The peach-potato aphid Myzus persicae and the tobacco aphid Myzus nicotianae have the same esterase-based mechanisms of insecticide resistance

Biochemical and molecular studies have established that in the peach-potato aphid, Myzus persicae, insecticide resistance is conferred by amplification of genes encoding the insecticide-detoxifying esterases E4 or FE4. Here we report that two insecticide-resistant clones of the closely related tobacco aphid Myzus nicotianae have elevated esterases indistinguishable from E4 and FE4 and amplified esterase DNA sequences, and flanking regions, with identical restriction maps to the M. persicae genes. Furthermore, the DNA sequences of c. 630 bp fragments of the E4 and FE4 genes of M. persicae are different from each other but identical to the fragment from corresponding M. nicotianae clones. The existence of apparently identical insecticide resistance genes in the two species can be best explained by the selection of the amplified genes in M. persicae, transfer to hybrids of M. persicae and M. nicotianae by sexual reproduction and subsequent spread through M. nicotianae populations.     

Cloning and characterisation of a prenyltransferase from the aphid Myzus persicae with potential involvement in alarm pheromone biosynthesis

The majority of aphid species release an alarm pheromone with the most common component being the sesquiterpene ( E )-β-farnesene, sometimes accompanied by other sesquiterpenes or monoterpenes. The genes/enzymes involved in the production of these compounds have not been identified in aphids although some components of isoprenoid biosynthesis have been identified in other insect species. Here we report the cloning, expression and characterisation of a prenyltransferase from the aphid Myzus persicae which can act as a farnesyl pyrophosphate synthase or a geranyl pyrophosphate synthase to produce both sesquiterpenes and monoterpenes and hence could be responsible for the biosynthesis of the observed components of the alarm pheromones. In addition, the enzyme can utilise geranyl pyrophosphate to produce farnesyl pyrophosphate showing that the synthesis of the latter involves the sequential condensation of isoprenyl pyrophosphate units.     

Three odorant binding proteins may regulate the behavioural response of Chrysopa pallens to plant volatiles and the aphid alarm pheromone (E)‐β‐farnesene

Artificial Chrysopa pallens release is a well‐known method for suppressing aphids, but it is difficult to establish lacewing populations in the field. Understanding the functions of C. pallens odorant‐binding proteins (CpalOBPs) and behavioural responses of C. pallens to plant volatiles and aphid alarm pheromone (E)‐ß‐farnesene has important implications for population establishment after lacewing release. Based on our previous study, five antennae‐enriched CpalOBPs were selected. Sequence alignment and phylogenetic analysis revealed that these five CpalOBPs were Classic OBPs and separated into different clades. Of them, CpalOBP10 clustered in the same clade with aphid OBP7, which mediates the perception of green leaf volatiles and (E)‐ß‐farnesene. Ligand‐binding assays showed 31 compounds, including plant‐derived compounds, pest‐induced volatiles and (E)‐ß‐farnesene, had high binding affinities for at least one of these five CpalOBPs. Of the 31 compounds, the pest‐induced volatiles (Z)?3‐hexenyl hexanoate and 2‐hexyl‐1‐decanol, used in host location by the black bean aphid, elicited significant attractive behavioural responses from C. pallens. Conversely, (E)‐ß‐farnesene elicited strongly repellent behavioural responses. It is conceivable that C. pallens utilizes plant‐derived compounds, pest‐induced volatiles and (E)‐ß‐farnesene as foraging cues. Our studies provide new insights into the interrelationships amongst C. pallens, its prey and the host plants. Compounds that elicited significant behavioural responses from C. pallens were also identified.     

Comparative analyses of salivary proteins from three aphid species

Saliva is a critical biochemical interface between aphids and their host plants; however, the biochemical nature and physiological functions of aphid saliva proteins are not fully elucidated. In this study we used a multidisciplinary proteomics approach combining liquid chromatography‐electrospray ionization tandem mass spectrometry and two‐dimensional differential in‐gel electrophoresis/matrix‐assisted laser desorption/ionization time‐of‐flight/mass spectrometry to compare the salivary proteins from three aphid species including Acyrthosiphon pisum, Megoura viciae and Myzus persicae. Comparative analyses revealed variability among aphid salivary proteomes. Among the proteins that varied, 22% were related to DNA‐binding, 19% were related to GTP‐binding, and 19% had oxidoreductase activity. In addition, we identified a peroxiredoxin enzyme and an ATP‐binding protein that may be involved in the modulation of plant defences. Knowledge of salivary components and how they vary among aphid species may reveal how aphids target plant processes and how the aphid and host plant interact.     

