OBJECTIVE

Despite the growing number of point-of-care (POC) tests available, little research has assessed primary care clinician need for such tests. We therefore aimed to determine which POC tests they actually use or would like to use (if not currently available in their practice).

METHODS

Cross-sectional survey.

METHODS

Primary care in Australia, Belgium (Flanders region only), the Netherlands, the UK and the USA.

METHODS

Primary care doctors (general practitioners, family physicians).

METHODS

We asked respondents to (1) identify conditions for which a POC test could help inform diagnosis, (<em>2</em>) from a list of tests provided: evaluate which POC tests they currently use (and how frequently) and (3) determine which tests (from that same list) they would like to use in the future (and how frequently).

RESULTS

<em>2</em>770 primary care clinicians across five countries responded. Respondents in all countries wanted POC tests to help them diagnose acute conditions (infections, acute cardiac disease, pulmonary embolism/deep vein thrombosis), and some chronic conditions (diabetes, anaemia). Based on the list of POC tests provided, the most common tests currently used were: urine pregnancy, urine leucocytes or nitrite and blood glucose. The most commonly reported tests respondents expressed a wish to use in the future were: D-dimer, troponin and chlamydia. The UK and the USA reported a higher actual and desired use for POC tests than Australia, Belgium and the Netherlands. Our limited data suggest (but do not confirm) representativeness.

CONCLUSIONS

Primary care clinicians in all five countries expressed a desire for POC tests to help them diagnose a range of acute and chronic conditions. Rates of current reported use and desired future use were generally high for a small selection of POC tests, but varied across countries. Future research is warranted to explore how specific POC tests might improve primary care.

BACKGROUND

Different coagulation abnormalities according to stroke subtypes have been reported. We have assessed the clinical utility of D-dimer, a product of fibrin degradation, in the early diagnosis of stroke subtypes.

METHODS

Patients hospitalized after an acute ischemic cerebrovascular event underwent D-dimer assay (STA Liatest D-Dimer) (reference level, <0.50 micro g/mL) on days 1, 6 +/- 1, and 12 +/- 1 and were studied to identify stroke subtypes.

RESULTS

We included 126 patients (mean age, 75.5 years) and 63 age-matched control subjects. Stroke subtypes were cardioembolic in 34 patients (27%), atherothrombotic in 34 (27%), lacunar in 31 (25%), and unknown in 27 (21%). At all 3 measurements, D-dimer levels were significantly higher in the cardioembolic group (mean +/- SEM, 2.96 +/- 0.51, 2.58 +/- 0.40, and 3.79 +/- 0.30 micro g/mL, respectively) than in the atherothrombotic (1.34 +/- 0.21, 1.53 +/- 0.26, and 2.91 +/- 0.23 micro g/mL, respectively) (P<.05) and lacunar (0.67 +/- 0.08, 0.72 +/- 0.15, and 0.64 +/- 0.06 micro g/mL, respectively) groups (P<.01). The difference was also significant between the latter 2 groups (P<.01). We found no difference between the lacunar group and controls (0.53 +/- 0.14 micro g/mL). According to day 1 measurements, the optimal cutoff point for predicting cardioembolic stroke was 2.00 micro g/mL, resulting in a specificity of 93.2% and in a sensitivity of 59.3%. For predicting lacunar stroke, the cutoff point was 0.54 micro g/mL, with a specificity of 96.2% and a sensitivity of 61.3%.

CONCLUSIONS

The increasing use of the D-dimer assay in clinical practice could be extended to patients presenting with acute cerebrovascular ischemic events to help predict stroke subtype.

<strong class="sub-title"> Importance: </strong> Coronavirus disease <em>2</em>019 (COVID-19) affects the nervous system in adult patients. The spectrum of neurologic involvement in children and adolescents is unclear.

Objective: To understand the range and severity of neurologic involvement among children and adolescents associated with COVID-19.

<strong class="sub-title"> Setting, design, and participants: </strong> Case series of patients (age <<em>2</em>1 years) hospitalized between March 15, <em>2</em>0<em>2</em>0, and December 15, <em>2</em>0<em>2</em>0, with positive severe acute respiratory syndrome coronavirus <em>2</em> test result (reverse transcriptase-polymerase chain reaction and/or antibody) at 61 US hospitals in the Overcoming COVID-19 public health registry, including 616 (36%) meeting criteria for multisystem inflammatory syndrome in children. Patients with neurologic involvement had acute neurologic signs, symptoms, or diseases on presentation or during hospitalization. Life-threatening involvement was adjudicated by experts based on clinical and/or neuroradiologic features.

<strong class="sub-title"> Exposures: </strong> Severe acute respiratory syndrome coronavirus <em>2</em>.

Main outcomes and measures: Type and severity of neurologic involvement, laboratory and imaging data, and outcomes (death or survival with new neurologic deficits) at hospital discharge.

<strong class="sub-title"> Results: </strong> Of 1695 patients (909 [54%] male; median [interquartile range] age, 9.1 [<em>2</em>.4-15.3] years), 365 (<em>2</em><em>2</em>%) from 5<em>2</em> sites had documented neurologic involvement. Patients with neurologic involvement were more likely to have underlying neurologic disorders (81 of 365 [<em>2</em><em>2</em>%]) compared with those without (113 of 1330 [8%]), but a similar number were previously healthy (195 [53%] vs 7<em>2</em>3 [54%]) and met criteria for multisystem inflammatory syndrome in children (1<em>2</em>6 [35%] vs 490 [37%]). Among those with neurologic involvement, 3<em>2</em><em>2</em> (88%) had transient symptoms and survived, and 43 (1<em>2</em>%) developed life-threatening conditions clinically adjudicated to be associated with COVID-19, including severe encephalopathy (n = 15; 5 with splenial lesions), stroke (n = 1<em>2</em>), central nervous system infection/demyelination (n = 8), Guillain-Barré syndrome/variants (n = 4), and acute fulminant cerebral edema (n = 4). Compared with those without life-threatening conditions (n = 3<em>2</em><em>2</em>), those with life-threatening neurologic conditions had higher neutrophil-to-lymphocyte ratios (median, 1<em>2</em>.<em>2</em> vs 4.4) and higher reported frequency of D-dimer greater than 3 μg/mL fibrinogen equivalent units (<em>2</em>1 [49%] vs 7<em>2</em> [<em>2</em><em>2</em>%]). Of 43 patients who developed COVID-19-related life-threatening neurologic involvement, 17 survivors (40%) had new neurologic deficits at hospital discharge, and 11 patients (<em>2</em>6%) died.

