Calcium-Dependent Activation of Protein C by Thrombin/Thrombomudulin: Role of Negatively Charged Amino Acids within the Activation Peptide of Protein C

1994 ◽  
Vol 72 (04) ◽  
pp. 567-572 ◽  
Author(s):  
Ute Friedrich ◽  
Bernd Pötzsch ◽  
Klaus T Preissner ◽  
Gert Müller-Berghaus ◽  
Hartmut Ehrlich
Keyword(s):  
Amino Acids ◽  
Calcium Binding ◽  
Cleavage Site ◽  
Protein C ◽  
Regulatory Element ◽  
Binding Properties ◽  
Calcium Dependent ◽  
Thrombin Cleavage ◽  
Charged Amino Acids ◽  
Activation Peptide

SummaryIn the absence of its cofactor thrombomodulin (TM) thrombin is only a poor activator of the anticoagulant serine protease protein C (PC). The TM-dependence of PC-activation has been restricted to a series of molecular structures of the PC molecule including high-affinity calcium binding sites and single amino acid residues. However, thrombin induced activation of a PC derivative altered in all these critical positions is markedly enhanced by TM indicating that additional structures of the PC molecule are involved in determining the TM specificity. Based on the hypothesis that such an additional regulatory element should be located near the thrombin cleavage site and should include negatively charged amino acids to ascertain calcium binding, we studied whether Glu and Asp in positions P7 and P6 relative to the thrombin cleavage site together with Asp in P3 are involved in formation of such a regulatory element. Three PC derivatives containing the neutral counterpart of the negatively charged amino acids in positions P3; P3 and P6; and P3, P6, and P7, respectively, were generated using site-directed mutagenesis. Compared to rPC-wt the initial rates of PC activation of all three mutants were increased 4.0-fold for thrombin/TM and 4.0-, 5.3-fold for activation by thrombin alone. However, compared to the PC derivative neutralized exclusively in P3, additional changes in P6 and P7 showed no increase in the thrombin activation kinetics and calcium binding properties were identical in all of the three mutants. We conclude that .1) among the three negatively charged amino acids at the COOH-terminal end of the activation peptide of PC, only Asp in P3 is involved in calcium-dependent inhibition of PC activation by thrombin; 2) the residues in P7 and P6 do not contribute to the calcium binding properties of PC; 3) P7 and P6 sites are not required for calcium-dependent activation of PC by the thrombin/TM complex.

DIRECTED MUTAGENESIS IN THE STUDY OF THE REQUIREMENTS FOR FACTOR VIII ACTIVITY IN VITRO AND IN VIVO

1987 ◽  
Author(s):  
Randal J Kaufman ◽  
Debra D Pittman ◽  
Louise C Wasley ◽  
W Barry Foster ◽  
Godfrey W Amphlett ◽  
...  
Keyword(s):  
Amino Acids ◽  
Factor Viii ◽  
Cleavage Site ◽  
Factor Xa ◽  
Cleavage Sites ◽  
Thrombin Cleavage ◽  
Terminal Fragment ◽  
Cofactor Activity

