Cross induced coagulations between human and crustacean clotting factors. Considerations on the clotting processes

Comp. Biochem. Physid. Printed

in Great

Vol. 72A, No. 4, pp. 741 to 745, 1982

0300-9629/82/080741-05 SO3.00/0 6 1982 Pergamon PressLtd

Britain.

CROSS INDUCED COAGULATIONS BETWEEN HUMAN AND CRUSTACEAN CLOTTING FACTORS. CONSIDERATIONS ON THE CLOTTING PROCESSES W. GHIDALIA, R. VENDRELY, C. MONTMORY and Y. COIRAULT Immunochimie et Strologie des Arthropodes, Laboratoire de Zoologie, Universite Paris VI, 7 Quai Saint Bernard, 75005 Paris, France (Receioed 7 December 1981)

Abstract-l. Cross induced coagulations show that human factor XIII and crustacean coagulin are to some extent functionally equivalent and may be substituted for each other. 2. In crustacea, fibrinogen and coagulin appear as in situ activated products since they are both able to react with non-activated human clotting factors. 3. The coagulin catalyzed transamidation which stabilizes the clot and renders it insoluble in l-5% monochloroacetic acid solutions seems to be the basic reaction of the clotting process in the animals in which coagulation occurs. 4. The possibility of a two step clotting in crustacea is discussed.

1NTRODUffION

blood cell extract is able to clot a solution of fibrinogen. This is due to the presence in this extract of an enzyme called coagulin, acting as a transglutaminase (Fuller & Doolittle, 1971). This enzyme appears to be non specific since that of Macropipus puber, for instance, initiates clotting of fibrinogen from several other species of decapods (Ghidalia et al., 1981). Attempts have been made to establish whether this lack of specificity observed for coagulins from various genera and families of the class crustatea may also be encountered at a higher systematic level, namely that of phyla. In vertebrate coagulation, a transglutaminase termed factor XIII is known to operate. Consequently, the functional equivalence of human factor XIII and crustacean coagulin towards human and crustacean fibrinogens has been tested through cross-induced clotting experiments. Among crustacea,

MATERIALS AND METHODS Preparation of the reagents Crustacea_/%rinogen. This was prepared according to the procedure of Zuch (1969). Hemolymph was taken from crabs (Macropipus puber (L.)) of both sexes. Blood was withdrawn from the sinus at the base of the pereiopods. After rapid centrifugation at low speed (6,000 rpm for 5 min) the blood cells were put aside for further extraction of the clotting enzyme. The supernatant plasma was heated at 55°C for 0.5 hr to inactivate enzyme possibly freed during withdrawal and centrifugation. Plasma was then diluted ten times with cooled distilled water and the whole adjusted to pH 5.3 with l/6 N acetic acid. The precipitate collected after centrifugation (J.B. 21 Beckmann; 20,000 rpm for 0.25 hr) was dissolved into a volume of borate buffer (0.7% NaCl, 0.25% sodium borate) equal to 1/7th of the initial volume. &a&in. Blood cells previously separated from 10 ml of plasma were rapidly washed three times with saline and the

pellet crushed into 400 ~1 of saline. After 0.5 hr of contact, a second extraction of the pellet was performed under the same condi!ions: 400~1, 0.5 hr of contact. The two supernatants were collected after centrifugation at 17,500 rpm for 6 min in an Eppendorf microcentrifuge 3200. The solution of clotting enzyme was kept in liquid nitrogen. Human factor XIII. Dr Lafav from Hoechst-Behring Laboratories kindly provided Fidrogammine, a lyophilizei factor XIII extracted from human placenta. The contents of one flask, corresponding to a fibrin-stabilizing activity of 250 ml of fresh human plasma, was dissolved into 2.0 ml of saline. Factor XIII-free human jbrinogen solution (3 g/l). (Rapid test reagent for clotting factor XIII, Hoechst-Behring TXS.) The flask contents were reconstituted with 5 ml of diethylbarbiturate-acetate buffer pH 7.6 (Hoechst-Behring RHW) and incubated at 37°C for 5 min. Ca-ihrombin solution (for crustacean fibrinogen clotting). The contents of one flask (Hoeschst-Behring RHI 30 UI) was dissolved into 1 ml of calcium chloride, 0.025 mol/l (Hoechst-Behring RHO). Ca-thrombin-kaolin solution (for human fibrinogen clotting). The contents of one flask of thrombin reagent (Hoechst-Behring RHI 30 UI) were reconstituted with 1 ml of calcium chloride 0.025 mol/l and mixed with 1 ml of kaolin suspension (5 g/l in 5 g NaCI/I saline solution). Factor XIII activation. 80 ~1 of fibrogammine and 50 ~1 of Ca-thrombin solution were mixed together and incubated at 37°C for 10 min before addition of the fibrinogen solution. In some experiments fibrinogen was added before incubation, activation is then carried out in contact with it. Electrophoretical analysis

