Thrombin has dual trypsin-like and chymotrypsin-like specificity

Abstract

Background:

The residue at the site of activation of protein C is Arg in all species except the ray-finned fish, where it is Trp. This feature raises the question as to whether thrombin is the physiological activator of protein C across vertebrates.

Objective:

Establish if thrombin can cleave at Trp residues.

Methods:

The activity of thrombin wild-type and mutant D189S was tested with a library of chromogenic substrates and toward protein C wild-type and mutants carrying substitutions at the site of cleavage.

Results:

Thrombin has trypsin-like and chymotrypsin-like specificity and cleaves substrates at Arg or Trp residues. Cleavage at Arg is preferred, but cleavage at Trp is significant and comparable to that of chymotrypsin. The D189S mutant of thrombin has broad specificity and cleaves at basic and aromatic residues without significant preference. Thrombin also cleaves natural substrates at Arg or Trp residues, showing activity toward protein C across vertebrates, including the ray-finned fish. The rate of activation of protein C in the ray-finned fish is affected by the sequence preceding Trp at the scissile bond.

Conclusions:

The results provide a possible solution for the paradoxical presence of a Trp residue at the site of cleavage of protein C in ray-finned fish and support thrombin as the physiological activator of protein C in all vertebrates. The dual trypsin-like and chymotrypsin-like specificity of thrombin suggests that the spectrum of physiological substrates of this enzyme is broader than currently assumed.

1. Introduction

Thrombin is a serine protease engaged in multiple functional roles in the blood [1]. As other proteases involved in blood coagulation, fibrinolysis and complement, thrombin features a trypsin-like specificity due to the presence of an Asp residue (D189) in the primary specificity pocket that confers preference for a basic residue (Arg or Lys) [2–4] at the P1 position of substrate [5]. In contrast, proteases featuring chymotrypsin-like specificity carry a Ser residue (S189) in the primary specificity pocket and cleave preferentially at aromatic residues (Phe or Trp) [2–4]. The two types of specificity, trypsin-like toward basic residues and chymotrypsin-like toward aromatic residues, have long been considered as mutually exclusive. In fact, trypsin cleaves basic residues 5–6 orders of magnitude faster than aromatic residues and the reverse is observed for chymotrypsin [6]. Pioneering studies have shown that the D189S replacement in trypsin is insufficient to achieve chymotrypsin-like specificity and additional replacements are needed in the 186- and 220- loops lining the primary specificity pocket and residue 172 [6, 7]. However, the same strategy fails to convert chymotrypsin to trypsin [8], suggesting that specificity is a distributed property arising from multiple domains of the structure. Whether such a conclusion extends to highly specialized enzymes like thrombin remains to be established.

In addition to its procoagulant and prothrombotic roles, i.e., cleavage of fibrinogen leading to clot formation and cleavage of protease activated receptor (PAR) 1 leading to platelet activation, thrombin functions as an anticoagulant by converting the zymogen protein C (PC) to its active form APC (Figure 1) with the assistance of the endothelial receptor thrombomodulin (TM) [1]. APC down-regulates the coagulation response by inactivating the cofactors V and VIII that are responsible for the amplification of thrombin generation from prothrombin [9]. APC also plays a cytoprotective role mediated by cleavage of PAR1 and PAR3 [10]. Blood coagulation evolved in the common vertebrate ancestor and the genes encoding prothrombin and PC have been identified in all classes of vertebrates [11, 12]. Thrombin activates PC in higher mammals [13], but it is unknown whether this is the case in other vertebrates. Specifically, it is unclear whether thrombin activates PC in ray-finned fish that carries a conserved Trp instead of Arg in the cleavage site (Figure 1), as observed in all other vertebrates [14]. Thrombin has a strong preference for synthetic and natural substrates carrying Arg at the P1 position [15], but can also cleave chromogenic substrates with Phe at P1 with significant specificity, unlike other trypsin-like proteases [16]. Furthermore, like chymotrypsin and unlike any other coagulation protease, thrombin is inhibited by heparin cofactor II that carries Leu at the P1 position [17]. Whether the broadened specificity of thrombin extends to natural substrates like PC in the ray-finned fish is investigated in this study to better understand its physiological importance.

