Fibrinogen and fibrin--proteins with complex roles in hemostasis and thrombosis

alpha-Thrombin cleavage of 30 polypeptide hormones and their derivatives were analysed by quantitative amino-terminal analysis. The polypeptides included secretin, vasoactive intestinal polypeptide, cholecystokinin fragment, dynorphin A, somatostatins, gastrin-releasing peptide, calcitonins and human parathyroid hormone fragment. Most of them were selected mainly on the ground that they contain sequence structures homologous to the well known tripeptide substrates of alpha-thrombin. All selected polypeptides have one single major cleavage site and both Arg-Xaa and Lys-Xaa bonds were found to be selectively cleaved by alpha-thrombin. Under fixed conditions (1 nmol polypeptide/0.5 NIH unit alpha-thrombin in 20 microliters of 50 mM ammonium bicarbonate at 25 degrees C), the time required for 50% cleavage ranges from less than 1 min to longer than 24 h. Heparin invariably enhanced thrombin cleavage on all polypeptide analysed. The optimum cleavage site for alpha-thrombin has the structures of (a) P4-P3-Pro-Arg-P1'-P2', where P3 and P4 are hydrophobic amino acid and P1', P2' are nonacidic amino acids and (b) P2-Arg-P1', where P2 or P1' are Gly. The requirement for hydrophobic P3 and P4 was further demonstrated by the drastic decrease of thrombin cleavage rates in both gastrin-releasing peptide and calcitonins after chemical removal of hydrophobic P3 and P4 residues. The requirement for nonacidic P1' and P2' residues was demonstrated by the drastic increase of thrombin cleavage rates in both calcitonin and parathyroid hormone fragments, after specific chemical modification of acidic P1' and P2' residues. These findings confirm the importance of hydrophobic P2-P4 residues for thrombin specificity and provide new evidence to indicate that apolar P1' and P2' residues are also crucial for thrombin specificity. It is concluded that specific cleavage of polypeptides by alpha-thrombin can be reasonably predicted and that chemical modification can be a useful tool in enhancing thrombin cleavage.

Thrombin specificity. Requirement for apolar amino acids adjacent to the thrombin cleavage site of polypeptide substrate.

Thrombin (EC 3.4.21.5) is the activation product of prothrombin. Unlike related serine proteinases of the blood coagulation pathways, procoagulant αthrombin has several seemingly diverse, yet central functions in hemostasis. It also participates in wound healing and is implicated in various disease processes. Human disability and death attributable to throm­ botic or hemostatic disorders (such as coronary throm­ bosis, pulmonary embolism, stroke, atherosclerosis) are of considerable social and economic concern. Yet, despite involvement in such maladies, its underlying functions appear to be those of controlling normal physiologic processes for the benefit and survival of higher animals. Hemostasis is a necessary adaptation for the con­ tainment of circulating blood in order to supply nu­ trients and remove wastes from dependent tissues. Thrombin functions at all three levels of hemostasis and does so by enzymic (proteolytic), nonenzymic (hormonal), or coupled mechanisms. In the case of coupled mechanisms, both the proteolytic active site and a noncontiguous site (exosite) are involved, such as a-thrombin cleavage of fibrinopeptide A from fi­ brinogen and stimulation of platelets, as well as other cells. Such mechanisms, including use of the anion-binding exosite, are among the distinguishing properties of α-thrombin from those of the pancreatic serine proteinases with primarily digestive functions (such as α-chymotrypsin, β-trypsin). Whereas α-thrombin is a multifunctional en­ zyme, its physiologic control is to a large part directed at regulation of thrombin generation. First of all, pro­ thrombin is a vitamin K-dependent protein synthe­ sized by the parenchymal cells in the liver, where the vitamin is required for postribosomal formation of ten 7-carboxylglutamic acids. The vitamin K antagonist warfarin has been a widely used rodenticide, as well as an orally administered pharmaceutical agent for treatment of patients with thrombotic ten­ dencies. Prothrombin circulates in plasma with a half-life of about 3 days. The desialidation of its three carbohydrate side chains is presumptively im­ portant in determining its resorption by the liver. On the other hand, the single carbohydrate side chain retained by thrombin (Fig. 1) has no known function and is removed from active-site regions. Plasma contains sufficient prothrombin to gener­ ate 150,000 US NIH units of α-thrombin per liter, corresponding to 1.37 μM. During the activation in whole blood or recalcified plasma only approximately 10% of the potential α-thrombin concentration is ever achieved, despite more than 80% consumption of the proenzyme.' This apparent consumption of αthrombin is believed to be largely due to its active incorporation into fibrin clots, where it is sequestered and can be released on fibrinolysis. Plasma con­ tains various proteinase inhibitors (such as antithrombin III, heparin cofactor II, α2-macroglobulin) that inactivate various thrombin forms. Cultured fibro­ blasts release into media various proteinase inhibitors, or "nexins," which also inhibit thrombin. Further­ more, the activities of the nexin PN1 are enhanced by heparin, analogous to antithrombin III. Heparin is a sulfonated, negatively charged, heterogeneous car­ bohydrate found on the surface of various cells (such as endothelial cells). It binds both α-thrombin and antithrombin III and does so in a catalytic manner, where a minimum of approximately 18 saccharide units are needed. In plasma, heparin greatly en­ hances the activity of antithrombin III in thrombin inactivation. Perhaps its primary function, when ad­ ministered therapeutically, is to lower α-thrombin concentrations to those where it no longer activates From the Wadsworth Center for Laboratories and Research, School of Public Health Sciences, New York State Department of Health, and the Department of Physiology, The Albany Medical College of Union University, Albany, New York. Reprint requests: Dr. Fenton, Wadsworth Center for Labo­ ratories and Research, New York State Department of Health, Empire State Plaza, Albany, NY 12201.

