Next Generation Sequencing of Human Platelet Antigens for Routine Clinical Investigations and Donor Screening.

Human platelet antigen (HPA) genotyping is performed in a number of clinical scenarios, including characterization of immune-mediated thrombocytopenia and provision of HPA-matched platelets. Current gold-standard methods for HPA genotyping utilize single nucleotide variant (SNV) based approaches. This review aims to ascertain if next generation sequencing (NGS) has reasonable grounds to replace SNV-based genotyping for HPA systems. A systematic review was conducted following a comprehensive literature search in accordance with the Preferred Reporting Items for Systematic Review and Meta-Analysis guidelines. Studies were subjected to screening based on a defined set of inclusion/exclusion criteria. Study quality, characteristics and results were extracted and a meta-analysis was performed to assess the concordance of HPA genotyping results between NGS and the SNV-based comparators for HPA-1,-2,-3,-4,-5,-15. In total, 3374 potentially eligible articles were identified, only 6 of which were included in the meta-analysis. The pooled proportion agreement for the overall concordance of the 6 included studies was shown to be 0.998, 95%CI [0.995, 0.999], P < .001. The discrepancies between HPA genotypes obtained by the two platforms were due to allele dropout in real-time PCR, thus discordant results were in favor of NGS over SNV-based comparators. Currently available platforms for NGS are not without their limitations, including high upfront and ongoing costs, data management and storage, accurate variant calling and availability of appropriately trained staff. Despite the high level of concordance between NGS and current gold-standard methods, these significant challenges mean that NGS is currently not viable as a stand-alone technique for HPA typing.

Evolution of the Fermi surface of BiTeCl with pressure.

We report measurements of Shubnikov-de Haas oscillations in the giant Rashba semiconductor BiTeCl under applied pressures up to ∼2.5 GPa. We observe two distinct oscillation frequencies, corresponding to the Rashba-split inner and outer Fermi surfaces. BiTeCl has a conduction band bottom that is split into two sub-bands due to the strong Rashba coupling, resulting in two spin-polarized conduction bands as well as a Dirac point. Our results suggest that the chemical potential lies above this Dirac point, giving rise to two Fermi surfaces. We use a simple two-band model to understand the pressure dependence of our sample parameters. Comparing our results on BiTeCl to previous results on BiTeI, we observe similar trends in both the chemical potential and the Rashba splitting with pressure.

Pressure-induced superconductivity in the giant Rashba system BiTeI.

At ambient pressure, BiTeI exhibits a giant Rashba splitting of the bulk electronic bands. At low pressures, BiTeI undergoes a transition from trivial insulator to topological insulator. At still higher pressures, two structural transitions are known to occur. We have carried out a series of electrical resistivity and AC magnetic susceptibility measurements on BiTeI at pressure up to ∼40 GPa in an effort to characterize the properties of the high-pressure phases. A previous calculation found that the high-pressure orthorhombic P4/nmm structure BiTeI is a metal. We find that this structure is superconducting with T c values as high as 6 K. AC magnetic susceptibility measurements support the bulk nature of the superconductivity. Using electronic structure and phonon calculations, we compute T c and find that our data is consistent with phonon-mediated superconductivity.

Early tumor response to intraarterial or intravenous administration of carboplatin to treat naturally occurring lower urinary tract carcinoma in dogs.

BACKGROUND: Survival times and tumor responses associated with malignant neoplasia of the lower urinary tract are poor despite the vast array of current treatments. Therefore, the evaluation of alternative treatments, such as intraarterial administration of chemotherapy (IAC) should be considered. OBJECTIVE: To describe a technique for superselective catheterization for IAC and to evaluate initial tumor response by ultrasonography after both IAC and intravenous administration of chemotherapy (IVC). ANIMALS: Client-owned dogs with lower urinary tract neoplasia treated with either IVC (n = 15) or IAC (n = 11). METHODS: Retrospective study. An arterial approach via the carotid or femoral artery was utilized to obtain superselective access and administer chemotherapy in the IAC cases. Medical record review was performed, data were recorded, and recorded variables were evaluated statistically. RESULTS: Intraarterial chemotherapy was successfully administered in all cases. There was a significantly greater decrease in longest unidimensional measurement in the IAC group as compared to the IVC group (P = .013). The IAC group was also significantly more likely to have a tumor response as assessed by modified RECIST guidelines (P = .049). Dogs in the IAC group were significantly less likely to develop anemia (P = .001), lethargy (P = .010) and anorexia (P = .024). CONCLUSION AND CLINICAL IMPORTANCE: This study demonstrated the feasibility and efficacy of performing IAC for lower urinary tract neoplasia. Further investigation is necessary as the follow-up time was short and the impact on long-term outcome and survival was not determined.

