N. Raphael Shulman

Adenyl cyclase activity in human platelets.

Adenyl cyclase activity occurs in all animal cells examined with the exception of non-nucleated erthrocytes. Prostaglandins known to affect platelet aggregation and NaF known to alter platelet metabolism have been reported to alter adenyl cyclase activity. This study attempts to determine whether human platelets (non-nucleated cells) contained adenyl cyclase activity which might be modified by some of these agents. Isolated human platelets obtained from blood of normal adults were centrifuged washed and sonically disrupted using a sonicator. Isolated red cells and concentrated leukocytes were likewise processed and analyzed. Adenyl cyclase activity was demonstrated in washed particles from human platelets. The control activity levels in EDTA-prepared platelets were increased approximately 10-fold by NaF and about 18-fold by PGE1. This is the first time a substance other than NaF has caused maximal stimulation of adenyl cyclase. Removal of calcium by EDTA appear to increase PGE stimulation of adenyl cyclase without altering NaF stimulation. The findings confirm the hypothesis that calcium plays a role in regulating the sentivity of cell membranes to hormones or the hormone-like prostaglandins.

The Use of Platelet Serotonin Release as a Sensitive Method for Detecting Anti‐Platelet Antibodies and a Plasma Anti‐Platelet Factor in Patients with Idiopathic Thrombocytopenic Purpura*

Summary. The method described in this paper can be used for detecting anti‐platelet iso‐, hetero‐, and drug‐antibodies and for detecting a factor in the plasma of patients with idiopathic thrombocytopenic purpura that attaches to platelets (ITP factor). Plasma from the test subject, anticoagulated with citrate, is added to a suspension of normal platelets labelled with [14C] serotonin and the amount of radioactivity (serotonin) released is measured after incubating the mixture for 45 min at 37°C. Anti‐platelet antibodies and drug antibodies that fix complement as well as isoantibodies not detectable in vitro by complement fixation or agglutination can be measured by this test. Plasma from 24 of 40 patients with ITP released significantly more serotonin than control plasmas (P < 0.025). False positive results were obtained in less than 2.5% of plasmas from normal individuals, from patients with a variety of diseases and from patients with secondary thrombocytopenia. However, plasmas from two of 13 patients with systemic lupus erythematosus and normal platelet levels gave positive results in the test. The 7S gamma globulin fractions of plasmas containing isoantibodies or ITP factor as well as acid eluates from platelets which had been incubated in these plasmas gave positive serotonin‐release tests.

Immunological Considerations Attending Platelet Transfusion

1 Iso‐immunization against platelets occurs infrequently with less than ten transfusions, but the frequency rises steadily with the number of transfusions until all patients eventually become immunized. 2 Characteristics of specific antigens and antibodies that have been identified to date are described briefly and the relative merits of various immunologic technics used to measure platelet iso‐antibodies are discussed. 3 More than half of the anti‐platelet iso‐anti‐bodies that arise after transfusion are “incomplete” and cannot be detected by conventional serologic tests. Some of these antibodies have been identified by “blocking” tests or by passive transfer. These findings explain why refractoriness to platelet transfusions is not always accompanied by serologic evidence of immunization. 4 Isoimmune destruction of platelets is a relatively innocuous occurrence and does not contra‐indicate further trials of platelet transfusion. 5 Although platelets can be matched under some circumstances when iso‐immunization has taken place, at present empirical selection of donors based on survival of transfused platelets appears to be the best approach to platelet transfusion in sensitized individuals.

Studies on the purification and characterization of human factor VIII

Factor VIII (antihemophilic globulin) has been prepared from Hyland method IV AHG and cryoprecipitate using limited chymotryptic digestion followed by Sepharose gel filtration. The activity of factor VIII is unaffected by the digestion procedure, while fibrinogen in converted to large noncoagulable fragments. The purified factor VIII has been found to be a macromolecular glycoprotein with a major subunit of 240,000, as shown by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Carbohydrate analysis of factor VIII gave values of 1% sialic acid, 2.8% hexosamine, and 1-2% hexose (mannose, galactose, and fucose). The lipid content was found to be less than 5% of the protein content, and included no detectable phospholipid. The amino acid content is also reported. Immunoelectrophoretic analysis using rabbit antibody to purified factor VIII produced a single precipitin line. The chymotrypsin digestion step facilitates the preparation of factor VIII by reducing the viscosity of fibrinogen in the crude starting material, thereby increasing fivefold the quantity of material which can be processed at one time. It also improves markedly the resolution between factor VIII and fibrinogen on gel filtration.

