The platelet receptor for type III collagen (TIIICBP) is present in platelet membrane lipid microdomains (rafts)

Platelet interactions with collagen are orchestrated by the presence or the migration of platelet receptor(s) for collagen into lipid rafts, which are specialized lipid microdomains from the platelet plasma membrane enriched in signalling proteins. Electron microscopy shows that in resting platelets, TIIICBP, a receptor specific for type III collagen, is present on the platelet membrane and associated with the open canalicular system, and redistributes to the platelet membrane upon platelet activation. After platelet lysis by 1% Triton X-100 and the separation of lipid rafts on a discontinuous sucrose gradient, TIIICBP is recovered in lipid raft-containing fractions and Triton X-100 insoluble fractions enriched in cytoskeleton proteins. Platelet aggregation, induced by type III collagen, was inhibited after disruption of the lipid rafts by cholesterol depletion, whereas platelet adhesion under static conditions did not require lipid raft integrity. These results indicate that TIIICBP, a platelet receptor involved in platelet interaction with type III collagen, is localized within platelet lipid rafts where it could interact with other platelet receptors for collagen (GP VI and α2β1 integrin) for efficient platelet activation. Pascal Maurice and Ludovic Waeckel have contributed equally to this work.     

Functional role of the calmodulin- and inositol 1,4,5-trisphosphate receptor-binding (CIRB) site of TRPC6 in human platelet activation

Background

All identified mammalian TRPC channels show a C-terminal calmodulin (CaM)- and inositol 1,4,5-trisphosphate receptors (IP₃Rs)-binding (CIRB) site involved in the regulation of TRPC channel function.

Objectives

To assess the basis of CaM/IP₃Rs binding to the CIRB site of TRPC6 and its role in platelet physiology.

Methods

Protein association was detected by co-immunoprecipitation and Western blotting, Ca²⁺ mobilization was measured by fluorimetric techniques and platelet function was analyzed by aggregometry.

Results

Co-immunoprecipitation of TRPC6 with CaM or the IP₃Rs at different cytosolic free Ca²⁺ concentrations ([Ca²⁺]_c) indicates that the association between these proteins is finely regulated by cytosolic Ca²⁺ via association of CaM and displacement of the IP₃Rs at high [Ca²⁺]_c. Thrombin-stimulated association of TRPC6 with CaM or the IP₃Rs was sensitive to 2-APB and partially inhibited by dimethyl BAPTA loading, thus suggesting that the association between these proteins occurs through both Ca²⁺-dependent and -independent mechanisms. Incorporation of an anti-TRPC6 C-terminal antibody, whose epitope overlaps the CIRB region, impaired the dynamics of the association of TRPC6 with CaM and the IP₃Rs, which lead to both inhibition and enhancement of thrombin- and thapsigargin-evoked Ca²⁺ entry in the presence of low or high, respectively, extracellular Ca²⁺ concentrations, as well as altered thrombin-evoked platelet aggregation.

Conclusions

Our results indicate that the CIRB site of TRPC6 plays an important functional role in platelets both modulating Ca²⁺ entry and aggregation through its interaction with CaM and IP₃Rs.     

Involvement of SNARE proteins in thrombin-induced platelet aggregation: evidence for the relevance of Ca2+ entry

Thrombin induces platelet activation through a variety of intracellular mechanisms, including Ca(2+) mobilization. The protein of the exocytotic machinery SNAP-25, but not VAMPs, is required for store-operated Ca(2+) entry, the main mechanism for Ca(2+) influx in platelets. Hence, we have investigated the role of the SNAP-25 and VAMPs in thrombin-induced platelet aggregation. Platelet stimulation with thrombin or selective activation of thrombin receptors PAR-1, PAR-4 or GPIb-IX-V results in platelet aggregation that, except for GPIb-IX-V receptor, requires Ca(2+) entry for full activation. Depletion of the intracellular Ca(2+) stores using pharmacological tools was unable to induce aggregation except when cytosolic Ca(2+) concentration reached a critical level (around 1.5 microM). Electrotransjection of cells with anti-SNAP-25 antibody reduced thrombin-evoked platelet aggregation, while electrotransjection of anti-VAMP-1, -2 and -3 antibody had no effect. These findings support a role for SNAP-25 but not VAMP-1, -2 and -3 in platelet aggregation, which is likely mediated by the regulation of Ca(2+) mobilization in human platelets.     

