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Sci., Wash., 28, 478.Hele, M. P. (1950). Nature, Lond., 166, 786.Hemingway, A. (1939). J. Phy8iol. 95, 3P.Hers, H. G. & Duve, C. de (1950). Bull. Soc. Chim. biol.,

Paris, 82, 20.Krebs, H. A. (1934). Tabul. biol. Berl. 9, 209.Leut'skii, K. H. (1946). Ukr. Biochem. J. 18, 87. Cited byChem. Abstr. (1947), 41, 6947.

Meyerhof, 0. & Geliazkowa, N. (1947). Arch. Biochem. 12,405.

Nelson, N. (1944). J. biol. Chem. 153, 375.Potter, V. R. & Elvejhem, C. A. (1936). J. biol. Chem. 114,

495.Reiner, J. M. (1947). Arch. Biochem. 12, 327.Sacks, J. (1951). Arch. Biochem. 80, 423.Saxton, J. A. & Miller, M. L. (1944). Arch. Path. 37, 34.

Slein, M. W., Cori, G. T. & Cori, C. F. (1950). J. biol. Chem.186, 763.

Somogyi, M. (1945). J. biol. Chem. 160, 69.Stadie, W. C. & Haugaard, N. (1949). J. biol. Chem. 177,

311.Stadie, W. C., Haugaard, N. &; Hills, A. G. (1950). J. biol.

Chem. 184, 617.Swanson, M. A. (1950). J. biol. Chem. 184, 647.Taylor, D. W. (1951). Private communication.Treadwell, C. R., Tidwell, H. C. & Grafa, B. G. jun. (1943).

J. biol. Chem. 149, 209.Utter, M. F. (1950). J. biol. Chem. 185, 499.Vestling, C. S., Mylroie, A. K., Irish, U. & Grant, N. H.

(1950). J. biol. Chem. 185, 789.Wiebelhaus, V. D. & Lardy, H. A. (1949). Arch. Biochem.

21, 321.Youngburg, G. E. (1944). Arch. Biochem. 4, 137.

Blood Clotting: the Function of Electrolytes and of Calcium

BY J. E. LOVELOCK AND B. M. PORTERFIELDMedical Re8earch Council, Common Cold Re8earch Unit, Harvard Ho8pital, Salisbury

(Received 10 May 1951)

The theory of blood clotting advanced by Morawitz(1904) and Fuld & Spiro (1904) postulated that thepresence of free calcium ions was a necessary con-dition of coagulation. This was based on theevidence of Arthus & Pag6s (1890) and Pekel-haring (1892) that certain anions, namely, oxalateand citrate, whose calcium salts have low solu-bility products, inhibit the clotting of blood. Vines(1921), and Stewart & Percival (1928) have com-mented on the unsatisfactory nature of thisevidence, and the recent work ofQuick & Stephanini(1948) has cast further doubt on these acceptedbeliefs. They have suggested that calcium functionsin blood clotting, not as a free ion, but in com-bination with prothrombin as an unionized com-plex. Also that the activity of citrate is not con-nected with the low solubility of its calcium salt.If these suggestions are accepted, it is of someinterest to consider the alternative theories whichcan be put forward to explain the anticoagulantactivity of oxalate and citrate anions.

(1) Oxalate and citrate ions form complexes withlow solubility products with other elements, inparticular, iron, cobalt and zinc. If it is assumedthat some enzyme incorporating one of theseelements is necessary for coagulation, then theanions might lower its activity by removal of anessential element from the enzyme.

(2) The process of coagulation involves the inter-action of electrically charged colloidal particles.Ions, particularly multivalent ions, are known to

exert an influence on such processes and the activityof oxalate and citrate ions may be due to this effect.The second theory, namely that ions such as

oxalate and citrate might act as anticoagulants bya non-specific salt effect was first proposed bySchmidt (1895). Interest in this possibility was,however, largely dropped when Morawitz (1904)suggested that ionized calcium acted catalyticallyin the coagulation of blood, and when Sabbatani(1908) explained the action of citrate ions in termsof the low solubility product of its calcium salt.The only systematic studies of the action of

neutral salts on coagulation are those of Glazko &Greenburg (1939), Astrup (1944) and Mommnaerts(1945). These authors investigated the anticoagu-lant activity of anions, including oxalate andcitrate ions, on the clotting of purified fibrinogen bythrombin. They showed that in this second phaseof blood coagulation, the anticoagulant activity ofthe anions tested was connected with their valency.The results reported in this paper were obtained

by observing the effect of various anions on thecoagulation of whole blood, and by determining theconcentration and relative proportions of electro-lytes needed to restore the coagulability of salt-freeplasma. Many physiological processes are known tobe highly sensitive to changes in the concentrationand species of ions present in the suspendingmedium. The results indicate the process of coagu-lation to be similarly sensitive to disturbances in theelectrolyte balance of the blood. In particular they

