Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.69 (APC)
 16,337 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Thrombomodulin is an endothelial cell thrombin receptor that serves as a cofactor for thrombin-catalyzed activation of protein C. Structural requirements for thrombin binding and cofactor activity were studied by mutagenesis of recombinant human thrombomodulin expressed on COS-7 and CV-1 cells. Deletion of the fourth epidermal growth factor (EGF)-like domain abolished cofactor activity but did not affect thrombin binding. Deletion of either the fifth or the sixth EGF-like domain markedly reduced both thrombin binding affinity and cofactor activity. Thrombin binding sequences were also localized by assaying the ability of synthetic peptides derived from thrombomodulin to compete with diisopropyl fluorophosphate-inactivated 125I-thrombin binding to thrombomodulin. The two most active peptides corresponded to (a) the entire third loop of the fifth EGF-like domain (Kp = 85 +/- 6 microM) and (b) parts of the second and third loops of the sixth EGF-like domain (Kp = 117 +/- 9 microM). These data suggest that thrombin interacts with two discrete elements in thrombomodulin. Deletion of the Ser/Thr-rich domain dramatically decreased both thrombin binding affinity and cofactor activity and also prevented the formation of a high molecular weight thrombomodulin species containing chondroitin sulfate. Substitutions of this domain with polypeptide segments of decreasing length and devoid of glycosylation sites progressively decreased both cofactor activity and thrombin binding affinity. This correlation suggests that increased proximity of the membrane surface to the thrombin binding site may hinder efficient thrombin binding and the subsequent activation of protein C. Membrane-bound thrombomodulin therefore requires the Ser/Thr-rich domain as an important spacer, in addition to EGF-like domains 4-6, for efficient protein C activation.
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PMID:Functional domains of membrane-bound human thrombomodulin. EGF-like domains four to six and the serine/threonine-rich domain are required for cofactor activity. 131 30

Single-stranded DNA molecules containing a 15-nucleotide consensus sequence have been reported to inhibit thrombin activity. The mechanism of the inhibition was studied using a consensus 15-mer oligonucleotide and two recombinant mutant thrombins: the anion-binding exosite mutant thrombin R70E, and thrombin K154A, in which the mutation was located in a surface loop outside of the exosite. The consensus 15-mer oligonucleotide inhibited both fibrinogen-clotting and platelet-activation activities of plasma-derived thrombin, recombinant wild type thrombin, and mutant thrombin K154A in a sequence-specific and dose-dependent manner, whereas it did not inhibit either activity of mutant thrombin R70E. The 15-mer oligonucleotide also inhibited thrombomodulin-dependent protein C activation by plasma-derived thrombin. In competition equilibrium binding experiments, binding of 125I-labeled diisopropyl phosphoryl-thrombin to thrombomodulin was completely inhibited by the consensus 15-mer oligonucleotide with a Kd value of 2.68 +/- 0.16 nM. These results suggest that Arg-70 in the anion-binding exosite of thrombin is a key determinant for interaction with specific single-stranded DNA molecules, and that binding of single-stranded DNA molecules to the exosite prevents the interaction of thrombin with fibrinogen, the platelet thrombin receptor, and thrombomodulin.
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PMID:Localization of the single-stranded DNA binding site in the thrombin anion-binding exosite. 133 57

Thrombomodulin is an endothelial surface thrombin receptor. Thrombin bound to thrombomodulin loses all procoagulant activity and instead activates the protein C anticoagulant pathway. We developed a recombinant thrombomodulin analog and compared the effects of recombinant thrombomodulin (100 micrograms/ea), saline (controls), recombinant hirudin (1.0 mg/kg), and heparin (100 units/kg) on thrombus formation, activated partial thromboplastin time, and tail transection bleeding time in a rat model of stasis-induced venous thrombosis. Results showed that thrombus was detected in the vena cava in six of the six rats treated with saline solution, in zero of the six rats treated with recombinant thrombomodulin (p less than 0.05), in one of six rats treated with recombinant hirudin (p less than 0.05), and in zero of six rats treated with heparin (p less than 0.05). The activated partial thromboplastin time in rats receiving recombinant thrombomodulin was slightly longer than controls (22 +/- 8 vs 37 +/- 6, p less than 0.05). The bleeding times in rats receiving recombinant thrombomodulin were approximately twice as long as controls (215 +/- 68 vs 545 +/- 173, p = 0.037). In all rats treated with recombinant hirudin or heparin, activated partial thromboplastin times were greater than 120 seconds and bleeding times were greater than 1200 seconds. We conclude that recombinant thrombomodulin inhibits venous thrombosis in a rat model with less prolongation of activated partial thromboplastin time and bleeding time than heparin or hirudin.
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PMID:Intravenous recombinant soluble human thrombomodulin prevents venous thrombosis in a rat model. 165 3

