rFVIIa and PCC partially improved laboratory parameters, but did not reverse rivaroxaban induced-bleeding.

Biochemistry ABSTRACTIn serine proteases, residue 192, three residues prior to the active site , plays an important role in determining substrate specificity. In trypsin (EC 3.4.21.4) and most trypsin-like enzymes with relatively broad specificity, this position is occupied by Gin. In thrombin (EC 3.4.21.5), an enzyme with restricted specificity, position 192 is occupied by Glu. The potential importance of Glu-192 in restricting the specificity of thrombin was investigated by isosterically replacing Glu-192 with Gin. Unlike trypsin, thrombin cleavage of peptides with acidic residues in positions P3 and P'3 [where P3 and P'3 refer to three residues removed from the Arg (P1) cleavage site on the amino and carboxyl side, respectively] is inefficient. Protein C, an an gulant zymogen, has Asp residues in positions P3 and P'3. Thrombomodulin, an endo-

Factor Xa (FXa) hydrolyzes two peptide bonds in prothrombin having (Glu/Asp)-Gly-Arg-(Thr/Ile) for P 3 -P 2 -P 1 -P 1 residues, but the exact preferences of its catalytic groove remain largely unknown. To investigate the specificity of FXa, we synthesized full sets of fluorescence-quenched substrates carrying all natural amino acids (except Cys) in P 3 , P 2 , P 1 , P 2 , and P 3 and determined the k cat /K m values of cleavage. Contrary to expectation, glycine was not the "best" P 2 residue; peptide with phenylalanine was cleaved slightly faster. In fact, FXa had surprisingly limited preferences, barely more pronounced than trypsin; in P 2 , the ratio of the k cat /K m values for the most favorable side chain over the least was 289 (12 with trypsin), but in P 1 , this ratio was only 30 (versus 80 with trypsin). This unexpected selectivity undoubtedly distinguished FXa from thrombin, which exhibited ratios higher than 19,000 in P 2 and P 1 . Thus, with respect to the catalytic groove, FXa resembles a low efficiency trypsin rather than the highly selective thrombin. The rates of cleavage of the peptidyl substrates were virtually identical whether or not FXa was in complex with factor Va, suggesting that the cofactor did not exert a direct allosteric control on the catalytic groove. We conclude that the remarkable efficacy of FXa within prothrombinase originates from exosite interaction(s) with factor Va and/or prothrombin rather than from the selectivity of its catalytic groove.At the confluence of the formerly named intrinsic and extrinsic pathways, factor Xa (FXa) 1 is the midway protease of the blood clotting waterfall (1). FXa belongs to clan SA of the S1 family of serine peptidases along with thrombin and trypsin (2-5). Without cofactors, activation of prothrombin by FXa is slow; it becomes efficient only when FXa complexes factor Va to form prothrombinase (6). Rapid inhibition of FXa by antithrombin also requires heparin as cofactor (7). However, tissue factor pathway inhibitor (TFPI) does not require any cofactor to rapidly neutralize FXa (8). FXa catalyzes a number of other reactions: activation of factor VII in a positive feedback within the tissue factor pathway (9), activation of factors V (10) and VIII (11), cleavage of protease-activated receptor 2 (12), and neutralization of protein S, albeit only in the presence of phospholipid and calcium (13). Thrombin (14) requires a cofactor for activation of protein C and factor XI, as well as for its inhibition by antithrombin and heparin cofactor II. In contrast to FXa, however, thrombin alone rapidly catalyzes a number of critical reactions in the cascade: cleavage of fibrinogen, activation of factors V and VIII, and activation of protease-activated receptor 1 (6, 10, 15). Trypsin, the archetypal endopeptidase of the digestive tract, does not require cofactors to rapidly hydrolyze (in appropriate conditions) most peptide bonds that follow an arginine or a lysine. The notable specificity of the blood coagulation peptidases result from at least four molecul...

