Specialist Palliative Care and Dementia: Staff Challenges and Learning Needs.

Objective: This study explored the perspectives of specialist palliative care (SPC) teams in Ireland, in relation to personal learning needs and education regarding dementia care. Methods: This mixed-methods study involved a survey and focus group. SPC staff were recruited through a professional palliative care society and via hospices in 4 regions. Survey items included challenges in clinical care, personal learning needs, and preferred modes of educational delivery. Quantitative data analysis was descriptive; open-answer survey questions and the focus group transcript underwent thematic analysis. Results: In total, 76 staff completed surveys and rated the following as most challenging: timely access to community agency and specialist support; and managing the needs of people with dementia (PwD). Respondents volunteered additional challenges around the timing/duration of SPC involvement, prognostication, and inadequate knowledge of local services. Staff ranked learning needs as highest in: nonpharmacological management of noncognitive and cognitive symptoms; differentiation of dementia subtypes; and pharmacological management of cognitive symptoms. The focus group (n = 4) gave deeper perspectives on these topics. Overall, 79.2% of staff preferred formal presentations by dementia-care specialists and 76.6% preferred e-learning. Conclusion: Several dementia-care challenges and learning needs are identified by SPC staff, as above. These can inform the design and delivery of tailored education programs for SPC staff. There is also a need for closer working between dementia services and SPC services to provide integrated, holistic care for PwD. One aspect of achieving this is greater awareness of local dementia-care services among SPC staff, and vice versa.

Dementia palliative care: A multi-site survey of long term care STAFF'S education needs and readiness to change.

Many people with dementia reside in long-term care, where limited staff knowledge of dementia palliative care has been identified, along with poor awareness that a palliative approach can assist in identifying unmet care needs. Evidence-based guidance in palliative care for people with dementia is available however, implementing this guidance requires staff engagement and a tailored educational approach. This pre-implementation situational analysis informed a tailored staff education intervention to support the implementation of national guidance on dementia palliative care in long term care. Using a cross-sectional study design, underpinned by the Consolidated Framework for Implementation Research, survey data were collected on site profile, staff demographics, learning needs, and readiness-to change at three residential care sites for older people in Ireland. In total, 69 staff (predominantly nurses and healthcare attendants) completed the surveys. Medication management and management of pain were the most frequently identified learning needs. Staff were confident in their ability to implement change but de-motivation and powerlessness were substantial factors as only one-third of staff were "ready for change". Staffing levels, managing risk during change and perceived reluctance in others were common barriers. These results informed an educational intervention to address the specific care context, staff learning needs and barriers to change prior to implementation.

Potentially modifiable determinants of malnutrition in older adults: A systematic review.

BACKGROUND & AIMS: Malnutrition in older adults results in significant personal, social, and economic burden. To combat this complex, multifactorial issue, evidence-based knowledge is needed on the modifiable determinants of malnutrition. Systematic reviews of prospective studies are lacking in this area; therefore, the aim of this systematic review was to investigate the modifiable determinants of malnutrition in older adults. METHODS: A systematic approach was taken to conduct this review. Eight databases were searched. Prospective cohort studies with participants of a mean age of 65 years or over were included. Studies were required to measure at least one determinant at baseline and malnutrition as outcome at follow-up. Study quality was assessed using a modified version of the Quality in Prognosis Studies (QUIPS) tool. Pooling of data in a meta-analysis was not possible therefore the findings of each study were synthesized narratively. A descriptive synthesis of studies was used to present results due the heterogeneity of population source and setting, definitions of determinants and outcomes. Consistency of findings was assessed using the schema: strong evidence, moderate evidence, low evidence, and conflicting evidence. RESULTS: Twenty-three studies were included in the final review. Thirty potentially modifiable determinants across seven domains (oral, psychosocial, medication and care, health, physical function, lifestyle, eating) were included. The majority of studies had a high risk of bias and were of a low quality. There is moderate evidence that hospitalisation, eating dependency, poor self-perceived health, poor physical function and poor appetite are determinants of malnutrition. Moderate evidence suggests that chewing difficulties, mouth pain, gum issues co-morbidity, visual and hearing impairments, smoking status, alcohol consumption and physical activity levels, complaints about taste of food and specific nutrient intake are not determinants of malnutrition. There is low evidence that loss of interest in life, access to meals and wheels, and modified texture diets are determinants of malnutrition. Furthermore, there is low evidence that psychological distress, anxiety, loneliness, access to transport and wellbeing, hunger and thirst are not determinants of malnutrition. There appears to be conflicting evidence that dental status, swallowing, cognitive function, depression, residential status, medication intake and/or polypharmacy, constipation, periodontal disease are determinants of malnutrition. CONCLUSION: There are multiple potentially modifiable determinants of malnutrition however strong robust evidence is lacking for the majority of determinants. Better prospective cohort studies are required. With an increasingly ageing population, targeting modifiable factors will be crucial to the effective treatment and prevention of malnutrition.

