Inhibition of the plasma contact system on endpoint immobilized heparin and human endothelium

Abstract: When blood is exposed to foreign materials or injured tissue, several defense systems are activated. The contact with an artificial surface leads to activation of FXII, the first proenzyme of the plasma contact activation system, with concomitant triggering of the intrinsic coagulation and also the fibrinolytic-, kallikrein- and complement systems. A successful technique to improve the blood compatibility of artificial materials is end point attachment of heparin. This technique for coupling preserves the functional properties of the heparin molecule, giving rise to a surface with highly thromboresistant properties. Ideally, a blood compatible material should prevent the triggering of defense mechanisms rather than counteracting the consequences of a continously ongoing activation process. The present thesis, therefore, deals with the activation of FXII on surfaces of end point attached heparin and the role of the plasma coagulation inhibitor antithrombin (AT) and of the specific heparin AT-binding sequence in this process. Comparative studies were made on the endothelial surface of the natural vascular wall which exposes heparan sulfate a substance structurally and functionally closely related to heparin, to the circulating blood. Surfaces of functionally active strandard heparin and low afffinity (LA) heparin, were prepared on the inside of 1 mm polyethylene tubing. The LA heparin lacks the specific AT-binding sequence and is essentially devoid of anticoagulant activity. FXII was determined as prokallikrein activating capacity, either in the presence (Total FXII) or absence (Spontaneously generated FXlla) of the FXII-activating agent ellagic acid. Both surfaces adsorbed FXII from plasma to a similar extent, measured as depletion from the liquid phase and as total FXII on the surfaces. On the standard heparin surface, FXII was recovered in its precursor form, FXII. In contrast, the major fraction of surface-associated FXII on the LA heparin surface had undergone spontaneous activation to FXlla. This activation process could not be prevented by addition of free standard heparin or low molar mass heparin to the plasma phase. Exposure of the surfaces to plasma depleted of AT led to extensive activation of FXII even on the standard heparin surface. This activation could be reversed by reconstituting the exposed plasma with AT. In contrast, the effect of removal of C1 esterase inhibitor (C1 INH), considred to be a main inhibitor of FXlla, was insignificant Similar results were obtained using solutions of purified FXII instead of plasma. Repeated cycles of activation of adsorbed FXII with ellagic acid led to consumption of bound FXII and also of AT. By preparing surfaces with a constant amount of heparin while varying the content of the specific AT-binding sequence, it could be demonstrated that a certain critical density of these functional units in the immobilized heparin is required for effective suppression of FXII activation. Moreover, free heparin in plasma decreased the uptake of AT on the immobilized heparin in a dose dependent manner, resulting in deterioration of the FXII inhibitory capacity of the surface as well as of the capacity to prevent clot formation. The adsorption and activation of FXII on the heparin surface is a rapid process with essentially complete uptake within one second. The FXlla activity, i.e. alpha-FXlla, observed only during the initial 12 seconds of plasma exposure indicates that the inhibitory process is slightly slower than the activation. Beyond 12 seconds the alpha-FXlla activity on the surface ceased and FXlla-AT complexes were formed. However, the activation never was transmitted to the liquid phase since neither ß-FXIIa nor FXla could be demonstrated in the exposed plasma. It is concluded that alpha-FXlla formed on the heparin surface is effectively inhibited by AT by a mechanism directly dependent on the specific AT-binding sequence in the immobilized heparin. Thereby the autocatalytic amplification of the contact activation mechanism is suppressed, and as a result, the triggering of the intrinsic coagulation as well as of the fibrinolytic-, kallikrein- and complement systems is prevented. In parallel to the heparin surface, the endothelial surface of human saphenous veins prevented spontaneous activation of adsorbed FXII. The amount of adsorbed proenzyme was about the same as for the heparin surface, as judged from the prokallikrein activating activity generated by exposure to ellagic acid. However, on vein segments harvested after administration of systemic heparin, the adsorbed FXII was present largely in its activated form, and in smaller quantities. The FXlla inhibitory activity of the endothelial surface could be restored by exposing it to a solution of purified AT. It seems evident that FXlla is under the control of an AT-dependent inhibitory mechanism on the endothelial surface similar to that described for the heparin surface. This mechanism probably renders the natural vascular wall compatible with the contact activation system and defense systems triggered thereby.