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Biomedical subjects

A T Nurden

Publications and source records attributed to A T Nurden.

At least 37 records · Page 2Linked to original sources
PubMed2000

The cleaved peptide of PAR1 results in a redistribution of the platelet surface GPIb-IX-V complex to the surface-connected canalicular system.

The only known function of the 41 amino acid cleaved peptide (TR1-41) of the seven transmembrane domain thrombin receptor (PARI) is to activate platelets (as determined by aggregation, surface P-selectin, and fibrinogen binding to activated GPIIb-IIIa). We now demonstrate that TR1-41 results in a concentration-dependent decrease in the platelet surface expression of each component of the GPIb-IX-V complex, as determined by flow cytometry with a panel of monoclonal antibodies (including 6D1, directed against the von Willebrand factor binding site on GPIbalpha, and TM60, directed against the thrombin binding site on GPIbalpha). TR1-41 also decreased ristocetin-induced platelet agglutination. Immunoblotting after incubation of platelets with TR1-41 revealed neither a loss of platelet GPIb nor increase in supernatant GPIb fragments. As demonstrated by immunoelectron microscopy, TR1-41 resulted in a redistribution of GPIb, GPIX, and GPV from the platelet surface to the surface-connected canalicular system (SCCS). In summary, the cleaved peptide (TR1-41) of PAR1 results in a redistribution of the platelet surface GPIb-IX-V complex to the SCCS, thereby negatively regulating the GPIbalpha binding sites for von Willebrand factor and thrombin.

Animals↗
PubMed1999

Analysis of the V genes coding for a monospecific human antibody to myosin and functional expression of single chain Fv fragments.

A monospecific human IgM monoclonal antibody (mAb), reactive with myosin from human heart, has been obtained by EBV transformation. This mAb may have a diagnostic potential in the imaging of myocardial necrosis. However, owing to the fact that the molecular mass of an IgM is 900 kDa, a poor diffusion and a slow penetration inside necrotic myocytes could reduce its capacity for scintigraphic detection. In order to alleviate these problems, we constructed the scFv by cloning the VH and VL domains into the pHOG21 vector. Analysis of the V genes proved an unmutated configuration showing that the immortalized B cell issued from the primary IgM repertoire. The expression product in Escherichia coli was a 35 kDa scFv fragment with the antigen-binding specificity of the parental mAb.

Amino Acid Sequence↗
PubMed1999

Labeling of the internal pool of GP IIb-IIIa in platelets by c7E3 Fab fragments (abciximab): flow and endocytic mechanisms contribute to the transport.

Abciximab is a new antiplatelet therapeutic in ischemic cardiovascular disease. The drug, chimeric Fab fragments of a murine monoclonal antibody (MoAb) (c7E3), blocks GP IIb-IIIa function. However, its capacity to reach all receptor pools in platelets is unknown. Electron microscopy and immunogold labeling were used to localize abciximab in platelets of patients receiving the drug for up to 24 hours. Studies on frozen-thin sections showed that c7E3 Fab, in addition to the surface pool, also labeled the surface-connected canalicular system (SCCS) and alpha-granules. Analysis of gold particle distribution showed that intraplatelet labeling was not accumulative and in equilibrium with the surface pool. After short-term incubations of platelets with c7E3 Fab in vitro, gold particles were often seen in lines within thin elements of the SCCS, some of which appeared in contact with alpha-granules. Little labeling was associated with Glanzmann's thrombasthenia platelets, confirming that the channels contained bound and not free c7E3 Fab. Endocytosis of abciximab in clathrin-containing vesicles was visualized by double staining and constitutes an alternative mechanism of transport. The remaining free pool of GP IIb-IIIa was evaluated with the MoAb AP-2; flow cytometry showed it to be about 9% on the surface of nonstimulated platelets but 33% on thrombin-activated platelets. The ability of drugs to block all pools of GP IIb-IIIa and then to be associated with secretion-dependent residual aggregation must be considered when evaluating their efficiency in a clinical context.

Abciximab↗
PubMed1999

Annexin V relocates to the periphery of activated platelets following thrombin activation: an ultrastructural immunohistochemical approach.

We have previously shown biochemically that the physiological agonist thrombin can cause translocation of endogenous annexin V to a fraction containing all platelet membranes. This paper reports ultrastructural immunohistochemical data revealing that annexin V molecules localize with plasma membranes of blood platelets following thrombin activation. When ultrathin sections of resting platelets were examined by immunogold staining, annexin V was found to be cytosolic, having a generalized distribution throughout the platelet. After thrombin activation, annexin V became peripheral in location and plasmalemma association increased. Morphometric analysis of gold particles shows that annexin V relocates specifically to the plasma membrane and its underlying cytoskeleton following treatment with thrombin. In control platelets 6.1% +/- 0.78 of annexin V is present at the plasma membrane and 15.0% +/- 0.82 in the region corresponding to the membrane cytoskeleton (10-80 nm); after stimulation with 0.5 unit/ml thrombin for 2 min this increased to 16.7% +/- 0.22 and 40.4% +/- 0.53, respectively.