Conclusions and relevance: In this study, many children and adolescents hospitalized for COVID-19 or multisystem inflammatory syndrome in children had neurologic involvement, mostly transient symptoms. A range of life-threatening and fatal neurologic conditions associated with COVID-19 infrequently occurred. Effects on long-term neurodevelopmental outcomes are unknown.

Confusion in the nomenclature of vascular malformations has been a major obstacle to the un<em>d</em>erstan<em>d</em>ing of these con<em>d</em>itions, so that mis<em>d</em>iagnosis an<em>d</em> treatment inconsistencies are common. Coagulation abnormalities occurring in combination with venous malformations (VM) have been mis<em>d</em>iagnose<em>d</em> as Kasabach-Merritt syn<em>d</em>rome (KMS), <em>d</em>espite marke<em>d</em> <em>d</em>ifferences in clinical features, pathology an<em>d</em> treatment. A homogenous group of <em>2</em>4 patients with <em>d</em>iffuse limb VM was entere<em>d</em> into a retrospective chart review stu<em>d</em>y. The VM affecte<em>d</em> an upper limb in 1<em>2</em> patients, a lower limb in 10 an<em>d</em> both in two. Localize<em>d</em> intravascular coagulation (LIC) was characterize<em>d</em> by a <em>d</em>ecrease in fibrinogen (0.5-1 g/l), an increase in <em>d</em>-<em>dimers</em> (<em>2</em>-64 micro g/ml) an<em>d</em> presence of soluble complex of fibrin (+ to +++). Platelet counts were normal or slightly <em>d</em>ecrease<em>d</em>. Higher VM severity scores were associate<em>d</em> with more severe LIC. A number of events such as sclerotherapy, surgery, bone fracture, prolonge<em>d</em> immobilization an<em>d</em> pregnancy or menstruation triggere<em>d</em> conversion of the LIC to <em>d</em>isseminate<em>d</em> intravascular coagulation (DIC), with blee<em>d</em>ing relate<em>d</em> to factor consumption an<em>d</em> multiorgan failure relate<em>d</em> to <em>d</em>isseminate<em>d</em> microvascular thrombosis. Clinical symptoms associate<em>d</em> with worsening of LIC were pain, thrombosis an<em>d</em> blee<em>d</em>ing at woun<em>d</em> sites or <em>d</em>uring surgery. None of the patients ha<em>d</em> the large ecchymotic an<em>d</em> inflammatory tumours seen in KMS. Gra<em>d</em>e<em>d</em> permanent elastic compression with heparin therapy was the only effective treatment. In conclusion, VM-associate<em>d</em> LIC is a <em>d</em>istinctive lifelong coagulopathy that must be <em>d</em>ifferentiate<em>d</em> from KMS, which is characterize<em>d</em> by platelet trapping within a vascular tumour of infancy. The treatment of the two con<em>d</em>itions is very <em>d</em>ifferent.

Insect glutathione-S-transferases (GSTs) are grouped in three classes, I, II and recently III; class I (<em>Delta</em> class) enzymes together with class III members are implicated in conferring resistance to insecticides. Class II (Sigma class) GSTs, however, are poorly characterized and their exact biological function remains elusive. Drosophila glutathione S-transferase-<em>2</em> (GST-<em>2</em>) (DmGSTS1-1) is a class II enzyme previously found associated specifically with the insect indirect flight muscle. It was recently shown that GST-<em>2</em> exhibits considerable conjugation activity for 4-hydroxynonenal (4-HNE), a lipid peroxidation product, raising the possibility that it has a major anti-oxidant role in the flight muscle. Here, we report the crystal structure of GST-<em>2</em> at 1.75A resolution. The GST-<em>2</em> <em>dimer</em> shows the canonical GST fold with glutathione (GSH) ordered in only one of the two binding sites. While the GSH-binding mode is similar to other GST structures, a distinct orientation of helix alpha6 creates a novel electrophilic substrate-binding site (H-site) topography, largely flat and without a prominent hydrophobic-binding pocket, which characterizes the H-sites of other GSTs. The H-site displays directionality in the distribution of charged/polar and hydrophobic residues creating a binding surface that explains the selectivity for amphipolar peroxidation products, with the polar-binding region formed by residues Y<em>2</em>08, Y153 and R145 and the hydrophobic-binding region by residues V57, A59, Y<em>2</em>11 and the C-terminal V<em>2</em>49. A structure-based model of 4-HNE binding is presented. The model suggest that residues Y<em>2</em>08, R145 and possibly Y153 may be key residues involved in catalysis.

BACKGROUND

Obstructive sleep apnoea (OSA) is associated with increased cardiovascular risk, however the mechanisms are not well established.

OBJECTIVE

This study aimed to determine whether treatment of OSA with nasal continuous positive airway pressure (CPAP) would favourably alter coagulability across the sleep-wake cycle.

METHODS

In a randomised crossover trial, <em>2</em>8 patients received therapeutic or placebo CPAP, each for <em>2</em> months with a 1 month washout between treatments. After each treatment period, a <em>2</em>4 h coagulation study was conducted in the laboratory. Plasminogen activator inhibitor-1 (PAI-1), <em>D</em>-<em>dimer</em>, fibrinogen, von Willebrand Factor (vWF), factor VIII (FVIII), factor VII (FVII) and factor V (FV) were determined at seven time points over the day and night.

RESULTS

At baseline, patients had severe OSA (Apnoea Hypopnoea Index 37.9 ± <em>2</em>3.9 events/h). Treatment of OSA with CPAP compared with placebo resulted in lower <em>2</em>4 h levels of vWF (-3.9%, p=0.013), FVIII (-6.<em>2</em>%, p=0.007) and FV (-4.<em>2</em>%, p<0.001). The greatest difference occurred during the nocturnal and early morning periods. In contrast, fibrinogen, <em>D</em>-<em>dimer</em>, FVII and PAI-1 did not differ between treatments, however all markers displayed diurnal variability independent of treatment.

CONCLUSIONS

In this randomised, placebo-controlled crossover trial, treatment of OSA with CPAP reduced the early morning level of vWF, and nocturnal levels of FVIII and FV. These findings suggest that CPAP may reduce cardiovascular risk in OSA, in part through reducing risk of thrombosis.

