Leslie A. Bush

Protective Signaling by Activated Protein C Is Mechanistically Linked to Protein C Activation on Endothelial Cells

Activated protein C (APC) has endothelial barrier protective effects that require binding to endothelial protein C receptor (EPCR) and cleavage of protease activated receptor-1 (PAR1) and that may play a role in the anti-inflammatory action of APC. In this study we investigated whether protein C (PC) activation by thrombin on the endothelial cell surface may be linked to efficient protective signaling. To minimize direct thrombin effects on endothelial permeability we used the anticoagulant double mutant thrombin W215A/E217A (WE). Activation of PC by WE on the endothelial cell surface generated APC with high barrier protective activity. Comparable barrier protective effects by exogenous APC required a 4-fold higher concentration of APC. To demonstrate conclusively that protective effects in the presence of WE are mediated by APC generation and not direct signaling by WE, we used a PC variant with a substitution of the active site serine with alanine (PC S360A). Barrier protective effects of a low concentration of exogenous APC were blocked by both wildtype PC and PC S360A, consistent with their expected role as competitive inhibitors for APC binding to EPCR. WE induced protective signaling only in the presence of wild type PC but not PC S360A and PAR1 cleavage was required for these protective effects. These data demonstrate that the endogenous PC activation pathway on the endothelial cell surface is mechanistically linked to PAR1-dependent autocrine barrier protective signaling by the generated APC. WE may have powerful protective effects in systemic inflammation through signaling by the endogenously generated APC.

Molecular mapping of thrombin-receptor interactions

In addition to its procoagulant and anticoagulant roles in the blood coagulation cascade, thrombin works as a signaling molecule when it interacts with the G‐protein coupled receptors PAR1, PAR3, and PAR4. We have mapped the thrombin epitopes responsible for these interactions using enzymatic assays and Ala scanning mutagenesis. The epitopes overlap considerably, and are almost identical to those of fibrinogen and fibrin, but a few unanticipated differences are uncovered that help explain the higher (90‐fold) specificity of PAR1 relative to PAR3 and PAR4. The most critical residues for the interaction with the PARs are located around the active site where mutations affect recognition in the order PAR4 > PAR3 > PAR1. Other important residues for PAR binding cluster in a small area of exosite I where mutations affect recognition in the order PAR1 > PAR3 > PAR4. Owing to this hierarchy of effects, the mutation W215A selectively compromises PAR4 cleavage, whereas the mutation R67A abrogates the higher specificity of PAR1 relative to PAR3 and PAR4. 3D models of thrombin complexed with PAR1, PAR3, and PAR4 are constructed and account for the perturbations documented by the mutagenesis studies. Proteins 2001;45:107–116.

Redesigning the monovalent cation specificity of an enzyme

Monovalent-cation-activated enzymes are abundantly represented in plants and in the animal world. Most of these enzymes are specifically activated by K+, whereas a few of them show preferential activation by Na+. The monovalent cation specificity of these enzymes remains elusive in molecular terms and has not been reengineered by site-directed mutagenesis. Here we demonstrate that thrombin, a Na+-activated allosteric enzyme involved in vertebrate blood clotting, can be converted into a K+-specific enzyme by redesigning a loop that shapes the entrance to the cation-binding site. The conversion, however, does not result into a K+-activated enzyme.

The Thrombin Epitope Recognizing Thrombomodulin Is a Highly Cooperative Hot Spot in Exosite I

The functional epitope of thrombin recognizing thrombomodulin was mapped using Ala-scanning mutagenesis of 54 residues located around the active site, the Na+ binding loop, the 186-loop, the autolysis loop, exosite I, and exosite II. The epitope for thrombomodulin binding is shaped as a hot spot in exosite I, centered around the buried ion quartet formed by Arg67, Lys70, Glu77, and Glu80, and capped by the hydrophobic residues Tyr76 and Ile82. The hot spot is a much smaller subset of the structural epitope for thrombomodulin binding recently documented by x-ray crystallography. Interestingly, the contribution of each residue of the epitope to the binding free energy shows no correlation with the change in its accessible surface area upon formation of the thrombin-thrombomodulin complex. Furthermore, residues of the epitope are strongly coupled in the recognition of thrombomodulin, as seen for the interaction of human growth hormone and insulin with their receptors. Finally, the Ala substitution of two negatively charged residues in exosite II, Asp100 and Asp178, is found unexpectedly to significantly increase thrombomodulin binding.