IMMUNOREACTIONS INVOLVING PLATELETS. VI. REACTIONS OF MATERNAL ISOANTIBODIES RESPONSIBLE FOR NEONATAL PURPURA. DIFFERENTIATION OF A SECOND PLATELET ANTIGEN SYSTEM

Although there is some evidence that neonatal thrombocytopenic purpura, which occurs in otherwise normal children born of healthy mothers, is caused by maternal antibodies formed against fetal platelets (1-3), lack of reliable serologic techniques generally has prevented convincing demonstration of the responsible antibodies and has precluded thorough investigation of the properties and inheritance of specific platelet antigens presumed to be involved. Because experience in this laboratory has shown that complement fixation is the most sensitive technique for detecting several different types of antiplatelet antibodies (4-6), this technique was applied in a search for antibodies in the sera of normal mothers who had given birth to thrombocytopenic infants. In the present report the isoantibodies which were found are characterized, and the sensitivity of the complement fixation technique is compared with that of other techniques employed in attempts to detect antiplatelet antibodies.

DISTINCTIONS IN CHRONIC ACTIVE HEPATITIS BASED ON CIRCULATING HEPATITIS-ASSOCIATED ANTIGEN

Abstract Thirty cases of chronic active hepatitis in a general hospital population were screened for circulating hepatitis-associated antigen (H.A.A.), antinuclear antibodies (A.N.A.), and the L.E.-cell phenomenon. On the basis of these tests, the patients could be divided into three groups: group I (seven) were positive for H.A.A.; group II (nine) had either A.N.A. or L.E. cells; and group III (fourteen) were negative for all three tests. There was no overlap between groups I and II in that no patient positive for H.A.A. demonstrated antinuclear antibodies or L.E. cells. It was noteworthy that six group-I patients had a history compatible with parenteral transmission of hepatitis virus (shared needles or exposure to blood products) and five had an abrupt rather than insidious onset to their illness. In contrast, group-II patients had no such exposure history, and their illness tended to have an insidious onset. Whether the separation of chronic active hepatitis patients in this manner has significance in regard to course, prognosis, or therapy remains to be determined.

Multiple neoplasms in two siblings with a variant form of Fanconi's anemia

Two siblings with a variant form of Fanconis anemia developed multiple neoplasms after prolonged survival and treatment with androgens. One of the siblings developed two separate oral squamous cell carcinomata, and the other developed acute leukemia and hepatoma. Androgens may have had a carcinogenic role in the appearance of the hepatic neoplasm. There is an increased incidence of neoplasm associated with Fanconis anemia. This may be related to frequent spontaneous chromosomal aberrations and/or to increased cellular susceptibility to viral transformation.

Malaria: Host-Defense Mechanisms and Complications

Abstract The importance of specific diagnosis needs emphasis in relation to the increased prevalence of malaria at civilian medical facilities in the United States. In considering host-defense mech...

Inhibition of platelet energy production and release reaction by PGE1, theophylline and cAMP

Thrombin induces a rapid, simultaneous release from platelets of adenine nucleotides and calcium in a constant molar ratio. PGE1, theophylline, and dibutyryl cyclic AMP inhibit this release reaction, the attendant lactate production, and the subsequent aggregation of platelets. Compounds which result in increased intracellular levels of cAMP appear to block aggregation by inhibiting the release reaction and energy production.

Chromosomal Aberrations in Two Cases of Inherited Aplastic Anemia with Unusual Clinical Features

Abstract Two brothers had aplastic anemia similar to Fanconi anemia but without associated congenital abnormalities. Both had mild clinical courses and prolonged response to androgen therapy, one h...