Five short screening tests in the detection of prevalent delirium: diagnostic accuracy and performance in different neurocognitive subgroups.

BACKGROUND: Delirium is prevalent and serious, yet remains under-recognised. Systematic screening could improve detection; however, consensus is lacking as to the best approach. Our aim was to assess the diagnostic accuracy of five simple cognitive tests in delirium screening: six-item cognitive impairment test (6-CIT), clock-drawing test, spatial span forwards, months of the year backwards (MOTYB) and intersecting pentagons (IPT). METHODS: A cross-sectional study was conducted. Within 36 h of admission, older medical patients were assessed for delirium using the Revised Delirium Rating Scale. They also underwent testing using the five cognitive tests outlined above. Sensitivity, specificity, positive and negative predictive values (PPV; NPV) were calculated for each method. Where appropriate, area under the receiver operating characteristic curve (AUC) was also calculated. RESULTS: Four hundred seventy patients were included, and 184 had delirium. Of the tests scored on a scale, the 6-CIT had the highest AUC (0.876), the optimum cut-off for delirium screening being 8/9 (sensitivity 89.9%, specificity 62.7%, NPV 91.2%, PPV 59.2%). The MOTYB, scored in a binary fashion, also performed well (sensitivity 84.6%, specificity 58.4%, NPV 87.4%, PPV 52.8). On discriminant analysis, 6-CIT was the only test to discriminate between patients with delirium and those with dementia (without delirium), Wilks' Lambda = 0.748, p < 0.001. CONCLUSION: The 6-CIT measures attention, temporal orientation and short-term memory and shows promise as a delirium screening test. This study suggests that it may also have potential in distinguishing the cognitive impairment of delirium from that of dementia in older patients. Copyright © 2016 John Wiley & Sons, Ltd.

Frequency of delirium and subsyndromal delirium in an adult acute hospital population.

Background The frequency of full syndromal and subsyndromal delirium is understudied. Aims We conducted a point prevalence study in a general hospital. Method Possible delirium identified by testing for inattention was evaluated regarding delirium status (full/subsyndromal delirium) using categorical (Confusion Assessment Method (CAM), DSM-IV) and dimensional (Delirium Rating Scale-Revised-98 (DRS-R98) scores) methods. Results In total 162 of 311 patients (52%) screened positive for inattention. Delirium was diagnosed in 55 patients (17.7%) using DSM-IV, 52 (16.7%) using CAM and 58 (18.6%) using DRS-R98⩾12 with concordance for 38 (12.2%) individuals. Subsyndromal delirium was identified in 24 patients (7.7%) using a DRS-R98 score of 7-11 and 41 (13.2%) using 2/4 CAM criteria. Subsyndromal delirium with inattention (v. without) had greater disturbance of multiple delirium symptoms. Conclusions The point prevalence of delirium and subsyndromal delirium was 25%. There was modest concordance between DRS-R98, DSM-IV and CAM delirium diagnoses. Inattention should be central to subsyndromal delirium definitions.

Delirium: a key challenge for perioperative care.

Delirium is highly prevalent, occurring in 20% of acute hospital inpatients and up to 62% of surgical patients. It is a significant predictor of poor outcomes including mortality and institutionalisation, however it is often viewed as simply a marker of underlying illness and is frequently overlooked in older adults. Although delirium is commonly comorbid with dementia, it represents a more urgent diagnosis, requiring prompt intervention. Delirium presents most commonly with hypoactive features (e.g. withdrawal and reduced spontaneous movement and speech). The common stereotype of hyperactive delirium tremens (e.g. agitation, hallucinations), although more visible, is less common. All presentations share acute disimprovement of cognitive function. Delirium is a highly predictable and preventable occurrence, however a major barrier to improving delirium care and impacting upon outcomes is that it remains poorly detected, particularly in surgical populations and especially in patients with hypoactive presentations. Routine ward-based screening for delirium, particularly in high-risk populations, and improved staff awareness of the significance of the problem can improve detection rates. Preventative strategies, particularly multicomponent approaches, have been most efficacious in improving patient outcomes. Optimising perioperative risk factors can lead to reduced incidence. Appropriate treatment of delirium requires thorough investigation, management of the underlying illness, avoidance of complications and simplification of the care environment. Studies suggest a role for pharmacological prophylaxis, particularly in relation to anaesthetic and sedative agents used intra- and post-operatively. Furthermore, gathering evidence suggests that judicious use of antipsychotic medications may be helpful in delirium prevention and treatment.

