Adenosine diphosphate stimulation of cultured hematopoietic cell lines.

Adenosine diphosphate (ADP) plays a critical role in platelet activation both by exogenous stimulation and the release of endogenous intracellular stores. As the platelet ADP receptor is not well defined, we have chosen to identify and characterize several cell lines that possess functional receptors for this nucleotide. Rat promegakaryoblasts (RPM), human erythroleukemia cells (HEL), U937, and K562 leukemia cells responded to ADP, as measured by a rapid increase in intracellular calcium. In the case of RPM cells, ADP was the only naturally occurring platelet agonist capable of eliciting this response. Binding studies with [3H]ADP and fixed cells showed 3.99 +/- 1.77 x 10(5) binding sites/cell for RPM cells (apparent dissociation constant [kd] = 7.75 +/- 2.3 x 10(-8) mol/L), 8.19 +/- 3.25 x 10(5) sites/cell for HEL cells (kd = 2.15 +/- 0.84 x 10(-7) mol/L, 1.15 +/- 0.23 x 10(6) sites/cell for U937 cells (kd = 2.20 +/- 0.53 x 10(-7) mol/L) and 5.39 +/- 2.80 x 10(5) sites/cell for K562 cells (kd = 1.37 +/- 0.39 x 10(-7) mol/L), Inhibition studies with unlabeled nucleotides and analogues showed that binding was approximately 85% specific and the inhibitory pattern was similar to that seen with mature platelets. The purine base adenosine resulted in little or no inhibition. These studies indicate that both human and rat hematopoietic cell lines possess intact ADP receptors and may be useful tools in future studies of the structure and function of this important platelet-activation system.

The production of a factor X activator by a methylcholanthrene-induced rat fibrosarcoma.

The association between neoplasia and thrombosis has been well documented. We have studied the production of a procoagulant which is a factor X activator in a rat fibrosarcoma model. Extracts of excised tumor were assayed in a one stage clotting assay using normal and factor deficient human plasmas. The activity was not due to tissue factor, as acceleration of clotting was observed in FVII deficient plasma. No activity was noted in FX deficient plasma. The activator was capable of cleaving 125I-FX in the absence or presence of calcium. A radioimmunoassay (RIA) demonstrated a 5,100-fold increase in the levels of FX activation peptide after exposure to sarcoma extract. The FX activation occurs in the absence of calcium although the effect is greatly accelerated in the presence of 5 mM CaCl2. Using a two-stage amidolytic assay, functional activation of FX by the sarcoma was demonstrated. Inhibitor studies suggest that the sarcoma-derived procoagulant is a serine protease. The methylcholanthrene-induced rat sarcoma may serve as a useful model for investigating the regulation and effects of cancer procoagulants.

The effects of doxorubicin and mitoxantrone on wound healing.

The goal of the present study was to determine whether mitoxantrone would impair wound healing to a similar degree as doxorubicin when given in equally cytotoxic doses. On day 0, male Fischer rats were wounded and treated with 5% dextrose (control), 6 mg/kg doxorubicin, or 1.2 or 2.4 mg/kg mitoxantrone. On day 5, WBCs for the doxorubicin group and the group that had been treated with 1.2 mg/kg mitoxantrone were 33% and 43% lower than control values, respectively. All rats that had been given 2.4 mg/kg mitoxantrone died within 1 week of being wounded due to drug toxicity. On day 21, wound-breaking strength (WBS) analysis was performed: two skin specimens were taken from each dorsal skin incision perpendicular to the scar axis and were subjected to wound disruption (grams of force) by uniaxial extension. The WBS analysis indicated significant differences between the doxorubicin treated group (1183 +/- 96 g) and the control group (2422 +/- 247 g). However, no significant difference was found between the group that had been given 1.2 mg/kg mitoxantrone (2140 +/- 191 g) and control animals. Thus, mitoxantrone seems to exert myelosuppressive effects similar to those displayed by doxorubicin, but the former drug results in significantly less impairment of wound healing in the rat model.

The binding of radioiodinated thrombin to rat promegakaryoblasts.

Rat promegakaryoblasts have recently been shown to possess a number of properties of mature platelets. We have studied the binding of 125I-thrombin to these cells grown in culture. The binding was found to be saturable, specific, reversible, and of high affinity. Such cell lines may be useful models for the study of platelet-agonist interactions.

Patients with prolonged bleeding time of undefined etiology, and their response to desmopressin.

