Localization and roles of coagulogen and transglutaminase in hemolymph coagulation in decapod crustaceans

Comp. Biochem. Physiol. Vol. 100B,No. 3, pp. 517-522, 1991

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LOCALIZATION A N D ROLES OF COAGULOGEN A N D TRANSGLUTAMINASE IN HEMOLYMPH COAGULATION IN DECAPOD CRUSTACEANS GARY G. MARTIN,* Jo ELLENHOSE, SIDNEOMORI,~ CELESTECHONG,~ TANYAHOODBHOYand NANCYMCKRELL Department of Biology, Occidental College, Los Angeles, CA90041, USA (Received 7 May 1991)

Abstract--1. Of several anticoagulants tested, only N-ethyl maleimide (NEM) prevents lysis of shrimp hemocytes and maintains their normal morphology. 2. Coagulogen, the clotting protein, is a plasma protein with a molecular mass of ~400 kDa. 3. Transglutaminase, the enzyme that cross links coagulogen to form a visible clot, is more abundant in the hemocytes than in plasma. 4. Hemocytes can be separated into two bands by differential centrifugation. The upper band is enriched with hyaline cells, which initiate clot formation, and contains most of the transglutaminase activity. 5. The mechanisms of hemolymph coagulation are discussed.

INTRODUCTION

Coagulation of the bemolymph is an essential defense response of crustaceans. It prevents both the loss of blood (hemolymph) through breaks in the exoskeleton and the dissemination of bacteria throughout the body. Tait (1911) described three patterns of hemolymph coagulation in crustaceans. Type A coagulation is characterized by rapid agglutination of hemocytes without clotting of the plasma. Type B involves cell aggregation coupled with limited clotting of the plasma, and in type C, lysis of "explosive corpuscles" causes clotting of the plasma with little cell aggregation. These three types are, most likely, variations of the basic mechanism involving both hemocyte aggregation and coagulation of hemolymph proteins. The clottable protein, coagulogen, is present in the plasma of species with coagulation types A, B and C, and can be induced to form a gel by the addition of hemocyte lysate (Ghidalia et al., 1981). In fact, hemocyte lysate from a type A crab which normally shows very weak gelation of the hemolymph, is effective in coagulating plasma from crustaceans with any type of coagulation. The active component of the cell lysate is a calcium-dependent transglutaminase (Fuller and Doolittle, 1971b; Lorand and Conrad, 1984) which is also active in the coagulation of vertebrate blood. The basic difference between the two processes is that crustacean coagulogen, unlike fibrinogen, does not require proteolysis to initiate polymerization (Fuller and Doolittle, 1971b). *Author to whom correspondence should be addressed. tPresent address: Department of Microbiology and Immunology, UCLA, School of Medicine, Los Angeles, CA 90024, USA. :IPresent address: Department of Biophysics, Columbia University, New York, NY 10025, USA. 517

Identification of the hemocyte type which initiates coagulation has been complicated by past confusion over hemocyte classification. Some reports identify granulocytes as initiating coagulation (Toney, 1958; Hearing and Vernick, 1967; Mengeot et al., 1977; Madaras et al., 1981), whereas others claim that hyaline cells are responsible (Wood et al., 1971; Ravindranath, 1980). Our lab has undertaken a detailed study of hemocyte function, supporting hemocyte identifications with light and electron microscopy, and cytochemistry. In all decapod species we have examined, including representatives from all three of Tait's categories, hyaline hemocytes lyse, and subsequent clotting is initiated by the release of cytoplasmic constituents. We base the identification of hyaline cells on several features and avoid relying on the feature traditionally used to separate hyaline cells, that is, the absence of cytoplasmic granules (Hose et al., 1990). Hyaline cells are typically the smallest bemocytes, have a high nucleocytoplasmic ratio, and the cytoplasm stains with Sudan Black B. At the TEM level, the cytoplasm contains numerous small ( ~ 50 nm diameter), electron-dense deposits which are released during cell lysis. The identity of these cytoplasmic deposits has not been established, but they are probably a protein crucial to hemolymph coagulation, possibly transglutaminase. Granulocytes, which lack these distinctive cytoplasmic deposits, remain essentially unchanged during the process of clotting. These cells are responsible for phagocytosis of small foreign agents and encapsulation of large ones. The enzyme prophenoloxidase, contained within their cytoplasmic granules, is a marker for cells with defensive capabilities (Hose et al., 1987). Hyaline cells are most abundant in crustaceans with type C coagulation ( > 50%) and least abundant in crustaceans with type A coagulation (<30%). The amount of clotted material produced reflects the

