Per Olsson

Synthetic chromogenic substrates for determination of trypsin, thrombin and thrombin-like enzymes

Abstract New, synthetic substrates for trypsin-like enzymes are described. These substrates consist in general of arginine p-nitroanilides in which the NH2-group of arginine is acylated with hydrophobic amino acids or peptides. The susceptibility of the peptide substrates is enhanced by benzoylation of the free NH2-terminal group. One of the most reactive substrates is Nα-benzoyl-phenylalanyl-valyl-arginine p -nitroanilide (Bz-Phe-Val-Arg-pNA). Some kinetic parameters for the substrates with trypsin, thrombin and Reptilase have been studied. Trypsin has a much higher affinity for Bz-Phe-Val-Arg-pNA than for benzoyl-DL-arginine p-nitroanilide (BAPNA). Bz-Phe-Val-Arg-pNA also has a high susceptibility toward thrombin and Reptilase. The narrow substrate specificity of thrombin as compared with trypsin is reflected by the fact that Bz-L-Phe-L-Val-L-Arg-pNA is rapidly hydrolyzed by thrombin while the rate of hydrolysis of Bz-D-Phe-L-Val-L-Arg-pNA is very slow. On the other hand the action of trypsin on the D-isomer is only slightly reduced. The synthetic peptide substrates are useful for determination of enzymes of the trypsin type, and they can also be used in biological fluids. Small amounts of trypsin, thrombin and Reptilase can be determined.

Studies of Adsorption, Activation, and Inhibition of Factor XII on Immobilized Heparin

The aim of the present investigation was to clarify whether immobilized heparin does, as previously suggested, prevent triggering of the plasma contact activation system. Purified FXII in the absence or presence of antithrombin and/or C1 esterase inhibitor as well as plasma was exposed for 1 to 600 seconds to a surface modified by end-point immobilization of heparin. With purified reagents, a process including surface adsorption and activation of FXII occurred within 1 second. In the presence of antithrombin, the resulting surface-bound alpha-FXIIa was inhibited within that time. Likewise, the adsorption of native FXII from plasma was a rapid process. However, the inhibition of surface-bound alpha-FXIIa was slightly slower than with purified components. Nevertheless, neither beta-FXIIa nor FXIa were found in the plasma phase. Exposure of a surface prepared from heparin molecules, lacking antithrombin binding properties, to plasma resulted in surface-bound alpha-FXIIa within 1 second. In the liquid phase, beta-FXIIa was detected after 2.5 seconds and, 12 seconds later, FXIIa and FXIa in complex with the C1 esterase inhibitor appeared. Addition of heparin to plasma prior to surface exposure did not prevent activation of surface-bound FXII, nor did it increase the beta-FXIIa inhibition rate and prevent FXI activation in plasma, although beta-FXIIa and FXIa-AT complex formation occurred. It is concluded that surface-immobilized heparin, unlike heparin in solution, effectively inhibits the initial contact activation enzymes by an antithrombin-mediated mechanism, thereby suppressing the triggering of the intrinsic plasma coagulation pathway.

The assay of antithrombin using a synthetic chromogenic substrate for thrombin

Abstract A rapid method for the assay of antithrombin activity is described in which a chromogenic synthetic tripeptide, benzoylPheValArgp.nitroanilide, is used as substrate for thrombin. During the reaction p-nitroaniline is released. The optimal conditions of the thrombin-antithrombin interaction with respect to pH, ionic strength and temperature have been determined. The results obtained with this method compare well with antithrombin assays using coagulation or immunological procedures.

Thrombin inactivation on surfaces with covalently bonded heparin.

