(Received for publication, November 11, 1994; and in revised form, January 30, 1995)
From the
An inhibitor of tissue-type plasminogen activator (tPA)-mediated and plasminogen-dependent fibrinolysis was isolated from human neutrophils. On a G-50 gel filtration column, the antifibrinolytic activity present in neutrophil homogenates comigrated with proteins of <13 kDa. The inhibitory fraction had only a slight effect on urokinase with plasminogen- or plasmin-mediated fibrinolysis and no effect on urokinase- or plasmin-mediated cleavage of H-D-valyl-L-leucyl-L-lysine-p-nitroanilide (S-2251). The neutrophil-derived fraction inhibited tPA with plasminogen activity on S-2251 but not on H-D-isoleucyl-L-prolyl-L-arginine-p-nitroanilide (S-2288). The inhibition of tPA-mediated and plasminogen-dependent fibrinolysis or S-2251 cleavage showed a competitive pattern and could be relieved by increasing the concentration of plasminogen. The same fraction also inhibited binding of plasminogen to fibrin. Consecutive purification steps revealed that the molecular mass of the inhibitor was 1-5 kDa. Polylysine-Sepharose affinity chromatography indicated that the inhibitor is a protein of 4 kDa, migrating as one band on SDS-polyacrylamide gel electrophoresis. Amino acid sequence analysis of this band showed the presence of two sequences, differing by one amino acid, which are identical to defensin I and II. Comparison of the sequences of plasminogen and defensin showed homology of defensin to the plasminogen kringles known to contain the lysine binding sites. The close structural similarity between defensin and plasminogen kringles and the ability of defensin to compete with plasminogen on binding to fibrin explain the ability of defensin to inhibit tPA-mediated, plasminogen-dependent fibrinolysis. These results suggest that the antifibrinolytic activity of defensin may have a biological function in preventing the spread of infection.
Fibrin deposition is the end result of normal hemostasis. Fibrin deposition is also a component of the delayed type hypersensitivity reactions (1, 2) and an integral feature of the inflammatory response to nonspecific stimuli(3) . Infiltration of inflammatory sites by leukocytes results in the release of mediators, which, among other functions, promote blood coagulation(4) .
Fibrin formed in the inflammatory response is subject to the known regulatory processes of coagulation. For example, anticoagulant therapy has been shown to prevent local fibrin deposition after delayed type hypersensitivity skin tests(5) . Furthermore, in afibrinogenemic subjects, such skin tests did not elicit characteristic indurations(2, 5, 6) . The role of fibrin formation during the immune response is not completely clear, although Menkin (7) suggested that its role was to immobilize the irritant.
In parallel with the coagulation
system, there is an efficient fibrinolytic system. Physiological
fibrinolysis, which results in local lysis of fibrin clot
deposits(8, 9, 10, 11, 12) ,
is coordinated by the interaction of enzymes, zymogens, and inhibitors.
Fibrin acts not only as a substrate of the fibrinolytic system but also
as a cofactor, which increases the catalytic efficiency of plasmin
production from plasminogen by the endogenous tissue-type plasminogen
activator
(tPA)()(13, 14, 15, 16, 17) .
The presence of this highly effective fibrinolytic system raises the question why the fibrin deposits appearing during the immune response are not immediately dissolved.
In an attempt to answer this question, we looked for an endogenous inhibitor of the fibrinolytic system among the components of the immune system. By inhibiting the cleavage of fibrin deposits that appear during the inflammatory response, two advantages may be gained: the first is better confinement of pathological processes, and the second is prevention of a vicious cycle involving the inflammatory process through inhibition of the release of thrombin and of fibrin(ogen) degradation products, which are known to express chemotactic and mitogenic properties(4, 18, 19, 20) .
The observation that neutrophil infiltration retarded the resolution of acute ischemic renal failure in an animal model (21) prompted us to direct our attention to these cells. We presently report that neutrophils do indeed contain an antifibrinolytic factor capable of inhibiting tPA-mediated fibrinolysis.
All experiments were performed in triplicate and repeated at least three times. Results are means ± S.D. from representative experiments.
Application of neutrophil homogenate to a Sephadex G-50 gel
filtration column revealed two protein peaks: a high molecular weight
peak and a peak with a molecular mass <13 kDa (Fig. 1).