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proportion of hyaline cells in the hemolymph. The morphological events of clotting have been most thoroughly studied in the ridgeback prawn, Sicyonia ingentis (Omori et al., 1989), but several key questions remain unanswered regarding the interplay between hemocyte constituents and plasma proteins in the process of hemolymph gelation. The purpose of this paper is: (1) describe the effect of anticoagulants on preventing gelation of shrimp hemolymph; (2) characterize the plasma coagulogen in shrimp and compare it with coagulogen from other decapods; (3) examine transglutaminase activity in the plasma and hemocytes of shrimp throughout the molt cycle; and (4) discuss the role of hyaline hemocytes in the coagulation of crustacean hemolymph. MATERIALS AND METHODS

Animals Ridgeback prawns, Sicyonia ingentis, were collected by otter trawls in 60 m of water off the Palos Verdes Peninsula near Los Angeles, California. Spiny lobsters (Panulirus interruptus) and sheep crabs (Loxorhynchus grandis) were collected by divers in shallow water. The Maine lobster (Homarus americanus) was purchased at local fish markets and crayfish (Procambarus clarkii) were obtained from Carolina Biological Supply Co. Crayfish were kept in freshwater aquaria and used within 2 weeks of purchase. The marine species were maintained in recirculating aquaria at 18°C. Molt stages of the shrimp were determined according to criteria described by Anderson (1985).

Anticoagulant tests Equal volumes (usually 0.1 ml) of hemolymph and test anticoagulant solutions were gently mixed in 2 ml plastic microfuge tubes at room temperature and examined at 2min intervals for a total of 10min. Any hemolymph coagulation was noted. If a test solution remained fluid after 10min, an excess amount of fixative (3% glutaraldehyde containing 0.1 M sodium cacodylate pH 7.6 and 12% glucose) was added and mixed with the contents of the tube. The tube was centrifuged (9000 g for 1 min), and the pellet was rinsed for 10 min in 0.i M sodium cacodylate containing 24% sucrose, and post-fixed in 1% OsO4 in 0.1 M sodium cacodylate at room temperature for 1 hr. Pellets were dehydrated in ethanol and embedded in Spurr's (1969) plastic. Thin (90 nm) sections were stained with uranyl acetate and lead citrate and viewed in a Zeiss EM 109 transmission electron microscope. Anticoagulant solutions tested included many previously described in the literature: 12.5% sodium citrate in distilled water (Stutman and Dolliver, 1968), 10% potassium oxalate in distilled water, saturated solution of sodium heparin in distilled water, artificial seawater substituting strontium chloride for calcium chloride (Vella and Tripp, 1983), EDTA-citric acid solution as described by S6derh/ill and Smith (1983), 5mM histamine in distilled water and 5 mM serotonin in distilled water (Lorand et al., 1964), dansylcadaverine (Lorand and Conrad, 1984), 0.2M N-ethylmaleimide in 3% NaC1 (NEM), and Sicyonia culture medium (Brody and Chang, 1989).

