Heparinized, thromboresistance

A semi-interpenetrated network was obtained by bulk polymerization of 2-hydroxye-thyl methacrylate incorporated in DMF treated PET films by solvent-exchange technique, followed by treatment of films in e-lectrical discharges. Heparinization was accomplished by reacting glutaraldehyde with heparin and poly(2-hydroxyethyl methacrylate) present on the surface of modified polyester films. The immobilization of heparin was indirectly evidenced by chromatographying the silylated hydrolyza-tes of heparinized PET films and heparin, respectively. In vitro experiments demonstrated the enhanced thromboresistance of heparinized films. 

The thromboresistance of heparinized films, as shown in Table VI, was enhanced by higher hydrogel content since the concentration of hydroxylic groups on modified PET film surface, able to react with glutaralde-hyde in order to bind heparin, was higher. 

Table VI. Thromboresistance of Heparinized pHEMA Containing PET Films...

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. 

Of these the last one has been most widely used, since heparin-modified polymeric materials exhibit the highest and by today unsurpassed effects of thromboresistance enhancement. Many of these materials have not only proved to be potent in trials on animals, but have already found clinical application. These achievements have stimulated continuous interest in heparin-containing polymers (HCP) which is best manifested by listing the investigations performed in the field in recent years and still under way. They involve the new procedures for the synthesis of HCP providing minimal loss of activity of bound heparin, the studies of interactions of HCP with blood and its individual components, as well as on the mechanism of enhanced thromboresistance of HCP, and the search for new tasks for HCP. 

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 heparin content of the materials involving anion-exchange resins goes up to 800 ng/cm2, whereas the maximal heparin content of graphite-based polymers is 0.78 ng/cm2. All of the synthesized polymers, although exhibiting sufficiently high thromboresistance in in vitro tests (Table 3), drastically varied in their stability in various model media (Table 4). 

Table 5. Thromboresistance of heparinized poly(tetrafluoroethylene) as a function of the diameter of uncoated regions62 ...

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 authors of the above-discussed results consider the constant rate of heparin elution to be absolutely necessary for a successful functioning of the implant. Catheters made of these copolymers are much better, in respect of their thromboresistant properties, than the commercially available poly(tetrafluoroethylene) polyethylene, and plasticized polyvinyl chloride catheters (Table 8). The tested catheters were clotted in 9 cases of 81 (11 %), whereas usual silicone rubber catheters were clotted in 5 cases of 8 (63 %)70). 

Grafting ethyleneimine onto cellophane followed by conventional heparinization yielded a product used in making the membranes for artificial kidney which displayed good thromboresistance 80). 

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. 

It seemed reasonable to anticipate that the synergism of these two features (high heparin content and stability of the resultant materials) would result in long-term thromboresistant polymers. The in vivo tests revealed, however, their extremely low thromboresistance as compared to the ionically bound heparin-containing polymers, in particular. The effect is assumed to be caused by a lack of sufficient mobility of the polymer-bound heparin molecules, which prevents the performance of the intrinsic anticoagulant properties of heparin. 

The prospects for this method as well as for the method involving the generation of free macroradicals by y-irradiation of heparin 95), are provided for by the variety of polymerizable monomers, which makes it possible to produce materials with various physico-mechanical and chemical properties. The in vitro thromboresistance of the copolymers obtained in this way was proportional to the heparin content (Table 9). 

Table 9. Thromboresistance of heparin-(methyl methacrylate) copolymers 93 ...

A similar approach was used in Ref. 103) to obtain the fat-soluble derivatives of heparin by its copolymerization with butyl methacrylate or vinyllaurate and to get high-molecular products with a molecular mass of over 200000 by homopolymerization of the unsaturated heparin derivative. The products were used as thromboresistant... 

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. 

Mechanism of Enhanced Thromboresistance of Polymeric Materials in the Presence of Heparin... 

The results discussed in the previous Section permits to make some assumptions regarding the mechanism of enhanced thromboresistance of HCP. Two self-excluding viewpoints concerning the problem coexist in the literature. The first one implies the elution of heparin from a polymeric surface into the bloodstream which prevents clotting in a way common for the anticoagulant itself64,67 73). According to the second, the... 

What are the experimental data underlying the first hypothesis It is common knowledge today that the covalently bound heparin (if bound to the polymer rigidly and inexorably) is usually less active than ionically attached heparin which is capable of elution. In a series of works a group of Japanese investigators, who are today probably the only supporters to this concept, has disclosed the correlation between thromboresistance of the polymer and the amount of heparin released into the bloodstream 68 71). The minimal elution rate of heparin providing sufficient thromboresistance was found to be 4 x 10s g/cm2 min. The decrease of the rate of elution resulted in a drastic decrease of thromboresistance. 

Table 18. Thromboresistance of polyethylene modified with heparin and trypsin 137)...

To clarify the mechanism of enhanced thromboresistance of such polymers, the state of the blood clotting system before and after the contact with the polymer was examined I36) (Table 19). Obviously, the changes of the blood clotting system parameters are of the same type as those accompanying the interaction of blood with HCP (see Table 16) but essentially exceeding the latter. Consequently, the heparin-protease-... 

Optimal stent implantation and new antiplatelet therapy have reduced the thrombotic complication after stent implantation, dramatically. However, thrombosis remains a challenge in some lesions and patient subgroups. As an initial and unavoidable event during stent implantation, thrombosis and platelet activation are also involved in the development of neointimal hyperplasia. Stents coated with heparin and other antithrombotic drugs have been demonstrated to decrease thrombotic complications, although their effect on neointimal hyperplasia remains uncertain. As heparin is attached to the stent surface, we divide thromboresistant stents as heparin-coated stents and drug-eluting thromboresistant stents. 

Wilson, at Bishop College, and Eberhart and Elkowitz at University of Texas (27) have irradiated a silicone substrate in the presence of chloromethylstyrene monomer to produce a reactive graft polymer that can be quarternized with pyridine and reacted with sodium heparin to produce a thromboresistant heparinized product that has a higher blood compatibility than the untreated silicone. The same group has used essentially the same methods to create a heparin grafted polyethylene surface. 

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