Coagulation factor mutations and thrombosis

The coagulation system is governed by a subtle balance between clotting activators and inhibitors. Many genes can contribute to the overall phenotype, and polymorphisms may act to up regulate or down regulate the generation of thrombin, the coagulation-key enzyme. An increase in coagulation factor (gain function) or/and a decrease in coagulation inhibitors (loss of function) may favor venous thromboembolism (VTE). It has been observed since a long time that VTE may be a familial disease, but it was only in 1965 that Egeberg published the first case of inherited antithrombin (AT) deficiency. This was followed by similar reports of protein C (PC) and protein S (PS) deficiencies. Hereditary thrombophilia was thus initially considered as a rare monogenic disorder with incomplete penetrance. AT, PC and PS deficiencies are due to multiple and mostly private mutations of the corresponding genes. Most patients are heterozygous and experience VTE at adult age. Homozygosity associated with severe thrombosis at birth has been observed in newborns with undetectable PC or PS concentrations. The discovery of factor (F) V Leiden and F2 g.20210 G>A, two gain of function mutations, challenged the view of thrombophilia as a rare monogenic disorder. FV Leiden and F2 g.20210 G>A are due to a founder effect and affect populations of European descent with frequencies at 5% and 3% respectively. These two mutations are moderate of risk factor for thrombosis and paved the way for gene-gene and gene-environment interactions. Patients carrying more than one genetic risk factor are at higher risk to develop VTE. The exposition to acquired risk factors such as estrogen based oral contraception may also have a synergistic effect favoring thrombosis in patients with FV Leiden or other genetic risk factors.     

两例抗凝血蛋白缺乏患者的基因诊断

目的:探讨导致蛋白C、蛋白S、抗凝血酶缺乏症的分子发病机制。方法:检测蛋白C活性(PC:C)、蛋白S活性(PS:C)以及抗凝血酶活性(AT:C);PCR法分别扩增患者PC、PS、AT基因序列,寻找突变点。结果:蛋白C合并蛋白S合并抗凝血酶AT缺乏患者PC基因启动子区域存在C4867T杂合突变(NG_016323.1),为蛋白C基因的多态性位点;在蛋白S基因第四号外显子区域有G68395T杂合突变(NG_009813.1),导致Arg90Leu(NP_000304.2),为国际首次报道。遗传性PS缺陷在家系:四名家系成员均检测到PS基因第四号外显子区域一个杂合(错义)突变,G68395T(NG_009813.1)。结论:PC基因启动子的多态性位点C4867T杂合突变(NG_016323.1),PS基因第四号外显子区域的G68395T杂合突变(NG_009813.1),可能是导致患者PC、PS联合缺乏的原因。PS基因第四号外显子区域G68395T(NG_009813.1)杂合突变,可能是导致PS缺陷症家系成员PS缺乏的原因。     

THE BOOK CORNER

In order to increase the yield of prothrombin complex concentrates (PCCs) and to reduce their associated thrombotic risks, the influence of washing conditions on the yield, purity, and balance of coagulation factors (FII, FVII, FIX, and FX), and inhibitor proteins (PC, PS, PZ, and AT [antithrombin]) in PCCs was investigated by orthogonal testing, in which three variables (sodium citrate, NaCl, and pH) and their three levels were selected. It was found that AT yield and purity were extraordinarily low, and at lower NaCl content, the general yield, purity, and balance were higher, lower, and better, respectively; however, the results became contrary at higher NaCl. Moreover, within the investigated levels, NaCl was the first determinant for the yield except AT and the purity except FVII, PC, PS, and AT. Sodium citrate was the first determinant for AT yield and FVII, PS, and AT purity. The yield except FII, PS, and AT decreased and the purity except PC increased with increase of sodium citrate content. Just for PC purity, pH was the first determinant. The effect with pH fluctuation on the yield and purity was characteristically unobvious. The outcome undoubtedly supplies the guidance to further improve PCCs.     