Electrophoresis Hemolymph (0.3 ml) was mixed with 0.7 ml NEM and the hemocytes immediately pelleted by centrifugation (1 min at 9000g), leaving the supernatant as the plasma sample. The cell pellet was washed twice with NEM, homogenized by two freeze-thawing cycles in 1 ml of distilled water, and centrifuged (5 min at 12,000g). The resulting supematant was the cell (hemocyte) lysate. An additional 0.3 ml of hemolymph was removed from each shrimp and allowed to clot in a microfuge tube. After 5 min, the clotted sample was

centrifuged (5 min at 12,000g) and the serum sample was diluted 6-fold with NEM. Polyacrylamide gel electrophoresis was performed on plasma, serum and cell lysates according to the procedures of Laemmli (1970) using 6% polyacrylamide gels for denatured proteins and 4% gels for native proteins. Gels were stained with Coomassie Brilliant Blue.

Transglutaminase activity Transglutaminase activity was assessed colormetrically using a commercial kit (Sigma Chem. Co. No. 418). A small sample (0.2 ml) of hemolymph was drawn into a syringe containing 0.2 ml of NEM. A drop was added to a brightline hemocytometer and a total hemocyte count (number of hemocytes/ml of hemolymph) was determined. The remaining sample was centrifuged at 12,000 rpm for 10min at 0°C to separate the plasma and cell fractions. The plasma sample was removed and stored at -20°C. The hemocyte pellet was washed twice in a solution containing 0.4 M CaC12 and 20 mM NaC1. The cells were resuspended in this solution and then lysed by rapid freezing and thawing twice. The samples were then assayed following standard instructions and the activity (in units/1 according to Sigma brochure) recorded using a Hewlett-Packard Vectra C5 spectrophotometer at 405 nm.

Cell separation Metrizoic acid (Sigma M4762) was used to separate hemocytes into two populations. Hemolymph (2 ml pooled from five shrimp) was mixed with 5 ml of NEM. A small sample was removed to obtain a total hemocyte count. The remaining sample was placed in a 15 ml disposable centrifuge tube containing the following layers of metrizoic acid diluted with NEM: 1 ml of 40%, 1 ml of 37.5%, and 1 ml of 35%. The tube was then centrifuged at room temperature at 600 rpm for 10min followed by 1000 rpm for 10min. Cell layers were removed using micropipettes and differential counts were performed. In addition, the two layers were assayed for transglutaminase (described above) and prophenoloxidase activity (see Srderh/ill and Smith, 1983), recorded as change in absorbance/min, and examined at the light and transmission electron microscope levels. This experiment was repeated three times and the data analyzed using analysis of variance.

Hemolymph volume Hemolymph volumes were measured using the radioisotope dilution technique (Smith and Dall, 1982). Twentyfive/~1 of t4C-inulin (Sigma) was injected into the ventral sinus of each shrimp and the animals were maintained in 200 ml seawater. After 30 min, a sample of hemolymph was obtained and dilution of the radioisotope was determined and compared with the level of radioactivity in the seawater. RESULTS

Effect of anticoagulants on hernolymph coagulation Solutions of heparin and artificial seawater with SrCI2 replacing CaC12 did not prevent coagulation; the entire test solution clotted so the hemocytes could not be examined morphologically. Sodium citrate, potassium oxalate, and E D T A - c i t r a t e prevented gelation of the test solution, but sections through pelleted hemocytes showed that numerous small areas of clot material had formed between the packed hemocytes. Sodium citrate is the preferred anticoagulant in this group because it produced the smallest amount of coagulated hemolymph (Fig. 1) and did not cause the precipitation of grainy material between the packed hemocytes that is seen with potassium oxalate. With all these anticoagulants, lysed hyaline

Coagulation in decapod crustaceans cells were often seen in the center of the coagulated areas. Granulocytes and non-lysed hyaline cells showed filopodia, swelling of the nuclear envelope and rough endoplasmic reticulum (RER), cytoplasmic vacuoles suggesting exocytosis of granules, and homogeneous nuclei with no separation into hetero- and euchromatin.