Electrophoreses were performed using cellulose acetate strips (17.5 x 2.5 cm Cellogel) and borate buffer (0.05 M sodium tetraborate solution adjusted to pH 9 with hydrochloric acid). They were run at 150 V, 2 mA per strip for 1 hr at room temperature. Staining, by Amido-black, and destaining were performed as per usual. Strips were made transparent with a mixture of diacetone alcohol (5 ml), methanol (75 ml) and acetic acid (20 ml). Electrophoretograms examined under white light were scanned with a TRD 5 Vernon spcctrophotometer.

741

Table

1. Human

factor

XIII as coagulin

substitute

Test tube number -Reagents

1

2

3*

4+

s

Crustacean fibrinogen Coagulin Human factor XIII CaCl, saturated solution Thl-ombin Clotting Clot stabilization * Activation t Activation

of human of human

factor factor

XIII performed XIII performed

RESULTS

Two sets of experiments were carried out simultaneously to test the functional equivalence of human factor XIII and crustacean coagulin. Addition of human factor XIII (fibrogammine), in definite proportions (Table 1). at room temperature, to a solution of fibrinogen from Mncropipus puber induces coagulation. Electrophoretical analysis of the reagents before and after they have been mixed shows that clotting causes the disappearance of a fraction corresponding to fibrinogen, as happens when one uses M. puher coagulin (Vendrety et al., 1977), while that of fibrogammine appears as lessened (Fig. 1). Clotting times range from 0.5 hr to several hours, clot stabilization occurring in less than twelve hours

before addition of fibrinogen. in contact with fibrinogen.

(Fig. 2). Differences between the recorded clotting times may be ascribed to two factors, at least: (a) Fibrinogen content of the solutions-A value which is still difficult to determine accurately, since there is at present no method both swift and sure to estimate this in the case of crustacea. (b) Factor XIII ageing--During our experiments. the contents of each flask of lyophilized fibrogammine were used two or three times, owing to the small amounts of product we had. In between successive uses, the open flask, carefully recorked, was kept at 4°C in a refrigerator. Despite these cares, it was observed that clotting times increased in proportion to time after unstoppering of the flask. Thus, in M. puber, as is likely in other crustacea, human factor XIII is able to trigger the fibrinogenfibrin conversion--at temperatures lower than that of

Fig. 1. Electrophoretograms and scans of: (A) (-----). a solution of crustacean fibrinogen; (B) ( ---), solution of fibrogammine (human factor XIII); (C), both solutions after coagulation; (D), both solutions after coagulation (activation of human factor XIII performed before addition of fibrinogen),

a

Human and crustacean clotting factors

743

Fig. 2. Human factor XIII as coagulin substitute (cf Table 1 for the contents of the test tubes).

the human body-without having been converted into fibrinoligase, its thrombin-activated form, which is the only effective one for stabilization of a vertebrate clot. This feature suggests that, in crustacea, fibrinogen would be naturally reactive. Previous activation of factor XIII constitutes a favouring condition, since coagulation then occurs more rapidly. Clotting time is longer when factor XIII activation is performed in contact with fibrinogen, but these times are always shorter than those observed for test tubes containing non-activated factor XIII. Coagulin as human factor XIII substitute

In vertebrates, factor XIII acts as a fibrin stabilizer, rapidly rendering fibrin monomer networks insoluble in l-S% monochloroacetic acid solutions. Normal factor XIII-free human fibrinogen, Ca-thrombin kaolin (kaolin allowing a better visualization of the clot) and factor XIII, when mixed together in definite proportions and incubated at 37°C for 10 min, give a slightly yellow coloured clot. Addition of 5% monochloroacetic acid, followed by an incubation of the whole at 37°C for 2min after the clot has been removed from the tube wall by a vigorous shaking is ineffective on the latter. In the absence of factor XIII, on the other hand, the clot is dissolved and only fibrin strands are visible in the test tube. Using coagulin, in place of factor XIII, gives results which depend on the time interval separating formation of the clot from addition of the monochloroacetic acid solution. If acid addition takes place after 2 min, as is standard practice in detecting factor XIII deficiency in man, the clot is dissolved, but if it is done 12hr later, the clot remains undissolved. Coagulin from crustacea thus appears capable of inducing cross-linking of factor XIII-free human fibrinogen monomers, but seems to be less efficient than fibrinoligase since it needs a longer time to trigger transamidation. This functional equivalence, even if only relative, between coagulin and human factor XIII makes possible the evaluation of coagulin levels in crustacean blood cells, either in different species or in the same species at various stages in the intermoult cycle; this