Prediction of Recurrent Venous Thromboembolism by Clot Lysis Time: A Prospective Cohort Study

Venous thromboembolism (VTE) is a chronic disease, which tends to recur. Whether an abnormal fibrinolytic system is associated with an increased risk of VTE is unclear. We assessed the relationship between fibrinolytic capacity (reflected by clot lysis time [CLT]) and risk of recurrent VTE. We followed 704 patients (378 women; mean age 48 yrs) with a first unprovoked VTE for an average of 46 months after anticoagulation withdrawal. Patients with natural coagulation inhibitor deficiency, lupus anticoagulant, cancer, homozygosity for factor V Leiden or prothrombin mutation, or requirement for indefinite anticoagulation were excluded. Study endpoint was symptomatic recurrent VTE. For measurement of CLT, a tissue factor-induced clot was lysed by adding tissue-type plasminogen activator. Time between clot formation and lysis was determined by measuring the turbidity.135 (19%) patients had recurrent VTE. For each increase in CLT of 10 minutes, the crude relative risk (RR) of recurrence was 1.13 (95% CI 1.02–1.25; p = 0.02) and was 1.08 (95% CI 0.98–1.20; p = 0.13) after adjustment for age and sex. For women only, the adjusted RR was 1.14 (95% CI, 0.91–1.42, p = 0.22) for each increase in CLT of 10 minutes. CLT values in the 4^th quartile of the female patient population, as compared to values in the 1^st quartile, conferred a risk of recurrence of 3.28 (95% CI, 1.07–10.05; p = 0.04). No association between CLT and recurrence risk was found in men. Hypofibrinolysis as assessed by CLT confers a moderate increase in the risk of recurrent VTE. A weak association between CLT and risk of recurrence was found in women only.     

Effect of light and protein phosphorylation on photoreceptor rod outer segment acyltransferase activity

Rod outer segments (ROS) exhibit high acyltransferase (AT) activity, the preferred substrate of which being lysophosphatidylcholine. To study factors possibly regulating ROS AT activity purified ROS membranes were assayed under conditions under which protein kinase C (PKC), cAMP-dependent protein kinase (PKA), and phosphatases were stimulated or inhibited. PKC activation produced a significant increase in the acylation of phosphatidylethanolamine (PE) and phosphatidylinositol (PI) with oleate, it inhibited phosphatidylcholine (PC) acylation, and phosphatidylserine (PS) and phosphatidic acid (PA) acylation remained unchanged. ROS PKA activation resulted in increased oleate incorporation into PS and PI while the acylation of PC, PE, and PA remained unchanged. Inhibition of ROS PKC or PKA produced, as a general trait, inverse effects with respect to those observed under kinase-stimulatory conditions. ROS phosphatase 2A was inhibited by using okadaic acid, and the changes observed in AT activity are described. These findings suggest that changes in ROS protein phosphorylation produce specific changes in AT activity depending on the phospholipid substrate. The effect of light on AT activity in ROS membranes was also studied and it is reported that acylation in these membranes remains unchanged independent of the illumination condition used.     

Men had a Higher Risk of Recurrent Venous Thromboembolism than Women: A Large Population Study

BackgroundMost reports of sex differences in the risk of recurrent venous thromboembolism (VTE) are based on small or moderate sized cohorts of selected patients with VTE.MethodsWe aimed to determine the effect of sex on recurrent VTE in a large non-selected, real-world population of men and women with incident VTE. Using the linked administrative health care databases of the province of Québec, Canada, we constructed a cohort of patients with a first-time diagnosis of VTE between January 1, 1996 and December 31, 2004. Patients were followed forward in time for the occurrence of recurrent VTE until the earliest of either death, termination of health coverage, or end of study period (December 31, 2005). The cohort comprised 55,314 patients (43% men and 57% women) with incident VTE and the mean age was 61.9 years.ResultsDuring a mean follow-up of 3.9 years, 5243 (9.5%) of patients developed recurrent VTE. Men had a significantly higher rate of recurrence than women (adjusted hazard ratio = 1.13; 95% CI, 1.07–1.19), and this difference persisted when women with hormonally mediated VTE were excluded from the analysis (adjusted hazard ratio = 1.15; 95% CI, 1.08–1.21). At 5 years, the cumulative probability of recurrent VTE was 12.4% among men versus 10.9% among women (P = 0.0001).ConclusionsOur study is the largest to date to report an effect of sex on risk of VTE recurrence, with men having about a 13% higher risk of recurrence than women. This provides further evidence that sex is a significant predictor of VTE recurrence.     

Dissociation of peripheral protein-membrane complexes by high pressure.