A comprehensive ovine model of blood transfusion.

BACKGROUND: The growing awareness of transfusion-associated morbidity and mortality necessitates investigations into the underlying mechanisms. Small animals have been the dominant transfusion model but have associated limitations. This study aimed to develop a comprehensive large animal (ovine) model of transfusion encompassing: blood collection, processing and storage, compatibility testing right through to post-transfusion outcomes. MATERIALS AND METHODS: Two units of blood were collected from each of 12 adult male Merino sheep and processed into 24 ovine-packed red blood cell (PRBC) units. Baseline haematological parameters of ovine blood and PRBC cells were analysed. Biochemical changes in ovine PRBCs were characterized during the 42-day storage period. Immunological compatibility of the blood was confirmed with sera from potential recipient sheep, using a saline and albumin agglutination cross-match. Following confirmation of compatibility, each recipient sheep (n = 12) was transfused with two units of ovine PRBC. RESULTS: Procedures for collecting, processing, cross-matching and transfusing ovine blood were established. Although ovine red blood cells are smaller and higher in number, their mean cell haemoglobin concentration is similar to human red blood cells. Ovine PRBC showed improved storage properties in saline-adenine-glucose-mannitol (SAG-M) compared with previous human PRBC studies. Seventy-six compatibility tests were performed and 17·1% were incompatible. Only cross-match compatible ovine PRBC were transfused and no adverse reactions were observed. CONCLUSION: These findings demonstrate the utility of the ovine model for future blood transfusion studies and highlight the importance of compatibility testing in animal models involving homologous transfusions.

Treatment with quercetin and 3',4'-dihydroxyflavonol inhibits platelet function and reduces thrombus formation in vivo.

Flavonols are polyphenolic compounds with reported cardiovascular benefits and have been shown to exhibit antiplatelet properties in vitro. While some studies have shown inhibition of platelet aggregation following dietary supplementation with flavonol rich foods, few studies have assessed the ability of flavonols to inhibit platelet mediated thrombus generation in vivo. Furthermore, the duration of benefit and the influence of different dosing regimens remain unclear. In this study we investigate the ability of two structurally related flavonols; quercetin (Que) and 3',4'-dihydroxyflavonol (DiOHF) to inhibit platelet aggregation, platelet granule exocytosis and vessel occlusion in a well characterized mouse model of platelet mediated arterial thrombosis. We investigated the effect of a single 6 mg/kg intravenous bolus and daily 6 mg/kg intraperitoneal doses over seven consecutive days. Carotid artery blood flow after injury was better maintained in mice treated with both Que and DiOHF when compared to the vehicle for both dosage regimens. This improved blood flow corresponded to inhibition of platelet aggregation and platelet dense granule exocytosis following chemical stimulation of PAR4. We therefore provide evidence of inhibition of platelet-mediated arterial thrombosis by flavonols in vivo, and demonstrate that this effect persists for at least 24 h after the last intraperitoneal dose. These data suggest a potential clinical role for flavonols as anti-platelet therapy.

A platelet tetraspanin superfamily member, CD151, is required for regulation of thrombus growth and stability in vivo.

BACKGROUND: This study was designed to determine the role of CD151 in platelet thrombus formation in vivo and define the contribution of platelet vs. endothelial CD151 in regulating platelet thrombus formation in vivo. METHODS AND RESULTS: Using intravital microscopy and ferric chloride (FeCl(3)) injury of mesenteric arterioles, we found that thrombi formed in CD151(+/-) and CD151(-/-) mice were smaller and less stable, than those formed in CD151(+/+) mice, with a tendency for embolization. Similarly, in Folt's FeCl(3)-induced carotid injury model, both CD151(+/-) and CD151(-/-) mice showed more prolonged times to 95% vessel occlusion than CD151(+/+) mice. In addition, laser-induced injury of cremaster muscle arterioles showed that thrombi formed in CD151(+/-) and CD151(-/-) mice were smaller and less stable than those formed in CD151(+/+) mice. Following platelet depletion/reconstitution with ex vivo-labeled donor platelets, platelet-depleted CD151(+/+) mice that received reconstitution with CD151(-/-) platelets had smaller thrombi that were unstable and embolized. In contrast, platelet-depleted CD151(-/-) mice that received reconstitution with CD151(+/+) platelets had normal thrombi that were stable. CONCLUSIONS: These data provide evidence that platelet CD151 is required for regulating thrombus formation in vivo.

