A prothrombinase-based assay for detection of resistance to activated protein C

In this paper we present a new method for the detection of resistance to activated protein C (APC) that is based on direct measurement of the effect of APC an the cofactor activity of plasma factor Va. The factor V present in a diluted plasma sample was activated with thrombin and its sensitivity towards APC was subsequently determined by incubation with phospholipids and APC; The loss of factor Va cofactor activity was quantified in a prothrombinase system containing purified prothrombin. factor Xa and phospholipid vesicles and using a chromogenic assay for quantitation of thrombin formation. The reaction conditions were optimized in order to distinguish normal, heterozygous and homozygous APC-resistant plasmas. Maximal differences in the response of these plasmas towards ATC were observed when factor Va was inactivated by APC in the absence of protein S and when the: cofactor activity of factor Va was determined at a low factor Xa concentration (0.3 nM).Addition of 0.2 nM APC and 20 mu M phospholipid vesicles to a 1000-fold diluted sample of thrombin-activated normal plasma resulted in loss of mon than 85% of the cofactor activity factor Va within 6 min. Under the same conditions, APC inactivated similar to 60% and similar to 20% of the factor Va present in plasma samples from APC-resistant individuals that were heterozygous or homozygous for the mutation Arg(506)-->Gln in factor V, respectively. Discrimination between the plasma samples from normal and heterozygous and homozygous APC-resistant individuals was facilitated by introduction of the so-called APC-sensitivity ratio (APC-sr). The APC-sr was defined as the ratio of the factor Va cofactor activities determined in thrombin-activated plasma samples after 6 min incubation with or without 0.2 nM APC and was multiplied by as 100 to obtain integers (APC-sr = {factor Va(+APC)/factor Va(-APC)} x 100). Clear differences were observed between the APC-sr of plasmas from normal healthy volunteers (APC-sr: 8-20, n = 33) and from individuals that were heterozygous (APC-sr: 35-50, n = 17) or homozygous APC resistant (APC-sr: 82-88, n = 7). There was no mutual overlap between the APC-sr of normal plasmas and plasmas from heterozygous or homozygous APC resistant individuals (p < 0.0001), In all cases our test gave the same result as the DNA-based assay. Since the test is performed on a highly diluted plasma sample there is no interference by conditions that affect APC resistance tests that are based on clotting time determinations (e.g. coagulation factor deficiencies, oral anticoagulation, heparin treatment. the presence of lupus anticoagulants, pregnancy or the use of oral contraceptives). Furthermore, we show that part of the factor Va assay can be performed on an autoanalyzer which increases the number of plasma samples that can be handled simultaneously

Erratum to: 36th International Symposium on Intensive Care and Emergency Medicine

[This corrects the article DOI: 10.1186/s13054-016-1208-6.]

Theoretical and Experimental Study of the D2194G Mutation in the C2 Domain of Coagulation Factor V

Coagulation factor V (FV) is a large plasma glycoprotein with functions in both the pro- and anticoagulant pathways. In carriers of the so-called R2-FV haplotype, the FV D2194G mutation, in the C2 membrane-binding domain, is associated with low expression levels, suggesting a potential folding/stability problem. To analyze the molecular mechanisms potentially responsible for this in vitro phenotype, we used molecular dynamics (MD) and continuum electrostatic calculations. Implicit solvent simulations were performed on the x-ray structure of the wild-type C2 domain and on a model of the D2194G mutant. Because D2194 is located next to a disulfide bond (S-S bond), MD calculations were also performed on S-S bond depleted structures. D2194 is part of a salt-bridge network and investigations of the stabilizing/destabilizing role of these ionic interactions were carried out. Five mutant FV molecules were created and the expression levels measured with the aim of assessing the tolerance to amino acid changes in this region of molecule. Analysis of the MD trajectories indicated increased flexibility in some areas and energetic comparisons suggested overall destabilization of the structure due to the D2194G mutation. This substitution causes electrostatic destabilization of the domain by ∼3 kcal/mol. Together these effects likely explain the lowered expression levels in R2-FV carriers

Factor Va alternative conformation reconstruction using atomic force microscopy.

