Membrane-dependent reactions of blood coagulation: classical view and state-of-the-art concepts

Although the viscoelastic testing (VET) method was first described by Hartert1 more than seven decades ago, its application as point-of-care (POC) instrumentation in the surgical and trauma settings is relatively recent. Short turnaround times and delivery of results in real time provide clinicians with a relevant and rapid global assessment of hemostasis. Recent advances have further simplified and parsed out the multiple components of hemostasis and clotting to assess for large alterations in clotting factors, inhibitors, fibrinogen, and fibrinolysis. As such, many have reported the use of various VET variables in managing perioperative bleeding and have developed transfusion protocols for specific blood products and pharmacological intervention based on these testing results.2-6 A summary of the VET method is discussed in previous articles within this supplementary edition. This article specifically focuses on the use of VET in the context of platelet function tests (PFTs), and its advantages and limitations compared to other PFT in various clinical settings. For his invention Hartert coined the designation thromboelastography. For the sake of this review we will use the term thromboelastography (TEG) to designate the principle of a testing system in which a sample of whole blood is introduced into a cup that contains a pin or cylinder. Initially the pin and the cup move relative to one another in clockwise and counterclockwise rotations. After activation, fibrin and platelet (PLT) strands form between the cup and the cylinder resulting in an inhibition of motion or an impedance. This impedance or viscoelastic element is quantified and displayed as an optical signal (Figure 1 and Table 1). Currently there are two types of VET, the original form based on the cup-and-pin technology and the more recent form based on ultrasound. In fixed-motion TEG the cup moves, while the detection of altered viscoelasticity and the elastic coupling are on the side of the pin. Fixed-motion TEG has the disadvantage of being sensitive to shock and vibrations. Each dislocation of a device necessitates a calibration of the machine. In elastic-motion TEG, generally, the pin is motile and the site of detection and elastic coupling. There is one exception to this rule with the ClotPro technology where the cup is motile and detection and elastic coupling are linked to the cup. The elastic-motion technology is relatively vibration and shock resistant. Devices are readily transportable. To improve on fixed-motion technology, producers developed resonance viscoelastography, where alterations in clot viscoelastic properties are assessed by ultrasound. Clots are made to resonate by ultrasound stimulation. Wave modeling allows for analysis of viscoelastic properties. "Platelet function" can be defined as a complex process including all activities mediated by PLTs or by PLT-derived products. Before elaborating on how PLT function integrates into VET, we shall focus on relevant aspects of PLT function. In its simplest sense, PLT function can be categorized into energy-independent vs energy-dependent processes. The classic energy-independent process is PLT agglutination in the presence of the antibiotic ristocetin. Under basal in vivo conditions, the PLT von Willebrand factor (VWF) receptor, glycoprotein Ib (GPIb) cannot interact with circulating plasmatic VWF. However, ristocetin interacts with PLTs and VWF and can promote agglutination in a passive VWF-dependent mechanism. As such, this interaction has been used in in vitro assays for diagnostic purposes.7 In contrast, the energy-dependent mechanisms refer to the biomechanical processes that are governed by signal transduction pathways, such as glycolysis and oxidative phosphorylation, as well as the ATP/ADP stored in dense granules. These energy sources are critical to support and sustain energy intensive processes requiring PLT cytoskeletal arrangements, such as adhesion, spreading, aggregation and contraction.8, 9 In vivo, one can define different stages of PLT activation, namely the initiation phase with adhesion, the extension phase with secretion, the consolidation phase with aggregation and clot retraction, and finally the termination and elimination phases with fibrinolysis.10-12 It is important to note that each phase is its own series of multi-step processes that occur simultaneously and may subsequently repeat over time (Figure 2). In the presence of endothelial damage single PLTs may adhere to the newly exposed adventitial collagen fibers and freshly deposited VWF. Through "outside-in" signaling, mediated by the stimulation of the PLT surface collagen receptors and GPIb (VWF receptors), PLT activation is initiated. Through various signaling pathways, soluble and solid mediators are produced and secreted for further recruitment of PLTs to the site of injury and propagation of coagulation. During PLT activation, a flip-flop of the inner leaflet phospholipids into the outer leaflet provides a negatively charged and activated surface on which coagulation factors can bind and generate thrombin in a localized fashion. Coagulation is further propagated by the tissue factor–rich, PLT-derived microparticles generated and released into circulation. The membrane-bound and eventual activated coagulation factors lead to the generation of thrombin (FIIa). In addition to its role as activator of multiple coagulation factors, circulating blood cells, and cells located in the blood vessel wall, thrombin is also a key activator of PLTs. The thrombin-dependent PLT activation is mediated by thrombin's specific PLT receptor, the protease activator receptors (PARs).13, 14 The binding of ligands and/or activators to PLT surface receptors play a major role in PLT function. Receptors have high specificity for their respective ligands, which can be solutes, such as thromboxane A2 (TXA2) and adenosine diphosphate (ADP); VWF; or solids, such as collagen. The interaction of the ligands with their specific receptor results in the outside-in activation of PLTs, with each receptor associated with either a single particular or a multiple signaling pathway, forming intricate intracytoplasmic networks. For instance, as a safeguard against any basal level PLT aggregation, in its resting state the GPIIb/IIIa receptor does not bind any ligand. Once the signaling threshold for activation is reached, an inside-out activation occurs, and with subsequent allosteric changes of the PLT GPIIb/IIIa receptor, there is increased affinity for fibrinogen/fibrin binding. The resultant PLT aggregation is defined by this GPIIb/IIIa–fibrinogen interaction culminating in multiple PLTs binding the same fibrinogen molecule and subsequent bridging of adjacent PLTs. The thrombin generated in the immediate vicinity will transform the PLT-bound fibrinogen into fibrin followed by the stabilization of the primary fibrin clot by activated coagulation factor XIII (FXIIIa).15 A main contributor to PLT function are the PLT granules, whose secreted contents play roles in the amplification of PLT activation, the recruitment of circulating PLTs into aggregates, and clot stabilization. Alpha-granules contain adhesion proteins such as fibrinogen and VWF as well as coagulation and fibrinolytic factors, cytokines, and growth factors. Dense granules contain activating nucleotides such as ADP and ATP, serotonin, histamine, pyrophosphates, and divalent cations.10, 13 No PFT can truly recapitulate the in vivo environment and anatomy of the differing arterial, capillary, and venous vascular beds and the relationship of single PLTs to the vessel (taking into account variables such as shear rate, relative proximity to vessel wall, and the underlying endothelium). Of note, these underlying mechanisms promoting PLT aggregation and clot formation can vary with shear rate conditions. Under the relatively low shear conditions, typically found in the low flow/pressure of venous vascular beds, coagulation factors dominate the hemostatic