About 8000 Daltons porcine mucosa heparin fragments were covalently bonded by end-point attachment to polyethylene. The interaction between the immobilized heparin, added thrombin, and antithrombin III [AT] was investigated. The heparin surface was adsorbed with either albumin, AT dissolved in albumin or Tyrode, or platelet free plasma. Irrespective of the pre-treatment procedure, exposure of the surface to thrombin resulted in the same substantial decrease of thrombin in solution and the same degree of surface-confined thrombin activity. It was concluded that the heparin surface has a large capacity to bind thrombin and that the thrombin inhibitory capacity of high affinity heparin fragments is limited. On exposure of the thrombin-loaded surfaces to defibrinogenated plasma or AT, the surface-confined thrombin was inhibited within 30 seconds. Successive dilutions of plasma or AT decreased the inhibition rate but not the inhibition capacity. It is concluded that inhibition of thrombin adsorbed on the heparin surface occurs as follows: Added AT adheres to high affinity heparin fragments on the surface whereupon adsorbed thrombin migrates in the hydrophilic heparin coating towards the reaction site of AT and becomes inhibited. The inactivated thrombin-AT complex leaves then the surface, thus enabling the process to be repeated.

Measuring the degree of plasma contact activation induced by artificial materials

Exposure of blood to foreign materials or to tissues other than the vascular endothelial lining leads to activation of the plasma contact activation system. This system includes the proenzymes coagulation factor XII (FXII), coagulation factor XI (FXI), prokallikrein and cofactor high-molecularweight kininogen (HMWK). On adsorption, FXII immediately undergoes a conformational change and the active enzyme a-FXIIa is formed. An autocatalytic process thereby commences, leading to triggering of the intrinsic coagulation, fibrinolytic and kallikrein systems as well as of the complement system with cell reactions that follow [1]. In recent publications, certain aspects of the mechanisms controlling the contact activation system have been revealed. The specific antithrombin (AT)-binding sequence, which is expressed by heparan sulphate in the glycocalyx layer on the endothelial lining and can be immobilized on materials by attachment of heparin, takes part in this process [2–4]. By being negatively charged, both the heparin surface and the endothelium adsorb and activate FXII on exposure to plasma. The formed a-FXIIa, however, is instantaneously inhibited by AT, the inhibitory effect of which is greatly accelerated by binding to the specific ATbinding sequences on heparan sulphate or heparin [4,5]. The required acceleration of AT for the fast inhibition of a-FXIIa in the protein layer on the surface cannot be substituted by heparin in plasma [6]. Since the degree of contact activation may play a key role with regard to blood compatibility of artificial materials, it seems important that materials used for circulatory support, mechanical heart valves, vascular prostheses, stents, etc., should have very low or ideally no potential to trigger the plasma contact activation system. Methods to estimate contact activation potential of materials have not been available. A test device, which in itself does not trigger the plasma contact activation system, would open the possibility to study the degree of contact activation exerted by test materials on exposure to plasma. In the present investigation, it is shown that a stable heparin surface does not induce contact activation. The present investigation was designed to clarify if disposable polystyrene cuvettes modified with immobilized functional heparin would be useful in assaying the contact activation potential of other artificial materials.

Inhibition of the plasma contact activation system of immobilized heparin: Relation to surface density of functional antithrombin binding sites

End-point immobilization of heparin to artificial materials gives rise to a surface that prevents triggering of the plasma contact activation system and, presumably as a result thereof, generally has thrombo-resistant properties. The present investigation was undertaken to determine what density of immobilized heparin molecules expressing functionally intact antithrombin binding sites is required to achieve these blood compatible properties. Six different heparin surfaces were prepared on polyethylene tubing and studied in contact with human plasma. The content of bound heparin was the same on all surfaces while the densities of antithrombin binding sites ranged from 1 to 28 pmol/cm2. The surfaces expressing 4 pmol/cm2 or more of specific antithrombin binding sites generated no measurable enzymatic activity in contact with plasma, either on the exposed surfaces or in the plasma phases. Below this level, the degree of activation gradually increased with decreasing densities, and in parallel the thrombo-resistant properties deteriorated. Addition of heparin to the plasma phase reduced the capacity of the heparin surfaces to bind antithrombin, leading to a diminished ability of the surfaces to prevent contact activation. This finding supports the hypothesis that antithrombin is the critical coagulation inhibitor for the suppression of contact activation on end-point immobilized heparin.

On the platelet-fibrinogen interaction.

