Further characterization of platelet-type von Willebrand's disease in Japan

We studied four patients who showed aggregation of platelets in platelet-rich plasma at lower concentrations of ristocetin than those required for normal platelet-rich plasma and who demonstrated an increased capacity of the platelets to bind normal von Willebrand factor. The four patients were from two Japanese families. Platelets from one family aggregated spontaneously in vitro, and platelets from both families aggregated upon the addition of normal plasma and cryoprecipitate, in the absence of ristocetin or other agonists. Analysis of the multimeric composition of von Willebrand factor by sodium dodecyl sulfate-agarose gel electrophoresis revealed a decrease in large multimers or a decrease in both large and intermediate multimers in plasma, but normal multimers in platelets. 1-Deamino-[8-D- arginine]-vasopressin caused by an immediate appearance of larger multimers in plasma, followed by the rapid disappearance of these multimers from circulating plasma. Analysis of platelet membrane glycoproteins from the patients showed that there were two distinct bands in the glycoprotein I region; one migrated in a slower region and the other in a faster region than normal glycoprotein Ib. We suggest that the platelet receptor abnormality in these patients is related to this abnormality of glycoprotein Ib.     

Real-time analysis of shear-dependent thrombus formation and its blockade by inhibitors of von Willebrand factor binding to platelets

Two likely mechanisms for the initiation of arterial platelet thrombus formation under conditions of elevated fluid shear stresses are: (1) excessive adhesion and aggregation of platelets from rapidly flowing blood onto the exposed sub-endothelium of injured, atherosclerotic arteries; or (2) direct, fluid shear stress-induced aggregation of platelets in constricted arteries with intact endothelial cells. Mechanism (1) was simulated using a parallel plate flow chamber, fibrillar collagen type I-coated slides, and mepacrine-labeled (fluorescent) platelets in whole blood anticoagulated with citrate, hirudin, unfractionated porcine heparin, or low molecular weight heparin flowing for 1 to 2 minutes at wall shear rates of 100 to 3,000 seconds-1 (4 to 120 dynes/cm2). The precise sequence of interactions among von Willebrand factor (vWF), glycoprotein (GP)Ib, and GPIIb-IIIa during platelet adhesion and subsequent aggregation were resolved by direct real-time observation using a computerized epifluorescence video microscopy system. Adhesion at high shear rates was the result of the adsorption of large vWF multimers onto collagen and the binding of platelet GPIb to the insolubilized vWF. Aggregation occurred subsequently and required the binding of ligands, including vWF via its RGD binding domain, to GPIIb-IIIa. Mechanism (2) was modeled by producing shear stresses of 90 to 180 dynes/cm2 in a rotational cone and plate viscometer, which aggregates platelets from platelet-rich- plasma (PRP) anti-coagulated with citrate, hirudin, or either type of heparin in reactions that require large vWF multimers, Ca2+, adenosine diphosphate, and both GPIb and GPIIb-IIIa. Both vWF-mediated shear- aggregation in PRP and platelet-collagen adhesion in flowing whole blood (anticoagulated with citrate and hirudin) are inhibited by two potentially useful anti-arterial thrombotic agents: polymeric aurin tricarboxylic acid (ATA; 28.5 to 114 micrograms/mL), which binds to vWF and inhibits its attachment of GPIb, and a recombinant vWF fragment (rvWF445-733; 30 to 200 micrograms/mL) that binds to platelet GPIb (in the absence of any modulator) and blocks attachment of vWF multimers. Unfractionated heparin, but not low molecular weight heparin, apparently binds to rvWF445-733 and counteracts the inhibitory effects of the vWF fragment in vitro on shear-aggregation and platelet-collagen adhesion.     