Factor VIII is a high molecular weight plasma glycoprotein that functions in the blood clotting cascade as the cofactor for factor DCa proteolytic activation of factor X. Factor VIII does not function proteolytically in this reaction hut itself can be proteolytically activated by other coagulation enzymes such as factor Xa and thrombin. In the plasma, factor VIII exists as a 200 kDa amino-terminal fragment in a metal ion stabilized complex with a 76 kDa carboxy-terminal fragment. The isolation of the cENA for human factor VIII provided the deduced primary amino acid sequence of factor VIIT and revealed three distinct structural domains: 1) a triplicated A domain of 330 amino acids which has homology to ceruloplasmin, a plasma copper binding protein, 2) a duplicated C domain of 150 amino acids, and 3) a unique B domain of 980 amino acids. These domains are arranged as shown below. We have previously reported the B domain is dispensible far cofactor activity in vitro (Toole et al. 1986 Proc. Natl. Acad 5939). The in vivo efficacy of factor VIII molecules harboring the B domain deletion was tested by purification of the wildtype and modified forms and infusion into factor VIII deficient, hemophilic, dogs. The wildtype and the deleted forms of recombinant derived factor VIII exhibited very similar survival curves (Tl/2 = 13 hrs) and the cuticle bleeding times suggested that both preparations appeared functionally equivalent. Sepharose 4B chromatography indicated that both factor VIII molecules were capable of binding canine plasma vWF.Further studies have addressed what cleavages are necessary for activation of factor VIII. The position of the thrombin, factor Xa, and activated protein C (AFC) cleavage sites within factor VIII are presented below, site-directed ENA medicated mutagenesis has been performed to modify the arginine at the amino side of each cleavagesite to an soleucine. In all cases this modification resulted in molecules that were resistant to cleavage by thrombin at the modified site. Modification of the thrombin cleavage sites at 336 and 740 and modification of the factor Xa cleavage site at 1721 resulted in no loss of cofactor activity. Modification of the thrombin cleavage site at either 372 or 1689 destroyed oofactor activity. Modification of the thrombin cleavage site at 336 resulted in a factor VIII having an increased activity, possibly due to resistance to inactivation. These results suggest the requirement of cleavage at residues 372 and 1689 for cofactor activity.

scholarly journals Immunologic analysis of the cloned platelet thrombin receptor activation mechanism: evidence supporting receptor cleavage, release of the N-terminal peptide, and insertion of the tethered ligand into a protected environment

Blood ◽  
1993 ◽  
Vol 82 (7) ◽  
pp. 2125-2136 ◽  
Author(s):  
KJ Norton ◽  
RM Scarborough ◽  
JL Kutok ◽  
MA Escobedo ◽  
L Nannizzi ◽  
...  
Keyword(s):  
Amino Acids ◽  
Platelet Aggregation ◽  
Platelet Activation ◽  
Cleavage Site ◽  
Receptor Activation ◽  
Activation Mechanism ◽  
Thrombin Receptor ◽  
Thrombin Cleavage ◽  
Thrombin Activation ◽  
Tethered Ligand

The recently cloned functional thrombin receptor is thought to be activated by thrombin cleavage of the bond between R41 and S42, followed by the insertion of the new N-terminal region (“tethered ligand”) into an unknown site in the receptor. Antibodies to peptides at or near the cleavage site have been reported to inhibit thrombin- induced platelet activation to varying extents, but the precise mechanism(s) of their inhibition is unknown. We have produced: (1) a polyclonal antibody in rabbits to a peptide containing amino acids 34 to 52 (anti-TR34–52); enzyme-linked immunosorbent assays (ELISA) indicate that anti-TR34–52 contains antibodies to regions on both sides of the thrombin cleavage site; (2) two murine monoclonal antibodies (MoAbs) to a peptide containing amino acids 29 to 68; one antibody reacts primarily with residues N-terminal to the thrombin cleavage site, and the other reacts primarily with residues C-terminal to the cleavage site; and (3) a polyclonal rabbit antibody to a peptide containing amino acids 83 to 94 (anti-TR83–94). Anti-TR34–52 binds to platelets as judged by flow cytometry, and pretreating platelets with a thrombin receptor peptide ligand does not lead to loss of antibody reactivity, suggesting that platelet activation does not initiate redistribution or internalization of surface thrombin receptors. In contrast, pretreating platelets with thrombin leads to complete loss of anti-TR34–52 binding. Similarly, the binding of both MoAbs to platelets is dramatically reduced by pretreatment with thrombin. However, the binding of anti-TR83–94 is not decreased by thrombin activation, confirming that the receptor is not internalized. Anti-TR34–52 profoundly inhibits low dose thrombin-induced platelet shape change and aggregation, but the inhibition can be overcome with higher thrombin doses. However, anti-TR34–52 does not inhibit platelet aggregation induced by tethered ligand peptides. The TR34–52 peptide is a thrombin substrate, with cleavage occurring at the R41-S42 bond as judged by high performance liquid chromatography (HPLC) and platelet aggregation analysis. Anti-TR34–52 prevented cleavage of the TR34–52 peptide, suggesting that the antibody prevents platelet activation, at least in part, by preventing cleavage of the thrombin receptor. These data, although indirect, provide additional support for a thrombin activation mechanism involving thrombin cleavage of the receptor; in addition, they provide new evidence indicating that receptor cleavage is followed by loss of the N-terminal peptide, and insertion of the tethered ligand into a protected domain.