Platelet endothelial cell adhesion molecule-1 is a negative regulator of platelet-collagen interactions.

The functional importance of platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) in platelets is unclear. Because PECAM-1 represents a newly assigned immunoglobulin-ITIM superfamily member expressed on the surface of platelets, it was hypothesized that it may play an important regulatory role in modulating ITAM-bearing receptors such as collagen (GP)VI receptor and FcgammaRIIA. To examine the functional role of PECAM-1 in regulating platelet-collagen interactions, 2 different approaches were applied using recombinant human PECAM-1-immunoglobulin chimeras and platelets derived from PECAM-1-deficient mice. Stimulation of platelets by collagen-, (GP)VI-selective agonist, collagen-related peptide (CRP)-, and PECAM-1-immunoglobulin chimera induced tyrosine phosphorylation of PECAM-1 in a time- and dose-dependent manner. Activation of PECAM-1 directly through the addition of soluble wild-type PECAM-1-immunoglobulin chimera, but not mutant K89A PECAM-1-immunoglobulin chimera that prevents homophilic binding, was found to inhibit collagen- and CRP-induced platelet aggregation. PECAM-1-deficient platelets displayed enhanced platelet aggregation and secretion responses on stimulation with collagen and CRP, though the response to thrombin was unaffected. Under conditions of flow, human platelet thrombus formation on a collagen matrix was reduced in a dose-dependent manner by human PECAM-1-immunoglobulin chimera. Platelets derived from PECAM-1-deficient mice form larger thrombi when perfused over a collagen matrix under flow at a shear rate of 1800 seconds(-1) compared to wild-type mice. Collectively, these results indicate that PECAM-1 serves as a physiological negative regulator of platelet-collagen interactions that may function to negatively limit growth of platelet thrombi on collagen surfaces.

Src homology 2 domain-containing protein-tyrosine phosphatases, SHP-1 and SHP-2, are required for platelet endothelial cell adhesion molecule-1/CD31-mediated inhibitory signaling.

Platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) is a newly assigned member of the Ig immunoreceptor tyrosine-based inhibitory motif superfamily, and its functional role is suggested to be an inhibitory receptor that modulates immunoreceptor tyrosine-based activation motif-dependent signaling cascades. To test whether PECAM-1 is capable of delivering inhibitory signals in B cells and the functional requirement of protein-tyrosine phosphatases (PTPs) for this inhibitory signaling, we generated chimeric Fc gamma RIIB1-PECAM-1 receptors containing the extracellular and transmembrane portions of murine Fc gamma RIIB1 and the cytoplasmic domain of human PECAM-1. These chimeric receptors were stably expressed in chicken DT40 B cells either as wild-type or mutant cells deficient in SHP-1(-/-), SHP-2(-/-), SHIP(-/-), or SHP-1/2(-/-) and then assessed for their ability to inhibit B cell Ag receptor (BCR) signaling. Coligation of wild-type Fc gamma RIIB1-PECAM-1 with BCR resulted in inhibition of intracellular calcium release, suggesting that the cytoplasmic domain of PECAM-1 is capable of delivering an inhibitory signal that blocks BCR-mediated activation. This PECAM-1-mediated inhibitory signaling correlated with tyrosine phosphorylation of the Fc gamma RIIB1-PECAM-1 chimera, recruitment of SHP-1 and SHP-2 PTPs by the phosphorylated chimera, and attenuation of calcium mobilization responses. Mutational analysis of the two tyrosine residues, 663 and 686, constituting the immunoreceptor tyrosine-based inhibitory motifs in PECAM-1 revealed that both tyrosine residues play a crucial role in the inhibitory signal. Functional analysis of various PTP-deficient DT40 B cell lines stably expressing wild-type chimeric Fc gamma RIIB1-PECAM-1 receptor indicated that cytoplasmic Src homology 2-domain-containing phosphatases, SHP-1 and SHP-2, were both necessary and sufficient to deliver inhibitory negative regulation upon coligation of BCR complex with inhibitory receptor.

Platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) is associated with a naïve B-cell phenotype in human tonsils.

In B cells, signaling through the B-cell antigen receptor (BCR) is negatively modulated by the co-ligation of immunoglobulin (Ig)-immunoreceptor tyrosine-based inhibitory motif (ITIM)-bearing molecules such as FcgammaRIIB1, B-cell transmembrane protein CD72, paired immunoglobulin-like receptor PIR-B, leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1), Ig-like transcript ILT2, biliary glycoprotein BGP-1 and B-cell co-receptor CD22. The co-expression of multiple Ig-ITIM receptors may provide B cells with different mechanisms of regulating inhibitory pathways at different stages of differentiation. In this study, we have examined the expression of a newly defined Ig-ITIM receptor, PECAM-1 (CD31) on human B-cells. Human tonsillar B cells were purified using negative selection by depleting T cells with a combination of monoclonal antibodies and magnetic bead separation. Following purification, the pattern of PECAM-1 expression was analyzed in B-cell subpopulations using two- and three-colour fluorescence. To complement this work, PECAM-1 localization in the context of distinct areas of human tonsil was defined by immunohistochemical analysis of tonsil sections. Finally to investigate somatic mutation, Ig variable (V) region genes belonging to the nonpolymorphic VH6 family were amplified by polymerase chain reaction (PCR), subcloned and sequenced from sort-purified CD19+ PECAM-1+ and CD19+ PECAM-1- B cells. Our results demonstrate that PECAM-1 is associated with an unstimulated resting B-cell phenotype, localization to the follicular mantle and marginal zones of human tonsil and expression of unmutated Ig V region genes. These studies suggest that PECAM-1 appears on the cell surface at the naive B-cell stage and is lost as B cells differentiate into memory cells, indicating that PECAM-1 is primarily involved in naive or immature B-cell function.

Mass casualty triage knowledge of military medical personnel.

During battlefield and mass casualty incidents, triage has been traditionally performed by many different personnel, including medics, nurses, dentists, and physicians. The objective of this study was to determine which military medical providers are most knowledgeable in mass casualty triage. The design was a prospective, written, timed, case-based examination of triage knowledge. Participants were volunteers from the active duty medical (physician), dental, nursing, and enlisted corps of the three military services. Subjects completed a 16-minute written examination consisting of seven cases in each of three simulated mass casualty scenarios: combat; nuclear, biological, and chemical; and humanitarian. Tests were taken anonymously, although demographic data on medical specialty, training, and experience were collected. Participants were instructed to classify the cases using the NATO categories of immediate, delayed, minimal, or expectant. Scores were tabulated according to two grading scales: an absolute scale of number correct, and a weighted scale amplifying gross misclassifications. Median scores between groups were tested pairwise using the Kruskal-Wallis one-way analysis of variance with p < or = 0.05. Statistically significant differences were noted between the highest and lowest scoring groups in each scenario. Our conclusion is that among the subject groups tested, physicians were best at mass casualty triage. Dentists, nurses, and medics scored progressively less well on our examination.

Recruitment and activation of SHP-1 protein-tyrosine phosphatase by human platelet endothelial cell adhesion molecule-1 (PECAM-1). Identification of immunoreceptor tyrosine-based inhibitory motif-like binding motifs and substrates.

Stimulation of platelet aggregation leads to tyrosine phosphorylation of a number of receptors and signaling molecules including platelet endothelial cell adhesion molecule-1 (PECAM-1). In this report, we demonstrate that both protein-tyrosine phosphatases SHP-1 and SHP-2 physically associate with different kinetics of assembly with tyrosine-phosphorylated human PECAM-1 during integrin alphaIIbbeta3-mediated platelet aggregation. Peptido-precipitation analysis revealed that tyrosine-phosphorylated peptides encompassing residues 658-668 and 681-691 of PECAM-1 bound specifically to both protein-tyrosine phosphatases SHP-1 and SHP-2. We further show that the association of SHP-1 with PECAM-1 occurs through the direct interaction of the src homology region 2 domains of SHP-1 with two highly conserved phosphotyrosine binding motifs within PECAM-1 having the sequences NSDVQpY663TEVQV and DTETVpY686SEVRK (where pY represents phosphotyrosine). In vitro dephosphorylation experiments using phosphotyrosyl PECAM-1 peptides encompassing either Tyr-663 or Tyr-686 revealed induction of SHP-1 catalytic activity, suggesting that PECAM-1 serves as a SHP-1 substrate. Surface plasmon resonance studies reveal that recombinant SHP-2 binds PECAM-1 phosphopeptides with 5-fold higher affinity than recombinant SHP-1. These data suggest that in hematopoietic cells such as platelets, PECAM-1 cellular signaling is regulated by the selective recruitment and activation of two distinct protein-tyrosine phosphatases, SHP-1 and SHP-2, via a common immunoreceptor tyrosine-based inhibitory-like motif.

