Role of Shear Stress and tPA Concentration in the Fibrinolytic Potential of Thrombi.

Claire S Whyte,Kim M Baeten,David E Newby,Nicola J Mutch,Nuala A Booth,Hadj Ahmed Mostefai,Andrew J Lucking

doi:10.3390/ijms22042115

Abstract

The resolution of arterial thrombi is critically dependent on the endogenous fibrinolytic system. Using well-established and complementary whole blood models, we investigated the endogenous fibrinolytic potential of the tissue-type plasminogen activator (tPA) and the intra-thrombus distribution of fibrinolytic proteins, formed ex vivo under shear. tPA was present at physiologically relevant concentrations and fibrinolysis was monitored using an FITC-labelled fibrinogen tracer. Thrombi were formed from anticoagulated blood using a Chandler Loop and from non-anticoagulated blood perfused over specially-prepared porcine aorta strips under low (212 s−1) and high shear (1690 s−1) conditions in a Badimon Chamber. Plasminogen, tPA and plasminogen activator inhibitor-1 (PAI-1) concentrations were measured by ELISA. The tPA–PAI-1 complex was abundant in Chandler model thrombi serum. In contrast, free tPA was evident in the head of thrombi and correlated with fibrinolytic activity. Badimon thrombi formed under high shear conditions were more resistant to fibrinolysis than those formed at low shear. Plasminogen and tPA concentrations were elevated in thrombi formed at low shear, while PAI-1 concentrations were augmented at high shear rates. In conclusion, tPA primarily localises to the thrombus head in a free and active form. Thrombi formed at high shear incorporate less tPA and plasminogen and increased PAI-1, thereby enhancing resistance to degradation.

Highlights

We characterised the optimal time for thrombus formation to be 30 min, as after this point, substantial fluorescence release was detected in the Chandler loop serum which is indicative of ongoing fibrinolysis (Figure S1)
Our laboratories have been instrumental in investigating fibrinolysis of thrombi formed in the presence of physiologically-relevant levels of type plasminogen activator (tPA) and under a variety of continuous flow conditions [7,34,35,36,37,38]
Accumulation of tPA and plasminogen was observed in the head of Chandler model thrombi, directly aligning with localisation of fibrinolytic activity

Summary

Introduction

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Fibrinolysis is responsible for counterbalancing thrombus formation, promoting thrombus dissolution and facilitating wound healing. Plasmin is the key proteolytic enzyme responsible for degradation of fibrin and is formed by the cleavage of the zymogen form, plasminogen, at Arg561-Val562 [1,2]. The two principal physiological plasminogen activators are tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA). The circulating concentration of tPA is low but local concentrations rise markedly following release from vascular endothelial cells in response to stimuli including shear stress, fibrin deposition, thrombin and bradykinin [3,4,5,6]

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Conclusion