Examination of the effect of the protein fractions on tPA-mediated
fibrinolysis showed that the low molecular weight peak had significant
antifibrinolytic activity. In the presence of 80 µg/ml plasminogen
and 6.6 IU/ml tPA, 40.4 µg/ml of the low molecular weight protein
fraction produced a 94% inhibition (Fig. 1, inset). The
inhibitory effect of the low molecular weight protein fractions
(fractions 34-40) on tPA- and on urokinase-mediated fibrinolysis
was compared at a fixed plasminogen concentration of 80 µg/ml and
with either 6.6 IU/ml tPA or 6.3 IU/ml urokinase. Fig. 2shows
that the solubilization of I-labeled fibrin clots by the
two fibrinolytic systems was inhibited by the low molecular weight
protein fraction in a dose-dependent manner. The inhibitory activity
was proportional to protein concentration. The sensitivity of tPA to
inhibition by the protein was greater than that of urokinase; after 45
min of incubation, inhibition of tPA and urokinase-mediated
fibrinolysis was 91 and 18%, respectively (Fig. 2). The high
molecular weight peak contained considerable fibrinolytic activity that
was resistant to inhibition by
-antiplasmin, excluding
involvement of enzymes known to be involved in the fibrinolytic system.
Figure 1: The inhibitory effect of neutrophil-derived protein on tPA-mediated fibrinolysis. The neutrophil homogenate was applied to a Sephadex G- 50 gel filtration column. The second peak (molecular mass <13 kDa) had an inhibitory effect on tPA-mediated fibrinolysis (inset).
Figure 2:
The effect of neutrophil-derived
proteins from the second gel filtration peak on tPA- () and
urokinase-mediated (
) fibrinolysis.
In an attempt to elucidate the mechanism of the inhibitory effect on
the fibrinolytic cascade, the effect of the protein fraction on the
cleavage of I-labeled fibrin clot by plasmin was
examined. Fig. 3shows that the protein fraction had a small
inhibitory effect on the activity of plasmin upon fibrin, which was
observed in each of eight experiments. The extent of inhibition (20%)
was similar to the effect on urokinase-mediated fibrinolysis (Fig. 3). The results suggest that the inhibitory effect on
urokinase-mediated fibrinolysis may be due to inhibition of plasmin
activity rather than of plasminogen activation. In additional
experiments, the chromogenic substrate S-2251 was used instead of
fibrin. The protein fraction had no inhibitory effect on cleavage of
S-2251 by plasmin or by urokinase and plasminogen.
Figure 3:
The effect of neutrophil-derived proteins
on plasmin- (A) and urokinase (B)-mediated
fibrinolysis. Emptybars, activity without added
protein; hatchedbars, activity in the presence of
40.4 µg/ml of the second protein fraction. The time of incubation
was 45 min. Degradation of fibrin was measured by following release of
radioactivity from I-labeled
fibrin.
The effect of the
small molecular mass protein fraction upon tPA-mediated plasminogen
activation is shown in Fig. 4(A and B). In
the presence of a fixed concentration of the protein fraction (20.2
µg/ml) and increasing concentrations of plasminogen, the inhibition
of plasminogen activation was reduced. The curve of activity versus [plasminogen] was compatible with a competitive
inhibition pattern with a K of 1.67 µg/ml.
This pattern could have resulted from competition between plasminogen
and the inhibitor for the active site on tPA or on binding to fibrin.
To distinguish between these alternatives, we studied the displacement
of plasminogen from the surface of fibrin clots by the protein
fraction. Fig. 5shows that the inhibitory protein fraction
decreased the binding of plasminogen to fibrin.
Figure 4:
The
effect of plasminogen concentration on the inhibitory effect of
neutrophil proteins. A, tPA-mediated fibrinolytic activity was
assayed without () or with (
) 20.2 µg/ml of the
inhibitory protein fraction and varying concentrations of plasminogen. B, Lineweaver-Burk plot of the same data. The K
for plasminogen was 0.3 µM
in the
absence (
) and 4 µM in the presence (
) of the
inhibitor. The K
for the inhibitor was
1.67 µg/ml.
Figure 5: Displacement of plasminogen binding to a fibrin clot by neutrophil-derived proteins. Cleavage of S-2251 was measured after incubation of S-2251 with 160 µg/ml plasminogen (I), with the supernatant remaining after preincubation of the same amount of plasminogen with a fibrin clot, as described under ``Experimental Procedures'' (II), and after incubation with the same supernatant and 60 µg/ml of the inhibitory protein fraction (III).
To examine the alternative possibility, namely, competition between the inhibitor and plasminogen for the active site of tPA, we studied the effect of the inhibitor on tPA and plasminogen-mediated cleavage of S-2251. Fig. 6shows that the inhibitory effect of the protein fraction was also evident in this system. The pattern of the inhibition was also competitive; the curve obtained was similar to that shown in Fig. 4with the exception that in the case of Fig. 6, with plasminogen concentrations of 175 µg/ml, there was 8% inhibition, whereas in Fig. 4at the same concentration of plasminogen there was 70% inhibition with the same amount of inhibitor in both experiments. No inhibitory effect of the protein fraction on the direct activity of tPA on the chromogenic substrate S-2288 was found (not shown).
