Paroxysmal Nocturnal Hemoglobinuria : Plasma Factors of the Hemolytic System

This report demonstrates the role and to some extent the interrelations of various factors that are active in the PNH hemolytic system.

1. Activity of four plasma factors, probably protein in nature, has been demonstrated. Two of these factors are hemolytic against PNH red cells, but not against normal red cells. The other two inhibit PNH hemolysis. (a). The heat labile hemolytic factor is water soluble and is therefore present in the soluble fraction of serum that has been dialyzed against distilled water. It is almost completely destroyed by heating at 53 C. for 10 minutes. It is slowly inactivated by incubation at 37 C. with 100 units per ml. of thrombin. It is rapidly destroyed by concentrations of thrombin in excess of 200 units per ml. It is inactive unless the heat stable hemolytic factor is also present. (b). The heat stable hemolytic factor is insoluble in water and is therefore precipitated from serum by dialysis against distilled water. It is quite resistant to 100 units per ml. of thrombin and to incubation at 53 C. It is inactive unless the heat labile hemolytic factor is also present. (c). The heat labile inhibitor is insoluble in water and is therefore found in the insoluble fraction of serum dialyzed against distilled water. It is inactivated by heating at 53 C. for 10 minutes but not by incubation with 100 units per ml. of thrombin. (d). The heat stable inhibitor is found in the water-soluble fraction of dialyzed serum. It withstands dialysis poorly, but it is not affected by 30 minutes incubation at 53 C. Incubation at 37 C. with 100 units per ml. of thrombin for 10 minutes destroys its inhibitory activity. Apparently the inhibitors are not interdependent.

2. Calcium in small amounts is probably essential to the PNH hemolytic system. The concentration of calcium that is optimum for hemolysis lies in the neighborhood of 2.5 mM. The optimum is a little less than the amount normally present in the plasma. Calcium in excess inhibits hemolysis in vitro, and no hemolysis occurs when the concentration exceeds 25 mM. per liter.

3. Magnesium is also essential to the PNH hemolytic system. As magnesium is added to the system in vitro hemolytic activity increases until the concentration exceeds 10 mM. per liter. Amounts greater than that have some dampening effect. Magnesium appears to antagonize the heat stable inhibitor of the PNH hemolytic system.

4. Thrombin is involved in this system insofar as the heat stable inhibitor and the heat labile hemolytic factor may be destroyed by thrombic activity. The inhibitor is rapidly destroyed, the hemolytic factor slowly. Therefore, the sum of the reaction to small amounts of thrombin in the PNH hemolytic system is to increase hemolytic activity.

5. Dilute heparin and protamine increase the activity of the PNH hemolytic system in vitro, probably by blocking the two inhibitors. Heparin appears to work against the heat stable inhibitor, protamine against the heat labile inhibitor.

6. The intensity of PNH hemolytic activity whether in vitro or in vivo is probably related to a balance that exists between the inhibitors and the hemolytic factors. Hemolytic crises may occur when the hemolytic factors are increased or when their antagonists are depressed.