Immobilized heparins

Immobilizing heparin and heparan sulfate onto fused-silica capillaries using biotin-neutravidin conjugation has been applied to ACE analysis (73). These capillaries exhibit markedly reduced electroosmotic flow and were able to distinguish heparin-binding peptides that differed only in the stereochemistry or type of amino acid residue. No resolution of these peptides was observed if heparin or heparan sulfate was included in the buffer phase. 

Methods for preparing heparin-containing polymeric materials by means of ionic and covalent immobilization of heparin on various polymers are surveyed. The data on the biological activity of heparin are discussed as well as the probable mechanisms of thromboresistance enhancement endowed to polymeric materials by this anticoagulant. Some approaches toward an increased efficiency of anticoagulant properties of immobilized heparin are analyzed, and the position of heparin-containing polymers among other biomedical polymers is discussed. 

The year of 1961, when Vincent Gott11 observed the inhibition of thrombus formation by immobilized heparin for the first time, was marked as the second birth date of heparin, since it was for the first time isolated from liver tissue. Its anticoagulant action was detected in 1892. Although more than 20 years have passed since Gott s publication, there is still much confusion concerning the views on the mechanism of enhanced thromboresistance of heparin-modified polymers, which greatly hinders the introduction of HCP into clinical practice. 

The aim of the present review is to systematize and analyze the results of investigations concerning the synthesis and properties of HCP on the basis of literature data and own results. The probable mechanism of the effect of polymer-bound heparin on the blood clotting system will be discussed. A survey of the most promising pathways for further development of the chemistry of HCP involving the development of methods of their synthesis as well as the increase of the activity of immobilized heparin itself will be given. 

Polystyrene itself is not used for endoprosthetic purposes and its application is accounted for only because of easy substitutions in benzene rings. The method was subsequently modified for heparinization of silicone and natural rubber, polyethylene, polypropylene, polyethylene terephthalate), and other polymers. Styrene was first grafted onto the polymers by y-radiation and then the above-described reaction was performed in the second step. All the polymers synthesized in this way contained sufficiently large amounts of immobilized heparin (2.8—15.7 ng/cm2) and displayed good thromboresistance when tested in vitro — recalcified blood was not clotted for several hours. 

The thromboresistant properties of polystyrene with ionically immobilized heparin is unambiguously inferior compared to the covalently bound one. For instance, the blood clotting time for some of the covalently immobilized heparincontaining samples was up to 720 min, while for polystyrene, poly-p-aminostyrene, and polytrimethyl aminostyrene with electrostatically bound heparin the blood clotting time did not exceed 35 min. 

The main reason for the increase of the clotting time is provided by the covalently immobilized heparin but not by the eluted one. This is illustrated by Fig. 3, showing the plasma clotting time vs the amount of immobilized heparin or leaking into plasma. 

Summarizing the data reported in the literature, one may conclude that the activity of immobilized heparin does not depend on the nature of the functional group engaged in the immobilization (OH or COOH), especially when the concentration of the blocked groups is relatively small. 

Thus, the activity of immobilized heparin is effected most by the mobility of its macromolecule and its accessibility for physiological substrates 96,97). These parameters may easily be controlled by changing the structure of the hydrophilic gel and... 

Table 10. Activity of immobilized heparin as a function of the immobilization procedure 811...

Matrix Conjugating agent Amount of immobilized heparin rng/g polymer Amount of heparin cofactor bound per 1 g of product, antithrombin units... 

The use of the spacer is seen to lead to the increase in the amount of immobilized heparin and to a less dramatic decrease of the activity of heparin due to immobilization (Table 10), although the activity of the immobilized heparin is still below 10% of the initial activity of the free heparin81). 

Table 11 demonstrates, as in the previous case, the increased amount of immobilized heparin when the spacer was used the anticoagulant activity of immobilized heparin being around 10% of the initial heparin activity. The reason for the observed loss of initial activity is probably the inaccessibility of the immobilized heparin for high-molecular proteins (thrombin with a molecular mass of 34000 and antithrombin III with a molecular mass of 65 000) as the permeability of the grafted gels, whose water content was only 55 %, [Pg.113]

At the same time there are cases reported where the amount of immobilized heparin... 

Table 11. Amount of Immobilized heparin as a function of the method of immobilization 981...

In all of the reported cases, anticoagulant activity of immobilized heparin, as was already stressed above, was less than the activity of heparin in solution, which was attributed to the low accessibility of heparin molecules for physiological substrates. Increasing the porosity of the polymer, i.e., increasing the accessibility of the immobilized heparin led to an increase in its anticoagulant activity 100). 

