von Willebrand factor and von Willebrand disease [published erratum appears in Blood 1988 Mar;71(3):830]

Progress has occurred in the past several years in the understanding of the structure and function of von Willebrand factor (vWF). This multimeric glycoprotein exhibits a dual role, that of mediating platelet adhesion and aggregation onto thrombogenic surfaces, and that of functioning as carrier in plasma for the factor VIII procoagulant protein. New insights into the nature of the several functional domains of vWF have led to the identification of the regions of the molecule that interact with factor VIII, heparin, the glycoprotein lb of platelets, and collagen. Alterations of vWF are the cause of von Willebrand disease (vWD), a congenital bleeding disorder. In the majority of patients, the plasma levels of vWF are decreased, but there is no demonstrable structural or functional alteration of the protein. In other patients, however, the structure of vWF is abnormal. This review summarizes the current knowledge on vWF and vWD.     

Identification of platelet glycoprotein IIb/IIIa as the major binding site for released platelet-von Willebrand factor [published erratum appears in Blood 1987 Jun;69(6):1789]

We studied the effects(s) of two monoclonal antibodies, 6D1 and 10E5 (directed against platelet glycoprotein Ib [GPIb] and the GPIIb/IIIa complex, respectively), and purified human plasma fibrinogen on the binding of released platelet-von Willebrand factor (vWf) to the platelet surface. Neither of the monoclonal antibodies nor fibrinogen had any effect on the amount of platelet-vWf expressed on unstimulated platelets or on the amount expressed on platelets stimulated in the absence of extracellular Ca++. However, the antibody directed against GPIIb/IIIa inhibited 72% of the thrombin-induced increase in the platelet-vWf bound to the platelet surface when platelets were stimulated in the presence of 5 mmol/L Ca++. The antibody against GPIb did not inhibit the surface expression of platelet-vWf on stimulated platelets in the presence of Ca++. Purified normal human fibrinogen inhibited the surface binding of platelet-vWf to thrombin-stimulated platelets to a degree similar to that observed with the monoclonal antibody directed against the GPIIb/IIIa complex. These data indicate that platelet-vWf released from platelets binds primarily to the GPIIb/IIIa complex at or near the plasma fibrinogen binding site.     

The isolation of human platelet factor V [published erratum appears in Blood 1987 Jul;70(1):339]

Human platelet factor V has been isolated using either a monoclonal or polyclonal antibody directed against human plasma factor V. The largest peptide observed upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of purified human platelet factor V comigrates with purified human plasma factor V. However, a significant portion of the isolated protein is represented by peptides of lower apparent molecular weight (Mr). These lower Mr species that copurify with platelet factor V have been shown to be platelet factor V components by their immunological cross-reactivity with monoclonal and polyclonal antibodies to purified human plasma factor V. Platelets isolated from whole blood drawn directly into inhibitors to prevent proteolysis and platelet activation demonstrate the pattern of fragmented platelet factor V. The components of purified platelet factor V demonstrate apparent Mr ranging between 115 K and 330 K and are detectably different from the intermediates and end products observed during the thrombin cleavage of single-chain plasma factor V. Upon treatment with thrombin the platelet factor V components are cleaved and the end products are indistinguishable from those obtained upon thrombin activation of plasma factor V to plasma factor Va. Examination of the components by immunoblotting demonstrates that some of the cleavages which have occurred in the platelet factor V molecule are within the 150-K activation peptide. Bioassay indicates that platelet factor V exists as a procofactor and cleavage by thrombin yields the active cofactor, platelet factor Va. These data suggest that human platelet factor V is stored in the platelet as a partially fragmented procofactor that can be activated by thrombin to yield human platelet factor Va, the active cofactor in the human prothrombinase complex.     

Rearranged antigen receptor genes in Hodgkin's disease [published erratum appears in Blood 1987 Sep;70(3):893]

Despite intensive efforts using a wide variety of approaches, the cellular lineage and clonality of the abnormal cells of Hodgkin's disease have remained an enigma. In the present study, cell separation techniques that enriched for Reed-Sternberg cells and their variants were used to generate sufficient percentages of abnormal cells to allow detection of rearrangements in these cell fractions. DNA from the involved tissues of eight Hodgkin's disease patients was subjected to Southern blot analysis to detect rearrangements of T cell antigen receptor genes and immunoglobulin genes. Immunoglobulin gene rearrangements were found in three of five cases in which Reed- Sternberg cells and their variants were enriched by cell separation techniques to cell frequencies greater than 1%. Rearrangements of immunoglobulin heavy chain genes occurred in two cases, and a lambda light chain gene rearrangement occurred in a third case. Rearrangements were not detected in lymphocyte fractions or in unseparated cells prepared from the same tissues. The putative Hodgkin's cell line, L428, also contained rearrangements of immunoglobulin heavy and kappa and lambda light chain genes and, in addition, harbored a single T cell receptor beta gene rearrangement. These findings indicate that Reed- Sternberg cell-enriched fractions contain clonal cell populations and provide a lead, at the molecular genetic level, to a possible lymphoid derivation of the Reed-Sternberg cell.     

