IXFLCENCE OF ELECTROLYTES ON T H E SYSERESIS AND CLOTTING O F BLOOD B Y S.4TYA P R A K A S H AND N . R. DHAR
I n a previous communication,' we have advanced the view that clotting of blood and jelly formation are essentially similar processes. Blood may be regarded as an unstable colloidal system which remains fluid in the body partly due to its motion and partly to the capillary action of the blood vessels. We2 have investigated the influence of various electrolytes on the time of setting of jellies as well as on the extent of syneresis with numerous inorganic and some organic jellies. hloreover we have shown that a sol is stabilised by the adsorption of similarly charged ions and is sensitised by the oppositely charged ions, and under similar conditions, the uncharged particles are more hydrated than the charged ones. By the adsorption of similarly charged ions, the charge on the particles increases and the system becomes more stable, less viscous, and less hydrated. Blood is regarded as a negatively charged fibrin sol, which exists in the liquid condition in some mysterious way. When it is collected in a glass vessel, it spontaneoiisly forms a solid clot within a few minutes, but it is wellknown that if it is received in solutions of sodium, or ammonium oxalate, fluoride or citrate, it can remain liquid indefinitely, and further if calcium chloride is added in excess to the oxalated or citrated blood, the clotting occurs normally. As normal blood also contains calcium, this behaviour was ascribed to the formation of insoluble or undissociated calcium salts of citrated and oxalated blood and thus to the removal of calcium ions. We have shown in the previous communication that blood has a great tendency to adsorb similarly charged ions from such electrolytes as sodium acetate, tartrate and citrates, and also potassium fluoride and oxalate, and the stabilising influence of the salt is due to this fact and not to the removal of calcium ions. I n the present communication, we have investigated the influence of the concentration of electrolytes on the extent of syneresis and have shown that blood is markedly stabilised by the addition of calcium chloride. For these experiments on the syneresis of blood-clot, goat's blood was received in 250 C.C.glass bottles containing different amounts of electrolytes and made up to a definite volume. A blank experiment was always performed with the same blood, as the blood from different animals behaves slightly differently. After definite intervals, synerised serum was carefully collected in a graduated measuring cylinder and the volume was measured. The experiments were carried a t the room temperature (27°-290). The results are recorded in the following tables: J. Phys. Chem., 33, 459 (1929). J. Indian. Chem. SOC.,6, 587 (1929); 7 (1930).
630
SATYA PRAKASH A N D N. R . DHAR
TABLE I Influence of Dilution of Syneresis Amount of syneresis blood 230 C.C. blood 20 C.C. water
Time
2 j o C.C.
30 minutes I hour I hour 30 minutes 2 hours 30 minutes 3 hours 30 minutes 4 hours 30 minutes 5 hours 30 minutes I 7 hours z z hours
+
28
9 C.C.
30
C.C.
58 77 99
47
67 83 93 98
I22
127
151
129
141
I12
I
18
The serum when no water was mixed was straw-coloured, but in the presence of water, blood wsbs hemolysed and the serum was dark red.
TABLE I1 Influence of Potassium Chloride on Syneresis 5 c.c., I O C.C. and 15 C.C. of 3 3 potassium chloride made up to 20 C.C. were taken into bottles in which 230 C.C. blood were received. In the blank bottle, 20 C.C. of water and 230 C.C. of blood were taken. Time
Amount of syneresis
KCl
0
5
30 min.
IO C.C.
16
hr. hr. 30 min. z hr. 3 0 min. 3 hr. 30 min. 4 hr. 30 min. 5 hr. 30 min. 2 1 hr.
38
30
I
I
C.C. C.C.
IO C.C.
15 C.C.
2 C.C.
0.5 C.C.
5 5 7
I
3
7.5
4.5
50
48
67 83 93
63 74 81
a
98
83
8
I28
127
8
.o
4 5 7 8
Hemolysis was checked in the presence of potassium chloride and the serum was straw coloured.
63 1
SYNERESIS AND CLOTTING O F BLOOD
TABLEI11 Influence of Calcium Chloride on Syneresis Concentration of calcium chloride = 1.76 M Total volume = Blood = 230 C.C. Time CaCI2
5
0
min. I hr. I hr. 3 0 min. 2 hr. 30 min. 3 hr. 3 0 min. 4 hr. 3 0 min. j hr. 30 min. 13 hr. 3 0 min. 2 1 hr. 3 0 min. 48 hrs.
2 C.C.
30
Amount of syneresis 6
C.C.
7
C.C.