A sodium channel point mutation is associated with resistance to DDT and pyrethroid insecticides in the peach-potato aphid, Myzus persicae (Sulzer) (Hemiptera: Aphididae)

The voltage-gated sodium channel is the primary target site of DDT and pyrethroid insecticides, and point mutations in the domain II region of the channel protein have been implicated in the knockdown resistant (kdr ) phenotype of several insect species. Here, we report that one of these mutations, a leucine-to-phenylalanine replacement in transmembrane segment IIS6, is also found in certain insecticide-resistant clones of the peach-potato aphid, Myzus persicae. The mutation was present in four clones with amplified E4 esterase genes, but was absent from both susceptible clones and those with amplified FE4 genes. The inferred presence of kdr-type resistance in the four E4 clones was subsequently confirmed by bioassays that showed this to be the primary mechanism of resistance to deltamethrin and DDT, although the esterase-based mechanism also contributes to the overall level of deltamethrin resistance. The kdr mutation on its own conferred 35-fold resistance to deltamethrin and this was enhanced up to 540-fold when it was present in a high (E4) esterase background. The esterase (FE4) mechanism was far less effective without the kdr mutation, conferring just 3–4-fold resistance to deltamethrin. These findings, and the linkage disequilibrium of the kdr mutation within clones overproducing the E4 esterase, have important implications for the evolution of resistance in this insect and for the use of pyrethroid sprays in the management of M. persicae populations in the field.     

Cross-species transferability of microsatellite markers from six aphid (Hemiptera: Aphididae) species and their use for evaluating biotypic diversity in two cereal aphids

The abundance and distribution of microsatellites, or simple sequence repeats (SSRs) were explored in the expressed sequence tag (EST) and genomic sequences of the pea aphid, Acyrthosiphon pisum (Harris), and the green peach aphid, Myzus persicae (Sulzer). A total of 108 newly developed, together with 40 published, SSR markers were investigated for their cross-species transferability among six aphid species. Genetic diversity among six greenbug, Schizaphis graminum (Rondani) and two Russian wheat aphid, Diuraphis noxia (Kurdjumov) biotypes was further examined with 67 transferable SSRs. It was found that the pea aphid genome is abundant in SSRs with a unique frequency and distribution of SSR motifs. Cross-species transferability of EST-derived SSRs is dependent on phylogenetic closeness between SSR donor and target species, but is higher than that of genomic SSRs. Neighbor-joining analysis of SSR data revealed host-adapted genetic divergence as well as regional differentiation of greenbug biotypes. The two Russian wheat aphid biotypes are genetically as diverse as the greenbug ones although it was introduced into the USA only 20 years ago. This is the first report of large-scale development of SSR markers in aphids, which are expected to have wide applications in aphid genetic, ecological and evolutionary studies.     

Influence of membrane-bound tumor necrosis factor (TNF)-α on obesity and glucose metabolism

Summary.  Objective : To investigate the influence of transmembrane tumor necrosis factor (TNF)-α on adipose tissue development and insulin-mediated glucose metabolism. Methods and results : TNF-α and lymphotoxin-α-deficient mice expressing non-cleavable transmembrane TNF-α (Tg-tmTNF-α) and TNF-α/lymphotoxin-α double knockout (control) mice were kept on high-fat diet for 15 weeks. The food intake and feeding efficiency of Tg-tmTNF-α mice were significantly higher compared with control mice. At the end of the study, Tg-tmTNF-α mice had a significantly higher total body weight, as well as subcutaneous and gonadal adipose tissue mass. Histological analysis revealed that the expression of Tg-tmTNF-α resulted in a significantly increased adipocyte area and blood vessel density. Plasma leptin levels correlated positively with adipose tissue mass. The plasma levels of total cholesterol and HDL-cholesterol were significantly increased and LDL-cholesterol levels significantly decreased in Tg-tmTNF-α mice. Fasting blood glucose and plasma insulin levels were not different between the two genotypes and intraperitoneal glucose and insulin tolerance tests did not show significant differences. Conclusions : Transmembrane TNF-α enhances adipose tissue formation without altering insulin-mediated glucose metabolism in mice with nutritionally induced obesity.     