Physical properties of membrane fractions isolated from human platelets: implications for chilling induced platelet activation

In previous studies, it has been suggested that chilling induced activation of human platelets is related to a lipid phase transition seen in membrane lipids. Those studies showed a single, surprisingly cooperative transition in human platelets, as determined by Fourier transform infrared (FTIR) spectroscopy, findings that are confirmed here with calorimetric measurements. Such transitions have now been studied in membrane fractions obtained from the platelets and it is reported that all fractions and purified phospholipids show similar transitions. In order to obtain these data it was necessary to develop means for separating these fractions. Therefore, a novel method for isolation and separation of dense tubular system (DTS) and plasma membranes in human platelets is described here. Lipid analysis showed that phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were the dominant phospholipids in both fractions, whereas cholesterol and sphingomyelin (SM) were predominantly located in the plasma membranes. Thermotropic phase transitions in the two membrane fractions, determined by differential scanning calorimetry (DSC) and FTIR spectroscopy were found to occur at about 15 degrees C, similar to the Tm of intact human platelets. These data are discussed in relation to the role of the DTS and plasma membranes in the cold-induced activation of human platelets.     

Mating aggregation of a stink bug,Megacopta punctissimum (Montandon) (Heteroptera: Plataspidae)

Summary Adults of a stink bug,Megacopta punctissimum, form aggregations on stems of the bush-clover,Lespedeza crytobotria, in spring. They do not oviposit there, but feed and mate within aggregations. Oviposition is made on other leguminous plants such as the kudzu-vine,Pueraria lobata. Mean size of aggregations (groups of two or more individuals which sit on a stem at distance less than 3 cm from their nearest neighbors) consisted of 4.4 individuals. The sex ratios within aggregations were similar to overall sex ratio of the population including solitary individuals (0.58). More than 50% of females found in aggregations were copulating. Percentage of femalesin copula in larger aggregations was higher than that in smaller aggregations, and this difference was considered to be caused by the higher chance of sexual enconters in larger aggregations. It was suggested that the aggregation of this species on the bush-clover is not a ‘harem’ (a male monopolizes a group of females reported in some other stink bugs) but is mating aggregation to raise the chance of mating. This work was partially supported by Grant-in-Aid (No. 56480039) from the Ministry of Education, Science and Culture.     

Discovery of a novel potent GABA(B) receptor agonist

Structure-activity studies have led to a discovery of 3-(4-pyridyl)methyl ether derivative 9d that has 25- to 50-fold greater functional potency than R-baclofen at human and rodent GABA(B) receptors in vitro. Mouse hypothermia studies confirm that this compound crosses the blood-brain barrier and is approximately 50-fold more potent after systemic administration.     

Phosphorylation of a tropomyosin-like (30 KD) protein during platelet activation

In this study, we have used the tumor promoter 12-o-tetradecanoylphorbol-13-acetate (TPA), as well as its biologically inactive analogue 4 alpha-phorbol 12,13-didecanoate (4 alpha-PDD), to investigate platelet protein phosphorylation and its possible correlation with platelet activation. Our data show that TPA, but not 4 alpha-PDD, induces a preferential phosphorylation of a 30,000 dalton (30 KD) protein. This phosphoprotein is found to be physically associated with an actomyosin-containing platelet cytoskeleton complex. Further analysis using both standard two-dimensional gel electrophoresis and one-dimensional urea-SDS gel electrophoresis reveals that this 30 KD protein has several tropomyosin-like properties. Most importantly, the degree of TPA-induced phosphorylation of the 30 KD protein is directly proportional to the extent of platelet granule release and the shape change of the platelet, as well as to the degree of aggregation. We speculate that this phosphorylated tropomyosinlike protein may play a pivotal role in the regulation of actomyosin-mediated platelet contractility, which has been previously implicated in a variety of platelet functions.     

