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Stability of gels formed following coagulation of limulus amebocyte lysate: Lack of covalent crosslinking of coagulin
THROMBOSIS RESEARCH 55; 25-36, 1989 0049-3848/89 $3.00 + .OO Printed in the USA. Copyright (c) 1989 Maxwell Pergamon Macmillan plc.
STABILITY AMEBOCYTE
OF GELS FORMED FOLLOWING LYSATE: LACK OF COVALENT
All rights reserved.
COAGULATION CROSSLINKING
OF LIMULUS OF COAGULIN.
Robert I. Roth, Joseph C.-R. Chen and Jack Levin From the Departments of Laboratory Medicine and Medicine, University of California of Medicine and Veterans Administration Medical Center, San Francisco, CA, USA
School
(Received 17.3.1989; accepted in original form 30.3.1989 by Editor W.H. Seegers)
ABSTRACT.
Incubation of lysates prepared from amebocytes of the horseshoe crab (Limulus polyphemus) with bacterial endotoxin results in coagulation and formation of a solid gel. Although Limulus gels remained solid indefinitely, if undisturbed, they were easily disrupted by mechanical agitation. Chemical solubility studies of gelled lysates demonstrated rapid solubilization of gels in monochloroacetic acid, a property of clots that have not been covalently stabilized; but in contrast stabilized demonstrated resistance to solubilization by urea, a property of clots. Analysis of solubilized proteins by polyacrylamide gel electrophoresis in SDS demonstrated coagulin, the designation for the activated form of coagulogen (the clottable protein) that forms a gel, only in samples derived from clotted lysate that had been previously incubated with monochloroacetic acid, but not in samples following incubation with urea, confirming the results of the chemical solubility studies. Enzymatic assays for transpeptidase (Factor XIII-like) activity in either native or gelled Limulus lysates were negative. Furthermore, analysis for covalently crosslinked peptides in gelled coagulin confirmed the absence of intermolecular y-glutamyl-e-lysyl bonds. Therefore, the stable endotoxin
gels formed following coagulation are not covalently crosslinked.
of Limulus
lysate
by bacterial
INTRODUCTION Limulus polyphemus, the horseshoe crab, has a coagulation mechanism that can be activated by bacterial endotoxin (l-6), the lipopolysaccharide component of the cell wall of gram-negative bacteria. After activation of coagulation, a solid gel is formed which is stable. However, the basis for structural stability of the gel in Limulus has not been investigated. We conducted this study to determine whether this stability was the result of chemical crosslinking of the coagulation proteins. Blood coagulation in the ancient invertebrate Limulus polyphemus, the horseshoe crab,
Key Words: Limulus,
amebocyte
lysate, coagulin, 25
clot stability,
crosslinking
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STABILITY OF LIMULUS LYSATE GELS
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is based on an enzymatic cascade, which includes one or more trypsin-like serine proteases and is similar in many respects to that of mammalian coagulation (l-57-10). However, coagulation of Limulus blood is mediated by cellular rather than plasma factors. The amebocyte, the only type of circulating blood cell in Limulus polyphemus, contains all of the factors necessary for coagulation of the blood (11,12). Plasma factors are not required for blood coagulation. Amebocytes react to a breach of hemostasis in the animal by aggregation and degranulation (8,12). Degranulation leads to release of the components of the coagulation cascade, and their subsequent activation results in the formation of a solid gel composed primarily of the Coagulogen is functionally analogous to fibrinogen, and clottable protein, coagulogen. contains a segment with sequence homology to the mammalian clotting protein (13,14). After activation by bacterial endotoxin, the clottable protein, coagulogen, undergoes limited proteolysis to become coagulin (14-17) and then structural reorganization into an opaque gel. Gels formed in vitro from amebocyte lysates (1 l), of which 50% of the protein is coagulogen (4,16), are easily disrupted by vortexing or stirring, yet remain indefinitely intact in a test tube if they are not mechanically disrupted (personal observation). There is no evidence for a plasmin-like proteolytic activity in Limulus, and dissolution of clots has not been noted in other invertebrates with coagulable blood (18). Covalent crosslinking is a process which greatly enhances the structural stability of clots, i.e., increased rigidity, greater internal cohesion and lower stress relaxation (19-22). Clottable proteins of many diverse vertebrates, including many species of mammal, bird, reptile, amphibia and fish (23,24), undergo covalent crosslinking following the formation of a clot. Crosslinking of the clottable protein also has been described in the invertebrate, Homarus The crosslinking reaction characteristically is mediated by americanus (lobster) (2526). transpeptidase activities which involve the creation of intermolecular y-glutamyl-e-lysyl bonds. The marked stability of the mammalian clot is associated with a very limited degree of crosslinking, one mole of yglutamyl-e-lysyl peptide per 80,000 grams of protein (22). The transpeptidase reaction has been well described in the lobster (26) as a function of a single polymerization/crosslinking enzyme and in mammals as a function of Factor XIIIa (Fibrin Stabilizing Factor, FSF) (20,27-31). Congenital deficiencies of Factor XIII, which can result in a significant hemorrhagic diathesis, have been described in man (32-34). Presumptive evidence for presence of a clot stabilizing factor in mammals is the insolubility of a solid clot in dilute acid (1% monochloroacetic acid) or concentrated (5M) urea (20,28,33-36). Congenital absence of FSF results in clots which dissolve readily in these solubilizing agents (33,34). Further presumptive evidence for clot stabilizing activity is the detection of crosslinked fibrin (primarily gamma-chain dimers in vertebrate species) by polyacrylamide gel electrophoresis in SDS (23). Enzymatic assays for transpeptidase activity, such as the in vitro incorporation into protein substrates of the fluorescent molecule dansylcadavarine (37) or radiolabeled putrescine (27), and the isolation of chemically-crosslinked amino acids (e-(yglutamyl)lysine peptides) following proteolytic digestion of fibrin (26,38), provide direct evidence for a clot stabilizing activity. The experiments described in this report were undertaken to characterize the basis for stability of gelled lysates of Limulus amebocytes.
MATERIALS
AND METHODS
Materials. Endotoxin used was Escherichia coli lipopolysaccharide B, 026:B6 (Difco Laboratories, Detroit, MI). Sterile, endotoxinfree water and saline solutions were purchased from Travenol Laboratories (Deerfield, IL.). Chemicals were reagent grade. Glassware. All glassware was rendered endotoxin-free then heating at 190°C in a dry oven for 4 hours.
