ABS
A0
).
1. The clotting mechanism of Balanus nubilt
was found to be affected by calcium ion with a
threshold value for induced clot formation of blood
i
diluted 1:3:l w
h 10% EDTA and 3% Naol respectively
falling between 0.025M and O.1M calcium chloride.
2. The clotting mechanism of B. nubilus was
also found to be affected by hydrogen ion concen¬
tration with an optimum value for clot formation at
a ph value of approximately 6.7.
TNA
INTRODUCTION
The blood clot formed in decapod crustaceans
was described by Fredericg (1879) and Halliburton
(1665) as a jelly-like aglutination, consisting of
a cross-hatching of fibers to which is attached an
amorphous, granulated gel. Although
avind (1948)
obse
ved an effect induced by variations in hydrogen
ion conent
ation and alium io on the clot
ng mech¬
anism 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 Balanus nubilus (Darwin, 1854) and
the effect of calcium ion and hydrogen ion on the
process.
MATERIALS AND
THODS
Specimens of B. nubilus were collected with

their basal calcareous plates intact from the pliings
of Fisherman's Wharf, Monterey, California, and kept
29
in running sea water until used.
The blood was removed by puncturing the basal
plate, removing excess debris w
ith forceps, and
mantle cavity fluid with a water aspirator. Blood
was withdra
n from the ventral thoracic sinus using

aone ml. plas
i syringe contain
ing 0.4 ml. of a
10 solution of Ethylenediaminetetraacetic acid
disodium salt (EDTA) adjusted to a pH of 7.1 and fitted
with a 22-gauge needle coated with Beckman's
sicote, to minimize contact with wettable su
faces.
Generally, 0.6 ml. samples of blood were withdrawn
rred to a glass centrifuge tube conta:
ining
and transfe:
10% EDTA and 3% Nacl so that the final ratio of blood,
EDTA, and Nacl was 1:3:l respectively.
iis sample was then centrifuged immediately at
approximately 1500xG to remove any
particulate matter,
including any clot formed during the collect
tion of
blood. One ml. aliquots of the supernatant were used
in the eper
ntal procedures described below and
amounted to a l:5 dilution of the blood.
Acetate and tris (hydroxymethyl) a
inomethane
(Tris) buffers were prepared acco
ing to the method
of Gomori (1955); except buffers with a pH from 2.0
rom a mixture of 50 ml. of 0.2M
to 3.5 were preparedi
acetic acid (diluted to a total volume of 100 ml.)
and 5 ml. of 0.2M sodium acetate to which was added
glacial acetic acid until the mixture reached the
desired pH values. Similarly, buffers in the range from
6.7 to 10.0 were prepared from 50 ml. of a 0.2M Tris
solution (diluted to a total volume of 200 ml.) to
which was added Tris u
til the desired pH values were
reached.
56
The amount of clot was estimated in terms of its
protein content as measured by the method of Lowry (1951)
after dissolving the clot in 2% Naco, in O.1N NaoH.
RESULT
he ef?
slect of calcium ion on clot formation
To a one ml. sample of diluted blood containing
2TA was added an additional ml. of 3% Nagl, plus one
ml. of a solution of calcium chloride of molarities
ranging from 0.00625M to O.5M. Samples were allowed to
stand at room temperature for 6 hours, then centrifuged.
the supernatant removed, and the clot washed twice
in a 3% Nacl solution.. The clot was then dissolved
in 5 ml. of a 2% Naco, in O.lN NaoH solution and one
mi. aliquots were removed for protein analysis.
Fig. 1. presents the results. Even without the
addition of calcium chloride, a small amount of par-
ticulate protein was formed. This small amount remained
constant until the solution of calcium chloride added
exceeded 0.025M. The addition of levels in excess of
this amount induced clot formation.
The
fect of hydrogen ion concentration on clot
0
ion
To a one ml. sample of diluted blood containing
EDTA was added one ml. of buffer and one ml. of 0.2M
calcium chloride solution, an amount sufficient to
yield near maximum clot. The procedure follows exactly
the protocol used for calcium dependency above.
Fig. 2. relates pH values to the effectiveness
of a 0.2M calcium chloride solution to induce clot
formation. Between pH 2.0 and 4.5 the amount of
O
particulate protein remains at a low level approx-
lately equal to the amount of protein obtained with
the pre-threshold concentrations of calcium in Fig. 1.
At pil values above 4.5 the amount of clot increases
steadily to a maximum value at a pH equal to 6.7.
hen decreases at approximately the same rate, reaching
a zero clot value at a pH of 10.0.
relationship of clot
protein to
Sreod
Changes in the soluble protein content of the
blood were analyged as indicated in Fig. 3. An anal-
ysis of the first separation (Supe
rnatant I and Sediment
1) indicates a total protein content of approxi
mately
35
ng. protein/ ml. of which 19% to 27% was in the form
of clot protein formed during the collection of blood.
lus any
proteinaceous substances and any cellular
mponents attached to the clot. Although repeated
microscopic examinations of the blood failed to reveal
discreet cellular elements, any cellular blood com¬
ponents present in low concentrations would be contained
thin Sediment I. A further breakdown of Sedi
ent I
shows that 65% is in the form of an insoluble protein.
while the remaining proteins could be washed away by
either 103 EDTA or 3% Nacl. The clot formed in Sedi-
ment I could not be reversed by treatment with EDTA.
No di
erences in the solvent property of Nagl and
WDTA was observed. This treatment with
TA correlates
with parallel experi.
ments done on the reversibilit
of washed Sediment II. In as
ilar treatment y
3 ml. of 3% Nacl and 3 ml. of 10% EDTA on a clot in¬
duced by 0.2M calcium chloride, the values were found
to be equal. Treatment with 6 ml. and 9 ml. of 10%
32
0
D
failed to lessen the protein content si
nificantly.
n analysis o
Super
natant I shows that 29% of the
proteins in this solution are capable of
ming clot.
and that there is a negl:
jible amount of proteinaceous
substances attached to the clot at this point (Sediment
—
nally,t
tio of the
1).
votal clo protein
th to
oolten ren
OT.
een 32% and 35%.