What radiation dose for DLA-identical canine marrow grafts? [published erratum appears in Blood 1989 Feb;73(2):624]

In view of reported attempts at marrow grafting after nuclear accidents with a broad range of radiation exposures, the present study explored the total-body irradiation (TBI) conditions needed for engraftment in a canine model by using marrow from DLA-identical littermates. Previous studies have shown that such grafts are consistently successful when recipients are exposed to 920 cGy of TBI delivered at a rate of 7 cGy/min from opposing dual cobalt sources. The present TBI doses were all in the lethal range. Five dogs were administered 450 cGy; seven dogs, 600 cGy; five dogs, 700 cGy; and five dogs, 800 cGy of TBI administered at 7 cGy/min. They received a median of 3.3 x 10(8) marrow cells/kg intravenously after completion of radiation. Results showed transient allogeneic marrow engraftment in all dogs administered the lowest dose of TBI studied (450 cGy). Importantly, transient grafts permitted four of five dogs to live long enough for autologous marrow recovery to occur. At increasing radiation doses, 600, 700, and 800 cGy, the risk of graft failure lessened, with 3 of 7, 2 of 5, and 1 of 5 dogs, respectively, showing graft rejection. Fewer dogs survived with autologous marrow recovery, and more showed sustained allogeneic engraftment (4 of 7, 3 of 5, and 4 of 5 dogs, respectively). We conclude that DLA-identical littermate marrow grafts are beneficial in the setting of otherwise lethal radiation exposures, with most dogs either experiencing sustained allogeneic engraftment or surviving with autologous marrow recovery due to the extended support provided by a transient allogeneic graft.     

von Willebrand disease in the RIIIS/J mouse is caused by a defect outside of the von Willebrand factor gene [published erratum appears in Blood 1995 Sep 15;86(6):2461]

An animal model for human type I von Willebrand disease (vWD) has been previously described in the inbred mouse strain RIIIS/J. Murine vWD is characterized by a prolonged bleeding time, normal von Willebrand factor (vWF) multimer distribution, autosomal dominant inheritance, and proportionately decreased plasma vWF antigen, ristocetin cofactor, and factor VIII (FVIII) activities. To study the molecular genetics of murine vWD, a portion of the vWF gene surrounding exon 28 was cloned, sequenced, and used to develop two informative DNA sequence polymorphisms for rapid genotyping by DNA polymerase chain reaction. RIIIS/J mice were crossed with PWK/Ph mice, an inbred line of Mus musculus musculus, and the F1 progeny backcrossed to the parental PWK/Ph strain. vWF antigen levels in F1 mice were not significantly different from the parental RIIIS/J strain but were markedly decreased compared with the parental PWK/Ph mice. Genetic linkage analysis of 104 backcross progeny showed no correlation between vWF antigen level and vWF genotype. These data indicate that murine vWD is caused by a defect at a novel genetic locus, distinct from the murine vWF gene. The distribution of vWF antigen levels among backcross progeny suggests the presence of one major dominant vWD gene in the RIIIS/J mouse with possible modifying contributions from one or more additional minor loci. These observations may provide new insights into the molecular basis and variable expressivity of human vWD.     

Interrelationship between mitosis and endomitosis in cultures of human megakaryocyte progenitor cells [published erratum appears in Blood 1987 May;69(5):1547]

