scholarly journals Enhancing the anticoagulant profile of meizothrombin

2018 ◽  
Vol 9 (1) ◽  
pp. 169-175 ◽  
Author(s):  
Bosko M. Stojanovski ◽  
Leslie A. Pelc ◽  
Xiaobing Zuo ◽  
Nicola Pozzi ◽  
Enrico Di Cera
Keyword(s):  
Single Molecule ◽  
Protein C ◽  
Coagulation Cascade ◽  
Proteolytic Activation ◽  
Single Molecule Fret ◽  
Functional Aspects ◽  
Active Intermediate ◽  
Thrombotic Complications ◽  
Gla Domain ◽  
Structural Architecture

AbstractMeizothrombin is an active intermediate generated during the proteolytic activation of prothrombin to thrombin in the penultimate step of the coagulation cascade. Structurally, meizothrombin differs from thrombin because it retains the auxiliary Gla domain and two kringles. Functionally, meizothrombin shares with thrombin the ability to cleave procoagulant (fibrinogen), prothrombotic (PAR1) and anticoagulant (protein C) substrates, although its specificity toward fibrinogen and PAR1 is less pronounced. In this study we report information on the structural architecture of meizothrombin resolved by SAXS and single molecule FRET as an elongated arrangement of its individual domains. In addition, we show the properties of a meizothrombin construct analogous to the anticoagulant thrombin mutant W215A/E217A currently in Phase I for the treatment of thrombotic complications and stroke. The findings reveal new structural and functional aspects of meizothrombin that advance our understanding of a key intermediate of the prothrombin activation pathway.

scholarly journals Zymogen and activated protein C have similar structural architecture

2020 ◽  
Vol 295 (45) ◽  
pp. 15236-15244
Author(s):  
Bosko M. Stojanovski ◽  
Leslie A. Pelc ◽  
Xiaobing Zuo ◽  
Enrico Di Cera
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Single Molecule ◽  
Protein C ◽  
Conformational Transition ◽  
Activated Protein C ◽  
Coagulation Cascade ◽  
Clotting Factors ◽  
Epidermal Growth ◽  
Structural Architecture

Activated protein C is a trypsin-like protease with anticoagulant and cytoprotective properties that is generated by thrombin from the zymogen precursor protein C in a reaction greatly accelerated by the cofactor thrombomodulin. The molecular details of this activation remain elusive due to the lack of structural information. We now fill this gap by providing information on the overall structural organization of these proteins using single molecule FRET and small angle X-ray scattering. Under physiological conditions, both zymogen and protease adopt a conformation with all domains vertically aligned along an axis 76 Å long and maximal particle size of 120 Å. This conformation is stabilized by binding of Ca2+ to the Gla domain and is affected minimally by interaction with thrombin. Hence, the zymogen protein C likely interacts with the thrombin-thrombomodulin complex through a rigid body association that produces a protease with essentially the same structural architecture. This scenario stands in contrast to an analogous reaction in the coagulation cascade where conversion of the zymogen prothrombin to the protease meizothrombin by the prothrombinase complex is linked to a large conformational transition of the entire protein. The presence of rigid epidermal growth factor domains in protein C as opposed to kringles in prothrombin likely accounts for the different conformational plasticity of the two zymogens. The new structural features reported here for protein C have general relevance to vitamin K-dependent clotting factors containing epidermal growth factor domains, such as factors VII, IX, and X.

Mice Engineered To Express a Form of Thrombin Favoring Protein C Are Resistant to S. aureus-Induced Sepsis.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 267-267
Author(s):  
Matthew J. Flick ◽  
Anil K. Chauhan ◽  
Sara Welch ◽  
Maureen A. Shaw ◽  
Kathryn E. Talmage ◽  
...  
Keyword(s):  
Protein C ◽  
Thrombus Formation ◽  
Activated Protein C ◽  
Catalytic Efficiency ◽  
Coagulation Cascade ◽  
Bleeding Events ◽  
Spontaneous Bleeding ◽  
Proteolytic Activation ◽  
Tail Tip

