A polyelectrolyte synthesized from cis-1,4-polyisoprene, containing aminosulfonate and carboxylate groups was shown to have an anticoagulant activity of about 1/30 compared with heparin. Because the substance prevents the coagulation of plasma in the presence of thrombin it is assumed that it acts as an antithrombin

Bantjes, A.

Beugeling, T.

Does, L. van der

Sederel, W.L.

English

van der Does, L.

University of Twente Research Information

J. BIOMED. MATER. RES. VOL. 8, PP. 375-379 (1974) Antithrombin Activity of a Polyelectrolyte Synthesized from cis-1,P-Polyisoprene T. BEUGELING, L. VAN DER DOES, A. BANTJES, and W. L. SEDEREL, Polymer Chemistry Laboratory, Twente University of Technology, Enschede, The Netherlands Summary A polyelectrolyte synthesized from cis-1,4-polyisoprene, containing amino- sulfonate and carboxylate groups was shown to have an anticoagulant activity of about 1/30 compared with heparin. Because the substance prevents the coagula- tion of plasma in the presence of thrombin it is assumed that it acts as an anti- thrombin. Recently, the addition reaction of N-chlorosulfonyl isocyanate and cis-1,4-polyisoprene has been reported.' It was shown that the addition product upon treatment with alkali gave a water soluble polyelectrolyte (A) containing the following structural unit : I NH SOtNa ONa I \  The aminosulfonate and carboxylate groups are also present in the tetrasaccharide unit of the anticoagulant heparin, for which the following structure has been suggested:2 CH,OSO; COOH CH,OSOC COOH Ij?-Jio*oj@oqL 0- H NHSOC H OH H NHSOC H OSO; 375 @ 1974 by John Wiley & Sons, Inc. 376 BEUGELING ET AL. In  seeking new thromboresistant polymeric materials we in- vestigated the activit!y of A as an anticoagulant. The present results indicate that A acts as an antithrombin, with an activity of about 1/30 compared with heparin. MATERIALS AND METHODS The polyelectrolyte A was synthesized as described.' Citrated human and bovine plasma were stored a t  - 26°C before use. Human plasma had a concentration of about 1.8.10-2 M disodium citrate and 2.0.10-2 M glucose. Prior to use the pH of the plasma was adjusted to 7.35 (unless otherwise stated) with the aid of 0.15 M HCl or 0.15 M Na2C03. Recalcification times of plasma were measured in Pyrex tubes with an outer diameter of 1.0 cm at 37°C. Part of the experiments were followed in an optical way as described by Matthews et al.3 For this purpose a Vitatron colorirneter, Model UC 200 S, was used, equipped with a special thermostated vessel holder for the test tubes. Further, a 2 mm thick heat filter was placed between the lamp and vessel holder. Recalcification was carried out with 0.3 M CaClz (pH 7.35) to a final concentration of 1.9.10-2 M to 3.5.10-2 M of added CaC12. Heparin, 152 E/mg, was purchased from Diosynth (Oss, Holland). Thrombin in 0.05 M phosphate buffer (pH 7.0), 250 NIH units/ml, was obtained from Sigma. Stock solutions of 12 mg/ml for both heparin and A were used for all measurements. The pH of these solutions was adjusted to 7.35 prior to use. RESULTS AND DISCUSSION The anticoagdant activity of A compared with heparin in human plasma, is shown in Table I. At concentrations up to about 0.4 mg A/ml plasma a real coagula- tion of the plasma took place after recalcification. In the case of 0.4 mg A/ml plasma the coagulation started after approximately 20 min and was completed in about 40 min. When the concentra- tion was 0.8 mg A/ml, only some precipitate was formed at  the inter- face of plasma and air after about 5 hr. When the experiment was continued longer than 8 hr a precipitate was also observed in the liquid. A similar behavior was found when heparin was present in SYNTHESIZED ELECTROLYTE AS ANTITHROMBIN 377 TABLE I Anticoagulant Activity of A Compared With Heparin8 Concentration of Concentration of A Beginning of heparin in plasma in plasma coagulation or (mg/ml) (mg/ml) precipitation no anticoagulant 0.04 0.06 0.4 0.8 1.2 clotting completed within 5 min 21-28 min 5-8 hr >8 hr > 6  hr >8 hr Conditions: aliquots of 1.5 ml citrated human plasma, pH 7.35, were incu- Recalcification was carried bated with the anticoagulant during 5 min at 37°C. out to a final concentration of 3.5.10-2 M of added CaC12. a concentration of 0.04 mg/ml plasma. When the concentration of A was 1.2 mg/ml no clotting or precipitation occurred even after 8 hr. From the experiments it follows that the activity of A in pre- venting clot formation is about 1/20 compared with heparin. In Table I1 data on some coagulation experiments with bovine plasma in the presence of A are listed, for different concentrations of added CaC12 and different pH values. At low Ca2+ concentrations the coagulation times of plasma in the presence of A were less than those at high Ca2f concentrations. The anticoagulant effect of A cannot be