Regulation of thrombin generation and functions

Activation of prothrombin to alpha-thrombin generates not only the catalytic site and associated regions but also an independent site (an exosite) which binds anionic substances, such as Amberlite CG-50 resin [cross-linked poly(methylacrylic acid)]. Like human alpha-thrombin with high fibrinogen clotting activity (peak elution at I = 0.40 +/- 0.01 M, pH 7.4, approximately 23 degrees C), catalytically inactivated forms (e.g., i-Pr2P-alpha- and D-Phe-Pro-Arg-CH2-alpha-thrombins) were eluted with only slightly lower salt concentrations (I = 0.36-0.39 M), while gamma-thrombin with very low clotting activity was eluted with much lower concentrations (I = 0.29 M) and the hirudin complex of alpha-thrombin was not retained by the resin. In a similar manner, hirudin complexes of alpha-, i-Pr2P-alpha-, and gamma-thrombin were not retained by nonpolymerized fibrin-agarose resin. Moreover, the ionic strengths for the elution from the CG-50 resin of seven thrombin forms were directly correlated with those from the fibrin resin (y = 0.15 + 0.96x, r = 0.95). In other experiments, the 17 through 27 synthetic peptide of the human fibrinogen A alpha chain was not an inhibitor of alpha-thrombin, while the NH2-terminal disulfide knot (NDSK) fragment was a simple competitive inhibitor of alpha-thrombin with a Ki approximately 3 microM (0.15 M NaCl, pH 7.3, approximately 23 degrees C). These data suggest that alpha-thrombin recognizes fibrin(ogen) by a negatively charged surface, noncontiguous with the A alpha cleavage site but found within the NDSK fragment. Such interaction involving an anion-binding exosite may explain the exceptional specificity of alpha-thrombin for the A alpha cleavage in fibrinogen and alpha-thrombin incorporation into fibrin clots.

Anion-binding exosite of human alpha-thrombin and fibrin(ogen) recognition.

The structure of residues 7-16 of the A alpha-chain of human fibrinogen bound to bovine thrombin at 2.3-A resolution.