Risk factors for pulmonary embolism in an Irish patient cohort.

INTRODUCTION: The prevention of pulmonary embolism (PE) is an important component of medical care. AIM: To examine the risk factors for venous thromboembolism in an Irish patient cohort with acute PE, and identify cases that may have been preventable. METHODS: Retrospective review of 60 consecutive cases of computed tomography (CT)-confirmed acute PE. RESULTS: The primary thromboembolic risk factors were elective surgery (27%), medical illness (20%), primary immobility (13%) and isolated distal lower limb fracture (7%). A significant proportion (43%) had been hospitalised within the six weeks prior to PE onset. Some patients had undergone 'low risk' procedures, without prophylaxis, but had other significant thromboembolic risk factors that indicated a requirement for prophylaxis. CONCLUSIONS: Hospital- and ward-based thromboprophylaxis guidelines, based on certain categories of patient or procedure, need to be routinely supplemented by an individual risk factor assessment for each patient, to determine those at particularly high risk for venous thromboembolism.

Effects of fiberoptic bronchoscopy on intracranial pressure in patients with brain injury: a prospective clinical study.

BACKGROUND: Fiberoptic bronchoscopy (FB) plays an important role in making the diagnosis of nosocomial pneumonia and resolving lobar atelectasis in critically injured trauma patients. It has been shown to be a safe procedure with only occasional complications. However, in patients with head injuries, FB can lead to intracranial hypertension. Sustained increases in intracranial pressure (ICP) leads to poor outcome in these patients. Because of this, a prospective study was done not only to assess the effect of FB on ICP and cerebral perfusion pressure (CPP) in patients with brain injuries, but also to identify a regimen of sedation and anesthesia that could prevent significant increases in ICP during FB. METHODS: Twenty-six FB were performed in 23 patients with ICP monitors or ICP monitors and ventriculostomy drains in place for Glasgow Coma Scale score < 8 or management of postcraniotomy trauma. FB was performed to aid in the diagnosis of nosocomial pneumonia or to aid in resolving lobar atelectasis. Before FB, all patients received a standard anesthetic regimen consisting of vecuronium (10 mg), morphine sulfate (4 mg), and midazolam (2.5 mg). Patients with diminished cranial compliance, defined as ICP > 10 mm Hg, also received a nebulizer treatment of 3 mL of 4% lidocaine before FB. All patients were preoxygenated with FIO2 = 1.0 for 10 minutes. Intracranial pressure, mean arterial pressure, and CPP were monitored continuously throughout the procedure. These same variables were also recorded at baseline and at 2-minute intervals during the procedure. The time to return to baseline ICP was also recorded. RESULTS: The mean ICP at baseline (immediately before FB) was 12.6 mm Hg. After introduction of the bronchoscope, the ICP rapidly increased in 21 procedures (81%) and the mean highest ICP was 38.0 mm Hg. There was also a concomitant increase in mean arterial pressure such that there was no substantial change in CPP. The mean lowest CPP was 73.1 mm Hg. The average time for return of ICP to baseline was 13.9 minutes. In the subgroup of patients with ICP > 10, attempting to blunt the tracheal stimulation by anesthetizing the trachea with 4% nebulized lidocaine did not seem to be successful. The mean highest ICP in this subgroup was 41.8 mm Hg. The CPP changed in a similar manner, as the mean lowest CPP was 74.0 mm Hg. The mean time to return to baseline was 12.5 minutes. No patient had acute neurologic deterioration secondary to FB. CONCLUSIONS: Although FB is an important procedure in the pulmonary care of head injured patients, it produces substantial, but transient, increases in ICP and should be used with caution in patients with diminished cranial compliance. Sedation, analgesia, paralysis, and topical tracheal anesthesia did not completely prevent the rise in ICP. Although no acute deterioration in condition occurred, secondary brain injury caused by localized cerebral ischemia is certainly possible. Because of the substantial increases in ICP, herniation may be precipitated in an occasional patient. Further study is needed to identify a regimen that will confer protection.