Management of oral complications of disease-modifying drugs in rheumatoid arthritis

Stomatitis is a troublesome adverse effect of disease-modifying anti- rheumatic drug (DMARD) therapy in rheumatoid arthritis (RA) patients. This review presents data to examine the incidence, clinical features and consequences of DMARD-related stomatitis, and suggests an algorithm for its clinical management. The specific objectives of the two studies presented here were to determine the incidence of DMARD-related stomatitis and its effect on DMARD continuation, and secondly to identify the clinical and laboratory risk factors. We investigated two cohorts of patients: (i) a retrospective survey of data collected from drug monitoring clinics run for patients on DMARDs from 1987 to 1994 involving 1539 patients and 2394 drug exposures; (ii) a prospective study of 25 consecutive RA patients presenting with DMARD-related stomatitis compared to 29 RA controls with no history of DMARD stomatitis. The retrospective survey showed that 2% of DMARD patients stopped therapy because of stomatitis, but 55% of these were able to resume the same therapy. In the case control study. 24% of patients discontinued temporarily and 8% permanently. Cases of DMARD-related stomatitis differed from controls in that they had a higher incidence of previous mouth ulcers (40% vs 14%), they smoked less (8% vs 31%) and Schirmer's test was more often abnormal (44% vs 21%). There were no differences in RA severity, disease activity or oral hygiene. Haematinic deficiencies were equally common in cases and controls: 30% for iron, 8% for vitamin B12 and 24% for folic acid. Herpes simplex virus was involved in a minority (8%) of cases. In conclusion, the occurrence of stomatitis in RA patients on DMARD should not lead to cessation of drug therapy, but to a careful evaluation so that patients may be maintained on effective treatment.      

Analysis of Epstein-Barr virus gene polymorphisms in normal donors and in virus-associated tumors from different geographic locations

While Epstein-Barr virus (EBV) infection is associated with the development of certain lymphoid and epithelial tumors, the role of the virus in the pathogenesis of these malignancies remains unknown. It has been suggested that EBV strain variation may contribute to tumor development. Two major strains of EBV, type 1 and type 2, have been identified on the basis of genetic polymorphisms and other minor genetic variations give rise to distinct EBV isolates. We analyzed EBV strain variation in healthy individuals and compared them with EBV isolates present in lymphoid and epithelial neoplasms from the same geographic regions. In particular, the incidence of the 30-bp latent membrane protein (LMP1) gene deletion, recently implicated in the development of more aggressive forms of virus-positive lymphomas and Hodgkin's disease [HD], was examined in the normal population. While a preferential association of the LMP1 deletion with the type 2 strain of EBV was observed, there was no increased incidence of virus isolates carrying this deletion in HD, Burkitt's lymphoma, or virus-associated carcinomas compared with the appropriate normal population. A polymorphism in the BamHI F region of the EBV genome, previously identified in Chinese populations, was found at increased incidence in European HD biopsies. Overall, we found that most of the EBV gene polymorphisms detected in EBV isolates from healthy virus carriers occurred with similar frequency in virus-associated tumors from the same geographical region.     

BCR-ABL protein expression in peripheral blood cells of chronic myelogenous leukemia patients undergoing therapy

Chronic myelogenous leukemia (CML) is a myeloproliferative disorder associated with the Philadelphia chromosome (Ph1) in more than 95% of these patients. The Ph1 and the resulting BCR-ABL fused genes are markers for this type of leukemia. In CML, the product of the fused BCR- ABL gene is typically a protein of approximately 2,000 amino acids termed P210 BCR-ABL. We have developed an assay for the BCR-ABL protein involving Western blotting of circulating white blood cells (WBC) with an anti-ABL monoclonal antibody that can detect P210 BCR-ABL and P145 ABL in peripheral blood cells from chronic phase Ph1-positive leukemia patients. This assay was used to analyze the BCR-ABL protein content of circulating WBC from CML patients before and after various treatments. In parallel to changes in percentages of Ph1-positive blood cells as determined by cytogenetic analyses of bone marrow samples, BCR-ABL protein expression in blood cells decreased or increased as patients entered remission or underwent relapse. Of interest, six Ph1-negative CML patients were BCR-ABL protein-positive. All except one had a rearrangement in the major breakpoint cluster region and that patient expressed P185 BCR-ABL and not P210. Our results indicate that the BCR- ABL Western blotting assay has clinical applications for both diagnosis and prospective evaluation of Ph1-positive and Ph1-negative CML patients.     

