Originally published online as doi:10.1189/jlb.0502257 on July 26, 2004

Published online before print July 26, 2004

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(Journal of Leukocyte Biology. 2004;76:571-576.)
© 2004 by Society for Leukocyte Biology

Polymorphonuclear leukocytes from patients with severe sepsis have lost the ability to degrade fibrin via u-PA

E. Moir^*, M. Greaves^*, G. D. Adey and B. Bennett^*,1

^* Departments of Medicine and Therapeutics, University of Aberdeen, Institute of Medical Science, and
Anaesthetics, Grampian Hospitals NHS Trust, Scotland, United Kingdom

¹ Correspondence: Department of Medicine and Therapeutics, Institute of Medical Science, Polwarth Building, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, Scotland, UK. E-mail: b.bennett{at}abdn.ac.uk

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Fibrin persistence in the vasculature is an important complication of sepsis that can often lead to mortality. We have previously established that polymorphonuclear leukocytes (PMN) from healthy individuals have the capacity to degrade fibrin via urokinase-type plaminogen activator (u-PA). We have also demonstrated an increase in u-PA antigen in the plasma of patients suffering from septic shock. In this study, we investigate the hypothesis that PMN from patients with sepsis have lost their fibrinolytic ability and that this might contribute to the persistence of fibrin deposits. We show here that PMN from these patients do not express any u-PA activity, despite retaining some u-PA antigen. Additionally, thrombi prepared from the whole blood of the patients exhibit reduced endogenous lysis compared with those from healthy individuals. These data indicate that loss of fibrinolytic activity from PMN may be a contributing factor in fibrin persistence in the microvasculature in sepsis.

Key Words: fibrinolysis • PMN • urokinase

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The pathological deposition of fibrin is often an early event in episodes of sepsis [¹ , ² ]. Nonclearance of such fibrin deposits contributes to the tissue damage that frequently causes complications such as acute respiratory distress syndrome and acute renal failure in sepsis. The mechanisms underlying excess fibrin formation within the microvasculature of these patients have been well-studied and result from the inappropriate activation of coagulation as a result of increased tissue-factor activity and decreased levels of the coagulation inhibitors ATIII and protein C (reviewed in ref. [³ ]). Other groups have suggested that persistence of fibrin is mainly a result of retarded fibrinolysis in the plasma of septic patients. The fibrinolytic system is shown in Figure 1 . Elevation of PAI-1 in these patients is most striking [^{4 5 6 7} ]. PAI-1 inhibits the PAs, t-PA and u-PA, thus preventing generation of active plasmin. Increased PAI-1 has been identified in some studies as a marker for mortality in sepsis [⁸ , ⁹ ]. However, elevated plasma PAI-2 levels have also been observed and can be associated with nonsurvival [¹⁰ ].

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Figure 1. Schematic of fibrinolytic cascade. Inhibition is indicated by dotted lines. Thrombin-activatable fibrinolysis inhibitor (TAFI)* acts as an indirect inhibitor of fibrinolysis. t-PA, Tissue-type plasminogen activator; u-PA, urokinase-type PA; PAI, PA inhibitor; 2-AP, 2-antiplasmin.

Sepsis can also affect the plasma concentrations of plasminogen and its physiological activators. Plasma plasminogen is decreased [¹¹ ], and the more severe the sepsis, the greater the depletion [⁷ ]. Plasma levels of t-PA antigen are raised in sepsis [⁴ , ⁶ , ⁷ , ¹² ], presumably reflecting its release from endothelial cells, but it appears mainly in complex with PAI-1, with decreased ability to activate plasminogen. It has also been demonstrated that plasma u-PA antigen can be raised in sepsis in some cases, with u-PA activity sometimes being detected [¹⁰ , ¹³ ]. Alterations in activators and inhibitors may not follow the same time-course, introducing the potential for dysregulation of activity.

