Recombinant thrombomodulin inhibits arterial smooth muscle cell proliferation induced by thrombin.

PURPOSE: Restenosis after angioplasty or bypass grafting to restore circulation to ischemic organs is still an unsolved problem. Thrombin generated in high concentrations at the sites of vascular injury plays a central role in thrombosis and hemostasis. alpha-Thrombin has also been implicated as a mitogen for smooth muscle cell (SMC) proliferation that contributes to arterial restenosis. Thrombomodulin has a high affinity of binding with thrombin and converts thrombin from a procoagulant to an anticoagulant. This study was designed to examine whether thrombomodulin could also moderate the thrombin-mediated SMC proliferative response. METHODS: Porcine carotid artery SMCs (passages 4-7) were plated onto 96-well plates and incubated for 3 days. After growth arrest in a defined serum-free medium for 2 to 3 days, SMCs were subjected to the reagents as follows: (1) human alpha-thrombin, (2) recombinant human soluble thrombomodulin containing a chondroitin sulfate moiety, (3) thrombin receptor agonist peptide (SFLLRNPNDKYEPF), and (4) alpha-thrombin or thrombin receptor agonist peptide combined with recombinant thrombomodulin (rTM). The viability and proliferation status of SMCs were quantified with MTT (thiazolyl blue) mitochondrial function and bromodeoxyuridine (BrdU)-DNA incorporation assays. RESULTS: Human alpha-thrombin increased SMC proliferation in a dose dependent manner by more than 25% and 30% with thrombin 1 U/mL to 3 U/mL compared with control groups on day 7 (P <.006). rTM concentrations from 0.5 microg/mL to 3 microg/mL have no significant effect on SMC growth. The stimulation of SMC proliferation induced by alpha-thrombin at 0.5 U/mL, 1 U/mL, and 2 U/mL was significantly inhibited with rTM at 2 microg/mL and 3 microg/mL on days 3, 7, and 10 as evaluated with MTT assay (P <.01 to <.05) and BrdU-DNA incorporation assay on day 3 (P <.008). Thrombin receptor agonist peptide increased SMC BrdU-DNA incorporation at 48 hours (P <.007), and its effect was not altered by rTM. CONCLUSION: rTM containing all of the extracellular domains of thrombomodulin inhibits the effect of thrombin on SMC proliferation in vitro. Because thrombin is a mitogenic mediator of SMC in vascular injury, inhibition of its function in vivo could help to prevent SMC hyperplasia. The success of further studies in vivo may lead to use of rTM for decreasing or preventing arterial restenosis.

Antithrombotic efficacy in the guinea pig of a derivative of human protein C with enhanced activation by thrombin.

Conversion by alpha-thrombin of the zymogen human protein C (HPC) to activated protein C (aPC) is an important physiologic feedback control mechanism for the coagulation cascade. Although activation of HPC by thrombomodulin-bound thrombin is relatively rapid, activation by free thrombin occurs at a significantly slower rate. Previously, we generated a "hyper-activatable" derivative of HPC (FLIN-Q3) with an increased activation rate by free alpha-thrombin in vitro. In this study, the antithrombotic efficacy of FLIN-Q3 was compared with both native zymogen and aPC in an arteriovenous shunt model of thrombosis in the guinea pig. Recombinant proteins were infused 15 minutes before and throughout a 15-minute period while blood was circulated from carotid to jugular through tubing that enclosed a thread on which fibrin was deposited. Parallel dose-dependent antithrombotic responses were observed. Under these non-steady-state conditions, the calculated infusion doses associated with a 50% reduction of thrombus mass were 2.7, 24, and 250 mg/kg/h for aPC, FLIN-Q3, and HPC, respectively. Thrombus weight correlated inversely with plasma concentration of aPC, measured amidolytically, from either direct infusion of aPC or that generated from the zymogens in the animal, and similarly correlated inversely with anticoagulant activity measured by whole blood aPTT. Neither zymogen form showed significant aPC activity before shunt circulation, suggesting a requirement for exposure to thrombin. After the infusion was discontinued for 15 minutes, a second period of thrombus formation in the shunt demonstrated the ability of zymogen forms of PC, unlike aPC, to provide "on-demand" anticoagulant responses to repeated thrombotic stimuli. Thus, a "hyper-activatable" PC molecule such as FLIN-Q3 may represent a superior form of anticoagulant therapy than either the native zymogen or aPC.

Mutation of protease domain residues Lys37-39 in human protein C inhibits activation by the thrombomodulin-thrombin complex without affecting activation by free thrombin.

