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Distinctive Role of Histidine-16 of the B,8 Chain of Fibrinogen in the End-To

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Distinctive Role of Histidine-16 of the B,8 Chain of Fibrinogen in the End-To Proc. Nati. Acad. Sci. USA Vol. 83, pp. 591-593, February 1986 Biochemistry Distinctive role of histidine-16 of the B,8 chain of fibrinogen in the end-to-end association of fibrin (polymerization/chemical modification/affinity chromatography) AKIRA SHIMIZU, YUJI SAITO, AND YUJI INADA Laboratory of Biological Chemistry, Tokyo Institute of Technology, Ookayama, Meguro-ku, Tokyo 152, Japan Communicated by John D. Ferry, September 12, 1985 ABSTRACT Photooxidation of fibrinogen reduced the includes 17th to 19th amino acid residues in Aa chain of batroxobin-induced fibrin polymerization. The fibrin fragment fibrinogen, specifically inhibits fibrin association and has des-AB N-DSK, which contains the binding sites termed A and proved itselfuseful for the elucidation ofassociation sites (7). B, lost the ability to bind to the site termed a in fibrinogen- These examples stress the critical role played by arginine-19 Sepharose upon the oxidation of histidine-16 in the BP chain of of Aa chain and some residues adjacent to it in the NH2- fibrinogen [Shimizu, A., Saito, Y., Matsushima, A. & Inada, terminal domain. On the other hand, very short fragments Y. (1983) J. Biol. Chem. 258, 7915-7917]. Some of the derived from the y chain of the COOH-terminal domain fragments, which became unable to bind to fibrinogen-Seph- (threonine-374 to valine-411 or threonine-374 to glutamic arose due to the destruction of site A, however, retained the acid-396) quite efficiently inhibited the association and sug- ability to bind to D-dimer-Sepharose, which contains both sites gested the importance ofthat region for the association (8, 9). a and b. This shows that histidine-16 of the BfP chain of We used a technique of chemical modification to locate an fibrinogen is essential for site A but may not be essential for site amino acid residue directly involved in the association. The B. It is of interest that histidine-16 of the B,8 chain, which is problem of the lack of specificity generally blamed on only one residue away from the thrombin-susceptible bond, chemical modifications was circumvented by the combined makes a part ofthe site A for the end-to-end association created use of affinity chromatography and sequence analyses (10, by the release of fibrinopeptide A. 11). We showed unequivocally that histidine-16 of BP chain was involved in the association offibrin. It has been reported The ultimate reaction ofthe blood coagulation cascade is the that the specific elimination of a part of BP chain from conversion of soluble fibrinogen to insoluble fibrin. Although fibrinogen, which includes histidine-16, diminishes its ability it is over-simplified and is too far from being complete, to polymerize, while the conformation of fibrinogen is re- current understanding about this process can be summarized tained (12). This result supports our claim that histidine-16 as the following. An activated protease, thrombin, removes plays an important role for the association (11). a pair of hexadecapeptides (called fibrinopeptide A) from the Although there are two kinds of fibrin association, end-to- NH2 termini ofAa chains ofthe antiparallel dimeric molecule end and lateral, it has been difficult to delineate the involve- with Mr 340,000 containing three pairs of polypeptides (1). ment of particular amino acid residues or regions in each The newly exposed association site in the NH2-terminal specific type of association. In this report we show that region (called site A) binds specifically to a corresponding histidine-16 of BP3 chain is specifically involved in the initial site always available in the COOH-terminal region (called site end-to-end association, but that it may not be involved in the a) (2). This association proceeds in an end-to-end fashion lateral association of protofibrils. with half-molecule staggered overlaps and makes linear protofibrils. After the formation of some protofibrils, a pair MATERIALS AND METHODS of tetradecapeptides (called fibrinopeptide B) are removed from BP chains. This reveals another association site in the Materials. Fibrinogen (95% clottable), plasminogen, and NH2-terminal region (called site B) (3). On the other hand, the urokinase were kind gifts of Green Cross (Osaka, Japan). end-to-end association in protofibrils aligns the COOH- Fibrinogen was further purified with lysine-Sepharose to terminal domains of two adjacent molecules and creates a remove plasminogen. Thrombin, batroxobin, and factor XIII new association site (called site b) (4). This alignment of the concentrate were kindly provided by Mochida Pharmaceu- COOH-terminal domains is stabilized by one of the blood tical (Tokyo), Tobishi Pharmaceutical (Tokyo), and Hoechst coagulation factors, factor XIII (a transglutaminase), which Japan (Tokyo), respectively. Contortrix protease was kindly is activated by thrombin in the presence of (5). Sites B