Neutrophil elastase acts as a biased agonist for proteinase-activated receptor-2 (PAR2)

Human neutrophil proteinases (elastase, proteinase-3, and cathepsin-G) are released at sites of acute inflammation. We hypothesized that these inflammation-associated proteinases can affect cell signaling by targeting proteinase-activated receptor-2 (PAR(2)). The PAR family of G protein-coupled receptors is triggered by a unique mechanism involving the proteolytic unmasking of an N-terminal self-activating tethered ligand (TL). Proteinases can either activate PAR signaling by unmasking the TL sequence or disarm the receptor for subsequent enzyme activation by cleaving downstream from the TL sequence. We found that none of neutrophil elastase, cathepsin-G, and proteinase-3 can activate G(q)-coupled PAR(2) calcium signaling; but all of these proteinases can disarm PAR(2), releasing the N-terminal TL sequence, thereby preventing G(q)-coupled PAR(2) signaling by trypsin. Interestingly, elastase (but neither cathepsin-G nor proteinase-3) causes a TL-independent PAR(2)-mediated activation of MAPK that, unlike the canonical trypsin activation, does not involve either receptor internalization or recruitment of β-arrestin. Cleavage of synthetic peptides derived from the extracellular N terminus of PAR(2), downstream of the TL sequence, demonstrated distinct proteolytic sites for all three neutrophil-derived enzymes. We conclude that in inflammation, neutrophil proteinases can modulate PAR(2) signaling by preventing/disarming the G(q)/calcium signal pathway and, via elastase, can selectively activate the p44/42 MAPK pathway. Our data illustrate a new mode of PAR regulation that involves biased PAR(2) signaling by neutrophil elastase and a disarming/silencing effect of cathepsin-G and proteinase-3.     

High Definition Oscillometry: a novel technique for non-invasive blood pressure monitoring in the cynomolgus monkey (Macaca fascicularis)

Background  Current approaches for accurate blood pressure determination rely predominantly on invasive techniques. High Definition Oscillometry (HDO) was evaluated as a potential non-invasive approach for accurate blood pressure recordings in cynomolgus monkeys.
Methods  In conscious animals, systolic, diastolic, mean arterial blood pressure (MAP) and pulse/minute were determined 15 times within approx. 9 minutes per individual. This session was performed during 3 consecutive days. Anaesthesia induced hypotension was controlled simultaneously with HDO and telemetry as reference.
Results  Repeated measurements were highly reproducible. After procedural habituation, mean MAP was 96.2 ± 13.7 mmHg in males and 86.9 ± 4.3 mmHg in females. Mean intraindividual coefficients of variation ranged between 10.8% and 2.4% depending on the session and parameter. Values determined by HDO corresponded to those reported for invasive techniques.
Conclusion  Our results demonstrate, using telemetry as reference, the accuracy of HDO-based non-invasive blood pressure measurements in macaques to detect drug-related cardiovascular changes.     

Pulsed electric field treatment as a potential method for microbial inactivation in scaffold materials for tissue engineering: the inactivation of bacteria in collagen gel

Aims: To investigate the effectiveness of pulsed electric field (PEF) treatment as a new method for inactivation of micro-organisms in complex biomatrices and to assess this by quantifying the inactivation of Escherichia coli seeded in collagen gels. Methods and Results: PEF was applied to E. coli seeded collagen gels in static (nonflowing) chambers. The influence of electric field strength, pulse number and seeded cell densities were investigated. The highest level of inactivation was obtained at the maximum field strength of 45 kV cm⁻¹. For low levels of E. coli contamination (10³ CFU ml⁻¹), PEF treatment resulted in no viable E. coli being recovered from the gels. However, PEF treatment of gels containing higher cell densities (≥10⁴ CFU ml⁻¹) did not achieve complete inactivation of E. coli. Conclusions: PEF treatment successfully inactivated E. coli seeded in collagen gels by 3 log₁₀ CFU ml⁻¹. Complete inactivation was hindered at high cell densities by the tailing effect observed. Significance and Impact of the Study: PEF shows potential as a novel, nondestructive method for decontamination of collagen-based matrices. Further investigation is required to ensure its compatibility with other proteins and therapeutic drugs for tissue engineering and drug delivery applications.     