Inflammatory neutrophils retain susceptibility to apoptosis mediated via the Fas death receptor.

Apoptosis and clearance of neutrophils is essential for successful resolution of inflammation. Altered signaling via the Fas receptor could explain the observed prolongation of neutrophil lifespan and associated tissue injury at inflammatory sites. We therefore compared inflammatory neutrophils extracted from joints of rheumatoid arthritis patients, with peripheral blood neutrophils. Inflammatory neutrophils underwent constitutive apoptosis in culture more rapidly than peripheral blood neutrophils; this was not explained by changes in surface expression of Fas or by induction of Fas ligand. Inflammatory neutrophils remained sensitive to Fas-induced death, at levels comparable to those seen in peripheral blood neutrophils. Similarly, granulocyte-macrophage colony-stimulating factor reduced apoptosis but did not abolish signaling via Fas. These data provide evidence for the rate of apoptosis in inflammatory neutrophils being continually modulated by death and survival signals in the inflammatory milieu. This allows for rapid resolution of inflammation as levels of survival factors fall, and suggests new strategies for inducing resolution of inflammation.

Peptide-directed suppression of a pro-inflammatory cytokine response.

Signal-dependent nuclear translocation of transcription factor nuclear factor kappaB (NF-kappaB) is required for the activation of downstream target genes encoding the mediators of immune and inflammatory responses. To inhibit this inducible signaling to the nucleus, we designed a cyclic peptide (cSN50) containing a cell-permeable motif and a cyclized form of the nuclear localization sequence for the p50-NF-kappaB1 subunit of NF-kappaB. When delivered into cultured macrophages treated with the pro-inflammatory agonist lipopolysaccharide, cSN50 was a more efficient inhibitor of NF-kappaB nuclear import than its linear analog. When delivered into mice challenged with lipopolysaccharide, cSN50 potently blocked the production of proinflammatory cytokines (tumor necrosis factor alpha and interferon gamma) and significantly reduced the lethality associated with ensuing endotoxic shock. Based on specificity studies conducted with a mutated form of cSN50, a functional nuclear localization motif is required for this protective effect. Taken together, our findings demonstrate effective targeting of a cell-permeable peptide that attenuates cytokine signaling in vivo. This new class of biological response modifiers may be applicable to the control of systemic inflammatory reactions.

IkappaB kinase complex is an intracellular target for endotoxic lipopolysaccharide in human monocytic cells.

Endotoxic lipopolysaccharide (LPS) is a proinflammatory agonist produced by gram-negative bacteria and a contributor to the majority of the 400,000 septic shock cases recorded annually in US hospitals. The primary target cells for LPS are monocytes and macrophages. Their response consists of massive production of proinflammatory cytokines, reactive oxygen- and nitrogen-intermediates, procoagulants, and cell adhesion molecules. In turn, expression of these LPS-responsive factors contributes to collapse of the circulatory system, to disseminated intravascular coagulation, and to a 30% mortality rate. A common intracellular mechanism responsible for the expression of septic shock genes in monocytes and macrophages involves the activation of NF-kappaB. This transcription factor is regulated by a family of structurally related inhibitors including IkappaBalpha, IkappaBbeta, and IkappaBepsilon, which trap NF-kappaB in the cytoplasm. In this report, the investigators show that LPS derived from different gram-negative bacteria activates cytokine-responsive IkappaB kinases containing catalytic subunits termed IKKalpha (IKK1) and IKKbeta (IKK2). The kinetics of IKKalpha and IKKbeta activation in LPS-stimulated human monocytic cells differ from that recorded on their stimulation with tumor necrosis factor-alpha, thereby implying a distinct activation mechanism. LPS-activated IKK complexes phosphorylate all 3 inhibitors of NF-kappaB: IkappaBalpha, IkappaBbeta, and IkappaBepsilon. Moreover, LPS activates IKKbeta preferentially, relative to IKKalpha. Thus, IKK complex constitutes the main intracellular target for LPS-induced NF-kappaB signaling to the nucleus in human monocytic cells to activate genes responsible for septic shock.

Preparation of functionally active cell-permeable peptides by single-step ligation of two peptide modules.

