Calcium and pH dependency in the clotting of the blood of Balanus nubilus (Darwin, 1854)

Comp. Biochem, PhysioL, 1968, Vol. 24, pp. 1055 to 1059. PergamonPr*ss. Printed in Great Britain

SHORT COMMUNICATION CALCIUM AND pH DEPENDENCY IN THE CLOTTING OF THE BLOOD OF B A L A N U S NUBILUS (DARWIN, 1854) ROBERT T. FITZGERALD* Hopkins Marine Station of Stanford University, Pacific Grove, California (Received 19 ~une 1967)

Abstract--1. T h e clotting mechanism of Balanus nubilus was found to be affected by calcium ion with a threshold value for induced clot formation of blood diluted 1 : 3 : 1 with 10% E D T A and 3% NaC1 respectively falling between 0"025 M and 0"1 M calcium chloride. 2. T h e clotting mechanism of B. nub//us was also found to be affected by hydrogen ion concentration with an optimum value for clot formation at a p H value of approximately 6"7.

INTRODUCTION

THE BLOODclot formed in decapod crustaceans was described by Fredericq (1879) and Halliburton (1885) as a jelly-like agglutination, consistingof a cross-hatchingof fibers to which is attached an amorphous, granulated gel. Although Glavind (1948) observed an effect induced by variations in hydrogen ion concentration and calcium ion on the clotting mechanism in some decapod crustaceans, no investigation of the effect of hydrogen ion and calcium ion on the process in cirripeds has been reported. This is a report of a study of the clotting mechanism of Balangs nubil~ (Darwin, 1854) and the effect of calcium ion and hydrogen ion on the process. MATERIALS AND METHODS Specimensof B. nubilu.~were collectedwith their basal calcareousplatesintactfromthe pilings of Fisherman's Wharf, Monterey, California, and kept in running sea water until used. T h e blood was removed by puncturing the basal plate, removing excess debris with forceps and mantle cavity fluid with a water aspirator. Blood was withdrawn from the ventral thoracic sinus using a 1-ml plastic syringe containing 0"4 ml of a 10% solution of ethylenediaminetetraacetic acid disodium salt (EDTA) adjusted to a p H of 7'1 and fitted with a 22-gauge needle coated with Beckman's New Desicote, to minimize contact with wettable surfaces. Generally, 0.6-ml samples of blood were withdrawn and transferred to a glass centrifuge tube containing 10% E D T A and 3% NaC1 so that the final ratio of blood, E D T A and NaC1 was 1 : 3 : 1 respectively. * Permanent address: 4202 W o o d m a n Avenue, Sherman Oaks, California. 1055

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ROBERT T. FITZGERALD

This sample was then centrifuged immediately at approximately 1500 g to remove any particulate matter, including any clot formed during the collection of blood. One-ml aliquots of the supernatent were used in the experimental procedures described below and amounted to a I : 5 dilution of the blood. Acetate and tris (hydroxymethyl) aminomethane (Tris) buffers were prepared according to the method of Gomori (1955); except buffers with a p H from 2.0 to 3"5 were prepared from a mixture of 50 ml of 0.2 M acetic acid (diluted to a total volume of 100 ml) and 5 ml of 0.2 M sodium acetate to which was added glacial acetic acid until the mixture reached the desired pH values. Similarly, buffers in the range from 8"7 to 10"0 were prepared from 50 ml of a 0"2 M Tris solution (diluted to a total volume of 200 ml) to which was added Tris until the desired p H values were reached. T h e amount of clot was estimated in terms of its protein content as measured by the method of Lowry et al. (1951) after dissolving the clot in 2% NaCO3 in 0'1 N NaOH. RESULTS

The effect of calcium ion on clot formation To a 1-ml sample of diluted blood containing EDTA was added an additional ml of 3% NaC1, plus 1 ml of a solution of calcium chloride of molarities ranging from 0.00625 M to 0.5 M. Samples were allowed to stand at room temperature for 6 hr, then centrifuged, the supernatant removed and the clot washed twice in a 3% NaC1 solution. The clot was then dissolved, in 5 ml of a 2% NaCO3 in 0.1 N NaOH solution and 1-ml aliquots were removed for protein analysis. Figure 1 presents the results. Even without the addition of calcium chloride, a small amount of particulate protein was formed. This small amount remained 175 --

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FIo. 1. T h e e f f e c t s o f c a l c i u m i o n o n t h e a m o u n t o f c l o t t e d p r o t e i n p r o d u c e d i n 6hr. Each aliquot of the blood and E D T A mixture represents a 1 : 75 dilution of Balanus blood.

CALCIUMANDpH IN BLOODCLOTTINOOF BAC,~IVUSIVVBILVS

1057

constant until the solution of calcium chloride added exceeded 0.025 M. The addition of levels in excess of this amount induced clot formation.

