Synthetic substrates for human factor VIIa and factor VIIa-tissue factor

Butenas, Saulius; Ribarik, N; Mann, Kenneth G.

doi:10.1021/bi00077a006

Cited by 55 publications

(58 citation statements)

References 35 publications

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“…Factor VIIa functions physiologically in complex with Ca 2ϩ and tissue factor. Ca 2ϩ is required by factor VIIa to become conformationally active (23), and tissue factor enhances the amidolytic efficiency of factor VIIa by 60-to 100-fold (23,24) by causing a conformational change in factor VIIa (23,25). As expected, we found that in the absence of tissue factor and CaCl 2 , factor VIIa showed essentially no cleavage with our sublibraries (data not shown), consistent with literature reports of poor activity in the absence of these activators (11,20,23,26,27).…”

Section: Resultsmentioning

confidence: 99%

High Throughput Substrate Specificity Profiling of Serine and Cysteine Proteases Using Solution-phase Fluorogenic Peptide Microarrays

Gosalia

Salisbury

Ellman

et al. 2005

Molecular & Cellular Proteomics

152

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Proteases regulate numerous biological processes with a degree of specificity often dictated by the amino acid sequence of the substrate cleavage site. To map protease/substrate interactions, a 722-member library of fluorogenic protease substrates of the general format AcAla-X-X-(Arg/Lys)-coumarin was synthesized (X ‫؍‬ all natural amino acids except cysteine) and microarrayed with fluorescent calibration standards in glycerol nanodroplets on glass slides. Specificities of 13 serine proteases (activated protein C, plasma kallikrein, factor VIIa, factor IXa␤, factor XIa and factor ␣ XIIa, activated complement C1s, C1r, and D, tryptase, trypsin, subtilisin Carlsberg, and cathepsin G) and 11 papain-like cysteine proteases (cathepsin B, H, K, L, S, and V, rhodesain, papain, chymopapain, ficin, and stem bromelain) were obtained from 103,968 separate microarray fluorogenic reactions (722 substrates ؋ 24 different proteases ؋ 6 replicates). This is the first comprehensive study to report the substrate specificity of rhodesain, a papain-like cysteine protease expressed by Trypanasoma brucei rhodesiense, a parasitic protozoa responsible for causing sleeping sickness. Rhodesain displayed a strong P 2 preference for Leu, Val, Phe, and Tyr in both the P 1 ‫؍‬ Lys and Arg libraries. Solution-phase microarrays facilitate protease/substrate specificity profiling in a rapid manner with minimal peptide library or enzyme usage. Molecular & Cellular Proteomics 4:626 -636, 2005.Because of their critical roles in biological pathways like hormone activation, proteasomal degradation, and apoptosis, proteases are essential for cellular function and viability. Proteases regulate hormonal activation, cellular homeostasis, apoptosis, and coagulation and play an important role in the pathogenicity and progression of many diseases (1). Proteases comprise one of the largest protein families in organisms from Escherichia coli to humans (2-4). Improved understanding of proteases will provide insight into biological systems and will likely provide a number of important new therapeutic targets (1).To properly function, proteases must preferentially cleave their target substrates in the presence of other proteins. While many factors impact protease substrate selection, one of the key aspects is the complementary nature of the enzymeactive site with the residues surrounding the cleaved bond in the substrate. As such, determination of the residues that comprise the preferred cleavage site of a protease provides critical information regarding substrate selection. Furthermore, determination of substrate specificity also provides a framework for the design of potent and selective inhibitors.Here we exploit solution-phase substrate nanodroplet microarrays (5), in which fluorogenic substrates suspended in glycerol droplets are treated with aerosolized aqueous enzyme solutions, to provide protease substrate specificity profiles (6). These arrays allow high throughput characterization of the preferred residues on the P side (7) of the substrate in a highl...

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Section: Resultsmentioning

confidence: 99%

High Throughput Substrate Specificity Profiling of Serine and Cysteine Proteases Using Solution-phase Fluorogenic Peptide Microarrays

Gosalia

Salisbury

Ellman

et al. 2005

Molecular & Cellular Proteomics

152

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“…Analysis by quantitative densitometry indicated that possible cleavage of X R15Q by VIIa-TF/PC was below detection limits or at least 50-fold slower than that of plasma-derived factor X. This result is expected based on the established preference of factor VIIa for substrates with an arginine side chain at the P 1 position and with the presumed importance of substrate interactions at the primary specificity pocket for bond cleavage (15,47).…”

Section: Kinetics Of Inhibition By Active Site-directed Ligands-pabmentioning

confidence: 90%

Exosite Interactions Determine the Affinity of Factor X for the Extrinsic Xase Complex