A Ser162-->Leu mutation within glycoprotein (GP) IIIa (integrin beta3) results in an unstable alphaIIbbeta3 complex that retains partial function in a novel form of type II Glanzmann thrombasthenia.

Platelets from Glanzmann thrombasthenia patient BL express approximately 30% of the normal alphaIIbbeta3 content and support fibrin-mediated clot retraction, but fail to bind fibrinogen or aggregate following cellular activation. BL platelets bind neither activation-dependent nor activation-independent ligands. DNA sequence analysis of BL platelet mRNA revealed a homozygous C583-->T point mutation in a conserved region of beta3, resulting in a Ser162Leu amino acid substitution. This mutation appears to produce destabilizing effects on the alphaIIbbeta3 complex, as evidenced by the fact that (1) the BL alphaIIbbeta3 complex exhibited altered sedimentation velocity through sucrose gradients, (2) alphaIIb and beta3 was not recognized by complex-dependent monoclonal antibodies or co-precipitated by integrin subunit-specific antibodies, and (3) biosynthesis and trafficking of the alphaIIbbeta3Leu162 complex was delayed relative to that of the wild-type control. Taken together, these data implicate the region encompassing Ser162 in the stabilization and ligand binding properties of the alphaIIbbeta3 complex.

Outcome analysis of noninstrumented anterior cervical discectomy and interbody fusion in 348 patients.

STUDY DESIGN: Twenty-one outcome and outcome-relevant variables (fusion and patient satisfaction) were evaluated in a subset of 348 of 514 patients operated on by one surgeon during a 22-year period, using Cloward's anterior cervical discectomy and dowel interbody fusion. Minimum patient follow-up was 2 years; average length of follow-up was 5.2 years. This retrospective analysis is accompanied by a comprehensive review of the literature (1975-1996) of noninstrumented anterior cervical fusions, excluding fibular grafts alone in the interbody space. OBJECTIVES: To provide data on outcome (with regard to patient satisfaction and radiologically supported fusion) and risks of noninstrumented anterior cervical discectomy and fusion for intractable cervical nerve root and spinal cord compression symptoms at single or multiple levels, using the results from a single surgeon. METHODS: Three experienced spine radiologists determined fusion rates in one to five levels in 348 patients on the basis of the results of plain film analysis. Patient self-assessment was used to determine degree of patient satisfaction and other related variables. From a comprehensive review of the literature, 43 clinical reports meeting requirements for comparison of findings with those in the current study were selected from more than 1600 reports. RESULTS: The mean fusion rate for 348 patients in the current study ranged from 75% (multilevel) to 88% (one level; n = 202). The overall fusion rate was 83%. The persistent complication rate was 0.1%, and patient self-assessments showed that 78% were satisfied with the outcome and that 83% returned to work. The overall fusion rate for 2037 patients evaluated in the comprehensive literature review is 92%. CONCLUSIONS: Results of this study indicate that better outcome in noninstrumented anterior cervical discectomy and fusion is associated with solid fusion, fewer fused levels, nonsmoking patients, higher education levels, and absence of secondary economic gain. There was no correlation between fusion status and bone graft source or use of cervical collar.

Platelet/endothelial cell adhesion molecule-1 serves as a costimulatory agonist receptor that modulates integrin-dependent adhesion and aggregation of human platelets.

