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main physiological plasminogen activators are the serine proteases tissue plasminogen activator (tPA) and uroki- nase plasminogen activator (uPA). Efficient plasminogen activation by tPA requires fibrin as a cofactor, acting as a template for its own dissolution by binding tPA and plas- minogen.11 uPA is fibrin-independent and efficiently acti- vates plasminogen in solution, but is often found in asso- ciation with its cellular receptor, urokinase protease acti- vated receptor (uPAR).12 uPAR does not have a catalytic role but acts to localize plasminogen and uPA to the cell surface increasing local reactant concentration.
We have previously shown that binding of tPA and plas- minogen to fibrin is downregulated by polyP, in turn, delaying tPA-mediated fibrinolysis.4 Here, we characterize the impact of polyP on uPA-mediated plasminogen activa- tion. Our data reveal that polyP has a strong accelerating effect on uPA-mediated plasminogen activation that is highly dependent on concentration of polyP and plas- minogen. Enhanced uPA-mediated plasminogen activa- tion in the presence of polyP translates as a robust profib- rinolytic effect in clot lysis assays. These data exemplify the complexity of polyP’s action on hemostasis and illus- trate that the presence of different activators and sub- strates in the local milieu can direct functional activity.
Methods
Additional experimental details can be found in the Online Supplementary Appendix.
Ethical Consent
Ethical approval was obtained from the University of Aberdeen College Ethics Review Board.
Materials
PolyP70 was a kind gift from Dr Thomas Staffel BK Giulini GmbH (Ludwigshafen, Germany). PolyP 14, 60, 130 were a kind gift from Dr Toshikazu Shiba Regenetiss Inc. Medium chain (p100) polyP with and without biotin-labeling was from Kerafast Inc (Boston, MA, USA). PolyP concentrations are expressed as monomer con- centrations throughout (monomer formula NaPO3). All reactions were carried out in TBST (50 mM Tris, 100 mM NaCl, 0.01% Tween-20, pH 7.4).
plates. Clotting was initiated by thrombin (0.25 U/mL) and CaCl (5 mM), and activity quantified using the fluorogenic substrate D- Val-Leu-Lys 7-amido-4-methylcoumarin (D–VLK-AMC [0.35 mM]) by measuring fluorescence release (excitation 360/40 nm, emission 460/40 nm) every minute (min) for 5 hours (h) in a Biotek Flx800 fluorescence microplate reader at 37 °C. The rate of plas- min generation was calculated using; Longstaff C, 2016, Shiny App for calculating zymogen activation rates, version 0.6 (https://drclongstaff.shinyapps.io/zymogenactnCL/).
PolyP enhances uPA-mediated plasminogen activation
biotin-labelled polyP (71 mM) were performed using an adaptation of the protocol described by Choi et al.13 Bound tPA, uPA or plas- min was detected with chromogenic substrates (1.2 mM S2288, CS-61 44, or 0.6 mM S2251, respectively). FXII(a) was detected using a peroxidase conjugated goat anti-human FXII antibody.
Confocal Microscopy
Clots were prepared with human plasminogen-free fibrinogen
Plasmin generation and uPA activity
Purified human plasminogen-free fibrinogen (2.4 mM), Glu- or
Lys-plasminogen (0–1 mM), tPA (20 pM) or uPA (180 pM) ± polyP (328 mM) in TBST was added in triplicate to 96-well polystyrene
UPA activity was determined by incubating the enzyme (180
pM) ±328 mM polyP 65
with the chromogenic substrate CS-61 44 (1.25 mM). Change in absorbance was measured every 30 seconds
(s) at 405 nm for 200 min.
Protein binding assays to biotin-labelled polyP
Binding of tPA, uPA, plasmin, FXII and activated FXII (FXIIa) to
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Turbidimetric fibrinolysis assays
Purified human plasminogen-free fibrinogen (2.4 mM), Glu- or Lys-plasminogen (0–1 mM), tPA (20 pM) or uPA (180 pM) with or without polyP (0–1.3 mM) in TBST was added in triplicate to 96- well polystyrene plates. Clotting was initiated by thrombin (0.25 U/mL) and CaCl2 (5 mM), and turbidity monitored every min at 340 nm for 5 h at 37°C in a FLX-800 plate reader (Biotek Instruments).
(2.65 mM) of which 9% was DyLight 488-labeled, Glu-plasmino-
gen (1.25 mM) of which 20% was DyLight 633-labeled, ±328 mM
polyP 65
0.4
in TBST in Ibidi -slides VI . Thrombin (0.25 U/mL) and
CaCl2 (5 mM) were added and fibrinolysis initiated by addition of tPA or uPA (75 nM).
Statistical analysis
Statistical analysis was performed in GraphPad Prism® 5.04 using one-way analysis of variance or two-way analysis of vari- ance with Bonferroni post hoc test or an unpaired Student’s t-test (2- tailed). P<0.05 was considered to be significant.
Results
PolyP promotes uPA-mediated plasmin generation
Our work has previously established that polyP inter- feres with the plasminogen activator function of tPA4 while augmenting plasminogen activation by FXIIa.14 Here we address the role of polyP on uPA-mediated plasmino- gen activation using the fluorogenic substrate D–VLK- AMC. PolyP dramatically enhanced the rate of plasmin generation (3.96±0.34 vs. 0.84±0.08 pM/s; P<0.001) during uPA-mediated fibrinolysis (Figure 1A). In contrast, and consistent with our previous results4, a decrease in the rate (0.74±0.06 vs. 1.17±0.14 pM/s in P<0.05) and amount of plasmin generation was observed during tPA-mediated fibrinolysis (Figure 1B). The ability of polyP to enhance the rate of uPA-mediated lysis was dose-dependent up to 32.8 mM after which it decreased before increasing again at 164 mM (Figure 1C). Above this concentration the rate of plasmin generation was greatly enhanced with a 6-fold increase at 328 mM polyP. In marked contrast, downregu- lation of tPA-mediated plasmin generation required con- centrations of greater than 70 mM and was not strongly does-dependent (Figure 1D).
Plasminogen concentration attenuates the cofactor function of polyP in uPA-mediated plasmin generation
We next analysed the impact of plasminogen concentra- tion on the cofactor function of polyP. Plasmin generation was monitored during clot lysis at various concentrations of plasminogen. The rate of plasmin generation by uPA and tPA in clots was directly proportional to the Glu-plas-
Cascade blue ethylenediamine (CB)-labeled polyP was pre- 70
pared as described.13 Clots were formed ±328 mM CB-polyP by polymerizingfibrinogen(2.65 mM,with9%labeledwithDL550- fibrinogen) as above.
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