Studies on the response of patients with classic hemophilia to transfusion with concentrates of antihemophilic factor. A difference in the half-life of antihemophilic factor as measured by procoagulant and immunologic techniques

Antihemophilic factor (AHF, factor VIII) levels were measured by a standard coagulation method and by an immunologic technique before and after infusion of AHF concentrates into patients with classic hemophilia. After infusion of AHF concentrates, the half-life of the AHF procoagulant (i.e., clot-promoting) activity varied from 12 to 14 hr, whereas that of the antigen ranged from 24 to 40 hr. The half-life of the antigen was similar in patients with and without circulating anticoagulants to AHF. The data are compatible with the suggestion that the antigen may be carried on a precursor molecule which the patient with hemophilia produces but cannot convert to the functional clot-promoting agent. Other explanations of the observations are, however, recognized.     

Studies on a proteolytic enzyme in human plasma; some factors controlling the rate of fibrinolysis

Methods are described for the assay of chloroform-activated and fibrinolysin-activated plasma proteolytic enzyme, and for the determination of the inhibitory activity of plasma or other substances against chloroform-activated enzyme. The inhibitory activities of plasma, serum, and crude plasma albumin against chloroform-activated plasma proteolytic enzyme were proportional to the logarithm of the concentration of the inhibitory substance tested. This suggests that enzyme and inhibitor react in accordance with the law of mass action. The rate of fibrinolysis of recalcified plasma clots could not be related to the total proteolytic activity available in the plasma, nor to the inhibitory activity of fresh plasma or serum against plasma proteolytic enzyme. During the incubation of a recalcified plasma clot at 37 degrees C., the inhibitory activity of its serum against plasma proteolytic enzyme decreased until a minimal stationary level was reached. The clot lysis time could be correlated directly with the time which elapsed until this minimal level was reached.     

Studies on the purification of antihemophilic factor (factor VIII). II. Separation of partially purified antihemophilic factor by gel filtration of plasma

A high degree of purification of antihemophilic factor was achieved by filtration of chylomicronpoor human plasma through columns of agarose. The final product contained, on the average, 67 units of antihemophilic activity per mg of protein, and was 3360-fold purified compared with the filtered plasma. The molecular weight of antihemophilic factor appeared to be at least two million. Preparations separated by gel filtration were contaminated with appreciable amounts of plasma thromboplastin antecedent (PTA), and traces of Christmas factor and Hageman factor, but no detectable fibrinogen was present. Similar fractions of plasma prepared from the blood of patients with classic hemophilia, von Willebrand's disease, or a circulating anticoagulant directed against antihemophilic factor contained, on the average, somewhat less protein than normal plasma; whether this difference was significant is not yet known. The purified fractions were partially stabilized by the addition of 1% gelatin. Adaptation of the technique of gel filtration to purification of antihemophilic factor for clinical use remains to be explored.     

Studies on the inhibition by propranolol of some human erythrocyte membrane enzymes and plasma cholinesterase

Human erythrocyte acetylcholinesterase and the plasma cholinesterase variants are not only inhibited by propranolol but have been found to show stereospecificity for its isomers. The erythrocyte enzyme has a greater affinity for the L-isomer than either the racemate or the D-isomer. In contrast the plasma cholinesterases have greater specificity for the D-isomer than the other isomer or racemate. The usual enzyme shows greater stereospecificity than the atypical enzyme and these findings present additional evidence that these enzyme variants differ in structure at the catalytic active site. Neither Na+ + K+ -ATPase nor Mg2+-ATPase show stereo-specificity for the isomers of propranolol although both enzymes are inhibited by the drug. The action of the drug on the four enzymes in blood samples obtained from patients having Huntington's disease was found to be identical to those observed on the enzymes in blood samples from healthy controls.     

Immunoelectron microscopic observations on the localization of fibronectin in normal human lung

The localization of fibronectin was examined in normal human lung using immunoelectron microscopy. Fibronectin staining was present not only in the endothelial, alveolar epithelial and bronchial epithelial basal laminae and associated with interstitial collagen fibrils and elastic fibers, but also in basal laminae of fibroblasts and smooth muscle cells. There was a periodicity in the staining of fibronectin on collagen fibrils. Reaction products against fibronectin were present in intracellular organelle including cisternae of smooth endoplasmic reticulum of endothelial cells and those of rough endoplasmic reticulum of fibroblasts. No other cells contained reaction products. The localization of fibronectin was compared with that of ruthenium red staining in normal human lung. The localization of fibronectin was consistent with that of proteoglycan.     

