Bloconjugate Chem. 1002,3, 363-365
383
LETTERS Immobilization of Human Thrombomodulin on Glass Beads and Its Anticoagulant Activity Mitsuru Akashi,'y+ Ikuro Maruyama,*J Norihiro Fukudome,s and Eiji Yashimat Department of Applied Chemistry and Chemical Engineering, Faculty of Engineering, Kagoshima University, 1-21-40 Korimoto, Kagoshima 890, Japan, The Third Department of Internal Medicine, Faculty of Medicine, Kagoshima University, Usuki-cho 1208, Kagoshima 890, Japan, and Department of Industrial Chemistry, Miyakonojo National College of Technology, 473-1 Yoshio, Miyakonojo, Miyazaki 885, Japan. Received May 27, 1992
Human thrombomodulin (TM) was for the first time immobilized on glass beads by the reaction between the carboxyl group of T M and the amino group of glass beads using water-soluble carbodiimide. Immobilized human T M exhibited both anticoagulant activity and inhibition of platelet aggregation of human blood.
Thrombomodulin (TM) is a newly described endothelial cell associated protein that functions as a potent natural anticoagulant by converting thrombin from a procoagulant protease to an anticoagulant (1,2). Very recently, the preparation of human TM was achieved by using gene technology ( 3 , 4 ) ,and since then biomaterials containing human T M have been of interest. The antithrombogenicity of immobilized T M has previously been demonstrated by Miura et al. using bovine lung T M that had been bound to agarose (5). In this study we were able to use recombinant human TM. In a previous paper (61, we showed that poly(viny1 chloride) (PVC) containing heparin-immobilized hydrogels provides high antithrombogenicity and PVC containing prostaglandin 12-immobilized hydrogels inhibits platelet aggregation sufficiently. The results show that the combination of pharmaceutically active compounds and polymer materials is important for development of antithrombogenic biomaterials. In this article, we report the immobilization of human T M on glass beads as a first part of our study on human T M conjugated biomaterials. As shown in Scheme I, T M is an integral membrane protein and the active site of it has been thought to be located on the surface of blood vessel endothelia. Because only the amino terminal part of T M is involved in the interaction with thrombin, T M was immobilized on the surface of the glass by means of its carboxyl terminal part. The condensation between the amino group of N42aminoethyl)-3-aminopropyl-substitutedglass and the carboxyl group of T M using water-soluble carbodiimide gave glass-immobilized T M in a 0.2 M phosphate buffer
* To whom correspondence should be addressed. + Department of Applied Chemistryand Chemical Engineering,
Kagoshima University. The Third Department of Internal Medicine, Kagoshima University. 5 Department of Industrial Chemistry, Miyakonojo National College of Technology.
Scheme I. Antithrombogenicity of the Endothelial Cell Surface Protein
TM : PG12 Va : Vllla
c
--+
-+
A C ~ ~ ~ ~ & ? ~inactivation
o f Va,Viila
7
Heparin Thrombomodulin, ATllI : A n t i t h r o m b i n 111 : P r o s t a g l a n d i n 12 A c t i v a t e d Coagulation F a c t o r V : Activated Coagulation Factor Vlll
Table I. Activated Partial Thromboplastin Time (aPTT) of Glass-TM aF'TT (s) sample 3.3 x 10-3 rg/pLa 6.6 X pg/pLa glass-TM 32 35 glass 28 31 Final concentration of glass-TM or glass.
solution. After washing with phosphate buffer solution and drying in vacuo, the antithrombogenic activity of the resulting glass-immobilizedhuman T M was first evaluated by the method previously reported (6). As shown in Table I, no significant difference between glass and glass-TM was observed. When lipid was added first, the lipid prevented the complex formation between thrombin and T M on the glass. Therefore, thrombus formation started in the vicinity of lipid and it was difficult for thrombin occurring with activation of coagulatic factor to make a complex with T M on the glass bead as shown in Scheme 0 1992 American Chemical Society
304
Akashl et al.
Bloconjugate Chem., Vol. 3, No. 5, 1992
Scheme 11. Mechanism of Measurement for the Data Outlined in Tables I (a, aPTT Measurement System) and I1 (b, Modified Plasma Recalcification Time Measurement System) (b)
(a)
Thrombus Formation Place
3
y
: Thrombomodulin. T : Thrombin V a : A c t i v a t e d Coagulation F a c t o r V .
Xa : A c t i v a t e d Coagulation F a c t o r X
Table 11. Modified Plasma Recalcification Time of Glass-TM
(I
modified plasma material recalcification timen (s) 225.0 f 4.8 (n = 4) glass-TMb 172.6 f 1.5 (n = 4) glass Mean f SE. Final concentration was 6.7 mg/mL.
