1. In pig embryos of 100 to 270 mm. the average coagulation time of the blood was found to be about 23 minutes. This represents a coagulation time six to eight times greater than that obtained for the adult.
2. The first evidence of coagulation in the blood of these embryos consisted in the appearance of small masses of fibrin deposited almost invariably at the side of the test-tube. The ensuing coagulum was, as a rule, in the nature of a sliding clot, never attaining any marked degree of density or firmness.
3. In an analysis of the possible factors involved in this greater coagulation time of embryonic blood, it was found that numerically the blood platelets varied from 415,000 to 800,000 per c.mm., a content not differing in any significant degree from that of the adult, in which the average was found to be about 588,000 with a variation from 544,000 to 932,000.
4. The addition of platelet material obtained from adult pig blood reduced the coagulation time for embryonic blood to an average of 8.4 minutes, a decrease of 75 per cent.
5. The addition of two drops of 0.5 per cent calcium chloride reduced the coagulation time for embryonic blood to an average of 10.3 minutes, a reduction of over 50 per cent.
6. The addition of tissue extract to embryonic blood reduced the coagulation time to an average of 3.7 minutes, a time essentially equivalent to that obtained for adult blood. The clot was of a much firmer character than that obtained either in the normal coagulation or in the calcium experiments.
7. Chemical analysis demonstrated a calcium content in embryonic blood in excess of that of the adult, in the proportion of 7 : 5.
8. In view of the results indicated above, it became evident that the calcium in embryonic blood must be present in some combined form. This conclusion is supported by experiments in which barium and magnesium brought about a reduction in coagulation time in non-oxalated embryonic blood, but did not have this effect when added to oxalated blood, indicating in the former case, on the basis of the specificity of calcium, a liberation of free calcium ions. With oxalated blood, it was also found that under certain conditions coagulation could be brought about by the addition of tissue extract.
9. That the fibrinogen content plays no important part in coagulation time is shown by the fact that whereas the maximum amount of fibrin obtained by defibrinating embryonic blood was about 12 per cent of that obtained from the adult, nevertheless upon the addition of tissue extract the coagulation time of embryonic blood becomes equivalent to that of the adult.
10. The presence of bile was demonstrated in the circulating blood of these embryos. Since it was further demonstrated that by the addition of bile to adult blood conditions could be produced essentially identical with those of embryonic blood, and since in the preceding analysis of the factors involved in coagulation no significant differences could be demonstrated between the blood of the embryos and that of the adult other than the presence of bile in the former, the data justify the conclusion that the bile content in the circulation of 100 to 270 mm. pig embryos constitutes the primary factor accounting for the greater coagulation time in the blood of these embryos. A condition in embryonic blood in some respects comparable with that of icterus is also indicated.
11. In the calcium experiments the results are apparently due primarily to the introduction of calcium ions in excess of the amount which enters into chemical combination with the bile present. With tissue extract it appears that the free calcium ions essential for the initiation of coagulation must be liberated through some interaction with the constituents of bile.
12. The results of this study suggest that the normal coagulation of embryonic blood, as far as bile is concerned, involves a process comparable with that obtained after the addition of tissue extract or cephalin, but on a smaller scale. In the embryonic blood in vitro, through the gradual disintegration of cellular elements, a certain amount of tissue substance (cephalin (?)) is slowly set free in the plasma, neutralizing the bile and ultimately liberating a sufficient amount of calcium to bring about coagulation.