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Biomedical subjects

G C White

Publications and source records attributed to G C White.

At least 19 recordsLinked to original sources
PubMed2001

Characterization of high-risk HIV-1 seronegative hemophiliacs.

Mechanisms that protect most high-risk HIV-1 seronegative (HRSN) persons are not well understood. Among hemophiliacs from the Multicenter Hemophilia Cohort Study who remained HIV-1 seronegative despite a high (94%) risk for acquisition of HIV-1 infection, only 7/43 were homozygous for the protective CCR5 Delta32 polymorphism. Among the remainder, neither CCR5 density nor beta-chemokine production, nor in vitro susceptibility to infection with the HIV-1 isolate JR-FL could distinguish HRSN hemophiliacs from healthy controls. When compared to lymphocytes of healthy controls not at risk for HIV-1 infection, diminished spontaneous lymphocyte proliferation was seen in lymphocytes of HRSN hemophiliacs as well as in lymphocytes of hemophiliacs not at risk for HIV-1 infection. Surprisingly sera/plasmas obtained from high-risk HIV-1 seropositve hemophiliacs prior to seroconversion more often contained alloreactive antibodies than date-matched sera/plasmas obtained from HRSN hemophiliacs. Thus alloreactivity may predispose to acquisition of HIV-1 infection after parenteral exposure.

Adolescent↗
PubMed2000

Megakaryocyte-targeted synthesis of the integrin beta(3)-subunit results in the phenotypic correction of Glanzmann thrombasthenia.

Glanzmann thrombasthenia is an inherited bleeding disorder characterized by qualitative or quantitative defects of the platelet-specific integrin, alphaIIbbeta(3). As a result, alphaIIbbeta(3) cannot be activated and cannot bind to fibrinogen, leading to a loss of platelet aggregation. Thrombasthenia is clinically characterized by mucocutaneous hemorrhage with episodes of intracranial and gastrointestinal bleeding. To develop methods for gene therapy of Glanzmann thrombasthenia, a murine leukemia virus (MuLV)-derived vector, -889Pl(A2)beta(3), was transduced into peripheral blood CD34(+) cells from 2 patients with thrombasthenia with defects in the beta(3) gene. The human alphaIIb promoter was used in this vector to drive megakaryocyte-targeted expression of the wild-type beta(3) subunit. Proviral DNA and alphaIIbbeta(3) biosynthesis were detected after in vitro differentiation of transduced thrombasthenic CD34(+) cells with megakaryocyte growth and development factor. Flow cytometric analysis of transduced patient samples indicated that 19% of megakaryocyte progeny expressed alphaIIbbeta(3) on the surface at 34% of normal receptor levels. Treatment of transduced megakaryocytes with a combination of agonists including epinephrine and the thrombin receptor-activating peptide induced the alphaIIbbeta(3) complex to form an activated conformation capable of binding fibrinogen as measured by PAC-1 antibody binding. Transduced cells retracted a fibrin clot in vitro similar to megakaryocytes derived from a normal nonthrombasthenic individual. These results demonstrate ex vivo phenotypic correction of Glanzmann thrombasthenia and support the potential use of hematopoietic CD34(+) cells as targets for alphaIIb promoter-driven MuLV vectors for gene therapy of platelet disorders. (Blood. 2000;95:3645-3651)

Antigens, CD↗
PubMed2000

Expression and function of calcium binding domain chimeras of the integrins alpha(IIb) and alpha(5).

To further identify amino acid domains involved in the ligand binding specificity of alpha(IIb)beta(3), chimeras of the conserved calcium binding domains of alpha(IIb) and the alpha subunit of the fibronectin receptor alpha(5)beta(1) were constructed. Chimeras that replaced all four calcium binding domains, replaced all but the second calcium binding domain of alpha(IIb) with those of alpha(5), or deleted all four calcium binding domains were synthesized but not expressed on the cell surface. Additional chimeras exchanged subsets or all of the variant amino acids in the second calcium binding domain, a region implicated in ligand binding. Cell surface expression of each second calcium binding domain mutant complexed with beta(3) was observed. Each second calcium binding domain mutant was able to 1) bind to immobilized fibrinogen, 2) form fibrinogen-dependent aggregates after treatment with dithiothreitol, and 3) bind the activation-dependent antibody PAC1 after LIBS 6 treatment. Soluble fibrinogen binding studies suggested that there were only small changes in either the K(d) or B(max) of any mutant. We conclude that chimeras of alpha(IIb) containing the second calcium binding domain sequences of alpha(5) are capable of complexing with beta(3), that the complexes are expressed on the cell surface, and that mutant complexes are capable of binding both immobilized and soluble fibrinogen, suggesting that the second calcium binding domain does not determine ligand binding specificity.