Activated protein C (aPC) is an important feedback regulator of the clotting cascade. In vivo, the conversion of protein C (PC) from its zymogen to activated form is mediated primarily by thrombin bound to thrombomodulin (TM), an endothelial cell surface protein. Molecular modeling suggests residues Lys37-Lys38-Lys39 of protein C's serine protease domain reside in a surface-exposed loop (variable region 1) whose high concentration of positive charge might be involved in protein-protein interactions. In this study, we have examined the role of the conserved tribasic Lys37-39 charge center in human protein C activation. This sequence was changed to acidic by substitution with Asp37-Glu38-Asp39 (DED) and Glu37-Glu38-Glu39 (EEE), or to neutrality by substitution with Gly37-Gly38-Gly39 (GGG). These mutant PCs, expressed and purified from recombinant human 293 cells, appeared normal with regard to intracellular processing, ability to be secreted, and formation of a viable active site for tripeptidyl-p-nitroanilide substrate cleavage. For activation by free thrombin, wild-type (wt) and mutant PCs displayed equivalent activation rates, as well as identical calcium-dependent inhibition of such activation. Activation of wt-PC with a soluble TM-thrombin complex yielded a 2,000-fold faster rate compared with that by free thrombin at the same (physiological) calcium level. In contrast, the acidic mutants DED and EEE exhibited virtually no TM-mediated increase in activation rate, while the neutral mutant GGG was somewhat intermediate with a 30-fold stimulation of activation rate. These reductions in activation rate were independent of the presence of chondroitin sulfate on TM. Our observations represent the first identification of residues whose mutation essentially uncouples activation by the TM-thrombin complex without affecting activation by free thrombin. Further, our results suggest that VR1 residues within the zymogen form of a serine protease can be important for recognition by physiological activators.

Identification of a region in protein C involved in thrombomodulin-stimulated activation by thrombin: potential repulsion at anion-binding site I in thrombin.

During coagulation human protein C is activated by thrombin; however, this cleavage reaction is slow unless thrombin is complexed with a cofactor, thrombomodulin. Near the thrombin cleavage site in protein C is a cluster of basic residues, at positions P5' (Lys-174), P8' (Arg-177) and P9' (Arg-178). We have explored the role of this basic cluster in the activation of protein C by thrombin, and by thrombin-thrombomodulin complex, by substitution of glutamic acid at each position to generate the acidic protein C derivative P'-EEE. The activation rate of P'-EEE by free alpha-thrombin was approx. 12-fold faster than that observed for wild-type (wt) human protein C zymogen (HPC) in the presence of calcium, but unchanged in the absence of calcium. While the thrombin-catalysed activation of wt-HPC was stimulated approx. 300-fold by thrombomodulin, we observed no effect of thrombomodulin on thrombin-catalysed activation of the P'-EEE derivative. Using synthetic peptides that bind to anion-binding site I of thrombin (thrombin-receptor sequence 52-66 and hirudin sequence 54-65 SO4 Tyr), we found that the rate of thrombin-catalysed activation of wt-HPC in the presence of calcium could be increased severalfold in a dose-dependent manner. However, the enhanced rate of thrombin-catalysed activation of P'-EEE could be progressively reduced to wt-HPC levels with increasing concentrations of both synthetic peptides. Our data suggest that the P' basic cluster in protein C reduces interaction with free alpha-thrombin through electrostatic repulsion with anion-binding site I, a site that is masked when thrombomodulin binds thrombin. Further, the lack of thrombomodulin cofactor activity with thrombin-catalysed activation of P'-EEE suggests that the basic cluster in protein C forms a contact site with thrombomodulin.

Charge reversal at the P3' position in protein C optimally enhances thrombin affinity and activation rate.

We have examined the properties of several human protein C (HPC) derivatives with substitutions for acidic residues near the thrombin cleavage site, including changing the P3' Asp to Asn (D172N), Gly (D172G), Ala (D172A), or Lys (D172K). The rate of thrombin-catalyzed activation of D172N, D172G, and D172A was increased 4-9-fold compared to wild-type HPC, primarily due to a reduction in the inhibitory effect of calcium and a resulting increase in affinity for free alpha-thrombin. There was no significant increase in activation rate or affinity with these 3 derivatives in the absence of calcium, confirming that P3' Asp affects calcium dependency in the native protein C molecule. With charge reversal at P3' (D172K), there was a 30-fold increase in activation rate in the presence of calcium, but unlike the other derivatives, there was a substantial effect (5-fold) on the activation rate and affinity for free alpha-thrombin in the absence of calcium. Thus, protein C affinity for thrombin appears to be influenced by a combination of calcium-dependent and -independent effects of the acidic P3' residue.

Identification of the predominant glycosaminoglycan-attachment site in soluble recombinant human thrombomodulin: potential regulation of functionality by glycosyltransferase competition for serine474.