Abstract Thrombin is central in thrombus formation as both a positive mediator of thrombus formation through the proteolytic activation of PARs, fibrinogen, fXI and other prothrombotic substrates, and a negative modulator of the coagulation cascade through the activation of protein C. Detailed structure-function studies have revealed that thrombin can be redesigned to favor either procoagulant or anticoagulant substrates. The introduction of W215A/E217A substitutions in the murine thrombin active site (fIIWE) results in a pronounced “specificity switch” that reduces catalytic efficiency with fibrinogen by at least 3-orders-magnitude while only modestly reducing activity for protein C activation. To evaluate the effects of fIIWE activity in vivo, we have used a gene-targeting strategy to generate mice carrying the W215A/E217A mutations in the endogenous murine prothrombin gene. The mutant allele was transmitted through the germline and was found to support the expression of normal levels of hepatic fII mRNA and plasma fII in both heterozygous and homozygous neonates. Unlike fII knockout mice, homozygous fIIWE mice were observed at term with the expected Mendelian frequency. Nevertheless, homozygous fIIWE offspring uniformly succumbed to spontaneous bleeding events within days of birth. Heterozygous fIIWE/WT animals generally survived to adulthood, were capable of carrying multiple liters to term, and unchallenged mice displayed a hematological profile similar to wildtype mice. However, consistent with a predicted anticoagulant phenotype, adult fIIWE/WT heterozygotes exhibited significantly delayed thrombus formation following ferric chloride injury of mesenteric arterioles and extended bleeding times following tail tip excision relative to control mice expressing wildtype fII. Given that activated protein C has been shown to be efficacious in the treatment of sepsis, we explored whether the shift in thrombin specificity in heterozygous fIIWE/WT mice would confer the benefit of rendering animals tolerant to acute septic challenges. Kaplan-Meier analyses following intravenous administration of S. aureus revealed that fIIWE/WT mice exhibited a significant survival advantage over littermate wildtype animals challenged in parallel and tracked over a 7-day observation period. Notably, extended thrombus formation and bleeding times as well as resistance to sepsis was not simply a function of half normal wildtype fII expression. When these analyses were performed in animals carrying one wildtype allele and one null mutation allele, results were similar to wiltype. These studies further underscore the interplay between the hemostatic and inflammatory systems in vivo and highlight the possible therapeutic utility of recombinant (pro)thrombin derivatives with selected alterations in substrate specificity.

Mutations which Introduce Free Cysteine Residues in the Gla-Domain of Vitamin K Dependent Proteins Result in the Formation of Complexes with α1-Microglobulin

1996 ◽  
Vol 75 (01) ◽  
pp. 070-075 ◽  
Author(s):  
E G C Wojcik ◽  
P Simioni ◽  
M v d Berg ◽  
A Girolami ◽  
R M Bertina
Keyword(s):  
Vitamin K ◽  
Protein C ◽  
Cysteine Residue ◽  
Factor Ix ◽  
Disulphide Bond ◽  
Clotting Factors ◽  
High Molecular Weight Complex ◽  
Low Concentrations ◽  
Gla Domain ◽  
Formation Of Complexes

SummaryWe have previously described a genetic factor IX variant (Cys18→Arg) for which we demonstrated that it had formed a heterodimer with armicroglobulin through formation of a disulphide bond with the remaining free cysteine residue of the disrupted disulphide bond in the Gla-domain of factor IX. Recently, we observed a similar high molecular weight complex for a genetic protein C variant (Arg-1→Cys). Both the factor IX and the protein C variants have a defect in the calcium induced conformation. In this study we show that the aminoterminus of this protein C variant is prolonged with one amino acid, cysteine. This protein C variant, as well as protein C variants with Arg9→Cys and Ser12→Cys mutations which also carry a free cysteine residue, are shown to be present in plasma as a complex with α1-microglobulin. A prothrombin variant with a Tyr44→Cys mutation, had not formed such a complex. Furthermore, complexes between normal vitamin K-dependent clotting factors and α1-microglobulin were shown to be present in plasma at low concentrations. The data suggest that the presence of an unpaired cysteine residue in the propeptide or the N-terminal half of the Gla-domain has strongly promoted the formation of a complex with α1-microglobulin in the variants.

