A B S T R A C T Immunoprecipitates containing guinea pig Factor VIII antigen were prepared from guinea pig plasma with a cross-reacting rabbit anti-human Factor VIII. Monospecific antisera to guinea pig Factor VIII antigen were produced in rabbits by using these washed immunoprecipitates as immunogens. The resulting antisera to guinea pig Factor VIII antigen detected Factor VIII antigen in guinea pig plasma and inhibited the von Willebrand factor activity in guinea pig plasma. This antibody also detected Factor VIII antigen in a solubilized protein mixture prepared from isolated cultured guinea pig megakaryocytes. Cultured guinea pig megakaryocytes were labeled with radioactive leucine. By radioautography, 96.2% of the radioactivity was present in megakaryocytes. The radioactive Factor VIII antigen present in the solubilized cell protein mixture was isolated by immunoprecipitation and characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The results demonstrate that cultured guinea pig megakaryocytes synthesize Factor VIII antigen which contains the same polypeptide subunit (mol wt 200,000) present in guinea pig plasma Factor VIII antigen.

To determine the locus of platelet production, we sought to determine if sufficient megakaryocytes reach the lungs in a state that could produce platelets. Elutriation was used to isolate megakaryocytes from blood reaching and leaving the lungs of 20 patients undergoing routine cardiac catheterizations. A mean of 5.0 intact megakaryocytes/ml were found in pulmonary artery blood, compared to only 0.5 megakaryocytes/ml, with partial cytoplasmic content, in aortic samples. The megakaryocytes in central venous and aortic samples were all mature. The identity of these cells as megakaryocytes, their maturity and normal morphology were confirmed by standard and immunoelectron microscopy. Cardiac outputs were obtained for each patient at the time of blood sampling, allowing an extrapolation that 40 × 106 intact, mature megakaryocytes were being delivered to the lungs every day in the average patient, compared to only 4.0 × 106 partially spent megakaryocytes exiting the lungs daily. About 98% of megakaryocyte cytoplasm reaching the lungs did not exit as recognizable megakaryocytes or fragments. The number and state of the megakaryocytes apparently filtered in the lungs is consistent with the hypothesis that megakaryocytes may shed platelets within the pulmonary microvasculature, which may be the primary site of platelet production.

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The in vitro effect of corticosterone in insulin and glucagon secretion has been examined in the isolated perfused rat pancreas preparation. COrticosterone at physiological concentrations was found to inhibit acutely and strongly the secretion of insulin induced by both glucose and arginine and to potentiate the output of glucagon in a glucose-free medium or when induced by arginine. Phentolamine, an alpha-adrenergic blocking agent, diminished the strong inhibitory effect of corticosterone on insulin secretion. The present results demonstrate that corticosterone has direct effects on pancreatic islet cells, and they suggest that the inhibition of insulin secretion is to some extent related to the alpha-adrenergic receptors.

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Human megakaryocytes have been isolated from marrow obtained from ribs removed at thoracotomy. All but one of the patients had normal pre-operative platelet and leucocyte counts. Megakaryocytes averaged 0.37% of all cells in marrow cell suspensions from nine consecutive subjects. A 283-fold purification (to 10.3%) was achieved by a density gradient centrifugation followed by two successive velocity sedimentations at unit gravity. The net yield, 12 800 megakaryocytes per specimen, was sufficient for many kinds of morphological study. Bright-field, phase contrast, and electron microscopy were used to characterize the younger and smaller megakaryocytes. Ploidy analyses were carried out on 100--235 megakaryocytes per specimen; 8N was the predominant ploidy class in isolated megakaryocyte populations from three individuals. The mean megakaryocyte diameter was 24 micrometers in three other specimens and the range was 10--48 micrometers. This data had a normal distribution and overlapped minimally with the size range of all other marrow cells. The presence of a distinct size threshold (at 11.5 micrometers) implied that size alone may be a sufficient objective criterion for identification of human megakaryocytes.

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