Annexin A5↗
PubMed1999

Accessibility of abciximab to megakaryocytes and endothelial cells in the bone marrow compartment: studies on a patient receiving antithrombotic therapy.

Abciximab, chimaeric Fab fragments of the monoclonal antibody 7E3 (c7E3 Fab), has achieved widespread use as an anti-platelet agent for blocking GP IIb-IIIa (alphaIIbbeta3) function and preventing ischaemic complications after coronary artery angioplasty. However, its accessibility to the bone marrow compartment during therapy is unknown, as is its ability to bind alphavbeta3 in vivo. Using electron microscopy and immunogold labelling, we have looked for abciximab in the bone marrow of a patient who became thrombocytopenic during treatment. The presence of abciximab was assessed on ultrathin frozen sections of a marrow aspirate, the drug being revealed by a rabbit antibody to c7E3 Fab. Labelling was maximal on fragmenting megakaryocytes (MK) and proplatelets in the vascular sinus and in direct access to the blood compartment. Not only the plasma membrane but also the demarcation membrane system (DMS) and the membranes of alpha-granules were labelled. Abciximab was also revealed on the luminal surface of endothelial cells lining the marrow sinuses, thereby confirming for the first time its ability to bind to alphavbeta3 in vivo. The study revealed no signs that abciximab had accumulated in the marrow.

Abciximab↗
PubMed1999

Expression of markers of platelet activation and the interpatient variation in response to abciximab.

Our study concerns the biological effects of abciximab (c7E3 Fab, ReoPro), a powerful new antiplatelet drug that blocks glycoprotein (GP) IIb-IIIa complexes. Samples were examined from 6 patients with coronary artery disease who received a bolus of abciximab followed by a 10- microg/min infusion for at least 18 hours before percutaneous transluminal coronary angioplasty. Inhibition of ADP-induced PA was maximal for 4 patients but partial (79% and 53%) for 2 others during the infusion. Flow cytometry performed with monoclonal antibodies (PAC-1, AP-6, and F26) specific for the "activated" GP IIb-IIIa complex revealed large decreases in the expression of activation markers on platelets during therapy, but these decreases were less marked when inhibition of ADP-induced PA was incomplete. Residual aggregation was seen for all patients during the infusion when TRAP 14-mer peptide or thrombin was the stimulus. Unblocked GP IIb-IIIa complexes were detected on thrombin-stimulated platelets from the patients by immunoelectron microscopy performed using the monoclonal antibody AP-2. Unblocked GP IIb-IIIa complexes were also detected by flow cytometry when platelets preincubated for 1 hour in vitro with abciximab under saturating conditions were (1) incubated with TRAP 14-mer or (2) permeabilized with Triton X-100. In confirming interpatient variation in the platelet response to a standard dose of abciximab, our results also show that an uninhibited internal pool of GP IIb-IIIa complexes may mediate a residual response to strong agonists.

Abciximab↗
PubMed1999

Homozygous Cys542-->Arg substitution in GPIIIa in a Swiss patient with type I Glanzmann's thrombasthenia.

Glanzmann's thrombasthenia (GT) arises from a qualitative or quantitative defect in the GPIIb-IIIa complex (integrin alphaIIbbeta3), the mediator of platelet aggregation. We describe a patient in whom clinical and laboratory findings typical of type I GT were found together with a second pathology involving neurological and other complications symptomatic of tuberous sclerosis. Analysis of platelet proteins by Western blotting revealed trace amounts of normally migrating GPIIb and equally small amounts of GPIIIa of slightly slower than normal migration. Flow cytometry confirmed a much decreased binding to platelets of monoclonal antibodies to GPIIb, GPIIIa or GPIIb-IIIa, and an antibody to the alphav subunit also showed decreased binding. Nonradioactive PCR single-strand conformation polymorphism analysis followed by direct sequencing of PCR-amplified DNA fragments showed a homozygous point mutation (T to C) at nucleotide 1722 of GPIIIa cDNA and which led to a Cys542-->Arg substitution in the GPIIIa protein. The mutation gave rise to a HinP1 I restriction site in exon 11 of the GPIIIa gene and allele-specific restriction enzyme analysis of family members confirmed that a single mutated allele was inherited from each parent. This amino acid substitution presumably changes the capacity for disulphide bond formation within the cysteine-rich core region of GPIIIa and its study will provide new information on GPIIb-IIIa and alphavbeta3 structure and biosynthesis.