processes. With endothelial damage, PLT adhesion and aggregation are primarily influenced by the supporting role of fibrinogen, with its binding of PLTs via GPIIb/IIIa. Contrast this to the high-shear conditions of the arterial vascular system, wherein VWF plays the more prominent role and PLT aggregation becomes more heavily influenced by the VWF engagement of GPIb.16 Although these underlying mechanisms are key concepts in defining "platelet function," no one assay completely replicates the full complexity of in vivo PLT activity. For PFT, one may consider PLT function sensu stricto as the processes involved in PLT aggregation. However, as it shall become apparent, disregarding or discounting other components of PLT function can result in possible misinterpretation or misunderstanding of the underlying PLT defect or dysfunction. A multitude of systems assess PLT functions in vitro, most of which evaluate PLT aggregation (Figure 2). Light transmission aggregometry (LTAl; turbidimetric-based assay) and whole blood PLT aggregometry (WBA; impedance-based aggregometry assay) measure low-shear, GPIIb/IIIa-dependent PLT-to-PLT aggregation in response to agonists in PLT-rich plasma and whole blood, respectively. Although considered the gold standard in PLT defect diagnostics, with standardized guidelines for sample preparation, agonist concentration, and interpretation of results,17-20 LTA is considered labor- and time-consuming and difficult to standardize in practice. Despite omitting the labor-intensive step of PLT-rich plasma preparation, WBA continues to be plagued by large sample volumes and time commitments. As an assay supplement to LTA or WBA, lumiaggregometry evaluates dense granule secretion of activated PLTs in response to various agonists. Secretion is measured by detecting luminescence emitted when secreted ATP reacts with a bioluminescent reagent. The multiple-electrode aggregometry (MEA) method, a cartridge-based POC version of WBA touting minimal technical training, still requires skilled interpretation of the assay result. Its measurements are based on a combination of PLT activation and adhesion of activated PLTs to a surface. As with LTA and WBA, MEA utilizes multiple agonists and may be suitable for diagnosis of PLT dysfunction or for monitoring anti-PLT therapy, but as with other PLT aggregation tests, MEA is dependent on PLT count.21-24 With increased interest in monitoring anti-PLT therapy, studies have compared newer platforms, such as VerifyNow and TEG-PM, to MEA as a proxy for the gold-standard LTA. The whole blood, flow-dependent PFA-100/200 assays quantify adhesion/aggregation by measuring the time necessary for a PLT plug to occlude an aperture within a biologically active membrane (collagen–epinephrine vs collagen–ADP coated). Although considered a simple and rapid test, it requires a minimum hematocrit and a minimum PLT count,25 both of which are frequently unmet in the perioperative or acute bleeding setting, thereby limiting its applicability. This system is sensitive to VWF concentration and activity,26 and these aspects must be considered when evaluating PLT function deficits. VerifyNow is a turbidimetric, cartridge-based POC device assessing whole blood PLT aggregation upon fibrinogen-coated beads in response to agonists, whose measurement is proportional to the number of activated GPIIb/IIIa receptors. Modified versions of the assay are sensitive to the PLT response to aspirin or P2Y12 receptor blockers. As the VerifyNow P2Y12 assay blocks P2Y1-mediated aggregation it can measure P2Y12-mediated aggregation. This contrasts with standard PLT aggregometry, wherein the agonist ADP is used without inhibitors and both P2Y1- and P2Y12-mediated aggregation are measured.27 This may explain observed discrepancies between VerifyNow and standard PLT aggregometry when assessing clopidogrel responses.28-30 Monitoring anti-PLT therapy in cardiac patients with VerifyNow had been a low-level recommendation.31 Large prospective randomized controlled trails had failed to demonstrate the use of POC assessments of PLT function in reducing ischemic events and did not provide support for changing anti-PLT therapy based on these tests.32-35 Although initial studies showed that the utilization of VerifyNow for PLT transfusion guidance led to improvement in measured PLT activity for patients with intracranial hemorrhage,36 larger studies focusing on intracranial bleed patients on COX inhibitors found an increase in the odds of death or dependence at 3 months in those who had PLT transfusion compared to the standard care group.37 Few patients on P2Y12 inhibitors were included in the study, and results may not be generalizable to that population. Such scenarios demonstrate the need of large well-designed clinical trials to validate extrapolations of in vitro PLT function studies. The POC Plateletworks also assesses PLT aggregation by comparing the baseline count of nonaggregated PLTs in agonist-untreated samples to those treated with agonists such as collagen, ADP, and arachidonic acid. The degree of PLT aggregation tends to be higher in Plateletworks in comparison to that of LTA, with speculation that the single PLT counting of Plateletworks is more sensitive to microaggregation as opposed to the LTA-measured macroaggregation.38, 39 Evaluation for use in clinical practice to monitor the use of the anti-PLT drugs such as aspirin and clopidogrel39-41 demonstrated good agreement with LTA in acute care settings. Unfortunately, due to a measurement window of only a few minutes, the test has been utilized primarily within surgical settings and clinical data are lacking on its effect on clinical outcomes.42 Flow cytometry's utilization of agonists and antibodies against glycoproteins, such as P-selectin, activated GPIIb/IIIa, and GPIb, profiles structural and functional PLT variables of 1000 of individual PLTs. Granule secretion and aggregation are quantified by gating the amount of P-selectin and activated GPIIb/IIIa receptors on membranes.43-45 Primarily used for research, assessment of PLT surface glycoprotein expression has potential clinical applications in monitoring GPIIb/IIIa antagonist therapy and diagnosing glycoprotein deficiencies or storage pool disease, among other applications.46 Viscoelastic testing is often referred to as a global assay of hemostasis. VET analyzes changes in the viscoelastic forces during clot formation upon the addition of activators or inhibitors to whole blood (Table 2). The VET method is discussed extensively in previous modules within this supplementary edition and we refer to those articles for detailed description. Polybrene Cyclochalasin D Polybrene Tranexamic acid Reptilase FXIIIa ADP Reptilase FXIIIa Arachidonic acid Cytochalasin D, GPIIb/IIIa antagonist Polybrene Polybrene Abciximab By design there is a major difference in VET vs in vivo physiology. In VET or in vitro, there is no contribution of the vascular system. Furthermore, activation occurs inversely, in that initially secondary hemostasis is activated either intrinsically or extrinsically before subsequently primary hemostasis gets activated—mostly by thrombin. In vivo, the sequence of cardinal events is endothelial damage, VWF deposition, PLT adhesion, outside-in signaling, secretion, inside-out signaling, aggregation, clot retraction, and fibrinolysis. In summary in vitro the chain of events is secondary hemostasis, primary hemostasis, and fibrinolysis, while in vivo, vascular hemostasis precedes primary and secondary hemostasis before fibrinolysis sets in. In VET, PLT function is assayed as a maximal stimulation or an all or nothing response. This is due to the design of the testing systems. After recalcification of the citrated whole blood sample, activation of secondary or plasmatic hemostasis by potent activators of the intrinsic or extrinsic system will lead to traces of thrombin being generated followed by a massive thrombin "burst." The thrombin burst will strongly stimulate PLTs leading to a maximal PLT signal. This maximal signal will correlate with the PLT concentration.47 