The following peptide was synthesized by classical methods in solution: Ac-Asp-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg-Gly-Pro-Arg-Val-NHCH3 (F-8). The Michaelis-Menten parameters for the hydrolysis of the Arg-Gly bond in F-8 by thrombin were determined to be Kcat = 31 X 10(-11) M [(NIH unit/L) s]-1 and KM = 310 X 10(-6) M. Comparison of these values with those determined previously for native fibrinogen and for a series of similar synthetic peptides, together with information about the amino acid sequences of this portion of the A alpha chain of abnormal fibrinogens, suggests an important role for Asp at position P10. Differences in the Michaelis-Menten parameters between F-8 and the 51-residue N-terminal CNBr fragment of the A alpha chain of fibrinogen correspond to only 1-2 kcal/mol in binding affinity.

Mechanism of action of thrombin on fibrinogen. Kinetic evidence for involvement of aspartic acid at position P10.

The following peptides were synthesized by classical methods in solution: Ac-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg-Gly-Pro-NHCH3 (F-6) and Ac-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg-Gly-Pro-NHCH3 (F-7). The rates of hydrolysis of the Arg-Gly bond in these peptides by thrombin were measured, and the rate for the Phe-containing peptide F-6 was found to be much larger than that for F-7. Previous work [van Nispen, J. W., Hageman, T. C., & Scheraga, H. A. (1977) Arch. Biochem. Biophys. 182, 227] has demonstrated the importance of Phe-Leu at positions P9-P8 of the A alpha chain of fibrinogen for the thrombin-fibrinogen interaction. This work demonstrates that the presence of Leu (P8) alone is insufficient to account for the enhanced hydrolysis rates and that the presence of Phe (P9) is essential for normal action of thrombin on the A alpha chain of fibrinogen.

Mechanism of action of thrombin on fibrinogen. Direct evidence for the involvement of phenylalanine at position P9.

The following synthetic linear A alpha fibrinogen-like peptides were studied by NMR spectroscopy in aqueous solution: Ac-Asp(P10)-Phe(P9)-Leu-Ala-Glu-Gly(P5)-Gly(P4)-Gly(P3)-Val- Arg(P1)-Gly-(P1)-Pro-Arg(P3')-Val-NHCH3 (F-8), Ac-Phe-Leu-Ala-Glu-Gly-Gly(P4)-Gly(P3)-Val-Arg-Gly-Pro-NHCH3 (F-6), Ac-Leu-Ala-Glu-Gly-Gly(P4)-Gly(P3)-Val-Arg-Gly-Pro-NHCH3 (F-7), and Ac-Gly-Gly(P4)-Gly-(P3)-Val-Arg-Gly-Pro-NHCH3 (F-9). The temperature dependence of the amide proton chemical shift is smaller by approximately 22% for the Gly(P3) amide proton in F-9, F-6, and F-8 and is similarly smaller for the Gly(P4) amide proton in F-6 and F-8, but not F-9, relative to the other amide protons in these peptides. The exchange rates with solvent water for the Gly(P3) amide proton in each of these four peptides were determined by solvent spin saturation transfer experiments. The exchange rate constant for the Gly(P3) amide proton of F-8 was half that of the rate constant determined for this proton in F-9, F-7, and F-6. In conjunction with previously reported data for the rate of hydrolysis of the Arg(P1)-Gly(P1') bond by thrombin, these results suggest that there is a beta-bend at Gly(P5)-Gly(P4), possibly stabilized by salt links between Asp(P10) and Arg(P3') and between phosphorylated Ser(P14) and Arg(P7'), which brings Phe(P9) close to the hydrolyzable Arg-Gly bonds.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanism of action of thrombin on fibrinogen: NMR evidence for a beta-bend at or near fibrinogen A alpha Gly(P5)-Gly(P4).

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Mechanism of action of thrombin on fibrinogen. Kinetic evidence for involvement of aspartic acid at position P10.

Mechanism of action of thrombin on fibrinogen. Direct evidence for the involvement of phenylalanine at position P9.

Mechanism of action of thrombin on fibrinogen: NMR evidence for a beta-bend at or near fibrinogen A alpha Gly(P5)-Gly(P4).