0
21
0.2
0
51
0 . 2
0.1
73
0.2
0.1
0
86
0.2
0.1
0
92 92
0.7
0.1
0
I
.o
0.1
0
108
2
.o
I IO
7 .O -
-
8
C.C.
not set just set
0 . 2 C.C.
I
2 5 0 C.C.
0
.o
0.5
-
-
TABLE IV Influence of Ammonium Sulphate on Syneresis Concentration of ammonium sulphate = 2h4 Blood = 230 C.C. Total volume = 250
C.C.
not set not set not set not set not set not set not set just set 3
C.C.
C.C.
Amount of syneresis
Time AmzSOd
j C.C.
8
29
2 C.C.
0
min. I hr. I hr. 30 min. 2 hr. 3 hr. 3 0 min. 3 hr. 30 min. 5 hr. 3 0 min. 13 hrs. I ; hrs.
28 59 70
30
C.C.
C.C.
56
15
71
17 64 83
81
82
IO2
103
I02
I08
I 1 C.C.
C.C.
not set just set 2
8 27
36
I08
113
92 93
119
I28
117
52
I22
I28
I18
55
41
TABLE V Influence of Potassium Oxalate on Syneresis Concentration of potassium oxalate = N Total volume = 250 C . C . Blood = 230 C.C. Time Potassium oxalate
min. I hr. I hr. 3 0 min. 2 hr. 3 hr. 30 min. 4 hr. 3 0 min. 5 hr. 3 0 min. 30
Amount of syneresis 2 C.C.
3
IO C.C.
IO C . C .
not set
57 85 96
57
2 C.C.
68 70
4
5
not set half set half set just set
- I
/3
8
2 C.C.
I18
i8
I2
4
121
78 5
I3
5
0
115
4 C.C.
C.C.
SATYA PRAK.4SH AND S . R. DHhR
632
TABLE VI Influence of Sodium Acetate on Syneresis Concentration of sodium acetate = IK Blood = 230 C.C. Total volume = 250 30 min. I hr. I hr. 30 min. 3 hr. 30 min. 4 hr. 30 min. 5 hr. 30 min. 2 0 hrs.
4
2 C.C.
0
C.C.
6
C.C.
32 C.C. 66
C.C.
39 62
2 0 C.C.
30
31 66
56 75
84
78
IO0
105
1x5
I12
I08
I08
I I8
117
115
111
I22
I21
130
I11
I35
I35
C.C.
VI1
T.4BLE
Influence of Sodium Tartrate on Syneresis Concentration of sodium tartrate = N Blood = 230 C . C . Total volume = 2 5 0
C.C.
Amount of syneresis
Time Sodium tartrate
30 min. I hr. I hr. 30 min. 2 hr. 2 hr. 30 min. 3 hr. 30 min. 4 hr. 30 min. 16 hrs.
C.C.
Amount of syneresis
Time Sodium acetate
0
6 64 77
4 C.C.
6 c.c.
42 C.C.
32 52
77
67 90
84
a6
2 C.C.
34
C.C.
C.C.
62
82
92 106
9.3
94
102
107
111
I Oj
113
I20
r 20
12.5
C.C.
66 74 80
9' 93 '13
TABLE VI11 Influence of Sodium Citrate on Syneresis Concentration of sodium citrate = N Blood = 230 C.C. Total volume = 2 5 0 C.C. Time Sodium citrate
30 min. I hr. I hr. 30 min. 2 hr. 30 min. 3 hr. 30 min. 4 hr. 30 min. 14 hrs.
2
0
26
C.C.
Amount of syneresis c.c 4 C.C.
,!I
61
62
86
io 91
109 I20
113
I21
I
-
16
-.
C.C.
half half half half just
set set set set set
0
IO C.C.
6 C.C.
not set not set not set not set not set not set loose clot disturbed
633
SYNERESIS AND CLOTTISG O F BLOOD
TABLE IX Influence of Potassium Fluoride on Syneresis Concentration of potassium fluoride = 4.08 r\; Total volume = 2 5 0 C.C. Blood = 230 C.C. Time
Amount syneresis
KzFz
30 min.
2 1 C.C.
hr. hr. 30 min. 2 hr. 30 min. 3 hr. 30 min. 4 hr. 30 niin. 14 hrs.
56 80
I
I
3
I C.C.
0
7
C.C.
I4 22
0
I11
30 39 44
I22
50
7
97 104
j C.C.
C.C.
not set not set not set not set not set setting begins
not set setting begins loose clot 0
0
0
TABLE X Influence of Sodium Hydroxide on Syneresis Concentration of sodium hydroxide = 2.47 iK Total volume = 2 5 0 C.C. Blood = 230 C.C. Amount of syneresis
Time NaOH
0
30 min.