Proteomic analysis of integrin αIIbβ3 outside-in signaling reveals Src-kinase-independent phosphorylation of Dok-1 and Dok-3 leading to SHIP-1 interactions

Summary.  Background and Objectives : Outside-in integrin αIIbβ3 signaling involves a series of tyrosine kinase reactions that culminate in platelet spreading on fibrinogen. The aim of this study was to identify novel tyrosine phosphorylated signaling proteins downstream of αIIbβ3, and explore their role in platelet signaling. Methods and Results : Utilizing proteomics to search for novel platelet proteins that contribute to outside-in signaling by the integrin αIIbβ3, we identified 27 proteins, 17 of which were not previously shown to be part of a tyrosine phosphorylation-based signaling complex downstream of αIIbβ3. The proteins identified include the novel immunoreceptors G6f and G6b-B, and two members of the Dok family of adapters, Dok-1 and Dok-3, which underwent increased tyrosine phosphorylation following platelet spreading on fibrinogen. Dok-3 was also inducibly phosphorylated in response to the GPVI-specific agonist collagen-related peptide (CRP) and the PAR-1 and -4 agonist thrombin, independently of the integrin αIIbβ3. Tyrosine phosphorylation of Dok-1 and Dok-3 was primarily Src kinase-independent downstream of the integrin, whereas it was Src kinase-dependent downstream of GPVI. Moreover, both proteins inducibly interacted with Grb-2 and SHIP-1 in fibrinogen-spread platelets. Conclusions : This study provides new insights into the molecular mechanism regulating αIIbβ3-mediated platelet spreading on fibrinogen. The novel platelet adapter Dok-3 and the structurally related Dok-1 are tyrosine phosphorylated in an Src kinase-independent manner downstream of αIIbβ3 in human platelets, leading to an interaction with Grb2 and SHIP-1.     

The inhibitory effect of resveratrol on leptin secretion from rat adipocytes

Background  Resveratrol was found to alleviate consequences of some metabolic disturbances which may be due to inappropriate dietary habits. It decreases mortality, increases insulin sensitivity and improves motor functions; these effects are accompanied by reduced plasma leptin and insulin. Leptin plays a significant role in the regulation of food intake and energy expenditure – elevated level in blood is one of the reasons of leptin-resistance and obesity. In this study, the direct effect of resveratrol on leptin secretion from isolated adipocytes was investigated.
Material and methods  Isolated rat adipocytes were incubated with resveratrol (62·5, 125 or 250 μM) and its effects on leptin secretion were studied. Cells were incubated with resveratrol in the presence of glucose (5 and 20 mM) and insulin (10 nM); glucose and nicotinic acid (1 mM); glucose and insulin in the presence of an inhibitor of protein kinase A (H-89, 50 μM) or alanine (10 mM) and insulin. The glucose uptake, glycerol release to the incubation medium, lactate and ATP produced by the cells were also measured.
Results  Resveratrol inhibited leptin secretion in all experimental designs in a dose-dependent manner. The effect was not accompanied by changes in glycerol release and glucose uptake. Adipocyte exposure to resveratrol enhanced the lactate formation. It was found that resveratrol dramatically reduced ATP in adipocytes.
Conclusion  The obtained results revealed the direct ability of resveratrol to reduce leptin secretion from isolated rat adipocytes. Resveratrol is therefore a compound affecting the endocrine function of adipocytes.     

Molecular basis of platelet activation by an αIIbβ3-CHAMPS peptide

Summary.  Background:  A novel method, known as computed helical anti-membrane protein (CHAMP), for the design of peptides that bind with high affinity and selectivity to transmembrane helices was recently described and illustrated using peptides that bind αIIb- and αv-integrin subunits, which induce selective activation of integrins αIIbβ3 and αvβ3, respectively [ 1 ]. Objectives:  In the present study, we have investigated the ability of an αIIb-CHAMPS peptide (termed integrin-activatory-peptide or IAP) to stimulate protein tyrosine phosphorylation and aggregation in human and mouse platelets. Methods:  The ability of IAP to stimulate platelet aggregation and dense granule secretion was measured in washed preparations of human and mouse platelets. Samples were taken for measurement of tyrosine phosphorylation. Results:  IAP stimulates robust tyrosine phosphorylation of the tyrosine kinase Syk and the FcR γ-chain, but only weak phosphorylation of PLCγ2. Aggregation to low but not high concentrations of IAP is reduced in the presence of the Src kinase inhibitor, PP1, or by inhibitors of the two feedback agonists, ADP and thromboxane A₂ (TxA₂) suggesting that activation is reinforced by Src kinase-driven release of ADP and TxA₂. Unexpectedly, aggregation by IAP is only partially inhibited in human and mouse platelets deficient in integrin αIIbβ3. Further, IAP induces partial aggregation of formaldehyde-fixed platelets. Conclusions:  The present study demonstrates that the αIIb-CHAMPS peptide induces platelet activation through integrin αIIbβ3-dependent and independent pathways with the former mediating tyrosine phosphorylation of FcR γ-chain and Syk. The use of the αIIb-CHAMPS peptide to study integrin αIIbβ3 function is compromised by non-integrin-mediated effects.     

