Polymorphonuclear leucocytes have two opposing roles in fibrinolysis.

Polymorphonuclear leucocytes (PMN) are important in the resolution of human thrombi, with u-PA as a key player. We have shown that the u-PA activity of PMN depends on the presence of plasma; the study presented here provides an explanation for that requirement. Here we show that PMN degraded scu-PA and also tcu-PA, t-PA and plasmin, resulting in loss of fibrinolytic activity. Plasma protected against this degradation; alpha1-antitrypsin was identified as a protective factor. Purified human neutrophil elastase mirrored the effects of PMN, again neutralized by plasma inhibitors. These findings illustrate the dual role of PMN in the breakdown of thrombi, in that they contribute both u-PA, which lyses fibrin, and other proteases, including elastase, which can cleave fibrin and plasminogen activators/plasmin. Similarly, plasma can potentiate fibrinolysis by neutralization of PMN elastase, in addition to direct inhibition of fibrinolytic proteases. Our previous studies show that PMN in thrombi are mostly pro-fibrinolytic; the anti-fibrinolytic role defined here may be important in other pathologies where fibrin persists.

Human thrombi contain an abundance of active thrombin.

This study assessed the abundance and activity of thrombin in human thrombi. removed at autopsy or during surgery. Arterial and venous thrombus sections showed thrombin activity by in situ zymography, based on conversion of fibrinogen to fibrin. Hirudin or antibodies to thrombin abolished the activity. Thrombin activity in extracts of 40 thrombi was quantified by cleavage of fibrinogen or small peptide substrates: the results correlated well (r = 0.87, p<0.0001) with a median activity of about 4.5 IU/g of thrombus (wet weight). Activity correlated poorly with total prothrombin (median 27 microg/g) and was inversely related to antithrombin, but not to PAI-1. Zymography showed two major active bands, thrombin at 37 kDa, and a 50 kDa form that probably corresponds to meizothrombin desF1. The abundant local thrombin demonstrated here has implications for thrombus lysis and extension: incomplete lysis and exposure of active thrombin may lead to re-occlusion of vessels.

Thrombin upregulates tissue transglutaminase in endothelial cells: a potential role for tissue transglutaminase in stability of atherosclerotic plaque.

Atherosclerosis is characterized by thickening of the vessel wall, smooth muscle cell proliferation, macrophage infiltration, and deposition of a fibrin network. Transglutaminases are a family of enzymes catalyzing the formation of stable covalent cross-links between proteins. Here, we show that tissue transglutaminase (tTG) synthesis by human umbilical vein endothelial cells is upregulated by thrombin, the serine protease that causes fibrin formation and many cellular inflammatory effects. Thrombin upregulated tTG 2-fold at the mRNA and protein level. Cellular cross-linking activity was increased to an even greater extent; antibody to tTG neutralized the increased activity. The effect on tTG expression required active thrombin and was mediated mainly through protease-activated receptor-1, a thrombin receptor. Increased tTG antigen and activity were evident in human umbilical vein endothelial cells and extracellular matrix in situ. Thrombin treatment also led to a cellular redistribution of tTG. Normal vessel wall stained positively for tTG in the smooth muscle cells and in the subendothelium. The intensity of staining increased in vessel walls with plaque, where there was a striking increase in tTG in the smooth muscle cells immediately below the plaque. These studies indicate a role for tTG in the stabilization of atherosclerotic plaques and suggest that its local expression can be controlled by thrombin.

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.

Effective lysis of model thrombi by a t-PA mutant (A473S) that is resistant to alpha2-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 alpha2-antiplasmin (alpha2-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 alpha2-AP. We examined these forms of t-PA in model systems that are physiologically relevant. Neutralization of alpha2-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 alpha2-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 alpha2-AP or PAI-1. A473S was more effective than wild-type t-PA without the addition of antibodies by virtue of its resistance to alpha2-AP. This resistance was remarkable, in that no complex formed between A473S t-PA and alpha2-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 alpha2-AP contribute approximately equally to the inhibition of thrombus lysis. This study underlines the functional significance of alpha2-AP as a direct inhibitor of t-PA and further explains the basis of the accepted role of alpha2-AP as a regulator of fibrin persistence and thrombus resistance to 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.

Monocyte plasminogen activator inhibitor 2 (PAI-2) inhibits u-PA-mediated fibrin clot lysis and is cross-linked to fibrin.

Plasminogen activator inhibitor 2 (PAI-2) is a major product of activated human monocytes. Here we show that monocytes inhibited u-PA- but not t-PA-mediated fibrinolysis, by secreting PAI-2 into an overlying fibrin clot. Extracts of arterial and venous human thrombi were found to contain active PAI-2. PAI-2 was cross-linked to fibrin in a reaction catalyzed by two major transglutaminases (TG), tissue TG and factor XIII. The activity of PAI-2 was not affected by such cross-linking. Cross-linking of PAI-2 to fibrin was inhibited by Tridegin, a specific inhibitor of TG, and also by EDTA and iodoacetamide. The use of competitive peptides mimicking the loop between helices C and D of PAI-2 identified Gln 83 and 86 as residues important in cross-linking. This study defines a mechanism by which PAI-2 is localized to fibrin, where it acts as an effective inhibitor of u-PA-mediated fibrinolysis.