Respiratory failure and acute kidney injury (AKI) are associated with high mortality in SARS-CoV-<em>2</em>-associated Coronavirus disease <em>2</em>019 (COVI<em>D</em>-19). These manifestations are linked to a hypercoaguable, pro-inflammatory state with persistent, systemic complement activation. Three critical COVI<em>D</em>-19 patients recalcitrant to multiple interventions had skin biopsies documenting deposition of the terminal complement component C5b-9, the lectin complement pathway enzyme MASP<em>2</em>, and C4d in microvascular endothelium. Administration of anti-C5 monoclonal antibody eculizumab led to a marked decline in <em>D</em>-<em>dimers</em> and neutrophil counts in all three cases, and normalization of liver functions and creatinine in two. One patient with severe heart failure and AKI had a complete remission. The other two individuals had partial remissions, one with resolution of his AKI but ultimately succumbing to respiratory failure, and another with a significant decline in FiO_<em>2</em> requirements, but persistent renal failure. In conclusion, anti-complement therapy may be beneficial in at least some patients with critical COVI<em>D</em>-19.

<strong class="sub-title"> Keywords: </strong> COVI<em>D</em>-19; Complement; Coronavirus; Eculizumab; Lectin pathway; MASP<em>2</em>.

COVID-19 has been associated with a hypercoagulable state causing cardiovascular and neurovascular complications. To further characterize cerebrovascular disease (CVD) in COVID-19, we review the current literature of published cases and additionally report the clinical presentation, laboratory and diagnostic testing results of 12 cases with COVID-19 infection and concurrent CVD from two academic medical centers in Houston, TX, USA, between March 1 and May 10, 2020. To date, there are 12 case studies reporting 47 cases of CVD in COVID-19. However, only 4 small case series have described the clinical and laboratory findings in patients with COVID-19 and concurrent stroke. Viral neurotropism, endothelial dysfunction, coagulopathy and inflammation are plausible proposed mechanisms of CVD in COVID-19 patients. In our case series of 12 patients, 10 patients had an ischemic stroke, of which 1 suffered hemorrhagic transformation and two had intracerebral hemorrhage. Etiology was determined to be embolic without a clear cause identified in 6 ischemic stroke patients, while the remaining had an identifiable source of stroke. The majority of the patients had elevated inflammatory markers such as D-dimer and interleukin-6. In patients with embolic stroke of unclear etiology, COVID-19 may have played a direct or indirect role in the processes that eventually led to the strokes while in the remaining cases, it is unclear if infection contributed partially or was an incidental finding.

Keywords: APLS; COVID-19; Cerebrovascular disease; Coronavirus; SARS-CoV-2; Stroke.

<em>D</em>-Glycerate dehydrogenase (G<em>D</em>H) catalyzes the NA<em>D</em>H-linked reduction of hydroxypyruvate to <em>D</em>-glycerate. G<em>D</em>H is a member of a family of NA<em>D</em>-dependent dehydrogenases that is characterized by a specificity for the <em>D</em>-isomer of the hydroxyacid substrate. The crystal structure of the apoenzyme form of G<em>D</em>H from Hyphomicrobium methylovorum has been determined by the method of isomorphous replacement and refined at <em>2</em>.4 A resolution using a restrained least-squares method. The crystallographic R-factor is 19.4% for all <em>2</em>4,553 measured reflections between 10.0 and <em>2</em>.4 A resolution. The G<em>D</em>H molecule is a symmetrical <em>dimer</em> composed of subunits of molecular mass 38,000, and shares significant structural homology with another NA<em>D</em>-dependent enzyme, formate dehydrogenase. The G<em>D</em>H subunit consists of two structurally similar domains that are approximately related to each other by <em>2</em>-fold symmetry. The domains are separated by a deep cleft that forms the putative NA<em>D</em> and substrate binding sites. One of the domains has been identified as the NA<em>D</em>-binding domain based on its close structural similarity to the NA<em>D</em>-binding domains of other NA<em>D</em>-dependent dehydrogenases. The topology of the second domain is different from that found in the various catalytic domains of other dehydrogenases. A model of a ternary complex of G<em>D</em>H has been built in which putative catalytic residues are identified based on sequence homology between the <em>D</em>-isomer specific dehydrogenases. A structural comparison between G<em>D</em>H and L-lactate dehydrogenase indicates a convergence of active site residues and geometries for these two enzymes. The reactions catalyzed are chemically equivalent but of opposing stereospecificity. A hypothesis is presented to explain how the two enzymes may exploit the same coenzyme stereochemistry and a similar spatial arrangement of catalytic residues to carry out reactions that proceed to opposite enantiomers.

The EF-han<em>d</em> calcium-bin<em>d</em>ing protein S100B also bin<em>d</em>s one zinc ion per subunit with a relatively high affinity (K(<em>d</em>) approximately 90 nM) [Wil<em>d</em>er et al., (<em>2</em>003) Biochemistry 4<em>2</em>, 13410-134<em>2</em>1]. In this stu<em>d</em>y, the structural characterization of zinc bin<em>d</em>ing to calcium-loa<em>d</em>e<em>d</em> S100B was examine<em>d</em> using high-resolution NMR techniques, inclu<em>d</em>ing structural characterization of this complex in solution at atomic resolution. As with other S100 protein structures, the quaternary structure of Zn(<em>2</em>+)-Ca(<em>2</em>+)-boun<em>d</em> S100B was foun<em>d</em> to be <em>dimer</em>ic with helices H1, H1', H4, an<em>d</em> H4' forming an X-type four-helix bun<em>d</em>le at the <em>dimer</em> interface. NMR <em>d</em>ata together with mutational analyses are consistent with Zn(<em>2</em>+) coor<em>d</em>ination arising from His-15 an<em>d</em> His-<em>2</em>5 of one S100B subunit an<em>d</em> from His-85 an<em>d</em> Glu-89 of the other subunit. The a<em>d</em><em>d</em>ition of Zn(<em>2</em>+) was also foun<em>d</em> to exten<em>d</em> helices H4 an<em>d</em> H4' three to four resi<em>d</em>ues similar to what was previously observe<em>d</em> with the bin<em>d</em>ing of target proteins to S100B. Furthermore, a kink in helix 4 was observe<em>d</em> in Zn(<em>2</em>+)-Ca(<em>2</em>+)-boun<em>d</em> S100B that is not in Ca(<em>2</em>+)-boun<em>d</em> S100B. These structural changes upon Zn(<em>2</em>+)-bin<em>d</em>ing coul<em>d</em> explain the 5-fol<em>d</em> increase in affinity that Zn(<em>2</em>+)-Ca(<em>2</em>+)-boun<em>d</em> S100B has for pepti<em>d</em>e targets such as the TRTK pepti<em>d</em>e versus Ca(<em>2</em>+)-boun<em>d</em> S100B. There are also changes in the relative positioning of the two EF-han<em>d</em> calcium-bin<em>d</em>ing <em>d</em>omains an<em>d</em> the respective helices comprising these EF-han<em>d</em>s. Changes in conformation such as these coul<em>d</em> contribute to the or<em>d</em>er of magnitu<em>d</em>e higher affinity that S100B has for calcium in the presence of Zn(<em>2</em>+).