Amino Acid Substitution↗
PubMed1999

Three cases of acquired von Willebrand disease associated with systemic lupus erythematosus.

Acquired von Willebrand disease associated with systemic lupus erythematosus (SLE) has been detected in three middle-aged women. In each case the first clinical manifestation was a bleeding syndrome. Plasma levels of von Willebrand factor (VWF) and ristocetin-induced platelet agglutination were as found in type 1 von Willebrand disease for the first patient, type 3 for the second patient, and type 2 for the third patient. Intraplatelet levels of VWF were normal for all three patients. In all cases a mixture of patient's plasma with normal plasma resulted in inhibition of ristocetin-induced binding of VWF to normal platelets. Intravenous immunoglobulin given to patients 2 and 3 corrected the plasma VWF level of the second patient but not that of the third. Therapy with corticosteroids was partially beneficial for patient 3 and patient 2. For patient 2, the severity of the cutaneous lesions also led to the use of cyclophosphamide, and this therapy resulted in total correction of VWF levels. Our observations confirm previous reports of acquired von Willebrand syndrome associated with SLE and show heterogeneity both in the phenotypic form and in the response to treatment.

Aged↗
PubMed1998

R to Q amino acid substitution in the GFFKR sequence of the cytoplasmic domain of the integrin IIb subunit in a patient with a Glanzmann's thrombasthenia-like syndrome.

The integrin IIbbeta3 mediates platelet aggregation through its fibrinogen and adhesive protein-binding properties. Particular interest concerns the role of the cytoplasmic domains of IIb and beta3. We now report the molecular analysis of IIbbeta3 from a patient with a Glanzmann's thrombasthenia-like syndrome for whom the principal characteristics are an approximate 50% total platelet content of IIbbeta3 but with a much lower proportion in the surface pool (Hardisty et al, Blood 80:696, 1992). Polymerase chain reaction (PCR) single-strand conformational polymorphism and DNA sequencing showed a heterozygous mutation giving rise to amino acid substitution R995 to Q in the GFFKR sequence of the cytoplasmic domain of IIb. Reverse transcriptase-PCR and polymorphism analysis only detected mRNA for the mutated allele of the IIb gene and a single allele of the beta3 gene in his platelets, suggesting other unidentified defects. Site-directed mutagenesis followed by transient expression of the mutated IIb together with wild-type beta3 in Cos-7 cells resulted in a markedly decreased expression of the complex at the cell surface when compared with cells transfected with wild-type IIb and beta3. Flow cytometry with PAC-1 and a stable Chinese hamster ovary-transfected cell line showed that the mutated receptor was not locked into a high activation state, although it became so in the presence of the activating antibody, anti-LIBS6. This is the first reported natural mutation in the highly conserved GFFKR sequence of the IIb cytoplasmic domain.

Amino Acid Substitution↗
PubMed1998

Microvesicle release is associated with extensive protein tyrosine dephosphorylation in platelets stimulated by A23187 or a mixture of thrombin and collagen.

Phosphatidylserine exposure and microvesicle release give rise to procoagulant activity during platelet activation. We have previously shown that whereas the Ca2+ ionophore A23187 and 2,5-di-(t-butyl)-1, 4-benzohydroquinone, a Ca2+-ATPase inhibitor, induce phosphatidylserine exposure, only the former triggers microvesicle release. We now report that microvesicle formation with ionophore A23187 is specifically associated with mu-calpain activation, increased protein tyrosine phosphatase (PTP) activity and decreased tyrosine phosphorylation. The degree to which calpain and individual PTPs were activated in response to A23187 depended on the extent of bivalent cation chelation in the external medium. EGTA (2 mM) blocked or severely retarded their activation, and addition of extracellular Ca2+ in excess (2 mM) resulted in virtually immediate tyrosine dephosphorylation. Dephosphorylation was correlated with an increase in total PTP activity in platelet lysates. In platelets stimulated by a combination of thrombin and collagen, only the subpopulation undergoing microvesicle release and isolated by their binding to annexin-V-coated magnetic beads exhibited protein tyrosine dephosphorylation. Detection of PTP activity in an 'in-gel' assay showed the Ca2+-dependent appearance of active low-molecular-mass bands at 38, 36 and 27 kDa. Individual PTPs varied in their protease sensitivity to changes in intracellular Ca2+ levels. For example, PTP1B was a more sensitive substrate than SH2-domain-containing tyrosine phosphatase-1 for mu-calpain cleavage. Incubation of platelets with the PTP inhibitors, phenylarsine oxide and benzylphosphonic acid acetoxymethyl ester, led to increased tyrosine phosphorylation and the surface expression of aminophospholipids but little microvesicle formation. Furthermore, microvesicle release in response to ionophore A23187 was inhibited. We conclude that platelet microvesicle formation is associated with extensive protein tyrosine dephosphorylation.