Sera from dogs rendered aplastic by total-body irradiation stimulate human bone marrow megakaryocyte progenitors to form megakaryocyte colonies in plasma clot cultures. In this investigation, we evaluated the effects of varying concentrations of such sera on both the mitotic and endomitotic phases of human megakaryocyte development in vitro. When low concentrations of aplastic canine sera (2.5% to 5.0% [vol/vol]) were added to cultures of human peripheral blood mononuclear cells in place of normal AB serum, megakaryocyte colony formation was augmented fivefold, cell numbers per colony increased approximately 2.5- fold, and the geometric mean megakaryocyte ploidy almost doubled. Further increasing the aplastic canine serum concentration from 10% to 30% (vol/vol) stimulated no additional colony formation. However, there was a further augmentation of cell numbers per colony associated with a progressive decrease in the mean megakaryocyte ploidy. Megakaryocyte cultures were harvested after 7, 12, 15, and 19 days of incubation, and these demonstrated that the lower mean ploidy values found at the higher concentrations of aplastic canine serum did not result from delayed endoreduplication. At all aplastic serum concentrations evaluated, there existed a strong correlation between nuclear ploidy and cell diameter. We conclude that both the mitotic and endomitotic events in human megakaryocytopoiesis may be influenced by a factor or factors present in aplastic canine serum. At lower in vitro concentrations, such sera stimulate both mitosis and endomitosis, which promotes the development of megakaryocyte colonies composed of larger cells with a higher mean ploidy. With increasing aplastic serum concentrations, colony formation plateaus and mitosis is favored over endomitosis. This results in colonies composed of more numerous but smaller megakaryocytes with a lower mean ploidy. Our data suggest that the size and extent of polyploidization that can be achieved by a developing megakaryocyte may be influenced by the mitotic prior history of its immediate precursor cell.     

Ektacytometric measurement of sickle cell deformability as a continuous function of oxygen tension [published erratum appears in Blood 1987 Apr;69(4):1272]

In an effort to study the rheologic effects of small amounts of hemoglobin S (HbS) polymer in sickle red cells, we have used the ektacytometer, a laser diffraction couette viscometer, to measure sickle cell deformability as a function of oxygen tension. Sickle cell populations of defined intracellular hemoglobin concentration (MCHC) were isolated using Stractan density gradients and were resuspended in buffered polyvinylpyrrolidone solutions for deformability measurements. Using a gas-porous, hollow fiber gas exchange system to establish a linear gradient in oxygen tension, deformability was measured over a pO2 range of 76 to 0 mm Hg. Parallel spectroscopic determinations of oxygen saturation permitted determination of cell deformability as a function of oxygen saturation for each discrete MCHC population. From these measurements the level of oxygen saturation at which a loss in cell deformability was first detected could be defined. Then, using the data of Noguchi and Schecter, the amount of polymerized HbS in the cells at that defined level of oxygen saturation was estimated. The results of this analysis suggested that the quantity of polymer that caused a detectable loss in cell deformability increased with increasing MCHC. In addition, for MCHC above 30 g/dL, this represented a substantial fraction of the total HbS in the cell.     

Recombinant adeno-associated virus-mediated gene transfer into hematopoietic progenitor cells [published erratum appears in Blood 1995 Feb 1;85(3):862]

Recombinant adeno-associated viruses (rAAV) containing only the inverted terminal repeats (ITR) from the wild-type virus are capable of stable integration into the host cell genome, and expression of inserted genes in cultured cells. We have now defined the ability of rAAV to introduce genes into primary hematopoietic progenitors. A vector was constructed containing the coding sequences for beta- galactosidase (beta-gal), including a nuclear localization signal, under the control of a strong viral promotor. Infectious vector particles were prepared by cotransfection of the vector plasmid with a second plasmid that contained the coding sequences for AAV proteins into adenovirus-infected human embryonic kidney cells. These vector preparations transferred and expressed the beta-gal gene in human K562 erythroleukemia and Detroit 6 cells. Positive immunoselection yielded a population of enriched CD34+ cells that were transduced with the rAAV beta-gal vector. Nuclear localized enzyme expression was documented in 60% to 70% of infected cells. Progenitor-derived colonies that developed after 2 weeks in clonogenic cultures were shown to have viral- associated DNA at an estimated copy number of 1 to 2 per cell using a semiquantitative polymerase chain reaction (PCR) method. Integration of AAV into hematopoietic progenitors was documented using wild-type virus, as its genome may integrate at a preferred site on chromosome 19. Our data suggest that rAAV will transfer and express genes in primitive hematopoietic progenitors with high frequency, and support the development of this vector system for therapeutic gene transfer.     