Background: COVID-19 is associated with a prothrombotic state leading to adverse clinical outcomes. Whether therapeutic anticoagulation improves outcomes in patients hospitalised with COVID-19 is unknown. We aimed to compare the efficacy and safety of therapeutic versus prophylactic anticoagulation in this population.

Methods: We did a pragmatic, open-label (with blinded adjudication), multicentre, randomised, controlled trial, at 31 sites in Brazil. Patients (aged ≥18 years) hospitalised with COVID-19 and elevated D-dimer concentration, and who had COVID-19 symptoms for up to 14 days before randomisation, were randomly assigned (1:1) to receive either therapeutic or prophylactic anticoagulation. Therapeutic anticoagulation was in-hospital oral rivaroxaban (20 mg or 15 mg daily) for stable patients, or initial subcutaneous enoxaparin (1 mg/kg twice per day) or intravenous unfractionated heparin (to achieve a 0·3-0·7 IU/mL anti-Xa concentration) for clinically unstable patients, followed by rivaroxaban to day 30. Prophylactic anticoagulation was standard in-hospital enoxaparin or unfractionated heparin. The primary efficacy outcome was a hierarchical analysis of time to death, duration of hospitalisation, or duration of supplemental oxygen to day 30, analysed with the win ratio method (a ratio >1 reflects a better outcome in the therapeutic anticoagulation group) in the intention-to-treat population. The primary safety outcome was major or clinically relevant non-major bleeding through 30 days. This study is registered with ClinicalTrials.gov (NCT04394377) and is completed.

Findings: From June 24, 2020, to Feb 26, 2021, 3331 patients were screened and 615 were randomly allocated (311 [50%] to the therapeutic anticoagulation group and 304 [50%] to the prophylactic anticoagulation group). 576 (94%) were clinically stable and 39 (6%) clinically unstable. One patient, in the therapeutic group, was lost to follow-up because of withdrawal of consent and was not included in the primary analysis. The primary efficacy outcome was not different between patients assigned therapeutic or prophylactic anticoagulation, with 28 899 (34·8%) wins in the therapeutic group and 34 288 (41·3%) in the prophylactic group (win ratio 0·86 [95% CI 0·59-1·22], p=0·40). Consistent results were seen in clinically stable and clinically unstable patients. The primary safety outcome of major or clinically relevant non-major bleeding occurred in 26 (8%) patients assigned therapeutic anticoagulation and seven (2%) assigned prophylactic anticoagulation (relative risk 3·64 [95% CI 1·61-8·27], p=0·0010). Allergic reaction to the study medication occurred in two (1%) patients in the therapeutic anticoagulation group and three (1%) in the prophylactic anticoagulation group.

Interpretation: In patients hospitalised with COVID-19 and elevated D-dimer concentration, in-hospital therapeutic anticoagulation with rivaroxaban or enoxaparin followed by rivaroxaban to day 30 did not improve clinical outcomes and increased bleeding compared with prophylactic anticoagulation. Therefore, use of therapeutic-dose rivaroxaban, and other direct oral anticoagulants, should be avoided in these patients in the absence of an evidence-based indication for oral anticoagulation.

Funding: Coalition COVID-19 Brazil, Bayer SA.

Background: The aim of this study was to analyze and summarize the clinical characteristics of elderly patients with coronavirus disease 2019 (COVID-19) and compare the differences of young-old patients (60-74 years old) and old-old patients (≥75 years old).

Methods: In thisretrospective, multicenter study, the medical records of elderly patients who were diagnosed with COVID-19 in Hunan province, China, from January 21 to February 19, 2020 were reviewed. The characteristics of young-old patients and old-old patients were compared.

Results: Of the 105 elderly patientsconfirmed withCOVID-19, 81.0% were young-old patients, and 19.0% were old-old patients; 54.3% of elderly patients were females. Overall, 69.5% of elderly patients had underlying diseases, and the most common comorbidities included hypertension (43.8%), diabetes (25.7%), and cardiac disease (16.2%). Of the elderly patients, 22.9% were severe and 10.5% were critical severe cases. On admission, the most frequent symptoms in elderly patients included fever (66.7%), cough (64.8%), and fatigue (33.3%). Lymphopenia (31.4%), increased D-dimer (38.1%), depressed albumin (36.2%), elevated lactate dehydrogenase (41.0%), and a high level of C-reactive protein (79.0%) were common among elderly patients with COVID-19. The median prothrombin time (PT) and the activated partial thromboplastin time (APTT) were longer in old-old patients than young-old patients (PT median 12.3 vs. 13.1 s, p = 0.007; APTT median 39.0 vs. 33.5 s, p = 0.045). Young-old patients showed fewer complications (14.1%) than old-old patients (40.0%; p = 0.0014) and fewer received invasive ventilator support (3.5 vs. 25.0%, p = 0.006). As of March 11, 2020, 85.7% of elderly patients had been discharged, 3 deaths had occurred, and 11.4% were still hospitalized.

Conclusions: Elderly patients usually have chronic medical illness and are likely to have a severe or critically severe condition. They could show atypical symptoms without fever or cough and multiple organ dysfunction. Old-old patients tend to have more complications than young-old patients during hospitalization. Careful nursing, observation, and systemic treatment are very important in elderly patients.

Keywords: Coronavirus disease 2019; Elderly; Epidemiology; Older people; SARS-CoV-2.

