344 VEHICLES OF CHEMICAL CORRELATION 



Since calcium is necessary for the coagulation of freshly-shed blood, 

 it might seem reasonable to suppose that the thrombin solution 

 contains combined or associated calcium, which suffices to permit the 

 process to go forward. This is, however, not at all the case, for throm- 

 bin may be purified by dialysis and precipitation with Acetone, and 

 when this has been done twice the thrombin is found to be perfectly 

 free from calcium. 



The true secret of the essentiality of calcium in the clotting of 

 recently shed blood lies in the fact that thrombin, as such, is absent 

 from the circulating blood, and from oxalated plasma. Instead, we 

 have a mother-substance, Prothrombin which is converted by calcium 

 salts into thrombin. This fact may be shown in a variety of ways, 

 among which the following may be cited: Wooldridge showed that 

 if peptone plasma be cooled for some time to zero degrees centigrade 

 a precipitate of minute discoidal particles collects at the bottom of the 

 container. They resemble very greatly the Blood-platelets and may, 

 indeed, actually be identical with them. When these are removed from 

 the plasma, clotting of the fluid is now very difficult to induce by the 

 customary agents, by carbon dioxide, calcium chloride and so forth. 

 Wright subsequently showed that the same precipitate occurs in oxa- 

 lated plasma and Hammarsten showed that its removal prevented the 

 subsequent clotting of the plasma by the addition of sufficient calcium 

 chloride to precipitate the oxalate and furnish a favorable excess of 

 calcium ions. If, however, this precipitate be treated with lime salts 

 and the calcium subsequently removed by oxalates, it now is found to 

 contain very active Thrombin which quickly induces coagulation in 

 Oxalated Plasma. A portion at least of the material in the discoidal 

 particles was, therefore, converted by the calcium salts into thrombin. 

 This constituent is prothrombin. 



Another method of preparing prothrombin is that which has been 

 devised by Howell. Oxalated blood is centrifugalized and the plasma 

 is heated to 54 Centigrade. This coagulates the fibrinogen. The 

 filtered plasma is treated with Acetone, and the precipitate is collected 

 upon a filter and dried. When the prothrombin is required for use the 

 filter paper is cut into small pieces and extracted for about one hour 

 with dilute sodium bicarbonate solution. This solution does not cause 

 clotting of pure fibrinogen or of oxalated plasma unless it is first treated 

 with calcium chloride (0.2 per cent.). 



The circulating blood contains prothrombin, therefore, and it also 

 contains calcium salts, and the question necessarily arises why the 

 prothrombin is not converted into thrombin in the vessels, thus 

 leading to intravascular coagulation? The reason that this does not 

 occur is that the conversion of prothrombin into thrombin requires 

 not only the presence of- calcium salts but also another factor, derivable 

 from tissue extracts, which Morawitz termed Thrombokinase, but which 

 has recently been identified by Howell as a phospholipin, namely, 

 Kephalin. 