scholarly journals Functional and Biochemical Analysis of the C2 Domains of Synaptotagmin IV

1999 ◽  
Vol 10 (7) ◽  
pp. 2285-2295 ◽  
Author(s):  
David M. Thomas ◽  
Gregory D. Ferguson ◽  
Harvey R. Herschman ◽  
Lisa A. Elferink
Keyword(s):  
Pc12 Cells ◽  
Membrane Trafficking ◽  
Calcium Binding ◽  
Neurotransmitter Release ◽  
Biochemical Properties ◽  
Binding Properties ◽  
Independent Manner ◽  
C2 Domains ◽  
Calcium Dependent ◽  
C2a Domain

Synaptotagmins (Syts) are a family of vesicle proteins that have been implicated in both regulated neurosecretion and general membrane trafficking. Calcium-dependent interactions mediated through their C2 domains are proposed to contribute to the mechanism by which Syts trigger calcium-dependent neurotransmitter release. Syt IV is a novel member of the Syt family that is induced by cell depolarization and has a rapid rate of synthesis and a short half-life. Moreover, the C2A domain of Syt IV does not bind calcium. We have examined the biochemical and functional properties of the C2 domains of Syt IV. Consistent with its non–calcium binding properties, the C2A domain of Syt IV binds syntaxin isoforms in a calcium-independent manner. In neuroendocrine pheochromocytoma (PC12) cells, Syt IV colocalizes with Syt I in the tips of the neurites. Microinjection of the C2A domain reveals that calcium-independent interactions mediated through this domain of Syt IV inhibit calcium-mediated neurotransmitter release from PC12 cells. Conversely, the C2B domain of Syt IV contains calcium binding properties, which permit homo-oligomerization as well as hetero-oligomerization with Syt I. Our observation that different combinatorial interactions exist between Syt and syntaxin isoforms, coupled with the calcium stimulated hetero-oligomerization of Syt isoforms, suggests that the secretory machinery contains a vast repertoire of biochemical properties for sensing calcium and regulating neurotransmitter release accordingly.

Downstream regulatory element antagonistic modulator regulates islet prodynorphin expression

2006 ◽  
Vol 291 (3) ◽  
pp. E587-E595 ◽  
Author(s):  
David A. Jacobson ◽  
Julie Cho ◽  
Luis R. Landa ◽  
Natalia A. Tamarina ◽  
Michael W. Roe ◽  
...  
Keyword(s):  
Calcium Binding ◽  
Regulatory Element ◽  
Glucagon Secretion ◽  
Dependent Manner ◽  
Β Cells ◽  
Dynorphin A ◽  
Calcium Dependent ◽  
Low Glucose ◽  
Stimulation Of

Calcium-binding proteins regulate transcription and secretion of pancreatic islet hormones. Here, we demonstrate neuroendocrine expression of the calcium-binding downstream regulatory element antagonistic modulator (DREAM) and its role in glucose-dependent regulation of prodynorphin (PDN) expression. DREAM is distributed throughout β- and α-cells in both the nucleus and cytoplasm. As DREAM regulates neuronal dynorphin expression, we determined whether this pathway is affected in DREAM−/− islets. Under low glucose conditions, with intracellular calcium concentrations of <100 nM, DREAM−/− islets had an 80% increase in PDN message compared with controls. Accordingly, DREAM interacts with the PDN promoter downstream regulatory element (DRE) under low calcium (<100 nM) conditions, inhibiting PDN transcription in β-cells. Furthermore, β-cells treated with high glucose (20 mM) show increased cytoplasmic calcium (∼200 nM), which eliminates DREAM's interaction with the DRE, causing increased PDN promoter activity. As PDN is cleaved into dynorphin peptides, which stimulate κ-opioid receptors expressed predominantly in α-cells of the islet, we determined the role of dynorphin A-(1–17) in glucagon secretion from the α-cell. Stimulation with dynorphin A-(1–17) caused α-cell calcium fluctuations and a significant increase in glucagon release. DREAM−/− islets also show elevated glucagon secretion in low glucose compared with controls. These results demonstrate that PDN transcription is regulated by DREAM in a calcium-dependent manner and suggest a role for dynorphin regulation of α-cell glucagon secretion. The data provide a molecular basis for opiate stimulation of glucagon secretion first observed over 25 years ago.