Abstract It is shown that platelets in platelet rich plasma or platelets isolated by gel filtration will adsorb to fibrinogen or fibrinmonomer coupled to Sepharose. The adsorption appears to require divalent cations. In comparison with control columns the adsorption is not accompanied by significant release of serotonin or ADP and it occurs in the presence of indomethacin. In the presence of PGE1 the adsorption is decreased. The adsorbed platelets release serotonin in the presence of thrombin and also in the presence of ADP but to a lesser extent. It is suggested that the adsorption is due to exposure of new binding sites for platelets on the fibrinogen molecule as a result of conjugation to Sepharose. The adsorption process is distinguishable from that involving collagen but may be similar to the adsorption of platelets to fibrinogen on artificial surfaces.

The effect of plasma antithrombin concentration on thrombin generation and fibrin gel structure

Congenital deficiency of antithrombin (AT) is associated with thrombotic events and AT consumption occurs in some severe disorders and after treatment with heparin. The aim of this study was to investigate whether variations in the level of plasma AT modify thrombin generation and the fibrin formation process after the intrinsic coagulation mechanism is triggered. Normal plasma was depleted of AT by immunoadsorption on CNBr-Sepharose coupled with the anti-AT-IgG fraction of antiserum. The AT-depleted plasma was reconstituted with AT (between 0.3 and 1.5 AT units per ml). Thrombin generation was measured as the development of thrombin-antithrombin complexes (TAT). The lag phase preceding fibrin formation depended on the concentration of AT. The short lag phase was seen in completely AT-depleted plasma and the long in plasma with 1.5 AT units per ml. TAT generation, determined in parallel consecutive samples, showed that the rate at which thrombin was generated was inverse to the AT concentration in plasma. The network structure of hydrated fibrin gels in the clotted plasma was studied by measuring the wavelength dependence of gel turbidity. The mass/length ratio value, -i.e. the thickness of fiber strands and porosity of the gel increased with increasing AT concentrations. It is concluded that plasma AT regulates the rate of prothrombin-thrombin conversion, the clotting time and the consequently network structure of the fibrin gel.

Uptake and inhibition of thrombin by the vascular wall

Thrombin activity and inhibition were assayed on the aortic surface of dogs and pigs. After sacrifice, the aortae were excised and thrombin activity was measured with an amidolytic assay. A small thrombin activity was found on the endothelium. Heparinization of the animals lowered endothelial thrombin activity. After exposure of the aortic endothelium to a thrombin/albumin solution in vitro, thrombin activity disappeared from the solution and was recovered on the surface. De-endothelialized vessels took up more thrombin than those with intact endothelium. Endothelium confined thrombin was rapidly inactivated when exposed to plasma. A slow inactivation was seen upon exposure to a modified Ringers solution. Thrombin confined to de-endothelialized aortae, i.e. to medial structures, was always inactivated at a slow rate irrespective as to whether it was exposed to plasma or Ringers solution. It is concluded that endothelium and subendothelium bind thrombin and subsequently inactivate it. The inactivation proceeds faster on endothelium when exposed to plasma. The possible role of glycosaminoglycans is discussed.

The stability of glutardialdehyde-stabilized 35-heparinized surfaces in contact with blood.

Heparin can be bound to polymer surfaces by precipitation of an ionic heparin-amine complex which can be stabilized against dissolution by treatment with glutardialdehyde. The aim of this investigation was to study the degree and course of desorption of heparin from such a heparinized surface on contact with blood. This desorption must be considered when analysing the interaction between the heparinized surface and blood. Different heparinized surfaces were prepared by using 35S-labelled heparin, and the desorption of heparin on exposure in vitro to citrated plasma or heparinized blood as well as during exposure at in vivo conditions was quantified. During in vitro experiments, the glutardialdehyde stabilized surface became stable with no further desorption of heparin after an initial loss of about 3% of the initial surface-bound heparin. Under in vivo conditions, there was an initial loss of about 12%. There was no further loss from surfaces inserted into the circulation of the dog for seven days as compared to those inserted for one hour.