High Affinity Binding Sites for Activated Protein C and Protein C on Cultured Human Umbilical Vein Endothelial Cells

1994 ◽  
Vol 72 (03) ◽  
pp. 465-474 ◽  
Author(s):  
Neelesh Bangalore ◽  
William N Drohan ◽  
Carolyn L Orthner
Keyword(s):  
Binding Sites ◽  
Protein C ◽  
Umbilical Vein ◽  
Activated Protein C ◽  
Affinity Binding ◽  
Cell Binding ◽  
High Affinity Binding ◽  
High Affinity ◽  
Gla Domain ◽  
Umbilical Vein Endothelial Cells

SummaryActivated protein C (APC) is an antithrombotic serine proteinase having anticoagulant, profibrinolytic and anti-inflammatory activities. Despite its potential clinical utility, relatively little is known about its clearance mechanisms. In the present study we have characterized the interaction of APC and its active site blocked forms with human umbilical vein endothelial cells (HUVEC). At 4° C 125I-APC bound to HUVEC in a specific, time dependent, saturable and reversible manner. Scatchard analysis of the binding isotherm demonstrated a Kd value of 6.8 nM and total number of binding sites per cell of 359,000. Similar binding isotherms were obtained using radiolabeled protein C (PC) zymogen as well as D-phe-pro-arg-chloromethylketone (PPACK) inhibited APC indicating that a functional active site was not required. Competition studies showed that the binding of APC, PPACK-APC and PC were mutually exclusive suggesting that they bound to the same site(s). Proteolytic removal of the N-terminal γ-carboxyglutamic acid (gla) domain of PC abolished its ability to compete indicating that the gla-domain was essential for cell binding. Surprisingly, APC binding to these cells appeared to be independent of protein S, a cofactor of APC generally thought to be required for its high affinity binding to cell surfaces. The identity of the cell binding site(s), for the most part, appeared to be distinct from other known APC ligands which are associated with cell membranes or extracellular matrix including phospholipid, thrombomodulin, factor V, plasminogen activator inhibitor type 1 (PAI-1) and heparin. Pretreatment of HUVEC with antifactor VIII antibody caused partial inhibition of 125I-APC binding indicating that factor VIII or a homolog accounted for ∼30% of APC binding. Studies of the properties of surface bound 125I-APC or 125I-PC and their fate at 4°C compared to 37 °C were consistent with association of ∼25% of the initially bound radioligand with an endocytic receptor. However, most of the radioligand appeared not to be bound to an endocytic receptor and dissociated rapidly at 37° C in an intact and functional state. These data indicate the presence of specific, high affinity binding sites for APC and PC on the surface of HUVEC. While a minor proportion of binding sites may be involved in endocytosis, the identity and function of the major proportion is presently unknown. It is speculated that this putative receptor may be a further mechanisms of localizing the PC antithrombotic system to the vascular endothelium.

Diabetes and Vascular Disease: Is It All About Glycemia?

2019 ◽  
Vol 25 (29) ◽  
pp. 3112-3127 ◽  
Author(s):  
Alessandra Vecchié ◽  
Fabrizio Montecucco ◽  
Federico Carbone ◽  
Franco Dallegri ◽  
Aldo Bonaventura
Keyword(s):  
Vascular Disease ◽  
Inflammatory Cell ◽  
Vascular Dysfunction ◽  
Microvascular Complications ◽  
Coagulation Cascade ◽  
Inflammatory Cell Infiltrate ◽  
Extracellular Traps ◽  
Thrombotic Complications ◽  
Independent Entity ◽  
Complications And Mortality