The structure of the T cell gamma chain gene in lymphoproliferative disorders and lymphoma cell lines [published erratum appears in Blood 1987 Jan;69(1):368]

During the development of B and T cells, a number of genes undergo rearrangement. In this paper we have studied the structure of the T cell gamma-chain gene in primary lymphomas and cell lines derived from patients with lymphoma. Samples derived from patients with angioimmunoblastic lymphadenopathy (AIL), Lennert's lymphoma, and Hodgkin's disease were also examined. TcR gamma was rearranged in all T cell lymphomas and in some B cell lymphomas and B cell lymphoma cell lines. Rearrangement of the gamma-chain gene was also found in AIL, Lennert's lymphoma, and four of eight cases of Hodgkin's disease. These studies indicate that rearrangement of TcR gamma is a useful clonal marker but does not aid in the identification of cell lineage.     

Increased resistance to membrane deformation of shape-transformed human red blood cells [published erratum appears in Blood 1987 Sep;70(3):893]

The study of shape-transformed human erythrocytes is of hematological interest since several clinical conditions are associated with erythrocyte shape changes. Using the micropipette technique, we examined the rheological behaviors of echinocytes induced by sodium salicylate or ATP depletion and of stomatocytes induced by chlorpromazine. Both the discocyte-echinocyte and discocyte-stomatocyte transformations resulted in an increased resistance to extension. Sodium salicylate-treated erythrocytes could be returned to normal morphology and normal membrane rheology by addition of chlorpromazine. We conclude that these morphological changes are associated with detrimental effects on the intrinsic membrane deformability of the human erythrocyte.     

Total body irradiation and high-dose etoposide: a new preparatory regimen for bone marrow transplantation in patients with advanced hematologic malignancies [published erratum appears in Blood 1987 Jun;69(6):1789]

In a phase I/II study, 47 patients (median age, 24 years) with hematologic malignancies (33 patients with acute leukemia not in first remission and 14 patients with other advanced malignant hematologic disorders) were treated with total body irradiation and high doses of etoposide (VP16-213) followed by bone marrow transplantation. At the time of analysis, 21 patients were alive, and 19 of them were in continued complete remission for 101 days to greater than 40 months (median, 12 months). The actuarial disease-free survival rate of the 33 acute leukemia patients is 43% (2 SEM, 18%) and the actuarial relapse rate is 32% (2 SEM, 20%). Five of the 14 patients with the other hematologic malignancies are alive, and four of them continue to be free of disease for 8 to 27 months. Pharmacokinetic studies established a strong correlation between the administered drug doses and their plasma levels and also demonstrated complete drug clearance prior to marrow grafting. An etoposide dose of 60 mg/kg body weight was found to be the maximum tolerated dose. This new preparatory regimen was well tolerated and was not associated with specific acute or long-term regimen-related toxicities. Our data suggest that total body irradiation with high-dose etoposide presents a viable alternative to other preparatory regimens. The role of this novel combination remains to be defined by future prospective randomized trials.     

Rearrangement of both immunoglobulin and T-cell receptor genes in a prolymphocytic variant of hairy cell leukemia patient resistant to interferon-alpha [published erratum appears in Blood 1989 Feb;73(2):624]

We describe a patient with the so-called "prolymphocytic variant" form of hairy cell leukemia (HCL) resistant to treatment with interferon- alpha (IFN-alpha). Analysis of immunoglobulin (Ig) and T-cell receptor- beta (TCR beta) gene rearrangements from serial peripheral blood mononuclear cell specimens (MNCs) confirmed not only the B-cell nature of the disease, but also the subsequent emergence of a morphologically indistinguishable population of cells with a clonal TCR beta rearrangement in addition to the original Ig gene rearrangement. With the exception of a transient increase in peripheral blood T cells during treatment with deoxycoformycin (DCF), the MNCs remained essentially constant throughout therapy with no evidence of a co- existing T-cell clone to account for the TCR beta rearrangement. Although MNCs from this patient bound significantly less IFN-alpha than did MNCs from other HCL patients, the binding was of high affinity with a kd similar to that of control cells. The number of IFN-gamma receptors on our patient's MNCs was four times higher than the number of IFN-alpha receptors and was similar to the number of IFN-alpha receptors on MNCs from HCL patients responsive to IFN-alpha. While various treatments including IFN-alpha, DCF, chlorambucil, splenectomy, leukopheresis, and IFN-gamma were not able to change the clinical progression of the disease, they may have provided an opportunity for the divergent TCR beta rearranged clone to expand and displace the initially dominant clone.     