Determination of human platelet antigen frequencies in the Dutch population by immunophenotyping and DNA (allele-specific restriction enzyme) analysis

Platelets from 200 random Dutch blood donors were typed for the human platelet alloantigens HPA-1 to -5 recognized at present and for Naka. Naka is an epitope on glycoprotein IV, not expressed on the platelet of individuals with hereditary GP IV deficiency. Platelet immunofluorescence and monoclonal antibody-specific immobilization of platelet antigens (MAIPA) were applied for this purpose. The observed phenotype frequencies were 97.86% and 28.64% for HPA-1a and -1b, 100% and 13.15% for HPA-2a and -2b, 80.95% and 69.84% for HPA-3a and -3b, 100% and 0% for HPA-4a and -4b, 100% and 19.7% for HPA-5a and HPA-5b, respectively. Platelets from all donors reacted with the anti-Naka antibodies. To determine the gene frequencies for the HPA-1, HPA-2 and HPA-3 systems directly, DNA from 98 of these donors was isolated from peripheral blood mononuclear leucocytes and specific fragments were amplified by polymerase chain reaction (PCR). The fragments were analyzed using allele-specific restriction enzymes (ASRA). In all amplified PCR products an "internal control" for each assay, ie, a restriction site for the applied enzyme independent from the phenotype of the donor was present. In all donors tested, phenotypes, as determined by serological methods and genotypes, directly determined by the ASRA, were identical. Thus, the PCR-ASRA described in this report is a practical and reliable technique for the determination of alleles that code for platelet antigen allotypes, at least in the Dutch population.     

Analysis of interphase cells for the Philadelphia translocation using painting probe made by inter-Alu-polymerase chain reaction from a radiation hybrid

Fluorescence in situ hybridization (FISH) probe for the identification of the Philadelphia (Ph) translocation [t(9;22) (q34;q11)] in chronic myelogenous leukemia cells was developed by inter-Alu-polymerase chain reaction of DNA from an interspecific somatic cell hybrid containing approximately 5 Mb of human DNA covering the ABL gene region on human chromosome 9q34. This probe was large enough to be effective in identifying the genomic domains yet small enough to resolve them in more than 90% of bone marrow interphase cells. Combination of the probe with a cosmid contig probe for the BCR region of chromosome 22 in two- color FISH reduced the frequency of false-positive identification of the Ph chromosome to less than 1%. The procedure allows detection of as few as 1% Ph+ cells independent of the cycling status or BCR/ABL expression level of cells, and the quantitation of non-Ph chromosome- containing interphase nuclei in the marrow of patients judged 100% Ph+ by standard cytogenetics.     

Histamine induces interleukin-8 secretion by endothelial cells

It has been shown that histamine induces early changes on endothelial cells (EC), such as a transient expression of P-selectin and secretion and/or surface expression of early mediators (eg, prostacyclin [PG1(2)], platelet-activating factor [PAF], and leukotriene B4 [LTB4]). However, delayed effects of histamine on EC and particularly on cytokine production are undefined. In this study, the effect of histamine on interleukin (IL)-8 production by EC was evaluated using an enzyme-linked immunosorbent assay (ELISA) method and mRNA expression. The results showed that histamine increased the secretion and the mRNA expression of IL-8 by EC. Histamine-induced IL-8 production was (1) dose-dependent (at a dose > or = 10(-6) mol/L), (2) potentialized by costimulation with tumor necrosis factor (TNF)-alpha, (3) inhibited by H1 or H2 histamine receptor antagonists, and (4) significantly increased 4 hours after the initial stimulation. These data suggest that histamine may be involved in the control of the late inflammatory reaction associated to allergic disorders through IL-8 secretion by EC.