Figure 6:
The effect of the neutrophil-derived
protein on tPA plasminogen-mediated S-2251 cleavage. tPA was incubated
with increasing concentrations of plasminogen, with () or without
(
) 20 µg/ml of the inhibitory
fraction.
To further identify the protein responsible for the inhibitory effect, each fraction containing inhibitory activity was analyzed by SDS-PAGE. Fig. 7shows that the predominant band in each fraction was a low molecular mass protein (4 kDa). When neutrophils were disrupted without 50 mM 6-aminohexanoic acid, the inhibitory activity of the protein fraction was unstable. In several experiments in which the inhibitory activity was completely lost, no low molecular mass band was recovered from the SDS-PAGE preparation, which suggests a direct relationship between the protein band evident on SDS-PAGE and the inhibitory activity found in this fraction.
Figure 7: SDS-PAGE analysis of the protein fractions obtained from the second gel filtration peak shown in Fig. 1. The protein applied to lanes1 and 2 was from fraction 36, lanes3 and 4 from fraction 37, and lanes5 and 6 from fraction 38.
To isolate the inhibitory protein, the fraction with the highest optical density from the initial Sephadex G-50 gel filtration was concentrated in a Speed-Vac apparatus and applied to a Sephadex G-25 gel column. Fig. 8shows that the second peak, with a molecular mass of 1-5 kDa, contained all of the inhibitory activity. This inhibitory activity was not due to residual 6-aminohexanoic acid because the active fraction migrated on gel filtration between cytochrome c (13 kDa) and p-nitroanilide (140 Da). The specific inhibitory activity of the peak obtained from the Sephadex-G25 column was more than 200% compared with the peak from the first gel filtration.
Figure 8: The inhibitory effect of protein fractions obtained by gel filtration on a G-25 column. The second peak from the first gel filtration was concentrated under vacuum and applied to a Sephadex G-25 gel filtration column. A peak corresponding to a molecular mass of 1-5 kDa inhibited tPA-mediated fibrinolysis. In the presence of 80 µg/ml plasminogen and 6.6 IU/ml tPA, 90 ± 3% inhibition was obtained in the presence of 14 µg/ml of the protein fraction 18. Fraction 9 of the first peak induced 8 ± 4% inhibition at a concentration of 14 µg/ml. Fraction 39 had no inhibitory effect.
If the assumptions concerning the mechanism of inhibition of tPA-mediated fibrinolysis are correct, the inhibitory protein should bind to polylysine (which binds plasminogen in a manner that is similar to its binding of fibrin). To examine this possibility, the protein peak obtained from the Sephadex G-25 column was applied to a polylysine affinity chromatography column. Little protein was eluted by the addition of 6-aminohexanoic acid or 2 M NaCl. Fig. 9shows the protein that was eluted from polylysine using an HCl gradient. Electrophoretic analysis of this protein, after concentration, indicated a molecular mass of 4 kDa (Fig. 10). The amino acid sequence of this protein showed that it contained 2 sequences: A_Y_RIPA_IAGERRYGT_IY and *_Y_RIPA_IAGERRYGT. These sequences are identical with amino acid sequences in defensin 1 and 2, respectively(27, 28) . Comparison of the amino acid sequence of defensin with that of plasminogen shows a degree of similarity between defensin and part of the repeated sequences in plasminogen kringles. The greatest similarity was with kringle 5 (Fig. 11); 8 out of 27 amino acids of defensin were identical, 6 were highly similar, 7 had some similarity, and only 6 amino acids were totally different. Another similarity between plasminogen kringles and defensin is that both contain three intramolecular S-S bonds.
Figure 9: Elution pattern of the protein fractions from a polylysine-Sepharose affinity chromatography column. Protein fractions 15-20, obtained from the second gel filtration peak (Fig. 8), were pooled and applied to a polylysine-Sepharose column. Protein bound to the column was eluted with an HCl gradient. The protein could not be eluted with NaCl at concentrations up to 2 M.
Figure 10: SDS-PAGE analysis of the protein eluted by an HCl gradient from the polylysine-Sepharose column. Lanes1 and 2 are duplicates.
Figure 11: Comparison of the amino acid sequence of defensin (first line) with part of the sequence of kringle 5 of plasminogen (second line).
The results clearly show that human neutrophils contain an antifibrinolytic activity, which acts by inhibiting tPA, an important physiological plasminogen activator. This inhibitory factor appears to compete with plasminogen for binding to fibrin as well as to compete for the active site of tPA.