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Histamine, serotonin and dansylcadaverine prevented hemolymph coagulation but examination of the pellets showed that nearly all of the hemocytes had lysed (Fig. 2). The few cells that remained intact showed degenerative changes; condensation of the nuclear material, swelling of organelles and leaching of the cytoplasm.

Figs 1-4. Light micrographs of sections through hemocytes (H, hyaline; SG, small granule; LG, large granule) treated with the following anticoagulants: Fig. 1. Sodium citrate. Note the homogeneous nucleus (HN), vacuoles (V) and dilated RER (arrow). Not all hemocytes can be classified. Fig. 2. Dansylcadaverine. Note the material from lysed ceils (M), irregular nuclei with dense chromatin around the nuclear envelope (arrow) and hemocytes with leached cytoplasm (C). Fig. 3. N-Ethyl maleimide. Hemocytes appear the same as when fixed immediately after removal from shrimp. Fig. 4. Sicyonia culture medium. The hemocytes are well preserved but are more densely packed than in other preparations and hemocytes have irregular shapes. All figures x 3300.

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A

B

C

D

E

F

G

H

205 kDa~

116 kDa ~

--

97.4 kDa 6 6 kDG

Fig. 5. S D S ~ A G E of hemolymph samples. Lane A contains molecular mass standards (from top to bottom: myosin, B-galactosidase, phosphorylase B and bovine albumin). Lane B contains shrimp plasma (note hemocyanin, HCN; coagulogen, C and two associated bands, arrows). Lane C contains shrimp serum showing hemocyanin band and loss of coagulogen and top thinner associated band. Lane D contains shrimp hemocyte lysate. Note hemocyanin band no longer overloaded and coagulogen is absent. Lanes E - H contain plasma samples from Panulirus interruptus, Homarus americanus, Loxorhynchus grandis and Procambarus clarkii.

When hemolymph was collected in NEM, less than 1% of the hyaline hemocytes showed signs of cytolysis. The cytoplasm of all hemocytes appeared dense with good preservation of organelle morphology (Fig. 3). Hemocytes retained the smooth outlines typical of circulating cells and were indistinguishable from hemocytes fixed immediately upon removal from the body as described previously (Martin and Graves, 1985; Martin et al., 1987; Omori et al., 1989). Sicyonia culture medium (SCM) was also an effective anticoagulant. Cell pellets showed very few lysed hyaline hemocytes or regions of coagulated hemolymph (Fig. 4). However, the normal morphology of circulating cells was lost; hemocytes were packed more densely than in other preparations and they possessed numerous extensions of the plasma membrane which interdigitated with adjacent cells. S D S - P A G E of plasma, serum and cell lysates Figure 5 shows a typical SDS-PAGE gel. Coaguiogen (molecular mass ~180 kDa) was the second largest band in the plasma sample next to hemocyanin, and it was nearly absent in the serum.

In plasma samples, two bands (molecular mass 167 and 174 kDa) were routinely seen below the coagulogen. The heavier band was not seen in the serum samples. In cell lysates, an extremely faint band was sometimes seen at the location of coagulogen band and was interpreted as the result of incomplete washing. The pattern of major proteins shown in Fig. 5 was seen in the plasma, serum, and cell lysate throughout the molt cycle. This pattern of hemocyanin, coagulogen and the two thin bands beneath the coagulogen was also seen in four other decapods (Fig. 5). Gels of plasma samples not subjected to denaturing conditions showed the molecular mass of native coagulogen to be approximately 400 kDa, i.e. twice the value of the reduced form. Transglutaminase activity Transglutaminase activity was detected in hemocyte lysate and plasma fractions of shrimp hemolymph throughout the molt cycle (Table 1). Activity averaged 2.2 times higher in the hemocyte lysate than the plasma samples. Transglutaminase activity in both the cell lysate and the plasma was low in

Table 1. Transglutaminase activity (TGA), hemocyte counts and hemolymph volume throughout the molt cycle Molt stage Plasma TGA (units/l) Cell lysate TGA (units/l) Lysate TGA/plasma TGA Total hemocyte count (THC) (cells/ml) Lysate TGA/THC × 100 Hemolymph volume/weight of shrimp