Calpastatin domain L is a partial agonist of the calmodulin-binding site for channel activation in Cav1.2 Ca2+ channels

Cav1.2 Ca(2+) channel activity diminishes in inside-out patches (run-down). Previously, we have found that with ATP, calpastatin domain L (CSL) and calmodulin (CaM) recover channel activity from the run-down in guinea pig cardiac myocytes. Because the potency of the CSL repriming effect was smaller than that of CaM, we hypothesized that CSL might act as a partial agonist of CaM in the channel-repriming effect. To examine this hypothesis, we investigated the effect of the competitions between CSL and CaM on channel activity and on binding in the channel. We found that CSL suppressed the channel-activating effect of CaM in a reversible and concentration-dependent manner. The channel-inactivating effect of CaM seen at high concentrations of CaM, however, did not seem to be affected by CSL. In the GST pull-down assay, CSL suppressed binding of CaM to GST fusion peptides derived from C-terminal regions in a competitive manner. The inhibition of CaM binding by CSL was observed with the IQ peptide but not the PreIQ peptide, which is the CaM-binding domain in the C terminus. The results are consistent with the hypothesis that CSL competes with CaM as a partial agonist for the site in the IQ domain in the C-terminal region of the Cav1.2 channel, which may be involved in activation of the channel.     

Regulation of intracellular levels of NAD: a novel role for CD38

Nicotinamide adenine dinucleotide (NAD) plays key roles in many cellular functions. In addition to its well-known role in energy metabolism, NAD also plays a role in signal transduction, ageing, and cellular injury. NAD is also involved in many signal transduction pathways. Therefore, it is imperative to understand the mechanisms that control intracellular NAD levels. However, to date, the mechanisms that regulate intracellular levels of NAD have not been completely elucidated. CD38 is a multifunctional enzyme ubiquitously distributed in mammalian tissues. CD38 has been implicated as the enzyme responsible for the synthesis of the second messengers. However, its major enzymatic activity is the hydrolysis of NAD, in fact, CD38 will generate one molecule of cADPR for every 100 molecules of NAD hydrolyzed. To date, the role of CD38 as a modulator of levels of NAD has not been explored. We postulated that CD38 is the major NADase in mammalian cells and that it regulates intracellular NAD levels. In the current studies we examined the NADase activities and NAD levels in a variety of tissues from both wild-type and CD38 deficient mice. In accordance with our hypothesis, we found that tissue levels of NAD in CD38 deficient mice are 10- to 20-fold higher than in wild-type animals. In addition, NADase activity in the plasma membrane, mitochondria, sarcoplasmic reticulum, and nuclei is essentially absent in most tissues from CD38 deficient mice. These data support the novel concept that CD38 is a major regulator of cellular NAD levels. These findings have implications for understanding the mechanisms that regulate intracellular NAD levels and its role in energy homeostasis, signal transduction, and ageing.     

Furanocoumarins Are a Novel Class of Modulators for the Transient Receptor Potential Vanilloid Type 1 (TRPV1) Channel