However, this signal will not correlate with PLT function. Thus the VET signal will not be influenced by the presence or absence of antiaggregants therapy, because thrombin-induced and PAR-mediated outside-in signaling bypasses other activation routes and always produces a maximal response. PAR-mediated PLT activation represents a form of emergency bypass to all other signal transduction pathways, in situations where rapid and complete PLT activation is necessary. This kind of all-or-nothing activation situation is not sensitive to cofactors including antiaggregants such as aspirin or P2Y12 inhibitors. The only PLT inhibitor or situation to which this setting is sensitive to are GPIIb/IIIa inhibitors or defects in that receptor including Glanzmann thrombasthenia. In these cases, PLTs can no longer activate inside out, preventing multiple PLTs from interacting with fibrinogen or fibrin molecules leading to a reduced PLT signal in VET. Eli Cohen, the inventor of TEG and TEG PLT mapping (TEG -PM), had the idea of activating citrated whole blood samples with an array of different activators and inhibitors.48 He used a kaolin- and calcium-activated TEG to generate a maximum signal. This signal is composed of a fibrinogen (and FXIIIa) contribution and a PLT contribution. With the use of a cocktail of activators, VET was sensitized to PLT activators weaker than thrombin. This cocktail consisted of (a) reptilase (or botrobotoxin)—a snake poison that activates fibrinogen to fibrin but does not lead to activation of the thrombin-dependent PAR receptors on PLTs; (b) FXIIIa—to stabilize the fibrin clot by generating stable covalent bonds between fibrin monomers; and (c) heparin—which neutralizes any generated thrombin. Then, by adding either arachidonic acid, an activator of the TXA2 receptor/cyclooxygenase pathway (inhibited by aspirin), or ADP, an activator of the P2Y12 receptor (inhibited by the class of P2Y12 inhibitors including clopidogrel, prasugrel, and others), he generated tests sensitive to cyclooxygenase and P2Y12 inhibitors. Platelets play a key role in fibrinolysis, and fibrinolysis can be detected by VET. PLTs represent an activated surface suitable for the assembly of the entire plasminogen activator system. Furthermore, PLTs secrete plasmin in an autocrine fashion and bind it to their surface. The latter aspect is relevant in the sense that free plasmin is prone to inhibition through its primary plasmatic inhibitor alpha2 antiplasmin, whereas PLT-bound plasmin is not. Plasmin has also been reported to cleave GPIIIa, leading to a dysfunctional PLT state late on in the process of hemostasis. High numbers of PLTs in a clot lead to stable clots, whereas PLT-poor clots are more prone to fibrinolysis.49 Plasminogen activator inhibitor 1 can bind to the PLT surface, leading to inhibition of fibrinolysis early on in coagulation.50, 51 Thus, whenever fibrinolysis becomes apparent in VET, this too can be interpreted as a manifestation of PLT function. Most clinical studies have focused on global hemostasis monitoring and have evaluated the clinical usefulness of VET for the prediction, diagnosis, and transfusion guidance in coagulopathic-based bleeding in trauma, obstetrics, liver, and cardiac perioperative settings. Some have focused on the application of VET-guided transfusion strategies to reduce the use of blood products and improve morbidity in bleeding patients.3-6 Less is known on VET use in the evaluation of PLT function in these settings,52-55 because standard VET tracings give minimal information on PLT function's contribution to clot strength. Meta-analyses have been mixed, with at least two describing a significant effect of VET on the proportion of patients transfused with PLTs.52, 54, 56 There are data that suggest that the two platforms, Sonoclot and Quantra QPlus, can assay aspects of PLT function.57, 58 As detailed, and not unlike other VET systems, these two systems also fall under the all-or-nothing principle of thrombin-activated tests, wherein the resulting implications include a lack of sensitivity to antiaggregants including aspirin and P2Y12 inhibitors. However, when one defines PLT function solely by the interaction of the PLT's fibrinogen receptor with its substrate, then these VET systems may indeed be able to report on a limited aspect of PLT function–related The by and and the and is evaluated based on clot with the that clot occurs with PLT while clot is when the fibrinogen with and is from the in the extrinsic pathway activated treated with Unfortunately, only a of and perioperative studies have focused on the clinical and of or measurements in PLT function PLT were found to be associated with Sonoclot measurements in a prospective study, which compared TEG to Sonoclot variables in cardiac It was that the degree of a that such a be of an immediate POC in vitro studies found and influenced by PLT and of the to PLT was in studies are to validate PLT function and is not a of PLT early that Quantra with function demonstrated by to the and associated for PLT studies are to for potential clinical use by first assessing PLT contribution to clot in patients cardiac and with studies comparing its to those of other in vitro studies have assessed to monitor PLT function and GPIIb/IIIa such had found to be to PLT aggregometry in its to pharmacological GPIIb/IIIa inhibition by studies are to demonstrate potential use in assessing the of anti-PLT In a prospective wherein Quantra was compared to TEG for use in transfusion for patients cardiac it had been described that the P2Y12 receptor inhibitor therapy and may the clinical need for PLT is because these early and in vitro studies not coagulation changes in clinical trials are still to evaluate both and in monitoring PLT function in the trauma, and settings. The of VET for anti-PLT detection of and have been However, standard VET the major in their to measure PLT adhesion and to the effect of aspirin or P2Y12 receptor due to the thrombin generation as a response to the This response any to the inhibition of the TXA2 receptor/cyclooxygenase and P2Y12 receptor at only the detection of GPIIb/IIIa In of the increased clinical use of P2Y12 inhibitors and the of previous VET to their studies the of for anti-PLT monitoring and its use in bleeding or transfusion have compared to other PFT and evaluated in its to but there have been clinical For two prospective focusing on bypass patients described PLT inhibition TEG-PM, had to correlate with or blood In contrast, a prospective of bypass patients may as a for PLT inhibition as the PLT function measured by was associated with increased transfusion to other aspects including and transfusion the of testing may be key to the in these The studies that PLT function found a with the primary or transfusion whereas those that did not. Furthermore, a factor may be that bypass can be associated with a PLT as by that may or may not be associated with an increased bleeding in evaluating for its in monitoring anti-PLT therapy in trauma, and agreement and between and other PFT as and have been larger prospective studies are to the clinical of for In are to the many role in hemostasis. PLT function may be defined in a of and the activation and inhibition of a of As such there is no standard to define PLT function and no gold standard in the VET PLT between different VET and the of PFT a The of VET and its relative turnaround time have led to increased utilization among clinical for the evaluation of anti-PLT effect on hemostasis during trauma or surgical situations or in the of for upon the of the Although most VET measuring PLT function correlate with PLT many low and for PLT due to the thrombin activation of PLTs through the receptor, a that standard anti-PLT cannot be detected standard VET. VET ADP or arachidonic acid may the assessment of the of P2Y12 inhibitors and clinical trials are to evaluate these can the inhibition of the anti-PLT of and improve clinical The their to from the and from for the real time of a whole blood no potential of has from and no other potential of