Platelet/endothelial cell adhesion molecule-1 (PECAM-1) is a 130-kD member of the Ig gene superfamily that is expressed on the surface of circulating platelets, monocytes, neutrophils, and selective T-cell subsets. It is also a major component of the endothelial cell intercellular junction. Previous studies have shown that cross-linking PECAM-1 on the surface of leukocytes results in the activation of adhesion molecules of both the beta 1 and beta 2 integrin family. In addition, the process of leukocyte transendothelial migration appears to be mediated, at least in part, by homophilic adhesive interactions that take place between leukocyte and endothelial cell junctional PECAM-1 molecules. However, little is known about the functional role of this membrane glycoprotein in human platelets. In the present study, we examined the effects of PECAM-1 engagement on integrin-mediated platelet-extracellular matrix or platelet-platelet interactions. Bivalent, but not monovalent, anti-PECAM-1 monoclonal antibodies (MoAbs) specific for membrane-proximal Ig-homology domain 6 significantly augmented platelet deposition (increased surface coverage) and aggregation (increased average size) onto extracellular matrix, under both oscillatory or defined low shear flow conditions (200 s-1) in a modified cone and plate viscometer. Moreover, bivalent anti-domain 6 MoAbs were capable of serving as costimulatory agonists to markedly enhance both adenosine diphosphate (ADP)- and platelet activating factor (PAF)-induced platelet aggregation resonses. These antibodies appeared to act via outside-in signal transduction through PECAM-1, as evidenced by the fact that their binding (1) led to conformational changes in the alpha IIb beta 3 integrin complex, (2) induced surface expression of P-selectin, and (3) resulted in the tyrosine phosphorylation of PECAM-1. Together, these data support a role for PECAM-1 in cellular activation and suggest that PECAM-1 may serve as a costimulatory agonist receptor capable of modulating integrin function in human platelets during adhesion and aggregation.

Characterization of phosphotyrosine binding motifs in the cytoplasmic domain of platelet/endothelial cell adhesion molecule-1 (PECAM-1) that are required for the cellular association and activation of the protein-tyrosine phosphatase, SHP-2.

Recent studies have shown that the Src homology-2 (SH2) domain-containing protein-tyrosine phosphatase, SHP-2, associates with the cytoplasmic domain of PECAM-1 as it becomes tyrosine-phosphorylated during platelet aggregation: a process that can be mimicked in part by small synthetic phosphopeptides corresponding to the cytoplasmic domain of PECAM-1 encompassing tyrosine residues Tyr-663 or Tyr-686. To further examine the molecular requirements for PECAM-1/SHP-2 interactions, we generated human embryonic kidney (HEK)-293 cell lines that stably expressed mutant forms of PECAM-1 harboring tyrosine to phenylalanine (Tyr --> Phe) mutations in the cytoplasmic domain. Y663F and Y686F forms of PECAM-1 were tyrosine-phosphorylated to a somewhat lesser extent than wild-type PECAM-1, and a doubly substituted Y663,686F form of PECAM-1 failed to become tyrosine-phosphorylated, suggesting that the PECAM-1 cytoplasmic domain tyrosine residues 596, 636 and 701 do not serve as substrates for cellular kinases. Interestingly, SHP-2 binding was lost when either Tyr-663 or Tyr-686 were changed to phenylalanine, indicating that both residues are required for SHP-2/PECAM-1 association. Although PECAM-1 phosphopeptides NSDVQpY663TEVQV and DTETVpY686SEVRK stimulated the catalytic activity of the phosphatase to a similar extent, surface plasmon resonance studies revealed that the Tyr-663-containing peptide had approximately 10-fold higher affinity for SHP-2 than did the Tyr-686 peptide. Finally, peptido-precipitation analysis showed that the NH2-terminal SH2 domain of SHP-2 reacted preferentially with the Tyr-663 PECAM-1 phosphopeptide, while the Tyr-686 phosphopeptide associated only with the COOH-terminal SH2 domain of the phosphatase. Together, these data provide a molecular model for PECAM-1/SHP-2 interactions that may shed light on the downstream events that follow PECAM-1-mediated interactions of vascular cells.

Identification and characterization of functional cation coordination sites in platelet endothelial cell adhesion molecule-1.

We have employed 45CaCl2 binding studies, terbium (Tb3+) luminescence spectroscopy, and electrospray mass spectroscopy (ESI-MS) to identify divalent metal binding properties of soluble recombinant human PECAM-1 (srPECAM-1), and to define unique cation binding domains using short, linear peptide sequences from the protein. PECAM-1 was found to directly interact with 45CaCl2, binding 2.3 nmol of Ca2+/nmol of srPECAM-1 with a Kd of 1.17 nM. PECAM-1 was found to contain high-affinity cation binding sites involving amino acids Asp443, Asp444, and Glu446 of Ig-domain 5 and residues Glu487, Glu490, Asp491, Glu538, Glu540, and Glu542 of Ig-domain 6. The PECAM cation binding sites demonstrated broad specificity for all divalent cations, with Mn2+ having a higher affinity than Ca2+ or Mg2+. Direct binding of Tb3+ to these PECAM peptides was confirmed by ESI-MS. Modeling studies predict that the six cation binding residues within Ig-domain 6 are proximal to each other in three-dimensional space, and may form a single cation coordination site. The identification of cation binding sites in PECAM-1 will direct further work in examining its cation-dependent roles in cellular signaling.