Furanocoumarin imperatorin is the major active component of Angelica dahurica root extracts, widely used in traditional medicine to treat headache, toothache, and orbital eye pain. In this study, we investigated the mechanisms that may underlie the pain-relieving effects of the compound. We found that imperatorin significantly inhibited formalin- and capsaicin-induced nocifensive responses but did not alter baseline thermal withdrawal thresholds in the rat. We established that imperatorin is a weak agonist of TRPV1, a channel implicated in detecting several noxious stimuli, exhibiting a 50% effective concentration (EC₅₀) of 12.6 ± 3.2 μm. A specific TRPV1 antagonist, JNJ-17203212 (0.5 μm), potently inhibited imperatorin-induced TRPV1 activation. Site-directed mutagenesis studies revealed that imperatorin most likely acted via a site adjacent to or overlapping with the TRPV1 capsaicin-binding site. TRPV1 recovery from desensitization was delayed in the presence of imperatorin. Conversely, imperatorin sensitized TRPV1 to acid activation but did not affect the current amplitude and/or the activation-inactivation properties of Na_v1.7, a channel important for transmission of nociceptive information. Thus, our data indicate that furanocoumarins represent a novel group of TRPV1 modulators that may become important lead compounds in the drug discovery process aimed at developing new treatments for pain management.     

Bioefficacy of a novel calcium-potassium salt of (-)-hydroxycitric acid

Obesity is associated with cardiovascular disease, diabetes and certain forms of cancer. Popular strategies on weight loss often fail to address many key factors such as fat mass, muscle density, bone density, water mass, their inter-relationships and impact on energy production, body composition, and overall health and well-being. (−)-Hydroxycitric acid (HCA), a natural plant extract from the dried fruit rind of Garcinia cambogia, has been reported to promote body fat loss in humans without stimulating the central nervous system. The level of effectiveness of G. cambogia extract is typically attributed solely to HCA. However, other components by their presence or absence may significantly contribute to its therapeutic effectiveness. Typically, HCA used in dietary weight loss supplement is bound to calcium, which results in a poorly soluble (<50%) and less bioavailable form. Conversely, the structural characteristics of a novel Ca²⁺/K⁺ bound (−)-HCA salt (HCA-SX or Super CitriMax) make it completely water soluble as well as bioavailable. An efficacious dosage of HCA-SX (4500 mg/day t.i.d.) provides a good source of Ca²⁺ (495 mg, 49.5% of RDI) and K⁺ (720 mg, 15% of RDI). Ca²⁺ ions are involved in weight management by increasing lipid metabolism, enhancing thermogenesis, and increasing bone density. K⁺, on the other hand, increases energy, reduces hypertension, increases muscle strength and regulates arrhythmias. Both Ca and K act as buffers in pH homeostasis. HCA-SX has been shown to increase serotonin availability, reduce appetite, increase fat oxidation, improve blood lipid levels, reduce body weight, and modulate a number of obesity regulatory genes without affecting the mitochondrial and nuclear proteins required for normal biochemical and physiological functions.     

Interactions in solution of calcium(II) and copper(II) with nucleoside monophosphates: a calorimetric study

The thermodynamic parameters enthalpy and entropy of the interaction between calcium(II) or copper(II) with 5′-UMP, 5′-CMP, 5′-AMP, 5′-GMP or 5′-IMP in aqueous solution were determined calorimetrically (ionic strength adjusted to 0.1 with tetramethylammonium bromide) at 25 °C and pH 7 for Ca(II) or pH 3–5 for Cu(II). The experimental conditions were carefully selected to avoid polynuclear complex formation and nucleotide self-stacking. The calorimetric data confirm the tendency toward macrochelation which was indicated by Sigel after very precise potentiometric studies, and which follows the order Cu(II)>Ca(II) for the metal ions and GMP>IMP>AMP>CMP=UMP for the nucleotides. Macrochelate formation for these metal-nucleoside monophosphate complexes is energetically favorable and entropically unfavorable. Received: 13 August 1999 / Accepted: 1 February 2000     

Cutting edge: T cells trigger CD40-dependent platelet activation and granular RANTES release: a novel pathway for immune response amplification

Platelets, in addition to exerting hemostatic activity, contribute to immunity and inflammation. The recent report that platelets express CD40 led us to hypothesize that CD40 ligand (CD40L)-positive T cells could bind to platelets, cause their activation, and trigger granular RANTES release, creating a T cell recruitment feedback loop. Platelets were cocultured with resting or activated autologous T cells and their activation was assessed by P-selectin expression. RANTES binding to endothelial cells was assessed by confocal microscopy, and its biological activity was demonstrated by a T cell adhesion assay. CD40L-positive T cells induced platelet activation through a contact-mediated, CD40-dependent pathway resulting in RANTES release, which bound to endothelial cells and mediated T cell recruitment. Soluble CD40L induced the same events via p38, but not extracellular signal-regulated kinase, phosphorylation. These results show the existence of a novel platelet-dependent pathway of immune response amplification which brings these nonimmune cells close to the level of pathogenic relevance traditionally attributed to classical immune cells.     