AI

A2

B

C

Do

D14

1.01 +0.56 1.11 +0.51 1.92 + 1.01 2.46 + 1.81 1.90 2.21 5374 + 3522 5908+ 2702

1.31 +0.21 2.61 + 1.40 3.05 + 2.26 3.77 + 1.49 2.39 1.44 9196+ 5724 10,027+ 4249

1.85+_0.49 1.15+0.39 3.14 + 2.38 3.35 + 2.38 1.69 2.93 7843+- 5698 10,485+ 7193

0.357 0.416 30.98 _-4-6.01 31.51 _4-7.22

0.349 27.04+ 3.51

0.399 28.78+- 8.44

0.376 32.29+ 9.34

0.320 28.56+ 7.56

All values, except ratios, are given as means + SD; n = 8 for all values except hemolymph volume where n = 10.

Dr~ 0.94+0.81 2.68 + 2.48 2.86 7273+ 3893 0.368 26.51 +- 5.64

Coagulation in decapod crustaceans molt stage A1, peaked in stages C, and then declined by stage D3_4. When the activity in the cell lysates was standardized by dividing the cell lysate activity by the number of hemocytes in the hemolymph sample, there was no significant difference in transglutaminase activity throughout the molt cycle. These values are not affected by changes in the hemolymph volume throughout the molt cycle because although our study showed changes in hemolymph volume throughout the cycle (Table 1), the changes were not statistically significant (P < 0.05).

Localization of transglutaminase activity In three experiments, 2ml of hemolymph was collected from five shrimps and pooled. The average differential hemocyte count from these experiments was as follows: hyaline cells 56.24%_ 3.40, small granule hemocytes 34.38% _ 3.34, and large granule hemocytes 9.38% __+3.99. Differential centrifugation of the hemolymph pooled for each experiment resulted in two distinct bands. Combining data from the three experiments yielded the following differential counts for the (A) top band: hyaline cells 71.02%_ 3.95, small granule hemocytes 26.68% + 3.60, and large granule hemocytes 2.30% + 0.57, and (B) bottom layer: hyaline cells 17.7% +3.05, small granule hemocytes 49.02% + 6.07, and large granule hemocytes 33.28%_ 4.40. Enzyme assays performed on the separated cell layers showed that transglutaminase activity was 7.95 times higher in the upper layer, whereas prophenoloxidase activity was 3-4 times higher in the bottom layer (Table 2). DISCUSSION Results of this study on shrimp (Sicyonia ingentis), crab (Loxorhynchus grandis), and crayfish (Procambarus clarkii), support previous work on lobsters (Panulirus interruptus and Homarus americanus) (Fuller and Doolittle, 1971; Doolittle and Fuller, 1972; Durliat and Vranckx, 1981) that coagulogen is a plasma protein with a molecular mass of ~400 kDa composed of two identical subunits with molecular masses of 180-200kDa. Plasma coagulogen was not detected in cell lysates from shrimp when the hemocyte pellets were adequately washed prior to homogenization. The site of synthesis of plasma coagulogen is currently unresolved but because it was not detected by SDS--PAGE in hemocyte lysates, it is unlikely that the hemocytes produce this plasma protein. A second form of coagulogen has been described within hemocytes, yet it is not abundant, has a lower molecular mass ( ~ 70 kDa) than plasma coagulogen, and its function is not clear (Madaras et al., 1981; Durliat, 1985; Durliat and Vranckx, 1989). Various anticoagulants have been used to prevent or at least delay the rapid clotting of decapod hemolymph. Calcium chelators such as sodium citrate, potassium oxalate and EDTA solutions have been used in the majority of previous studies on hemocyte function. Our results demonstrate that these solutions are not completely effective and significant hemocyte lysis may occur, resulting in formation of small regions of coagulated hemolymph. Therefore, these anticoagulants should not be used in biochemical