Pathophysiology of Anti-PL Syndrome in Thrombosis.

Phosphatidylserine is known to significantly accelerate the blood coagulation reaction. In a previous communication submitted for publication, we demonstrated that phosphatidylcholine, phosphatidylethanolamine and lysophosphatidylcholine showed effects on the blood coagulation reaction using the factor Xa-prothrombin reaction system, and discuss a new function of membrane phospholipids. The present study examined the role of phospholipids in the blood coagulation regulatory reaction (anticoagulation system), by studying the effects of phospholipids on the protein C/protein S reaction. We have established quantitative methods for measuring activated protein C activity and protein S activity, and used them to measure their activity after the addition of liposomes with different phospholipid compositions. We found that phosphatidylcholine inhibited activated protein C and protein S activities in a dose-dependent manner, as in the factor Xa-prothrombin reaction system. On the other hand, phosphatidylethanolamine and lysophosphatidylcholine showed no effect on activated protein C activity. Phosphatidylethanolamine inhibited and lysophosphatidylcholine accelerated coagulation activity in the factor Xa-prothrombin system, but such effects were not observed in the protein C/protein S reaction system. The coagulation and anticoagulation reactions are exquisitely balanced by thrombin, with a role both as a procoagulant and anticoagulant. Therefore, it is understandable that phosphatidylethanolamine and lysophosphatidylcholine show different effects in the factor Xa-prothrombin and protein C/protein S reaction systems. It appears that coagulation and anticoagulation reactions are co-ordinated and controlled by changes in phospholipid composition of the cellular membrane where the coagulation reaction takes place.

