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Biomedical subjects

D E Jackson

Publications and source records attributed to D E Jackson.

At least 19 recordsLinked to original sources
PubMed2001

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.

Animals↗
PubMed2001

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.

Amino Acid Motifs↗
PubMed2000

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.

Antigens, CD↗
PubMed1999

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.

Analysis of Variance↗
PubMed1998

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.

Binding Sites↗
PubMed1998

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.

Adult↗
PubMed1998

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.

Blood Platelets↗
PubMed1998

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.

Amino Acid Sequence↗
PubMed1997

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.

Binding Sites↗
PubMed1997

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.

Amino Acid Sequence↗
PubMed1997

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.

Amino Acid Sequence↗
PubMed1996

Inherited mutations within the calcium-binding sites of the integrin alpha IIb subunit (platelet glycoprotein IIb). Effects of the amino acid side chain and the amino acid position on cation binding.

To examine the effects of naturally occurring inherited mutations on the ability of the integrin alpha-subunit, also termed glycoprotein IIb (GPIIb), to bind metal ions, we prepared small synthetic peptides that encompassed individual cation-binding domains, and recombinant GPIIb poly peptides that encompassed all four Ca(2+)-binding domains, and examined their interactions with divalent cations by means of Tb3+-luminescence spectroscopy. Replacement of the highly conserved Gly418 residue, located within the flanking region of the fourth Ca(2+)-binding domain of GPIIb, with a negatively charged Asp residue resulted in marked reduction in the ability to bind divalent cations. A variant form of GPIIb with a deletion of two amino acids at the -1 and X positions of the fourth Ca(2+)-binding domain of GPIIb also failed to bind metal ions in a normal manner. In contrast, a Glanzmann mutation at the -1 position of the first Ca(2+)-binding domain of GPIIb had no effect on divalent-cation-binding ability with either synthetic peptides or recombinant GPIIb polypeptides. These data support the hypothesis that the highly conserved Gly normally found 7-8 residues N-terminal to integrin metal-binding domains plays a critical role in the maintenance of the conformation or orientation of surrounding EF-hand structures so that they can effectively interact with and bind divalent cations. Furthermore, inherited mutations at or near the divalent-cation-binding domains of GPIIb do not necessarily exert their biochemical effects by disruption of cation binding, but can interfere with integrin biogenesis in a Ca(2+)-independent manner.

Amino Acid Sequence↗
PubMed1996

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.

Acantholysis↗
PubMed1996

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.

Animals↗
PubMed1995

Immunohistochemical localization of thrombomodulin in normal human skin and skin tumours.

Thrombomodulin (TM) expression has been investigated in sections of normal human skin, in cultured normal human keratinocytes, and in a variety of skin tumours. TM was present in squamous epithelial cells in the spinous layer of normal epidermis and in the outer root sheath of hair follicles, but was absent in the cells of the basal layer. It appeared to be predominantly localized to the cell membrane and the intercellular bridges in these areas. Cultured normal human keratinocytes demonstrated functionally active constitutive TM expression on their cell surface. Immunoperoxidase staining of skin tumours using anti-human TM antibodies demonstrated a typical cell membrane positivity in tumours with squamous or hair follicle differentiation. Basal cell carcinomas showed TM expression only in areas where incomplete squamoid metaplasia occurred. Sweat gland tumours and lesions of the melanogenic system failed to express TM. The localization of TM by immunostaining in various benign and malignant skin tumours typically correlated with their normal skin element of origin. The physiological significance of TM expression in the epidermis is currently undefined.

Basal Cell Carcinoma↗
PubMed1994

Purification and characterization of two forms of soluble thrombomodulin from human urine.

We have isolated and characterized two forms of soluble thrombomodulin from human urine. The purification procedure consisted of ultrafiltration, chromatography on DEAE-Sepharose, affinity chromatography on diisopropyl-phosphate-thrombin and/or monoclonal anti-thrombomodulin IgG affigel followed by reverse-phase HPLC. An active soluble form of thrombomodulin was purified 1600-fold from 34-l urine. The purified protein migrated as a doublet, with molecular mass 76/72 kDa under reducing conditions and 63/57 kDa under non-reducing conditions as determined by SDS/PAGE. Amino acid analysis of the 63/57-kDa soluble thrombomodulin confirmed sequence identity with human thrombomodulin and demonstrated N-terminal heterogeneity. Compared to membrane-type thrombomodulin, the purified 63/57-kDa soluble thrombomodulin was more active as a cofactor for protein-C activation. The second major thrombomodulin fragment urine is an inactive 35-kDa thrombomodulin polypeptide derived from the N-terminal extracellular region of thrombomodulin.

Chromatography, Affinity↗
PubMed1994

A scanning tunnelling microscopy comparison of passive antibody adsorption and biotinylated antibody linkage to streptavidin on microtiter wells.

An antiferritin antibody was either, (a) passively adsorbed to microwells or (b) biotinylated and immobilised to streptavidin coated microwells. Scanning tunnelling microscope (STM) imaging of these well surfaces coated with a platinum (95%) carbon (5%) coating (Pt/C) conductive layer showed a randomly oriented array of antibodies for passive adsorption whereas for biotin-streptavidin immobilisation there was a more uniform and even distribution of antibodies on the well surface. On further incubation with ferritin STM imaging showed that for passive adsorption approximately 5% of the surface was functional, while for the biotinylated antibody it was greater than 60%. The images presented in this paper show graphically the loss of functionality that occurs using passive adsorption and, conversely, the preservation of antibody functionality using the biotin-streptavidin linkage for antibody immobilisation. These results correlate well with the work of others in the field.

Adsorption↗