C.&P.(A) 7214-l

by testing its stabilizating ability towards clots proceeding from factor XIII-free human fibrinogen. When coagulin is serially diluted before being added to such a fibrinogen, the resultant clots remain undissolved when in contact with 5% monochloroacetic acid solution above a certain dilution (Table 2). Below this concentration fibrin strands visible in the test tubes point to insufficient amounts of coagulin, which consequently, is unable to stabilize the fibrin monomer network (Fig. 3). Coagulin concentration is thus given by the last undissolved clot of the dilution series and may be estimated by reference to the factor XIII content of normal human plasma, as may be determined according to its stabilizing ability. Under the chosen experimental conditions (Behringwerke “Rapid test reagent for clotting factor XIII”) normal human plasma gave clots insoluble up to a dilution of l/20, the value corresponding to 100% of the norm. Thus, in the experiment of which the results are given as an example (Table 2 and Fig. 2) blood cell extract from M. puber at the C4 stage gives rise to undissolved clots up to a dilution of l/4. Its coagulin concentration is, therefore, 0.2 of the factor XIII content of normal human plasma. Experiments are presently in progress in our laboratory to detect, using this method, possible quantitative variations of coagulin concentration in blood cells during the intermoult cycle, and to see if they may be related to the variation of clot consistency observed through this cycle. DISCUSSION

The reported experiments have shown that coagulin and factor XIII may be substituted for each other, appearing therefore as functionally equivalent, at least to a certain extent, since in both cases clotting times were noticeably longer for the crossed reaction than for the homologous one. This means that mammals share a part of their clotting process with crustacea, the one in which a transglutaminase operates. Clot formation in the two groups may be as outlined below. In vertebrates, a clot results from a two step procedure-First, fibrinogen molecules present in plasma are converted into fibrin monomers through a series

W. GHIINLIA t'rtrl.

744

Table 2. Coagulin

as human

factor

XIII substitute

Test tube number 1

2

3

4

5

15 nl (1121

75 ,ul (1141

75/J (19)

75pl (I:161

75 /(I (132)

15Opl

150 /ll

150/A

is0 1’1

150/A

Reagents Coagulin diluted in F XIII-free human fibrinogen Thrombin-Ca-Kaolin Incubation 5% monochloroacetic acid

10 min at 37’C, then 12 hr at room

1.6 ml

1.6ml Incubation

Clot stabilization

+

as human

factor

1.6 ml

1.6ml

2 min at 37 C +

of hydrolytic reactions leading to the release of two kinds of peptides: a from the c( chain and b from the p chain. These monomers join together in a reversible manner to form an unstable network. In a second step, stabilization of the network occurs through the agency of activated factor XIII (fibrinoligase) which catalyses formation of l-(‘t-glutamyl)lysine bonds between the tl and y chains of fibrin monomers. In crustacea, the process may be simpler in that only the second step, fibrin stabilization through a transglutaminase, exists. The rapidity with which coagulase clots and stabilizes fibrinogen (most of the time within a few seconds, and often almost instantaneously) is in favour of this, suggesting a process involving few reactions, if not a single one. The fact that both coagulin and fibrinogen appear in crustacea as spontaneously reactive products reacting with nonactivated human factors would indicate such a one step process. In the present state of the subject, however, the possibility of coagulin and fibrinogen activation following their extraction should not be ruled out. In this view of a one step process, the transglutaminase reaction appears as phylogenetically earlier than the proteolytic one and would consequently

Fig. 3. Coagulin

1.6 ml

temperature

XIII substitute

the basic process of coagulation. The first stage, which in vertebrates leads to thrombin activation and fibrinopeptide release rendering fibrin monomers reactive, might then correspond to a safety device preventing in situ blood clotting. In vertebrates, factor XIII and fibrinogen are both plasma constituents and the former is a strong contaminant of the latter with which it may constitute a complex. In crustacea, such a preventive reaction is not necessary since coagulase and fibrinogen are not in contact, the former occurring in blood cells and the latter in plasma. Interaction between these two factors happens only when blood is shed, producing blood cell bursting and coagulin release. However, the question is likely to be more involved, for one observation at least does not fit with such a pattern. Very frequently, coagulation needed several minutes to occur. One then noticed that during this interval plasma appeared to form a gel, but even a gentle shake of the test tube rendered it liquid again; clot stabilization only occurred if the tube was left motionless for a longer time. Such a momentary gelification of plasma preceding clot stabilization is detected in the case of species of groups B and C during cross-induced clotting experiments using fibrinoconstitute

(cf: Table 2 for the contents

of the test tubes).