explained, however, by assuming a complex between A and Ca2+. For instance, in the case of 0.2 mg A/ml plasma and 3.5.10-2 M CaClz only 2% of the Ca2+ can be complexed, when it is assumed that the whole polymer consists of those units shown in the preceding structure (based on a ratio 1 unit: 1 Ca2+). Furthermore, a conductometric titration of A and Ca2+ did not indicate any complex formation. The anticoagulant activity is also not caused by a “soap-eff ect” of the polyelectrolyte. Comparable amounts of sodium dodecylsulfate even enhanced the clotting of plasma. No significant differences in recalcification times were found for the experiments in which pH values of 7.37 and 7.98 were used. The coagulation of bovine plasma is prevented at much lower con- centrations of both the anticoagulants A and heparin compared to 378 BEUGELING ET AL. TABLE I1 Anticoagnlant Activity of A in Bovine Plasma at Different Concentrations of Added CaClz and Different pH Values* Concentration of heparin in Concentration plasma of A in plasma Concentration Beginning of (mg/ml) (mg/ml) of CaClp (10-2 M) pH coagulation no anticoagulant 0.008 0.04 0.08 0 . 2  0.04 0.04 0.06 0.08 no anticoagulant 0.002 0.003 3 .5  3 .5  3 .5  3.5 3 .5  1.9 1.9 1.9 1 . 9  1 . 9  1.9 1 .9  7.76 7.76 7.76 7.76 7.76 7.37 7.37 7.98 7.98 7.98 7.98 7.98 5 . 5  min 7 min 21-26 rnin 70 min > 8  hr 4 min 6-9 rnin 8 min 12 min 18-20 rnin <12.5 min 11.5 rnin a Aliquots of 1.5 ml plasma were incubated with A or heparin during 5 min at 37°C before recalcification. human plasma. Because more precipitate is formed in bovine plasma than in human plasma during freezing, larger amounts of denaturated clotting factors may be present in the precipitate of bovine plasma. A comparable polymer was synthesized, starting from 3,4-poly- isoprene and N-chlorosulfonyl isocyanate. From the IR spectrum of the addition product it was expected that the alkaline hydrolysis product should contain less NH-S03Na-groups. In agreement with this assumption, the anticoagulant activity of this compound was found to be 4 to 5 times lower compared to A. A similar polymer derived from poly(2,3-dimethylbutadiene), which probably contains even fewer NH-S03Na-groups, was found to be even less active. In Table I11 data from some experiments are given, in which thrombin, together with a small amount of CaC12, was added to bovine plasma that had been incubated with A or heparin. In the presence of 1.67 NIH units thrombin/ml plasma and 1.9.10-2 M Ca2+ the coagulation time of plasma without an anticoagulant was about 30 sec. In the presence of A or heparin the coagulation of the plasma is strongly delayed. The fact that A prevents the SYNTHESIZED ELECTROLYTE AS ASTITHROMBIN 379 TABLE I11 Anticoagulant Activity of A in the Presence of Thrombin* Concentration of heparin Concentration of A Beginning of in plasma (mg/ml) in plasma (mg/ml) coagulation no anticoagulant 0.08 0.16 0.002 0.004 0.008 <30 sec 9-11 min > 2 . 5  hr 5 min 19 rnin > 2 . 5  hr a Aliquots of 1.5 ml bovine plasma were incubated with A or heparin during Then 1.67 NIH units thrombin/ml plasma were added together 5 min a t  37°C. with a solution of CaC12 to a final concentration of 1.9.10-2 M of added CaC12. clotting of plasma in the presence of thrombin indicates that i t  acts as an antithrombin. Whether A also interferes in earlier stages of the blood coagulation process is not yet known. From the experi- ments, listed in Table 111, in which the course of the clotting was also considered, it followed that the antithrombin activity of A is 1/20 to  1/40 compared with heparin. This order of magnitude is the same as the anticoagulant activity that can be derived from the Tables I and 11. Attempts will be made to synthesize an insoluble polymer derived from A. This can be done either by starting from a partly cross- linked cis-1,4-polyisoprene or by crosslinking the final product A. The authors wish to thank Dr. H. W. Krijnen of the “Bloedtransfusiedienst” in Amsterdam for the gift of human blood plasma and the firm Poviet in Amster- dam for their gifts of bovine plasma. Thanks are also due to Mr. F. Kuypers for the synthesis of the polyelectrolytes derived from 3,4-polyisoprene and poly(2, 3-dimethylbutadiene). Further, they thank Dr. Ir. M. Bos and Mr. W. Lengton for the conductometric titrations. References 1. L. van der Does, J. Hofman, and T. E. C. van Utteren, J .  Polym. Sci. : Polym. Lett., Ed. 11, 169 (1973). 2. J .  E. Jorpes, Anticoagulantien, Handbuch der experimentellen Pharmakologie, F. Markwardt, Ed., Vol. 27, Springer, Berlin, 1971, p. 154. 3. D. B. Matthews, S. I. Reger, and A. Catlin, J .  Biomed. Mater. Res., 2 ,  337 (1968). Received October 4, 1973 

Antithrombin activity of a polyelectrolyte synthesized from cis-1,4-polyisoprene

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