Amino Acid Sequence↗
PubMed2000

Intracellular function in rehydrated lyophilized platelets.

This study aimed to evaluate the effect of cross-linking and lyophilization on intracellular signalling processes in rehydrated, lyophilized (RL) platelets, which are under development as a platelet substitute for transfusion. Exposure of RL platelets to thrombin resulted in enhanced phosphorylation of several proteins, including 18 kDa and 42 kDa kinase substrates that were shown to be the substrates of myosin light chain and protein kinase C respectively. Cross-linking and lyophilization depleted the platelets of free cytoplasmic ADP and ATP, but had less effect on protein-bound nucleotides. The surface membrane of RL platelets was found to be permeable to poly dT probes less than approximately 3 kDa in size; larger nucleotide probes and proteins did not penetrate the surface membrane. Taken together, our results indicate that RL platelets retain some of the haemostatic stimulus-response functions of fresh platelets and are capable of feedback amplification in coagulation.

Adenosine Diphosphate↗
PubMed2000

Seventeen years' experience with Autoplex/Autoplex T: evaluation of inpatients with severe haemophilia A and factor VIII inhibitors at a major haemophilia centre.

A retrospective case analysis of the use of Autoplex T (anti-inhibitor coagulant complex [heat treated]) at a major haemophilia treatment centre is described. Twenty-three haemophilia A patients with a history of high-titre (>10 BU), high-response inhibitors were treated for a total of 54 bleeding episodes requiring hospital admission and/or surgical intervention. Effective or partially effective haemostatic control was achieved within 72 h in 94% of reported cases (effective 85%; partially effective 9%). The incidence of adverse reactions was low and there were no thrombotic complications. In bleeding episodes for which BU titres were determined within 6 months pre- and 6 months post-treatment, there was little tendency for Autoplex T to cause an anamnestic response (1 of 19 bleeding episodes). In the three invasive surgical procedures included in the case review, coverage with Autoplex T resulted in no postoperative complications, no bleeding at the surgical site, and no postoperative haematoma. The results support the efficacy and safety of Autoplex T in the treatment of bleeding episodes and haemostatic coverage of surgical procedures in haemophilia A patients with inhibitors.

Adolescent↗
PubMed2000

Issues with the assay of factor VIII activity in plasma and factor VIII concentrates.

A review of the literature suggests that assays accurate for the determination of factor VIII in plasma samples may not necessarily retain this accuracy when used for the determination of factor VIII in high-purity factor VII concentrates such as Hemofil M. Review of assay data suggests that it is imperative to obtain maximal activation of the factor VIII in the sample with thrombin when using an assay system of isolated coagulation factors such as the two-stage assay or the various chromogenic substrate assays. Based on a combination of ease and reproducibility of performance and correlation of in vivo and in vitro measurements. it is recommended that the one-stage activated partial thromboplastin time performed with plasma from an individual with severe hemophilia A be used for the measurement of factor VIII potency. Chromogenic substrate assays can be used if care is taken to assure optimal activation of factor VIII by thrombin in the assay and the presence of sufficient factor IXa, phospholipid and calcium ions to stabilize factor VIIIa during the assay process.

Blood Coagulation Tests↗
PubMed2000

Hemophilia factor VIII therapy. Immunological tolerance. A clinical perspective.

Human alloantibodies to factor VIII occur in 15-50% of patients with severe hemophilia A. The development of these inhibitory alloantibodies prevents treatment with replacement factor VIII and places the patient at higher risk for complications from bleeding. Immune tolerance to factor VIII can be achieved in patients with inhibitors through the regular administration of factor VIII. The mechanism by which immune tolerance is achieved is unclear but the regular administration of factor VIII is believed to interrupt the normal immune mechanisms, perhaps by altering antigen presentation and inducing T or B cell anergy. The development of immune tolerance in hemophilia can be seen as a potential model for the development of methods for inducing tolerance to other alloimmune and autoimmune disorders.

Factor VIII↗
PubMed2000

Inhibition of CD40 ligand (CD154) in the treatment of factor VIII inhibitors.