by autoclaving
for 45 minutes
and
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STABILITY OF LIMULUS LYSATE GELS
27
Limulus Amebocvte Lvsate. Horseshoe crabs were obtained from the Department of Marine Resources of the Marine Biological Laboratory, Woods Hole, MA. Amebocyte lysates were prepared from Limulus polyphemus (the horseshoe crab) by disruption in distilled water of washed amebocytes (separated from hemolymph plasma in the presence of N-ethyl maleimide (NEM)), as described previously (11). Amebocyte lysate was stored at 4’C. For some experiments, lysates termed “Pre-gel” were prepared by disruption of amebocytes in samples of native hemolymph with release of the components of the coagulation mechanism, in the absence of NEM (39). Hemocyanin, the oxygen carrying protein of Limulus plasma, was separated from the remaining components of “Pre-gel” into a pellet by centrifugation at 18,500 rpm (41,280 x g) for 3 hours. By this procedure, the overlying plasma, which contained the components of the coagulation mechanism, was hemocyanin-free. Assavs for Stabilitv of Gelled Lvsate. Experimental conditions described previously to assess clot stability in vertebrates (28,30,34-36,40) vary considerably with regard to temperature of incubation, length of incubation, and presence or absence of shaking during incubation with solvent. Since Limulus coagulin gels do not retract from glass as an intact, solid clot (personal observation), not all gel stability tests useful in vertebrates can be applied to evaluation of the horseshoe crab, Therefore, in this study several experimental conditions were examined to help characterize gel stability in Limulus. I. Mechanical Fraailitv of Coaaulin Gels. 200 1.11of Limulus lysate was incubated with 10 ~1 of E.coli endotoxin
(final
concentration,
0.005 pg/ml)
at 37’C. Stages of gelation
of the
endotoxin-activated lysate (flocculation (F), heavy flocculation (F+), increased viscosity (V), Turbidity of the solutions (an increase in light and solid gel (G)) were assessed visually. scattering accompanies coagulation (11,41)) was monitored. At various stages of gelation, 300 1.11of SM urea was added to samples to aid in solubilization of any non-crosslinked proteins which had been reversibly aggregated, partially denatured or trapped in the gel. The samples then were subjected to either gentle stirring with a spatula or vigorous agitation by vortexing for 2 minutes. The residual turbidity was measured spectrophotometrically at 650 nm. Gels that were broken apart by mechanical means became less turbid. Accordingly, fragility of the coagulin gels was assessed by the extent to which mechanical agitation was capable of diminishing the turbidity of the solutions. II. Visible Clot Solubilization. A variety of solvents were examined for ability to completely dissolve gelled Limulus lysates. Tubes containing 0.5 ml of Limulus lysate and 0.05 ml of E.coli endotoxin (final concentration, 50 pg/ml) were incubated for two hours at 37’C. In some experiments, similar incubations were performed using “Pre-gel” lysates, centrifugally-concentrated hemocyanin (prepared as described above) or hemocyanin-free plasma, rather than standard amebocyte lysates. Under these conditions, both types of lysates and both subfractions of “Pre-gel” formed a solid opaque gel within ten minutes. Gelled lysates or “Pre-gel” subfractions then were overlaid with 1 ml of water, 10% SDS, 10% Triton X-100, 1% monochloroacetic acid or 5M urea. Incubations were performed at room temperature for up to one week. Gels were monitored for physical stability by observing whether gels maintained their original solid shape during tilting of the gel tubes. III. Splubilization of Proteins from Gelled Lvsate. Tubes containing 0.5 ml of Limulus lysate and 0.05 ml of E.coli endotoxin (final concentration, 50 pg/ml) were incubated for two hours at 37’C. Gelled lysates then were overlaid with 1 ml of water, 10% SDS, 10% Triton X-100, 1% monochloroacetic acid or SM urea. The samples were covered and incubated for 24 hours at room temperature or at 37’C. Each sample was observed during this 24 hour period, after which time the overlying solutions were removed from the gels and dialyzed against phosphate buffer (pH 7.6) with several buffer changes. The samples then were analyzed by polyacrylamide gel electrophoresis in SDS (42), using 12% acrylamide gels. Undialyzed samples of the supernatants also were characterized by measurement of their ultraviolet absorbance
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STABILITY OF LIMULUS LYSATE GELS
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spectra, from 200-350 nm, in a Gilford 2400-2 spectrophotometer (Gilford Laboratories, Oberlin, Ohio). Characterization of absorbance as predominantly nucleic acid was obtained from the ratio of A260nm / A2gOnm (43).
Instrument protein or
Enzvmatic Assay fer Transpeptidase Activitv. Aliquots of Limulus lysate were incubated with E.coli endotoxin (final concentrations, O.Ol,O.OOl or 0.0001 fig/ml) at 37’C for four hours, and then at room temperature for 20 hours. These conditions produced final stages of gelation that included flocculation, increased viscosity and gelled samples. Another aliquot of the same lysate was incubated with E.coli endotoxin (final concentration 0.1 pg/ml) for twenty minutes at 37’C, resulting in a solid gel, immediately prior to assay. These various samples, and aliquots of lysate not incubated with endotoxin, were assayed for transpeptidase activity using the incorporation of l4 C-putrescine into dimethylcasein (27). A preparation of “Pre-gel” was incubated with E.coli endotoxin (final concentration, 0.1 pg/ml) for 72 hours. Other aliquots were incubated with E.coli endotoxin (final concentrations, 0.1, 0.00001 or 0.000001 pg/ml) for 30 minutes immediately prior to assay. These samples of “Pre-gel”, all of which ultimately formed solid gels following addition of endotoxin, and aliquots of “pre-gel” not incubated with endotoxin, were assayed for transpeptidase activity, as described above. bs. Anal i for Samples of Limulus lysate incubated with endotoxin, as described above for the assay of transpeptidase activity, and aliquots of lysates not incubated with endotoxin, were assayed for the presence of covalent crosslinks by J. Wilson in the laboratory of L. Lorand, Northwestern University, by published procedures (44).
RESULTS Mechanical Fraailitv of Gelled Limulus Lvsate. Lysates were gelled with endotoxin (final concentration, 0.005 pg/ml) and the gels were observed for one week. Lysates originally were clear. After incubation with endotoxin, grey-white, opaque solid gels formed and remained unchanged during the one week period of observation. They were solidly attached to the glass tubes; no retraction from the inner surface of the tubes was noted. At all times during the observation period of one week, these gels were friable and easily disrupted by mechanical This property was distinctly different from the greater stability (e.g., no agitation. fragmentation with vigorous vortexing) of a human clot observed after retraction. Gelation of Limulus lysate into an opaque, solid gel was associated with an increase in turbidity (light scattering) of the solution as coagulin molecules polymerized, as has been reported previously (11,41). Increasing turbidity during the process of coagulation was determined spectrophotometrically by measurement of light scattering at 650 nm. We utilized a reversal of this opacity to assess the physical stability of the coagulin gel. Preliminary experiments established the concentration of endotoxin required to gel a selected batch of lysate at a moderately slow rate, i.e., 30-90 minutes, so that visible stages of gelation could be distinguished. Pairs of tubes of activated lysate were removed at various times from the 37OC waterbath and urea was added to solubilize any proteins that were non-specifically precipitated or trapped by the gel. One sample of each pair then was stirred gently with a spatula, following which light scattering was measured (Figure 1, line A). Turbidity (A6SOnm) increased slowly as the samples progressed from flocculation to viscous states, and then increased dramatically during the process of solid gel formation (Figure 1, line A). In contrast, when corresponding samples of each pair were mixed vigorously using a Vortex mixer (Figure 1, line B), the subsequent turbidity was greatly diminished at all stages of coagulation. The latter samples contained small residual particles which closely resembled the particulate
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29
material observed during early stages of flocculation. To determine whether these residual particles could re-associate into a solid gel structure, other gelled Limulus lysate samples were disrupted with the Vortex mixer but no urea was added. These appeared visually similar to the samples treated with urea and were replaced into the waterbath and observed for re-gelation. None of these vortexed samples demonstrated increased viscosity or gelation after reincubation, suggesting that the three-dimensional structure of the coagulin matrix had been irreversibly altered.
0.9, 0.80.70.6OS0.40.30.2O.l0.0 0
I 15
I 30
I 45
(F)
(VI
((3
Incubation
I 60
Figure 1. Turbidity of Limulus Lysate Following Activation by Endotoxin. Aliquots of Limulus lysate were activated by endotoxin, as described in Methods, such that gelation proceeded slowly and the various stages of activation were Turbidity was discernabIe. measured as A650 nm. Sequential alterations of visible appearance of initially clear Limulus lysate, following activation by endotoxin, are designated at flocculation (F), increased viscosity (V) or solid gel Individual samples were (G). examined at various times after activation and either stirred gently with a spatula (A) or mixed vigorously by vortex (B), prior to measurement of turbidity.
Time (min.)
Visible Clot Solubilitv. The solubilization of proteins of gelled lysates was evaluated by urea and acid solubility studies, the classic assays for the detection of mammalian Factor XIII (Fibrin Stabilizing Factor)(20,36), and by assays for solubilization by ionic (SDS) or non-ionic Gelled lysates were covered with each of these solvents and (Triton X-100) detergents. observed at 4 hours (Fig. 2A) or 48 hours (Fig. 2B). Gelled lysates which were covered with water, SDS, or Triton X-100 all maintained their initial opacity and remained solid after several days of incubation at room temperature, with no visible evidence of solubilization (Fig. 2A and 2B, tubes a,b,e, respectively). In some experiments, gelled lysates covered with Triton X-100 slowly became clear yet remained solid. The gel incubated with 1% monochloroacetic acid (MCA) completely dissolved within 30 minutes (Figure 2A, tube d), leaving only translucent liquid in the tube. Gelled lysates incubated with 5M urea remained solid at 4 hours (Fig. 2A, tube c) and at 24 hours, but were completely solubilized by 48 hours (Fig. 2B, tube c). Each of the five test solutions which overlay the gels for 4 hours demonstrated absorbantes at 280 nm greater than 3.0. Ultraviolet absorbance spectra of these solutions showed broad absorbance peaks between 256 and 270 nm; A260nm/A280nm for each solution was approximately 1.5-2.0. These spectral features are characteristic of nucleic acids, and were
30
STABILITY OF LIMULUS LYSATE GELS
consistent with predominant absorbance due to nucleic acids rather than proteins in these solutions. Even the totally solubilized gels had absorbance were dominated by the nucleic acid component of Limulus lysate.