Most studies of fibrinolysis only investigate the levels of fibrinolytic proteins and inhibitors in plasma. We are interested in the leukocyte contribution to spontaneous fibrin degradation and have recently demonstrated that in healthy individuals, polymorphonuclear leukocytes (PMN) are central to fibrinolysis, mediating the spontaneous breakdown of model thrombi via u-PA activation of plasminogen [¹⁴ ]. In sepsis, however, leukocytes may also contribute to the persistence of fibrin deposition by an increase in PAI-1 and PAI-2 in PMN and an increase in PAI-2 in monocytes [⁵ , ¹² ]. We have also shown a reduction in u-PA antigen from PMN in septic patients [¹⁰ ], but cellular u-PA activity has not been studied in sepsis.

This study was undertaken to determine whether in a situation characterized by fibrin deposition and persistence, PMN retain the ability to lyse fibrin via u-PA generation. A failure or loss of cell-associated lytic ability would represent an additional factor in persistence of intravascular and extravascular fibrin deposits.

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Preparation of plasma and leukocytes
Patients entering the Intensive Therapy Unit (Grampian Hospitals, NHS Trust, Aberdeen, Scotland) with established or presumptive diagnoses of major sepsis according to the systemic inflammatory response syndrome criteria [¹⁵ ] were studied within 36 h of admission to the unit. Informed consent was obtained from patients or relatives, and the Institutional Research Ethics Committee approved the study. Blood samples were collected into 0.1 vol of 0.13 M trisodium citrate. Platelet-poor plasma (PPP) was prepared by centrifugation as described previously [¹⁶ ]. Leukocytes were obtained by density gradient centrifugation over Polymorphoprep (Nycomed, Oslo, Norway) as described previously [¹⁴ ]. Blood was also obtained from healthy individuals as controls and treated identically.

Clot lysis assay
Purified fibrin clots were prepared, and their lysis was measured as described previously [¹⁷ ]. Briefly, fibrinogen (2.92 µM, Kabi), plasminogen (0.24 µM), and thrombin (0.4 IU/ml, Leo Pharmaceuticals, Ballerup, Denmark) were recalcified in a microtiter plate with CaCl₂ (5.3 mM), and absorbance was monitored over time. An increase in absorbance occurs during fibrin formation, and a decrease indicates fibrin lysis. PMN (1x10⁷/ml) and/or plasma (8%) were included as indicated. Antibodies to ₂-AP (Dako, Bucks, UK) were included in clots containing plasma at a final concentration of 250 µg/ml to allow lysis to be detected. Note that although these antibodies are required to allow lysis to be detected in this purified system, no such addition is necessary to allow spontaneous lytic activity to be detected in a model thrombus system [¹⁴ ]. Antibodies to u-PA or t-PA (Technoclone, Surrey, UK) were included in some clots at a final concentration of 5 µg/ml.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and zymography
PMN (1.6x10⁵) alone or incubated with plasma (4 µl) for 30 min at 37°C were separated on 10% SDS-PAGE gels, and zymography was carried out as described previously [¹⁸ ]. In some experiments, rabbit antibodies to human u-PA or t-PA (Technoclone) were incorporated into the fibrin detector plate at final concentrations of 12.5 µg/ml to identify the active bands.

Immunohistochemistry
Immunohistochemistry of isolated PMN was carried out by the alkaline phosphatase and antialkaline phosphatase method [¹⁹ ] for u-PA. Frozen smears were allowed to come to room temperature before fixing in 100% acetone for 10 min at 4°C. Slides were allowed to air-dry and then were rehydrated for 10 min in Tris-buffered saline (5 mM Tris, pH 7.6, 0.13 M NaCl) before staining. Primary antibody was a monoclonal antibody (mAb) to u-PA (methyl UK-1, Biopool, supplied through Porton Products, Berkshire, UK). Secondary antibody was rabbit anti-mouse immunoglobulin G (Dako) at 175 µg/ml in 10% normal human serum. Two negative controls for immunohistochemistry were used: secondary antibody alone or a nonspecific antibody [mouse antibody to glucose oxidase of Asperigillus niger (Dako)].