Figure 1AB. Structural architecture of PC.

(A) Schematic representation of the multidomain assembly of PC comprised of a γ-carboxyglutamate (Gla), epidermal growth factor (EGF) 1 and 2, AP linker and serine protease domains. (B) Sequence of the AP linker in human and herring PC. The AP linker is numbered based on human mature PC as the sequence between the zymogen activation site at R169 and the perfectly conserved C141 that connects EGF2 to the protease domain. The AP_FISH variant was engineered by replacing in the AP the human sequence (underlined) with that of herring. The zymogen activation site is indicated by an arrow.

2. Methods

Mutations in the plasmids of interest were introduced using a Quick Change Lightning mutagenesis kit (Agilent Technologies). The AP_FISH plasmid was constructed by deleting the sequence ¹⁴⁴PWKRMEKKRSHLKRDTEDQEDQVDPR¹⁶⁹ in the activation peptide (AP) linker of human PC and replacing it with the sequence ¹³⁷ILIHKSSFNTGPIQGLLPW¹⁵⁵ from the AP linker of herring (Clupea harengus; XP_031415396.1) (Figure 1). PC variants were expressed in baby hamster kidney cells and purified as described[18]. Human thrombin wild-type (WT) or mutant D189S and rat anionic trypsin were expressed, purified and tested for activity as described [19, 20]. α-chymotrypsin (0.5–2 nM) from bovine pancreas (C4129) was from Sigma-Aldrich, while the chromogenic substrates H-D-Phe-Pro-Phe-p-nitroanilide (FPF), H-D-Phe-Pro-Arg-p-nitroanilide (FPR) and H-D-Phe-Pro-Trp-p-nitroanilide (FPW) were from MidWest Biotech (Fisher, IN). Measurements of the Michaelis-Menten parameters k_cat and K_m for cleavage of chromogenic substrates were carried out from direct integration of progress curves of substrate hydrolysis taking into account product inhibition [21]. The activation of PC by thrombin (0.5–200 nM) in complex with rabbit TM (100–400 nM; Prolytix) was monitored by a discontinuous assay under pseudo-first order conditions where the activation reaction was quenched at different time points with hirudin and activity was measured using the S-2366 (Diapharma) chromogenic substrate [22]. Experimental conditions for PC activation were: 20 mM Tris, 145 mM NaCl, 10 mM CaCl₂, 0.1% PEG8000, 0.1% BSA, pH 7.5 at 37 °C. N-terminal sequencing was performed at the protein core facility of the Iowa State University. Intrinsic disorder was measured using the VSL2 algorithms [23] from the PONDR family of predictors.

3. Results

3.1. Cleavage of synthetic substrates with basic and aromatic residues at the P1 position

The specificity of thrombin was studied with a library of chromogenic substrates FPX, with residues FP at the P3-P2 positions fixed for optimal interaction with the enzyme and X at the P1 position carrying basic (Arg) or aromatic (Phe, Trp) residues. Thrombin shows highest specificity for FPR, as seen in previous studies [16, 24], but cleaves FPF with a k_cat/K_m comparable to that of chymotrypsin and FPW with a value about 50-fold lower (Table 1, Figure 2A). In contrast, trypsin cleaves FPF and FPW at rates 10⁵-fold slower than chymotrypsin and 10⁶-fold slower than FPR (Table 1, Figure 2A). These findings support the conclusion that thrombin is more versatile than trypsin in its P1 specificity and features a preference for basic residues at the P1 position that is not nearly absolute. An important corollary of this property is that the D189S substitution in thrombin reduces activity toward FPR but has no significant effect on the hydrolysis of FPF and FPW (Table 1, Figure 2A), thereby producing an enzyme with broad specificity toward basic and aromatic residues at the P1 position of substrate. This property is rare among proteases and only a few examples have been documented [25–27].