Murine thrombin lacks Na+ activation but retains high catalytic activity.

Human thrombin utilizes Na+ as a driving force for the cleavage of substrates mediating its procoagulant, prothrombotic, and signaling functions. Murine thrombin has Asp-222 in the Na+ binding site of the human enzyme replaced by Lys. The charge reversal substitution abrogates Na+ activation, which is partially restored with the K222D mutation, and ensures high activity even in the absence of Na+. This property makes the murine enzyme more resistant to the effect of mutations that destabilize Na+ binding and shift thrombin to its anticoagulant slow form. Compared with the human enzyme, murine thrombin cleaves fibrinogen and protein C with similar kcat/Km values but activates PAR1 and PAR4 with kcat/Km values 4- and 26-fold higher, respectively. The significantly higher specificity constant toward PAR4 accounts for the dominant role of this receptor in platelet activation in the mouse. Murine thrombin can also cleave substrates carrying Phe at P1, which potentially broadens the repertoire of molecular targets available to the enzyme in vivo.

Limited generation of activated protein C during infusion of the protein C activator thrombin analog W215A/E217A in primates.

Summary.  Anticoagulation with activated protein C (APC) reduces the mortality of severe sepsis. We investigated whether the circulating protein C (PC) pool could be utilized for sustained anticoagulation by endogenous APC. To generate APC without procoagulant effects, we administered the anticoagulant thrombin mutant W215A;E217A (WE) to baboons. In preliminary studies, administration of high dose WE (110 μg kg−1 i.v. bolus every 120 min; n = 2) depleted PC levels by 50% and elicited transient APC bursts and anticoagulation. The response to WE became smaller with each successive injection. Low dose WE infusion (5 μg kg−1 loading + 5 μg kg−1 h−1 infusion; n = 5) decreased plasma PC activity by 15%, from 105% to 90%, to a new equilibrium within 60 min. APC levels increased from 7.5 ng mL−1 to 86 ng mL−1 by 40 min, then declined, but remained elevated at 34 ng mL−1 at 240 min. A 22‐fold higher dose WE (n = 5) decreased PC levels to 60% by 60 min without significant further depletion in 5 h. The APC level was 201 ng mL−1 at 40 min and decreased to 20 ng mL−1 within 120 min despite continued activator infusion. Co‐infusion of WE and equimolar soluble thrombomodulin (n = 5) rapidly consumed about 80% of the PC pool with significant temporal increase in APC generation. In conclusion, low‐grade PC activation by WE produced sustained, clinically relevant levels of circulating APC. Limited PC consumption in WE excess was consistent with the rapid depletion of cofactor activity before depletion of the PC zymogen. Reduced utilization of circulating PC might have similar mechanism in some patients.

Interaction between thrombin mutant W215A/E217A and direct thrombin inhibitor.