Chandler model thrombi
Model thrombi were prepared from normal or septic whole blood spiked with fluorescein isothocyanate-labeled fibrinogen as described previously [¹⁴ ]. Basically, fresh citrated blood was recalcified, introduced into an artificial circulation, and allowed to circulate continuously for 90 min. Thrombi were then removed from the tubes, blotted on filter paper, placed in 0.5 ml 10 mM Tris buffer/0.01% Tween 20, pH 7.5, and incubated at 37°C. Samples of supernatant (0.02 ml) were removed at time intervals and diluted 1/50 in Tris/Tween buffer. The fluorescence released, indicating fibrin degradation, was measured with a Perkin Elmer luminescence spectrophotometer (LS-5B) with an excitation wavelength of 500 nm and emission wavelength 520 nm.

Patient data
PPP was analyzed by enzyme-linked immunosorbent assay (ELISA) for t-PA [²⁰ ] and PAI-1 [²¹ ]. Other variables were measured in the diagnostic haematology laboratory.

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Patients
Clinical parameters of the 12 patients studied are detailed in Table 1 . All patients fulfilled the criteria of septic shock [¹⁵ ] and required one or more forms of organ support in the form of assisted ventilation, extracorporeal membrane oxygenation, inotrope infusion, renal dialysis, or haemofiltration. Three patients in the same age range admitted to Intensive Care with similar disorders to sepsis patients (see below) but who did not develop the sepsis syndrome served as "ill controls". Comparison was also made with thrombi made from blood of normal individuals observed concurrently.

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Table 1. Patient Data

Clot lysis with normal and septic PMN
Normal PMN incorporated within purified clots did not lead to clot lysis (Fig. 2A ); however, when normal plasma was also present within the clot, lysis occurred. The plasminogen activator responsible was identified as u-PA by the inclusion of neutralizing antibodies. Plasma alone did not mediate clot lysis (data not shown). PMN from septic patients did not mediate clot lysis when alone, and there was also no lysis upon incubation with their own plasma (not shown; n=eight of 12). In some cases, there was slow lysis upon inclusion of neutralizing antibodies to PAI-1 and PAI-2 (four of 12; not shown). Septic PMN were included in clots in the presence of normal plasma. In eight of 12 patients, no clot lysis was detected (Fig. 2B) , but in some (four of 12), minimal lysis was detected at later time-points. Three patients in the Intensive Therapy Unit, without signs of sepsis (with cholelithiasis and acute pancreatitis or after major bowel surgery), served as ill controls for the sepsis patients. Their PMN supported clot lysis in the presence of plasma (data not shown). Normal PMN were incorporated into clots along with septic plasma. In some cases, lysis was detected (Fig. 3 ; n=six), but with plasma from other patients, there was only slow or no lysis (not shown; n=six).

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Figure 2. (A) Clot lysis by normal PMN is a result of u-PA. Purified clots were formed with normal PMN (); normal PMN, normal pool plasma, and antibodies to 2-AP (•); normal PMN, normal pool plasma, antibodies to 2-AP, and antibodies to u-PA (); or normal PMN, normal pool plasma, antibodies to 2-AP, and antibodies to t-PA (), and absorbance was measured over 240 min. The final concentrations in the clot were PMN, 1.6 x 107/ml; plasma, 8%; antibodies to 2-AP, 250 µg/ml; and antibodies to u/t-PA, 5 µg/ml. A decrease in absorbance indicates clot lysis. (B) Clot lysis with septic PMN. Purified clots were formed with septic PMN (); septic PMN, normal pool plasma, and antibodies to 2-AP (); or normal PMN, normal plasma, and antibodies to 2-AP (), and absorbance was measured over 240 min. The final concentrations in the clot were PMN, 1.6 x 107/ml; plasma, 8%; and antibodies to 2-AP, 250 µg/ml.

