Linda A. Robbie

Inhibitors of fibrinolysis are elevated in atherosclerotic plaque.

The proteins of the fibrinolytic system have been examined in the human normal and atherosclerotic arterial wall by immunohistochemical techniques and by quantitative immunoassay of extracts. The concentration of plasminogen activator inhibitor-1 (PAI-1) increased significantly during the progression from normal vessels to fatty streaks to the developed atherosclerotic plaque. Staining for PAI-1 was strongly positive, particularly in the areas adjacent to the plaque. In these areas, PAI-1 appeared to colocalized with its binding protein vitronectin. Alpha2-antiplasmin (alpha2-AP) was present in the aorta at even higher concentrations than PAI-1; a small but significant increase was seen in some atherosclerotic compared with normal vessel walls. Tissue plasminogen activator (TPA) showed the opposite trend, being lowest in lesions with plaque. Thus, higher concentrations of the two principal inhibitors of fibrinolysis, PAI-1 and alpha2-AP, together with lower levels of TPA, are characteristic of advanced atheromatous lesions. Alteration in the balance of the fibrinolytic system, favoring its inhibition, may predispose to the development or maintenance of atherosclerotic plaque.

Polymorphonuclear leucocytes mediate endogenous thrombus lysis via a u-PA-dependent mechanism

Many human thrombi lyse spontaneously without the administration of lytic drugs and cause no clinical symptoms. The mechanisms by which this occurs are incompletely understood. We found that model thrombi prepared from whole human blood in a Chandler loop also exhibited significant spontaneous lysis. Lysis was inhibited by chemical protease inhibitors, consistent with proteolysis resulting primarily from serine proteases, with a small contribution from matrix metalloproteinases. Whole blood was fractionated into platelet‐rich plasma and cell populations. Significant spontaneous lysis was observed in platelet‐rich thrombi enriched with polymorphonuclear leucocytes (PMNs), whereas mononuclear cells (MCs) and erythrocytes did not contribute to lysis. Incorporation of antibodies to urokinase (u‐PA) and its receptor u‐PAR neutralized a large proportion of the activity. Incubation of plasma with PMNs generated free u‐PA activity, which was also detectable in model thrombi and in vivo human thrombi. Purified neutrophils, free of eosinophils, generated activity identical to PMNs. Smaller contributions to lysis by tissue‐type plasminogen activator (t‐PA), elastase and cathepsin G were also identified. These findings suggest a major role for circulating PMNs in endogenous thrombus lysis.

Plasminogen activator inhibitor 2 and urokinase‐type plasminogen activator in plasma and leucocytes in patients with severe sepsis

Proteins influencing plasminogen activation to plasmin, namely plasminogen activators tissue‐type plasminogen activator (t‐PA) and urokinase‐type plasminogen activator (u‐PA) and their principal inhibitors, plasminogen activator inhibitor 1 (PAI‐1) and PAI‐2, were measured in the plasma, the polymorph and mononuclear cell fractions taken from patients with major sepsis who were entering a general intensive care unit. The purpose of this study was to elucidate the factors favouring the persistence of fibrin in the microvasculature and thus contributing to multiple organ failure. Levels of u‐PA antigen in plasma rose in sepsis and u‐PA activity, not detectable in normal plasma, appeared. Levels of u‐PA antigen in the cell fractions fell concomitantly. t‐PA antigen in plasma and in the mononuclear cell fraction rose in sepsis, but t‐PA activity was not detectable. Plasma PAI‐1 antigen levels were strikingly raised in sepsis, presumably accounting for the complete neutralization of t‐PA activity. PAI‐2 antigen, not normally detected in plasma, appeared in the plasma of some patients, whereas it disappeared from the cellular fractions. Appearance of PAI‐2 in plasma was associated with non‐survival of the patient. The observations indicate that all the agents involved in plasminogen activation are released into the plasma in major sepsis. The levels of PAI‐1 reached were quantitatively sufficient to suppress all activity of the released t‐PA, but the inhibitors did not prevent expression of u‐PA activity in the circulation. Circulating active u‐PA and PAI‐2 in the plasma of patients with severe sepsis may represent material originating from leucocytes. Leucocyte release of these agents within fibrin deposits may influence the persistence of fibrin and thus the development of multiple organ failure.