Human and crustacean clotting factors

745

gen and coagulin from different species. In group A from vertebrate fibrinogen are demonstrated as having no common origin, this may no longer be considered species also, in which plasma does not clot, such a as an argument in favour of the above cited opinion. gelification is observed, and constitutes the only clue to clotting processes in these species (Ghidalia et al., Clot stabilization appears then as a complex achieve1977). This may suggest the formation of an unstable ment that has apparently partly concealed, up to now, network before clot stabilization through coagulin; the similarity of the results in the two reactions. Now, fibrinoligase and coagulin being functionally equivalthat is to say, in this case too, a two step procedure, the first one perhaps more rudimentary than in verteent, one may suggest that they ought to work in the brates since it may sometimes occur in less than a few same way, which would imply some structural similarities and, consequently, some phylogenetic relationseconds. ship between vertebrate and crustacean fibrinogens. It has been assumed, from reference to published data concerning the molecular weight, the number of The problem is now to go further into their respective structure, to confirm or invalidate this hypothesis. polypeptide chains and the amino-acid composition, that crustacean and vertebrate fibrinogens derive from independent evolution (Doolittle, 1973). In the Acknowledgement--The authors are greatly indebted to same view, the formation of l-(y-glutamyl)lysine Dr Lafay from Hoechst-Behringwerke Laboratories who kindly provided them with fibrogammine. bonds between polypeptide chains of these fibrinogens during clot stabilization, was not considered as a valid argument in support of a phylogenetic relationship between these two molecules. It has been sugREFERENCES gested that, as regards vertebrate fibrinogen, the two different kinds of polypeptide chain of the same fibrin DCNILI~TLER. F. (1973) Structural aspects of the fibrinogen monomer (the u and y chains) involved in the stabilizto fibrin conversion. Adu. Protein Chem. 27, l-109. FULLER G. M. & DCKUTTLE R. F. (1971) Studies of inverteation process demonstrate this bond, although there brate fibrinogen. II. Transformation of lobster fibrinogen is no evidence that they have a common origin (Fuller into fibrin. Biochemistry 10, 1311-1315. & Doolittle, 1971). GHIDALIA W., VENDRELY R., COIRAULT Y., MON~MORY C. For us, however, these same facts, when considered & PROU-WARTELLE 0. (1977) Coagulation de l’htmofrom another viewpoint, might lead to an opposite lymphe’chez Macropipus puher (L.) CrustacC dCcapode. conclusion. It is well known that fibrin stabilization Mise en kvidence des rsles respectifs do plasma et des in vertebrates proceeds from two distinct processes organites cellulaires dans les mbcanismes de la coaguwhich affect the a and y chains differently, though lation. C.r. hebd. Sbanc. Acad. Sci. Paris 284D, 69-72. carried out by the same enzyme. The first one rapid, GHIDALIA W., VENDRELY R., MONTMORY C., COIRAULT Y. requiring only small amounts of fibrinoligase con& BROUARD M. 0. (1981) Coagulation in decapod Crustacea. Comparative study of the clotting process in specerns the y chains. It causes the formation of y dimers cies from groups A, B and C. J. camp. Physiol. 142, with the y chains from two different monomers of 473-418. fibrin. The second one slower, and using more fibrinoVENDRELY R., GHIDALIA W., COIRALJL~ Y., MONTMORY C., ligase than the preceding reaction, affects the c(chains BROUARD M. 0. & PROU-WARTELLE 0. (1977) Coagufrom several fibrin monomers which together form a: lation de I’himolymphe chez Macropipus puber (L.) multimers. Thus, if fibrinoligase effects on the two Crustaci dtcapode. Mise en kvidence par electrophortse kinds of polypeptide chains lead to the same result, d’un composant plasmatique intervenant dans I’hbmosi.e., the formation of +glutamyl)lysine cross links, tase. C.r. hebd. Sianc. Acad. Sci. Paris 285D, 1069-1072. the way the enzyme operates is markedly different in ZUCH A. (1969) The hemolymph coagulation system in the crayfish (Astacus astacus, L.). Zoologica Pol. 19, 27-45. each case. So, even if in the future, CI and y chains