J. E. LOVELOCK AND B. M. PORTERFIELDsuggest that coagulation can only occur when thetotal ionic strength of the blood is within pre-scribed limits, and only when a certain proportionof the cations present are of a suitable species.They indicate that the anticoagulant activity ofoxalate, citrate and fluoride ions is due not onlyto their ability to depress the calcium ion concen-tration of blood, but also to their ionic charge; withcitrate ions the latter effect predominates, withfluoride ions the former. They also suggest there isno need to ascribe any special function, such ascatalytic activity, to the element calcium in thecoagulation process, or the formation of definedcompounds with the elements of the blood, butinstead suggest that calcium functions by main-taining an electrolyte balance suitable for the inter-action of the various plasma colloids.

METHODSDetermination of the concentration of anion8 needed to

prevent the coagulation of whole blood. Human blood wascollected by venipuncture using a cold dry syringe andtransferred immediately to a polyethylene beaker kept at 0°.Portions (0-2 ml.) were transferred by Pasteur pipettes tosmall glass tubes each containing 0-2 ml. of a solution ofthe Na or K salt of the test anion. These tubes were kept at200 and examined after 12 hr. for clotting. The syringe andpipettes were treated before use with dimethyldichloro-silane to render their surfaces inert towards the blood(Jacques, Fidlar, Feldstedt & Macdonald, 1946).Thepreparation ofcalcium-free plasma. The decalcification

of blood by cation-exchange resins was first described bySteinberg (1944) and developed into a laboratory methodby Stephanini (1948). The method described below is similar

to that reported by Stephanini, and we can confirm that theuse of cation-exchange resins provides a convenient andeffective method of decalcifying blood without otherwisealtering its properties.Human blood was collected by the procedure described

earlier, and transferred to a polyethylene flask containingthe sodium form ofthe cation-exchange resin Zeo-Karb 225.For each 10 ml. of blood added 4 g. were used. The mixtureof blood and resin was kept at 0° and gently stirred for aperiod of 15 min. The blood was then separated from theresin by filtration and the plasma from the blood by centri-fugation. All glassware used in these operations was treatedbefore use with dimethyldichlorosilane. The plasma pro-duced by this method contained less than 0-2 itg. Ca/ml.and was stored until required in polyethylene flasks at 00.The Preparation of salt-free plasma. Plasma decalcified

by the method just described was dialysed for 24 hr. at 200against running distilled water to remove salts. It wasfound necessary to take considerable precautions to preventthe access of Ca to the plasma during dialysis. When theionic strength of plasma is 0-03 as little as 3 jug. Ca/ml. issufficient to cause coagulation. The dialysis apparatus wastherefore constructed of polyethylene and the cellophanmembrane washed thoroughly in N-HC1 before use to removetraces of Ca. In earlier experiments in which glass funnelswere used to support the dialysis membrane clottingfrequently occurred during dialysis. Experiments describedlater in this paper suggest this was probably due to theliberation of Ca from the glassware.

RESULTS

The inhibition of coagulation by anions. Table 1shows the concentrations of various anions neededto prevent coagulation. Also shown are the solu-bility products of the calcium salts of the anions

Table 1. The anticoagulant activity of anions with reference to their valencyand their ability to remove calcium ions from 8olution

(Measurements were made of the concentration of salt required to prevent the coagulation of human blood in Pyrexglass tubes at 200.) Ca salt

AnionAzideChlorideFluorideGlycollateIodateIodoacetateMandelateMalateMalonateOxalateSuccinateSulphateThiosulphateTartrateCitrateFerricyanideMethanetrisulphonateNaphthalenetrisulphonate