Thrombomodulin (TM) is an endothelial cell thrombin receptor that converts thrombin from a procoagulant to an anticoagulant enzyme. It has previously been shown that TM is expressed in both a high-M(r) form containing chondroitin sulphate and a low-M(r) form lacking this modification. Site-directed mutagenesis of a soluble human TM derivative (TMD1) was employed to determine the attachment site(s) of this functionally important oligosaccharide on the core protein. Although there are four serine residues within the Ser/Thr-rich domain of TMD1 that might support glycosaminoglycan assembly, our analysis demonstrates that the primary site of attachment is at Ser474, and evidence is presented for low levels of attachment at Ser472. It was possible to improve the overall degree of attachment by mutating Ser472 to glutamic acid (so as to conform Ser474 to the xylosyltransferase acceptor consensus acidic-Gly-Ser-Gly-acidic); however, a significant proportion (approx. 35%) of the total TM still lacked a glycosaminoglycan moiety. Mutants that possess a substitution for Ser474 show an increased mobility of their low-M(r) form on SDS/PAGE compared with native TMD1. Isolation and sequencing of a C-terminal peptide demonstrated that this serine is modified in the low-M(r) form of native TMD1. An apparent 'acceptor consensus overlap' at Ser474 suggests that the mechanism behind the glycosaminoglycan split of TM may involve a competition for substrate between xylosyltransferase and N-acetylgalactosaminyltransferase.

Enhancing protein C interaction with thrombin results in a clot-activated anticoagulant.

Human protein C is a vitamin K-dependent plasma glycoprotein that circulates as an inactive zymogen. At the endothelial cell surface, thrombin in complex with the integral membrane protein thrombomodulin converts protein C to its active form by specific cleavage of an activation peptide. The activated form of protein C has potent anticoagulant activity as a feedback regulator of thrombin generation (reviewed in refs 4-6), and also has profibrinolytic, anti-ischaemic and anti-inflammatory properties. Protein C is effective in the treatment of model and human thrombotic diseases but, except when it has been used to treat genetic or acquired deficiencies and microvascular thrombosis, it is administered as the activated enzyme, which has a short biological half-life. We have altered two putative inhibitory acidic residues near the thrombin cleavage site, which results in a 30-fold increase in substrate utilization by alpha-thrombin. We combined these changes with a genetically altered glycoform to generate a zymogen protein C with a 60-fold increased cleavage rate by free alpha-thrombin, independent of its cofactor thrombomodulin. We show that this 'proform' of protein C, unlike the natural circulating zymogen, can be activated by thrombin generated in clotting human plasma, resulting in an inhibition of further clot formation. Our data therefore show that we have engineered a site-activated agent, which only has anticoagulant activity when significant amounts of thrombin are being generated.

Glycosylation of human protein C affects its secretion, processing, functional activities, and activation by thrombin.

Human protein C (HPC) is an antithrombotic serine protease that circulates in the plasma as several glycoforms. To examine the role of glycosylation in the function of this protein, we singly eliminated each of the four potential N-linked glycosylation sites by site-directed mutagenesis of Asn to Gln at amino acid positions 97, 248, and 313 (HPC derivatives Q097, Q248, and Q313) or at the unusual consensus sequence Asn-X-Cys at 329 (HPC derivative Q329). The cDNAs for wild type and each derivative were inserted into expression vectors and expressed both transiently and stably in human 293 and hamster AV12-664 cells. We demonstrate that N-linked glycosylation at position 97 in the light chain of HPC is critical for efficient secretion and affects the degree of core glycosylation at Asn-329. Glycosylation at position 248 affects the intracellular processing of the internal Lys-Arg (KR) KR cleavage site, and partial glycosylation at the sequence Asn-329-X-Cys is responsible for the natural alpha-glycoform. Altering the glycosylation pattern of the protein had no significant effect on the level of fully gamma-carboxylated HPC secreted from the 293 cell line. However, elimination of glycosylation sites in the heavy chain resulted in a 2- to 3-fold increase in anticoagulant activity. Utilizing synthetic substrate, both the Km and kcat were affected, depending on the specific glycosylation site eliminated. However, there were no significant differences in the inhibition kinetics by alpha-1-antitrypsin (association rate constants of 10-11 M-1s-1 and t1/2 of 27-29 min at 40 microM alpha-1-antitrypsin) or t1/2 in human plasma (17-18 min). A comparison of the rate of activation of each derivative by thrombin alone or in complex with thrombomodulin revealed that Q313 was activated approximately 2.5-fold faster than wt HPC, independent of calcium concentration. This increase in rate was due to an enhanced affinity of thrombin-thrombomodulin for Q313, as indicated by a 3-fold reduction in Km. Overall, our studies demonstrate that glycosylation at different sites in HPC affects distinct properties of this complex protein. Furthermore, we demonstrate the ability to improve the catalytic efficiency of this enzyme through carbohydrate modifications.