LC8 dynein light chain (<em>D</em>YNLL) is a eukaryotic hub protein that is thought to function as a <em>dimer</em>ization engine. Its interacting partners are involved in a wide range of cellular functions. In its dozens of hitherto identified binding partners <em>D</em>YNLL binds to a linear peptide segment. The known segments define a loosely characterized binding motif: [<em>D</em>/S](-4)K(-3)X(-<em>2</em>)[T/V/I](-1)Q(0)[T/V](1)[<em>D</em>/E](<em>2</em>). The motifs are localized in disordered segments of the <em>D</em>YNLL-binding proteins and are often flanked by coiled coil or other potential <em>dimer</em>ization domains. Based on a directed evolution approach, here we provide the first quantitative characterization of the binding preference of the <em>D</em>YNLL binding site. We displayed on M13 phage a naïve peptide library with seven fully randomized positions around a fixed, naturally conserved glutamine. The peptides were presented in a bivalent manner fused to a leucine zipper mimicking the natural <em>dimer</em> to <em>dimer</em> binding stoichiometry of <em>D</em>YNLL-partner complexes. The phage-selected consensus sequence V(-5)S(-4)R(-3)G(-<em>2</em>)T(-1)Q(0)T(1)E(<em>2</em>) resembles the natural one, but is extended by an additional N-terminal valine, which increases the affinity of the monomeric peptide twentyfold. Leu-zipper <em>dimer</em>ization increases the affinity into the subnanomolar range. By comparing crystal structures of an SRGTQTE-<em>D</em>YNLL and a <em>dimer</em>ic VSRGTQTE-<em>D</em>YNLL complex we find that the affinity enhancing valine is accommodated in a binding pocket on <em>D</em>YNLL. Based on the in vitro evolved sequence pattern we predict a large number of novel <em>D</em>YNLL binding partners in the human proteome. Among these EML3, a microtubule-binding protein involved in mitosis contains an exact match of the phage-evolved consensus and binds to <em>D</em>YNLL with nanomolar affinity. These results significantly widen the scope of the human interactome around <em>D</em>YNLL and will certainly shed more light on the biological functions and organizing role of <em>D</em>YNLL in the human and other eukaryotic interactomes.

We evaluate the performance of ten functionals (B3LYP, M05, M05-<em>2</em>X, M06, M06-<em>2</em>X, B<em>2</em>PLYP, B<em>2</em>PLYPD, X3LYP, B97D, an<em>d</em> MPWB1K) in combination with 16 basis sets ranging in complexity from 6-31G(<em>d</em>) to aug-cc-pV5Z for the calculation of the H-bon<em>d</em>e<em>d</em> water <em>dimer</em> with the goal of <em>d</em>efining which combinations of functionals an<em>d</em> basis sets provi<em>d</em>e a combination of economy an<em>d</em> accuracy for H-bon<em>d</em>e<em>d</em> systems. We have compare<em>d</em> the results to the best non-<em>d</em>ensity functional theory (non-DFT) molecular orbital (MO) calculations an<em>d</em> to experimental results. Several of the smaller basis sets lea<em>d</em> to qualitatively incorrect geometries when optimize<em>d</em> on a normal potential energy surface (PES). This problem <em>d</em>isappears when the optimization is performe<em>d</em> on a counterpoise (CP) correcte<em>d</em> PES. The calculate<em>d</em> interaction energies (ΔEs) with the largest basis sets vary from -4.4<em>2</em> (B97D) to -5.19 (B<em>2</em>PLYPD) kcal/mol for the <em>d</em>ifferent functionals. Small basis sets generally pre<em>d</em>ict stronger interactions than the large ones. We foun<em>d</em> that, because of error compensation, the smaller basis sets gave the best results (in comparison to experimental an<em>d</em> high-level non-DFT MO calculations) when combine<em>d</em> with a functional that pre<em>d</em>icts a weak interaction with the largest basis set. As many applications are complex systems an<em>d</em> require economical calculations, we suggest the following functional/basis set combinations in or<em>d</em>er of increasing complexity an<em>d</em> cost: (1) D95(<em>d</em>,p) with B3LYP, B97D, M06, or MPWB1k; (<em>2</em>) 6-311G(<em>d</em>,p) with B3LYP; (3) D95++(<em>d</em>,p) with B3LYP, B97D, or MPWB1K; (4) 6-311++G(<em>d</em>,p) with B3LYP or B97D; an<em>d</em> (5) aug-cc-pVDZ with M05-<em>2</em>X, M06-<em>2</em>X, or X3LYP.

BACKGROUND

Cl-inhibitor (C1-INH) deficiency leads to recurrent attacks of mucocutaneous edema and may be inherited (hereditary angioedema [HAE]) or acquired (acquired angioedema [AAE]), which have the same clinical picture characterized by angioedema involving the skin, gastrointestinal tract, and larynx. Although cutaneous swelling is evident, abdominal angioedema is still a diagnostic challenge and attacks can mimic surgical emergencies. There is currently no laboratory marker for identifying angioedema attacks.

OBJECTIVE

As coagulation and fibrinolysis are activated during angioedema attacks, we assessed if plasma measurements of prothrombin fragment F1 + <em>2</em> (marker of thrombin generation) and <em>D</em>-<em>dimer</em> (marker of fibrin degradation) can be useful for the diagnosis of angioedema because of C1-INH deficiency, especially in case of hidden locations as abdominal attacks.

METHODS

In addition to complement, we measured plasma levels of F1 + <em>2</em> and <em>D</em>-<em>dimer</em> in <em>2</em>8 patients with C1-INH deficiency during acute attacks and remission, 35 patients without C1-INH deficiency during abdominal colics, and <em>2</em>0 healthy subjects.

RESULTS

Plasma F1 + <em>2</em> levels were higher in patients with C1-INH deficiency during remission than in healthy controls (P = 0.001), and further increased during cutaneous and abdominal attacks (P = 0.0001); patients without C1-INH deficiency had normal F1 + <em>2</em> levels during abdominal colics. Plasma <em>D</em>-<em>dimer</em> levels were higher in patients with C1-INH deficiency during remission than in controls (P = 0.01<em>2</em>) and increased during angioedema attacks, reaching higher levels than in patients without C1-INH deficiency during colics (P = 0.00<em>2</em>).

CONCLUSIONS

<em>D</em>uring acute angioedema attacks, patients with C1-INH deficiency have high prothrombin fragment F1 + <em>2</em> and <em>D</em>-<em>dimer</em> levels, the measurement of which may have an important diagnostic value.

OBJECTIVE

Hemostasis and thrombosis may be important contributors to cognitive decline and dementia. Certain blood markers may assist in diagnosis or management.

OBJECTIVE

To collate evidence for the association of circulating hemostatic variables and dementia or cognitive impairment.

METHODS

A systematic review of studies describing blood markers of hemostatic function and cognition/dementia. Abstracts were reviewed by two independent assessors and studies selected based on pre-specified criteria. We described methodological quality and performed meta-analyzes where data allowed.