The ability of high pressure to dissociate several peripheral protein-membrane complexes was investigated. Three vitamin K-dependent proteins (factor X, protein Z, and prothrombin) dissociated from small unilamellar vesicles (SUVs, 30 nm diameter) composed of 25% phosphatidylserine (PS) and 75% phosphatidylcholine (PC) at comparable pressures (midpoints of 0.3-0.6 kbar). The pressure-induced dissociation curves for the factor X-SUV interaction followed the expected behavior for an interaction with an apparent dissociation equilibrium constant at atmospheric pressure, KD(atm), of 9 x 10(-7) M and a change in volume of association, delta Va, of 88 mL/mol. Factor X also dissociated from large unilamellar vesicles (LUVs, 100 nm diameter, 25% PS:75% PC) with a midpoint of 0.5 kbar. A second group of calcium-dependent membrane-binding proteins included protein kinase C (PKC), a 64-kDa protein, and a 32-kDa protein. The 32-kDa protein dissociated from SUVs (midpoint of 0.8 kbar), whereas PKC and the 64-kDa protein did not dissociate to a significant degree. The differences in dissociability of these proteins appeared to be a result of the differences in their KD(atm)'s (decreased dissociability with decreased KD(atm)). This pattern was further demonstrated by the relatively high midpoint of dissociation (1.1-1.4 kbar) of serum amyloid P component (SAP; KD(atm) ca. 10(-11)) and the limited dissociation of factor Va light chain (KD(atm) ca. 10(-11)). Changing the vesicle composition to phosphatidylethanolamine in place of PC gave higher affinity and decreased dissociation of the 32-kDa protein and SAP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Increased concentrations of phosphatidylinositol (PI) and decreased esterification of arachidonic acid into phospholipids in platelets from patients with schizoaffective disorders or atypic phasic psychoses.

The concentration of various phospholipids (PLs) and sphingomyelin in platelets and the amount of [14C] arachidonic acid ([14C]-AA) esterified in phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylethanolamine (PE), and phosphatidylcholine (PC) were measured. The platelet-rich plasmas from unmedicated patients with psychiatric disorders and healthy controls were incubated for 30 min with 1 microM [14C]-AA. Platelets from patients with a schizoaffective disorder according to RDC criteria, a schizophreniform disorder (DSM III criteria) or an atypical phasic psychosis according to FC criteria contained twice as much PI and had significantly increased concentrations of PC as compared to controls (p less than 0.05, t-test). A highly significant (40-70%) reduced rate of esterification of [14C]-AA into PI/PS, PC and PE was found in platelets from patients with schizophreniform, schizoaffective and major depressive disorders but not in platelets from patients with chronic schizophrenia. The largest reduced esterification of [14C]-AA (about 70%) was found in PI/PS of platelets from patients with schizoaffective disorders (1.9 +/- 0.7 vs 6.3 +/- 1.7 mumol [14C]-AA/mol PI/PS; p less than 10(-4), t-test). The results indicate that changes in the metabolism of arachidonic acid and phosphatidylinositol and, to a lesser degree, of phosphatidylcholine in platelets are characteristic of patients with a likely favorable outcome of a psychotic episode.     

Salinity-Dependent Impacts of ProQ,Prc, and Spr Deficiencies on Escherichia coli Cell Structure

ProQ is a cytoplasmic protein with RNA chaperone activities that reside in FinO- and Hfq-like domains. Lesions at proQ decrease the level of the osmoregulatory glycine betaine transporter ProP. Lesions at proQ eliminated ProQ and Prc, the periplasmic protease encoded by the downstream gene prc. They dramatically slowed the growth of Escherichia coli populations and altered the morphologies of E. coli cells in high-salinity medium. ProQ and Prc deficiencies were associated with different phenotypes. ProQ-deficient bacteria were elongated unless glycine betaine was provided. High-salinity cultures of Prc-deficient bacteria included spherical cells with an enlarged periplasm and an eccentric nucleoid. The nucleoid-containing compartment was bounded by the cytoplasmic membrane and peptidoglycan. This phenotype was not evident in bacteria cultivated at low or moderate salinity, nor was it associated with murein lipoprotein (Lpp) deficiency, and it differed from those elicited by the MreB inhibitor A-22 or the FtsI inhibitor aztreonam at low or high salinity. It was suppressed by deletion of spr, which encodes one of three murein hydrolases that are redundantly essential for enlargement of the murein sacculus. Prc deficiency may alter bacterial morphology by impairing control of Spr activity at high salinity. ProQ and Prc deficiencies lowered the ProP activity of bacteria cultivated at moderate salinity by approximately 70% and 30%, respectively, but did not affect other osmoregulatory functions. The effects of ProQ and Prc deficiencies on ProP activity are indirect, reflecting their roles in the maintenance of cell structure.     

