K. M. Brinkhous

Macroscopic Studies of Platelet Agglutination; Nature of Thrombocyte Agglutinating Activity of Plasma.∗

Summary and conclusions 1. Optimal conditions for platelet agglutination were determined in the dog by a macroscopic test. Most rapid agglutination was observed with Mg++ or Mn++, undiluted plasma, and a platelet concentration above 200,000/cmm. Frozen or heated platelets were not agglutinable. 2. The thrombocyte agglutinating (TAg) factor found in plasma was thermolabile and non-dialyzable. Under optimal conditions it caused gross platelet clumping in 5-15 seconds. This factor was active in the absence of fibrin coagulation, Ca++, fibrinogen, AHF, prothrombin and related coagulant factors, suggesting the TAg may act independently of the coagulation process.

Antihemophilic Factor (AHF) Levels Following Transfusions of Blood, Plasma and Plasma Fractions.∗

Summary 1. Canine and human hemophilic subjects were transfused with homologous blood or plasma, Dogs were also transfused with AHF-rich plasma fractions. The increase in plasma AHF levels following transfusions was proportional to the amount of AHF administered. Over half of the AHF activity disappeared within a few hours, although traces of AHF may persist for nearly a week. Frequent replacement transfusions are required if AHF levels as high as 5-10% are to be maintained. 2. Injection of potent plasma fractions into normal dogs resulted in transient supernormal AHF levels. Half of the injected AHF was lost in about 2 hours. 3. The minimum amount of AHF required to give normal values is not uniform with different clotting tests. AHF levels above 1% resulted in normal values for the clotting time and prothrombin utilization tests. Levels above 15% resulted in normal values for the PTT test.

Prothrombin Utilization during Clotting Comparison of Results with the Two-Stage and One-Stage Methods.

Summary 1. A comparison was made of the changes in prothrombin during clotting, as indicated by the one- and 2-stage methods. 2. By the 2-stage method, progressive disappearance of prothrombin from serum was observed. Prothrombin utilization was slower in human blood than in dog blood, and was delayed greatly in canine hemophilic blood, platelet-poor human plasma, and in blood clotting in silicone-treated glassware. 3. By the one-stage method, an initial period of hypoactivity in the plasma was followed by a hyperactive phase in the serum. At the peak of hyperactivity, “prothrombin” values were about 180% of the control plasma. In slowly clotting bloods, the hyperactive phase developed less rapidly and persisted for a longer period than in normal blood. The abnormally high serum prothrombin values obtained by the one-stage test appear to be due to the evolution and persistence of the recently recognized serum factor which accelerates thrombin formation. Apparently this factor does not influence the 2-stage serum prothrombin values.

Assay of Plasma Antihemophilic Activity in Normal, Heterozygous (Hemophilia) and Prothrombinopenic Dogs.

Summary 1. A method is described for the assay of antihemophilic factor; results may be expressed in per cent of a control plasma or in units. Hemophilic blood, canine or human, is used as substrate. 2. In normal dogs, regardless of sex, and in females heterozygous for hemophilia, a steady state of plasma AHF appears to exist. The antihemophilic activity of heterozygotes is approximately the same as that of normal dogs. 3. The antihemophilic activity of plasma is maintained at normal levels following acute liver injury, as well as after dicumarol administration.

Antihemophilic factor (AHF): plasma levels after administration of AHF preparations to hemophilic dogs.

Summary 1. The plasma tide of AHF was measured after subcutaneous and intramuscular injections of bovine AHF fractions into hemophilic dogs. Better results were obtained with intramuscular than with subcutaneous administration; higher plasma AHF levels were maintained for longer times. Citrate in adequate concentration in the AHF solutions appeared to have an adjuvant action. 2. The rate of fall off of plasma AHF was followed after moderate levels of this clotting factor had been maintained for several hours. It is suggested that the biologic half-life of AHF in the dog may be at least 23 hours.

Plasma Antihemophilic Activity Following Total Body Irradiation.

Summary 1. Plasma antihemophilic activity of dogs subjected to total body irradiation remained at normal levels. 2. Whole hemophilic blood with a full complement of platelets accelerated the clotting of irradiated dogs blood, while platelet-poor hemophilic plasma did not.

Cation specificity of thrombocyte agglutinating activity (TAg) of canine plasma.

Summary The effect of each of 15 divalent or trivalent cations on the thrombocyte agglutinating activity (TAg) of canine plasma was tested. Adsorption of plasma with BaSO4 had little, if any, effect on TAg activity. In adsorbed resin-treated plasma TAg was active only in the presence of Mg++; Mn++, Fe++, Co++, or Ni++, with Mg++ being effective in the lowest concentration. Seeming differences in cation specificity of TAg in plasmas containing the chelating agents, oxalate and EDTA, are discussed.

Antiaccelerator (Anticonvertin) Activity of Canine Plasma and Serum.

Summary 1. Canine plasma and serum possess potent antiaccelerator activity, probably anticonvertin. This activity is retained after dialysis, but is thermolabile and is inactivated by multiple ether extractions. It is found in the precipitate obtained between 50 and 80% saturation with ammonium sulfate. Separation from antithrombin has not been accomplished. 2. An assay procedure for determining relative antiaccelerator activity is described. 3. Normal and hemophilic plasma and normal serum all have approximately the same antiaccelerator activity. 4. It is suggested that this new activity limits the tide of SA during clotting, analogous to antithrombin and the thrombin tide.

Serum Accelerator Factors and Antihemophilic Factor (AHF) in Early Phases of Clotting.

Summary 1. An eluate from BaSO4-ad-sorption of normal serum is shown to have a marked effect on the clotting time in hemophilia without altering the defect in prothrombin utilization. 2. The eluate is shown to operate chiefly by reducing the latent period of prothrombin utilization. 3. The effect of this eluate on AHF assays is discussed. 4. The necessity of platelets for prothrombin conversion is demonstrated again under different experimental conditions. 5. AHF is shown to be required for the demonstration of maximal prothrombic activity in the prothrombin time test.