Effect of L-asparaginase administration on coagulation and platelet function in children with leukemia.

The use of L-asparaginase during remission induction in patients with leukemia is associated with coagulation abnormalities, which may present either as thrombosis or hemorrhage. However, because of the multiple pharmacologic and hematologic variables present in these patients, the exact contribution of L-asparaginase to these coagulation abnormalities is unclear. We studied platelet function and plasma coagulation parameters in 12 pediatric patients with acute lymphoblastic leukemia (ALL) receiving daily L-asparaginase as a single agent when in complete remission. Changes in the prothrombin time (PT), partial thromboplastin time (PTT), and fibrinogen, while statistically significant, remained within or close to the normal range during the study. Platelet function also remained normal during the study. In contrast, levels of protein C antigen decreased to a mean of 42%, a significant change from pretreatment values. Levels of antithrombin III (AT III) were likewise depressed to 15 mg/dL (34% of pretreatment value). Despite these changes in the levels of physiologic inhibitors of coagulation, this schedule of L-asparaginase administration was associated with only rare clinical thrombosis, and this study suggests that the development of this complication may be dependent on the presence of additional factors.

Amrinone-induced thrombocytopenia.

The frequency and characteristics of thrombocytopenia resulting from administration of amrinone, a new inotropic and vasodilator agent, was evaluated in 43 patients. Thrombocytopenia attributable to amrinone developed in eight patients (18.6%). The thrombocytopenia was due to accelerated peripheral loss of platelets. There appeared to be a dose relationship with regard to the rapidity of onset and degree of thrombocytopenia. Although platelet-associated IgG levels were elevated when measured in patients with thrombocytopenia, the clinical features were suggestive of a direct, perhaps nonimmunologic effect of amrinone on platelets. Thrombocytopenia was mild in most cases and bleeding attributable to thrombocytopenia did not occur. Several patients continued amrinone therapy over long periods despite low platelet counts, showing that mild to moderate thrombocytopenia is not necessarily an indication that therapy should be discontinued, but that platelet counts should be observed closely.

Repetitive bone marrow transplantation in nonmyeloablated recipients.

Transplantation of 200 million male BALB/c marrow cells into normal nonmyeloablated female BALB/c hosts results in relatively high levels of engraftment, whether the cells are infused repetitively over time or in a single infusion. These high engraftment rates suggested that repetitive injections of high levels of male BALB/c cells might be able to totally replace host marrow. Accordingly, we transplanted 40x10(6) male BALB/c bone marrow cells into female BALB/c recipients over a 7-week period for a total of 20 injections (800x10[6] marrow cells). Engraftment in this experimental group was compared to that seen when female recipients received 2x10(6) male marrow cells or phosphate-buffered saline (PBS) over the same injection schedule. Engraftment was quantitated at 5 and 7 weeks after the final infusion by detection of male-specific sequences in female host marrow, spleen, and thymus by Southern blot analysis using a Y-specific cDNA probe. Male DNA levels were quantitated with a Molecular Dynamics phosphorimager. Engraftment of male cells into female marrow at 5 and 7 weeks posttransplantation ranged from 19 to 88%, whereas that in spleen and thymus ranged between 30 and 100% and 28 and 50%, respectively. The mean percent engraftments for marrow, spleen, and thymus were 41, 69, and 39%, respectively. Mean percent engraftments for 2x10(6) cell infusions at 5 and 7 weeks for marrow, spleen, and thymus were 4, 6, and 4%, respectively. Marrow and spleen cellularity and total high proliferative potential colony-forming cell numbers were determined in PBS- and cell-injected mice. No significant differences between these groups were observed. For marrow engraftment, 20 injections of 40x10(6) cells was not more effective than five, but donor DNA in thymus and spleen was increased with 20 injections. Primitive progenitor cell levels and marrow cellularity do not increase in mice injected with large numbers of marrow cells, suggesting that host marrow cells are replaced rather than augmented by infused donor cells.

Characterization and experimental use of a monospecific antiserum to factor IX.

Purified human factor IX was used to develop xenogeneic neutralizing and precipitating antibodies. The final antiserum (R2) neutralized only factor IX and was equivalent to 220 Bethesda-inhibitory units. It showed two precipitating lines, one of which disappeared after absorption with human albumin. On immunodiffusion and Laurell immunoelectrophoresis, the albumin-absorbed R2 antiserum showed one precipitin line of identity, or one rocket respectively, with normal plasma, a Red Cross factor IX preparation (rich in factors IX, II and X), the original antigen, and Hemophilia-B antigen-positive plasmas. No line or rocket developed with normal plasma absorbed with aluminum hydroxide or with antigen-negative contained only factor IX neutralizing and precipitating antibodies. Experiments with various factor IX concentrates revealed that the majority contained excess factor IX antigen compared to their coagulant activity. In addition, crossed antigen-antibody electrophoresis uncovered differences in the migration of the factor IX of Konyne preparations, when done in the presence of EDTA or calcium. This monospecific antiserum to human factor IX was subsequently used to investigate a large population of hemophilia B patients and carriers.

Laboratory prediction of the carrier state in hemophilia B: role of replication of assays.

The authors studied the effect of averaging replicated assays of Factor IX coagulation activity and Factor IX antigen on each plasma specimen in improving the ability to detect carriers of hemophilia B. The improvement resulting from this procedure is particularly marked in tests depending on the linear regression of one characteristic on another to diagnose the carrier state. The effects of averaging assays on the ratio of Factor IX coagulation activity to Factor IX antigen were also explored. The benefits of averaging replications were not greatly increased by use of more than four replications.

Measurement of the activated partial thromboplastin time from a capillary (fingerstick) sample of whole blood. A new method for monitoring heparin therapy.