DNA methylation in insects

Cytosine DNA methylation has been demonstrated in numerous eukaryotic organisms and has been shown to play an important role in human disease. The function of DNA methylation has been studied extensively in vertebrates, but establishing its primary role has proved difficult and controversial. Analysing methylation in insects has indicated an apparent functional diversity that seems to argue against a strict functional conservation. To investigate this hypothesis, we here assess the data reported in four different insect species in which DNA methylation has been analysed more thoroughly: the fruit fly Drosophila melanogaster, the cabbage moth Mamestra brassicae, the peach-potato aphid Myzus persicae and the mealybug Planococcus citri.     

Correlation of haloperidol levels between saliva and plasma of acutely ill schizophrenic patients

ObjectiveClinical usefulness of monitoring haloperidol in salivary samples based on plasma:saliva correlation.Design and MethodsPlasma and saliva samples of schizophrenic patients [N = 105] were analyzed by highly sensitive reverse phase liquid chromatographic method to measure haloperidol at 240 nm using UV-PDA detector. Mobile phase consist of acetonitrile and water [50:50], pH 2.5 (0.1% acetic acid and 0.05 M KHPO₄) at flow rate 1.4 mL/min. Method was linear over 3–200 ng/mL.ResultsObserved therapeutic range was 5–19 ng/mL [11.66 ± 3.97] and 17–54 ng/mL [27.52 ± 11.51] for plasma and saliva respectively. Mean S:P was found to be 2.36.ConclusionCurrent study showed significantly high correlation [r = 0.93, p < 0.0001] between haloperidol levels in saliva and plasma with linear relationship. It is therefore concluded that monitoring of salivary concentration can be a clinically beneficial substitute. Patients showing clinical improvement [N = 90] were within salivary concentration range of 17–54 ng/mL, which can be an appropriate steady state monitoring range for haloperidol in saliva.     

Identification of mutations conferring insecticide-insensitive AChE in the cotton-melon aphid, Aphis gossypii Glover

We have identified two mutations in the ace1 gene of Aphis gossypii that are associated with insensitivity of acetylcholinesterase (AChE) to carbamate and organophosphate insecticides. The first of these, S431F (equivalent to F331 in Torpedo californica), is associated with insensitivity to the carbamate insecticide pirimicarb in a range of A. gossypii clones. The S431F mutation is also found in the peach-potato aphid, Myzus persicae (Sulzer), and a rapid RFLP diagnostic allows the identification of individuals of both aphid species with a resistant genotype. This diagnostic further revealed the presence of S431 in several other pirimicarb-susceptible aphid species. The serine at this position in the wild-type enzyme has only been reported for aphids and provides a molecular explanation of why pirimicarb has a specific aphicidal action. A less specific insensitivity to a wide range of carbamates and organophosphates is associated with a second mutation, A302S (A201 in T. californica).     

Quantitative EEG power and asymmetry increase 36 h before a migraine attack

The aim was to estimate ictal, pre- and postictal brain function changes in migraine in a blinded paired quantitative EEG (QEEG) study. EEG recordings ( n  = 119) from 40 migraineurs were retrospectively classified as ictal, interictal, preictal or postictal. δ, θ, α and β power, and hemispheric asymmetry in frontocentral, temporal and occipitoparietal regions were calculated from artefact-free EEG. Power and power asymmetry were calculated for two time-windows, 36 and 72 h before/after the attack, and compared with the interictal values. Frontocentral δ power increased ( P  = 0.03), whereas frontocentral θ and α power tended to increase ( P  < 0.09) within 36 h before the next attack compared with the interictal period. Occipitoparietal (α and θ) and temporal (α) power were more asymmetric before the attack compared with the interictal baseline ( P  < 0.04). Ictal posterior α power increased slightly ( P  = 0.01). Postictal power and power asymmetry were not significantly different from interictal baseline. EEG activity seems to change shortly before the attack. This suggests that migraineurs are most susceptible to attack when anterior QEEG δ power and posterior α and θ asymmetry values are high. Changed activity patterns in cholinergic brainstem or basal forebrain nuclei and thalamo-cortical connections before the migraine attack are hypothesized.     