D
SION
The effect of e
eleium sug
os involvement of
this ion in the clotting
mechanism, but the di
terms
i
oft
involvement w
vill require fu
ther investigation.
The possible displacemen
it by calcium of some othe:
dio:
r trivalent ion from a chelated complex canno¬
be ruled ou
The effect of hy
drogen ion concentration canno
e eo
ned as the effet of phont
he chelat


ablll
of
4. Mal
riel and Calvin (1952) point out
th
the chelating
power of EDTA is zero between a


phof 1.0 and 4.8, acceler
maximum at a
ating to a
of 7.8, and then remaining at this level.
his
relationship between pH and chelat
power does not
explain the high amounts of clottis
induced by calciu
between a ph of 7.0 and 8.5 where chelation is at a
um; nor the pH optimum of 6.7 for clott
which
is almost at the m
mum for chelating power; nor the
reasing amounts of clot fo
ation beween a ph of
5.0 and 6.7 which corresponds to a range of
aidly
increusing chelating ability; nor the low amounts of
lot
ig observed between a pH of 2.0 and 4.5 where
27-
chelatir
agower is esse
tially zero. The
efore, it
38
O
appoars, that there are pi-dependent factors inherent
in the clotting
mechanism.
Mlood clotting in cirripeds, as studied in 2.
nubilus, shows similarities to the process as
studied in other crustaceans, especially in decapods
(Glavind, 1948).


ACKNOWLED
GEMENT
Dhil7
I am grateful to Dr. John Filllips, whose advice
and assistance made this study possible.
.
O


O

C
+
S
t



9

S
.
12-282
8
C
O
2
O 0
8
O
8
0
O

59.
12-282





20 Squares to the Inch



Ste
I
O1
The
s
falum
ion
lotted
rouce
6 hour
rotein
ure
preser
the blood and ED
TA
i
uion of
Balanus bloo
7 -
igure2
he effet.
ion

of
ogen
amount of clotted
podu
ed:
ot
nthe
sence of added calci
e
aliquot of the blood a
mitu

5 dilution of

lood.

++-
f clot
anvere
lationshi
o total blood protein.
unt
aliqu
Wach
sal
a
6 h
a
orot
C
R
ERENCES
Darwin, C.H. 1854 A Monograph on the sub-class
Cirripe
Verrucidae,etc). Ray
lia (Balanidae
Society, London, p. 307.
Fredericg, L. (187
Note sur le sang de l'Homar
Sull. acad. roy. Bel
ique (ser. 2) 47: 409-413.
Glavind, J. (1948) Studies on the coagulation of
tacean blood. 137pp. Nyt Nordisk Forlag,
Cry
Arnold Busck, Copenhagen.
Gomori, G. (1955) Preparations of buffers used in
f Enzymology,
enzyme studies, cited in Methods
Vol. 1 ed. by Colowick, S. and Kaplan, ..
Academic Press, Inc. New York, pp. 138-146.
Halliburton, W.D. (1885) On the blood of decapod
Crustacea. J.
ysiol., 6: 300-335.
Lowry, Rosebrough, Farr, and Randall (1951) Protein
measurement with the Folin Phenol Reagent. J.
Biochem. 193: 265-275.
Martell, A. and Calvin, M. (1952) Chemis
of the

Metal Chelate Compound pp. 494-495. Prentice-
Hall, Inc, New lork.