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C.S. Whyte and N.J. Mutch
minogen concentration (Figure 2A-B). The presence of polyP significantly increased the rate of uPA-mediated plasmin generation by around 75–82% at all Glu-plas- minogen concentrations (Figure 2A). In marked contrast, polyP attenuated tPA-mediated plasmin generation at all Glu-plasminogen concentrations, with a maximal reduc- tion at 1 mM Glu-plasminogen (Figure 2B). Cleavage of the activation peptide of Glu-plasminogen by plasmin forms an intermediate form, Lys-plasminogen, which displays increased affinity for fibrin15 and is more readily cleaved by plasminogen activators.7,16,17 Overall plasmin genera- tion by uPA occurs at a significantly faster rate in clots formed with Lys-plasminogen compared to Glu-plasmino- gen and is directly proportional to the concentration (Figure 2C-D). Interestingly, Lys-plasminogen overcame the cofactor function of polyP on uPA-mediated plasmin generation, significantly decreasing the magnitude of the effect from 79% at 0.125 mM Lys-plasminogen to 9% at 1 mM Lys-plasminogen (Figure 2C). With tPA there was a similar attenuation of plasmin generation (approximately 37%) at all Lys-plasminogen concentrations analysed (Figure 2B, D).
polyP binds with a higher affinity to uPA than tPA
We have previously demonstrated that polyP binds to Glu-plasminogen.14 Here we use biotin-labelled polyP in a plate-based assay to determine the binding affinity to (A) uPA and (B) tPA. polyP bound with approximately 25-fold
=9.8 nM) than tPA (K
Figure 3). Interestingly, binding of polyP did not alter the activity of uPA and no specific binding of polyP to plasmin was detected (data not shown). We have shown that polyP stimulates autoactivation of FXII to an active single chain
FXII (scFXIIa)18 and enhances the plasminogen activator capacity of FXIIa.14 We found that the high affinity inter- action of uPA and polyP was similar to the interaction of polyP with FXII (Kd=7.9 nM) and FXIIa (Kd=4.0 nM) (Online Supplementary Figure S1).
polyP enhances uPA-mediated lysis in a concentration and polymer length dependent manner
Incorporation of polyP during clot formation shortened the uPA-mediated 50% clot lysis time by 43.7±1.7 min (P<0.0001; Figure 4A). In contrast, tPA-mediated clot lysis was delayed on inclusion of polyP (Figure 4B), as previous- ly reported.4 The cofactor function of polyP on uPA-medi- ated lysis was concentration dependent and required 66 mM or higher to significantly stimulate the rate of lysis (Figure 4C). The use of polyP of more refined chain length revealed that polymers of >14-mer were required to pro- mote uPA-mediated lysis (Figure 4D).
Plasminogen modulates polyP-mediated effects on fibrinolysis
The mode of action of tPA and uPA differ significantly, with tPA relying on the interaction with fibrin for efficient plasminogen activation, while uPA is fibrin independent. We investigated the relationship between plasminogen and polyP-mediated effects on fibrinolysis. A clear con- centration dependence was observed with Glu-plasmino- gen on uPA-mediated lysis, with maximal lysis at 0.75 mM plasminogen (Figure 5A). The impact of Glu-plasminogen on tPA-mediated lysis was less pronounced, but a small effect was observed with maximal lysis at 0.5 mM plas- minogen (P<0.01; Figure 5B). polyP significantly accelerat- ed uPA-mediated lysis at all Glu-plasminogen concentra-
higher affinity to uPA (K d
=245.3 nM; d
AB
CD
Figure 1. PolyP stimulates uPA-mediated plasmin generation in a concentration dependent manner. Fibrin clots were prepared containing 2.4 mM fibrinogen, 0.24
). Clotting was initiated with thrombin (0.25 U/mL) and CaCl
quantified by incorporating the fluorogenic substrate D–VLK-AMC and monitoring fluorescence release (FU; Ex 360 nm Em 460 nm). (A) uPA and (B) tPA mediated
mM Glu-plasminogen, 20 pM tPA or 180 pM uPA ± 328 mM (polyP
) for (C) uPA and (D) tPA. Data are expressed as mean ± standard error of the mean, n≥ 3. PolyP: polyphosphate; tPA: tissue plasminogen activator; uPA: urokinase plasminogen activator; FU: fluorescence units,
plasmin generation curves. Rate of plasmin generation in clots formed with varying polyP concentration (0-1.3 mM polyP Ex: excitation; EM: emission.
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(5 mM) and plasmin generation was
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