We isolated a cDNA encoding a functional human thrombin receptor by direct expression cloning in Xenopus oocytes. mRNA encoding this receptor was detected in human platelets and vascular endothelial cells. The deduced amino acid sequence revealed a new member of the seven transmembrane domain receptor family with a large amino-terminal extracellular extension containing a remarkable feature. A putative thrombin cleavage site (LDPR/S) resembling the activation cleavage site in the zymogen protein C (LDPR/I) was noted 41 amino acids carboxyl to the receptor's start methionine. A peptide mimicking the new amino terminus created by cleavage at R41 was a potent agonist for both thrombin receptor activation and platelet activation. "Uncleavable" mutant thrombin receptors failed to respond to thrombin but were responsive to the new amino-terminal peptide. These data reveal a novel signaling mechanism in which thrombin cleaves its receptor's amino-terminal extension to create a new receptor amino terminus that functions as a tethered ligand and activates the receptor.
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PMID:Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation. 167 65

Thrombomodulin, an endothelial thrombin receptor, acts as a cofactor for the thrombin-catalyzed activation of anticoagulant protein C. The extracellular region of human thrombomodulin consists of three tentative domains, a NH2-terminal domain (D1), a domain involving six consecutive epidermal growth factor-like structures (D2), and an O-glycosylation-rich domain (D3). To identify the domain onto which thrombin binds, a series of recombinant proteins corresponding to the entire protein, D1, D2, D1 + D2, D1 + D2 + D3, and D2 + D3 were expressed in simian COS-1 cells. The proteins were partially purified by rabbit anti-thrombomodulin-F(ab')2-agarose chromatography. Western blotting analysis showed the expression of the respective recombinant proteins. All proteins involving D2, as well as D2 alone, had cofactor activity that allowed binding directly to thrombin, but D1 did not. The cofactor activity of the entire protein but not the mutants is increased in the presence of phospholipids and this is the only protein that binds to the phospholipid layer. These results indicate that the domain involving the epidermal growth factor-like structures of thrombomodulin is essential for thrombin binding and expression of the cofactor activity for protein C activation and that none of the extracellular domains interact with phospholipids.
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PMID:A domain composed of epidermal growth factor-like structures of human thrombomodulin is essential for thrombin binding and for protein C activation. 253 65

The gene coding for human thrombomodulin, a thrombin receptor on endothelial cells and a cofactor for the activation of anticoagulant protein C zymogen, was isolated from a human genomic library by employing human thrombomodulin cDNA as a probe. The nucleotide sequences of the gene and the adjacent 5' and 3' flanking regions were then determined. The nucleotide sequence of this gene with approximately 3.7 kilobase pairs was identical to that of the cDNA, indicating that the gene for human thrombomodulin is free of introns. Hybridization data showed that there is only a single thrombomodulin gene in the human genome.
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PMID:Gene structure of human thrombomodulin, a cofactor for thrombin-catalyzed activation of protein C. 283 77

Thrombin receptor cleavage at the Arg41- decreases -Ser42 peptide bond in the receptor's amino-terminal exodomain is necessary and sufficient for receptor activation. The rate of receptor cleavage at this site is a critical determinant of the magnitude of the cellular response to thrombin. These observations underscore the importance of defining the molecular basis for thrombin-receptor interaction and cleavage. We report that chimeric proteins bearing only thrombin receptor amino-terminal exodomain residues 36-60 are cleaved at rates similar to the wild-type thrombin receptor when expressed on the cell surface. A soluble amino-terminal exodomain protein was also cleaved efficiently by thrombin with a Km of 15-30 microM and k(cat) of approximately 50 s-1, with cleavage occurring only at the Arg41- decreases -Ser42 peptide bond. In the context of previous studies, these data suggest that the receptor's LDPR cleavage recognition sequence and DKYEPF hirudin-like domain account for thrombin-receptor interaction. Because a P3 aspartate in protein C's cleavage site inhibits cleavage by free thrombin, we investigated the role of the P3 aspartate in the receptor's LDPR sequence. Studies with mutant receptors revealed an inhibitory role for this residue only in the absence of the receptor's hirudin-like domain. These and other data suggest that the receptor's hirudin-like domain causes a conformational change in thombin's active center to accommodate the LDPR sequence and promote efficient receptor cleavage. Taken together, these studies imply that the thrombin receptor's amino-terminal exodomain contains all the machinery needed for efficient recognition and cleavage by thrombin. Thrombin appears to bind and cleave this domain independently of the rest of the receptor, with one thrombin molecule probably activating multiple receptors.
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PMID:Determinants of thrombin receptor cleavage. Receptor domains involved, specificity, and role of the P3 aspartate. 760 15