The importance of substrate residues P2' and P3' on thrombin catalysis has been investigated by comparing the hydrolysis of a series of fluorescence-quenched substrates. Each consisted of a 10-residue peptide, carrying a 2-aminobenzoyl (Abz) group at the N-terminus, and a penultimate 2,4-dinitrophenyl (Dnp) derivatized lysine. Cleavage of such a peptide relieves the intramolecularly-quenched fluorescence, allowing determination of the kinetic parameters. The nature of the P2' residue was found to have a major influence on the rate of cleavage: the Kcat/Km value for the hydrolysis of the Arg-Ser bond in Abz-Val-Gly-Pro-Arg-Ser-Phe-Leu-Leu-Lys(Dnp)-Asp-OH was nearly 3 orders of magnitude higher than that for the hydrolysis of the same substrate with aspartate instead of phenylalanine at the P2' position. Comparatively, the P3' side chain was less important: the kcat/Km value for the substrate with the least effective residue (aspartate) was only 33 times lower than that of the substrate with the most favorable amino acid (lysine). The role of thrombin residues Arg35, Lys36, Glu39 and Lys60f in the putative P2' and P3' binding sites was also examined. Replacement of Lys60f by glutamine improved the rate of cleavage for peptides with P2' lysine or leucine. Compared with thrombin, mutants E39K and E39Q hydrolyzed faster substrates with an acidic residue in P2' or P3', but slightly slower those with a lysine at either position. Mutations R35Q and K36Q only improved the hydrolysis of substrates with an acidic P2' residue. Overall, thrombin prefers bulky hydrophobic side chains in subsite S2' and positively charged residues in S3', whereas acidic residues are markedly antagonistic to both subsites.

No abstract

factor pathway inhibitor (TFPI) (Ascenzi et al., 1988; Max-Planck-Institut für Biochemie, Abteilung Strukturforschung, Guinto et al., 1994 (Bode et al., 1989 parison of structures of thrombin in complex with a wide † This paper is dedicated to the memory of our friend and colleague variety of substrates and inhibitors indicates that the 148-Professor Stuart R.Stone, whose untimely death (December 16, 1996) loop can adopt a range of conformations (Priestle et al., is a tragic loss to us and to the scientific community.1993; Stubbs and Bode, 1993); the 60-loop, on the other hand, appears to possess a rigid structure that differs by Previous crystal structures of thrombin indicate that Ͻ1.5 Å in previously determined structures (Engh et al., the 60-insertion loop is a rigid moiety that partially 1996). Haematophages such as the medicinal leech Hirudo occludes the active site, suggesting that this structural medicinalis, the assassin bug Rhodnius prolixus and the feature plays a decisive role in restricting thrombin's soft tick Ornithodoros moubata all possess potent thrombin specificity. This restricted specificity is typified by the inhibitors that display novel inhibition strategies, accomexperimental observation that thrombin is not inhibited modating themselves to the restricted active site with by micromolar concentrations of basic pancreatic trypconcomitant binding to the other unique feature of thromsin inhibitor (BPTI). Surprisingly, a single atom mutabin, the basic fibrinogen recognition exosite (Rydel et al., tion in thrombin (E192Q) results in a 10 -8 M affinity 1991; van de Locht et al., 1995van de Locht et al., , 1996. for BPTI. The crystal structure of human thrombinThe actions of thrombin have hitherto been explained mutant E192Q has been solved in complex with BPTI in terms of a static molecular model (Stubbs and Bode, at 2.3 Å resolution. Binding of the Kunitz inhibitor is 1993, 1995). Although there have been some reports on accompanied by gross structural rearrangements in allosteric regulation of thrombin activity [linkage between thrombin. In particular, thrombin's 60-loop is found the active site and the fibrinogen recognition exosite (Parry in a significantly different conformation. Concomitant et al., 1993; De Cristofaro et al., 1995)], the sodiumreorganization of other surface loops that surround mediated fast-slow transition (Wells and Di Cera, 1992), the active site, i.e. the 37-loop, the 148-loop and the and the thrombomodulin-dependent activation of protein C 99-loop, is observed. Thrombin can therefore undergo (Ye et al., 1991), movements in the thrombin molecule major structural reorganization upon strong ligand have been assumed to be minimal. In particular, it has binding. Implications for the interaction of thrombin been an implicit assumption that the 60-loop represents a with antithrombin and thrombomodulin are discussed. rigid feature of thrombin, which would explain its poor Keywords: antithrombin/conformational change/Kunitz inhibition by the paradigmatic serine pr...

