Phospholipid Barrier to Fibrinolysis

Abstract

The massive presence of phospholipids is demonstrated in frozen sections of human arterial thrombi. Purified platelet phospholipids and synthetic phospholipids retard in vitro tissue-type plasminogen activator (tPA)-induced fibrinolysis through effects on plasminogen activation and plasmin function. The inhibition of plasminogen activation on the surface of fibrin correlates with the fraction of anionic phospholipid. The phospholipids decrease the amount of tPA penetrating into the clot by 75% and the depth of the reactive surface layer occupied by the activator by up to 30%, whereas for plasmin both of these parameters decrease by ϳ50%. The phospholipids are not only a diffusion barrier, they also bind the components of the fibrinolytic system. Isothermal titration calorimetry shows binding characterized with dissociation constants in the range 0.35-7.64 M for plasmin and tPA (lower values with more negative phospholipids). The interactions are endothermic and thermodynamically driven by an increase in entropy, probably caused by the rearrangements in the ordered gel structure of the phospholipids (in line with the stronger inhibition at gel phase temperatures compared with liquid crystalline phase temperatures). These findings show a phospholipid barrier, which should be overcome during lysis of arterial thrombi.The basis of the current thrombolytic therapy in acute myocardial infarction or ischemic stroke is the administration of plasminogen activators (e.g. tPA 1 and its recombinant variants), which convert the plasminogen in blood plasma or on the surface of the thrombi to plasmin and the latter dissolves fibrin, the solid matrix of thrombi (1). However, persistent recanalization of the occluded blood vessels often fails (in 15-40% of patients), and efficient doses of thrombolytics have significant bleeding side effects (2). The limitations of existing fibrinolytic therapy indicate that thrombolysis is not identical to fibrinolysis and direct attention to the variety of cellular and molecular factors present in thrombi that modulate the function of the basal fibrinolytic machinery (recently reviewed in Ref.3).Platelets render thrombi resistant to lysis with tPA (4), which can be partially explained by their PAI-1 content (5), but evidence for PAI-1-independent inhibition of fibrinolysis also exists (6). When arterial thrombi are formed, the platelet content of 10 ml of whole blood is compacted in a volume of 400 l, whereas the fibrin content of the same thrombi corresponds to the fibrinogen concentration in blood plasma (7), the intrathrombus plasminogen (0.4 M), and ␣ 2 -PI (0.25 M) concentrations are just fractions of the respective plasma values (8). After activation, the platelets release their cytosolic content and lose the majority of their phospholipid (9). By taking into account the average amount of phospholipids in platelets (10), the phospholipid content of arterial thrombi (7.5 g/liter) is expected to be higher than their fibrin content (1.7 g/liter). Phospholipids are known to bind fibrinogen a...

Fibrinolysis at the Fluid-Solid Interface of Thrombi

“…Conventional assays in free solution miss these effects, because there are certain structural requirements for the inhibiting action of phospholipids on the fibrinolytic system. The plasminogen activation assay shows correlation between the degree of inhibition and the fraction of anionic phospholipid [129]. The anionic charge, however, is not a sufficient prerequisite for the phospholipid effects.…”

“…After activation the platelets release the majority of their phospholipid [128]. Histological examination of arterial thrombi provides evidence for the presence of phospholipids and the intensity of their staining suggests phospholipid concentration of approximately 5 g/L [129], which is even higher than the fibrin content of the thrombi (1.7 g/L) [124]. The phospholipid membrane of activated platelets is an essential site for the assembly of complexes (tenase, prothrombinase) responsible for the generation of thrombin (reviewed in [130]).…”

“…Thus, phospholipids contribute to the organization of the fibrin network and the subsequent modulation of fibrinolysis as discussed above. In addition phospholipids (at concentration relevant to the thrombi) directly inhibit fibrinolysis induced by tPA [129]. Although phospholipids slow down the inhibition of plasmin by 2 -PI [129], the combined presence of phospholipids and 2 -PI exerts stronger inhibition of fibrinolysis than each of the inhibitors alone.…”

“…In addition phospholipids (at concentration relevant to the thrombi) directly inhibit fibrinolysis induced by tPA [129]. Although phospholipids slow down the inhibition of plasmin by 2 -PI [129], the combined presence of phospholipids and 2 -PI exerts stronger inhibition of fibrinolysis than each of the inhibitors alone. This suggests that the phospholipids impair the well-known protection of plasmin against its inhibitors on the fibrin surface [51].…”

“…The ordered gel-structure adopted by dipalmitoyl phospholipids at 37 °C inhibit the tPAinduced fibrinolysis. If the membrane is in the liquidcrystalline structure at temperatures far from the melting point, the inhibiting effect is completely eliminated [129]. The phase-structure requirement explains why studies approaching the dissolution of alveolar fibrin deposits and modeling the action of lung surfactant with dipalmitoylphospholipids (gel-phase) detected inhibition [131,132], whereas others working with natural phospholipid mixtures (with abundant unsaturated fatty acyl derivatives) missed this effect [133].…”