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the solubilized spectra which
Figure 2. Solubilization of Gelled Limulus Lysates by Detergents, Acid or Urea. Samples of gelled lysate (final concentration of endotoxin, 50 /.&g/ml) were overlaid with water (a), 10% SDS (b), 5M urea (c), 1% monochloroacetic acid (d) or 10% Triton X-100 (e), as described in Methods, and observed after incubation for 4 hours (A) or 48 hours (B). Solutions overlaying the gels were removed prior to photography. Undissolved gels remained opaque, whereas solubilized gels resulted in translucent liquid.
When similar solubility experiments were performed using whole “Pre-gel” or the hemocyanin-rich subfraction of “Pre-gel” (prepared as described in Methods) rather than standard amebocyte lysate (which contains no hemocyanin), gels remained solid in all the solvents tested for greater than 24 hours. In contrast to the rapid solubilization of standard Limulus lysate gels with 1% monochloroacetic acid, gels formed from whole “Pre-gel” or the centrifugally-obtained hemocyanin-rich subfraction of “Pre-gel” were still solid after one week of incubation with 1% monochloroacetic acid. These gels also remained solid for greater than one week when incubated with water, SDS or Triton X-100. Addition of “Pre-gel” to standard Limulus lysate prior to gelation by endotoxin resulted in resistance to solubilization of the mixture by monochloroacetic acid. This result was obtained when as little as IO%, by volume, of “Pre-gel” or its hemocyanin-rich subfraction was added to lysate. Gels formed from the hemocyanin-free subfraction of “Pre-gel” dissolved readily in monochloroacetic acid but not in water, SDS or Triton X-100, comparable to the results obtained using Limulus lysate. Similar to the experiments using standard lysate, gels which were formed from “Pre-gel” and then incubated with SM urea were softened and partially dissolved at 48 hours, and were completely dissolved by 12-96 hours. Solubilization of Gelled Proteins. Polyacrylamide gel electrophoresis in SDS, performed on dialyzed samples of the above test solutions which overlay the gelled lysates, demonstrated low concentrations of numerous different proteins (Figure 3). Gels which completely dissolved in 1% MCA (Fig. 3, lane A) demonstrated a major broad band (labeled C), plus multiple other minor protein bands, in a pattern similar to that of non-gelled, whole Limulus lysate. The
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31
major band can be identified as coagulin because of its characteristic irregular shape, great predominance, and position of migration (molecular weight equivalent to approximately 20,000 when a IO-fold diluted sample was electrophoresed to obtain a sharp band with discrete migration). All four test solutions in which gelled lysates were visibly unchanged during a 4-hour incubation (Fig. 3, lane B-urea; lane C-Triton X-100; lane D-SDS; lane E-water) showed only a very minor band in the molecular weight range of coagulin. These polyacrylamide gels confirmed the observations described above which suggested that little solubilization of coagulin occurred during incubation for 4 hours with any of the solvents other than 1% MCA.
A
6
C
D
E
_.__ ___. --’
Figure 3. Polyacrylamide Gel Electrophoresis in SDS of Proteins Solubilized by Detergents, Acid or Urea. Gelled lysates were overlaid with detergents, monochloroacetic acid or urea, as described in Methods, and solubilized proteins were analyzed by SDS-gel electrophoresis. Proteins were solubilized in monochloroacetic acid (lane A), urea (lane B), Triton X-100 (lane C), SDS (lane D) or water (lane E). Migrations of molecular weight marker proteins (66,000; 29,000; 12,000) are indicated by the arrows. The migration of coagulin is identified as C. Enzvmatic Assav for Transoevtidase Activitv. Limulus lysates, activated by endotoxin, were analyzed for the presence of transpeptidase activity using the incorporation of 14C-labeled putrescine into the protein acceptor dimethylcasein, as described in Methods. No transpeptidase activity was detectable in lysates which had reached various final stages of activation of coagulation after 24 hours incubation (including flocculation, increased viscosity, and gelled samples), in fresh, solid gels (less than one hour of incubation prior to assay), or in control lysates in the absence of endotoxin. Transpeptidase assays also were performed using “Pre-gel”, a source of Limulus coagulation factors which was obtained without the use of NEM, a compound thought to potentially interfere with the enzymatic assay. “Pre-gel” demonstrated sensitivity to endotoxin comparable to standard lysates prepared from washed amebocytes. Furthermore, since in
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addition to amebocyte enzymes “Pre-gel” contained all soluble proteins present in native hemolymph, this preparation also contained potential crosslinking factors which might be extracellular (i.c., in the hemolymph). No transpeptidase activity was detected in aliquots of freshly endotoxin-activated “Pre-gel” (30 minutes incubation prior to assay) or in aliquots after 72 hours incubation with endotoxin. p ideq. Limulus lysates, activated by bacterial endotoxin, were assayed for the presence of covalently crosslinked e-(yglutamyl)lysine peptides as described in Methods. Measurable amounts of crosslinks were not detected in samples of lysate at flocculation or gelled stages, or in aliquots of lysate not incubated with endotoxin.
DISCUSSION In comparison to mammalian coagulation, the ancient coagulation mechanism in Limulus polyphemus is a simplified biochemical cascade. However, remarkable similarities to more complex mammalian systems can be demonstrated (45). A single protein, coagulogen, forms the solid gel after activation of coagulation. Isolated coagulogen also can form a gel when proteolytically activated by trypsin (17). This is analogous to the ability of mammalian fibrinogen to form a clot following limited proteolysis by a single enzyme such as purified thrombin or trypsin. Mammalian clots formed from the reaction between fibrinogen and thrombin in vitro (fibrin clot), in the absence of Fibrin Stabilizing Factor (FSF, Factor XIII), are extremely fragile and easily solubilized in acid, base or urea (28,46), whereas clots formed in whole plasma (plasma clot) are stabilized by crosslinking produced by FSF (36). In contrast to this difference between mammalian fibrin clots and plasma clots, we had observed in preliminary studies of Limulus lysate that gels formed from isolated coagulogen and partially purified clotting enzyme, a system analogous to that of the fibrin clot, were similar in mechanical fragility to those formed from whole lysate. The studies described in this report were performed to determine whether the clottable protein of Limulus lysates undergoes covalent stabilization after gelation. In comparison to human clots, Limulus gels are extremely fragile. The turbidity associated with formation of a solid gel is easily decreased by mechanical agitation. Therefore, potential covalent crosslinking must be considered very limited in its ability to stabilize the three dimensional structure of Limulus gels. The solubility studies with urea, acid and detergents should be interpreted with caution. Mammalian clots are more easily solubilized by dilute acid than by urea (36). By convention, mammalian clots that are insoluble in monochloroacetic acid (MCA) for at least 1-3 hours or in urea for 24 hours or longer demonstrate enhanced resistance to solubilization by these agents, and are presumed to be stabilized by Factor XIII (28,33-36). On the basis of the experiments conducted in this study, Limulus amcbocyte lysate gels can be characterized as resistant to solubilization by urea (insoluble for at least 24 hours) but susceptible to solubilization by acid (soluble in MCA in less than one hour). The visible differences in solubilization of Limulus gels by the various solvents tested was confirmed by analysis of the solubilized proteins. When solubilized proteins from these lysate gels were analyzed by polyacrylamide gel electrophorcsis in SDS, coagulin was demonstrated only from the gel incubated with monochloroacetic acid. Since acid solubility is characteristic of absence of covalent crosslinking but urea insolubility is characteristic of such bonding, the solubility tests and electrophoretic analyses of solubilized proteins do not definitively establish whether Limulus The results of these studies are consistent either with the gels are covalently stabilized. presence of strong intermolecular interactions which are non-covalent yet not easily disrupted by urea, or with covalent crosslinking which is susceptible to rapid acid hydrolysis. Rapid hydrolysis by monochioroacetic acid of peptide bonds in human clots, without disruption of the crosslinks, has been attributed to activation of pcpsinogen at low pH (40).