Abundant nuclear copies of mitochondrial origin (NUMTs) in the Aedes aegypti genome

A portion of the Aedes aegypti mitochondrial NADH dehydrogenase subunit 4 gene (ND4) was amplified using PCR with a 42 °C annealing temperature. Amplified fragments from individual mosquitoes were similar to ND4 but contained multiple segregating sites. We suspected that nuclear copies of mitochondrial origin (NUMTs) exist in the Ae. aegypti genome. A BlastN search in VectorBase with the entire Ae. aegypti mitochondrial genome identified 233 NUMTs comprising 110 178 bp in 145 supercontigs. At a density of 0.080 bp/kb, this represents the second highest density of NUMTs in an insect genome and the highest in Diptera. Analyses of flanking sequences suggested that Ae. aegypti NUMTs arise through mtDNA leakage from damaged mitochondria followed by breakage and nonhomologous recombination, rather than through duplicative processes such as transposition or molecular drive.     

In vivo relevance for platelet glycoprotein Ibα residue Tyr276 in thrombus formation

Summary.  Background:  Platelet glycoprotein (GP) Ib-IX-V supports platelet adhesion on damaged vascular walls by binding to von Willebrand factor (VWF). For several decades it has been recognized that the α-subunit of GP (GPIbα) also binds thrombin but the physiological relevance, if any, of this interaction was unknown. Previous studies have shown that a sulfated tyrosine 276 (Tyr276) is essential for thrombin binding to GPIbα. Objectives:  This study investigated the in vivo relevance of GPIbα residue Tyr276 in hemostasis and thrombosis. Methods:  Transgenic mouse colonies expressing the normal human GPIbα subunit or a mutant human GPIbα containing a Phe substitution for Tyr276 (hTg^Y276F) were generated. Both colonies were bred to mice devoid of murine GPIbα. Results:  Surface-expressed GPIbα levels and platelet counts were similar in both colonies. hTg^Y276F platelets were significantly impaired in binding α-thrombin but displayed normal binding to type I fibrillar collagen and human VWF in the presence of ristocetin. In vivo thrombus formation as a result of chemical damage (FeCl₃) demonstrated that hTg^Y276F mice have a delayed time to occlusion followed by unstable blood flow indicative of embolization. In models of laser-induced injury, thrombi developing in hTg^Y276F animals were also less stable. Conclusions:  The results demonstrate that GPIbα residue Tyr276 is physiologically important, supporting stable thrombus formation in vivo .     

Glutathione, vitamin E and oxidative stress in coronary artery disease: relevance of age and gender

Background  Observational studies suggest that low levels of antioxidants are associated with high risk for coronary artery disease (CAD). We investigated whether the biomarkers of oxidative balance undergo the same modifications in all CAD patient groups, regardless of gender and age.
Materials and methods  One hundred sixty-eight CAD patients and 107 healthy controls were assayed for plasma levels of reduced glutathione (GSH), α- and γ-tocopherol (α- and γ-T) as endogenous antioxidants. A damage score (DS), representative of oxidative stress status, was calculated. ancova models were used to test the association between antioxidants, DS and CAD and its modulation by age and gender.
Results  The DS was higher in CAD than in controls. GSH levels, were lower in CAD patients (mean ± SEM: 57·61 ± 1·87 μmol 10 g⁻¹ haemoglobin vs. 68·55 ± 2·23 in controls, P  < 0·0006) in males and in older subjects. Levels of other antioxidants exhibited a complex pattern. Overall, no difference was found in α- and γ-T contents between CAD and controls, but lower α-T values were observed in CAD females. A significant interaction between CAD status and gender was observed ( P  = 0·003).
Conclusions  Our study shows that the involvement of antioxidants in CAD is related to patients' characteristics. These findings may be relevant in planning antioxidant therapies.     

