240 PRACTICAL PHYSIOLOGY 



EXPERIMENT III. In each of four tubes, a, b, c and d, place 5 c.c. 

 oxalated plasma. Leave a untouched ; to 6 add a few drops of 

 serum ; to c a few drops 1 per cent, calcium chloride solution ; 

 and to d an equal volume of saturated sodium chloride solution. 

 Filter off the flocculent precipitate and keep for the next experi- 

 ment, which should be performed as soon as possible. To the nitrate 

 add some calcium chloride solution. Place all in a water-bath as 

 above. It will be found that a has not clotted, that b may have 

 clotted, c has clotted, and that the nitrate d has not clotted. 



From these experiments, we gather (1) that blood will not clot 

 when the calcium salts have been removed by an oxalate ; (2) that 

 serum can clot oxalated blood (that is, blood without the presence 

 of calcium salts), because it contains the necessary enzyme already 

 formed in it ; (3) that oxalated blood will clot when calcium is added 

 to it, because with free calcium available the coagulating enzyme 

 can act ; (4) that the body coagulated is a protein thrown out 

 of solution by half saturation with sodium chloride solution. This 

 body is known as fibrinogen. It is insoluble in distilled water and 

 easily thrown out of solution by saturation with salts, and, there- 

 fore, belongs to the globulin class of proteins. 



EXPERIMENT IV. Quickly redissolve in water the precipitate 

 obtained in Experiment III, d. The salt adhering to the precipitate 

 forms a dilute saline solution, in which the precipitate dissolves. 

 Test the solution obtained for protein by the colour tests. 



EXPERIMENT V. Into the bent capillary tubes provided collect some of 

 your own blood, first introducing a small quantity of anti-coagulant fluid, 

 preferably 10 per cent, sodium citrate, since sedimentation is most rapid 

 with this solution ; 1 per cent, potassium oxalate or 3 per cent, sodium 

 fluoride may also be used. Having sealed off the ends, under the demon- 

 strator's supervision, hang it upon the centrifuge by the bent end. With the 

 plasma so obtained, perform experiments such as those given in Experiment 

 III. Sodium citrate, it will be found, acts like oxalate. This is not because 

 it precipitates the calcium, but because it combines with it to form a soluble 

 citrate, a salt which does not ionise (dissociate) in solution, and therefore 

 leaves no calcium free to aid in the formation of thrombin. If fluoride has 

 been used, it will be found that the addition of calcium salts to the plasma does 

 not cause a clot to form, showing that the fluoride in some way prevents the 

 formation of the enzyme from the pro-enzyme. 



Conditions which retard Clotting. (1) Coldr- -receive the blood into a vessel 

 placed in ice (i.e. keep it at a temperature a little above freezing point). The 

 enzyme action is inhibited by cold. The blood clots on warming. 



(2) Contact with blood-vessel wall. " The living test tube." This is made 

 by ligaturing in two places a vein of a large animal, such as the jugular vein 

 of the horse. In the tube thus formed the blood does not clot, and if it be 

 hung up the corpuscles gradually sink to the bottom, leaving the unclotted 

 plasma above. 



(3) Addition of certain neutral salts. " Salted plasma " (cf. Experiment II). 



(4) Addition of a soluble oxalate. " Oxalate plasma " (cf. Experiment III). 



(5) Addition of a soluble citrate. " Citrate plasma " (see Experiment V). 



(6) Addition of a soluble fluoride. "Fluoride plasma." This plasma will 

 not clot upon the addition of calcium (see Experiment V). 



(7) Addition of leech extract (Hirudin). This is a secretion produced by the 

 salivary glands of the leech, and which can be obtained by extracting the 

 heads with water. It acts because it contains an antithrombin. 