Between 1981 and 1986, we evaluated 59 patients who presented with isolated prolongation of bleeding time with normal platelet counts, platelet aggregation and coagulation parameters (including von Willebrand's factor), and without evidence of liver or kidney disease, or exposure to anti-platelet agents. These patients, termed as vascular fragility syndrome (VFS), were analyzed to further characterize their bleeding patterns. The pattern of bleeding manifestations was similar to that of patients with platelet dysfunction, such as mucocutaneous bleeding or excessive post-operative bleeding. In 16 patients, desmopressin (1-desamino-8-d-arginine vasopressin, DDAVP) was infused to control active bleeding or as a part of pre-surgical evaluation. Bleeding time improved (pre-DDAVP bleeding time 15.3 +/- 3.4 min, mean +/- S.D.; post-DDAVP bleeding time 10.7 +/- 3.9 min; p less than 0.01) within 30 minutes following the DDAVP infusion with either satisfactory arrest of acute bleeding or good control of subsequent hemostasis with surgery. Side effects with DDAVP were transient and minor, i.e. facial flushing, or conjunctival erythema. These findings indicate that VFS with isolated prolongation of bleeding time is a frequently encountered bleeding disorder and that DDAVP is effective in achieving hemostasis for the management of acute bleeding and may be useful prior to surgical procedures.

Adverse reactions to trimethoprim-sulfamethoxazole in hemophiliacs.

Adverse reactions to trimethoprim-sulfamethoxazole are very prevalent in patients with acquired immunodeficiency syndrome (AIDS). Recently we have observed severe toxicities associated with trimethoprim-sulfamethoxazole in three hemophiliacs, a group known to be at risk for developing AIDS. At the time of these reactions to the antibiotic, none of the patients had yet manifested any stigmata of AIDS per se. We advise caution in the use of trimethoprim-sulfamethoxazole in hemophiliacs and other patients at high risk for the development of AIDS.

Natural history of acquired immunodeficiency syndrome in hemophilic patients.

During the 5-year period from 1981 to 1985, we have observed 8 cases of acquired immunodeficiency syndrome (AIDS) among our 85 patients with hemophilia A. Thus, the prevalence of AIDS with hemophilia A is 9.4% in our patient population. By utilizing stored serum or plasma samples dating back to 1978, antibody against HTLV-III was detected in all 8 cases with AIDS. Based on the time interval from the appearance of antibody to HTLV-III to the diagnosis of AIDS in these patients, the incubation period ranged from 27 months to 60 months, with a median of 36 months. Before the diagnosis of full-blown AIDS, all patients exhibited a variety of prodromal manifestations of non-specific nature, including weight loss, oral candidiasis, unexplained non-productive chronic cough, generalized lymphadenopathy, and thrombocytopenia lasting several months to several years. Serial T-lymphocyte subset studies were available in some patients during the HTLV-III seropositive period and showed progressive lymphopenia, depletion of T4 cells with an average absolute count of 94 +/- 128 per mm3 (mean +/- 1 S.D.), and a markedly reversed T4/T8 ratio of 0.26 +/- 0.19 (mean +/- 1 S.D.). These findings suggest that the incubation period of AIDS is considerably long and that prospective study of serial immunologic markers and HTLV-III markers may be warranted in hemophilic patients at risk.

The role of tryptophan residues in heparin-antithrombin interactions.

A single tryptophan residue on antithrombin has been modified with dimethyl-(2-hydroxy-5-nitrobenzyl)sulfonium bromide. This alteration led to a 500-fold reduction in the heparin-dependent acceleration of thrombin-modified antithrombin interactions, as well as a 10-fold decrease in the avidity of the modified protease inhibitor for mucopolysaccharide. Preincubation of antithrombin with the octasaccharide binding domain of heparin prior to treatment with dimethyl-(2-hydroxy-5-nitrobenzyl) sulfonium bromide was able to suppress modification of the critical tryptophan and preserve the functional capacities of the protease inhibitor. Fluorescence quenching experiments indicated that the modifiable tryptophan groups of antithrombin were exposed to the solvent environment. Based upon these data, it was proposed that the loss of "heparin cofactor" activity of antithrombin must be predominantly due to an inability of the modified protease inhibitor to undergo a conformational transition required for mucopolysaccharide-dependent "activation" of the macromolecule.

Doxorubicin in pregnancy: possible transplacental passage.

Two pregnant women with lymphoproliferative disorders were treated with doxorubicin-containing regimens. Both patients delivered shortly after a dose of doxorubicin. One child was healthy and the other was stillborn. Measurements of anthracycline levels in placental, cord, and fetal tissues by high-performance liquid chromatography suggest that doxorubicin may be transported across the placenta.

Monarticular arthritis: early manifestation of a rhabdomyosarcoma.

We describe a case in which monarticular arthritis was a presenting manifestation of a rhabdomyosarcoma. Arthritis as a complication of rhabdomyosarcoma is extremely rare. Rheumatic manifestations of malignancy are also reviewed.