Table 1.

k_cat/K_m values (in μM⁻¹s⁻¹) for cleavage of chromogenic substrate by various serine proteases

	Trypsin	Chymotrypsin	WT Thrombin	D189S Thrombin
FPR	7.9±0.4	0.028±0.002	80±3	0.47±0.01
FPF	1.6±0.1 10−5	0.81±0.01	0.33±0.01	0.46±0.01
FPW	3.2±0.1 10−6	1.8±0.1	0.035±0.001	0.058±0.002

Experimental conditions: 20 mM Tris, 200 mM NaCl, 0.1% PEG8000, pH 8.0 at 25 °C

Figure 2AB. Protease specificity toward synthetic and natural substrates.

(A) Logarithm of the values of the specificity constant s=k_cat/K_m for the hydrolysis of the chromogenic substrates FPR, FPF and FPW (as indicated) by trypsin, chymotrypsin and thrombin, wild-type and mutant D189S (see also Table 1). (B) Logarithm of the values of the specificity constant s=k_cat/K_m for the hydrolysis of PC constructs (as indicated) carrying R169 (wild-type, 6A, AP_FISH-P1Arg) or W169 (R169W, 6A-R169W, AP_FISH) at P1 by thrombin wild-type and mutant D189S (see also Table 2).

3.2. Cleavage of physiological substrates with Trp at the P1 position

The ability of thrombin to cleave aromatic residues at the P1 position was tested with PC by replacing R169 at the site of cleavage with Trp, which is the residue found in all ray-finned fish (Figure 1). Earlier studies reported that thrombin would not activate the R169W mutant of Gla-domainless PC [28], but the assays were performed with low concentrations of thrombin and monitored over a short period of time (5–10 min). Longer incubation (5 hr) and higher thrombin concentration (80 nM) produce a detectable activation of the R169W mutant of PC (Figure 3) which is abolished by the thrombin specific inhibitor hirudin (Figure 3A), ruling out possible contamination by other proteases. Activation of the R169W of PC is also confirmed by SDS-PAGE analysis, where a progressive decrease in the concentration of single chain PC is accompanied by increased appearance of the heavy and light chains of APC (Figure 3B). N-terminal sequencing of the heavy chain reveals the sequence ¹⁷⁰LIDGKM¹⁷⁵ expected for cleavage at W169 and rules out potential contamination by bovine PC whose sequence is instead ¹⁷⁹IVDGQE¹⁷⁵. Finally, the R169W mutant of PC could be activated by chymotrypsin under conditions where no cleavage of wild-type PC would be observed (Figure 3C), ruling out the possibility of contamination of the R169W variant by forms carrying Arg at P1.

Figure 3ABC. Activation of the PC mutant R169W by thrombin.

(A) Activation by thrombin (80 nM) in the presence of TM (200 nM) measured after 0 hr (black) and 5 hr incubation, with (red) or without (blue) excess hirudin. APC activity was measured after quenching the reaction with hirudin and following cleavage of S-2366 at 405 nm. (B) SDS-PAGE analysis under reducing conditions following incubation with thrombin (IIa, 200 nM) and TM (400 nM). Activation is evident from the decrease of single chain (S.C.) PC and concomitant increase of heavy chain (H.C.) and light chain (L.C.) bands. (C) Progress curve of activation of PC wild-type (black) and mutant R169W (blue) by chymotrypsin (0.2 nM) monitored by following the hydrolysis of the APC specific substrate H-D-Asp-Arg-Arg-p-nitroanilide [45] at 405 nm. Experimental conditions are: 20 mM Tris, 145 mM NaCl, 10 mM CaCl₂, 0.1% PEG8000, pH 7.5 at 37 °C.

Although thrombin in complex with TM can cleave the R169W mutant of PC, the rate of activation remains >1200-fold slower compared to wild-type PC (Table 2, Figure 2B). The observation is consistent with the results of chromogenic substrates (Table 1, Figure 2A) and confirms the preference of thrombin for basic residues at P1. On the other hand, the D189S mutant of thrombin activates the R169W mutant of PC at a rate nearly 20-fold faster than that of wild-type thrombin and 6-fold faster than that of wild-type PC (Table 2, Figure 2B), confirming its broad specificity (Table 1, Figure 2A).