RESULTS

From 7103 titles, 485 abstracts and included <em>2</em>1 studies (n = 3<em>2</em>,773) were assessed. In two longitudinal studies, the incident of vascular dementia risk was greater for higher <em>D</em>-<em>dimer</em> [hazard ratio (HR): 1.50, 95% confidence interval (CI): 1.15-1.96]. For case-control data, we calculated standardized mean differences (SM<em>D</em>) and 95% CI. Higher levels of: factor (F)VII (SM<em>D</em>: 0.93; 95% CI: 0.60-1.<em>2</em>6), fibrinogen (SM<em>D</em>: 1.53; 95% CI: 1.17-1.87), prothrombin fragment 1 and <em>2</em> (SM<em>D</em>: 0.64; 95% CI: 0.3<em>2</em>-0.96), plasminogen activator inhibitor (SM<em>D</em>: 0.68; 95% CI: 0.<em>2</em>6-1.10), <em>D</em>-<em>dimer</em> (SM<em>D</em>: <em>2</em>.00; 95% CI: 1.59-<em>2</em>.40) and von Willebrand factor (VWF) (SM<em>D</em>: 1.68; 95% CI: 1.30-<em>2</em>.06) showed modest but significant associations with vascular dementia. For patients with any dementia diagnosis, associations were with higher <em>D</em>-<em>dimer</em> (SM<em>D</em>: 0.36; 95% CI: 0.15-0.56) and VWF (SM<em>D</em>: 0.31; 95% CI: 0.11-0.51). For specific cognitive domains, significant (P < 0.001) positive correlations were fibrinogen and speed of processing (0.76; 95% CI: 0.67-0.84), verbal memory (0.69; 95% CI: 0.59-0.79) and non-verbal reasoning (0.57; 95% CI: 0.49-0.65).

CONCLUSIONS

The present results suggest a modest association between hemostasis and vascular dementia including increased levels of thrombin generation markers (<em>D</em>-<em>dimer</em> and prothrombin fragment 1 + <em>2</em>) and endothelial dysfunction (VWF and plasminogen activator inhibitor). Associations are weaker for specific cognitive tests and when all dementias are combined.

The Tp34 (TP0971) membrane lipoprotein of Treponema pallidum, an obligate human pathogen and the agent of syphilis, was previously reported to have lactoferrin binding properties. Given the non-cultivatable nature of T. pallidum, a structure-to-function approach was pursued to clarify further potential relationships between the Tp34 structural and biochemical properties and its propensity to bind human lactoferrin. The crystal structure of a nonacylated, recombinant form of Tp34 (rTp34), solved to a resolution of 1.9A(,) revealed two metaloccupied binding sites within a <em>dimer</em>; the identity of the ion most likely was zinc. Residues from both of the monomers contributed to the interfacial metal-binding sites; a novel feature was that the <em>delta</em>-sulfur of methionine coordinated the zinc ion. Analytical ultracentrifugation showed that, in solution, rTp34 formed a metal-stabilized <em>dimer</em> and that rTp34 bound human lactoferrin with a stoichiometry of <em>2</em>:1. Isothermal titration calorimetry further revealed that rTp34 bound human lactoferrin at high (submicromolar) affinity. Finally, membrane topology studies revealed that native Tp34 is not located on the outer surface (outer membrane) of T. pallidum but, rather, is periplasmic. How propensity of Tp34 to bind zinc and the iron-sequestering lactoferrin may relate overall to the biology of T. pallidum infection in humans is discussed.

Warfarin, like all the 4-hydroxycoumarin compounds, has an asymmetric carbon atom. The clinically available warfarin preparations consist of a racemic mixture of equal amounts of <em>2</em> distinct S and R isomers, the former being 4-times more potent as anticoagulant and more susceptible to drug interaction. Warfarin is highly water soluble and rapidly absorbed from the stomach and the upper gastrointestinal tract; its plasma concentrations peak 60 to 90 minutes after oral administration. Warfarin binds to the enzyme vitamin K <em>2</em>,3-epoxide reductase in liver microsomes, stopping the cycle of vitamin K and reducing gamma-carboxylation of the precursors of vitamin <em>D</em>-dependent pro- and anticoagulant factors. A variable fraction of the binding with the target enzyme, albeit small, can be reversed by competitive displacers, such as dithiol-reducing agent activity. <em>D</em>ifferences in dithiol-reducing activity have been suggested as a contributing factor to the wide interindividual differences in sensitivity to oral anticoagulants. The anticoagulant effect is caused by a small fraction of the drug, since most (97 to 99%) is protein bound (mainly to albumin) and ineffective. <em>D</em>rugs that can displace the albumin binding will increase the action of warfarin, even though this effect is counteracted by a more rapid elimination of the drug. The elimination half-life of warfarin varies greatly among individuals, ranging from 35 to 45 hours; the S isomer has, however, an average half-life shorter than the R isomer. The plasma levels of vitamin K-dependent proteins are determined by a dynamic equilibrium between their synthesis and half-life times. The delay before warfarin takes effect reflects the half-life of the clotting proteins; the levels of factor VII and protein C (with shorter half-lives) are reduced earlier, reaching steady inhibited levels in about 1 day, whereas factor II takes more than 10 days. Oral anticoagulant therapy (OAT) with warfarin or other coumarin derivatives is increasingly administered to patients for primary or secondary prevention of various arterial or venous thromboembolic diseases. If in some clinical conditions OAT is given indefinitely, in others--such as venous thromboembolism or after tissue heart valve replacement--anticoagulants are usually given only for the high risk period of thrombotic complication. A recent large prospective study performed by the Italian Federation of Anticoagulation Clinics showed that about 30% of the patients who began OAT for various clinical indications stopped treatment at different times, confirming that withdrawal from OAT is an occurrence that affects a large number of patients. The expression 'rebound phenomenon' was adopted to indicate a hypercoagulant condition occurring after warfarin withdrawal. A possible more frequent recurrence of thromboembolism after cessation of anticoagulation became a matter of controversy and many clinical studies, mostly observational and noncontrolled, reported on the issue with inconsistent results. Most authoritative commentators agreed that rebound phenomenon, though possible, was not clinically relevant and did not differ in frequency and intensity according to mode of withdrawal. Scientific interest in the topic waned until more sensitive methods for investigating blood hypercoagulability became available. In recent years, many studies (reviewed in the text) have investigated the levels of different markers of hypercoagulability [fibrinopeptide A, activated factor VII, prothrombin fragments F1+<em>2</em>, thrombin-antithrombin complexes, <em>D</em>-<em>dimers</em> (<em>D</em><em>D</em>)], consistently finding an increase in their values after cessation of anticoagulation. Changes in the levels of markers of activated blood coagulation were prospectively investigated by our group in 3<em>2</em> patients with venous thromboembolism who were randomly withdrawn abruptly or gradually from warfarin treatment. Our results indicate that interruption of anticoagulant treatment frequently elicits low grade acti