Although inhibition by two different mechanisms seems less likely, this is the most probable explanation for our results. The first mechanism involves competition for fibrin binding between plasminogen and the inhibitor (Fig. 5). This competition may partially explain the observation that inhibition was observed in tPA- but not urokinase-mediated fibrinolysis, since activation of plasminogen by tPA is greatly enhanced in the presence of fibrin(13, 14, 15, 16, 17) , whereas a comparable effect of fibrin on urokinase-mediated proteolysis is controversial(13, 14, 29, 30, 31) . The presence of fibrin increases plasminogen activation by tPA through the formation of a ternary complex composed of fibrin, tPA, and plasminogen(15) . Displacement of plasminogen from the surface of the fibrin clot by the inhibitor could impede this complex formation and thus reduce the stimulatory effect of fibrin on plasminogen activation by tPA. This displacement of plasminogen from the surface of the fibrin clot can have additional consequences. Since both plasmin and plasminogen bind to fibrin through the same lysine binding sites, occupation of the binding sites on the fibrin clot would also prevent plasmin binding. This would reduce plasmin activity by decreasing the local concentration of the enzyme on the surface of the clot. The importance of lysine binding sites for inhibitor activity is supported by the observation that the inhibitor had no effect on plasmin-induced cleavage of S-2251. Interaction with lysine binding sites is not required for plasmin activity on this type of low molecular weight substrates(24) .
The inhibitor also appears to act through a second mechanism, namely by competing with plasminogen for the active site on tPA. Its inhibitory effect on tPA-plasminogen-mediated S-2251 cleavage cannot be explained by competition for binding to fibrin, since neither fibrin nor the lysine binding sites of plasmin and plasminogen are involved in this reaction. Instead, our data suggest that the inhibition of S-2251 cleavage is mediated through competitive inhibition of the activation of plasminogen by tPA. Although the absence of an inhibitory effect on S-2288 tPA-mediated cleavage supports our interpretation of a competitive inhibition mechanism, the results also suggest that the exact mechanism is more complicated (see below).
We have been able to purify and characterize this antifibrinolytic activity present in neutrophils. The activity copurifies with a low molecular mass protein, which comigrates with proteins of <13 kDa in G-50 gel filtration and between 1 and 5 kDa in G-25 gel filtration. Electrophoretic analysis demonstrated the presence of a protein with the characteristics of the above-described inhibitor, in the second peaks emerging from the G-50 and G-25 gel filtration columns, respectively. The protein also binds strongly to lysine, further supporting its identity as the inhibitor. The amino acid sequence of this purified protein is identical to that of defensin for the regions sequenced. Comparison of the sequences of plasminogen and defensin shows 53.8% similarity to the plasminogen kringles known to contain the lysine binding sites(32) . The binding of plasminogen to fibrin as well as to cellular binding sites is dependent on the presence of the kringle(33) . Other proteins with structures similar to the kringles of plasminogen, such as apolipoprotein(a) present in lipoprotein(a), compete with plasminogen for binding in a dose-dependent manner (33, 34) and compete with plasminogen activation by tPA in the presence of fibrin(33, 35) .
The sequence homology between defensin and the lysine binding regions of plasminogen supports the conclusion that defensin plays an important role in modulation of fibrinolysis by competing with plasminogen for binding to fibrin. This similarity between defensin and the plasminogen kringle may explain the ability of the inhibitor to compete with plasminogen for the active site of tPA, as well as the absence of such competition between the inhibitor and S-2288. Results from other studies suggest that kringle 5, present in the miniplasminogen, is involved in the activation of plasminogen by tPA, even though it is located at a distance from the cleavage site(36) . Therefore, the structural similarity between defensin and kringle 5 of plasminogen may explain the ability of defensin to interfere with the binding of plasminogen to the active site of tPA. The inhibitor probably binds to a portion of the active site somewhat distant from the amino acids directly involved in catalytic cleavage, since it competitively inhibited plasminogen activation but failed to inhibit cleavage of S-2288. Such a mechanism would be in accordance with the suggested involvement of kringle 5 in the activation of plasminogen by tPA.
Defensins are antimicrobial and cytotoxic peptides, which are present in the azuriphilic granules of neutrophils and appear to contribute to mammalian as well as to invertebrate immunity(37) . Defensins constitute more than 5% of the total cellular proteins of human and rabbit neutrophils and are produced by human small intestinal Paneth cells, among others(38) . Plasma defensin concentrations are very low in healthy volunteers (0-53 ng/ml) but are markedly elevated in patients with sepsis (up to 170,000 ng/ml)(39) . Therefore, the antifibrinolytic activity of defensin, which we observed in this study, may contribute to the thrombotic complications experienced by some patients with disseminated intravascular coagulation due to sepsis or other infections(40) . Defensin released from neutrophils may also stabilize fibrin deposition, thereby helping to prevent the spread of infection and modulating the inflammatory process. Further investigation of the involvement of defensin in the immune response and fibrinolysis-related diseases will better define its role in these processes.