Presenilin-1 and intracellular calcium stores regulate neuronal glutamate uptake

Glutamate uptake by high affinity glutamate transporters is essential for preventing excitotoxicity and maintaining normal synaptic function. We have discovered a novel role for presenilin-1 (PS1) as a regulator of glutamate transport. PS1-deficient neurons showed a decrease in glutamate uptake of approximately 50% compared to wild-type neurons. Gamma-secretase inhibitor treatment mimicked the effects of PS1 deficiency on glutamate uptake. PS1 loss-of-function, accomplished by PS1 deficiency or gamma-secretase inhibitor treatment, caused a corresponding decrease in cell surface expression of the neuronal glutamate transporter, EAAC1. PS1 deficiency is known to reduce intracellular calcium stores. To explore the possibility that PS1 influences glutamate uptake via regulation of intracellular calcium stores, we examined the effects of treating neurons with caffeine, thapsigargin, and SKF-96365. These compounds depleted intracellular calcium stores by distinct means. Nonetheless, each treatment mimicked PS1 loss-of-function by impairing glutamate uptake and reducing EAAC1 expression at the cell surface. Blockade of voltage-gated calcium channels, activation and inhibition of protein kinase C (PKC), and protein kinase A (PKA) all had no effect on glutamate uptake in neurons. Taken together, these findings indicate that PS1 and intracellular calcium stores may play a significant role in regulating glutamate uptake and therefore may be important in limiting glutamate toxicity in the brain.     

Characteristic and Prognostic Implication of Venous Thromboembolism in Ovarian Clear Cell Carcinoma: A 12-Year Retrospective Study

Purpose

To profile the characteristic and prognostic implications of venous thromboembolism (VTE) in Chinese ovarian clear cell carcinoma (CCC) patients.

Methods

We identified all of the cases between 2000 and 2012 by searching our institutional Ovarian CCC Database. A comprehensive review of the medical documentation was performed to collect relevant data. Kaplan-Meier models and Cox regression were employed for survival analysis.

Results

Of the 227 patients, 33 (14.5%) experienced VTE events. There was no significant difference between VTE and non-VTE group patients regarding age, serum cancer antigen 125 or tumor size. The optimal cytoreduction rate was higher in patients without VTE (70.1%) than in those with VTE (51.5%). VTE events were more likely to occur at presentation (36.4%) and recurrence (33.3%), followed by an adjuvant chemotherapy period (18.2%). VTE was more common in patients with advanced-stage disease than those with early-stage disease (P=0.003), whereas pulmonary embolism (PE) was 10-fold as common in advanced-stage disease as in early-stage disease (8.6% vs. 0.8%, P = 0.012). Patients with advanced disease tended to have thrombi in the proximal veins. Two patients died of PE, as confirmed by autopsy. Patients with VTE had reduced survival compared to those without VTE (median overall survival 54 vs. 140 months, P<0.001; median progression-free survival 17 vs. 43 months, P<0.001).

Conclusions

Overall, 14.5% of the patients with ovarian CCC experienced VTE, mainly before their cancer diagnosis or at a time of recurrence. VTE adversely impacted patient survival.     

Mechanism of protein-induced membrane fusion: fusion of phospholipid vesicles by clathrin associated with its membrane binding and conformational change