Cecropins are positively charged antibacterial polypeptides that were originally isolated from insects. Later on a mammalian homologue, cecropin P1 (CecP), was isolated from pig intestines. While insect cecropins are highly potent against both Gram-negative and Gram-positive bacteria, CecP is as active as insect cecropins against Gram-negative but has reduced activity against Gram-positive bacteria. To gain insight into the mechanism of action of CecP and the molecular basis of its antibacterial specificity, the peptide and its proline incorporated analogue (at the conserved position found in insect cecropins), P22-CecP, were synthesized and labeled on their N-terminal amino-acids with fluorescent probes, without significantly affecting their antibacterial activities. Fluorescence studies indicated that the N-terminal of CecP is located on the surface of phospholipid membranes. Binding experiments revealed that CecP binds acidic phosphatidylserine/phosphatidylcholine (PS/PC) vesicles better than zwitterionic PC vesicles, which correlates with its ability to permeate the former better than the latter. The shape of the binding isotherms suggest that CecP, like insect cecropin, binds phospholipids in a simple, noncooperative manner. However, resonance energy transfer (RET) measurements revealed that, unlike insect cecropins, CecP does not aggregate in the membrane even at relatively high peptide to lipid ratios. The stoichiometry of CecP binding to vesicles suggests that amount of CecP sufficient to form a monolayer causes vesicle permeation. In spite of the incorporation of the conserved proline in P22-CecP, the analogue has reduced antibacterial activity, which correlates with its reduced alpha-helical structure and its lower partitioning and membrane permeating activity with phospholipid vesicles. Taken together, our results support a mechanism in which CecP disrupts the structure of the bacterial membrane by (i) binding of peptide monomers to the acidic surface of the bacterial membrane and (ii) disintegrating the bacterial membrane by disrupting the lipid packing in the bilayers. These results, combined with data reported for other antibacterial polypeptides, suggest that the organization of peptide monomers within phospholipid membranes contributes to Gram-positive/Gram-negative antibacterial specificity.