Background: Diabetes is increasing over time, mainly driven by obesity, aging, and urbanization. Classical macro- and microvascular complications represent the final result of a complex interplay involving atherosclerosis at all stages. Methods: In this review, we aim at focusing on current updates in the pathophysiology of vascular disease in diabetes and discussing how new therapies might influence the management of these patients at high cardiovascular risk. Diabetes shows accelerated atherosclerosis with a larger inflammatory cell infiltrate, thus favoring the development of heart failure. ‘Diabetic cardiomyopathy’ perfectly describes a specific ischemia- and hypertension- independent entity due to diabetes-related metabolic alterations on myocardial function. Moreover, platelets from subjects with diabetes display a typical hyperreactivity explaining the stronger adhesion, activation, and aggregation. Additionally, diabetes provokes an exaggerated stimulation of the endothelium, with an increased release of reactive oxygen species and a reduced release of nitric oxide, both key elements of the endothelial dysfunction. Also, the coagulation cascade and leukocytes activate contributing to this pro-thrombotic environment. Neutrophils have been recently recognized to play a pivotal role by releasing neutrophil extracellular traps. Finally, microparticles from platelets, neutrophils or monocytes are detrimental effectors on the vessel wall and are involved both in vascular dysfunction and in thrombotic complications. Conclusion: In light of these findings, the therapeutic management of diabetes needs to be mostly focused on limiting the progression of complications by targeting precise pathophysiological mechanisms rather than the mere glycemic control, which failed to markedly reduce the risk for macrovascular complications and mortality.

scholarly journals Encoding Multiple Virtual Signals in DNA Barcodes with Single-Molecule FRET

Nano Letters ◽  
2021 ◽  
Vol 21 (4) ◽  
pp. 1694-1701 ◽  
Author(s):  
Sung Hyun Kim ◽  
Hyunwoo Kim ◽  
Hawoong Jeong ◽  
Tae-Young Yoon
Keyword(s):  
Single Molecule ◽  
Dna Barcodes ◽  
Single Molecule Fret

Single-Molecule FRET-Based Dynamic DNA Sensor

ACS Sensors ◽  
2021 ◽  
Author(s):  
Anoja Megalathan ◽  
Kalani M. Wijesinghe ◽  
Soma Dhakal
Keyword(s):  
Single Molecule ◽  
Dna Sensor ◽  
Single Molecule Fret

scholarly journals Inhibition of human coagulation factor VIII by monoclonal antibodies. Mapping of functional epitopes with the use of recombinant factor VIII fragments

1989 ◽  
Vol 263 (1) ◽  
pp. 187-194 ◽  
Author(s):  
A Leyte ◽  
K Mertens ◽  
B Distel ◽  
R F Evers ◽  
M J M De Keyzer-Nellen ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Factor Viii ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Procoagulant Activity ◽  
Coagulation Factor Viii ◽  
Coagulation Cascade ◽  
Restriction Enzyme Digestion ◽  
Proteolytic Activation

The epitopes of four monoclonal antibodies against coagulation Factor VIII were mapped with the use of recombinant DNA techniques. Full-length Factor VIII cDNA and parts thereof were inserted into the vector pSP64, permitting transcription in vitro with the use of a promoter specific for SP6 RNA polymerase. Factor VIII DNA inserts were truncated from their 3′-ends by selective restriction-enzyme digestion and used as templates for ‘run-off’ mRNA synthesis. Translation in vitro with rabbit reticulocyte lysate provided defined radiolabelled Factor VIII fragments for immunoprecipitation studies. Two antibodies are shown to be directed against epitopes on the 90 kDa chain of Factor VIII, between residues 712 and 741. The 80 kDa chain appeared to contain the epitopes of the other two antibodies, within the sequences 1649-1778 and 1779-1840 respectively. The effect of antibody binding to these sequences was evaluated at two distinct levels within the coagulation cascade. Both Factor VIII procoagulant activity and Factor VIII cofactor function in Factor Xa generation were neutralized upon binding to the region 1779-1840. The antibodies recognizing the region 713-740 or 1649-1778, though interfering with Factor VIII procoagulant activity, did not inhibit in Factor Xa generation. These findings demonstrate that antibodies that virtually inhibit Factor VIII in coagulation in vitro are not necessarily directed against epitopes involved in Factor VIII cofactor function. Inhibition of procoagulant activity rather than of cofactor function itself may be explained by interference in proteolytic activation of Factor VIII. This hypothesis is in agreement with the localization of the epitopes in the proximity of thrombin-cleavage or Factor Xa-cleavage sites.

Sign in / Sign up

Export Citation Format

Share Document