Influence of Serum Incubation Conditions on Asolectin Thromboplastin Generation Results

SummaryThromboplastin generation in a system employing Asolectin, a soybean phosphatide, suspension as a platelet substitute is greatly influenced by the time and temperature of serum incubation.Reproducible results and good separation of populations of normal and PTC deficient sera were obtained in the asolectin TGT system when test sera were incubated for 1 hour at 20° C prior to the thromboplastin generation test. Longer time or higher temperatures of serum incubation resulted in progressively less reliable, separation of pathologic and normal populations in asolectin TGT systems than in fresh platelet TGT systems. Cephalin suspension was shown in a smaller number of tests to behave similarly to asolectin suspension when used as a platelet substitute in TGT systems.The 0.14 M sodium citrate eluate of citrated normal or Hemophilia B sera (incubated 1 hour at 37° C) had the ability to shorten the prolonged asolectin TGT minimal time of “over-incubated” normal sera to values obtained with the same sera in fresh platelet TGT systems. This correction, however, was probably non-specific since Hemophilia B sera results were sometimes similarly normalized. Citrated (but not oxalated) plasma incubated at 37° C for 24 hours, then recalcified to manufacture serum, and incubated an additional hour at 20° C showed no loss of the fresh 1 hour 20° C serum activity in asolectin thromboplastin generation, further suggesting that the thermolabile component of normal serum influencing asolectin TGT is a reaction product evolved during clotting.

HYDROLYSIS OF GLYCEROLPHOSPHATIDES BY PLASTID PHOSPHATIDASE C

Studies of the influence of structural variation in the glycerolphosphatide molecule on the hydrolysis of this class of compounds by plastid phosphatidase C showed that the presence of both fatty acid ester groups is necessary for enzymatic reaction; that release of nitrogenous bases occurred, in the presence of ethyl ether, from phosphatidyl cholines, phosphatidyl ethanolamine, and phosphatidyl serine; and that a phosphatidyl choline was hydrolyzed more rapidly than the corresponding phosphatidyl ethanolamine or phosphatidyl serine. The rate of hydrolysis of phosphatidyl choline was influenced greatly by the chain length and degree of unsaturation of the fatty acids. The corresponding phosphatidic acid formed in the hydrolysis of (dipalmitoyl)- or (dipalmitoleyl)-lecithin by carrot phosphatidase C was isolated. Studies on the hydrolysis of crude soybean phosphatide by phosphatidase C showed that both choline and ethanolamine were liberated in the absence of ethyl ether, at an optimum pH of 4.8; in the presence of ether, the rate of liberation of each base was increased, and the pH optimum was between 4.8 and 6. Soybean phosphatide probably contains a substance that stimulates the enzymatic hydrolysis.

HYDROLYSIS OF GLYCEROLPHOSPHATIDES BY PLASTID PHOSPHATIDASE C

Studies of the influence of structural variation in the glycerolphosphatide molecule on the hydrolysis of this class of compounds by plastid phosphatidase C showed that the presence of both fatty acid ester groups is necessary for enzymatic reaction; that release of nitrogenous bases occurred, in the presence of ethyl ether, from phosphatidyl cholines, phosphatidyl ethanolamine, and phosphatidyl serine; and that a phosphatidyl choline was hydrolyzed more rapidly than the corresponding phosphatidyl ethanolamine or phosphatidyl serine. The rate of hydrolysis of phosphatidyl choline was influenced greatly by the chain length and degree of unsaturation of the fatty acids. The corresponding phosphatidic acid formed in the hydrolysis of (dipalmitoyl)- or (dipalmitoleyl)-lecithin by carrot phosphatidase C was isolated. Studies on the hydrolysis of crude soybean phosphatide by phosphatidase C showed that both choline and ethanolamine were liberated in the absence of ethyl ether, at an optimum pH of 4.8; in the presence of ether, the rate of liberation of each base was increased, and the pH optimum was between 4.8 and 6. Soybean phosphatide probably contains a substance that stimulates the enzymatic hydrolysis.