The development of persistent, high titer inhibitors represents a serious complication of the treatment of patients with severe hemophilia A. Elimination of these inhibitory antibodies is usually attempted through repeated administration of high doses of factor VIII. Such regimens are costly, time-consuming and often fail when the inhibitor is of very high titer or of longstanding duration. A potential alternative approach to inhibit the production of antifactor VIII antibodies is blockade of the T-cell/B-cell collaboration that is required to generate humoral responses. One cognate receptor pair that is required for T-cell-dependent B-cell activation consists of CD40, which is expressed on B-lymphocytes and other antigen presenting cells, and CD40 ligand (CD40L, CD154), which is transiently expressed on activated T-cells. To determine whether blockade of the CD40-CD40L pathway can inhibit the production of anti-factor VIII antibodies, a clinical study has been designed in which patients with hemophilia A and a high titer inhibitor (> 10 BU) receive monthly exposures to factor VIII in the presence of a humanized mouse monoclonal antibody to human CD40L (hu5c8*). Subjects must be between the ages of 5 and 60 years old and be HIV seronegative. To date, three subjects have received at least three doses of hu5c8 at the initial protocol dose of 10 mg/kg. Preliminary results suggest that anti-CD40L inhibition may be effective in blocking anamnestic responses to factor VIII in some patients. It remains to be determined whether this effect will persist and whether patients may eventually become tolerant to factor VIII in the absence of hu5c8 co-administration.

Adolescent↗
PubMed1999

Integrin alphaIIb promoter-targeted expression of gene products in megakaryocytes derived from retrovirus-transduced human hematopoietic cells.

Megakaryocyte-specific expression of the platelet-adhesion receptor, integrin alphaIIbbeta3, is caused by the presence of regulatory elements of the alphaIIb promoter that direct high-level, selective gene transcription early in megakaryocytopoiesis. To develop methods for targeted expression of transgenes, we transduced human CD34+ peripheral blood cells with a murine leukemia virus (MuLV) vector controlled by the human integrin alphaIIb promoter (nucleotides -889 to +35). A naturally occurring cDNA encoding the Pl(A2) alloantigen form (Pro(33)) of the integrin beta3 subunit was subcloned into this construct (-889Pl(A2)beta3) and transduced into cells that endogenously synthesized Pl(A1)beta3 (Leu(33)) as a marker for detection of provirus-derived beta3. The ability of this vector to target expression of Pl(A2)beta3 to megakaryocytes was first examined in cell lines. Immunoblot analysis with human anti-Pl(A2) alloserum detected synthesis of Pl(A2)beta3 in transduced promegakaryocytic cells; however, Pl(A2)beta3 protein was not detected in transduced epithelial cells. Human hematopoietic CD34+ cells were transduced with -889Pl(A2)beta3 virions and induced to differentiate with megakaryocyte growth and development factor. A hybrid alphaIIbbeta3 complex was formed in progeny megakaryocytes where provirus-derived Pl(A2)beta3 was detected associated with endogenous alphaIIb subunit. Another alphaIIb promoter-driven MuLV vector (-889nlacZ) encoding Escherichia coli beta-galactosidase was used to demonstrate that transgene expression was selectively targeted to the megakaryocyte progeny of transduced CD34+ cells. These studies demonstrate the feasibility of using alphaIIb promoter-driven MuLV vectors for gene transfer of hematopoietic CD34+ cells to target transgene expression in developing megakaryocytes and platelets and indicate potential applications toward human gene therapy for platelet disorders.

Antigens, CD34↗
PubMed1999

ERGIC-53 gene structure and mutation analysis in 19 combined factors V and VIII deficiency families.

Combined factors V and VIII deficiency is an autosomal recessive bleeding disorder associated with plasma levels of coagulation factors V and VIII approximately 5% to 30% of normal. The disease gene was recently identified as the endoplasmic reticulum-Golgi intermediate compartment protein ERGIC-53 by positional cloning, with the detection of two founder mutations in 10 Jewish families. To identify mutations in additional families, the structure of the ERGIC-53 gene was determined by genomic polymerase chain reaction (PCR) and sequence analysis of bacterial artificial chromosome clones containing the ERGIC-53 gene. Nineteen additional families were analyzed by direct sequence analysis of the entire coding region and the intron/exon junctions. Seven novel mutations were identified in 10 families, with one additional family found to harbor one of the two previously described mutations. All of the identified mutations would be predicted to result in complete absence of functional ERGIC-53 protein. In 8 of 19 families, no mutation was identified. Genotyping data indicate that at least two of these families are not linked to the ERGIC-53 locus. Taken together, these results suggest that a significant subset of combined factors V and VIII deficiency is due to mutation in one or more additional genes.

Amino Acid Substitution↗
PubMed1998

SH2- and SH3-mediated interactions between focal adhesion kinase and Src.