Valency

2

3

Concn.required

to preventcoagulation

(mm)30030080

3001503003001001005

100100100305

303020

Solubility productSolubleSoluble

3-4 x 10-Soluble

2-2 x 10-7SolubleSoluble

2-5 x 10-86-25 x 10-41-8 x 10-96-4 x 10-36-1 x lo-Soluble

7-7 x 10-71-7 x 10-8SolubleSolubleSoluble

Calcium ion concn.of saturatedaqueoussolution(mm)

0-2

6-0

50250-0680810-90-4

416

BLOOD CLOTTING: ELECTROLYTES AND CALCIUM

tested and the calcium ion concentration of theirsaturated aqueous solutions. The values of theirsolubility products are those given in the Inter-national Critical Tables (1929) except the value forcalcium citrate which was calculated from the dataof Schubert & Lindenbaum (1950).The results show a close connexion between anti-

coagulant activity and valency for the anionstested. The concentrations of uni-, bi-, and ter-valent ions needed to prevent clotting fell in theorder 10: 3: 1 respectively. There appears to be nodirect connexion between anticoagulant activityand ability to remove calcium ions from solution;the concentration of fluoride ions needed to preventclotting is sixteen times that of citrate ions,although the former are more active in removingcalcium from solution. A close examination of thetable reveals, however, that all of the anions testedhaving calcium salts with solubility products lessthan 10-7 were more active in preventing coagu-lation than were other ions of the same valency.

The concentration of electrolytes and calcium re-quired to cause coagulation of 8alt-free blood. Theresults shown in Fig. 1 were obtained by succes-sively adding salts and calcium ions to plasma de-calcified and dialysed as described previously. Thefigure shows that coagulation will not occur if theionic strength of blood is less than 0-01. As the saltconcentration is raised the quantity of calciumrequired for coagulation falls to a minimum valueat an ionic strength of 0-03. When the salt concen-tration is increased beyond this point there is anapproximately proportional increase in the quantityof calcium needed for coagulation until an ionicstrength of 0-3 is reached. Beyond this there is anupper limit of ionic strength, varying with saltadded, at which coagulation will no longer occur.For the most part the data from which Fig. 1 was

constructed were obtained by adding sodium andcalcium chlorides to salt-free plasma. It was found,however, as the diagram shows, that for ionicstrengths below 0-10 closely similar results could beobtained by adding sodium sulphate, or potassium

ferricyanide in place of sodium chloride. The differ-ence in behaviour of these salts at ionic strengthsabove 0-10 is to be expected, since their solutionscan then no longer be regarded as dilute.

20

NaCIK3FeCN6 Na2SO4

E 1

0

E 1.0

0.5

01 02 03 04Ionic strength of plasma

Fig. 1. The calcium concentration required for the coagu-lation of human plasma of varying ionic strength. De-calcified salt-free human plasma was treated with CaCl2solution, and a solution of a neutral salt NaCl, Na2SO4or K3FeCN .- Tests were made in Pyrex glass tubes at200 and the calcium concentration needed for coagulationwithin 12 hr. observed.

The effect of dilution upon the concentration ofcalcium required for coagulation. The results so fargiven suggest that the presence of free calcium ionsexerts a considerable influence on the coagulationprocess. They do not, however, provide directevidence concerning the condition of the element incoagulating plasma, in particular to what extentthe element functions in the adsorbed, combinedand ionized states. In view of the importanceattached by Quick & Stephanini (1948) to the non-ionized calcium in coagulating plasma, the followingexperiment was made to determine if calcium didfunction solely in the non-ionized state as Quick &Stephanini suggest.

Table 2. The concentration of calcium ions required for the coagulation of pla8ma diluteduwth varying volumes of 8alt 8olution

(Decalcified, salt-free, human plasma was diluted with 0415 and 0-03m-NaCl solution containing Ca ions at the con-centrations listed in the table. The coagulation time at 200 in Pyrex tubes was observed.)

Dilution Ionic strengthof plasma of plasma1:2 0-151:5 0-151:10 0-151:20 0-151:2 0-031:5 0-031:10 0-031:20 0-03

Biochem. 1952, 50

Coagulation time (hr.) at calcium concentrations (mM)

2-00-50-50-50-50-50-50-50-5

1-00-50-50-54-00-50-50-50-5

0-54-04-04-04-01114

0-2>24>24>24>24

4444

0-1>24>24>24>241212121227

VoI. 50 417

J. E. LOVELOCK AND B. M. PORTERFIELD

Table 3. The coagulation of decalcified plasma by varioUs 8ub8tance8

(Measurements were made of the quantity of test material required to cause the coagulation of decalcified humanplasma within 12 hr. at 200 in Pyrex glass tubes.)