Electrostatic interactions between the thrombin anion-binding exosite-I (ABE-I) and the hirudin C-terminal tail play an important role in the formation of the thrombin-hirudin inhibitor complex and serves as a model for the interactions of thrombin with its many other ligands. The role of each solvent exposed basic residue in ABE-I (Arg(35), Lys(36), Arg(67), Arg(73), Arg(75), Arg(77a), Lys(81), Lys(109), Lys(110), and Lys(149e)) in electrostatic steering and ionic tethering in the formation of thrombin-hirudin inhibitor complexes was explored by site directed mutagenesis. The contribution to the binding energy (deltaG(degrees)b) by each residue varied from 1.9 kJ mol(-)(1) (Lys(110)) to 15.3 kJ mol(-1) (Arg(73)) and were in general agreement to their observed interactions with hirudin residues in the thrombin-hirudin crystal structure [Rydel, T. J., Tulinsky, A., Bode, W., and Huber, R. (1991) J. Mol. Biol. 221, 583-601]. Coupling energies (delta deltaG(degrees) int) were calculated for the major ion-pair interactions involved in ionic tethering using complementary hirudin mutants (h-D55N, h-E57Q, and h-E58Q). Cooperativity was seen for the h-Asp(55)/Arg(73) ion pair (2.4 kJ mol(-1)); however, low coupling energies for h-Asp(55)/Lys(149e) (deltadeltaG(degrees)int 0.6 kJ mol(-1)) and h-Glu(58)/Arg(77a) (deltadeltaG(degrees)int 0.9 kJ mol(-1)) suggest these are not major interactions, as anticipated by the crystal structure. Interestingly, high coupling energies were seen for the intermolecular ion-pair h-Glu(57)/Arg(75) (deltadeltaG(degrees)int 2.3 kJ mol(-1)) and for the solvent bridge h-Glu(57)/Arg(77a) (deltadeltaG(degrees)int 2.7 kJ mol(-1)) indicating that h-Glu(57) interacts directly with both Arg(75) and Arg(77a) in the thrombin-hirudin inhibitor complex. The remaining ABE-I residues that do not form major contacts in tethering the C-terminal tail of hirudin make small but collectively important contributions to the overall positive electrostatic field generated by ABE-I important in electrostatic steering.

Basic fibroblast growth factor (FGF-2) activates its high-affinity receptors (FGFRs) but also acts through interaction with heparan sulfate proteoglycans (HSPG). Exogenous polysaccharides also modulate the angiogenic activity of FGF-2. We investigated the effect and mechanism of action of a low molecular weight fucoidan derivative (LMWF) on tube formation by human endothelial cells. LMWF has a better arterial antithrombotic potential in animals than low molecular weight heparin (LMWH). After stimulation of human umbilical vein endothelial cells (HUVEC) by FGF-2 and LMWF (or LMWH), we observed 1) using flow cytometry, an increase in the amount of the ␣6 integrin subunit; 2) using quantitative reverse transcriptionpolymerase chain reaction, an increase in ␣6 mRNA (higher with LMWF than with LMWH); and 3) using a Matrigel model, an increase in vascular tube formation (also higher with LMWF than with LMWH). A direct link between ␣6 overexpression and vascular tube formation was confirmed by use of an anti-␣6 antibody: in its presence, there was no capillary network formation on Matrigel. Unexpectedly, an anti-FGFR blocking antibody had no effect on ␣6 over-expression, whereas stripping off the heparan sulfate with heparitinases abolished overexpression. Overall, our data suggest that FGF-2 stimulates ␣6 over-expression in HUVEC, through HSPG but independently from FGFR, and that LMWF (or LMWH) modulates this interaction. Expression of heparan sulfate proteoglycan increases after ischemic injury. Given its antithrombotic properties and its ability to potentiate tube formation of endothelial cells, LMWF may have to be considered for revascularization of ischemic areas.