Identification of a novel point mutation in platelet glycoprotein Ibα, Gly to Ser at residue 233, in a Japanese family with platelet-type von Willebrand disease

Summary.  Background : Interaction between platelet glycoprotein (GP)Ibα and von Willebrand factor (VWF) has critical roles in both physiological hemostasis and thrombosis. Platelet-type von Willebrand disease (plt-VWD) is a congenital bleeding disorder characterized by gain-of-function mutations of GPIbα. To date, two mutations in GPIbα, G233V and M239V, have been reported in four unrelated families with plt-VWD. Objective : The present study aimed to determine whether G233S of GPIbα, a new mutation observed in plt-VWD patients, causes the plt-VWD phenotype and to examine whether conversions to other residues at this position affect VWF binding. Patients and methods : The propositus was a 3-year-old Japanese male. He displayed bleeding symptoms and moderate thrombocytopenia. His brother was similarly affected. Platelets from both patients were analyzed by ristocetin- or shear-induced platelet aggregation. DNA sequencing was performed to analyze the GPIbα sequence. We examined the ¹²⁵I-labeled VWF binding using a series of recombinant GPIbα fragments with different residues at position 233 (G233S, G233A, G233K, and G233D) together with naturally occurring mutations previously reported in patients (G233V and M239V). Results : Platelet function analysis indicated that platelets from both patients had a typical plt-VWD phenotype. DNA sequencing analysis showed a heterozygous mutation of Gly to Ser at residue 233 of GPIbα in both patients. The ¹²⁵I-labeled VWF binding to mutant compared with the wild type displayed three patterns, gain-of-function (G233S, G233V, and M239V), equivalent function (G233A), and loss-of-function (G233K and G233D). Conclusions : The G233S is a molecular basis of plt-VWD, and residue 233 plays critical roles in regulating VWF binding.     

Association of the antagonism of von Willebrand factor but not fibrinogen by platelet αIIbβ3 antagonists with prolongation of bleeding time

Summary.  Background:  The α _IIb β ₃ antagonists inhibit platelet aggregation and are used as antithrombotic agents for cardiothrombotic disease. The present study investigates the correlation of inhibition of fibrinogen and von Willebrand factor (VWF) binding by α _IIb β ₃ antagonists with the inhibition of platelet aggregation and prolongation of bleeding time (BT). Methods:  Inhibition of fibrinogen and VWF binding were assessed in a purified α _IIb β ₃-binding assay. As an in vitro cell-based assay, platelet aggregation and VWF-mediated adhesion studies were performed using human platelets. In vivo effects on BT were measured using a template device in dogs at the same time as an ex vivo aggregation study was performed. Results:   In vitro studies demonstrated that the antiaggregatory effects of α _IIb β ₃ antagonists correlate with their inhibition of fibrinogen binding, but not VWF. Interestingly, the effects of α _IIb β ₃ antagonists on BT could be differentiated from the inhibition of platelet aggregation. Furthermore, this differentiation was strongly correlated with the different inhibitory potencies between fibrinogen and VWF binding, as well as that between VWF-mediated adhesion and aggregation. Conclusions:  Our study provides novel evidence showing that the inhibitory effect of α _IIb β ₃ antagonists on VWF, but not fibrinogen binding, correlates with their ability to prolong BT.     

Evidence that α2-antiplasmin becomes covalently ligated to plasma fibrinogen in the circulation: a new role for plasma factor XIII in fibrinolysis regulation

Summary.  Background : Plasma alpha₂-antiplasmin (α₂AP) is a rapid and effective inhibitor of the fibrinolytic enzyme plasmin. Congenital α₂AP deficiency results in a severe hemorrhagic disorder due to accelerated fibrinolysis. It is well established that in the presence of thrombin-activated factor XIII (FXIIIa), α₂AP becomes covalently ligated to the distal α chains of fibrin or fibrinogen at lysine 303 (two potential sites per molecule). Some time ago we showed that α₂AP is covalently linked to plasma fibrinogen . That singular observation led to our hypothesis that native plasma factor XIII (FXIII), which is known to catalyze covalent cross-linking of fibrinogen in the presence of calcium ions, can also incorporate α₂AP into fibrinogen in the circulation. Results and Conclusions : We now provide evidence that FXIII incorporates I¹²⁵-labelled α₂AP into the Aα-chain sites on fibrinogen or fibrin. We also measured the content of α₂AP in isolated plasma fibrinogen fractions by ELISA and found that substantial amounts were present (1.2–1.8 moles per mole fibrinogen). We propose that α₂AP becomes ligated to fibrinogen while in the circulation through the action of FXIII, and that its immediate presence in plasma fibrinogen contributes to regulation of in vivo fibrinolysis.