Abstract. Binding of '25I-Factor XIa to platelets required the presence of high molecular weight kininogen, was enhanced when platelets were stimulated with thrombin, and reached a plateau after 4-6 min of incubation at 370C. Factor XIa binding was specific: 50-to 100-fold molar excesses of unlabeled Factor XIa prevented binding, whereas Factor XI, prekallikrein, Factor XIIa, and prothrombin did not. When washed erythrocytes, added at concentrations calculated to provide an equivalent surface area to platelets, were incubated with Factor XIa, only a low level of nonspecific, nonsaturable binding was detected. Factor XIa binding to platelets was partially reversible and was saturable at concentrations of added Factor XIa of 0.2-0.4 ug/ml (1.25-2.5 MM).

We tested whether vasorelaxation of coronary arteries is altered after overnight (18-22 h) exposure to physiological levels of 17 beta-estradiol. Ring segments of left circumflex coronary artery from six female and six castrated male pigs were incubated in vials of sterile Dulbecco's modified Eagle's medium with 1 nM 17 beta-estradiol, 1 nM 17 beta-estradiol + 10 nM tamoxifen, 1 nM 17 alpha-estradiol, or estrogen vehicle (ethanol) under normoxic conditions in an O2-CO2 incubator at 37 degrees C for 18-22 h. Coronary rings, with and without endothelium, were then suspended in vessel baths for measurement of isometric force. Vasorelaxation responses to the calcium ionophore A-23187, ADP, and nitroglycerin were examined in the rings after prostaglandin synthesis blockade and precontraction with U-46619. Sensitivity to A-23187 (-log M concentration required for 50% of maximal relaxation) was significantly enhanced in coronary rings with endothelium from females and castrated males when rings were incubated with 17 beta-estradiol but not when they were incubated with 17 alpha-estradiol or 17 beta-estradiol+tamoxifen. Acute (2h) exposure of coronary arteries to 1 nM 17 beta-estradiol did not alter responses to A-23187. 17 beta-Estradiol (1 nM) was not itself directly vasoactive in coronary arteries with or without prior incubation with the steroid. Vasorelaxation of rings with and without endothelium to ADP and nitroglycerin was not significantly different among the treatment groups. Relaxation to A-23187, but not ADP, was abolished by removal of the endothelium or exposure to 100 microM NO2-L-arginine.(ABSTRACT TRUNCATED AT 250 WORDS)

No abstract

We have recently shown that the triton X-100 insoluble cytoskeletons from thrombin-activated platelets contain coagulation Factor Va (Tuszynski, G.P. et al. J. Cell. Biol. 87, 219, 1980). Factor Va activity was measured by two independent techniques, a one-stage clotting assay, and a two- stage chromogenic assay utilizing purified Factor Xa, prothrombin, and the chromogenic substrate S-2238. Further analysis of these cytoskeletons by SDS-PAGE and by immunodiffusion revealed the presence of fibrin. These findings suggest that a common mechanism may link alpha granular proteins to the platelet cytoskeleton. To investigate this mechanism, platelet cytoskeletons prepared from platelets treated under various conditions were assayed for Factor Va activity. Both the rate of Factor Va appearance and the final level of activity associated with the cytoskeleton were diminished 30-50% in platelets that were treated with aspirin (500μM) or with indomethacin (20μM). When secretion of Factor V, serotonin and nucleotides was inhibited greater than 90% by incubation of a platelet suspension with 2-de- oxyglucose (30μM), gluconolactone (40μM) and antimycin-A (9μM), cytoskeletons prepared from this platelet suspension contained less than 10% Factor Va activity of controls. The rate of appearance of cytoskeletal Factor Va activity increased with increasing thrombin concentration while the final level remained constant. Calcium ionophore, A-23187 (1μM), released Factor V but less than 2% was associated with the cytoskeleton suggesting that Factor Va, the activated from of Factor V, was required for binding. We conclude that prior secretion of Factor V and its further activation by thrombin are necessary for the association of coagulation Factor Va with the cytoskeleton. These results are consistent with the hypothesis that released Factor Va becomes associated with the cytoskeleton via cell surface components.

No abstract