Free energies of oxygen-linked subunit assembly and cooperative interaction have been determined for 34 molecular species of human hemoglobin, which differ by amino acid alterations as a result of mutation or chemical modification at specific sites. These studies required the development of extensions to our earlier methodology. In combination with previous results they comprise a data base of 60 hemoglobin species, characterized under the same conditions. The data base was analyzed in terms of the five following issues. (1) Range and sensitivity to site modifications. Deoxy tetramers showed greater average energetic response to structural modifications than the oxy species, but the ranges are similar for the two ligation forms. (<em>2</em>) Structural localization of cooperative free energy. Difference free energies of <em>dimer</em>-tetramer assembly (oxy minus deoxy) yielded <em>delta</em> Gc for each hemoglobin, i.e., the free energy used for modulation of oxygen affinity over all four binding steps. A structure-energy map constructed from these results shows that the alpha 1 beta <em>2</em> interface is a unique structural location of the noncovalent bonding interactions that are energetically coupled to cooperativity. (3) Relationship of cooperativity to intrinsic binding. Oxygen binding energetics for dissociated <em>dimers</em> of mutants strongly indicates that cooperativity and intrinsic binding are completely decoupled by tetramer to <em>dimer</em> dissociation. (4) Additivity, site-site coupling and adventitious perturbations. All these are exhibited by individual-site modifications of this study. Large nonadditivity may be correlated with global (quaternary) structure change. (5) Residue position vs. chemical nature. Functional response is solely dictated by structural location for a subset of the sites, but varies with side-chain type at other sites. The current data base provides a unique framework for further analyses and modeling of fundamental issues in the structural chemistry of proteins and allosteric mechanisms.

This report <em>d</em>escribes bio<em>d</em>istribution characteristics of three ternary ligan<em>d</em> complexes [(99m)Tc(SQ168)(tricine)(L)] (SQ168 = [<em>2</em>-[[[5-[carboonyl]-<em>2</em>-pyri<em>d</em>inyl]hy<em>d</em>razono]methyl]-benzenesulfonic aci<em>d</em>]-Glu(cyclo{Lys-Arg-Gly-Asp-<em>d</em>-Phe})-cyclo{Lys-Arg-Gly-Asp-<em>d</em>-Phe}; L = TPPTS (triso<em>d</em>ium triphenylphosphine-3,3',3' '-trisulfonate), ISONIC (isonicotinic aci<em>d</em>) an<em>d</em> PDA (<em>2</em>,5-pyri<em>d</em>ine<em>d</em>icarboxylic aci<em>d</em>)) in athymic nu<em>d</em>e mice bearing MDA-MB-435 human breast cancer xenografts. Ternary ligan<em>d</em> complexes [(99m)Tc(SQ168)(tricine)(L)] (L = TPPTS, ISONIC an<em>d</em> PDA) were prepare<em>d</em> an<em>d</em> were analyze<em>d</em> by a reverse<em>d</em> HPLC metho<em>d</em>. Surprisingly, coligan<em>d</em>s have little impact on log P values of their ternary ligan<em>d</em> (99m)Tc complexes even though HPLC retention times suggest that [(99m)Tc(SQ168)(tricine)(PDA)] an<em>d</em> [(99m)Tc(SQ168)(tricine)(ISONIC)] are more hy<em>d</em>rophilic than [(99m)Tc(SQ168)(tricine)(TPPTS)]. The results from bio<em>d</em>istribution stu<em>d</em>ies in<em>d</em>icate<em>d</em> that excretion kinetics of the (99m)Tc-labele<em>d</em> cyclic RGDfK <em>dimer</em> can be mo<em>d</em>ifie<em>d</em> by the choice of coligan<em>d</em>. The fact that all three ra<em>d</em>iotracers show high tumor uptake <em>d</em>uring the <em>2</em> h stu<em>d</em>y perio<em>d</em> suggests that the coligan<em>d</em> has minimal effect on the tumor targeting capability of the (99m)Tc-labele<em>d</em> cyclic RGDfK <em>dimer</em>. Results from the blocking experiment suggest that the tumor localization of the (99m)Tc-labele<em>d</em> cyclic RGDfK <em>dimer</em> is integrin alpha(v)beta(3)-me<em>d</em>iate<em>d</em>. On the basis of their liver uptake an<em>d</em> tumor/liver ratios, we believe that PDA has the a<em>d</em>vantage over TPPTS an<em>d</em> ISONIC for the (99m)Tc-labeling of HYNIC-biomolecule conjugates.

The mannan-binding lectin (MBL) activation pathway of complement plays an important role in the innate immune defense against pathogenic microorganisms. In human serum, two MBL-associated serine proteases (MASP-1, MASP-<em>2</em>) and MBL-associated protein 19 (MAp19) were found to be associated with MBL. With a view to investigate the interaction properties of these proteins, human MASP-1, MASP-<em>2</em>, MAp19, as well as the N-terminal complement subcomponents C1r/C1s, Uegf, and bone morphogenetic protein-1-epidermal growth factor (CUB-EGF) segments of MASP-1 and MASP-<em>2</em>, were expressed in insect or human kidney cells, and MBL was isolated from human serum. Sedimentation velocity analysis indicated that the MASP-1 and MASP-<em>2</em> CUB-EGF segments and the homologous protein MAp19 all behaved as homo<em>dimers</em> (<em>2</em>.8-3.<em>2</em> S) in the presence of Ca(<em>2</em>+). Although the latter two <em>dimers</em> were not dissociated by E<em>D</em>TA, their physical properties were affected. In contrast, the MASP-1 CUB-EGF homodimer was not sensitive to E<em>D</em>TA. The three proteins and full-length MASP-1 and MASP-<em>2</em> showed no interaction with each other as judged by gel filtration and surface plasmon resonance spectroscopy. Using the latter technique, MASP-1, MASP-<em>2</em>, their CUB-EGF segments, and MAp19 were each shown to bind to immobilized MBL, with K:(<em>D</em>) values of 0.8 nM (MASP-<em>2</em>), 1.4 nM (MASP-1), 13.0 nM (MAp19 and MASP-<em>2</em> CUB-EGF), and <em>2</em>5.7 nM (MASP-1 CUB-EGF). The binding was Ca(<em>2</em>+)-dependent and fully sensitive to E<em>D</em>TA in all cases. These data indicate that MASP-1, MASP-<em>2</em>, and MAp19 each associate as homo<em>dimers</em>, and individually form Ca(<em>2</em>+)-dependent complexes with MBL through the CUB-EGF pair of each protein. This suggests that distinct MBL/MASP complexes may be involved in the activation or regulation of the MBL pathway.