Thrombin mutant W215A/E217A acts as an anticoagulant when administered intravenously by activating plasma protein C in concert with endothelial thrombomodulin [1,2]. The double mutation in the catalytic domain of the W215A/E217A molecule leads to compromised interactions with fibrinogen and chromogenic thrombin substrates [1,3]. Among direct thrombin inhibitors (DTIs), argatroban exerts antithrombotic activity by a rapid binding to the catalytic domain of thrombin, while lepirudin and bivalirudin occupy both exosite I and catalytic domains [4]. We hereby report on the changes in DTI-interaction with W215A/E217A, using thrombin substrate hydrolysis [1,3], activated partial thromboplastin time (aPTT) and the Thrombinoscope methods (Thrombinoscope BV, Maastricht, Netherlands) [5]. Based on the inhibition of H-D-Phe-Pro-Arg-p-nitroanilide hydrolysis, argatroban and lepirudin interact with the catalytic domain of W215A/E217A with a 7000-fold reduced affinity relative to wild-type thrombin (Table 1) [6]. This reduction coincides with the 19 000-fold decrease in affinity for fibrinogen [1]. On the contrary, lack of inhibition of the substrate hydrolysis with bivalirudin suggests that W215A/E217A binds to bivalirudin utilizing exosite I without blocking the active site. This finding was confirmed in studies looking at the competitive inhibitory effect of bivalirudin on the activation of protein C by W215A/E217A in the presence of thrombomodulin (Table 1). Table 1 Binding of wild-type and mutant thrombin to thrombin inhibitors and substrates Although the direct interaction between W215A/E217A and DTIs seems to be weak, addition of W215A/E217A in DTI-treated plasma corrected prolongation of aPTT toward normal (Fig. 1). Prolonged aPTT with DTIs was not reversed with active site mutant S195A [7] or active-site blocked thrombin (wild-type thrombin inactivated with H-D-Phe-Pro-Arg-CH2Cl; FPR-WT). Inactive thrombin species, S195A and FPR-wild-type, occupy thrombin binding sites on fibrin(ogen) and cause dose-dependent inhibition of clotting [8]. It is thus speculated that the catalytic domain of W215A/E217A exerts some procoagulant function through activation of factor V [9]. Anticoagulant effects of DTIs have been shown to be mitigated in the presence of factor Va [10,11]. Fig. 1 Activated partial thromboplastin time, measured in normal volunteer plasma, spiked with a direct thrombin inhibitor (n = 8). Mean values (±SD) of activated partial thromboplastin time(aPTT) (in seconds). Note aPTT values were shortened by W215A/E217A ... Using the Thrombinoscope system that continuously monitors thrombin generation based on the hydrolysis of fluorogenic substrate, Z-Gly-Gly-AMC (Bachem Bioscience, King of Prussia, Pennsylvania, USA), we showed that in plasma the combination of W215A/E217A (5µg/ml~135 nmol/l) and thrombomodulin (0.75µg/ml; Asahi Kasei Pharma, Oh-hito, Japan) [12], but not W215A/E217A alone exerted anticoagulant effects based on the reduced peak levels of thrombin generation (Fig. 2a). The addition of W215A/E217A was found to reverse prolonged thrombin generation lag time for all DTIs (Fig. 2). Reduced peak thrombin levels were not significantly recovered with W215A/E217A at therapeutic levels (0.5–1µg/ml) of argatroban (Fig. 2b) [13]. Our present data are in agreement with the reversal of melagatran-prolonged lag time in thrombin generation with factor Va [10]. For bivalent inhibitors, it is plausible that bivalirudin and lepirudin reduce the interaction of factor V with W215A/E217A by binding to exosite I [9]. FPR-wild-type (30µg/ml) reversed delayed onset of thrombin generation with bivalirudin, but clotting was extensively inhibited due to occupation of thrombin binding sites of fibrin with FPR-wild-type (Fig 1 and Fig 2c) [8]. Lepirudin obliterated thrombin generation, but this was restored toward normal after the addition of W215A/E217A, especially at therapeutic level (1µg/ml) of lepirudin (Fig. 2d). Fig. 2 Effects of W215A/E217A on thrombin generation. Panel a: a representative series of thrombin generation curves. The peak thrombin generation is slightly decreased by the addition of W215A/E217A, 5µg/ml, and modestly decreased by recombinant human ... In summary, the catalytic-site double mutant, W215A/E217A, shortens DTI-prolonged aPTT and the onset of endogenous thrombin generation in vitro. Further modifications of the catalytic domain or exosites of thrombin molecule or both may lead to a development of antidotes for DTI-related bleeding complications [10].