The protein-tyrosine phosphatase SHP-2 binds platelet/endothelial cell adhesion molecule-1 (PECAM-1) and forms a distinct signaling complex during platelet aggregation. Evidence for a mechanistic link between PECAM-1- and integrin-mediated cellular signaling.

Platelet/endothelial cell adhesion molecule-1 (PECAM-1) is a homophilic adhesion receptor that mediates leukocyte/endothelial cell interactions that take place during transendothelial migration. Recent reports have shown that the binding of certain anti-PECAM-1 antibodies results in up-regulation of integrin function on the surface of leukocytes and platelets, suggesting that PECAM-1 may be capable of transmitting information into the cell following its engagement. PECAM-1 isolated from resting or activated but nonaggregated platelets was phosphorylated predominantly on serine residues; however, PECAM-1 derived from activated, aggregated platelets was strongly phosphorylated on tyrosine. Synthetic tyrosine-phosphorylated peptides derived from five different regions within the cytoplasmic domain of PECAM-1 were screened for their ability to associate with cytoplasmic signaling molecules. The protein-tyrosine phosphatase SHP-2 was found to interact specifically with two different PECAM-1 phosphopeptides containing highly conserved phosphatase-binding motifs on PECAM-1 with the sequences VQpY663TEV and TVpY686SEV. More important, SHP-2 bound not only PECAM-1 phosphopeptides, but also became associated with full-length cellular PECAM-1 during the platelet aggregation process, and this interaction was mediated by the amino-terminal Src homology 2 domains of the phosphatase. Since SHP-2 normally serves as a positive regulator of signal transduction, its association with activated PECAM-1 suggests a number of potential mechanisms by which PECAM-1 engagement might be coupled to integrin activation in vascular cells.

Studies to show that with podophyllotoxin the early replicative stages of herpes simplex virus type 1 depend upon functional cytoplasmic microtubules.

The antiviral activity of podophyllotoxin against herpes simplex type 1 virus (HSV-1) grown in Vero cells was studied by a simple microtitration assay. Antiviral effects were induced at similar concentrations as direct cellular toxicity, as characterised by a time-dependent loss of cell monolayer. Podophyllotoxin-mediated toxicity arises from cytoplasmic microtubular, and hence cytoskeletal, decay. Some degree of selectivity was seen for inhibition of virus replication over direct cellular toxicity. Podophyllotoxin acted against an early viral process, as an antiviral effect was still seen if drug was removed 2 h after infection. Similar effects were seen with colchicine, a classical tubulin-binding compound, but not with bromovinyldeoxyuridine. Podophyllotoxin was capable of inducing a cytoprotective effect in Vero cells, as pre-treatment of cells abrogated virus growth for up to 90 min after removal of drug. This is coincident with the repolymerisation of cellular microtubules and re-formation of the cytoskeleton. We conclude that HSV-1 relies upon a functional cellular cytoskeleton for efficient completion of an early replicative event. Such a process may be the transport of viral material to the nucleus or inhibition of the formation of intranuclear viral 'replication factories', bodies containing cytoskeletal fragments constructed after viral infection.

Altered thrombomodulin staining in blistering dermatoses.

The immunocytochemical distribution of thrombomodulin (TM) was examined in sections of skin from patients with blistering dermatoses occurring in the presence and absence of acantholysis. Skin sections were stained using polyclonal and monoclonal anti-human TM antibodies and were correlated with the staining pattern that resulted when using the monoclonal antibody 32-2B, which recognises the chief desmosomal adhesion molecule desmoglein I (DG I). Our study demonstrates a loss of TM staining in acantholytic dermatoses, occurring only in the region of actual disruption of the intercellular bridging between keratinocytes in the stratum spinosum. The thrombomodulin antigen expression paralleled the DG I expression. The strong correlation between the DG I and TM immunostaining pattern in both normal skin and acantholytic dermatoses supports the concept that TM has a role in mediating adhesion processes between keratinocytes.