Importance of number and shape of platelets for contact activation of the blood kallikrein-kinin systemin vitro

Abstract

Activation of Hageman factor (HF) on contact with artificial surfaces is a widely used model for the investigation of the blood kallikrein--kinin system (KKS).It has been shown that activation of HP systems in the micricirculation can be mediated by proteinases or functionally modified blood and endothelial cell membranes [6,7,9]. The role of platelets, structurally and functionally the most labile blood cells, carrying both HF itself [3,4] on their surface and also a number of specific proteins which participate in activation of the blood clotting system, is particularly important in this respect.We know, in particular, that purified HF can be activated by platelets stimulated by ADP and collagen, and also that platelet factor 4 prevents contact activation of HF [7,8]. The character of the activating principle is one of the key factors in the biochemical mechanism of regulation of KKS, linked functionally with clotting and fibrinolysis sytems [i].The role of number and shape of platelets for contact activation of KKS was investigated. The main aim was an attempt to separate the plasma (biochemical) and cellular (membrane) components of activation of this system. EXPERIMENTAL METHODMale Wistar rats weighing 180-300 g, kept on a standard diet, were used.To obtain the different platelet f~actions, citrated blood (1:9), obtained from the right ventricle of animals anesthetized with pentabarbital, was used.The principal fraction containing platelets (i) was obtained by centrifugation of citrated blood at 1500 rpm for i0 min.This fraction contained from 5"i0 s to 3"10 ~ cells in 1 ~i. To obtain platelet-free plasma (2), fraction 1 was recentrifuged at 7000 rpm.This fraction contained from 0 to 25,000 cells/ml. Next, to obtain "washed" platelets the residue was washed with Tyrode solution not containing calcium or magnesium ions. Cells obtained from 2 ml of fraction 1 were resuspended in 0.5 ml of Tyrode solution.All the procedures indicated above were undertaken at room temperature, using plastic and siliconized vessels. The "washed" platelets were frozen and thawed 5 times, using dry ice.In this way up to 95% of the cells were disintegrated. The suspension of disintegrated platelets was ultracentrifuged at i00,000 g for 20 min.The supernatant fraction, into which the soluble contents of the platelets has passed, was used; the membrane fraction was resuspended in Tyrode solution.This fraction contained the disintegrated bodies of the platelets, their fragments, and fragments of membranes.The initial arginine esterase activity and the prekallikrein (PKK) level in the blood plasma were determined by the method in [2], using TAME (N-a-tosyl-L-arginine methyl ester, from Reanal, Hungary) as the substrate. The composition of the incubation mixture was changed depending on the series of the investigation, but the conditions of determination on the whole (ratio of the components, conditions of incubation, quantity of added reagents, etc.) always corresponded to the method indicated above. The shape of the nature and modified...