Table 2.

k_cat/K_m values (in mM⁻¹s⁻¹) for PC activation by thrombin WT and mutant D189S in the presence of TM

	PC constructs with Arg at P1			PC constructs with Trp at P1
	WT	6A	APFISH_P1-Arg	R169W	6A R169W	APFISH
WT	220± 20	550±50	2000±300	0.18±0.04	0.7±0.1	7.8± 0.2
D189S	0.34±0.05	6.9±0.9	65±8	2.2±0.2	4±1	30±9

Experimental conditions: 20 mM Tris, 145 mM NaCl, 10 mM CaCl₂, 0.1% PEG8000, 0.1% BSA, pH 7.5 at 37 °C

3.3. Role of the extended sequence preceding the scissile bond in PC

The activity of several serine proteases decreases orders of magnitude upon progressive truncation of the amino acids comprising the P4-P2 positions of substrate [29–32], because of removal of important interactions that position the scissile bond for optimal cleavage [24]. The extended sequence around the scissile site also affects the zymogen activation rate in several coagulation factors. Residues that occupy the P5-P11 position of the R271 cleavage site of prothrombin guide preferential attack by fXa at this site over the alternative R320 cleavage site [33]. Charge neutralization of the cluster of acidic residues in PC near the scissile bond significantly enhances the rate of activation by thrombin [22, 34–37], increases the propensity of promiscuous activation by non-physiological coagulation proteases [22] and autoactivation [34]. Similarly, shortening the length of the AP linker [38] or mutating disorder promoting residues [39] and glycosylation sites [39, 40] in this region modulates the activation rate of fX and its propensity for autoactivation.

Comparative sequence analysis has shown that the least conserved region of PC is the AP linker immediately upstream to the zymogen activation site [14]. During the emergence of placental mammals, the AP linker of PC evolved extensive structural modifications that included a novel pro-protein convertase processing site at the N-terminus, a significant elongation, an increase in the number of charged residues and sequence convergence of the important Pro at the P2 position that was lost during the emergence of tetrapods [14]. Hence, differences in the extended sequence near the scissile bond of PC from fish may promote cleavage by thrombin of Trp at P1. To test this hypothesis, we generated the AP_FISH chimera by replacing the entire AP linker of human PC with the analogous sequence from the Atlantic herring (Clupea harengus; XP_031415396.1) (Figure 1). The AP linker connects the EGF2 and protease domains and its C-terminus ends at the P1 residue (Figure 1). The AP_FISH chimera is activated by the thrombin-TM complex 43-fold faster than the R169W mutant of PC (Table 2, Figure 2B). Adding the W169R replacement in AP_{FISH_P1-ARG} increases the rate further to 10-fold higher than wild-type PC (Table 2, Figure 2B). On the other hand, the D189S mutant of thrombin activates AP_FISH and AP_{FISH_P1-ARG} at comparable rates (Table 2, Figure 2B), again consistent with its broad specificity.

The AP of ray-finned fish lacks acidic residues found in human PC (Figure 1) [22]. The contribution of this difference was tested with the 6A/R169W mutant of PC carrying Ala substitutions of all six acidic residues in the AP linker, along with the R169W replacement. The rate of activation of 6A/R169W by thrombin (Table 2) was not comparable to that observed with AP_FISH, supporting the conclusion that the AP sequence in ray-finned fish makes specific contributions not recapitulated by removal of potential electrostatic clashes, as observed for human PC. We have recently shown that the AP linker of mammals is the most disordered region of the sequence of PC [22]. Analysis of the sequence in herring PC reveals significant differences in the predicted disorder disposition scores, with the AP linker being highly ordered compared to the analogous region of human PC (Figures 4A,B). Interestingly, there is a negative correlation between the disorder disposition of the AP linker and the rate of activation by the thrombin-TM complex (Figure 4C,D), regardless of the nature of the P1 residue, basic or aromatic.

Figure 4ABCD. Disorder disposition of the AP linker of PC.

The disorder disposition affects the cleavage rate of the residue at P1 by the thrombin-TM complex. (A) Analysis of the primary sequences of human (blue), herring (purple), 6A (green), and AP_FISH (orange) using the VSL2 algorithm. (B) Average disorder disposition of the AP linker sequence. Correlation of the disorder disposition of the AP linker of PC and the k_cat/K_m values for activation of PC variants by the thrombin-TM complex carrying Arg (C) or Trp (D) at P1.