Intramolecular SH2 and SH3 interactions mediate enzymatic repression of the Src kinases. One mechanism of activation is disruption of these interactions by the formation of higher affinity SH2 and SH3 interactions with specific ligands. We show that a consensus Src SH3-binding site residing upstream of the Src SH2-binding site in FAK can function as a ligand for the Src SH3 domain. Surface plasmon resonance experiments indicate that a FAK peptide containing both the Src SH2- and SH3-binding sites exhibits increased affinity for Src. Furthermore, the presence of both sites in vitro more potently activates c-Src. A FAK mutant (FAKPro-2) with substitutions destroying the SH3-binding site shows reduced binding to Src in vivo. This mutation also reduces Src-dependent tyrosine phosphorylation on the mutant itself and downstream substrates, such as paxillin. These observations suggest that an SH3-mediated interaction between Src-like kinases and FAK may be important for complex formation and downstream signaling in vivo.

Amino Acid Sequence↗
PubMed1998

Giant platelet disorder in a patient with type 2B von Willebrand's disease.

While patients with type 2B von Willebrand's disease often exhibit thrombocytopenia, platelet morphology is typically normal. We describe a 44-year-old Jamaican man with thrombocytopenia and a history of bleeding, who had giant platelets on his peripheral blood film. Functional studies and von Willebrand factor gene sequencing showed him to have type 2B von Willebrand's disease with a heterozygous point mutation resulting in a V553M (V1316M in the new von Willebrand factor gene mutation nomenclature) amino acid substitution. Family studies showed one of his two sisters to have an ill-defined giant-platelet-syndrome with mild thrombocytopenia, but not von Willebrand's disease, indicating that the association of giant platelets and von Willebrand's disease in our patient was most likely coincidental. This report describes the rare concurrence of two uncommon disorders. It also demonstrates how the thrombocytopenia of type 2B von Willebrand's disease can be misdiagnosed as ITP, leading to unnecessary and potentially harmful therapeutic interventions.

Adult↗
PubMed1998

Successful treatment of transient acquired factor X deficiency by plasmapheresis with concomitant intravenous immunoglobulin and steroid therapy.

Two patients with no history of previous bleeding diatheses presented with active bleeding from multiple body sites, declining hemoglobin levels, and markedly prolonged prothrombin times (PT) and activated partial thromboplastin times (aPTT) with incomplete correction on PT mix assays. Both patients demonstrated a severe deficiency of factor X (F.X) (<1%; reference range 60-150%). F.X levels and bleeding were refractory to multiple transfusions of fresh frozen plasma (FFP) in both patients. In contrast, daily therapeutic plasma exchange (PLEX) with concomitant administration of intravenous immunoglobulin (IV IgG) and steroids produced a rapid increase in F.X levels with cessation of bleeding, followed by stabilization and normalization of F.X levels and progressive correction of coagulation times. Neither patient has demonstrated a recurrence of the bleeding tendency following discontinuation of steroid therapy. These patients had transient acquired F.X deficiency, a rare coagulopathy, which can result in a lethal bleeding diathesis. An IgG inhibitor that selectively inhibited F.X activation in Russell's viper venom or tissue factor/F.VIIa assays was demonstrated in one patient's pretreatment plasma. Previous treatment of hemorrhage in transient acquired F.X deficiency has been prothrombin complex and/or activated clotting concentrates, which can be associated with transient hypercoagulable states. This is the first reported use of PLEX in transient acquired F.X deficiency. PLEX is safe, efficacious, and rapidly restores hemostasis in this rare acquired bleeding disorder.

Adult↗
PubMed1998

Mammalian recombinant coagulation proteins: structure and function.

Recombinant DNA technology has permitted the production of synthetic proteins which are potentially free of human infectious agents. Despite production in foreign cells, these proteins are structurally and functionally comparable to the native proteins. Recombinant clotting factors VIII, IX, VIIa, and von Willebrand factor have the same primary sequence as their plasma counterparts. The secondary and tertiary structures are similar, Post-translational modifications, including proteolytic processing, disulfide bonding, addition and processing of N- and O-linked glycans, gamma-carboxylation of glutamic acid residues, beta-hydroxylation of aspartic acid residues, sulfation of tyrosine residues, and phosphorylation of serine residues, are similar but not always identical, In some instances. these differences may cause significant functional differences. For example, reduced tyrosine sulfation and serine phosphorylation of recombinant factor IX have been correlated with reduced recovery following intravenous infusion. The specific clotting activity of the recombinant factors, an indication of their coagulant function, is equivalent to that of the plasma factors. Finally, these proteins have been used clinically and shown to correct clinical deficiencies of these proteins in a manner that is similar to replacement with plasma factors. All in all, the promise of recombinant DNA technology for coagulation and other disorders remains bright.

Animals↗