Baugh

Dickinson²,

Ruf³

et al. 2000

Journal of Biological Chemistry

106

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The initiation of coagulation results from the activation of factor X by an enzyme complex (Xase) composed of the trypsin-like serine proteinase, factor VIIa, bound to tissue factor (TF) on phospholipid membranes. We have investigated the basis for the protein substrate specificity of Xase using TF reconstituted into vesicles of phosphatidylcholine, phosphatidylserine, or pure phosphatidylcholine. We show that occupation of the active site of VIIa within Xase by a reversible inhibitor or an alternate peptidyl substrate is sufficient to exclude substrate interactions at the active site but does not alter the affinity of Xase for factor X. This is evident as classical competitive inhibition of peptidyl substrate cleavage but as classical noncompetitive inhibition of factor X activation by active site-directed ligands. This implies that the productive recognition of factor X by Xase arises from a multistep reaction requiring an initial interaction at sites on the enzyme complex distinct from the active site (exosites), followed by active site interactions and bond cleavage. Exosite interactions determine protein substrate affinity, whereas the second binding step influences the maximum catalytic rate for the reaction. We also show that competitive inhibition can be achieved by interfering with exosite binding using factor X derivatives that are expected to have limited or abrogated interactions with the active site of VIIa within Xase. Thus, substrate interactions at exosites, sites removed from the active site of VIIa within the enzyme complex, determine affinity and binding specificity in the productive recognition of factor X by the VIIa-TF complex. This may represent a prevalent strategy through which distinctive protein substrate specificities are achieved by the homologous enzymes of coagulation.The proteolytic activation of factor X by the extrinsic pathway is considered the initiating step of the blood coagulation cascade following vascular damage (4 -7). The conversion of the zymogen, factor X, to the serine protease, factor Xa, is accomplished by the extrinsic Xase complex that assembles through reversible interactions between the serine proteinase, factor VIIa, and the integral membrane cofactor protein, tissue factor (TF) 1 (4, 5). Although this complex can catalyze the proteolytic activation of either factors X or IX to their respective active enzymes with comparable catalytic efficiency, Xa formation is considered essential for the initiation of the coagulation cascade, whereas IXa formation may be important for sustained flux toward thrombin formation (4 -7).The catalytic domains of the serine proteinases of coagulation are highly homologous to each other as well as to trypsin, the archetypical arginine-specific serine proteinase (8, 9). Despite these similarities, the coagulation proteinases act on their protein substrates with narrow and distinctive specificity (10). The molecular basis for the defined protein substrate specificity of the coagulation enzyme complexes is not well understood. It is i...

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“…The fluorogenic 6-peptidylamino-1-naphthalenesulfonamide substrates (PNS substrates) were synthesized and characterized as described previously (23,34,35). For all assays, substrates were initially dissolved in dimethyl sulfoxide to a stock concentration of 10 mM.…”

Section: Methodsmentioning

confidence: 99%

“…proteases involved in blood coagulation and fibrinolysis have been established (23,34,35). In this study, we describe the use of 6-peptidylamino-1-naphthalenesulfonamide substrates with selective inhibitors in single enzyme microassays of the blood coagulation serine proteases.…”

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confidence: 99%

Evaluation of the Initiation Phase of Blood Coagulation Using Ultrasensitive Assays for Serine Proteases

Butenas

Veer

Mann

1997

Journal of Biological Chemistry

Self Cite

121

113

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The initiation phase of enzyme generation in a reconstituted model of the tissue factor (TF) pathway to thrombin was evaluated. At 1.25 pM added TF, no thrombin generation was observed in the absence of factor V. The substitution of factor Va for factor V increased the rate of thrombin generation. Factor X activation during the initiation phase was not influenced by the absence of factor VIII or thrombin, leading to the conclusion that initially factor Xa is generated exclusively by the factor VIIa-TF complex. When thrombin was eliminated from the system, no contribution of the factor IXa-factor VIIIa complex to factor X activation was observed during the propagation phase. Similarly, factor V activation was also not observed in the absence of thrombin, indicating that thrombin is the only enzyme responsible for factor V and factor VIII activation. Only subnanomolar amounts of factor VII were activated when prothrombin activation was almost complete. In the absence of coagulation inhibitors, factor XI did not influence thrombin generation initiated by 1.25 pM factor VIIa-TF complex. The termination of factor XIa generation by added hirudin in the factor XI experiment indicates that factor XI activation occurs exclusively by thrombin.The blood coagulation cascade is thought to be triggered when subendothelial tissue factor (TF) 1 is exposed as a consequence of vascular damage (1, 2). TF forms an enzymatic complex with preexistent plasma factor VIIa, and the resulting factor VIIa-TF complex activates factors X and IX (3-5). Factor IXa in complex with its cofactor, factor VIIIa, activates factor X at an ϳ50-fold higher rate than the factor VIIa-TF complex (6). In turn, factor Xa with phospholipids may activate factor VII (7) and further enhance factor IX and factor X activation. The major function of factor Xa is to form the prothrombinase complex with factor Va and a phospholipid membrane surface, leading to prothrombin activation (8). Thrombin cleaves soluble fibrinogen, forming fibrin, which polymerizes to form an insoluble clot (9). The blood coagulation cascade is down-regulated by the synergistic action of the natural inhibitors of blood coagulation: tissue factor pathway inhibitor, antithrombin III, and the activated protein C system (10, 11).The procoagulant processes described above may be divided into two phases (see Fig. 1): (a) an initiation phase, which is characterized by the appearance of small amounts of thrombin (Ͻ1 nM) and other enzymes (Ͻ10 pM) and by low rates of their generation; and (b) a propagation phase, which may be characterized by rapid, quantitative prothrombin activation. Coincidentally, when the inhibitors are present, thrombin generation is terminated, and extant thrombin is inhibited by the anticoagulation system. The propagation and termination processes have been investigated and described in studies in various reconstituted systems (10 -15), in blood plasma (16), and in minimally altered whole blood (17).The major regulatory steps that occur during the initiation phase are major ...

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Synthetic substrates for human factor VIIa and factor VIIa-tissue factor

Cited by 55 publications

References 35 publications

High Throughput Substrate Specificity Profiling of Serine and Cysteine Proteases Using Solution-phase Fluorogenic Peptide Microarrays

High Throughput Substrate Specificity Profiling of Serine and Cysteine Proteases Using Solution-phase Fluorogenic Peptide Microarrays

Exosite Interactions Determine the Affinity of Factor X for the Extrinsic Xase Complex

Evaluation of the Initiation Phase of Blood Coagulation Using Ultrasensitive Assays for Serine Proteases

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