The fact that thromboresistance of HCP is dependent on the method of immobilization of heparin, together with a rather low activity of covalently immobilized heparin, makes the idea of long-term enhancement of thromboresistance of polymers on their heparinization doubtful 54,70,71J. Naturally, the answer can be given only after a detailed analysis of the interaction of HCP with blood and its components and clarification of the mechanism of the effect of the immobilized heparin on the blood clotting system and relying on the results of in vivo tests of these materials are necessary. 

Although the scheme presented does not reflect all details of the blood-polymer interaction, it is possible to evaluate the effect of the immobilized heparin on the above or other steps of the thrombosis process. 

In general, the effect of immobilized heparin on the blood contacting a polymeric surface consists in an increase of the blood clotting time even when no heparin is eluted at all (Table 12) 104-108>. 

Heparinization, as is seen from Table 12, results not only in the increase in blood clotting time, but also in an increase of the platelet adhesion the increase in both cases being more intensive the higher the content of the immobilized heparin. It should be noted that there is a complete agreement between the scientists concerning the effect of heparin on the blood clotting time the result of the contact of blood with HCP is considered analogous to that of the direct injection of soluble heparin into the bloodstream, whereas the data on platelet adhesion is quite contradictory. 

Quite significant is the fact that the amount of platelets adhered depends on the synthesis procedure for HCP. For example, the platelet adhesion onto silicone rubbers heparinized via the TDMAC procedure was 150000 platelets/cm2, while the very same rubbers that were heparinized via y-aminopropyltriethoxysilane adhered only 90000 platelets/cm2 88). The platelets are to a greater extent adhered by the polymers containing covalently immobilized heparin than by those that elute heparin into the bloodstream n3) although the immobilized heparin itself does not interact with the platelets 21 . 

The reasons for the increased platelet adhesion onto covalently-immobilized heparin-containing polymers were disclosed in the studies of the protein adsorption onto HCP i.e., as stated above, the first step of the complicated blood-polymer interaction. 

The data on protein adsorption as affected by immobilized heparin is quite contradictory. In Refs.114> and115), serum albumin was shown to be predominantly adsorbed by the HCP, whereas in the majority of other works the observed dependence was the opposite. Table 14 compiles the results of the studies of the absorption of plasma proteins by HCP of two different types 64). Such thrombogenic proteins as fibrinogen and thrombin are seen to be the ones adsorbed most. 

It is noteworthy that a qualitatively similar dependence was observed for heparin in solution116>. The complexes of immobilized heparin with fibrinogen and thrombin are rather stable (the corresponding association constants for the complexes with fibrinogen and thrombin are (1.4 0.5) x 105 M and (8 2) x 10s A/-1) and are not subjected to dissociation in physiological solution. 

Hence, concerning the interaction with plasma proteins, covalently immobilized heparin performs identically to heparin in solution, and this results in the enrichment of the HCP surface with the most thrombogenic plasma components fibrinogen and thrombin. 

Fig. 6 a and b. Thromboelastograms of canine blood before (a) and after (b) contact with heparin-containing hydrogel (4 ml of blood were incubated with 2 g of gel containing 11.8 mg of covalently immobilized heparin per g) 10B)... 

Fig. 8. Kinetics of unstabilized fibrin hydrolysis with complexes of immobilized heparin with fibrinogen (1), thrombin (2), fibrinolysin (plasmin) (3), and serum albumin (4),06)...

The kinetics of the lytic effect displayed by the complexes of immobilized heparin with thrombin and fibrinogen, in distinction from those with plasmin, are described by their saturation curves. The observed slowing down of the dissolution of unstabilized fibrin is probably due to the inhibiton of the lytic activity of the complexes by the soluble products of the reaction. In fact, as it was shown in Ref. 106, further addition of immobilized heparin-protein complex to partially hydrolyzed fibrin results in a complete recovery of the dissolution rate. 

Some Approaches to Increase the Efficiency of Immobilized Heparin Action... 

The increased platelet adhesion onto HCP surfaces is mainly due to the high surfacial concentration of fibrinogen associated with immobilized heparin. Therefore one of the solutions to the problem could have been provided by simultaneous immobilization of heparin and proteolytic enzymes which could hydrolyze the adsorbed fibrinogen. The immobilized enzyme could also provide the lysis of the fibrin clot, in case it was formed, for instance in stagnation regions of the complicated devices. 

More extensive studies were carried out on polymeric materials modified with a hydrogel containing covalently immobilized heparin and trypsin 136,137>. Table 18 compiles the results of in vitro tests of the thus-modified low-density polyethylene. Obviously, co-immobilization of heparin and proteolytic enzyme onto polyethylene results in an increase of the blood clotting time and decreases the number on adhered platelets as compared to immobilization of heparin alone. 

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