MaterialPowdered soft glassPowdered Pyrex glassPowdered silica glassPowdered soft glassPowdered Pyrex- glassPowdered silica glassCalcium oxalate

Previous treatmentWashed with distilled waterWashed with distilled waterWashed with distilled waterWashed with N-HNO3Washed with N-HNO5Washed with N-HNO,Washed with distilled water

Quantity needed for coagulationin 12 hr. or less

(mg./ml.)20

200200

No coagulation with 1 g./ml.No coagulation with 1 g./ml.No coagulation with 1 g./ml.

0.1

Plasma, free of calcium and salts, was prepared as

described earlier in this paper. A series of dilutionsof this plasma was made in sodium chloride solutioncontaining various amounts of calcium chloride.The coagulation time of the diluted plasma was

observed and is recorded in Table 2. The tableclearly shows that the coagulation time is dependentupon the concentration of calcium, and not uponthe quantity present. This is the reverse of whatwould be expected if the calcium necessary forcoagulation were present solely in the non-ionizedcondition.The coagulaton of decalcified blood by powdered

g1a88, calcium oxalate and other 8ub8tance8. In experi-ments described previously (Lovelock & Burch,1951), it was found that decalcified plasma wouldoften coagulate if kept in soda-glass vessels, or

when in contact with powdered glass or silica.Similar observations were reported by Overman(1949), who found that diluted oxalated blood wouldclot when in contact with powdered glass and othersimilar materials. At the time it seemed that theseobservations indicated that coagulation could occur

in the absence of calcium. The possibility that suffi-cient calcium was liberated from the glass, etc., tocause coagulation was not seriously considered,since it was then believed that the minimum con-

centration of calcium at which coagulation couldnormally occur was 0-4 mm. It was suggested thatthe function of calcium and ofpowdered glass in thecoagulation of blood was respectively to combinewith, or inactivate, some inhibitor of coagulation.Reference to Fig. 1, however, shows that in blooddiluted to an ionic strength of 0 03 the concen-

tration of calcium required for coagulation is only0 04 mM ( 1-6 ,ug./ml.). There seemed a distinct possi-bility that a quantity of calcium as small as thiscould be liberated from glassware or powdered glass.

Table 3 shows the quantities of various materialsneeded to cause the coaguldtion of decalcifiedplasma, and the quantity of calcium which couldbe removed from the same quantity of material byboiling with N-nitric acid for 2 hr. It is evident fromthe results that sufficient calcium is available from

powdered glass and probably also from powderedsilica to cause coagulation. That glass and silicawashed with acid will not cause coagulation, furtherconfirms that these materials cause the coagulationof decalcified blood by liberating calcium adsorbedupon their surfaces.

20

E 15

0

.o 10

v

'a 5

,cSn0~ Mg Ca

010 015 020tonic strength of plasma

Fig. 2. The concentrations of barium, magnesium andcalcium ions required for the coagulation of humanplasma of varying ionic strength. Decalcified salt-freehuman plasma was treated with NaCl solution, and witha solution of CaC12, BaCl2 or MgCl,. Tests were made inPyrex glass tubes at 20° and the divalent cation concen-

tration needed for coagulation within 12 hr. observed.

The results shown in Table 3 also include theobservation that coagulation can occur when cal-cium oxalate is added to decalcified blood. Thisexperiment has been repeated successfully on

several occasions and with blood from differentpersons. The calcium oxalate used was precipitatedfrom a solution containing an excess of oxalate ionsand washed with dilute potassium oxalate solutionbefore use. It was unlikely, therefore, to havecalcium ions adsorbed upon its surface. This resultillustrates clearly the fallacy of assuming, as is

frequently done, that the precipitated calciumoxalate in oxalated blood is inert.