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An enzymatic assay for transpeptidase activity failed to demonstrate such an enzyme in Limulus lysate. The assay utilized in this study, involving the incorporation of putrescine into dimethylcaseine, is considered the most sensitive test for a transpeptidase (27). Amine inhibition or incorporation assays such as this have been capable of detecting crosslinking activities in numerous other species, including invertebrates such as the lobster (25), sponge (47) and sand crab (48), as well as in a wide range of mammals (24). In contrast to our studies, experiments performed using sonicated homogenates of amebocytes, a preparation of Limulus enzymes distinctly different from our own, have demonstrated the presence of transpeptidase activity (49). Other than the difference in sample preparation, we are unable to explain this However, it is clear from the present work that the endotoxin-sensitive discrepancy. coagulation mechanism of Limulus does not utilize such an activity. The results of the solubility experiments using “Pre-gel” were surprisingly different from those using standard lysates in that gels were extremely resistant to solubilization with monochloroacetic acid, whereas gradual solubility in urea was qualitatively similar for these two preparations of Limulus coagulation factors. Resistance to solubilization with monochloroacetic acid was demonstrated in gels derived from the hernocyanin-rich pellet of centrifuged “Pre-gel” but was not observed with the hemocyanin-free plasma. Furthermore, resistance to solubilization by monochloroacetic acid was observed when standard Limulus lysate and “Pre-gel” were mixed together prior to gelation, even when “Pre-gel” composed as little as 10% of the mixture. Since transpeptidase activity in “Pre-gel” was not demonstrated, insolubility in monochloroacetic acid does not appear to indicate covalent crosslinking of clots of “Pre-gel” formed from or in the presence of “Pre-gel”. The data obtained using subfractions implicate hemocyanin in this resistance to acid solubilization, but we are unable to satisfactorily explain the mechanism of this unexpected finding. The differential solubilities of Limulus and mammalian clots in various solvents, and the differences in mechanical strength of Limulus and mammalian clots, may be a reflection of the major dissimilarity in molecular weights between horseshoe crab and mammalian clottable proteins. Vertebrate fibrinogen molecules have molecular weights of several hundred thousand (23), and the gamma-chain dimers which characterize their crosslinked fibrins typically weigh 88,000-90,000 (23). The fibrinogen of the invertebrate, Panulirus interruptus (California spiny lobster), which also forms intermolecular covalent crosslinks, is similarly a large molecule (molecular weight 420,000) (26). In comparison, horseshoe crab coagulogen has a molecular weight of 19-27,000 (15,17,50-53), and monomer coagulin is approximately 16-20,000, representing a loss of a peptide of molecular weight 3,000-7,000 during activation of the clottable protein (15,17,50,52). It is possible that the evolutionary transition from small to large clottable proteins may have been accompanied by development of the covalent stabilizing enzyme of vertebrates. However, although the coagulation proteins of mammalian species, for which covalent crosslinking has been demonstrated, are much larger than Limulus coagulogen, it is unlikely that protein size is the sole determining factor for presence or absence of crosslinking. Other transpeptidase-linked molecules in mammals include small molecular weight proteins such as the pregnancy-related protein, uteroglobulin, MW 15,000 (54) and a seminal vesicle clottable protein, MW 17,900 (55) essential for vaginal plug formation. In contrast to the presumed necessity of crosslinking for the appropriate function of these small molecular weight mammalian proteins, the lack of detectable transpeptidase activity in lysates of washed Limulus amebocytes or in whole “Pre-gel” suggests that Limulus coagulin is capable of providing physiologically adequate hemostasis by polymerization, ACKNOWLEDGMENTS We wish to express our gratitude to Dr. L. Lorand and Mr. J. Wilson of Northwestern University for performance of analyses for covalent crosslinked peptides. We also wish to thank Reed Brozcn and Peter Sands for performance of the transpeptidase assays.
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14. NAKAMURA, S., TAKAGI, T., IWANAGA, S., NIWA, M. and TAKAHASHI, K. Amino Acid Sequence Studies on the Fragments Produced from Horseshoe Crab Coagulogen During Gel Formation: Homologies with Primate Fibrinopeptide B. Biochem.Biophys.Res.Comm. 2, 902-908, 1976. 15. NAKAMURA, S., SHISHIKURA, F., IWANAGA,S.,TAKAGI, T., TAKAHASHI, K., NIWA, M. and SEKIGUCHI, K. Horseshoe Crab Coagulogens: Their Structure and Gelation Mechanism. In: Frontiers in Protein Chemistry;_Dsve_lopments itt@ochemistrv 10. T-Y.Liu, G.Mamiya and EYasunoba,editors,Ei&&r-North Holland, New York, pp 495-514, 1980. 16. SOLUM N.O. Some Characteristics of the Clottable Cells. mmb.Diath.Haemorrh. 23, 170- 18 1, 1980.
Protein
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17. TAI, J.Y., SEID, R.C., HUHN, R.D. and LIU, T-Y. Studies on Limulua Amebocyte Lysate II. Purification of the Coagulogen and the Mechanism of Clotting. J.Biol.Chem. 252,4773-4776, 1977. 18. HAWKEY,
C.M. Fibrinolysis
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27, 133-150, 1970.
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STABILITY OF LIMULUS LYSATE GELS
35
19. FERRY, J.D., MILLER, M. and SHULMAN, S. The Conversion of Fibrinogen to Fibrin. VII. Rigidity and Stress Relaxation of Fibrin Clots; Effects of Calcium. Arch.Biochem.Biophys 34, 424-436, 1951. 20. LORAND,
L. Fibrin
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21. LORAND, L., Fibinoligase: The Ann.N.Y.Acad.Sci. 202, 6-30, 1962. 22. LORAND,
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26. FULLER, Transformation
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24. LOPACIUK, S., MCDONAGH, R.P. and MCDONAGH, J. Comparative Coagulation Factor XIII. Proc.Soc.Exper.Biol.Med. 15& 68-72, 1978. 25. DOOLITTLE, 48 l-482, 1962.
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29. LORAND, L. Properties and Significance of the Fibrin Diath. Haemorrh. z Supple. 1, 238-248, 1962.
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32. DUCKERT, F. and BECK, E.A. Clinical Disorders Due to the Deficiency (Fibrin Stabilizing Factor, Fibrinase). Sem.Hematol. a 83-90, 1968. 33. DUCKERT, F., JUNG, E. and SCHMERLING, Haemorrhagic Diathesis Probably Due to Thromb.Diath.Haemorrh. 2, 179-l 86, 1960.
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30. LORAND, L. and JACOBSEN, A. Studies on the Polymerization of Fibrin. the Globulin: Fibrin-Stabilizing Factor. J.Biol.Chem. m 421-434, 1958. 31. LORAND, L., KONISHI, K. and JACOBSEN, Clotting. Nature 194, 1148-l 149, 1962.
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37. LORAND, L., SIEFRING, G.E., TONG, Y.S., BRUNER-LORAND, J. and GRAY, A.J. Dansylcadaverine Specific Staining for Transamidating Enzymes. Ana1.Biochem.a 453-458, 1979. 38. GRIFFIN, M., WILSON, J. and LORAND, L. High-Pressure Liquid Chromatographic Procedure for the Determination of e-(y-Glutamyl)Lysine in Proteins. Anal.Biochem. 124. 406-413, 1982.
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39. LEVIN, J. and BANG, F.B. The Role of Endotoxin in the Extracellular Limulus Blood. Bull.Johns Hopkins Hosp. 115, 265-274, 1964. 40. IKEMORI, Fibrin Clots Am.J.Clin.Path.