Canonical Transient Receptor Potential (TRPC) 1 Acts as a Negative Regulator for Vanilloid TRPV6-mediated Ca2+ Influx

TRP proteins mostly assemble to homomeric channels but can also heteromerize, preferentially within their subfamilies. The TRPC1 protein is the most versatile member and forms various TRPC channel combinations but also unique channels with the distantly related TRPP2 and TRPV4. We show here a novel cross-family interaction between TRPC1 and TRPV6, a Ca²⁺ selective member of the vanilloid TRP subfamily. TRPV6 exhibited substantial co-localization and in vivo interaction with TRPC1 in HEK293 cells, however, no interaction was observed with TRPC3, TRPC4, or TRPC5. Ca²⁺ and Na⁺ currents of TRPV6-overexpressing HEK293 cells are significantly reduced by co-expression of TRPC1, correlating with a dramatically suppressed plasma membrane targeting of TRPV6. In line with their intracellular retention, remaining currents of TRPC1 and TRPV6 co-expression resemble in current-voltage relationship that of TRPV6. Studying the N-terminal ankyrin like repeat domain, structurally similar in the two proteins, we have found that these cytosolic segments were sufficient to mediate a direct heteromeric interaction. Moreover, the inhibitory role of TRPC1 on TRPV6 influx was also maintained by expression of only its N-terminal ankyrin-like repeat domain. Our experiments provide evidence for a functional interaction of TRPC1 with TRPV6 that negatively regulates Ca²⁺ influx in HEK293 cells.     

Characterization of a novel anti-human lymphocyte activation gene 3 (LAG-3) antibody for cancer immunotherapy

ABSTRACT

Lymphocyte activation gene 3 (LAG-3) is expressed on activated T cells, natural killer cells or B cells, and functions to negatively regulate homeostasis of these cells. Anti-LAG-3 antibodies might be useful for antitumor immunotherapy. In this study, we characterized a novel anti-LAG-3 antibody, LBL-007, which was isolated from a human antibody phage display library. LBL-007 was found to specifically bind to human LAG-3 antigen, but not to human CD4 or mouse LAG-3. LBL-007 bound activated T cells and promoted interleukin-2 secretion. LBL-007 internalization efficacy by endocytosis into different cells was better than that of another anti-LAG-3 antibody, relatlimab analog. Moreover, LBL-007 was able to block LAG-3 binding to MHC class II molecules and liver sinusoidal endothelial cell lectin, and block LAG-3-induced downstream signaling. In mice transplanted with colorectal cancer cells, treatment with either anti-PD-1 antibody or LBL-007 (10 mg/kg per mouse twice a week for three weeks) resulted in a significant delay in tumor growth compared with control IgG treatment, and their combination was even more effective. Serum LBL-007 levels were highly stable in monkeys after a single intravenous injection of LBL-007 at 3, 10, or 30 mg/kg. This study demonstrated that the combination of LBL-007 with an anti-PD-1 antibody is a promising antitumor regimen for immunotherapy of solid tumors in future that deserves further study.     

Evidence for competition between mudsnails (Hydrodiidae): a field experiment

The paper describes an experimental investigation of competition between Hydrobia ulvae and H. ventrosa using enclosed populations at a site at which the species coexist naturally. lntraspecific competition is more intense than interspecific competition and may have a regulatory influence on snail densities. Other experimental studies of competition between mudsnails are reviewed and found to infer strongly the occurrence of competition in natural field populations. However, features of the species' field distributions previously attributed to interspecific competition can be more convincingly explained by other processes.