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Figure 3. Clot lysis with septic plasma. Purified clots were formed with normal PMN (•); normal PMN, normal pool plasma, and antibodies to 2-AP (); or normal PMN, septic plasma, and antibodies to 2-AP (), and absorbance was measured over 240 min. The final concentrations in the clot were PMN, 1.6 x 107/ml; plasma, 8%; and antibodies to 2-AP, 250 µg/ml.

u-PA activity associated with PMN
Normal PMN alone exhibited plasminogen activator activity only when incubated with plasma prior to SDS-PAGE (Fig. 4A , track 3), mirroring the clot lysis results and our previous observations [¹⁴ ]. This band was not found in plasma alone (track 2) and was identified as u-PA by the incorporation of neutralizing antibodies [¹⁴ ]. In contrast to normal PMN, in six of 12 patients with sepsis, there was no band of u-PA when their PMN were incubated with normal plasma prior to analysis (track 5). In the other six patients, a very weak band of activity was detected upon incubation with normal plasma (not shown), but the activity was always significantly less than that detected with normal PMN. In this system, the PMN of the ill controls without sepsis generated the band of u-PA activity (not shown).

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Figure 4. SDS-PAGE and zymography. (A) PMN and normal plasma. Track 1 = 1.6 x 105 normal PMN; track 2 = 4 µl normal pool plasma; track 3 = 1.6 x 105 normal PMN and 4 µl normal pool plasma incubated at 37°C for 30 min; track 4 = 1.6 x 105 patient PMN; track 5 = 1.6 x 105 patient PMN and 4 µl normal pool plasma incubated at 37°C for 30 min; track 6 = 0.8 ng two-chain u-PA (tcu-PA). (B) PMN and patient plasma. Track 1 = 4 µl patient plasma; track 2 = 1.6 x 105 normal PMN and 4 µl patient plasma incubated at 37°C for 30 min; track 3 = 1.6 x 105 patient PMN and 4 µl patient plasma incubated at 37°C for 30 min; track 4 = 0.8 ng tcu-PA.

Septic plasma contained no or trace free plasminogen activator activity (Fig. 4B , track 1), although there was, as in normal plasma, high molecular weight activity reflecting t-PA complexed with inhibitors [²² ]; in some cases, these high molecular weight bands were very strong. Septic plasma allowed a band of free u-PA to be detected from normal PMN (Fig. 4B , track 2,) in 10/12 cases, of which two were very weak bands. Again, there was no activity from the septic patient cells with patient plasma (track 3, n=10/12). However, cells from the ill controls without sepsis did generate the low molecular weight u-PA band in the presence of plasma (not shown). These results broadly mirror those found in the clot lysis system.

Do septic PMN retain u-PA antigen?
Septic and normal PMN were analyzed by immunohistochemistry. Like normal PMN, those from the septic patients showed positive pink staining for u-PA (Fig. 5 ). Negative controls showed no staining. However, this method does not allow for quantification of antigen.

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Figure 5. Immunohistochemical staining. Smears of PMN stained with mAb to u-PA. (A) Normal; (B) patient. Original magnification, x566.

Lysis of thrombi generated from whole blood
We have previously shown that thrombi from normal individuals lyse spontaneously without the addition of plasminogen activators and that this is a result of PMN-associated u-PA activity [¹⁴ ]. In a subset of patients, lysis of model thrombi was assessed by fluorescence release. Thrombi from three of the septic patients were analyzed on two separate days and on each day, were compared with lysis of a control thrombus from a normal individual. The thrombi from all three septic patients analyzed exhibited less lysis than those from normal individuals (Fig. 6A and 6B ).

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Figure 6. Spontaneous lysis of normal versus patient thrombi. (A and B) Analysis of patient thrombi versus normal thrombi on two different days, with two different normal individuals. Model thrombi were made from normal (•) or septic (, , ) whole blood, and fluorescence release was measured over time. Lysis was plotted with lysis at 4 h of the normal thrombus standardized to 100 arbitrary units.

Relationship of activity to severity of disease
No patterns were observed in relation to activity and severity of disease. Only two of the 12 patients studied did not survive. PMN from one of these patients exhibited some activity in contact with normal plasma, but those from the other did not. Two of the PMN patients produced some u-PA activity with their own plasma and normal plasma, suggesting that their cells were less altered than those of the other patients. However, one of them survived, and one died, so no correlation can be made. Both nonsurvivors had plasma that allowed only a weak band of u-PA to be detected from normal PMN, suggesting defects in their plasma. All the patients whose plasma did not allow any u-PA activity to be detected from normal PMN ultimately survived.