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R., GRUHL, M., SHRIVASTAVA,*S. and SHANBERGE, J.N. Solubility of in Monochloroacetic Acid. A Reflection of Serum Pepsinogen Levels. 63, 49-56, 1975.
41. LEVIN, J., TOMASULO, P.A. and OSER, R.S. Detection of Endotoxin and Demonstration of an Inhibitor. J.Lab.Clin.Med. 2 903-911, 1970. 42. LAEMMLI, U.K. Cleavage of Structural Proteins Bacteriophage T4. Nature 227, 680-685, 1970.
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43. LAYNE, E. Spectrophotometric and Turbidimetric Methods for Measuring Proteins. In: Methods in Enzymology III. Colowick, S.P. and Kaplan, N.O., editors, Academic Press, N.Y., pp 447-454, 1957. 44. CARIELLO, L., WILSON, J. and LORAND, L. Activation Embryonic Development. Biochem. 23,6843-6850, 1984,
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45. LEVIN, J. The Role of Amebocytes in the Blood Coagulation Mechanism of the Horseshoe Crab Limulus polvDhemu& In: Blood Cells of Marine Invertebrates. Experimental SvstemSip Cell Biology and Comparative Physiology. Cohen, W.D., editor, Alan R. Liss, pp.145-163, 1985. 46. ROBBINS, K.C. A Study on the Conversion 581-588, 1944.
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47. BROZEN, R., SANDS, P., RIESEN, W., WEISSMANN, G. and LORAND, L. The Antiquity of Transglutaminase: An Intracellular Enzyme from Marine Sponge Cells Enhances Clotting of Lobster Plasma. BioLBull. l73, 423, 1987. (abstr.) 48. MADARAS, F., PARKIN, J.D. and CASTALDI, P.A. Coagulation (Ovalipes Bipustulatus). Thromb.Haem. 42_ 734-742, 1979.
in the Sand
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49. CAMPBELL-WILKES, L.K. Calcium-dependent Transamidating Enzymes and their Involvement in Protein Assembly. Development of New Methods of Study and the Application of these Methods to Various Transamidases with the Goal of Elucidating Physiological Function. Doctoral Thesis, Northwestern University,. 1973. 50. GAFFIN, S.L. The Clotting of the Lysed White Cells of Limulus Induced by Endotoxin-I: Preparation and Characterization of Clot-Forming Proteins. Biorheology I.& 273-280, 1976. 51. MOSESSON, M.W., WOLFENSTEIN-TODEL, C., LEVIN, J. and BERTRAND, 0. Characterization of Amebocyte Coagulogen from the Horseshoe Crab (Limulus PolvDhemuS). Thromb.Res. l4_ 765-779, 1979. 52. SOLUM, N.O. The Coagulogen of Limulus Polyphemus Hemocytes. A Comparison Clotted and Non-Clotted Forms of the Molecule. Thromb.Res. $ 55-70, 1973.
of the
53. SRIMAL, S., MIYATA, T., KAWABATA, S., MIYATA, T. and IWANAGA, S. The Complete Amino Acid Sequence of Coagulogen Isolated from Southeast Asian Horseshoe Crab, Carcinoscorpius rotundicauda. J.Bi0chem.s 305-318, 1985. 54. MANJUNATH, Transglutaminase.
R., CHUNG, S.I. and MUKHERJEE, A.B. Crosslinking Biochem.Biophys.Res.Comm. 121, 400-407, 1984.
of Uteroglobin
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A.C., PABALAN, S.S. and LORAND, L. 55. WILLIAMS-ASHMAN, H.G., NOTIDES, Transamidase Reactions Involved in the Enzymic Coagulation of Semen: Isolation of r_GlutamyI-e-Lysine Dipeptide from Clotted Secretion Protein of Guinea Pig Seminal Vesicle. Proc.Nat.Acad.Sci, @, 2322-2325, 1972.
STABILITY AMEBOCYTE
OF GELS FORMED FOLLOWING LYSATE: LACK OF COVALENT
All rights reserved.
COAGULATION CROSSLINKING
OF LIMULUS OF COAGULIN.
Robert I. Roth, Joseph C.-R. Chen and Jack Levin From the Departments of Laboratory Medicine and Medicine, University of California of Medicine and Veterans Administration Medical Center, San Francisco, CA, USA
School
(Received 17.3.1989; accepted in original form 30.3.1989 by Editor W.H. Seegers)
ABSTRACT.
Incubation of lysates prepared from amebocytes of the horseshoe crab (Limulus polyphemus) with bacterial endotoxin results in coagulation and formation of a solid gel. Although Limulus gels remained solid indefinitely, if undisturbed, they were easily disrupted by mechanical agitation. Chemical solubility studies of gelled lysates demonstrated rapid solubilization of gels in monochloroacetic acid, a property of clots that have not been covalently stabilized; but in contrast stabilized demonstrated resistance to solubilization by urea, a property of clots. Analysis of solubilized proteins by polyacrylamide gel electrophoresis in SDS demonstrated coagulin, the designation for the activated form of coagulogen (the clottable protein) that forms a gel, only in samples derived from clotted lysate that had been previously incubated with monochloroacetic acid, but not in samples following incubation with urea, confirming the results of the chemical solubility studies. Enzymatic assays for transpeptidase (Factor XIII-like) activity in either native or gelled Limulus lysates were negative. Furthermore, analysis for covalently crosslinked peptides in gelled coagulin confirmed the absence of intermolecular y-glutamyl-e-lysyl bonds. Therefore, the stable endotoxin
gels formed following coagulation are not covalently crosslinked.
of Limulus
lysate
by bacterial
INTRODUCTION Limulus polyphemus, the horseshoe crab, has a coagulation mechanism that can be activated by bacterial endotoxin (l-6), the lipopolysaccharide component of the cell wall of gram-negative bacteria. After activation of coagulation, a solid gel is formed which is stable. However, the basis for structural stability of the gel in Limulus has not been investigated. We conducted this study to determine whether this stability was the result of chemical crosslinking of the coagulation proteins. Blood coagulation in the ancient invertebrate Limulus polyphemus, the horseshoe crab,
Key Words: Limulus,
amebocyte
lysate, coagulin, 25
clot stability,
crosslinking
26
STABILITY OF LIMULUS LYSATE GELS
Vol. 55, No. 1
is based on an enzymatic cascade, which includes one or more trypsin-like serine proteases and is similar in many respects to that of mammalian coagulation (l-57-10). However, coagulation of Limulus blood is mediated by cellular rather than plasma factors. The amebocyte, the only type of circulating blood cell in Limulus polyphemus, contains all of the factors necessary for coagulation of the blood (11,12). Plasma factors are not required for blood coagulation. Amebocytes react to a breach of hemostasis in the animal by aggregation and degranulation (8,12). Degranulation leads to release of the components of the coagulation cascade, and their subsequent activation results in the formation of a solid gel composed primarily of the Coagulogen is functionally analogous to fibrinogen, and clottable protein, coagulogen. contains a segment with sequence homology to the mammalian clotting protein (13,14). After activation by bacterial endotoxin, the clottable protein, coagulogen, undergoes limited proteolysis to become coagulin (14-17) and then structural reorganization into an opaque gel. Gels formed in vitro from amebocyte lysates (1 l), of which 50% of the protein is coagulogen (4,16), are easily disrupted by vortexing or stirring, yet remain indefinitely intact in a test tube if they are not mechanically disrupted (personal observation). There is no evidence for a plasmin-like proteolytic activity in Limulus, and dissolution of clots has not been noted in other invertebrates with coagulable blood (18). Covalent crosslinking is a process which greatly enhances the structural stability of clots, i.e., increased rigidity, greater internal cohesion and lower stress relaxation (19-22). Clottable proteins of many diverse vertebrates, including many species of mammal, bird, reptile, amphibia and fish (23,24), undergo covalent crosslinking following the formation of a clot. Crosslinking of the clottable protein also has been described in the invertebrate, Homarus The crosslinking reaction characteristically is mediated by americanus (lobster) (2526). transpeptidase activities which involve the creation of intermolecular y-glutamyl-e-lysyl bonds. The marked stability of the mammalian clot is associated with a very limited degree of crosslinking, one mole of yglutamyl-e-lysyl peptide per 80,000 grams of protein (22). The transpeptidase reaction has been well described in the lobster (26) as a function of a single polymerization/crosslinking enzyme and in mammals as a function of Factor XIIIa (Fibrin Stabilizing Factor, FSF) (20,27-31). Congenital deficiencies of Factor XIII, which can result in a significant hemorrhagic diathesis, have been described in man (32-34). Presumptive evidence for presence of a clot stabilizing factor in mammals is the insolubility of a solid clot in dilute acid (1% monochloroacetic acid) or concentrated (5M) urea (20,28,33-36). Congenital absence of FSF results in clots which dissolve readily in these solubilizing agents (33,34). Further presumptive evidence for clot stabilizing activity is the detection of crosslinked fibrin (primarily gamma-chain dimers in vertebrate species) by polyacrylamide gel electrophoresis in SDS (23). Enzymatic assays for transpeptidase activity, such as the in vitro incorporation into protein substrates of the fluorescent molecule dansylcadavarine (37) or radiolabeled putrescine (27), and the isolation of chemically-crosslinked amino acids (e-(yglutamyl)lysine peptides) following proteolytic digestion of fibrin (26,38), provide direct evidence for a clot stabilizing activity. The experiments described in this report were undertaken to characterize the basis for stability of gelled lysates of Limulus amebocytes.