I1 (part a). In order to clarify the role of TM on the glass, modified plasma recalcificationtime was evaluated without lipid, and the results are summarized in Table 11. Glassimmobilized T M was found to exhibit excellent prolongation of modified plasma recalcification time compared with control glass. This result shows that the coagulation factor was activated on the glass surface to produce thrombin and then TM immobilized on the glass surface formed a complex with it (Scheme 11, part b). In blood-compatible materials, it is also required that platelet adhesion to endothelial cells be inhibited. It is known that biomaterials with immobilized prostaglandins show excellent antithrombotic effect by inhibiting platelet aggregation,regardless of either chemical (7-10)or physical i m m o b i l i z a t i o n ( 6 ) . T M on g l a s s is a l s o expected to play a role in inhibiting platelet aggregation induced by thrombin. Typical platelet aggregation curves for platelet plasma from human blood exposed to glassimmobilized TM are shown in Figure 1. As seen in the figure, it was found that immobilized TM had an effect on inhibition of platelet aggregation in a dose-dependent manner. The turbidity observed in the control experiments traced platelet aggregation and coagulation induced by thrombin. It is thought that fibrin clotting took place. In the system containing enough glass-immobilized TM, however, fibrin clotting was not observed. This fact suggests that immobilized TM could combine with thrombin as seen in blood vessels. It is concluded that glass-immobilized human TM is a very active inhibitor of both coagulation and platelet aggregation of human blood. Since chemically immobilized T M is active enough as seen on the surface of blood vessel endothelia, TM-immobilized biomaterials are expected to inhibit platelet adhesion to their surface. They are also important as a model system of endothelia. Further study on TM-immobilized biopolymers is now in progress. Experimental Procedures. Human T M was supplied by Asahi-Kasei Ltd. Aminopropyl glass beads were
r"l*y~ Control
G l a s s -TM
7.8mg/ml
0
2
4
6
Time(min) Figure 1. Inhibition of thrombin-induced platelet aggregation by glass-immobilized TM: control, only platelet plasma poured into a test tube; glass control, glass beads suspended in platelet-
rich plasma and poured into a test tube. purchased from Sigma Chemical Co. Turbidimetry was performed with an aggregometer (Syenco). Glass-Immobilized Human TM. Phosphate buffer solution (3.18 mL, pH 5.0) including 3 mg of human TM and 0.72 mg of 1-ethyl-3-[3-(dimethylamino)propyllcarbodiimide hydrochloride was added to 1 g of N42aminoethyl)-3-aminopropyl-glasssuspended in 8.0 mL of phosphate buffer solution. The immobilization reaction was performed at 0 "C for 48 h, and the obtained glassimmobilized TM was repeatedly washed with phosphate buffer solution (pH 7.4) and dried in vacuo over night. Measurement of Activated Partial Thromboplastin Time (aPTT). aPTT was measured similarly to the method described in the literature (6). Measurement of Modified Plasma Recalcification Time. Glass-TM suspension (100 pL) was added to 100
Letters
FL of platelet-rich plasma and the mixture was incubated for 2 min a t 37 OC. Then the clotting was started by 0.025 M CaC12 addition. Measurement of Platelet Aggregation. Platelet aggregation was measured by the turbidimetric assay (11) using thrombin (0.50 unit/mL) as an inducer in glassimmobilized T M suspended in platelet-rich plasma. ACKNOWLEDGMENT We thank Miss M. Uchikado for her experimental support. We are also grateful to Professor N. Miyauchi of Kagoshima University for helpful discussions. LITERATURE CITED (1) Maruyama, I., and Majerus, P. W. (1985)The turnover of
thrombin-thrombomodulin complex in cultured human umbilical vein endothelial cells and A549 lung cancer cells. Endocytosis and degradation of thrombin. J.Biol. Chem.260, 15432-15438. (2) Maruyama, I., and Majerus, P. W. (1987)Protein C inhibits endocytosis of thrombin-thrombomodulin complexes in A549 lung cancer cells and human umbilical vein endothelial cells. Blood 69,1481-1484. (3) Suzuki, K., Kusumoto, H., Deyashiki, Y., Nishioka, J., Maruyama, I., Zushi,M., Kawahara, S., Honda, G., Yamamoto, S.,and Horiguchi,S. (1987)Structure and expression of human thrombomodulin, a thrombin receptor on endothelium acting as a cofactor for protein C activation. EMBO J.6,1891-1897.
Bloconlugete Chem., Vol. 3, No. 5, 1992 38s
(4) Sirai, T., Shiojiri, S., Ito, H., Yamamoto, S., Kusumoto, H., Deyashiki, Y., Maruyama, I., and Suzuki, K. (1988)Gene
structure of human thrombomodulin, a cofactorfor thrombincatalyzed activation of protein C. J.Biochem. 103,281-285. (5) Yagi, K., Hirota, K., Yamasaki, S., Uwai, A,, and Miura, Y. (1989)Anticoagulant Activity of Immobilized Thrombomodulin. Chem. Pharm. Bull. 37,732-734. (6)Akashi, M.,Takeda, S.,Miyazaki,T., Yashima,E., Miyauchi, N., Maruyama, I., Okadome, T., and Murata, Y. (1989) Antithrombogenic Poly(viny1chloride) with Heparin- and/or Prostaglandin 12-Immobilizedin Hydrogels. J.Bioact. Compat. Polym. 4 , 4-16. (7) Grode, G. A., Pitman, J., Crowley, J. P., Leininger, R. I., and Falb, R. D. (1974)Surface-Immobilized Prostaglandin as a Platelet Protective Agent. Trans. Am. SOC. Artif. Intern. Organs 20,38-41. (8) Ebert, C. D.,Lee, E. S., and Kim, S. W. (1982)TheAntiplatelet Activityof ImmobilizedProstacyclin. J.Biomed. Mater.Res. 16,629-638. (9)Jacobs, H. A., Okano, T., and Kim, S. W. (1989)Antithrombogenic Surfaces: Characterization and Bioactivityof Surface Immobilized PGEI-heparin Conjugate. J.Biomed.Mater. Res. 23,611-630. (10) Chandy, T., and Sharma, C. P. (1984)The antithrombotic effectof prostaglandin E1immobilizedon albuminated polymer matrix. J. Biomed. Mater. Res. 18, 1115-1124. (11) O’Brien, J. R. (1962)Platelet aggregation I. Some effects of the adenosine phosphates, thrombin, and cocaine upon platelet adehesiveness. J. Clin. Pathol. 15,446-452.