The protein C anticoagulant system generates an "on demand" physiologic anticoagulant response. The pathway is initiated when thrombin binds to the endothelial cell thrombin binding protein, thrombomodulin. The complex exhibits dramatically altered macromolecular specificity. It rapidly cleaves the protein C zymogen to form the anticoagulant, activated protein C. Complex formation between thrombin and thrombomodulin also prevents thrombin, the enzyme responsible for clot formation and a potent platelet activator, from being able to clot fibrinogen or to activate platelets. Structural, kinetic, and competition studies suggest that thrombomodulin blocks these clotting reactions by masking the binding sites for fibrinogen and the platelet thrombin receptor. Stimulation of protein C activation appears to occur through conformational changes in the extended binding pocket of thrombin. This prevents repulsive interactions with protein C that exist when the free enzyme attempts to dock with this substrate. In addition to protein-protein interactions, thrombomodulin has a covalently associated chondroitin sulfate moiety. Chondroitin sulfate binds to a basic surface on thrombin that is also involved in heparin interaction. The chondroitin sulfate enhances the affinity of thrombin for thrombomodulin approximately 10- to 20-fold, making thrombomodulin a more potent inhibitor of coagulation, altering thrombin's conformation and specificity, and accelerating thrombin inhibition by the serpin, antithrombin. These properties make thrombomodulin a molecular switch ideally suited to trigger an anticoagulant response when too much thrombin is generated. The importance of the system is documented by the clinical observation that patients deficient in protein C often die of massive thrombotic complications that can be reversed or prevented by infusion of protein C.
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PMID:Thrombomodulin as a model of molecular mechanisms that modulate protease specificity and function at the vessel surface. 761 64

Protein C, a precursor to a natural plasma anticoagulant, and the platelet thrombin receptor, involved in cell activation, both require proteolytic cleavage to be activated. In humans, the sequences adjacent to the scissile bond of protein C, DPR/LID and the thrombin receptor DPR/SFL are similar. Previous studies with Asp-->Gly mutants indicated that both the P3 and the P3' Asp residues make either peptides or protein C a poor substrate for free thrombin, but thrombin interaction with thrombomodulin overcomes these inhibitory interactions. Similar mechanisms are probably operative in the thrombin receptor. In rodents, the P3 Asp residue of the human thrombin receptor is replaced by Asn and in protein C, the P3' residue is Asn. To determine the functional significance of these Asp-->Asn substitutions, the Asp in the P3 or P3' position of human protein C was changed to Asn. The resultant mutants were still resistant to activation by thrombin, and still required Ca2+ for activation by thrombin-thrombomodulin complex. We conclude that, unlike activation of the Asp-->Gly mutants by thrombin, activation of the P3 and P3' Asp-->Asn mutants is still potently inhibited by physiological Ca2+. Furthermore, even though the charge has been deleted, thrombomodulin acceleration is retained.
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PMID:Calcium inhibition of the activation of protein C by thrombin. Role of the P3 and P3' residues. 805 28

An autoantibody, developed by a patient with severe and recurrent arterial thrombosis, was characterized to be directed against the anion-binding exosite of thrombin, and inhibited all thrombin interactions requiring this secondary binding site without interfering with the catalytic site. The effect of the antibody was studied on thrombin interactions with platelets and endothelial cells from human umbilical veins (HUVEC). The autoantibody specifically and concentration-dependently inhibited alpha-thrombin-induced platelet activation and prostacyclin (PGI2) synthesis from HUVEC. It had no effect when gamma-thrombin or the thrombin receptor activation peptide SFLLR were the inducers. The effect of the antibody on protein C activation has been studied. The antibody blocked the thrombin-thrombomodulin activation of protein C. The inhibition of the activation was maximal with a low concentration of thrombomodulin. The fact that the autoantibody inhibited concentration-dependent alpha-thrombin-induced platelet and endothelial cell functions emphasizes the crucial role of the anion-binding exosite of thrombin to activate its receptor. In regard to the pathology, the antibody inhibited two vascular processes implicated in thrombin-antithrombotic functions, PGI2 secretion, and protein C activation, which could be implicated in this arterial thrombotic disease.
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PMID:An autoantibody directed against human thrombin anion-binding exosite in a patient with arterial thrombosis: effects on platelets, endothelial cells, and protein C activation. 808 Sep 90


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