The effect of hydrogen ion concentration on clot formation To a 1-ml sample of diluted blood containing E D T A was added 1 ml of buffer and 1 ml of 0.2 M calcium chloride solution, an amount sufficient to yield near maximum clot. The procedure follows exactly the protocol used for calcium dependency above. Figure 2 relates pH values to the effectiveness of a 0.2 M calcium chloride solution to induce clot formation. Between pH 2.0 and 4"5 the amount of particulate protein remains at a low level approximately equal to the amount of protein 175

150

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FIG. 2. T h e

effects o f h y d r o g e n i o n c o n c e n t r a t i o n o n t h e a m o u n t o f c l o t t e d

protein produced in 6 hr in the presence of added calcium chloride. Each aliquot of the blood and EDTA mixture represents a 1 : 75 dilution of Balanus blood. obtained with the pre-threshold concentrations of calcium in Fig. 1. At p H values above 4.5 the amount of clot increases steadily to a maximum value at a pH equal to 6"7, then decreases at approximately the same rate, reaching a zero clot value at a pH of 10.0.

Quantitative relationship of clot protein to total blood protein Changes in the soluble protein content of the blood were analyzed as indicated in Fig. 3. An analysis of the first separation (Supernatant I and Sediment I) indicates a total protein content of approximately 35 mg protein/ml of which 1927 per cent was in the form of clot protein formed during the collection of blood,

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ROBERTT. FITZGERALD

plus any proteinaceous substances and any cellular components attached to the clot. Although repeated microscopic examinations of the blood failed to reveal discrete cellular elements, any cellular blood components present in low concentrations would be contained within Sediment I. A further breakdown of Sediment I shows that 66 per cent is in the form of an insoluble protein, while the remaining Blood

5 4 - 5 7 m g protein/mL ~

I

Centrifucjation

Supernatant T 27'- 29mcj protein

Sediment I

Co.8-9-6mcj protein

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I

Washed with EDTA

I

Washed with

I

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Sediment 4-2mcJprotein

Supernatant IT Sediment Z 2 3 - 2 6 mcj protein (clot)

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O'044mcj protein

Sediment 7,8

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Maximum clot protein =12mcj protein/mL (washed sediments "I- +TF ) %clot protein =32-35% mAll protein values expressed in terms of I mL of bolonus blood

FIC. 3. Quantitative relationship of clot protein to total blood protein.

proteins could be washed away by either 10% EDTA or 3% NaCI. The clot formed in Sediment I could not be reversed by treatment with EDTA. No differences in the solvent property of NaC1 and EDTA was observed. This treatment with EDTA corrdates with parallel experiments done on the reversibility of washed Sediment II. In a similar treatment with 3 ml of 3% NaC1 and 3 ml of 10% EDTA on a clot induced by 0.2 M calcium chloride, the values were found to be equal. Treatment with 6 ml and 9 ml of 10% EDTA failed to lessen the protein content significantly. An analysis of Supernatant I shows that 29 per cent of the proteins in this solution are capable of forming clot, and that there is a negligible amount of proteinaceous substances attached to the clot at this point (Sediment I). Finally, the ratio of the total clot protein to the total protein content ranges between 32 and 35 per cent.

CALCIUM AND p H IN BLOOD C L O T T I N G OF BALANU,.g NUBILU8

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DISCUSSION The effect of calcium suggests involvement of this ion in the dotting mechanism, but the direct terms of this involvement will require further investigation. The possible displacement by calcium of some other di- or trivalent ion from a chelated complex cannot be ruled out. The effect of hydrogen ion concentration cannot be explained as the effect of pH on the chelating ability of EDTA. Martell & Calvin (1952) point out that the chelating power of E D T A is zero between a pH of 1"0 and 4"8, accelerating to a maximum at a pH of 7"8, and then remaining at this level. This relationship between pH and chelating power does not explain the high amounts of clotting induced by calcium between a pH of 7"0 and 8.5 where chelation is at a maximum; nor the pH optimum of 6"7 for clotting which is almost at the maximum for chelating power; nor the increasing amounts of d o t formation between a pH of 5.0 and 6"7 which corresponds to a range of rapidly increasing chelating ability; nor the low amounts of clotting observed between a pH of 2.0 and 4"5 where chelating power is essentially zero. Therefore, it appears that there are pHdependent factors inherent in the clotting mechanism. Blood clotting in cirripeds, as studied in B. nubilus, shows similarities to the process as studied in other crustaceans, especially in decapods (Glavind, 1948). ~'lcknowledgement--I am grateful to Dr. John Phillips, whose advice and assistance made this study possible.

REFERENCES DARWINC. H. (1854) A monograph on the sub-class Cirripedia (Balanidae, Verrucidae, etc.). Roy Soc., Lond., p. 30t-. FR~mmmQ L. (1879) Note star le sang de l'homard. Bull. Acad. r. Belg. (S~r. 2) 47, 409-413. GI~VnVD J. (1948) Studies on the Coagulation of Crustacean Blood. Nyt Nordisk Forlag, Arnold Busck, Copenhagen. GOMORIG. (1955) Preparations of buffers used in enzyme studies. In Methods of Enzymolofty (Edited by COLOWICKS. & KAPLA~'~N.). Vol. 1, pp. 138-146. Academic Press, New York. HALLmURTONW. D. (1885) On the blood of decapod Crustacea. ]. Physiol. 6, 300-335. LOWRY,ROSEBROUGH,FARR& RANDALL(1951) Protein measurement with the Folin phenol reagent. J. Biochem. 193, 265-275. MARTELLA. & CALVXNM. (1952) Chemistry of the Metal Chelate Compound. pp. 494-495. Prentice-Hall, New York.