Evidence for an active fibrinolytic system in normal human bone marrow

Normal human bone marrow from patients undergoing heart surgery was analysed quantitatively for components of the fibrinolytic system, using functional and immunological assays. Marrow was found to contain considerable fibrinolytic activity, reflecting high levels of t‐PA (tissue‐type plasminogen activator). The t‐PA was in an active form, despite the presence of the inhibitors PAI‐1 and PAI‐2. Plasminogen and α2‐antiplasmin (α2‐AP) were also present in marrow. The balance of proteases and inhibitors differed dramatically from that observed in plasma, with higher levels of t‐PA, PAI‐1 and PAI‐2, and lower levels of u‐PA (urokinase), plasminogen, α2‐AP and t‐PA‐PAI‐1 complex in bone marrow, and resulted in favourable conditions for fibrinolysis. The presence of plasmin–α2‐AP complex at concentrations of the same order of magnitude as total plasminogen and α2‐AP demonstrated that active generation of plasmin was indeed occurring. A role for the active fibrinolytic system in normal human bone marrow may be the removal of unnecessary fibrin deposits formed in the cavities of the marrow, in order to maintain flow through this tissue.

Plasminogen activator in acute myeloid leukaemic marrows: u-PA in contrast to t-PA in normal marrow

Leukaemic and normal bone marrow samples were compared in terms of their content of the fibrinolytic agents, tissue plasminogen activator (t‐PA) and urokinase‐type plasminogen activator (u‐PA) and their inhibitors, plasminogen activator inhibitors 1 and 2 (PAI‐1 and PAI‐2). Normal marrow contained t‐PA as the principal plasminogen activator, whereas in leukaemic marrow samples u‐PA was the predominant activator. Both normal and leukaemic marrows contained PAI‐1 in similar amounts, but whereas normal marrow contained significant amounts of PAI‐2 the leukaemic marrows contained very little. Plasminogen activators were present in uncomplexed, active forms and plasmin–α2‐antiplasmin complexes were generated locally more prominently in leukaemic marrows. u‐PA associated with blast cells may contribute to the severe forms of haemorrhage sometimes occurring in myeloid types of leukaemia.

Influence of white blood cells on the fibrinolytic response to sepsis: studies of septic patients with or without severe leucopenia

Summary. In septic patients capable of normal white cell responses, high plasma levels of PAI‐I, t‐PA antigen and t‐PA‐PAI‐I complex were observed. The ratios of t‐PA and PAI‐I were such that free PA activity was almost never observed. In patients severely leucopenic prior to becoming septic the changes were significantly less marked, so presence of leucocytes enhances the fibrinolytic inhibition occurring in sepsis. The non‐leucopenic septic group showed greater evidence of thrombin generation in that FPA levels were higher but fibrinogen levels were only slightly less and antithrombin levels not different from those in the leucopenic group. A greater tendency to fibrin deposition and the striking fibrinolytic inhibition noted in patients with normal white cell responses may contribute to the development of some of the complications of sepsis in which fibrin deposition participates and may explain their relative rarity in leucopenic patients. When shock supervened, levels of PAI‐I were high in both leucopenic and non‐leucopenic groups, indicating that a source of PAI‐I outwith the leucocytes themselves contributes to the phenomena observed.

Effective lysis of model thrombi by a t‐PA mutant (A473S) that is resistant to α2‐antiplasmin

This study used two mutants of tissue‐type plasminogen activator (t‐PA) with resistance to inhibitors of fibrinolysis to define the contribution of plasminogen activator inhibitor (PAI)‐1 and α2‐antiplasmin (α2‐AP) to the control of fibrin lysis. Wild‐type t‐PA was compared with KHRR296–299AAAA, which is resistant to PAI‐1, and with A473S, which is resistant to α2‐AP. We examined these forms of t‐PA in model systems that are physiologically relevant. Neutralization of α2‐AP was essential for lysis of plasma clots, irrespective of their platelet content, by either wild‐type t‐PA or KHRR296–299AAAA. In marked contrast, A473S lysed plasma clots without neutralization of α2‐AP. Model thrombi, with structures similar to in vivo thrombi, were lysed slowly by wild‐type t‐PA; the rate and extent of lysis were enhanced by the addition of antibodies to α2‐AP or PAI‐1. A473S was more effective than wild‐type t‐PA without the addition of antibodies by virtue of its resistance to α2‐AP. This resistance was remarkable, in that no complex formed between A473S t‐PA and α2‐AP, even after extended incubation, when 50% of wild‐type t‐PA could be converted to complex. Comparison of A473S and KHRR296–299AAAA mutants showed their similar effectiveness in lysis of platelet‐rich model thrombi. Thus, PAI‐1 and α2‐AP contribute approximately equally to the inhibition of thrombus lysis. This study underlines the functional significance of α2‐AP as a direct inhibitor of t‐PA and further explains the basis of the accepted role of α2‐AP as a regulator of fibrin persistence and thrombus resistance to lysis.