19 C.C.
hr. I hr. 30 min. 2 hr. 3 hr. 4 hr. 4 hr. 30 min. .j hr. 1 7 hr.
53
I
2 C.C.
6 '7
61
25
84 98 104
30 42 47
I IO
54 65
-
I22
-
C.C.
5
C.C.
not set not set not set not set not set not set setting begins sets firm clot, no syn. in 2 days
a C.C. not not not not not not not not not
set set set set set set set set set
Our results on the syneresis of blood recorded in the above tables show that as the concentration of electrolytes is increased, the amount of syneresis gradually decreases and in some cases the syneresis of blood clot is totally stopped. Even in two days, no marked syneresis is observed. In some cases, with the increase in the concentration of electrolytes, the blood becomes so stable that it either sets after a long time or it does not set at all. I n a previous communication,' we have investigated the influence of the concentration of electrolytes on the syneresis of various inorganic jellies. We have shown that as the concentration of the coagulating electrolytes, Le., those electrolytes from which ions containing charge opposite to that of the sol are mostly adsorbed, is increased, the amount of syneresis is increased. 1
J. Indian. Chem. SOC. (1930).
SATYA PRAKASH AND N . H. DHAR
634
From our results we have also shown, that by increasing the concentration of coagulating electrolytes the time of setting of jellies is much decreased. The behaviour of blood is quite contrary to that of the jelly-forming inorganic sols. It possesses a high tendency to adsorb similarly charged ions, Le., anions; and does not appear to adsorb cations to a marked extent. For this reason, the addition of electrolytes invariably stabilises blood. The function of stabilising ions in the cases of jellies is to increase the original time of setting and decrease the extent of syneresis. Thus on increasing the concentration of the stabilising electrolytes, the following may happen:(i) Up to a certain limit-no of the original clot.
marked influence on the extent of syneresis
(ii) Up to the second limit-gradual
decrease in the amount of syneresis.
(iii) Within the next limited range-total (iv) Above this limit-blood indefinitely.
inhibition of syneresis.
not clotting at all, but remaining fluid
Hence the function of stabilising electrolytes is just opposite to that of the coagulating electrolytes. From our observations, it will be seen that blood has a high tendency to adsorb chloride, sulphate, oxalate, citrate, fluoride and hydroxyl ions, and in presence of these, the amount of syneresis is markedly decreased. Acetate and tartrate ions do not possess much stabilising influence. I n the following table, the comparative influence of acetate, tartrate, citrate and oxalate has been recorded.
TABLE XI Concentration of salts = N Total volume = 2 5 0 C.C. Blood = 230cc. 4 C.C. of N salts have been mixed with blood Time 30 min.
hr. I hr. 3 hr. 4 hr. 5 hr.
Sodium acetate 32 C.C.
66
I
30 min. 30 min. 30 min. 30 min.
Amount of syneresis Sodium Sodium tartrate citrate 42
84
67 80
115
107
I18
1'3
I22
C.C.
-
half set half set half set just set set, no syn. 0
Potassium oxalate
not set half set half set 2 C.C.
4 C.C. 5
From this, it will be seen that the stabilising influence of these ions is in the following decreasing order: citrate
> oxalate > tartrate > acetate.
Our results on the influence of calcium chloride on the syneresis of blood clot show that even in presence of calcium, chloride ions are preferentially adsorbed, and the view that the stabilising influence of citrate, oxalate,
63 5
SYNERESIS AND CLOTTING O F BLOOD
fluoride, and other anions is due to the removal of calcium ions appears to be incorrect. We have shown that by increasing the concentration of calcium chloride also, blood is much stabilised, and the amount of syneresis is markedly decreased, and a t higher concentrations, blood does not set at all. This is due to the high tendency for the adsorption of chloride ions, as has been observed in the case of potassium chloride also. In Table XI1 we are indicating the approximate concentrations of various salts necessary to stabilise the blood to such an extent that no marked syneresis may occur after formation of the clot within 1 2 hours.