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

OBJECTIVE: 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-alpha 2-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-alpha 2-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-alpha 2-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.

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.

Thrombi formed in a Chandler loop mimic human arterial thrombi in structure and RAI-1 content and distribution.

We have previously shown that whole blood clots prepared under static conditions are a poor model for human thrombi formed in vivo. The most striking contrast is in the PAI-1 content, some 100 times lower in static clots than in human thrombi. This study aimed to evaluate thrombi formed in an artificial circulation (Chandler loop) using whole blood augmented with platelets. Citrated blood was recalcified and placed in tubing, which was sealed to form a closed loop and circulated. Immunohistochemical staining revealed morphology very similar to human thrombi formed in vivo, comprising dense platelet-rich heads and fibrin-rich tails. Strong positive staining for PAI-1 was observed in fibrin-rich areas of both the head and the tail. The PAI-1 content of the thrombi increased significantly on augmentation of blood with isolated platelets, and reached levels comparable with those in human thrombi. These Chandler thrombi thus provide an appropriate model system for the study of thrombus lysis. in contrast to static blood clots.

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.

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 alpha2-antiplasmin (alpha2-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, alpha2-AP and t-PA-PAI-1 complex in bone marrow, and resulted in favourable conditions for fibrinolysis. The presence of plasmin-alpha2-AP complex at concentrations of the same order of magnitude as total plasminogen and alpha2-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.

Proteins of the fibrinolytic system in human thrombi.

The proteins of fibrinolysis have been quantified in human thrombi, to assess the balance between plasminogen activators and their major inhibitor PAI-1. The relative roles of PAI-1 and alpha 2-AP were also examined since we have previously shown that both platelet PAI-1 and plasma alpha 2-AP are important determinants of clot lysis in vitro. Extracts and sections were prepared from human thrombi for quantitative immunoassay and immunohistochemical staining respectively. PAI-1 and alpha 2-AP were present at high concentrations. Levels of t-PA and t-PA-PAI-1 complex were relatively low. Staining confirmed the presence of abundant PAI-1, associated primarily with platelet material within the thrombus and also with fibrin. Staining for alpha 2-AP was also intense and demonstrated strong association with fibrin. The alpha 2-AP concentration was similar to its high plasma concentration, whereas PAI-1 levels were up to 30 times greater than that in circulating blood, suggesting that active recruitment of platelets contributes to the high PAI-1 concentration in thrombi.

Inhibitors of plasminogen activator in neutrophils and mononuclear cells from septic patients.

Leucocytes, both polymorphs and mononuclear cells, play a variety of roles in the evolution of human response to sepsis, both local and generalised. In this study, inhibitors of plasminogen activator were measured in leucocytes from normal and septic patients. Plasminogen activator inhibitor-1 (PAI-1) was identified in polymorphs from normal individuals and levels rose significantly in polymorphs from septic patients: neutrophils from normal subjects did not contain PAI-2 but this protein was detectable in significant quantities in polymorph preparations from septic patients. In contrast, mononuclear cells from normal and septic patients contained no detectable quantities of PAI-1. Significant amounts of PAI-2 were present in normal mononuclear cells, and the levels rose significantly in monocytes from septic patients. PAI-2 is thus here identified in human subjects, distinct from those with pregnancy or malignancy, as playing a role in a pathological process. The increased levels of both inhibitors produced by leucocytes may clearly contribute directly to the persistence of fibrin, a characteristic feature of the response to infection, local or general; they may thus participate in successful localisation of infections (abscess formation etc.) and in the evolution of the major systemic complications of disseminated sepsis characterised by microvascular occlusion by fibrin such as renal failure, shock lung or digital ischaemia.

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

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.

The roles of alpha 2-antiplasmin and plasminogen activator inhibitor 1 (PAI-1) in the inhibition of clot lysis.

The relative importance of the two major inhibitors of fibrinolysis, alpha 2-antiplasmin (alpha 2-AP) and plasminogen activator inhibitor (PAI-1), were investigated using a simple microtitre plate system to study fibrin clot lysis in vitro. Cross-linked fibrin clots contained plasminogen and tissue plasminogen activator (t-PA) at concentrations close to physiological. Purified alpha 2-AP and PAI-1 caused dose-dependent inhibition. All the inhibition due to normal plasma, either platelet-rich or poor, was neutralised only by antibodies to alpha 2-AP. Isolated platelets, at a final concentration similar to that in blood, 2.5 x 10(8)/ml, markedly inhibited clot lysis. This inhibition was neutralised only by antibodies to PAI-1. At the normal circulating ratio of plasma to platelets, alpha 2-AP was the dominant inhibitor. When the platelet:plasma ratio was raised some 20-fold, platelet PAI-1 provided a significant contribution. High local concentrations of PAI-1 do occur in thrombi in vivo, indicating a role for PAI-1, complementary to that of alpha 2-AP, in such situations.