International audienceProtein conformational variability (or dynamics) for large macromolecules and its implication for their biological function attracts more and more attention. Collective motions of domains increase the ability of a protein to bind to partner molecules. Using atomic force microscopy (AFM) topographic images, it is possible to take snapshots of large multi-component macromolecules at the single molecule level and to reconstruct complete molecular conformations. Here, we report the application of a reconstruction protocol, named AFM-assembly, to characterise the conformational variability of the two C domains of human coagulation factor Va (FVa). Using AFM topographic surfaces obtained in liquid environment, it is shown that the angle between C1 and C2 domains of FVa can vary between 40° and 166°. Such dynamical variation in C1 and C2 domain arrangement may have important implications regarding the binding of FVa to phospholipid membranes

Defining the structure of membrane-bound human blood coagulation factor Va

Background: Blood coagulation factor (F) Va is the essential protein cofactor to the serine protease FXa. Factor Va stimulates the thrombin-to-prothrombin conversion by the prothrombinase complex, by at least five orders of magnitude. Factor Va binds with very high affinity to phosphatidylserine containing phospholipid membranes, which allows the visualization of its membrane-bound state by transmission electron microscopy (EM). Methods: In this paper we present an averaged three-dimensional structure of FVa molecules attached to phosphatidylserine containing lipid tubes, as determined by EM and single particle analysis. The low-resolution FVa three-dimensional structure is compared with the available atomic models for FVa. Results: The experimental data are combined with the most suitable atomic model and a membrane-bound FVaEM model is proposed that best fits the protein density defined by EM. In the FVaEM model, the C1 and C2 membrane-binding domains are juxtaposed onto the membrane surface and the model geometries indicate a deeper insertion of both C domains into the lipid bilayer than has been previously suggested. Conclusion: The present structure is a first step towards a higher-resolution experimental structure of a human FVa molecule in its membrane-bound conformation, allowing the visualization of individual domains within FVa and its association with the membrane

The structure-function relationship of activated protein C Lessons from natural and engineered mutations

Protein C is the central enzyme of the natural anticoagulant pathway and its activated form APC (activated protein C) is able to proteolyse non-active as well as active coagulation factors V and VIII. Proteolysis renders these cofactors inactive, resulting in an attenuation of thrombin formation and overall down-regulation of coagulation. Presences of the APC cofactor, protein S, thrombomodulin, endothelial protein C receptor and a phospholipid surface are important for the expression of anticoagulant APC activity. Notably, APC also has direct cytoprotective effects on cells: APC is able to protect the endothelial barrier function and expresses anti-inflammatory and anti-apoptotic activities. Exact molecular mechanisms have thus far not been completely described but it has been shown that both the protease activated receptor 1 and EPCR are essential for the cytoprotective activity of APC. Recently it was shown that also other receptors like sphingosine 1 phosphate receptor 1, Cd11b/CD18 and tyrosine kinase with immunoglobulin-like and EGF-like domains 2 are likewise important for APC signalling. Mutagenesis studies are being performed to map the various APC functions and interactions onto its 3D structure and to dissect anticoagulant and cytoprotective properties. The results of these studies have provided a wealth of structure-function information. With this review we describe the state-of-the-art of the intricate structure-function relationships of APC, a protein that harbours several important functions for the maintenance of both humoral and tissue homeostasi

Inhibition of Neutral Sphingomyelinase 2 by Novel Small Molecule Inhibitors Results in Decreased Release of Extracellular Vesicles by Vascular Smooth Muscle Cells and Attenuated Calcification

Vascular calcification (VC) is an important contributor and prognostic factor in the pathogenesis of cardiovascular diseases. VC is an active process mediated by the release of extracellular vesicles by vascular smooth muscle cells (VSMCs), and the enzyme neutral sphingomyelinase 2 (nSMase2 or SMPD3) plays a key role. Upon activation, the enzyme catalyzes the hydrolysis of sphingomyelin, thereby generating ceramide and phosphocholine. This conversion mediates the release of exosomes, a type of extracellular vesicles (EVs), which ultimately forms the nidus for VC. nSMase2 therefore represents a drug target, the inhibition of which is thought to prevent or halt VC progression. In search of novel druglike small molecule inhibitors of nSMase2, we have used virtual ligand screening to identify potential ligands. From an in-silico collection of 48,6844 small druglike molecules, we selected 996 compounds after application of an in-house multi-step procedure combining different filtering and docking procedures. Selected compounds were functionally tested in vitro; from this, we identified 52 individual hit molecules that inhibited nSMase2 activity by more than 20% at a concentration of 150 mu M. Further analysis showed that five compounds presented with IC(50)s lower than 2 mu M. Of these, compounds ID 5728450 and ID 4011505 decreased human primary VSMC EV release and calcification in vitro. The hit molecules identified here represent new classes of nSMase2 inhibitors that may be developed into lead molecules for the therapeutic or prophylactic treatment of VC