Noninvasive cellular import of synthetic peptides can be accomplished by incorporating a hydrophobic, membrane-permeable sequence (MPS). Herein, we describe a facile method that expedites synthesis of biologically active, cell-permeable peptides by site-specific ligation of two free peptide modules: one bearing a functional sequence and the second bearing a MPS. A nonpeptide thiazolidino linkage between the two modules is produced by ligation of the COOH-terminal aldehyde on the MPS and the NH2-terminal 1, 2-amino thiol moiety on the functional sequence. This thiazolidine ligation approach is performed with stoichiometric amounts of fully unprotected MPS and functional peptide in an aqueous buffered solution, eliminating the need for additional chemical manipulation and purification prior to use in bioassays. Two different MPSs were interchangeably combined with two different functional sequences to generate two sets of hybrid peptides. One set of hybrid peptides, carrying the cytoplasmic cell adhesion regulatory domain of the human integrin beta3, inhibited adhesion of human erythroleukemia cells to fibrinogen-coated surfaces. A second set of hybrid peptides, carrying the nuclear localization sequence of the transcription factor NF-kappaB, inhibited nuclear import of transcription factors NF-kappaB, activator protein 1, and nuclear factor of activated T cells in agonist-stimulated Jurkat T lymphocytes. In each assay, these nonamide bond hybrids were found to be functionally comparable to peptides prepared by the conventional method. Cumulatively, this new ligation approach provides an easy and rapid method for engineering of functional, cell-permeable peptides and demonstrates the potential for synthesis of cell-permeable peptide libraries designed to block intracellular protein-protein interactions.

Identification of a functionally important sequence in the cytoplasmic tail of integrin beta 3 by using cell-permeable peptide analogs.

Integrins are major two-way signaling receptors responsible for the attachment of cells to the extracellular matrix and for cell-cell interactions that underlie immune responses, tumor metastasis, and progression of atherosclerosis and thrombosis. We report the structure-function analysis of the cytoplasmic tail of integrin beta 3 (glycoprotein IIla) based on the cellular import of synthetic peptide analogs of this region. Among the four overlapping cell-permeable peptides, only the peptide carrying residues 747-762 of the carboxyl-terminal segment of integrin beta 3 inhibited adhesion of human erythroleukemia (HEL) cells and of human endothelial cells (ECV) 304 to immobilized fibrinogen mediated by integrin beta 3 heterodimers, alpha IIb beta 3, and alpha v beta 3, respectively. Inhibition of adhesion was integrin-specific because the cell-permeable beta 3 peptide (residues 747-762) did not inhibit adhesion of human fibroblasts mediated by integrin beta 1 heterodimers. Conversely, a cell-permeable peptide representing homologous portion of the integrin beta 1 cytoplasmic tail (residues 788-803) inhibited adhesion of human fibroblasts, whereas it was without effect on adhesion of HEL or ECV 304 cells. The cell-permeable integrin beta 3 peptide (residues 747-762) carrying a known loss-of-function mutation (Ser752Pro) responsible for the genetic disorder Glanzmann thrombasthenia Paris I did not inhibit cell adhesion of HEL or ECV 304 cells, whereas the beta 3 peptide carrying a Ser752Ala mutation was inhibitory. Although Ser752 is not essential, Tyr747 and Tyr759 form a functionally active tandem because conservative mutations Tyr747Phe or Tyr759Phe resulted in a nonfunctional cell permeable integrin beta 3 peptide. We propose that the carboxyl-terminal segment of the integrin beta 3 cytoplasmic tail spanning residues 747-762 constitutes a major intracellular cell adhesion regulatory domain (CARD) that modulates the interaction of integrin beta 3-expressing cells with immobilized fibrinogen. Import of cell-permeable peptides carrying this domain results in inhibition "from within" of the adhesive function of these integrins.

Isolation and sequencing of cDNAs for proteins with multiple domains of Gly-Xaa-Yaa repeats identify a distinct family of collagenous proteins.