offered by K. F. Stocker at Pentapharm (Basel, Switzer- Ca2' land). All other reagents were of analytical grade. and b then make specific and complementary lateral associ- Preparation of the Fragment des-AB N-DSK. The NH2- ations that allow protofibrils to grow laterally (4). terminal disulfide knot devoid of both fibrinopeptides A and Due to the lack of crystals of the native form or its B termed fragment des-AB N-DSK was obtained by directly derivatives, the detailed three-dimensional structure of fi- treating crosslinked fibrin with CNBr (11). brinogen is not available at present. The precise sites and the Preparation of Contortrix N-DSK. Fibrinogen was treated mechanism for the association, therefore, have not been with a purified Contortrix protease (6 units/g of fibrinogen) adequately elucidated. Abnormal fibrinogens have suggested at 15'C for 2 hr as described by Shainoff and Dardik (13). the importance of some amino acid residues for fibrin After lyophilization and dialysis against 70% (vol/vol) formic polymerization. A good example is the replacement of acid, this treated fibrinogen was further cleaved by CNBr. arginine-19 in the Aa chain by serine in fibrinogen Detroit (6). The digested sample was purified by the two consecutive gel A synthetic short peptide, glycylprolylarginylproline, which filtrations used for the purification of the fragment des-AB N-DSK (11). The affinity chromatography with fibrinogen- The publication costs of this article were defrayed in part by page charge Sepharose was omitted. Contortrix protease has been report- payment. This article must therefore be hereby marked "advertisement" ed to preferentially release fibrinopeptide B from fibrinogen in accordance with 18 U.S.C. §1734 solely to indicate this fact. (13). 591 Downloaded by guest on October 2, 2021 592 Biochemistry: Shimizu et al. Proc. Natl. Acad. Sci. USA 83 (1986) Preparation of the Fragment D-Dimer. Fibrinogen was To investigate whether this histidine residue exclusively is polymerized and crosslinked by thrombin (2.5 units/ml) in involved in the end-to-end association, two different kinds of the presence of 10 mM CaCl2 and factor XIII (0.25 mg/ml). affinity columns, fibrinogen- and fragment D-dimer-Sepha- Finely cut fibrin was treated with plasminogen (0.35 casein rose, were synthesized. The lateral association takes place unit/ml) and urokinase (35 units/ml) at 370C for 16 hr in the between the B site in the NH2-terminal domain and the b site presence of 10 mM CaCl2. After plasminogen (plasmin) was created in the aligned COOH-terminal domains. Fragment removed by lysine-Sepharose, the fragment D-dimer was D-dimer obtained from crosslinked fibrin contains the b site obtained by DEAE-cellulose chromatography followed by (4). Although the affinity column D-dimer-Sepharose may gel filtration by Sephadex G-150 Superfine, essentially as contain both sites a and b, it should make a suitable tool to described by Purves et al. (14). exclusively investigate the lateral association, ifthe site A in Preparation ofFibrinogen- and D-Dimer-Sepharose. Fibrin- the applied sample is uniformly destroyed. Olexa and ogen and the fragment D-dimer were immobilized on CNBr- Budzynski (17) have reported that plasmic fragments, E1 and activated Sepharose 4B according to the method of Heene E2, bind to fragment D-dimer, but not to fibrinogen. We and Matthias (15). confirmed this by the use ofthese affinity columns; fragments Photooxidation. des-AB N-DSK was photooxidized with E1 and E2 did bind to D-dimer-Sepharose but did not bind to methylene blue (10) followed by removal of the photosensi- tizer by dialysis. Measurement of Batroxobin-Induced Fibrin Association. 0.20 - A Batroxobin (1.3 units/ml) was added to fibrinogen in 50 mM Tris HCl containing 100 mM NaCl (pH 7.0) with or without photooxidation. Methylene blue was not removed and was added also to the control sample. The association was 0.10 monitored by measuring absorbance at 350 nm with a Shimadzu (Kyoto, Japan) recording spectrophotometer, Buffer 2 model UV-200. 0 20 40 60 RESULTS AND DISCUSSION 0.50 , We have shown that the smallest, active fragment derived B from the NH2-terminal domain of fibrin, fragment des-AB N-DSK, lost its ability to bind to fibrinogen-Sepharose by the specific oxidation of histidine-16 of the BP chain (11). Since this binding is between the a site in fibrinogen and the A site 0.25- in fragment des-AB N-DSK (3, 4), this result indicates that histidine-16 is involved in the end-to-end association offibrin. Since an enzyme, batroxobin, preferentially releases fibrin- Buffer 2 opeptide A and induces exclusively end-to-end association of 0 fibrin (3, 16), we expected that photooxidation of fibrinogen 0 10 20 30 would quickly reduce batroxobin-catalyzed fibrin polymer- 0.50 , ization. This prediction was confirmed by testing the poly- C merization rate of various irradiated fibrinogens (Fig. 1). By increasing the irradiation time, polymerization was steadily inhibited. Five minutes of irradiation was enough to almost completely inhibit the polymerization.
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