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Proteases and inhibitors of the coagulation and fibrinolytic cascades have usually been investigated only in the plasma, and many studies ignore potential contributions from the cellular components of blood. In vivo thrombi contain fibrin, platelet, and red cell masses throughout which leukocytes, predominantly polymorphs, are dispersed [^{23 24 25} ]. The observation that PMN are essential for spontaneous lysis of model thrombi [¹⁴ ] suggests that they may play significant roles in the resolution or persistence of other fibrin deposits in the body.

This study identifies that in a clinical situation, frequently characterized by fibrin occlusion of the microcirculation, the ability of PMN to express u-PA activity is diminished or absent. This is despite there still being u-PA antigen present in the cells when analyzed by immunohistochemistry. The data indicate that the failure lies with the PMN themselves and not with the plasma, as cross-over experiments with normal plasma did not usually restore u-PA activity to PMN from sepsis patients. The converse observation that in some cases, normal PMN could support clot lysis and generate u-PA activity in the presence of sepsis plasma suggests that the plasma levels of inhibitors, although often very high, were not sufficient to inhibit u-PA activity by normal cells. This indicates that fibrinolytic activity generated on the cell surface may be relatively protected from inhibitors.

The lack of u-PA activity detected from sepsis PMN with apparent retention of antigen requires explanation. We have recently observed that the expression of u-PA activity from PMN depends on the presence of elastase inhibitor(s) that protect u-PA from elastase cleavage [²⁶ ]. These inhibitors are provided by plasma. In patients with sepsis, PMN degranulation frequently occurs, releasing cellular elastase [²⁷ ], which could lead to the cleavage of cellular u-PA and loss of activity, despite the presence of inhibitors. Elastase can cleave plasminogen to miniplasminogen, which consists of kringle 5 and the protease domain [²⁸ , ²⁹ ], and detection of miniplasminogen in septic patients [¹¹ ] indicates elastase is active in this condition. An alternative mechanism could also operate. Plasmin is required to convert inactive, single-chain u-PA (scu-PA) to the active tcu-PA form [³⁰ ]. If the reduced plasma plasminogen levels observed in sepsis [⁷ , ¹¹ ] are paralleled by decreased plasmin(ogen) bound to PMN, local activation of scu-PA could be impaired. It is, however, possible that an unidentified mechanism is involved.

In addition to demonstrating that in sepsis, PMN-associated u-PA activity is reduced, we also detected diminished lysis of whole blood model thrombi from septic patients compared with thrombi made from normal donors. In normal individuals, lysis is largely a result of u-PA from PMN; therefore, the decrease in thrombus lysis in patients correlates with the lack of u-PA activity detected in purified cells. However, there was some residual activity in the septic model thrombi, suggesting that fibrin breakdown in these patients is not completely suppressed.

Loss of the ability of PMN to express u-PA activity and support fibrin lysis occurred in most, but not all, cases of sepsis studied. This may reflect, in part, at least, the time-point in the evolution of the septic episode at which the patients were studied. The time of their entry into the Intensive Therapy Unit is dependent on factors outwith our control and will differ from patient to patient. This possibility is currently under investigation.

The PMN of our ill controls retained the ability to generate u-PA activity, suggesting that its loss is a feature of the septic state. However, many other disease states require to be examined, and such studies are also underway.

The ability of normal PMN to generate u-PA activity in the presence of plasma seems likely to be a process of physiological significance. Our conclusions from the data presented here are that in a variety of types of severe sepsis, the ability of PMN to contribute u-PA activity and lyse fibrin is diminished in the early stages of illness. The absence of this activity may contribute to fibrinous occlusion of the microcirculation and the multiple organ damage characteristic of this disorder.

 
We thank the British Heart Foundation and Tenovus (Scotland) for funding this work, Mrs. Susan Berry for carrying out the ELISA analysis, and Professor Nuala Booth for helpful discussion.

Received May 29, 2002; revised October 16, 2003; accepted March 26, 2004.

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