We have isolated overlapping mouse cDNAs encoding a collagenous polypeptide that we have designated alpha 1(XVIII) collagen. Nucleotide sequence analysis shows that alpha 1(XVIII) collagen contains 10 triple-helical domains separated and flanked by non-triple-helical regions. Within the non-triple-helical regions, there are several Ser-Gly-containing sequences that conform to consensus sequences for glycosaminoglycan attachment sites in proteoglycan core proteins. Northern blots show that alpha 1(XVIII) transcripts are present in multiple organs, with the highest levels in liver, lung, and kidney. We have also isolated overlapping cDNAs encoding human alpha 1(XV) collagen, and their sequence extends a published partial alpha 1(XV) sequence to the 3' end. Comparison of the alpha 1(XV) and alpha 1(XVIII) sequences reveals a striking similarity in the lengths of the six most carboxyl-terminal triple-helical domains. In addition, within the carboxyl non-triple-helical domain NC1 of the two chains, a region of 177 amino acid residues shows about 60% identity at the amino acid level. We suggest, therefore, that alpha 1(XV) and alpha 1(XVIII) collagens are structurally related. Their structure is different from that of other known collagen types. We conclude that they belong to a subfamily of extracellular matrix proteins and we suggest the designation multiplexins (for protein with multiple triple-helix domains and interruptions) for members of this subfamily.

Cloning of cDNA and genomic DNA encoding human type XVIII collagen and localization of the alpha 1(XVIII) collagen gene to mouse chromosome 10 and human chromosome 21.

Types XV and XVIII collagen belong to a unique and novel subclass of the collagen superfamily for which we have proposed the name the MULTIPLEXIN family. Members of this class contain polypeptides with multiple triple-helical domains separated and flanked by non-triple-helical regions. In this paper, we report the isolation of human cDNAs and genomic DNAs encoding the alpha 1(XVIII) collagen chain. Utilizing a genomic clone as probe, we have mapped the COL18A1 gene to chromosome 21q22.3 by fluorescence in situ hybridization. In addition, using an interspecific backcross panel, we have shown that the murine Col18a1 locus is on chromosome 10, close to the loci for Col6a1 and Col6a2.

Complications of enterocystoplasty.

Bladder reconstruction, either by augmentation or substitution enterocystoplasty, is a safe alternative to supravesical urinary diversion providing careful attention to preoperative selection, surgical technique, and postoperative review is observed. However, under the most optimal conditions an untoward outcome may occur. We reviewed our series of 100 intestinocystoplasties to categorize the types of complications encountered, and to identify preoperative risk factors that could potentially develop into an unfavorable sequela. Twenty-seven patients required either early or late surgical intervention, while 30 were managed nonoperatively. In our review we identified two groups, those with myelodysplasia and those with a solitary functioning kidney, who are at a higher risk for an unfavorable outcome to develop.

Management of type III stress urinary incontinence using artificial urinary sphincter.

Type III stress urinary incontinence due to severe intrinsic urethral weakness without significant urethrovesical descensus may be treated by periurethral injection, sling cystourethropexy, bladder neck reconstruction, or artificial urinary sphincter implantation. The rationale for procedure selection depends on a number of patient factors and the surgeon's experience. We herein report on 25 women who were identified as having such incontinence by evaluation which included videourodynamic study and lateral voiding cystography and who were managed by the implantation of an artificial urinary sphincter. The etiology of the severe intrinsic urethral weakness in most patients was multiple prior failed cystourethropexies. Postoperatively, 1 patient died of a cerebral vascular accident. The remaining 24 women had significantly improved continence and were completely satisfied at latest follow-up. No revisions have been required for patients receiving an artificial sphincter after 1983. No sphincter erosions or infections have occurred. Our experience and review of the literature shows that the artificial sphincter provides an excellent first option for women with type III urinary stress incontinence due to intrinsic urethral weakness of various etiologies.

Platelet receptor recognition domains on the alpha chain of human fibrinogen: structure-function analysis.

We have previously shown that the alpha chain of human fibrinogen interacts directly with ADP-activated human platelets [Hawiger, J., Timmons, S., Kloczewiak, M., Strong, D. D., & Doolittle, R. F. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 2068]. Now, we report that platelet receptor recognition domains are localized on two CNBr fragments of the human fibrinogen alpha chain. They encompass residues 92-147 and 518-584, which inhibit 125I-fibrinogen binding to ADP-stimulated platelets. The inhibitory CNBr fragment alpha 92-147 contains the RGD sequence at residues 95-97. Synthetic peptides encompassing this sequence were inhibitory while peptide 99-113 lacking the RGD sequence was inactive. The synthetic peptide RGDF, corresponding to residues alpha 95-98, inhibited the binding of 125I-fibrinogen to ADP-treated platelets (IC50 = 2 microM). However, the peptides containing sequence RGDF, with residues preceding Arg95 or following Phe98, were less inhibitory. It appears that the sequence alpha 95-98 constitutes a platelet receptor recognition domain which is constrained by flanking residues. The second inhibitory CNBr fragment, alpha 518-584, also contains the sequence RGD at positions 572-574. Synthetic peptides overlapping this sequence were inhibitory, while peptides lacking the sequence RGDS were not reactive. Thus, another platelet reactive site on the alpha chain encompasses residues 572-575 containing sequence RGDS. In conclusion, the platelet receptor recognition domains on the human fibrinogen alpha chain in the amino-terminal and in the carboxy-terminal zones contain the ubiquitous cell recognition sequence RGD shared with other known adhesive proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Platelet receptor recognition domain on the gamma chain of human fibrinogen and its synthetic peptide analogues.

