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C.S. Whyte and N.J. Mutch
the cofactor function of polyP on uPA-mediated lysis is lost at high Lys-plasminogen concentrations (Figure 5C). Whereas, polyP delayed tPA-mediated lysis by approxi- mately 20 % at all Lys-plasminogen concentrations (Figure 5B, D).
polyP localization during fibrinolysis
polyP is found to co-localize with fibrinogen, as previ- ously reported,19 and plasminogen in fibrin clots. During fibrinolysis polyP concentrates within the fibrin dense knots of the clot alongside plasminogen and fibrinogen (Figure 6A). Colocalization was observed with both uPA and tPA but only the data for uPA is shown. Lysis was ini- tiated at the edge of the clot, by addition of plasminogen activator, and as it progressed through the fibrin network polyP and plasminogen were found to remain colocalized within the lysis front (Figure 6B).
Visualization of the cofactor function of polyP during uPA-mediated lysis in real-time
Fluorescent confocal microscopy was used to visualize lysis in real-time following application of either uPA or tPA to the edge of fibrin clots. The rate of fibrinolysis in the control clot was similar in the presence of uPA or tPA (Figure 7A-B). As uPA-mediated lysis progresses plasmin(ogen) is dispersed throughout the clot, with a small concentrated zone at the leading edge (Figure 7A), in contrast plasminogen is restricted to a distinct zone at the leading edge in tPA-mediated lysis (Figure 7B). The incor-
poration of polyP significantly accelerates uPA-mediated lysis (Figure 7A) with full lysis observed on average at 6 min (Online Supplementary Video S1B) compared to greater than 9 min in the control (Online Supplementary Video S1A).
Discussion
Over the last decade a role for the biomolecule polyP in the regulation of hemostasis has been exposed. The ability of polyP to modulate fibrinolysis manifests on several levels. We and others4,20 have demonstrated altered fibrin structure in the presence of polyP, a consequence of which is tempered binding of tPA and plasminogen to fibrin and subsequent down-regulation of fibrinolysis.4 In contrast, in this study we show that that polyP significantly accelerates uPA-mediated plasminogen activation thereby augmenting fibrinolysis. The rate of plasmin formation was approximately 5-fold faster in the presence of polyP and was dependent on concentration and polymer length, with polymers of 60-mer or greater required to accelerate lysis. We determined that polyP binds with a 25-fold high- er affinity to uPA than tPA and potentially accelerates con- version of Glu-plasminogen to the intermediate form Lys- plasminogen via a template mediated effect. Importantly, for the first time we visualize polyP during real-time lysis of fibrin clots and find it to be localized within fibrin dense areas alongside plasminogen, consistent with its known binding to these proteins.4,14,19
AB
CD
Figure 4. polyP acts as a cofactor to accelerate uPA-mediated fibrinolysis in a concentration and polymer size dependent manner. Fibrin clots were prepared con-
(5 mM) or (D) polyP of various chain lengths at equivalent concentration of monomer (328 mm). *P<0.05, ***P<0.001 and ****P<0.0001 compared with control clots. Data are
1/2
taining 2.4 mM fibrinogen, 0.24 mM Glu-plasminogen, 20 pM tPA or 180 pM uPA ±328 mM polyP
and fibrinolysis monitored at 340 nm shown as percentage turbidity over time with (A) uPA or (B) tPA. (C) uPA-mediated fibrinolysis with 0-1.3 mM polyP
expressed as mean 50 % lysis time (t activator.
) ± standard error of the mean, n≥3. polyP: polyphosphate; tPA: tissue plasminogen activator; uPA: urokinase plasminogen
65
2 65
. Clotting was initiated with thrombin (0.25 U/mL) and CaCl
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haematologica | 2021; 106(2)