The clathrin-induced fusion of liposome membranes, the membrane binding of clathrin, and the conformational states of clathrin were investigated over a wide pH range using large unilamellar and multilamellar vesicles composed of phosphatidylserine (PS), phosphatidylcholine (PC), PS/PC (2:1), PS/PC (1:1), or PS/PC (1:2). The pH profiles of clathrin-induced fusion of all types of liposomes containing PS showed biphasic patterns. Their pH thresholds were found in the pH range of 5-6 and shifted to lower pH values with decrease in the PS content. Similar shifts were observed in the pH range of 5-6 and shifted to lower pH values with decrease in the PS content. Similar shifts were observed in the pH profiles of clathrin binding to these vesicles, but the pH profiles of binding were different from the biphasic fusion patterns. With PC vesicles, only small degrees of fusion and clathrin binding were observed at pH 2-4. The pH dependences of the conformation and hydrophobicity of clathrin were determined by measuring the extent of the blue shift of the fluorescence maximum of 1-anilinonaphthalene-8-sulfonate in the presence of the protein, the fluorescence intensity of N-(1-anilinonaphthyl-4)maleimide bound to the clathrin molecule, the resonance energy transfer from its tryptophan to anilinonaphthyl residues, the partitioning of the protein in Triton X-114 solution, and the hydrophobicity index of clathrin using cis-parinaric acid. These measurements indicated that conformational change and exposure of hydrophobic regions occur below pH 6 and suggested that clathrin may adopt different conformational states in the pH region where it induced membrane fusion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neutral glycosphingolipid-dependent inactivation of coagulation factor Va by activated protein C and protein S.

To test whether neutral glycosphingolipids can serve as anticoagulant cofactors, the effects of incorporation of neutral glycosphingolipids into phospholipid vesicles on anticoagulant and procoagulant reactions were studied. Glucosylceramide (GlcCer), lactosylceramide (LacCer), and globotriaosylceramide (Gb(3)Cer) in vesicles containing phosphatidylserine (PS) and phosphatidylcholine (PC) dose dependently enhanced factor Va inactivation by the anticoagulant factors, activated protein C (APC) and protein S. Addition of GlcCer to PC/PS vesicles enhanced protein S-dependent APC cleavage in factor Va at Arg-506 by 13-fold, whereas PC/PS vesicles alone minimally affected protein S enhancement of this reaction. Incorporation into PC/PS vesicles of GlcCer, LacCer, or Gb(3)Cer, but not galactosylceramide or globotetraosylceramide, dose dependently prolonged factor Xa-1-stage clotting times of normal plasma in the presence of added APC without affecting baseline clotting times in the absence of APC, showing that certain neutral glycosphingolipids enhance anticoagulant but not procoagulant reactions in plasma. Thus, certain neutral glycosphingolipids (e.g. GlcCer, LacCer, and Gb(3)Cer) can enhance anticoagulant activity of APC/protein S by mechanisms that are distinctly different from those of phospholipids alone. We speculate that under some circumstances certain neutral glycosphingolipids either in lipoprotein particles or in cell membranes may help form antithrombotic microdomains that might enhance down-regulation of thrombin by APC in vivo.     

Theoretical and experimental analysis of analyte transport in a fiber-optic, protein C immuno-biosensor

Protein C (PC) is an important anticoagulant in human blood plasma, and early diagnosis of PC deficiency is critical for preventing dangerous thromboembolic complications. A fiber-optic PC immuno-biosensor has been under development in our research group for real-time PC-deficiency diagnosis. The sensor has demonstrated a good sensitivity and specificity for quantifying PC in buffered solutions. However, for plasma samples, with a limited sample reaction time, the sensor produced only 30% of the signal intensity of PC in buffer. The high plasma viscosity (1.9 cP) was speculated as the major reason for signal intensity reduction. In this investigation, the sensing performance of the fiber-optic PC biosensor is systematically characterized in terms of physical and chemical properties of the sample media. Theoretical and experimental analyses indicate that the reduced diffusion rate of PC molecules in viscous samples caused the sensing system to be more mass-transfer-limited. Convective flow of sample/reagent solutions during immunoreactions can increase the rate of the analyte mass transport from the bulk solution to the sensor surface, with reaction kinetics changing from mass-transfer-limited to reaction-limited as flow velocity increases. It was shown that PC sensor performance was significantly improved for plasma samples with convection. The effect of the flow velocity and incubation times for samples and reagents on the sensor performance was also systematically analyzed to optimize the assay protocol for PC sensing. Currently, a 6-cm-long immuno-biosensor is capable of quantifying PC in plasma (1 mL) in the heterozygous PC deficiency range (0.5 to 2.5 microg/mL) within 5 minutes, at an average signal-to-noise ratio of 50.     