We have shown previously that the domain recognizing receptors on activated human platelets is located on the human fibrinogen gamma chain between residues 400 and 411 [Kloczewiak, M., Timmons, S., Lukas, T. J., & Hawiger, J. (1984) Biochemistry 23, 1767]. To study the correlation between the structure of this segment of the gamma chain and its reactivity toward receptors on ADP-activated human platelets, we designed a series of analogues containing replacements at 9 out of 12 positions. A double substitution of the normal His400-His401 sequence by Ala-Ala reduced the inhibitory potency of the dodecapeptide 3-fold. When Lys406 was replaced by Arg, the inhibitory potency of the dodecapeptide decreased 15 times. On the other hand, substitution of Ala408 with Arg increased the inhibitory potency of the dodecapeptide 6-fold. A drastic decrease in the reactivity of the dodecapeptide toward platelet receptors was observed when Val411 was replaced by leucine or cysteine or tyrosine. A 3-fold decrease in reactivity was noted when Val411 was substituted with phenylalanine. Amidation of the carboxy-terminal Val411 also produced a significant decrease in dodecapeptide reactivity. With seven residues (His400, His401, Leu402, Lys406, Gln407, Asp410, and Val411) preserved, substitution of the intervening five amino acids with nonpolar leucine or polar serine, increasing or decreasing the hydrophobicity of the dodecapeptide, reduced more than 16-fold its inhibitory potency. Rabbit antibody Fab fragments directed against the human fibrinogen gamma-chain peptide encompassing residues 385-411 inhibited 50% of 125I-fibrinogen binding at a 2:1 stoichiometry with regard to 125I-fibrinogen.(ABSTRACT TRUNCATED AT 250 WORDS)

Antiplatelet "hybrid" peptides analogous to receptor recognition domains on gamma and alpha chains of human fibrinogen.

Platelet receptor recognition domains are located on the gamma and alpha chains of human fibrinogen. The former encompasses residues 400-411 [Kloczewiak, M., Timmons, S., Lukas, T. J., & Hawiger, J. (1984) Biochemistry 23, 1767], and the latter is present in two loci on the alpha chain (alpha 95-97 and alpha 572-574) [Hawiger, J., Kloczewiak, M., Bednarek, M. A., & Timmons, S. (1989) Biochemistry (first of three papers in this issue)]. Peptide gamma 400-411 (HHLGGAKQAGDV) inhibited aggregation of ADP-treated platelets mediated not only by gamma-chain but also by alpha-chain multimers. Peptide alpha 572-575 (RGDS) inhibited aggregation of platelets mediated by alpha-chain as well as gamma-chain multimers. These results indicate that the platelet receptor for fibrinogen is isospecific with regard to the domain present on alpha and gamma chains. Subsequent "checkerboard" analysis of combinations of gamma 400-411 and alpha 572-575 showed that the inhibitory effect toward binding of 125I-fibrinogen was additive rather than synergistic. Next, a series of "hybrid" peptides was constructed in which the alpha-chain sequence RGDF (alpha 95-98) replaced the carboxy-terminal segment of gamma 408-411. The dodecapeptide HHLGGAKQRGDF was inhibitory with concentration, causing 50% inhibition of binding (IC50) at 6 microM, 5 times more potent than gamma 400-411. The shorter peptides AKQRGDF and KQRGDF were also more inhibitory than gamma 400-411. The second series of hybrid peptides was constructed with the alpha-chain sequence RGDS preceding the sequence of gamma 400-411 or sequence RGDV following it.(ABSTRACT TRUNCATED AT 250 WORDS)