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Table 2. Enzymeactivityin two populationsof hemocytesseparated by differentialcentrifugation(data combinedfrom fiveexperiments) Top layer Bottomlayer Transglutaminase activity(units/l) 1.51 +_0.16 0.19 +0.07 Phenoloxidase activity(AA/min)* 1.43 + 0.82 4.38 + 1.20 *Change in absorbance(at 490 nm) per min. studies attempting to localize proteins in either the hemocyte or plasma fractions of the hemolymph. Some of these solutions, such as EDTA combined with citrate and the substitution of strontium for calcium in artificial seawater (Vella and Tripp, 1983), appear more effective with some crustaceans such as crabs (S6derh/ill and Smith, 1983) than they are with shrimp (this paper). It is important to note that crabs tend to have the smallest proportion of circulating hyaline hemocytes and exhibit type A coagulation characterized not by massive clotting of the hemolymph proteins but by aggregation of hemocytes. A second category of anticoagulants, including serotonin, histamine and dansylcadaverine, do not prevent lysis of hemocytes but rather the crosslinking activity of transglutaminase liberated from the hemocytes (Lorand et al., 1964). The two anticoagulants that most effectively prevent lysis of shrimp hemocytes are NEM and Sicyonia culture medium. This culture medium maintains hemocytes derived from hematopoietic tissue of the lobster (Homarus americanus) viable in culture for several weeks (Brody and Chang, 1989). It is also effective in preventing cytolysis and granule exocytosis in shrimp hemocytes. However, hemocytes in culture medium rapidly lose their smooth surface typical of circulating cells as they adhere to and migrate over adjacent hemocytes. NEM appears to be the most effective anticoagulant for shrimp bemocytes in morphological and biochemical studies. Cell lysis is minimal and the morphology of the hemocytes remains identical to hemocytes in circulation. Lysis of lobster hemocytes releases transglutaminase which cross-links plasma coagulogen and causes coagulation of the hemolymph (Doolittle and Fuller, 1972; Lorand, 1972; Lorand and Conrad, 1984). Our results show that transglutaminase is also contained within shrimp hemocytes and there is no significant change in its abundance throughout the molt cycle. We also found transglutaminase activity in the plasma, although the activity is typically 2.2 times lower than in the hemocyte fraction. Because the hemolymph used in the transglutaminase assays was withdrawn into NEM, the activity in plasma is not thought to be due to cytolysis. The possible role of plasma transglutaminase in hemolymph coagulation requires further study. Which type of hemocyte contains the transglutaminase that is released to initiate coagulation of the hemolymph? Three lines of evidence implicate the hyaline cells. First, hyaline hemocytes are most abundant in crustaceans with type C coagulation, the pattern characterized by massive gelation of hemolymph proteins. Second, analysis of the two populations of hemocytes separated by differential centrifugation showed that transglutaminase activity was enhanced in the upper layer which was enriched in hyaline cells. The bottom layer contained primarily

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granulocytes and the majority of the phenoloxidase activity as previously shown by Srderh/ill and Smith (1983) and Tsing et aL (1989). Third, microscopic observations in shrimp (Omori et al., 1989) and other decapods (Hose et al., 1990) suggest that hyaline cells lyse rapidly (often within 2-5 sec) and release cytoplasmic materials into the hemolymph. In contrast, granulocytes show only minor changes for up to 30 min such as the extention of a small number of filopodia. Further work is necessary to localize the transglutaminase within the hyaline hemocytes, preferably using immunocytochemical techniques, and to understand the interplay of cellular and plasma coagulogen and transglutaminase in decapod hemostasis. Acknowledgements--We would like to thank the crew of the R/V Vantuna for help in collecting the crustaceans. Juan Carlos Becerra, Anna Lee, Olivia Rhee, Garrett Lam and Julia White for their technical help. This work was supported by NSF grant DCB-8502150 to GGM and JEH.

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