The Contributions of the P4-P2 Positions in PAR1 and 4 for Thrombin Recognition and Cleavage.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3574-3574
Author(s):  
Marvin T. Nieman ◽  
Alvin H. Schmaier
Keyword(s):  
Amino Acids ◽  
Cleavage Site ◽  
Receptor Activation ◽  
Breakdown Product ◽  
Wild Type ◽  
Binding Region ◽  
Alanine Scanning Mutagenesis ◽  
Important Amino Acid ◽  
Thrombin Cleavage

Abstract The angiotensin converting enzyme breakdown product of bradykinin, bradykinin 1–5 (RPPGF), inhibits thrombin induced human or mouse platelet aggregation by preventing proteolysis of PAR1 and PAR4 by binding adjacent to the thrombin cleavage site and thus preventing activation of these receptors (Am. J. Physiol.285:H183–H193, 2003; J. Pharmacol. Exp. Ther.311:492–501, 2004; FEBS Lett. 579:25–29, 2005). Alanine scanning mutagenesis determined that RPPGF binds to Pro46 at the P2 position of PAR4 to block thrombin cleavage. New studies determined the amino acids required for thrombin to bind and cleave PAR4. Wild type PAR4 exodomain is cleaved by thrombin with a Km of 17 μM, kcat of 3.5 s−1 and kcat/Km of 2x105 M−1 s−1. In contrast, PAR4 exodomain in which the P2 (PAR4-P46A) is changed to alanine, is not efficiently cleaved with 10 nM alpha thrombin. Alteration of PAR4’s P4 position (e.g., PAR4-P44A) does not influence the PAR4’s rate of cleavage. The ability of the PAR4 exodomain to inhibit thrombin proteolysis of H-D-Phe-Pip-pNA was also determined as an independent measure of the thrombin/PAR4 interaction. Wild type PAR4 exodomain and PAR4-P44A are competitive inhibitors of thrombin hydrolysis of the chromogenic substrate with a Ki of 23 ± 6 micromolar and 19.6 ± 4 μM respectively. In contrast, PAR4-P46A and PAR4-P44A/P46A have a Ki &gt; 300 μM nd the nature of the inhibition changes from competitive to noncompetitive. Taken together, these data demonstrate that Pro46 of PAR4 is important for alpha thrombin to bind and orient PAR4 in its active site for efficient cleavage. Further studies examined the role of the combined amino acids in the P4 to P3 positions of PAR4 and PAR1 to contribute to the rate of thrombin cleavage. Chimeric molecules were prepared in which the P4 and P3 positions of PAR4 (ProAla) are replaced with those from PAR1 (LeuAsp) to generate PAR4-LD. The reciprocal chimera was also made (PAR1-PA). PAR4-LD is proteolyzed by 10 nM alpha thrombin more efficiently than PAR4-wt. Alternatively, the rate of PAR1-PA proteolysis by alpha thrombin is less efficient than PAR1-wt but better than PAR4-wt due to the presence of the exosite I binding region in the PAR1 exodomain. However, if the exosite I binding domain is removed from PAR1-PA the rate of cleavage is like that seen with PAR4-wt exodomain. These data indicate PAR1 is cleaved by alpha thrombin at a faster rate than PAR4 due to the amino acids in the P4 and P3 positions as well as the exosite I binding region. In sum, these data demonstrate that the P2 position of PAR4 is the most important amino acid in thrombin binding to the region adjacent to the thrombin cleavage site. However the combined P4 and P3 positions are also important determinants of the rate of receptor cleavage. These data indicate that the amino acids around the thrombin cleavage site of both PAR4 and 1 influence its rate of cleavage. Designing thrombin receptor activation antagonists directed to the thrombin cleavage site on PAR1 and 4 should be effective anti-platelet agents.

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