TABLE XI1 Electrolyte
Potassium chloride Calcium chloride Ammonium sulphate Potassium oxalate Sodium citrate Potassium fluoride Sodium hydroxide
Concentration to give clot undergoing no marked syneresis 0.12
x
o 0845 N
0.176 o 016 0.016 0.049 0,039
N N
K r\’ ?;
From the table it will be seen that the stabilising influence of these salts is in the following decreasing order: NaaCit
> K20xalate > NaOH > KzF2 > CaC12 > KC1 > Am2S04
From this order it appears that the stabilising influence depends both on the valency of the ions and alkalinity of the medium. The clotting of blood has generally been regarded as the conversion of soluble fibrinogen to insoluble fibrin under the action of an enzyme known as thrombin. We are of the opinion that thrombin may assist the process of clotting but it is not essentially the cause of the phenomenon. The clotting of blood is guided by the same forces which bring out the gelation of other organic and inorganic jellies. Fibrin has a high hydration tendency and yields an unstable colloidal suspension. The clotting of blood is guided by the characteristic unstable nature of fibrin, its concentration, and the nature and concentration of electrolytes present in blood, and all this has been so well regulated in blood, that as soon as the capillary action of blood-vessels and circulatory motion are stopped, jelly-forming forces begin to act and finally a solid clot is obtained in a few minutes. The presence of an excessive amount of coagulating electrolyte causes the agglomeration of particles, and the contraction of the clot and the synerised serum is squeezed out of the network. Such syneresis has been observed with various inorganic jellies, such as those of vanadium pentoxide, silicic acid, ferric arsenate, borate and various zirconium jellies. These jellies on ageing, lose markedly the hydration capacity, and on account of the agglomeration of particles give out the solvent.
63 6
SATPA PRAKASH A S D S . R. DHAR
I n previous communications’ from these laboratories, we have mentioned that the process of jelly formation is guided by the agglomeration and hydration tendencies of the particles. In presence of coagulating electrolytes, the charge on the jelly-formirig sol decreases and the amount of hydration increases up t’o a limiting value. When the concentration of the electrolyte is increased, agglomeration of the particles begins, with the result that the jelly contracts and undergoes syneresis. The same is applicable to the blood clot also. Under the action of coagulating ions, blood forms a clot, and due to the presence of an excess of the same ions, its particles agglomerate and undergo contraction, and finally the serum is squeezed out. Waele2 has shown that the blood-fibrinogen exists in a highly buffered system, of which the pH, however! is subject, to variations. He has also observed that fibrinogen is precipitated at pH j-6, forms a gel at pH i-9, and remains dissolved a t pH I O . This dissolution of fibrinogen can be explained on the view that it is stabilised by the adsorbtion of similarly charged OH’ ions from the alkaline medium. The observations of Herzfeld and Klinger3 that acids accelerate the precipitation of fibrin and alkalis exert an inhibitory action can also be explained on the same basis, As we have mentioned in a previous paper: the clotting tendency of blood is most marked near the neutral point, On the acid side and alkaline side, the charge on the blood is increased and hence the hydration tendency is less. Stuber and Heim5 have observed that t,he coagulating action of fatty acids increases with the increasing number of carbon atoms in the acids, Le., in the decreasing order of the dissociation constants. The fall in the coagulating action of the lower members of the series is due to the fact that comparatively larger amounts of hydrogen ions are given out in the presence of these acids and the medium becomes acidic, whereby the charge on the plasma is increased and the system is stabilised.
summary The influence of different concentrations of potassium chloride, calcium chloride, ammonium sulphate, potassium fluoride, potassium oxalate, sodium acetate, tartrate, citrate, and hydroxide on the extent of syneresis of blood clot has been studied. 2. It has been observed that in all the cases, the amount of syneresis decreases as the concentration of the electrolytes is increased. I n some cases, the syneresis is totally stopped and in a few cases, the electrolytes prevent the clotting of blood. I.
2
J. Indian. Chem. Soc., 6,391 (1929). Ann. physiol. physic0 chim. biol., 3, 91 (1927) Biochem. Z., 71, 391 (191j). LOC.cit. Biochem. Z., 77, 333 (1916).
FTSERESIS A S D CLOTTING O F BLOOD
63 7
3. The influence of electrolytes is explained on the view that blood has a high tendency of adsorbing similarly charged ions from the salts, and thus the electric charge on blood is increased and it is stabilised. The stabilising influence of the salts is in the following order: Sodium citrate > potassium oxalate > KaOH > KzF2 > CaCh KCl > Xm2S04. 4. I t has been shown that the stabilising influence of fluorides, citrates or oxalates is not due to the removal of calcium ions from the blood, but it is due to the stabilising influence of anions which are largely adsorbed by blood.
5 . The syneresis and clotting of blood are guided by the same forces which give rise to the syneresis and formation of inorganic and organic jellies. Chemical Laboratones, Unzverszty o j Allahabad, Allahabad, Indza. June, 1930.