4. Discussion

Thrombin specificity has historically been defined as trypsin-like because of the presence of D189 in its primary specificity pocket [41] and a basic residue (Arg/Lys) at the P1 position of all known natural substrates [1]. A recent comparative sequence analysis has revealed that PC in all ray-finned fish carry Trp at P1, raising the question as to whether PC has activators other than thrombin and possibly other functions in ray-finned fish. The results presented here do not address these alternative possibilities but show that thrombin is more versatile than trypsin insofar as it is capable of cleaving synthetic and natural substrates with basic or aromatic residues at P1, even though it carries D189 in the primary specificity pocket of all species [14]. The trypsin-like specificity of thrombin predominates but it is not exclusive because of significant activity that is chymotrypsin-like. It is therefore possible that the dual trypsin-like and chymotrypsin-like specificity of thrombin allows the enzyme to function as the physiological activator of PC in mammals (carrying Arg at P1) and ray-finned fish (carrying Trp at P1). An intriguing possibility also arises for thrombin acting on other physiological targets, yet to be identified, by cleaving at aromatic residues. Interestingly, the GLLPW↓L sequence of the P5-P1’ residues around the scissile site (↓) cleaved by thrombin in herring PC is found in many human proteins (Supplementary Figure 1 and Table 3), including heparin binding EGF that plays an important role in hematopoiesis [42] and anoctamin-6 that regulates exposure of phosphatidylserine on membrane surfaces [43]. The GLLPW↓L sequence is also found in many bacterial proteins (Supplementary Figure 1 and Supplementary Table 1). Because clot formation is an integral part of host defense [44], we speculate that a thrombin with broad specificity would have been beneficial during the evolution of vertebrates if capable of proteolyzing and inactivating several prokaryotic proteins. Finally, an enzyme with broad specificity like the D189S mutant of thrombin may prove useful as a sequencing tool in proteomics.

Table 3.

Distribution of the GLLPW↓L sequence among human proteins

Protein	Accession ID	P5-P1’ Sequence
Fuzzy Protein	NP_001339191.1	XLLPWL
Autogenous vein graft remodeling associated protein	CAJ77183.1	XLLPWL
S-adenosylmethionine synthetase	BAA08355.1	GLLPWL
Heparin binding EGF	AAA50562.1	XLXPWX
Immunoglobulin heavy chain junction region	MBB2077539.1	GXXPWL
Anoctamin-6	NP_001191732.1	XLLPWX
hCG2045022	EAW54630.1	XXLPWL
Catenin-beta like	KAI2594789.1	GLLXWL
Secretory carrier-associated membrane protein 5 isoform X1	XP_047288174.1	XXLXWL
Lipid storage droplet protein 5	AAI31525.1	XXLPWL
Glucosaminyl (N-acetyl) transferase family member 7	KAI4006065.1	XXLPWL
Taste receptor, type 2, member 39, isoform CRA_c	EAW51886.1	GLXPWL
Zinc transporter ZIP13 isoform X1	XP_054226442.1	XXLPWL
2-amino-3-carboxymuconate-6-semialdehyde decarboxylase	NP_612199.2	XXLPWL
Bitter taste receptor T2R39	BAD98035.1	GLXPWL
Lysosomal amino acid transporter 1	NP_001035214.1	XXLPWL
Ig kappa light chain	CAA51128.1	XLLPWL
Hemicentin 2	KAI2554293.1	XLLPWL
Protein furry homolog	XP_047285955.1	XLLPWL
Stalled ribosome sensor GCN1	NP_006827.1	XLLPWL
GCN1 general control of amino-acid synthesis 1-like 1	AAI53882.1	XLLPWL
Retinal-specific phospholipid-transporting ATPase ABCA4	7E7I_A	GXLPWL
ATP-BINDING CASSETTE TRANSPORTER	AAC51144.1	GXLPWL
Clathrin	EAW94395.1	XLLPWL
GATOR complex protein DEPDC5	8FW5_A	LLPWLX

X=variable amino acid residue.