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Ferrata Storti Foundation
Haematologica 2021 Volume 106(2):522-531
Coagulation & its Disorders
uPA-mediated plasminogen activation is enhanced by polyphosphate
Claire S. Whyte and Nicola J. Mutch
Aberdeen Cardiovascular & Diabetes Center, School of Medicine, Medical Sciences & Nutrition, Institute of Medical Sciences, University of Aberdeen, Aberdeen, UK
ABSTRACT
Tissue plasminogen activator (tPA) and urokinase plasminogen acti- vator (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 urokinase protease activated 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 acce-ler- ated 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 polyP enhanced uPA-mediated fibri-
65
no-lysis. Varying the plasminogen concentration (0.125-1 mM) 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 addition- al 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-media-ted plasminogen activation. In contrast, polyP may facilitate the interaction between fibrin-independent uPA and plas- minogen thereby accelerating plasmin generation and downstream fibri- nolysis.
Introduction
Polyphosphate (polyP) is a biomolecule composed of orthophosphate residues (Pi) linked by phosphoanhydride bonds.1 polyP of average chain length of 60-100-mers is a constituent of platelet dense granules and is released following stimulation of platelets with different agonists.1,2 polyP acts at numerous points in the coagulation cascade to augment clot formation, including stimulating factor XII (FXII) activation, thrombin-mediated factor XI (FXI) activation and interfering with the function of TFPI.3 Our work has shown that polyP interferes with fibrin poly- merization by stunting protofibril growth, producing a heterogeneous network of dense ‘knotted’ regions interspersed by pores with altered mechanical properties.4
Plasmin is the serine protease responsible for degradation of fibrin. Two forms of the zymogen precursor plasminogen circulate in plasma, the native more abundant form, Glu-plasminogen, and the intermediate form Lys-plasminogen, formed by cleavage of the N-terminal peptide from Glu-plasminogen.5 Lys-plasminogen exists in flexible open conformation, with an approximately 10-fold higher binding affi- nity for plasminogen activators thereby facilitating its activation.6-9 Two-chain plas- min is formed by enzymatic cleavage of plasminogen at Arg561-Val562.5,10 The two
Correspondence:
NICOLA J. MUTCH
n.j.mutch@abdn.ac.uk
Received: September 12, 2019. Accepted: January 31, 2020. Pre-published: February 6, 2020.
https://doi.org/10.3324/haematol.2019.237966
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