MATERIALS
AND METHODS
Materials. Endotoxin used was Escherichia coli lipopolysaccharide B, 026:B6 (Difco Laboratories, Detroit, MI). Sterile, endotoxinfree water and saline solutions were purchased from Travenol Laboratories (Deerfield, IL.). Chemicals were reagent grade. Glassware. All glassware was rendered endotoxin-free then heating at 190°C in a dry oven for 4 hours.
by autoclaving
for 45 minutes
and
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STABILITY OF LIMULUS LYSATE GELS
27
Limulus Amebocvte Lvsate. Horseshoe crabs were obtained from the Department of Marine Resources of the Marine Biological Laboratory, Woods Hole, MA. Amebocyte lysates were prepared from Limulus polyphemus (the horseshoe crab) by disruption in distilled water of washed amebocytes (separated from hemolymph plasma in the presence of N-ethyl maleimide (NEM)), as described previously (11). Amebocyte lysate was stored at 4’C. For some experiments, lysates termed “Pre-gel” were prepared by disruption of amebocytes in samples of native hemolymph with release of the components of the coagulation mechanism, in the absence of NEM (39). Hemocyanin, the oxygen carrying protein of Limulus plasma, was separated from the remaining components of “Pre-gel” into a pellet by centrifugation at 18,500 rpm (41,280 x g) for 3 hours. By this procedure, the overlying plasma, which contained the components of the coagulation mechanism, was hemocyanin-free. Assavs for Stabilitv of Gelled Lvsate. Experimental conditions described previously to assess clot stability in vertebrates (28,30,34-36,40) vary considerably with regard to temperature of incubation, length of incubation, and presence or absence of shaking during incubation with solvent. Since Limulus coagulin gels do not retract from glass as an intact, solid clot (personal observation), not all gel stability tests useful in vertebrates can be applied to evaluation of the horseshoe crab, Therefore, in this study several experimental conditions were examined to help characterize gel stability in Limulus. I. Mechanical Fraailitv of Coaaulin Gels. 200 1.11of Limulus lysate was incubated with 10 ~1 of E.coli endotoxin
(final
concentration,
0.005 pg/ml)
at 37’C. Stages of gelation
of the
endotoxin-activated lysate (flocculation (F), heavy flocculation (F+), increased viscosity (V), Turbidity of the solutions (an increase in light and solid gel (G)) were assessed visually. scattering accompanies coagulation (11,41)) was monitored. At various stages of gelation, 300 1.11of SM urea was added to samples to aid in solubilization of any non-crosslinked proteins which had been reversibly aggregated, partially denatured or trapped in the gel. The samples then were subjected to either gentle stirring with a spatula or vigorous agitation by vortexing for 2 minutes. The residual turbidity was measured spectrophotometrically at 650 nm. Gels that were broken apart by mechanical means became less turbid. Accordingly, fragility of the coagulin gels was assessed by the extent to which mechanical agitation was capable of diminishing the turbidity of the solutions. II. Visible Clot Solubilization. A variety of solvents were examined for ability to completely dissolve gelled Limulus lysates. Tubes containing 0.5 ml of Limulus lysate and 0.05 ml of E.coli endotoxin (final concentration, 50 pg/ml) were incubated for two hours at 37’C. In some experiments, similar incubations were performed using “Pre-gel” lysates, centrifugally-concentrated hemocyanin (prepared as described above) or hemocyanin-free plasma, rather than standard amebocyte lysates. Under these conditions, both types of lysates and both subfractions of “Pre-gel” formed a solid opaque gel within ten minutes. Gelled lysates or “Pre-gel” subfractions then were overlaid with 1 ml of water, 10% SDS, 10% Triton X-100, 1% monochloroacetic acid or 5M urea. Incubations were performed at room temperature for up to one week. Gels were monitored for physical stability by observing whether gels maintained their original solid shape during tilting of the gel tubes. III. Splubilization of Proteins from Gelled Lvsate. Tubes containing 0.5 ml of Limulus lysate and 0.05 ml of E.coli endotoxin (final concentration, 50 pg/ml) were incubated for two hours at 37’C. Gelled lysates then were overlaid with 1 ml of water, 10% SDS, 10% Triton X-100, 1% monochloroacetic acid or SM urea. The samples were covered and incubated for 24 hours at room temperature or at 37’C. Each sample was observed during this 24 hour period, after which time the overlying solutions were removed from the gels and dialyzed against phosphate buffer (pH 7.6) with several buffer changes. The samples then were analyzed by polyacrylamide gel electrophoresis in SDS (42), using 12% acrylamide gels. Undialyzed samples of the supernatants also were characterized by measurement of their ultraviolet absorbance
28
STABILITY OF LIMULUS LYSATE GELS
Vol. 55, No. 1
spectra, from 200-350 nm, in a Gilford 2400-2 spectrophotometer (Gilford Laboratories, Oberlin, Ohio). Characterization of absorbance as predominantly nucleic acid was obtained from the ratio of A260nm / A2gOnm (43).
Instrument protein or
Enzvmatic Assay fer Transpeptidase Activitv. Aliquots of Limulus lysate were incubated with E.coli endotoxin (final concentrations, O.Ol,O.OOl or 0.0001 fig/ml) at 37’C for four hours, and then at room temperature for 20 hours. These conditions produced final stages of gelation that included flocculation, increased viscosity and gelled samples. Another aliquot of the same lysate was incubated with E.coli endotoxin (final concentration 0.1 pg/ml) for twenty minutes at 37’C, resulting in a solid gel, immediately prior to assay. These various samples, and aliquots of lysate not incubated with endotoxin, were assayed for transpeptidase activity using the incorporation of l4 C-putrescine into dimethylcasein (27). A preparation of “Pre-gel” was incubated with E.coli endotoxin (final concentration, 0.1 pg/ml) for 72 hours. Other aliquots were incubated with E.coli endotoxin (final concentrations, 0.1, 0.00001 or 0.000001 pg/ml) for 30 minutes immediately prior to assay. These samples of “Pre-gel”, all of which ultimately formed solid gels following addition of endotoxin, and aliquots of “pre-gel” not incubated with endotoxin, were assayed for transpeptidase activity, as described above. bs. Anal i for Samples of Limulus lysate incubated with endotoxin, as described above for the assay of transpeptidase activity, and aliquots of lysates not incubated with endotoxin, were assayed for the presence of covalent crosslinks by J. Wilson in the laboratory of L. Lorand, Northwestern University, by published procedures (44).