Blood Platelets↗
PubMed1998

Protease-activated receptor genes are clustered on 5q13.

The serine protease, thrombin, is both a potent agonist for platelet aggregation and a mitogen inducing the proliferation of other cell types. Many cellular responses to thrombin are mediated by a G-protein-coupled thrombin receptor (protease-activated receptor-1, PAR-1). This represents the prototype of a new family of proteolytically cleaved receptors that includes PAR-2 and the recently identified PAR-3. Like PAR-1, PAR-3 is a potential thrombin receptor. Their similar gene structure, mechanism of activation, and colocalization to 5q13 raises the question of a common evolutionary origin and of their belonging to a clustered gene family. Construction of a physical map of the 5q13 region by pulsed-field gel electrophoresis (PFGE) has allowed us to identify six potential CpG islands and to establish a linkage of the PAR genes. Southern blot analysis showed that they were in a cluster on a 560-kb Asc I fragment, in the order PAR-2, PAR-1, and PAR-3. PAR-1 and PAR-2 genes were contained within the identical 240-kb Not I fragment, thus confirming a tight linkage between them. The localization of other CpG islands suggested that more PAR-family genes may be present.

Chromosome Mapping↗
PubMed1998

Autoimmune thrombocytopenic purpura (AITP) and acquired thrombasthenia due to autoantibodies to GP IIb-IIIa in a patient with an unusual platelet membrane glycoprotein composition.

The subject (E.B.) is a 63-year-old woman with autoimmune thrombocytopenic purpura (AITP) who was first examined some 6 years ago with symptoms of epistaxis and gum bleeding, severe thrombocytopenia, and large platelets. Her serum tested positively with control platelets in the MAIPA assay performed using monoclonal antibodies (MoAb) to glycoprotein (GP) IIIa (XIIF9, Y2/51), yet was negative in the presence of MoAbs to GP IIb (SZ 22) or to the GP IIb-IIIa complex (AP2, P2). The patient's platelets failed to aggregate with all agonists tested except for ristocetin. IgG isolated from the patient's serum inhibited ADP-induced aggregation of control platelets. Unexpectedly, flow cytometry showed an altered expression of membrane glycoproteins on the patient's platelets. Levels of GP Ib-IX were much higher than previously located by us in platelets. In contrast, the expression of GP IIb-IIIa was about half that seen with control subjects. When Western blotting was performed, a striking finding was a strong band of 250 kDa recognized by a series of MoAbs to GP Ib alpha in addition to the band in the normal position of GP Ib alpha. Finally, ADP-stimulated (E.B.) platelets failed to express activation-dependent epitopes on GP IIb-IIIa as recognized by PAC-1, AP6, or F26 and additionally gave a reduced P-selectin expression after thrombin addition. In conclusion, we present a novel patient with a severely perturbed platelet function where an altered membrane GP profile is associated with the presence of an autoantibody recognizing a complex-dependent determinant on GP IIb-IIIa and inhibitory of platelet aggregation.

Autoantibodies↗
PubMed1998

Double heterozygosity of the GPIIb gene in a Swiss patient with Glanzmann's thrombasthenia.

Glanzmann's thrombasthenia (GT) results from a qualitative or quantitative defect of GPIIb-IIIa complexes (integrin alphaIIbbeta3). the fibrinogen receptor on platelets. This integrin plays a critical role in platelet aggregation. In this report we describe the molecular abnormalities of a patient with clinical and laboratory findings typical of type I Glanzmann's thrombasthenia. SDS-PAGE with Western blotting revealed an absence of GPIIb but small amounts of normally migrating GPIIIa in his platelets. A non-radioactive PCR-SSCP procedure and direct sequence analysis of PCR-amplified DNA fragments showed the patient to be a compound heterozygote for mutations in the GPIIb gene. A single point mutation (G to A) at nucleotide 1064 of the cDNA derived from the mother's allele led to a Glu324 to Lys amino acid substitution in GPIIb. It was responsible for a MscI restriction site in exon 12 of the GPIIb gene. This amino acid substitution changes the electric charge between the second and third Ca++-binding domains of GPIIb. The second mutation was inherited from his father and is in exon 18 of the GPIIb gene. It was a T --> C base transition at position 1787 of GPIIb cDNA and results in a Ile565 to Thr substitution. The two GPIIb mutations identified in this study will provide new information on GPIIb-IIIa structure and biosynthesis.

Blotting, Western↗