The coagulation of decalcified pla8ma by cationsother than calcium. Fig. 3 shows the relationship

uaiclumremovedby boiling

with N-HNO3(/hg./g.)230-010-080

418 I952

BLOOD CLOTTING: ELECTROLYTES AND CALCIUMbetween the electrolyte content of plasma and theconcentration of calcium, magnesium and barium,required for coagulation. The concentration of eachof these cations needed for coagulation varied withthe ionic strength of the plasma in a closely similarmanner. The most favourable ionic strength forcoagulation was the same for all three elements,namely 0-03 and above this the concentration ofeach cation needed for coagulation increased pro-portionately with increasing ionic strength. Theconcentrations of calcium, magnesium and bariumneeded for coagulation rose in the order 1 : 8: 60respectively.The values and sequence of activity of calcium,

magnesium and barium ions in the coagulation ofplasma are similar to those required to neutralizethe charge of certain phosphate colloids. Forexample, the charge on sphingomyelin is neutralizedby calcium, magnesium and barium ions at concen-trations of 0-9, 5-0 and 20 mM respectively (Bungen-berg de Jong, Teunissen-Van Zijp & Teunissen,1940). Bungenberg de Jong (1949) has furthershown that the activity ofbivalent cationsin neutra-lizing the charge on phosphatide colloids is reducedwhen the ionic strength of the suspending mediumis increased.

DISCUSSION

The experimental observations indicate that thecoagulation process requires the concentration andproportion of simple electrolytes in plasma to bewithin a prescribed range. In particular, the processis sensitive to changes in the proportion of the totalionic strengtfi contributed by calcium ions. Coagu-lation occurs most readily when the proportion isapproximately 1: 16 for calcium and for other ionsrespectively. The experimental establishment oftheidea that a certain electrolyte balance is necessaryfor normal coagulation has proved of use in re-solving some of the discrepancies concerning thefunction of calcium in coagulation and of oxalate,citrate and fluoride ions in preventing it. Forexample, the discrepancy between the concen-trations of oxalate, citrate and fluoride ions neededfor coagulation and their ability to decalcify plasmacan be resolved without recourse to complextheories concerning the function of calcium incoagulation. The addition of these ions to plasmadisturbs the electrolyte balance ina twofold manner;first by lowering the calcium ion concentration, andsecondly by raising the total ionic strength. Thatcitrate ions are more effective than fluoride ions inpreventing coagulation then follows as a naturalconsequence of their higher valency. With oxalateions their superior decalcifying activity compen-sates for their lower valency to give them approxi-mately the same anticoagulant activity as citrateions. That the decalcifying activity of these ions

alone is insufficient to prevent coagulation is furtherillustrated by the observation that decalcified bloodcan be coagulated by the addition of calciumoxalate, citrate or fluoride. It should be noted thatthe solubilities of the calcium salts listed in Table 1refer to their solutions in water only. They are ofuse in illustrating qualitatively the ability of theanions tested to decalcify blood, but are almostcertainly different quantitatively from the solu-bilities of the same salts in blood, especially in thepresence of an anticoagulant concentration of thetest anion. Norbdo (1936) has shown, for example,that the calcium ion content of a saturated solutionof calcium oxalate in serum ultrafiltrate is 0-18 mm.The calcium ion content of a saturated solution ofthe same salt in water is 0-06 mm. When precisevalues of the solubilities of calcium salts in wholeblood become available it should be possible, usingalso the data in Fig. 1, to calculate the concentrationof a given anion needed to inhibit the coagulationof blood. The comparison of the calculated and theobserved anticoagulant activity of salts such asthose listed in Table 1 would provide a criticalmeans of checking the validity of the theories out-lined above.

It is frequently stated that the coagulation ofcitrated or oxalated blood following the addition ofcalcium is further proof of the necessity of thatelement for coagulation. In spite of the obviousillogicality of this statement, in that the addition ofcalcium removes the oxalate and citrate ions, itis of some interest to compare it with the obser-vation illustrated in Fig. 1. Here the addition ofcalcium ions to plasma, prevented from coagulatingby the addition of sodium chloride, also causescoagulation. In both of these examples it is therestoration of the electrolyte balance which restoresthe coagulability of the plasma.The experiments show that even with the smallest

amount of calcium at which coagulation will occur,namely 0-08 mM, it is the concentration and not thequantity of calcium in plasma which is important.This is not in accord with those theories whichpostulate that some non-ionized calcium compoundis necessary for coagulation. Neither is the evidencethat the concentration of calcium required forcoagulation varies with the ionic strength of theplasma, nor the observation that the concentra-tions of other bivalent elements needed for coagula-tion are different from those of calcium. An acidradical giving a firm non-ionized compound with1 part of calcium, 8 of magnesium or 60 of barium,seems very improbable. In general terms the ex-perimental evidence does not support the idea thata defined compound of calcium and one of themacromolecules of plasma, presumably analogousto iron in haemoglobin, is necessary for coagula-tion.