A set of subsites in barnase has been propose<em>d</em> from kinetic stu<em>d</em>ies. A specific substrate analog, the tetra<em>d</em>eoxynucleoti<em>d</em>e, CGAC, has been <em>d</em>esigne<em>d</em> from this information. We report the crystal structure of its complex with barnase at 1.76-A resolution. The structure was solve<em>d</em> by molecular replacement from a mo<em>d</em>el of free barnase an<em>d</em> refine<em>d</em> to a crystallographic R factor of 19.0%. The stoichiometry of the asymmetric unit <em>dimer</em>ic complex is [barnase:<em>d</em>(CGAC)]<em>2</em>, with <em>2</em>-fol<em>d</em> noncrystallographic symmetry. Each barnase molecule bin<em>d</em>s one oligonucleoti<em>d</em>e whereby the recognition site is occupie<em>d</em> by guanine, an<em>d</em> all three phosphate groups of the nucleoti<em>d</em>e make electrostatic interactions with basic resi<em>d</em>ues in a strongly electropositive region at the bottom of the active site. The active-site His 10<em>2</em> packs against the a<em>d</em>enine base of the nucleoti<em>d</em>e in an almost i<em>d</em>entical manner to the guanine base in the barnase-<em>d</em>(GpC) complex an<em>d</em> <em>d</em>efines a possible subsite in the Michaelis complex. The overall protein structure is unchange<em>d</em> on forming the complex with <em>d</em>(CGAC), but there are small <em>d</em>ifferences in the active site an<em>d</em> in crystal packing regions. The protein coor<em>d</em>inates will be useful for theoretical calculations since some <em>d</em>isor<em>d</em>er in<em>d</em>uce<em>d</em> by packing constraints in the crystals of the free enzyme are absent in the crystals of the complex. The interface of the <em>dimer</em> is forme<em>d</em> by a His 10<em>2</em>-a<em>d</em>enine-a<em>d</em>enine-His 10<em>2</em> face-to-face ring stack <em>d</em>irectly on the <em>2</em>-fol<em>d</em> axis. The e<em>d</em>ge of the a<em>d</em>enine-a<em>d</em>enine stack packs closely onto the face of a 3'-cytosine-3'-cytosine interaction, which has a "base-pair"-like conformation but too great a separation of the bases to form hy<em>d</em>rogen bon<em>d</em>s. This unusual arrangement is the major stabilizing interaction within the <em>dimer</em>ic complex, since there are no <em>d</em>irect protein-protein interactions. Using the structure of the complex as a starting point for mo<em>d</em>el buil<em>d</em>ing, the nature of the enzyme-substrate an<em>d</em> enzyme-transition state complexes is investigate<em>d</em>.

The European Concerted Action on Thrombosis (ECAT) <em>D</em>VT Study was a collaborative study of preoperative haemostatic tests in prediction of <em>D</em>VT (diagnosed by routine bilateral venography) after elective hip replacement. 480 patients were recruited in 11 centres across Europe. Clinical risk factors were assessed, and stored citrated plasma aliquots were centrally assayed for <em>2</em>9 haemostatic factors according to the ECAT methodology. 1<em>2</em>0 (3<em>2</em>%) of 375 evaluable patients had <em>D</em>VT, and 41 (11%) had proximal <em>D</em>VT. Among clinical variables, <em>D</em>VT was significantly associated with increased age, obesity, and possibly non-use of stockings. Of the <em>2</em>9 haemostatic factors, mean preoperative levels were significantly higher in patients with subsequent <em>D</em>VT (on univariate analyses) for factor VIII activity, prothrombin fragment F1+<em>2</em>, thrombin-antithrombin complexes, and fibrin <em>D</em>-<em>dimer</em>; and significantly lower for APTT and APC sensitivity ratio. Factor V Leiden was also associated with <em>D</em>VT. Most of these variables were also associated with age, while <em>D</em>-<em>dimer</em> was higher in patients with varicose veins. On multivariate analyses including clinical variables, only a shorter APTT (locally but not centrally performed) and APC resistance showed a statistically significant association with <em>D</em>VT. We conclude that (a) <em>D</em>VT is common after elective hip replacement despite prophylaxis; (b) the study provides some evidence that <em>D</em>VT is associated with a preoperative hypercoaguable state; and (c) preoperative haemostatic tests do not add significantly to prediction of <em>D</em>VT from clinical variables, with the possible exception of APC resistance.

The well characterize<em>d</em> subunits of the bovine ATP synthase complex are the alpha, beta, gamma, <em>delta</em>, an<em>d</em> epsilon subunits of the catalytic sector, F1; the ATPase inhibitor protein; an<em>d</em> subunits a, b, c, an<em>d</em> <em>d</em>, OSCP (oligomycin sensitivity-conferring protein), F6, an<em>d</em> A6L, which are present in the membrane sector, F0, an<em>d</em> the 45-A-long stalk that connects F1 to F0. It has been shown recently that bovine ATP synthase preparations also contain three small polypepti<em>d</em>es, <em>d</em>esignate<em>d</em> e, f, an<em>d</em> g, with respective molecular masses of 8.<em>2</em>, 10. <em>2</em>, an<em>d</em> 11.3 kDa. To ascertain their involvement as bona fi<em>d</em>e subunits of the ATP synthase an<em>d</em> to investigate their membrane topography an<em>d</em> proximity to the above ATP synthase subunits, polyclonal antipepti<em>d</em>e antibo<em>d</em>ies were raise<em>d</em> in the rabbit to the COOH-terminal amino aci<em>d</em> resi<em>d</em>ues 57-70 of e, 75-86 of f, an<em>d</em> 91-10<em>2</em> of g. It was shown that (i) e, f, an<em>d</em> g coul<em>d</em> be immunoprecipitate<em>d</em> with anti-OSCP IgG from a fraction of bovine submitochon<em>d</em>rial particles enriche<em>d</em> in oligomycin-sensitive ATPase; (ii) the NH<em>2</em> termini of f an<em>d</em> g are expose<em>d</em> on the matrix si<em>d</em>e of the mitochon<em>d</em>rial inner membrane an<em>d</em> can be curtaile<em>d</em> by proteolysis; (iii) the COOH termini of all three polypepti<em>d</em>es are expose<em>d</em> on the cytosolic si<em>d</em>e of the inner membrane; an<em>d</em> (iv) f cross-links to A6L an<em>d</em> to g, an<em>d</em> e cross-links to g an<em>d</em> appears to form an e-e <em>dimer</em>. Thus, the bovine ATP synthase complex appears to have 16 unlike subunits, twice as many as its counterpart in Escherichia coli.