RESULTS Mechanical Fraailitv of Gelled Limulus Lvsate. Lysates were gelled with endotoxin (final concentration, 0.005 pg/ml) and the gels were observed for one week. Lysates originally were clear. After incubation with endotoxin, grey-white, opaque solid gels formed and remained unchanged during the one week period of observation. They were solidly attached to the glass tubes; no retraction from the inner surface of the tubes was noted. At all times during the observation period of one week, these gels were friable and easily disrupted by mechanical This property was distinctly different from the greater stability (e.g., no agitation. fragmentation with vigorous vortexing) of a human clot observed after retraction. Gelation of Limulus lysate into an opaque, solid gel was associated with an increase in turbidity (light scattering) of the solution as coagulin molecules polymerized, as has been reported previously (11,41). Increasing turbidity during the process of coagulation was determined spectrophotometrically by measurement of light scattering at 650 nm. We utilized a reversal of this opacity to assess the physical stability of the coagulin gel. Preliminary experiments established the concentration of endotoxin required to gel a selected batch of lysate at a moderately slow rate, i.e., 30-90 minutes, so that visible stages of gelation could be distinguished. Pairs of tubes of activated lysate were removed at various times from the 37OC waterbath and urea was added to solubilize any proteins that were non-specifically precipitated or trapped by the gel. One sample of each pair then was stirred gently with a spatula, following which light scattering was measured (Figure 1, line A). Turbidity (A6SOnm) increased slowly as the samples progressed from flocculation to viscous states, and then increased dramatically during the process of solid gel formation (Figure 1, line A). In contrast, when corresponding samples of each pair were mixed vigorously using a Vortex mixer (Figure 1, line B), the subsequent turbidity was greatly diminished at all stages of coagulation. The latter samples contained small residual particles which closely resembled the particulate
STABILITY OF LIMULUS LYSATE GELS
Vol. 55, No. 1
29
material observed during early stages of flocculation. To determine whether these residual particles could re-associate into a solid gel structure, other gelled Limulus lysate samples were disrupted with the Vortex mixer but no urea was added. These appeared visually similar to the samples treated with urea and were replaced into the waterbath and observed for re-gelation. None of these vortexed samples demonstrated increased viscosity or gelation after reincubation, suggesting that the three-dimensional structure of the coagulin matrix had been irreversibly altered.
0.9, 0.80.70.6OS0.40.30.2O.l0.0 0
I 15
I 30
I 45
(F)
(VI
((3
Incubation
I 60
Figure 1. Turbidity of Limulus Lysate Following Activation by Endotoxin. Aliquots of Limulus lysate were activated by endotoxin, as described in Methods, such that gelation proceeded slowly and the various stages of activation were Turbidity was discernabIe. measured as A650 nm. Sequential alterations of visible appearance of initially clear Limulus lysate, following activation by endotoxin, are designated at flocculation (F), increased viscosity (V) or solid gel Individual samples were (G). examined at various times after activation and either stirred gently with a spatula (A) or mixed vigorously by vortex (B), prior to measurement of turbidity.
Time (min.)
Visible Clot Solubilitv. The solubilization of proteins of gelled lysates was evaluated by urea and acid solubility studies, the classic assays for the detection of mammalian Factor XIII (Fibrin Stabilizing Factor)(20,36), and by assays for solubilization by ionic (SDS) or non-ionic Gelled lysates were covered with each of these solvents and (Triton X-100) detergents. observed at 4 hours (Fig. 2A) or 48 hours (Fig. 2B). Gelled lysates which were covered with water, SDS, or Triton X-100 all maintained their initial opacity and remained solid after several days of incubation at room temperature, with no visible evidence of solubilization (Fig. 2A and 2B, tubes a,b,e, respectively). In some experiments, gelled lysates covered with Triton X-100 slowly became clear yet remained solid. The gel incubated with 1% monochloroacetic acid (MCA) completely dissolved within 30 minutes (Figure 2A, tube d), leaving only translucent liquid in the tube. Gelled lysates incubated with 5M urea remained solid at 4 hours (Fig. 2A, tube c) and at 24 hours, but were completely solubilized by 48 hours (Fig. 2B, tube c). Each of the five test solutions which overlay the gels for 4 hours demonstrated absorbantes at 280 nm greater than 3.0. Ultraviolet absorbance spectra of these solutions showed broad absorbance peaks between 256 and 270 nm; A260nm/A280nm for each solution was approximately 1.5-2.0. These spectral features are characteristic of nucleic acids, and were
30
STABILITY OF LIMULUS LYSATE GELS
consistent with predominant absorbance due to nucleic acids rather than proteins in these solutions. Even the totally solubilized gels had absorbance were dominated by the nucleic acid component of Limulus lysate.
Vol. 55, No. 1
the solubilized spectra which
Figure 2. Solubilization of Gelled Limulus Lysates by Detergents, Acid or Urea. Samples of gelled lysate (final concentration of endotoxin, 50 /.&g/ml) were overlaid with water (a), 10% SDS (b), 5M urea (c), 1% monochloroacetic acid (d) or 10% Triton X-100 (e), as described in Methods, and observed after incubation for 4 hours (A) or 48 hours (B). Solutions overlaying the gels were removed prior to photography. Undissolved gels remained opaque, whereas solubilized gels resulted in translucent liquid.
When similar solubility experiments were performed using whole “Pre-gel” or the hemocyanin-rich subfraction of “Pre-gel” (prepared as described in Methods) rather than standard amebocyte lysate (which contains no hemocyanin), gels remained solid in all the solvents tested for greater than 24 hours. In contrast to the rapid solubilization of standard Limulus lysate gels with 1% monochloroacetic acid, gels formed from whole “Pre-gel” or the centrifugally-obtained hemocyanin-rich subfraction of “Pre-gel” were still solid after one week of incubation with 1% monochloroacetic acid. These gels also remained solid for greater than one week when incubated with water, SDS or Triton X-100. Addition of “Pre-gel” to standard Limulus lysate prior to gelation by endotoxin resulted in resistance to solubilization of the mixture by monochloroacetic acid. This result was obtained when as little as IO%, by volume, of “Pre-gel” or its hemocyanin-rich subfraction was added to lysate. Gels formed from the hemocyanin-free subfraction of “Pre-gel” dissolved readily in monochloroacetic acid but not in water, SDS or Triton X-100, comparable to the results obtained using Limulus lysate. Similar to the experiments using standard lysate, gels which were formed from “Pre-gel” and then incubated with SM urea were softened and partially dissolved at 48 hours, and were completely dissolved by 12-96 hours. Solubilization of Gelled Proteins. Polyacrylamide gel electrophoresis in SDS, performed on dialyzed samples of the above test solutions which overlay the gelled lysates, demonstrated low concentrations of numerous different proteins (Figure 3). Gels which completely dissolved in 1% MCA (Fig. 3, lane A) demonstrated a major broad band (labeled C), plus multiple other minor protein bands, in a pattern similar to that of non-gelled, whole Limulus lysate. The
Vol. 55, No. 1
STABILITY OF LIMULUS LYSATE GELS
31
major band can be identified as coagulin because of its characteristic irregular shape, great predominance, and position of migration (molecular weight equivalent to approximately 20,000 when a IO-fold diluted sample was electrophoresed to obtain a sharp band with discrete migration). All four test solutions in which gelled lysates were visibly unchanged during a 4-hour incubation (Fig. 3, lane B-urea; lane C-Triton X-100; lane D-SDS; lane E-water) showed only a very minor band in the molecular weight range of coagulin. These polyacrylamide gels confirmed the observations described above which suggested that little solubilization of coagulin occurred during incubation for 4 hours with any of the solvents other than 1% MCA.
