Hyaluronic acid (HA) is a large, non-sulfated glucosaminoglycan abundantly present at sites where fibrin is also formed (during wound healing, in arterial restenotic lesions and eroded atherosclerotic plaques). The aim of the present study was to characterize the structure of composite fibrin-HA clots with scanning electron microscopy (SEM), pressure-driven permeation and small-angle X-ray scattering (SAXS) and their viscoelastic properties with an oscillation rheometer. In addition the efficiency of fibrinolysis in these clots was investigated by kinetic turbidimetric and chromogenic assays for dissolution of fibrin and plasminogen activation by tissue-type plasminogen activator (tPA). Fibrin formed in the presence of native (1500 kDa) HA and its 500 kDa fragments had thicker fibers and larger pores according to the SEM and clot permeation data, whereas the 25 kDa HA fragments had only minor effects. SAXS evidenced a mild disarrangement of protofibrils. These structural alterations suggest that HA modifies the pattern of fibrin polymerization favouring lateral association of protofibrils over formation of branching points. Rheometer data showed softer fibrin structures formed with 1500 kDa and 500 kDa HA and these clots presented with lower dynamic viscosity values and lower critical stress values at gel/fluid transition. tPA-catalysed plasminogen activation was markedly inhibited by HA, both in free solution and on the surface of fibrin clots, in the presence and in the absence of 6-aminohexanoate suggesting a kringle-independent mechanism. HA of 1500 and 500 kDa size prolonged clot lysis with both plasmin and tPA and this inhibition was kringle-mediated, because it was abolished by 6-aminohexanoate and was not observed with des-(kringle1–4)-plasmin. Our data suggest that HA size-dependently modifies the pattern of fibrin polymerization with consequent inhibition of fibrinolysis. At sites of tissue injury and inflammation, HA could stabilize fibrin through modification of its structure and lysibility.
Bibliographical noteFunding Information:
The authors are grateful to Gy?rgyi Oravecz and Kriszti?n B?lint for technical assistance. This work was supported by the Hungarian Scientific Research Fund OTKA 112612.
- Hyaluronic acid
- Plasminogen activation