Regulation of the Binding of Myristoylated Alanine-Rich C Kinase Substrate (MARCKS) Related Protein to Lipid Bilayer Membranes by Calmodulin

It is well-known that sugars protect membrane structures against fusion and leakage. Here, we have investigated the interaction between different sugars (sucrose, trehalose, and maltose) and phospholipid membrane of 1,2-dimyristoyl-sn-glycero-3-phoshpocholine (DMPC) using dynamic light scattering (DLS), transmission electron microscopy (TEM), and other various spectroscopic techniques. DLS measurement reveals that the addition of sugar molecule results a significant increase of the average diameter of DMPC membrane. We have also noticed that in the presence of different sugars the rotational relaxation and solvation time of coumarin 480 (C480) and coumarin 153 (C153) surrounding DMPC membrane increases, suggesting a marked reduction of the hydration behavior at the surface of phospholipid membrane. In addition, we have also investigated the effect of sugar molecules on the lateral mobility of phospholipids. Interestingly, the relative increase in rotational, solvation and lateral diffusion is more prominent for C480 than that of C153 because of their different location in lipid bilayer. It is because of preferential location of comparatively hydrophilic probe C480 in the interfacial region of the lipid bilayer. Sugars intercalate with the phospholipid headgroup through hydrogen bonding and replace smaller sized water molecules from the membrane surface. Therefore, overall, we have monitored a comparative analysis regarding the interaction of different sugar molecules (sucrose, trehalose, and maltose) with the DMPC membrane through DLS, TEM, solvation dynamics, time-resolved anisotropy, and fluorescence correlation spectroscopy (FCS) measurements to explore the structural and spectroscopic aspect of lipid-sugar interaction.

Electrical phenomena at the surfaces of phospholipid membranes caused by the binding of ionic compounds

A recent chemical footprinting study in our laboratory suggested that region 1803-1818 might contribute to A2 domain retention in activated factor VIII (FVIIIa). This site has also been implicated to interact with activated factor IX (FIXa). Asn-1810 further comprises an N-linked glycan, which seems incompatible with a role of the amino acids 1803-1818 for FIXa or A2 domain binding. In the present study, FVIIIa stability and FIXa binding were evaluated in a FVIII-N1810C variant, and two FVIII variants in which residues 1803-1810 and 1811-1818 are replaced by the corresponding residues of factor V (FV). Enzyme kinetic studies showed that only FVIII/FV 1811-1818 has a decreased apparent binding affinity for FIXa. Flow cytometry analysis indicated that fluorescent FIXa exhibits impaired complex formation with only FVIII/FV 1811-1818 on lipospheres. Site-directed mutagenesis revealed that Phe-1816 contributes to the interaction with FIXa. To evaluate FVIIIa stability, the FVIII/FV chimeras were activated by thrombin, and the decline in cofactor function was followed over time. FVIII/FV 1803-1810 and FVIII/FV 1811-1818 but not FVIII-N1810C showed a decreased FVIIIa half-life. However, when the FVIII variants were activated in presence of FIXa, only FVIII/FV 1811-1818 demonstrated an enhanced decline in cofactor function. Surface plasmon resonance analysis revealed that the FVIII variants K1813A/K1818A, E1811A, and F1816A exhibit enhanced dissociation after activation. The results together demonstrate that the glycan at 1810 is not involved in FVIII cofactor function, and that Phe-1816 of region 1811-1818 contributes to FIXa binding. Both regions 1803-1810 and 1811-1818 contribute to FVIIIa stability.