Studies on a proteolytic enzyme in human plasma; some factors influencing the enzymes activated by chloroform and by filtrates coccal fibrinolysin

1. Some conditions for the optimal activation of plasma proteolytic enzyme by chloroform have been described. The activation proceeds slowly. The action of chloroform is probably to remove some substance which inhibits or inactivates the plasma proteolytic enzyme preparation, rather than a direct activation of the enzyme. 2. Plasma proteolytic enzyme is activated by filtrates of cultures of beta hemolytic streptococci. When streptococcal fibrinolysin is present in maximally effective amounts, the activation is almost instantaneous. When the globulin is prepared from heated serum or the globulin is treated with chloroform, the activation of enzyme by streptococcal fibrinolysin appears to be catalytic. If the globulin is not so treated, the reaction appears to involve a stoichiometric process. 3. The plasma proteolytic enzyme activated by chloroform or by streptococcal fibrinolysin digests casein in direct proportion to the concentration of enzyme and to the time of digestion, during the early period of incubation. 4. Fibrinolysin-activated enzyme deteriorates rapidly relative to chloroform-activated enzyme. This may be due to the removal by chloroform of some substance which inactivates plasma proteolytic enzyme.     

Studies on a proteolytic enzyme in human plasma; the relationship between the proteolytic activity of plasma and blood coagulation

A fraction of globulin was prepared from human plasma which was deficient in prothrombin, thrombin, fibrinogen, plasma thromboplastin, and accelerator globulin. The preparation of globulin contained considerable potential proteolytic activity which could be activated by streptococcal fibrinolysin. This fraction of globulin accelerated the clotting of normal platelet-deficient plasma. However, the clot-accelerating effect of the globulin fraction was the same whether or not its proteolytic property had been activated. The addition of streptococcal fibrinolysin to normal platelet-deficient plasma did not accelerate coagulation. Nor did the addition of streptococcal fibrinolysin to hemophilic platelet-deficient plasma promote its coagulation. The data presented suggest that proteolysis by activated plasma proteolytic enzyme is not an essential stage in the coagulation of the blood.     

Human recombinant DNA-derived antihemophilic factor in the treatment of previously untreated patients with hemophilia A: final report on a hallmark clinical investigation.

BACKGROUND: Development of recombinant factor VIII (rFVIII) replacement therapy represents a milestone in the treatment of hemophilia A. OBJECTIVE: The objective of this long-term, multicenter study was to assess the safety, efficacy and rate of inhibitor formation of rFVIII (Kogenate) in the treatment of hemophilia A in a group of previously untreated patients (PUPs). PATIENTS AND METHODS: Between January 1989 and October 1997, 102 evaluable patients (mean age 3.9 years) were treated with rFVIII as sole therapy for prophylaxis against bleeding or for hemorrhage. Patients with mild hemophilia were treated for > or =2 years, while those with moderate or severe hemophilia were treated for > or =5 years or 100 exposure days. RESULTS: All patients responded well to therapy, so that 82% of bleeding episodes required a single infusion for treatment. Only four mild drug-related adverse events were recorded during the study for an overall rate of 0.03% (4/13 464 infusions). No viral seroconversions were observed. The inhibitor incidence in PUPs with severe hemophilia was 29% (19/65). Overall, inhibitory antibodies developed in 21 patients (20.6%). Inhibitor titers were low (<10 Bethesda Units) in nine of the 21 patients despite continued episodic treatment with rFVIII and transient in eight patients receiving episodic treatment (seven low titer, one high titer). Eight high-titer inhibitor patients were treated with immune-tolerance induction therapy; five had successful outcomes. CONCLUSIONS: The observed incidence of inhibitor formation is similar to studies of PUPs receiving plasma-derived FVIII. These results demonstrate the safety and efficacy of rFVIII in long-term treatment of hemophilia A.     

The effect of formula diets with a modified fat content on human plasma phosphatide fractions

Variation of human plasma phospholipids by formula diets with different composition of fat are caused by changes of the lecithin fraction, which amounts to 70 to 80% of total phospholipids. The changes of lysolecithin correspond to that of lecithin. The absolute amounts of sphingomyelin are unchanged by the different formula diets, while the relative amounts are negatively correlated to the lecithin fraction.     

Serologic evidence that factor IX inhibitor in the plasma of hemophilia B patients detects factor IX on normal red cells

BACKGROUND: Patients with hemophilia B lack factor IX (F IX). These patients may become alloimmunized after the transfusion of F IX concentrates and may develop F IX inhibitors, which have been characterized as polyclonal IgG4 alloantibodies. Two cases in which F IX inhibitors caused difficulty in compatibility testing and antibody identification were encountered. It was hypothesized that, because F IX is present in normal plasma, it might be adsorbed by red cells in vivo and then be detected during antibody screening tests with serum containing F IX inhibitors. CASE REPORT: Sera from two African American half-brothers with hemophilia B were incompatible with all common and rare red cell phenotypes tested in the anti-human globulin test, but did not react with each other's red cells. The brothers' red cell antibodies were neutralized with both normal plasma and a commercially available F IX concentrate, which indicated that the red cell incompatibility was most probably caused by their F IX inhibitors. Red cells from an unrelated patient with hemophilia B and a very low titer of F IX inhibitor were tested against the half-brothers' sera and did not react. The compatible red cells from one of the half-brothers and the unrelated patient with hemophilia B adsorbed F IX from normal plasma or F IX concentrate after 37 degrees C incubation; this rendered them incompatible with the plasma containing F IX inhibitor from the other half-brother. CONCLUSION: F IX appears to be present on normal red cells and may be detected during compatibility and antibody identification procedures when serum or plasma containing F IX inhibitors is tested.