Life-threatening transient neonatal Behcet's disease [published erratum appears in Br J Rheumatol 1997 Sep;36(9):1032]

This case report describes transient neonatal Behcet's disease, with life-threatening complications in the neonate. Male Baby R developed blood-streaked diarrhoea 5 days after birth, followed by recurrent severe scarring orogenital ulceration and vasculitic skin lesions. In this sixth week of life, he developed stridor leading to a respiratory arrest and necessitating assisted ventilation. No infective cause was isolated. Baby R responded well to i.v. and subsequent oral steroid therapy. At 8 weeks old he had fully recovered and remains well. Baby R's mother was not previously known to have Behcet's disease. During the pregnancy, she began to suffer orogenital ulceration, associated with skin lesions typical of Behcet's disease. Mild orogenital ulceration has become recurrent.      

Evidence that large granular lymphocytes from B-CLL patients with hypogammaglobulinemia down-regulate B-cell immunoglobulin synthesis [published erratum appears in Blood 1989 Jun;73(8):2232]

B-chronic lymphocytic leukemia (CLL) patients frequently suffer from moderate to severe hypogammaglobulinemia. This complication is a serious cause of morbidity and mortality in this disorder. There is recent evidence that natural killer (NK) cells modulate B-cell immunoglobin (Ig) synthesis/secretion. The authors therefore evaluated the circulating NK cells from B-CLL patients on their ability to regulate mitogen-induced B-cell Ig synthesis. Blood, NK cells (CD16+, CD3-) from three B-CLL patients with hypogammaglobulinemia were able to clearly down-regulate the pokeweed mitogen (PWM)-induced-B-cell Ig secretion. In contrast, CD16+, CD3- cells from age-sex-matched controls or B-CLL patients with normal Ig were either nonregulatory or enhanced mitogen-induced B-cell Ig secretion. An alternative explanation for hypogammaglobulinemia in B-CLL patients is the immunomodulation of B- cell Ig production/secretion by CD16+, CD3- blood cells.     

Abnormal subcellular distribution of myosin and talin in Wistar Furth rat platelets [published erratum appears in Blood 1995 Aug 1;86(3):1242]

The roles of most cytoskeletal proteins in platelet formation and function remain largely undefined. We earlier detected megakaryocyte membrane blebbing and a unique antigenic determinant associated with a missense mutation in the cytoskeletal protein, talin, in an animal model of hereditary macrothrombocytopenia, the Wistar Furth (WF) rat, which led us to examine the distribution of talin and other cytoskeletal proteins in resting normal and WF rat platelets. In contrast to the conclusions of an earlier ultrastructural analysis, our biochemical and ultrastructural immunogold studies indicate a significant membrane-association of talin in both resting normal and WF rat platelets as found earlier for rat megakaryocytes. Talin was associated with plasma membranes, membranes of the surface-connected canalicular system, and with alpha-granule membranes of both normal and WF rat platelets, but as in WF megakaryocytes, talin was absent from the large membrane complexes of WF platelets. An even more striking difference was seen in the distribution of myosin in subcellular fractions of normal and WF rat platelets separated in density gradients, in which the proportion of myosin in the least dense WF rat platelet membrane fraction was one half that in the same normal platelet fraction. This difference was balanced by a fourfold increase in myosin in the most dense WF rat subcellular fraction, which is highly enriched for alpha-granules. These results support our hypothesis that the platelet abnormalities of the WF rat are related to defects in the megakaryocyte-platelet cytoskeleton.     

BCR-ABL maintains resistance of chronic myelogenous leukemia cells to apoptotic cell death [published erratum appears in Blood 1994 Jun 15;83(12):3835]

Apoptosis is the major form of cell death associated with the action of chemotherapeutic agents on tumor cells, and therefore the expression of genes that interfere with apoptosis can have important consequences for the efficacy of therapeutic approaches. Here we show that K562, a chronic myelogenous leukemia (CML) cell line expressing the BCR-ABL fusion protein, are resistant to the induction of apoptosis by a number of agents and conditions. Antisense oligodeoxynucleotides corresponding to the translation start of bcr downregulate bcr-abl protein in these cells and render them susceptible to induction of apoptosis by chemotherapeutic agents or serum deprivation. Expression of a temperature sensitive v-Abl protein reverses the effects of the antisense oligonucleotides, such that the cells remain resistant to apoptosis at the permissive temperature. These data indicate that bcr- abl acts as an anti-apoptosis gene in CML cells and suggests that the effect is dependent on the abl kinase activity in this chimeric protein. Inhibition of bcr-abl to render CML cells susceptible to apoptosis can be combined with therapeutic drugs and/or treatment capable of inducing apoptosis to provide an effective strategy for elimination of these cells.