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PolyP enhances uPA-mediated plasminogen activation
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Figure 5. The cofactor function of polyP in uPA-mediated fibrinolysis is modulated by plasminogen concentration and form. Fibrin clots were prepared containing
Despite their homology in structure, the mechanism of tPA and uPA-mediated plasminogen activation are very different. Efficient plasminogen activation by tPA requires its association with the fibrin surface, that is fibrin orches- trates its own destruction by plasmin. Binding of tPA to fibrin is largely attributed to its finger domain,21,22 while plasminogen associates with fibrin via kringle domains.23 The colocalization of enzyme and substrate on fibrin, as a surface, is crucial, as tPA is a poor plasminogen activator in solution. Circulating fibrinogen cannot accelerate plas- minogen activation by tPA, as the sites are only exposed in fibrin24 thereby localizing plasmin formation to the fi- brin clot. In marked contrast uPA does not bind fibrin and is reasonably efficient at activating plasminogen in solu- tion. However, its cellular receptor, uPAR, localizes uPA via its amino terminal fragment to the cell surface, thereby augmenting plasminogen activation due to an increase in local reactant concentration.
The high affinity interaction of uPA and plasminogen14 with polyP and dependence on substrate and template concentration are indicative of a template mechanism of activation. In line with this polyP polymers of around 60- mer were required to stimulate plasminogen activation by uPA, suggesting this length is critical to accommodate binding of both enzyme and substrate to the same tem- plate. Indeed, a template effect of polyP in thrombin- mediated activation of FV to FVa has previously been reported.25 Polymers of around 80-100-mer are secreted following platelet stimulation2 with insoluble divalent cation bound polyP nanoparticles remaining associated
. Clotting was initiated with throm- (5 mM) and fibrinolysis monitored at 340 nm. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001 compared with control clots. Data
2.4 mM fibrinogen, 0-1 mM (A-B) Glu-plasminogen or (C-D) Lys-plasminogen, (A, C) 180 pM uPA or (B, D) 20 pM tPA ±328 mM polyP
bin (0.25 U/mL) and CaCl
65
are expressed as mean ± standard error of the mean, n≥3. polyP: polyphosphate; tPA: tissue plasminogen activator; uPA: urokinase plasminogen activator.
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with the activated platelet membrane.26 Platelet-derived polyP could actively participate in these template-media- ted reactions within the milieu of a thrombus where local concentrations of the polymer will be high.
Previously an inhibitory effect of polyP on uPA-media- ted fibrinolysis was described in a plasma based system.25 The data presented here delineates the role of polyP specifically on plasminogen activation by uPA using a purified system. The roles of polyP in hemostasis are mul- tifaceted and coagulation and fibrinolysis are inextricably linked. It is therefore difficult, at this current time, to exclude the fact that these differences cannot be attributed to different experimental setups, reagents and indeed an impact of polyP on a different part of the pathway.
Intriguingly, the cofactor function of polyP described here in uPA-mediated plasminogen activation is markedly more pronounced than in uPA-mediated fibrinolysis. This can potentially be explained by an increase in the number of binding sites, that is plasminogen has the capacity to bind both polyP and lysine residues exposed on partially degraded fibrin generated following onset of lysis.23 Thus, the increase in surface binding sites on fibrin for plasmino- gen tempers the cofactor function of polyP. Therefore, there is a more evident effect at low plasminogen concen- trations, where fewer lysine residues will be generated. Given the anionic nature of polyP it is feasible that it asso- ciates with positively charged lysine residues on fibrin, thereby obscuring the kringle-dependent binding of plas- minogen to fibrin, however, high plasminogen concentra- tions eliminate the need for fibrin binding.
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