uPA-mediated plasminogen activation is enhanced by polyphosphate

Abstract

Tissue plasminogen activator (tPA) and urokinase (uPA) differ in their modes of action. Efficient tPA-mediated plasminogen activation requires binding to fibrin. In contrast, uPA is fibrin independent and activates plasminogen in solution or when associated with its cellular receptor uPAR. We have previously shown that polyphosphate (polyP), alters fibrin structure and attenuates tPA and plasminogen binding to fibrin, thereby down-regulating fibrinolysis. Here we investigate the impact of polyP on uPA-mediated fibrinolysis. As previously reported polyP of an average chain length of 65 (polyP65) delays tPA-mediated fibrinolysis. The rate of plasmin generation was also delayed and reduced 1.6-fold in polyP65-containing clots (0.74 ± 0.06 vs. 1.17 ± 0.14 pM/s in P < 0.05). Analysis of tPA-mediated fibrinolysis in real-time by confocal microscopy was significantly slower in polyP65-containing clots. In marked contrast, polyP65 augmented the rate of uPA-mediated plasmin generation 4.7-fold (3.96 ± 0.34 vs. 0.84 ± 0.08 pM/s; P < 0.001) and accelerated fibrinolysis (t1/2 64.5 ± 1.7 min vs. 108.2 ± 3.8 min; P < 0.001). Analysis of lysis in real-time confirmed that polyP65 enhanced uPA-mediated fibrinolysis. Varying the plasminogen concentration (0.125 to 1 μM) in clots dose-dependently enhanced uPA-mediated fibrinolysis, while negligible changes were observed on tPA-mediated fibrinolysis. The accelerating effect of polyP65 on uPA-mediated fibrinolysis was overcome by additional plasminogen, while the down-regulation of tPA-mediated lysis and plasmin generation was largely unaffected. PolyP65 exerts opposing effects on tPA- and uPA-mediated fibrinolysis, attenuating the fibrin cofactor function in tPA-mediated plasminogen activation. In contrast, polyP may facilitate the interaction between fibrin-independent uPA and plasminogen thereby accelerating plasmin generation and downstream fibrinolysis.

Figure 1.

PolyP stimulates uPA-mediated plasmin generation in a concentration dependent manner. Fibrin clots were prepared containing 2.4 mM fibrinogen, 0.24 mM Glu-plasminogen, 20 pM tPA or 180 pM uPA ± 328 mM (polyP65). Clotting was initiated with thrombin (0.25 U/mL) and CaCl2 (5 mM) and plasmin generation was 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 plasmin generation curves. Rate of plasmin generation in clots formed with varying polyP concentration (0-1.3 mM polyP65) 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, Ex: excitation; EM: emission.

Figure 2.

High concentrations of plasminogen attenuate the cofactor function of polyP in uPA-mediated plasminogen activation. Fibrin clots were prepared with 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 polyP65. Clotting was initiated with thrombin (0.25 U/mL) and CaCl2 (5 mM). Plasmin generation in clots was quantified by incorporating the fluorogenic substrate D–VLK-AMC and monitoring fluorescence release (FU; Ex 360 nm Em 460 nm). *P<0.05; **P<0.01, ***P<0.001 and ****P<0.0001 compared with control clots. 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; Ex: excitation; EM: emission.

Figure 3.

polyP binds to uPA with a significantly higher affinity than tPA. Binding of uPA or tPA (0–400 nM) to biotin-labelled polyP (71 mM) bound to streptavidin coated stripwells. Bound uPA (orange) or tPA (blue) was detected with chromogenic substrates (S2288 or CS-61 44 respectively) by reading the change in absorbance at 405 nm every 30 seconds (s) for 200 minutes (min). No unspecific binding was detected in the absence of biotin-labelled polyP (black lines). Data are expressed as baseline corrected nonlinear fit as mean ± standard error of the mean (SEM), n=4. PolyP: polyphosphate; tPA: tissue plasminogen activator; uPA: urokinase plasminogen activator.

Figure 4.

polyP acts as a cofactor to accelerate uPA-mediated fibrinolysis in a concentration and polymer size dependent manner. Fibrin clots were prepared containing 2.4 mM fibrinogen, 0.24 mM Glu-plasminogen, 20 pM tPA or 180 pM uPA ±328 mM polyP65. Clotting was initiated with thrombin (0.25 U/mL) and CaCl2 (5 mM) 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 polyP65 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 expressed as mean 50 % lysis time (t1/2) ± standard error of the mean, n≥3. polyP: polyphosphate; tPA: tissue plasminogen activator; uPA: urokinase plasminogen activator.

Figure 5.

The cofactor function of polyP in uPA-mediated fibrinolysis is modulated by plasminogen concentration and form. Fibrin clots were prepared containing 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 polyP65. Clotting was initiated with thrombin (0.25 U/mL) and CaCl2 (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 are expressed as mean ± standard error of the mean, n≥3. polyP: polyphosphate; tPA: tissue plasminogen activator; uPA: urokinase plasminogen activator.

Figure 6.

polyP colocalizes with fibrinogen and plasminogen during fibrinolysis. Fibrin clots were formed containing fibrinogen (2.65 mM, 9% DyLight 550-labeled), Glu-plasminogen (1.25 mM, 20% DyLight 633-labeled) 328 mM Cascade-blue labeled polyP70 (CB-polyP), thrombin (0.25 U/mL), CaCl2 (5 mM). (A) polyP and plasminogen largely accumulate in the knotted regions of fibrin. (B) Fibrinolysis was initiated by exogenous uPA (75 nM). Plasminogen and polyP strongly colocalize at the lysis front during clot lysis, as indicated by the arrows. Scale bars =10 mm. polyP: polyphosphate; uPA; urokinase plasminogen activator.

Figure 7.

Real-time lysis of fibrin clots reveals the cofactor function of polyP on uPA-mediated fibrinolysis. Fibrinolysis by exogenous PA (75 nM) was monitored by fluorescent confocal microscopy of clots formed from fibrinogen (2.65 mM, 9% DyLight 488-labeled), plasminogen (1.25 mM, 20% DyLight 633-labelled), thrombin (0.25 U/mL), CaCl2 (5 mM) ± 328 mM polyP65. Images were taken every 15 seconds (s). (A) uPA and (B) tPA mediated fibrinolysis over time showing fibrinogen (green), plasminogen (red) and merged images where co-localisation is visualised as yellow. Representative image from n=3, scale bar=10 mm. (C) Quantification of lysis time in s as determined by the time taken to lyse scan area (134.8 mm x 134.8 mm). Data expressed as mean ±standard error of the mean, n=3. polyA: polyphosphate; tPA: tissue plasminogen activator; uPA: urokinase plasminogen activator.