Fibrinolytic changes in a patient with toxic shock syndrome; release of active u-PA

AbstractObjective: Definition of the changes in the activators of plasminogen, u-PA and t-PA, and examination of the possible generation of plasmin in the circulation in overwhelming sepsis. Design: Serial blood analysis starting 4 h after development of symptoms of toxic shock syndrome. Setting: Intensive care unit. Patient: A previously healthy woman underwent endometrial ablation and rapidly thereafter developed staphylococcal toxic shock syndrome with organ failure. Measurement and result: t-PA, PAI-1, t-PA-PAI-1 complexes, plasminogen, fibrinogen and plasmin-α2-antiplasmin complexes were measured serially by ELISA and free u-PA by SDS-PAGE with zymography. The onset of symptoms was accompanied by a rise of t-PA antigen followed rapidly by PAI-1 antigen. Plasmin-α2-antiplasmin complex was generated in large amounts but disappeared within hours. From day 3, free u-PA was detectable in the circulation without plasmin generation. Conclusion: Despite the sustained presence of active u-PA in the circulation and of t-PA antigen at the onset of symptoms, plasmin-α2-antiplasmin generation was largely suppressed by high levels of PAI-1. The suppression of plasmin generation by u-PA and t-PA may ensure the persistence of fibrin in the microcirculation and so contribute to organ failure.

Myeloid leukaemic cells can lyse fibrin directly

Purified preparations of circulating leukaemic blast cells from patients with acute myeloid (M1–7) or acute lymphoblastic leukaemia, and the myeloid or lymphoid cells from patients with chronic myeloid or lymphocytic forms of leukaemia, were incorporated into clots prepared from fibrinogen and plasminogen. Patterns of lysis were followed and measured by light transmission in a microtitre plate reader. Mature polymorphonuclear and mononuclear cell fractions from normal individuals were studied concurrently for comparison. Blast cells from the myeloid forms of acute leukaemia (M2–4) and ‘myeloid’ cell fractions from patients with chronic myeloid leukaemia were capable of lysing plasminogen‐containing clots; this activity was neutralized by addition of immunoglobulin against urokinase plasminogen activator (u‐PA), but not by anti‐tissue plasmogen activator (t‐PA). Mature polymorphonuclear and mononuclear cells from normal individuals lacked lytic activity, as did the leukaemic cells from patients with acute lymphoblastic or chronic lymphocytic leukaemia. Lysed blast cells showed the presence of free plasminogen activator on sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS–PAGE) with overlay zymography, also neutralized by anti‐u‐PA, whereas normal polymorphonuclear and mononuclear cells did not. These observations suggest that mechanisms underlying some forms of severe bleeding in acute myeloid leukaemias have a critical fibrinolytic component generated by the blast cells themselves.

Tumour cell u-PA as a cause of fibrinolytic bleeding in metastatic disease.

Tumour cells may express urokinase type plasminogen activator (u‐PA). This may influence the invasive properties of the cells but has seldom been implicated in production of a systemic bleeding state. Two patients are described in whom severe bleeding occurred in association with disseminated malignancies. Thrombin generation was little disturbed and platelet numbers were insufficient to account for the bleeding. Florid plasmin generation was evident in the circulation and the fibrinolytic inhibitor tranexamic acid controlled the bleeding well. Free active u‐PA was demonstrated in the circulation and u‐PA antigen on the malignant cells which invaded the marrow of one of the patients. Tumour cell u‐PA may occasionally be responsible for a bleeding state.