The regulation of presenilin-1 by nerve growth factor

Presenilin-1 (PS1) protein concentration is linked to neuronal development and to the pathogenesis of Alzheimer's disease, yet little is known about the biological factors and mechanisms that control cellular levels of PS1 protein. As PS1 levels are highest in the developing brain, we tested whether neurotrophin-induced differentiation influences PS1 expression using neuronotypic pheochromocytoma (PC12) cells. Treatment of PC12 cells with nerve growth factor (NGF) caused approximately 60-75% increases in the steady-state levels of endogenous PS1 N- and C-terminal fragments. PS1 protein accumulation was dose-responsive to NGF and required the presence of the TrkA NGF receptor tyrosine kinase. NGF also induced PS1 fragment accumulation in cultured explants of rat dorsal root ganglia. Quantitative northern blot analysis using PC12 cultures indicated that NGF did not increase steady-state PS1 mRNA levels. However, pulse-chase experiments indicated that NGF slowed the degradation rate of endogenous PS1 fragments, increasing the half-life from t(1/2) @22.5 to @25.0 h. This increase in half-life was insufficient to account for the approximately 60-75% increase in PS1 fragment levels measured in NGF-treated cells. Thus, NGF may regulate PS1 protein concentration in NGF-responsive cells by a complex mechanism that increases PS1 fragment production independent of holoprotein synthesis.     

Role of the phospholipid environment in modulating the activity of the rat brain synaptic plasma membrane Ca2(+)-ATPase

The role of the phospholipid environment in modulating the activity of the rat brain synaptic plasma membrane (SPM) Ca2(+)-ATPase was investigated by its reconstitution into different phospholipids. Retention of activity of the solubilized Ca2(+)-ATPase depended on addition of exogenous phospholipids. As the cholate concentration used for solubilization of native SPM increased, a larger excess of exogeneous phospholipids, relative to membrane protein, had to be added to maintain optimal activity. Highest ATP-dependent Ca2+ transport activity was obtained when reconstitution was carried out in calf brain phospholipids (BPLs) followed by soybean phospholipids (SPLs) and the lowest in egg PC; reconstitution at a 40:1 weight ratio of exogenous phospholipids to native SPM protein resulted in ATP-dependent Ca2+ transport of 40.0 +/- 4.16, 23.4 +/- 8.48, and 11.54 +/- 2.31 nmol of Ca2+ (mg of protein)-1 (5 min)-1, respectively. Partial substitution of egg PC with BPLs led to an increase in the activity of the reconstituted Ca2+ pump. The highest ATP-dependent Ca2+ uptake was obtained when ratios of 15:25 or 10:30 egg PC to BPLs were used. Testing the individual phospholipids participating in the BPL mixture showed that addition of PS to egg PC led to a consistent increase in Ca2+ pump activity. Substitution of 50% of the PC with PS resulted in a 3.8-fold higher ATP-dependent Ca2+ uptake than that obtained in egg PC alone. No other phospholipid tested--PE, SM, or PI--had a similar effect. Increasing the proportion of PS within the BPL mixture above its original content led to a gradual decrease in the reconstituted SPM Ca2+ pump activity. Enrichment of asolectin with PS led first to increased Ca2+ pump activity; then, as the proportion of PS increased, Ca2+ transport of the reconstituted pump decreased. An increased proportion of PE, SM, or PI within the BPLs or asolectin, above their original contents, resulted in decreased Ca2+ transport. These results indicate that optimal SPM Ca2+ pump activity requires the combined presence of a critical amount of PC and PS within the reconstituted membrane.     

Temperature dependence of Na+/Ca2+ exchange activity in beef-heart sarcolemmal vesicles and proteoliposomes

