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During protein depletion produced by plasmapheresis and a very low protein diet there is a proportionately greater decrease in extravascular, extracellular fluid protein than in plasma protein. A shift in the normal ratio of protein in these 2 compartments, approximately 1 to 1 in the dog, to over 2 to 1 as a result of depletion indicates an important, labile source of reserve protein for the plasma in the interstitial fluids. This reserve source is limited since a maximum drop of 50 per cent in the total exchangeable pool and of 75 per cent in the extravascular, extracellular protein occurred after both shorter and longer periods of depletion. Under the rigid conditions of these experiments additional plasma protein removal was associated with loss of weight despite adequate caloric intake. Investigation of the status of the interstitial fluid proteins in other conditions associated with disturbed protein metabolism seems warranted.

Two mechanisms have been described whereby rabbit platelets in vitro may be induced to release their contained histamine by the reaction of antigen and antibody. Both processes require the participation of the complement system. In the first, the adherence of platelets to particulate antigens such as zymosan or erythrocytes which have fixed complement through the third component was followed by histamine release. Plasma lacking C6 activity was fully active in this system. In the second mechanism, the reaction of soluble antigen with antibody in the presence of plasma also caused release of histamine from platelets and platelet clumping was observed. The release process, which appeared to follow the adherence of platelets to the immune complex, required the action of C6 and perhaps the later-acting components. No evidence could be obtained that a soluble factor was produced which caused the release. Instead, an inhibitor of the release process was detected after incubation of antigen, antibody, and plasma. Antibody preparations capable of giving complement-dependent PCA reactions in rabbits were also shown to induce the release of histamine from platelets in vitro.

Four basic proteins that increase vascular permeability have been isolated in purified form from rabbit neutrophilic granules. These proteins are termed band 1, 2, 3, and 4 protein according to their electrophoretic migration in acrylamide gel. Molecular weights of band 1 and 2 protein derived from amino acid composition were 4800 and 5300, respectively. These values are in good agreement with those obtained for these proteins by gel diffusion techniques. The molecular weight of band 3 protein was also in the range of 5000 by the latter technique. The molecular weight of band 4 protein determined by ultracentrifugal analysis and amino acid composition was 12,000. Although all four proteins had the capacity to induce immediate increase in vascular permeability, only band 2 protein was found to release histamine from isolated rat peritoneal mast cells. Furthermore, it has been shown that the permeability-inducing activity of band 2 protein can be inhibited by pretreating rabbits with antihistamine. Band 2 protein did not release histamine from rabbit platelets and depletion of rabbit platelets from the circulation had no influence on the permeability-inducing activity of this protein. Band 1, 3, and 4 proteins did not release histamine from isolated rat peritoneal mast cells and their capacity to increase vascular permeability remained unaffected by treatment of rabbits with antihistamine. These investigations suggest that the histamine-releasing activity of band 2 protein is a specific phenomenon and is associated with particular amino acid grouping or spacial configuration of the molecules. By the same token, the increase in vascular permeability induced by the nonhistamine-releasing band 1, 3, and 4 proteins represents a specific phenomenon (or phenomena) not particularly related to the over-all charge of these molecules.

Anaphylatoxin activity was derived from both human C'5 and C'3 molecules. This was achieved in the case of C'5 by interaction with trypsin or with EAC'4, oxy2a, 3. The smooth muscle-contracting material obtained from the treated C'5 was found to be a fragment of approximately 9,000–11,000 molecular weight. Its action was inhibited with antihistamine. The trypsinized C'5 also increased vascular permeability in guinea pig skin. When human C'3 was incubated with C'3 inactivator complex, which consists of a cobra venom protein and a ß-globulin of human serum, anaphylatoxin activity was observed. The activity was associated with a fragment cleaved from the C'3 molecule, having a molecular weight of between 6,000 and 15,000 as determined by gel filtration techniques. Similar activity was derived from C'3 by the C'3-converting enzyme in free or in cell-bound form. The C'5 anaphylatoxin failed to cross-desensitize guinea pig ileum to the contracting capacities of C'3 and guinea pig anaphylatoxin and vice versa. Anaphylatoxin prepared from C'3 by all methods mentioned above caused cross-desensitization to the other C'3 derivatives, but failed to desensitize to guinea pig anaphylatoxin.

The isolation and characterization of the first component of the kinin-forming system in human and rabbit plasma are presented. Functionally, the molecule is the precursor of the activator of prekallikrein (Pre-PKA) and evidence is presented that it is identical with Hageman factor (clotting factor XII). The component from each plasma possessed similar characteristics. This molecule was found to have a mol wt of 110,000 and sedimentation rate of 4.6S. It migrated in electrophoresis as a ß-globulin, having an isoelectric point of 6.1. Upon activation with glass, kaolin, diatomaceous earth, ellagic acid, or trypsin, the activated molecule converted purified prekallikrein (prokininogenase) to the active enzyme. Clot-promoting activity was associated with the capacity to activate prekallikrein through each procedure of isolation. The clot-promoting factor was in precursor form, requiring treatment with kaolin or trypsin to gain activity. Evidence indicated that the protein was Hageman factor (factor XII): it promoted clotting of factor XII-deficient, but not Factor XI- or IX-deficient plasma, and did not convert fibrinogen to fibrin it bound to and was activated by kaolin or other negatively charged particles in the presence of chelating agents; the activation by kaolin could be prevented by pretreating the kaolin with hexadimethrine bromide (H Br); prekallikrein-activating and clot-promoting activities were identical in their physical properties; and the prekallikrein activator could not be detected in Hageman factor-deficient plasma. Activation of Hageman factor was accompanied by cleavage of the molecule into several fragments, one of which possessed prekallikrein-activating (PKA) and clot-promoting properties. The PKA fragment sedimented at 2.6S and by gel filtration was found to have a molecular weight of 32,000. The PKA possessed only 1/50 the clot-promoting capacity of the freshly activated native molecule.

By depletion of C3 from rabbits undergoing acute experimental immune complex disease with an anticomplementary factor in cobra venom, it has been possible to demonstrate that deposition of the complexes in arteries and glomeruli does not require the complement components reacting after C2. Immunological reactions, in which platelets release their vasoactive amines, have been examined in rabbits undergoing immune complex disease. A correlation was obtained between the presence of a complement-independent reaction which required blood leukocytes, antigen and platelets, the deposition of immune complexes, and the induction of glomerulonephritis. C3 depletion did, however, have a marked alleviating effect on the severity of the arterial lesions. Neutrophil accumulation and the subsequent necrotizing arteritis were prevented. In contrast, the character and severity of the glomerulonephritis was not altered by depletion of later-acting complement components.