27.2

Vol.50 419

420 J. E. LOVELOCK AND B. M. PORTERFIELD 1952Calcium is distributed in plasma in the form of

free ions, ions adsorbed upon the many colloidspecies present and probably also in non-ionizedcombination with the simpler anions of plasma.The equilibria controlling the proportion of theelement distributed in these various forms arelikely to be complex and easily disturbed by changesof the electrolyte and calcium ion content ofplasma. In these circumstances the discovery ofthe precise function, or functions, of calcium in thenormal coagulation process is likely to be difficult.Nevertheless, from a knowledge of the nature of thecoagulation process, namely the serial interactionofa number of electrically charged macromolecules,the following conclusions can be drawn: calciumions as such are unlikely to enter directly into thecoagulation reaction, but their presence is necessaryin order to maintain the requisite amount of cal-cium adsorbed upon the various plasma colloids.According to the evidence presented earlier, cal-cium in firm non-ionized combination with eitherthe molecules, or macromolecules of plasma doesnot appear to be necessary for coagulation.

It follows therefore that the most probable con-dition of the element calcium necessary for coagu-lation is the adsorbed state. It seems possible thatthe function of the adsorbed calcium is to maintainthe surface charge of one or more of the plasmacolloids at a value suitable for its proper interactionwith the other plasma colloids. There is as yet no

direct evidence to suggest which of the plasmacolloids require adsorbed calcium for their properfunction, although the sequence and degree ofactivity of calcium, magnesium and barium ionssuggests that a phosphate colloid may be involved.Overman (1949) has suggested from other evidencethat calcium may function by neutralizing aphosphatide inhibitor of coagulation.

SUMMARY

1. Experiments are described which indicate thatthe activity of oxalate, citrate and fluoride ions inpreventing coagulation is complex, and is due notonly to their ability to depress the calcium ion con-centration of blood, but also to their ionic charge.

2. The concentrations of calcium, magnesiumand barium ions needed for the coagulation ofplasma containing various concentrations of saltshas been measured. These measurements indicatethat human plasma will not coagulate if the ionicstrength is less than 0-01 or more than 0-5. Theconcentration of alkaline earth elements requiredfor coagulation was 'found to vary with ionicstrength, calcium concentration as low as 0-08 mMcausing coagulation in plasma ofionic strength 0 03.

3. The function of calcium in coagulation is dis-cussed. It is suggested that it is concerned with themaintenance of an electrolyte balance suitable forthe interaction of the plasma colloids.

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Astrup, T. (1944). Acta phy8iol. Scand. 7, 11.BungenbergdeJong, H. G. (1949). Colloid Science. London:

Elsevier.Bungenberg de Jong, H. G., Teunissen-Van Zijp, L. &

Teunissen, P. H. (1940). Kolloidz8chr. 91, 311.Fuld, E. & Spiro, K. (1904). Beitr. chem. Phy8iol. Path. 5,

171.Glazko, A. J. & Greenburg, D. M. (1939). Amer. J. Phy8iol.

125, 108.International Critical Tables (1929). 4, 229-31.Jacques, L. B., Fidlar, E., Feldstedt, E. T. & Macdonald,

A. G. (1946). Can. med. A88. J. 55, 26.Lovelock, J. E. & Burch, B. M. (1951). Biochem. J. 48,

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Morawitz, P. (1904). Beitr. chem. Phy8iol. Path. 5, 133.Norbdo, R. (1936). Skand. Arch. Phy8iol. 75, Suppl. 11.Overman, R. S. (1949). Blood Clotting and Allied Problem&.New York: J. Macey.

Pekelharing, C. A. (1892). Dt8ch. med. W8chr. 18, 1133.Quick, A. J. & Stephanini, M. (1948). J. gen. Physiol. 32,

191.Sabbatani, L. (1908). Mem. R. Accad. Torino, 51, 257.Schmidt, A. (1895). Weitere Beitrage zur Blutlehre. Wies-

baden: Bergmann.Schubert, J. & Lindenbaum, A. (1950). Nature, Lond., 166,

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124.Stephaniini, M. (1948). Proc. Soc. exp. Biol., N. Y., 67, 22.Stewart, C. P. & Percival, G. (1928). Biochem. J. 22, 559.Vines, H. W. C. (1921). J. Physiol. 55, 86.