Temperature dependence of Na+/Ca2+ exchange activity was studied in beef cardiac sarcolemmal vesicles in the absence and presence of the inhibitor amiloride and in proteoliposomes reconstituted with different lipid mixtures. Arrhenius plots for Na+/Ca2+ exchange activity in both control and amiloride-treated vesicles revealed an apparent energy of activation of 9665 +/- 585 (SE, n = 4) cal/mol, corresponding to a temperature coefficient (Q10) value of 1.70 +/- 0.05 (SE, n = 4) over the range 25-37 degrees C. When Na+/Ca2+ exchange was reconstituted into phosphatidylcholine (PC):phosphatidylserine (PS) (52:48, mol/mol), PC:PS:cholesterol (25:39:36, mol/mol), and PC:PS:distearoylphosphatidylcholine (DSPC) (31:48:21, mol/mol) proteoliposomes, the highest activity was found in PC:PS:cholesterol proteoliposomes. Arrhenius plots of Na+/Ca2+ exchange activity exhibited breakpoints at 23 degrees C (PC:PS), 33 degrees C (PC:PS:cholesterol), and 23 degrees C (PC:PS:DSPC). The increase in the thermotropic transition temperature with cholesterol could result from the condensing effect of this sterol, whereas the breaks observed with PC:PS and PC:PS:DSPC could be caused by a non-lipid-mediated membrane protein conformational change. These results indicate that the lipid microenvironment around the Na+/Ca2+ exchanger and the nature of the specific lipid-protein interactions influence the activity of this antiporter. Further evidence supporting the hypothesis that cholesterol behaves as a specific positive effector for the exchanger is also given.     

Catalytic properties of the yeast phospholipid transfer protein

The properties of the phosphatidylcholine (PC) transfer reaction catalyzed by the yeast phospholipid transfer protein (TP-I) were examined in vitro. Donor and acceptor membranes consisted of unilamellar (ULV) and multilamellar (MLV) vesicles, respectively. The phospholipid composition of the membranes participating in the transfer reaction, and in particular that of the MLV acceptors, have a tremendous effect upon the rate of PC-catalyzed transfer. Phosphatidylethanolamine (PE) is an essential component of the acceptor membrane, but it alone is not sufficient to sustain appreciable transfer rates. If combined in an equimolar ratio with PC, there is only a modest increase in transfer rates. On the other hand, when combined with alternate substrates such as phosphatidylinositol (PI) or phosphatidylserine (PS), very high rates of PC transfer occur. The measurement of transfer rates is not affected by the molecular species of PC used as the radioactive tracer. Evidence is also presented to indicate that the two forms of the transfer protein (TP-I and TP-II) are not identical in terms of their interactions with a membrane surface: differences occur in the levels of transfer of PC, PE, PI, and PS at equilibrium. Finally, by kinetic analysis, the mechanism of the protein-catalyzed transfer of PC is shown to conform to a ping-pong bibi model with excess substrate inhibition, analogous to ordinary two-substrate enzyme-catalyzed reactions. Both the rates of desorption and adsorption of the protein from the surface of the ULV are much greater than those describing the similar interactions of the protein with MLV.(ABSTRACT TRUNCATED AT 250 WORDS)     

Anticoagulation Management Practices and Outcomes in Elderly Patients with Acute Venous Thromboembolism: A Clinical Research Study

Whether anticoagulation management practices are associated with improved outcomes in elderly patients with acute venous thromboembolism (VTE) is uncertain. Thus, we aimed to examine whether practices recommended by the American College of Chest Physicians guidelines are associated with outcomes in elderly patients with VTE. We studied 991 patients aged ≥65 years with acute VTE in a Swiss prospective multicenter cohort study and assessed the adherence to four management practices: parenteral anticoagulation ≥5 days, INR ≥2.0 for ≥24 hours before stopping parenteral anticoagulation, early start with vitamin K antagonists (VKA) ≤24 hours of VTE diagnosis, and the use of low-molecular-weight heparin (LMWH) or fondaparinux. The outcomes were all-cause mortality, VTE recurrence, and major bleeding at 6 months, and the length of hospital stay (LOS). We used Cox regression and lognormal survival models, adjusting for patient characteristics. Overall, 9% of patients died, 3% had VTE recurrence, and 7% major bleeding. Early start with VKA was associated with a lower risk of major bleeding (adjusted hazard ratio 0.37, 95% CI 0.20–0.71). Early start with VKA (adjusted time ratio [TR] 0.77, 95% CI 0.69–0.86) and use of LMWH/fondaparinux (adjusted TR 0.87, 95% CI 0.78–0.97) were associated with a shorter LOS. An INR ≥2.0 for ≥24 hours before stopping parenteral anticoagulants was associated with a longer LOS (adjusted TR 1.2, 95% CI 1.08–1.33). In elderly patients with VTE, the adherence to recommended anticoagulation management practices showed mixed results. In conclusion, only early start with VKA and use of parenteral LMWH/fondaparinux were associated with better outcomes.