Fibrinogen biosynthesis in isolated guinea pig megakaryocytes

Fibrinogen synthesis was investigated in guinea pig megakaryocytes. Purified megakaryocytes were incubated with 35S-methionine in methionine-free incubation medium for 18 hours. Newly synthesized fibrinogen in megakaryocyte lysates enriched with purified carrier guinea pig fibrinogen was immunoprecipitated with a specific anti- guinea pig fibrinogen antiserum produced in rabbits. Proteins in the immunoprecipitates were analyzed with a 3.5% to 10.0% gradient polyacrylamide slab gel electrophoresis and auto-radiography. Radioactivity was detected in a protein band of 340,000 daltons. In order to verify fibrinogen synthesis, immunoprecipitate was analyzed by two-dimensional slab gel electrophoresis: (1) the first dimension separated unreduced fibrinogen using a 3.5% to 10.0% gradient gel; (2) following reduction by 2-beta-mercaptoethanol, fibrinogen chains were separated in the second dimension using a 10% gel. Alpha, beta, and gamma fibrinogen chains, which represented carrier guinea pig plasma fibrinogen, were visualized by Coomassie brilliant blue. Autoradiography identified the incorporation of radioactivity into the three fibrinogen chains. In control experiments, immunoprecipitates, produced by exposing megakaryocyte lysates to preimmune rabbit serum and goat anti-rabbit IgG, were also analyzed by the two-dimensional gel system. Radioactivity was not detected in sites corresponding to the migration of fibrinogen subunits. The study demonstrates that isolated guinea pig megakaryocytes can synthesize fibrinogen. The electrophoretic mobility of newly synthesized fibrinogen and subunits is similar to that of guinea pig plasma fibrinogen.     

Effects on the buoyant density of rabbit platelets of ADP and agents that increase the concentration of cyclic AMP

Rabbit platelets were aggregated by adenosine diphosphate (ADP), allowed to deaggregate and then separated into density subpopulations by centrifugation through discontinuous Stractan density gradients. Although ADP causes little or no release of the contents of the amine storage granules of rabbit platelets, ADP caused a decrease in platelet density as compared with control platelets subjected to the same procedures except for exposure to ADP. The density change persisted for at least four hours. The apparent size of platelets stimulated with ADP increased initially, but returned to control values during a one-hour period. A similar decrease in platelet density was observed with an albumin density gradient. Under conditions in which aggregation did not occur in response to ADP with ethylenediaminetetraacetic acid (EDTA) in the medium, little or no decrease in platelet density was observed. Agglutination with polylysine did not change platelet density. Thus, not only agents such as thrombin and plasmin that cause the release of the contents of the platelet granules decrease platelet density, but ADP also has this effect. Platelets would be exposed to all of these stimuli during thromboembolic processes, and their effect on platelets may account for the decrease in platelet density observed previously in experiments with rabbits with indwelling aortic catheters. Agents that increase the concentration of cyclic AMP (cAMP) in platelets (PGE1, adenosine, dibutyryl cAMP, forskolin, and papaverine) also decreased platelet density. This effect persisted when the platelets were washed and resuspended in fresh medium and was also demonstrable in plasma. Platelet size was gradually increased by prostaglandin E1 (PGE1) which maintains platelets in a disc shape and does not cause the release of granule contents, indicating that the decrease in platelet density caused by PGE1 may be attributable to platelet swelling.     

Glycoprotein IIb-IIIa complex and Ca2+ influx into stimulated platelets

Changes in intracellular Ca2+ concentrations [( Ca2+]i) in platelets stimulated with aggregating agents were measured with the fluorescent indicator dye quin 2. Ca2+ influx, but not intracellular mobilization, in response to adenosine diphosphate (ADP), platelet aggregating factor (PAF-acether), and sodium arachidonate was significantly inhibited by monoclonal antibodies against the glycoprotein (GP) IIb-IIIa complex; inhibition of thrombin-stimulated influx was inhibited to a lesser extent and reached statistical significance only at thrombin concentrations of 0.1 U/mL and below. Anti-GP Ib and HLA-ABC monoclonal antibodies had no effect on Ca2+ influx in response to any agonist. Thrombasthenic platelets gave normal [Ca2+]i responses to ADP and thrombin, which were not inhibited by an anti-GP IIb-IIIa antibody. It is suggested that Ca2+ influx in response to weak agonists occurs predominantly via a channel closely adjacent to the GP IIb-IIIa complex, but that higher concentrations of thrombin and A23187 also stimulate influx via another pathway.     

Effect of long term treatment with calcitriol on calcium absorption and mineral metabolism in postmenopausal osteoporosis

The mechanism of impaired calcium absorption in postmenopausal osteoporosis is controversial. If it is caused by calcitriol deficiency, it should be corrected by long term administration of physiological doses of this metabolite, whereas if it is caused by either a primary defect in calcium absorption or intestinal resistance to calcitriol action, it should not be. In 56 osteoporotic women, mean (+/- SE) fractional calcium absorption (0.52 +/- 0.02) was lower (P less than 0.001) than that in 20 age-matched normal women (0.61 +/- 0.02). Treatment with calcitriol (0.5-0.75 micrograms/day) increased calcium absorption after 6-12 months to 0.67 +/- 0.02 (P less than 0.001) and, in 29 patients, to 0.66 +/- 0.02 (P less than 0.001) after 24 months. After treatment, only 1 patient still had subnormal calcium absorption. In 26 patients treated with placebo for 6-12 months, there was no significant change in calcium absorption. For all studies, total calcium absorption (fractional absorption X estimated dietary intake) correlated directly with urinary calcium excretion (r = 0.61; P less than 0.001). Urinary hydroxyproline excretion, an index of bone resorption, was 31.0 +/- 1.5 mg/dl glomerular filtration rate (GFR) initially and decreased during treatment to 24.6 +/- 1.1 mg/dl GFR (P less than 0.001) after 6-12 months and to 27.9 +/- 1.3 mg/dl GFR (P less than 0.01) after 24 months. The finding that calcium absorption can be normalized by small doses of calcitriol supports the hypothesis that insufficient endogenous production of calcitriol is the major cause of decreased calcium absorption in postmenopausal osteoporosis.