in vitro-generated HSA-AGE were explored using chromogenic assays, plasma-clotting assays, and an in vitro flow model using whole blood. Results: Plasma obtained from patients with DM contained increased plasma AGEs, activated FXIIa, and resultant cleaved cleaved high-molecular-weight kininogen. Elevated constitutive plasma kallikrein enzymatic activity was identified, which positively correlated with glycated hemoglobin levels, representing the first evidence of this phenomenon. HSA-AGE, generated in vitro, triggered FXIIa-dependent PK activation but limited the intrinsic coagulation pathway activation by inhibiting FXIa and FIXa-dependent FX activation in plasma. Conclusion: These data indicate a proinflammatory role of HSA-AGEs in the pathophysiology of DM via FXII and kallikrein-kinin system activation. A procoagulant effect of FXII activation was lost through the inhibition of FXIa and FIXa-dependent FX activation by HSA-AGEs.</p