The 20 S proteasome is an essential proteolytic particle, responsible for degrading short-lived and abnormal intracellular proteins. The 700-kDa assembly is comprised of 14 alpha-type and 14 beta-type subunits, which form a cylindrical architecture composed of four stacked heptameric rings (alpha7beta7beta7alpha7). The formation of the 20 S proteasome is a complex process that involves a cascade of folding, assembly, and processing events. To date, the understanding of the assembly pathway is incomplete due to the experimental challenges of capturing short-lived intermediates. In this study, we have applied a real-time mass spectrometry approach to capture transient species along the assembly pathway of the 20 S proteasome from Rhodococcus erythropolis. In the course of assembly, we observed formation of an early alpha/beta-heterodimer as well as an unprocessed half-proteasome particle. Formation of mature holoproteasomes occurred in concert with the disappearance of half-proteasomes. We also analyzed the beta-subunits before and during assembly and reveal that those with longer propeptides are incorporated into half- and full proteasomes more rapidly than those that are heavily truncated. To characterize the preholoproteasome, formed by docking of two unprocessed half-proteasomes and not observed during assembly of wild type subunits, we trapped this intermediate using a beta-subunit mutational variant. In summary, this study provides evidence for transient intermediates in the assembly pathway and reveals detailed insight into the cleavage sites of the propeptide.

Differential effects of oleuropein, a biophenol from Olea europaea, on anionic and zwiterionic phospholipid model membranes

Phospholamban (PLB) and phospholemman (PLM, also called FXYD1) are small transmembrane proteins that interact with P-type ATPases and regulate ion transport in cardiac cells and other tissues. This work has investigated the hypothesis that the cytoplasmic domains of PLB and PLM, when not interacting with their regulatory targets, are stabilized through associations with the surface of the phospholipid membrane. Peptides representing the 35 C-terminal cytoplasmic residues of PLM (PLM(37-72)), the 23 N-terminal cytoplasmic residues of PLB (PLB(1-23)), and the same sequence phosphorylated at Ser-16 (P-PLB(1-23)) were synthesized to examine their interactions with model membranes composed of zwitterionic phosphatidylcholine (PC) lipids alone or in admixture with anionic phosphatidylglycerol (PG) lipids. Wide-line 2H NMR spectra of PC/PG membranes, with PC deuterated in the choline moiety, indicated that all three peptides interacted with the membrane surface and perturbed the orientation of the choline headgroups. Fluorescence and 31P magic-angle spinning (MAS) NMR measurements indicated that PLB(1-23) and P-PLB(1-23) had a higher affinity for PC/PG membranes, which carry an overall negative surface charge, than for PC membranes, which have no net surface charge. The 31P MAS NMR spectra of the PC/PG membranes in the presence of PLM(37-72), PLB(1-23), and P-PLB(1-23) indicated that all three peptides induced clustering of the lipids into PC-enriched and PG-enriched regions. These findings support the theory that the cytoplasmic domains of PLB and PLM are stabilized by interacting with lipid headgroups at the membrane surface, and it is speculated that such interactions may modulate the functional properties of biological membranes.

Exploring the free energy landscapes of metal cations on phospholipid membrane surfaces is important for the understanding of chemical and biological processes in cellular environments. Using metadynamics simulations we have performed systematic free energy calculations of sodium cations bound to DMPC phospholipid membranes with cholesterol concentration varying between 0% (cholesterol-free) and 50% (cholesterol-rich) at infinite dilution. The resulting free energy landscapes reveal the competition between binding of sodium to water and to lipid head groups. Moreover, the binding competitiveness of lipid head groups is diminished by cholesterol contents. As cholesterol concentration increases, the ionic affinity to membranes decreases. When cholesterol concentration is greater than 30%, the ionic binding is significantly reduced, which coincides with the phase transition point of DMPC-cholesterol membranes from a liquid-disordered phase to a liquid-ordered phase. We have also evaluated the contributions of different lipid head groups to the binding free energy separately. The DMPC's carbonyl group is the most favorable binding site for sodium, followed by DMPC's phosphate group and then the hydroxyl group of cholesterol.