A
6
C
D
E
_.__ ___. --’
Figure 3. Polyacrylamide Gel Electrophoresis in SDS of Proteins Solubilized by Detergents, Acid or Urea. Gelled lysates were overlaid with detergents, monochloroacetic acid or urea, as described in Methods, and solubilized proteins were analyzed by SDS-gel electrophoresis. Proteins were solubilized in monochloroacetic acid (lane A), urea (lane B), Triton X-100 (lane C), SDS (lane D) or water (lane E). Migrations of molecular weight marker proteins (66,000; 29,000; 12,000) are indicated by the arrows. The migration of coagulin is identified as C. Enzvmatic Assav for Transoevtidase Activitv. Limulus lysates, activated by endotoxin, were analyzed for the presence of transpeptidase activity using the incorporation of 14C-labeled putrescine into the protein acceptor dimethylcasein, as described in Methods. No transpeptidase activity was detectable in lysates which had reached various final stages of activation of coagulation after 24 hours incubation (including flocculation, increased viscosity, and gelled samples), in fresh, solid gels (less than one hour of incubation prior to assay), or in control lysates in the absence of endotoxin. Transpeptidase assays also were performed using “Pre-gel”, a source of Limulus coagulation factors which was obtained without the use of NEM, a compound thought to potentially interfere with the enzymatic assay. “Pre-gel” demonstrated sensitivity to endotoxin comparable to standard lysates prepared from washed amebocytes. Furthermore, since in
32
STABILITY OF LIMULUS LYSATE GELS
Vol. 55, No. 1
addition to amebocyte enzymes “Pre-gel” contained all soluble proteins present in native hemolymph, this preparation also contained potential crosslinking factors which might be extracellular (i.c., in the hemolymph). No transpeptidase activity was detected in aliquots of freshly endotoxin-activated “Pre-gel” (30 minutes incubation prior to assay) or in aliquots after 72 hours incubation with endotoxin. p ideq. Limulus lysates, activated by bacterial endotoxin, were assayed for the presence of covalently crosslinked e-(yglutamyl)lysine peptides as described in Methods. Measurable amounts of crosslinks were not detected in samples of lysate at flocculation or gelled stages, or in aliquots of lysate not incubated with endotoxin.
DISCUSSION In comparison to mammalian coagulation, the ancient coagulation mechanism in Limulus polyphemus is a simplified biochemical cascade. However, remarkable similarities to more complex mammalian systems can be demonstrated (45). A single protein, coagulogen, forms the solid gel after activation of coagulation. Isolated coagulogen also can form a gel when proteolytically activated by trypsin (17). This is analogous to the ability of mammalian fibrinogen to form a clot following limited proteolysis by a single enzyme such as purified thrombin or trypsin. Mammalian clots formed from the reaction between fibrinogen and thrombin in vitro (fibrin clot), in the absence of Fibrin Stabilizing Factor (FSF, Factor XIII), are extremely fragile and easily solubilized in acid, base or urea (28,46), whereas clots formed in whole plasma (plasma clot) are stabilized by crosslinking produced by FSF (36). In contrast to this difference between mammalian fibrin clots and plasma clots, we had observed in preliminary studies of Limulus lysate that gels formed from isolated coagulogen and partially purified clotting enzyme, a system analogous to that of the fibrin clot, were similar in mechanical fragility to those formed from whole lysate. The studies described in this report were performed to determine whether the clottable protein of Limulus lysates undergoes covalent stabilization after gelation. In comparison to human clots, Limulus gels are extremely fragile. The turbidity associated with formation of a solid gel is easily decreased by mechanical agitation. Therefore, potential covalent crosslinking must be considered very limited in its ability to stabilize the three dimensional structure of Limulus gels. The solubility studies with urea, acid and detergents should be interpreted with caution. Mammalian clots are more easily solubilized by dilute acid than by urea (36). By convention, mammalian clots that are insoluble in monochloroacetic acid (MCA) for at least 1-3 hours or in urea for 24 hours or longer demonstrate enhanced resistance to solubilization by these agents, and are presumed to be stabilized by Factor XIII (28,33-36). On the basis of the experiments conducted in this study, Limulus amcbocyte lysate gels can be characterized as resistant to solubilization by urea (insoluble for at least 24 hours) but susceptible to solubilization by acid (soluble in MCA in less than one hour). The visible differences in solubilization of Limulus gels by the various solvents tested was confirmed by analysis of the solubilized proteins. When solubilized proteins from these lysate gels were analyzed by polyacrylamide gel electrophorcsis in SDS, coagulin was demonstrated only from the gel incubated with monochloroacetic acid. Since acid solubility is characteristic of absence of covalent crosslinking but urea insolubility is characteristic of such bonding, the solubility tests and electrophoretic analyses of solubilized proteins do not definitively establish whether Limulus The results of these studies are consistent either with the gels are covalently stabilized. presence of strong intermolecular interactions which are non-covalent yet not easily disrupted by urea, or with covalent crosslinking which is susceptible to rapid acid hydrolysis. Rapid hydrolysis by monochioroacetic acid of peptide bonds in human clots, without disruption of the crosslinks, has been attributed to activation of pcpsinogen at low pH (40).
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STABILITY OF LIMULUS LYSATE GELS
33
An enzymatic assay for transpeptidase activity failed to demonstrate such an enzyme in Limulus lysate. The assay utilized in this study, involving the incorporation of putrescine into dimethylcaseine, is considered the most sensitive test for a transpeptidase (27). Amine inhibition or incorporation assays such as this have been capable of detecting crosslinking activities in numerous other species, including invertebrates such as the lobster (25), sponge (47) and sand crab (48), as well as in a wide range of mammals (24). In contrast to our studies, experiments performed using sonicated homogenates of amebocytes, a preparation of Limulus enzymes distinctly different from our own, have demonstrated the presence of transpeptidase activity (49). Other than the difference in sample preparation, we are unable to explain this However, it is clear from the present work that the endotoxin-sensitive discrepancy. coagulation mechanism of Limulus does not utilize such an activity. The results of the solubility experiments using “Pre-gel” were surprisingly different from those using standard lysates in that gels were extremely resistant to solubilization with monochloroacetic acid, whereas gradual solubility in urea was qualitatively similar for these two preparations of Limulus coagulation factors. Resistance to solubilization with monochloroacetic acid was demonstrated in gels derived from the hernocyanin-rich pellet of centrifuged “Pre-gel” but was not observed with the hemocyanin-free plasma. Furthermore, resistance to solubilization by monochloroacetic acid was observed when standard Limulus lysate and “Pre-gel” were mixed together prior to gelation, even when “Pre-gel” composed as little as 10% of the mixture. Since transpeptidase activity in “Pre-gel” was not demonstrated, insolubility in monochloroacetic acid does not appear to indicate covalent crosslinking of clots of “Pre-gel” formed from or in the presence of “Pre-gel”. The data obtained using subfractions implicate hemocyanin in this resistance to acid solubilization, but we are unable to satisfactorily explain the mechanism of this unexpected finding. The differential solubilities of Limulus and mammalian clots in various solvents, and the differences in mechanical strength of Limulus and mammalian clots, may be a reflection of the major dissimilarity in molecular weights between horseshoe crab and mammalian clottable proteins. Vertebrate fibrinogen molecules have molecular weights of several hundred thousand (23), and the gamma-chain dimers which characterize their crosslinked fibrins typically weigh 88,000-90,000 (23). The fibrinogen of the invertebrate, Panulirus interruptus (California spiny lobster), which also forms intermolecular covalent crosslinks, is similarly a large molecule (molecular weight 420,000) (26). In comparison, horseshoe crab coagulogen has a molecular weight of 19-27,000 (15,17,50-53), and monomer coagulin is approximately 16-20,000, representing a loss of a peptide of molecular weight 3,000-7,000 during activation of the clottable protein (15,17,50,52). It is possible that the evolutionary transition from small to large clottable proteins may have been accompanied by development of the covalent stabilizing enzyme of vertebrates. However, although the coagulation proteins of mammalian species, for which covalent crosslinking has been demonstrated, are much larger than Limulus coagulogen, it is unlikely that protein size is the sole determining factor for presence or absence of crosslinking. Other transpeptidase-linked molecules in mammals include small molecular weight proteins such as the pregnancy-related protein, uteroglobulin, MW 15,000 (54) and a seminal vesicle clottable protein, MW 17,900 (55) essential for vaginal plug formation. In contrast to the presumed necessity of crosslinking for the appropriate function of these small molecular weight mammalian proteins, the lack of detectable transpeptidase activity in lysates of washed Limulus amebocytes or in whole “Pre-gel” suggests that Limulus coagulin is capable of providing physiologically adequate hemostasis by polymerization, ACKNOWLEDGMENTS We wish to express our gratitude to Dr. L. Lorand and Mr. J. Wilson of Northwestern University for performance of analyses for covalent crosslinked peptides. We also wish to thank Reed Brozcn and Peter Sands for performance of the transpeptidase assays.
34
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STABILITYOF LIMULUS LYSATE GELS
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