Metal cations are ubiquitous components in biological environments and play an important role in regulating cellular functions and membrane properties. By applying metadynamics simulations, we have performed systematic free energy calculations of Na(+), K(+), Ca(2+), and Mg(2+) bound to phospholipid membrane surfaces for the first time. The free energy landscapes unveil specific binding behaviors of metal cations on phospholipid membranes. Na(+) and K(+) are more likely to stay in the aqueous solution and can bind easily to a few lipid oxygens by overcoming low free energy barriers. Ca(2+) is most stable when it is bound to four lipid oxygens of the membrane rather than being hydrated in the aqueous solution. Mg(2+) is tightly hydrated, and it shows hardly any loss of a hydration water or binding directly to the membrane. When bound to the membrane, the cations' most favorable total coordination numbers with water and lipid oxygens are the same as their corresponding hydration numbers in aqueous solution, indicating a competition between ion binding to water and lipids. The binding specificity of metal cations on membranes is highly correlated with the hydration free energy and the size of the hydration shell.

Introduction: Changes in blood coagulation during exposure to high altitude are not well understood and studies of activation and consumption of specific coagula-tion factors in hypoxic humans have yielded conflicting results. In this study we used thrombelastometry (TEM) which allows a global evaluation of clot formation and lysis process to study blood coagulation profiles in volunteers exposed to pro-longed hypobaric hypoxia at extreme altitudes. Material and methods: We conducted a prospective, observational study in 39 healthy volunteers during a research expedition up to an altitude of 7050 m. Plasma based thrombelastometric measurements and standard coagulation parameters were performed at different altitudes. Results: TEM measurements showed an increase in clotting time (CT) and maxi-mum clot firmness (MCF) at high altitudes, paralleled by an increase in international normalized ratio (INR) and activated partial thromboplastin time (aPTT). Fibrinogen concentration increased until 6022 m. D-Dimer and Thrombin-Antithrombin complex (TAT) increased with time exposed to severe hypoxia. For both measurements highest levels were found at 4844 m after acclimatization; in contrast, lower values were observed again at 7050m in the group of summiteers. Activated protein C resistance (APC-R) was slightly lowered at all altitudes. Conclusion: Our results suggest that activation of the coagulation and fibrinolytic system occurs with increasing hypobaric hypoxia with concurrent use of coagula-tion factors indicating the occurrence of a consumption-coagulopathy phenotype.

A vascular fissure requires a patch that must be provided by constituents of the cellular and fluid phases of flowing blood. The principal components involved in primary haemostasis are platelets, collagen and von Willebrand factor (vWF). Platelets, the cellular elements of the patch, are inert until they encounter conditions that trigger their activation. Platelet adhesion and aggregation at the site of vascular injury lead to the formation of a platelet plug and to a local activation of the coagulation cascade. The resulting final product of blood coagulation is a fibrin network that stabilises the primary platelet plug. Most coagulation factors are zymogens of serine proteases. They are converted from an inactive form to an active enzyme by limited proteolytic cleavage of one or a few peptide bonds. The coagulation reactions must become extinguished as soon as the patch in the injured blood vessel has been established. Several inhibitors, present in excess in plasma, neutralise the surplus of remaining proteases, and the fibrinolytic system dissolves the plug after the surrounding tissue has been repaired. In fulfilling their function to control the fluidity and integrity of the vascular system, the plasmatic and cellular haemostatic players undergo multiple interactions of two kinds: they recognize and bind, often irreversibly, to several partners which are present in their immediate environment. On the other hand, some haemostatic factors, such as fibrinogen and von Willebrand factor, enhance their stickiness by polymerisation of identical subunits carrying multiple adhesive sites. Several haemostatic plasma proteins and their cellular surface receptors are involved in or may be affected by other homeostatic systems, such as immune response, complement activation, cytokine release, cell proliferation, growth and differentiation. These diverse functions are only possible because of the modular structure of participating proteins. In the process of evolution a series of structural modules have been incorporated into protein molecules as their integral domains by exon duplication and shuffling. Owing to variable conformations of the resulting multi-domain proteins, the same modules may perform different tasks and be recognized only by specific ligands, thus controlling the delicately balanced system of haemostasis.