The monitoring of heparin anticoagulation is fraught with difficulties because of the need for repetitive venipunctures and the vagaries of sample handling and processing. The authors evaluated a new aPTT monitoring system with the potential to eliminate many of these difficulties. The Ciba Corning Diagnostics 512 Coagulation Monitor (CCD monitor) is a hand-held portable instrument that can measure an aPTT from a fingerstick sample of capillary whole blood. Fingerstick aPTTs from 319 subjects (including controls and individuals on heparin and/or warfarin) were compared to venipuncture-derived standard laboratory plasma aPTTs using different aPTT reagents on conventional instruments. The correlation coefficients between fingerstick and standard aPTTs (0.79-0.83) were the same as the correlation coefficient between standard laboratory aPTTs using different reagents (0.79). When venipuncture-derived whole blood was compared to fingerstick samples on the new instrument, the correlation coefficient was excellent (0.93). A high degree of precision, as demonstrated by low coefficients of variations, was shown for within-day and between-day testing using the CCD monitor and normal controls. This capillary whole blood, aPTT system is the first to provide the clinician with a means of rapidly and reliably assessing the anticoagulant response to heparin therapy at the bedside, and its use may ultimately lead to more efficient and effective therapy overall.

Identification of six functional clotting factor VIII:C epitopes by analysis of cross-reactive public idiotypes in murine monoclonal VIII:C inhibitors.

Six factor VIII:C epitopes which can elicit clotting factor inhibitory antibodies were demonstrated by analysis of public idiotypes of murine monoclonal anti-VIII:C inhibitors. Anti-idiotypic immunoradiometric assays were developed with rabbit antibodies to murine VIII:C inhibitors: Synbiotics, Hybritech, C7F7 and RFF-VIIIC/8. Crossreactions among 10 murine monoclonal antibodies (MoAbs) to VIII:C were tested, showing 6 functional and immunospecific VIII:C epitopes. One epitope on the C-terminal, 80,000 dalton fragment of VIII:C was identified with cross-reaction among 3 MoAbs (Synbiotics, Chemicon, and IB3). Another unique site on this same fragment was recognized with C7F7. Two MoAbs (RFF-VIIIC/6 and RFF-VIIIC/8) defined another site with cross-reactive idiotypes on the N-terminal, 90,000 dalton fragment. Hybritech MoAb identified a fourth functionally distinct site to which no other cross-reacting MoAbs bound. A fifth functional locus was defined with RFF-VIIIC/2 which reacted with an N-terminal site (distinct from the RFF-VIIIC/6 X RFF-VIIIC/8-defined site). A sixth functional locus was recognized with RFF-VIIIC/5 which reacted with a C-terminal site (distinct from the Synbiotics/Chemicon/IB3-defined site but possibly near the C7F7-defined site). RFF-VIIIC/10 identified a non-functional locus on the middle region of VIII:C. These MoAb-based assays resolve six sites to which high-titered inhibitors are directed and offer a path to further study the immunoregulation of human anti-VIII:C inhibitors.

Characterization of a factor VIII immunogenic site using factor VIII synthetic peptide 1687-1695 and rabbit anti-peptide antibodies.

A 9 amino acid peptide, Ser-Pro-Arg-Ser-Phe-Gln-Lys-Lys-Thr, corresponding to the clotting factor VIII (FVIII) sequence Ser1687-Thr1695, was synthesized in order to analyze a site on FVIII to which antibody inhibitors of FVIII may be directed. This sequence contained a thrombin cleavage site. It was predicted to be immunogenic because a Hopp-Woods hydrophilicity analysis of the amino acid sequence of FVIII showed it to be very hydrophilic, and it contained a proline. The HPLC-purified peptide was cleaved by thrombin at Arg1689-Ser1690, as determined by amino acid sequencing of the cleavage product. Thrombin which had been treated with a specific chloromethyl ketone inhibitor, did not cleave the peptide. Two rabbits immunized with the peptide/keyhole limpet hemocyanin conjugate generated FVIII inhibitory sera with titers of 5.4 and 4.8 Bethesda units. These rabbit anti-peptide antibodies reacted with a peptide/-BSA conjugate on immunodot blot analyses and with native, affinity-purified FVIII in Western blots. In competitive immunoradiometric assays, cryosupernatants of 38/82 patients with FVIII inhibitors reacted with the synthetic peptide. We conclude that FVIII peptide Ser1687-Thr1695 is cleaved by thrombin at the same peptide bond which is cleaved in FVIII, and the peptide contains a site to which patients' inhibitory antibodies can be directed.

Development of anti-idiotypic antibodies in a patient with a factor VIII autoantibody.

Antibodies against coagulation factor VIII were detected in a 33-year-old who developed severe bleeding 2 months postpartum. After treatment with plasmapheresis and immunosuppressive agents, the inhibitor was no longer detectable, but anti-idiotypic antibodies were detected, as demonstrated by binding in immunoradiometric assays, and by in vitro neutralization of the initial factor VIII autoantibodies. The anti-idiotypic titer subsequently declined but was still detectable 1 year later. Three commercial immunoglobulin preparations and pooled multiparous IgG also bound to (but did not neutralize) the patient's factor VIII antibody. These studies suggest that anti-idiotypic antibodies, arising in the face of immunosuppressive therapy, might suppress autoimmunity to factor VIII and confirm the presence of anti-idiotypic antibodies in pooled normal immunoglobulins.

The effect of trypsin and storage on aggregation and release of human gel-filtered platelets.

Trypsin was studied as an aggregating and release-inducing agent with gel-filtered platelets (GFP) and was compared with ADP, epinephrine and collagen. GFP aggregated irreversibly with final concentrations of 0.5-4 microgram/ml trypsin, 1.6-3.2 micrometer ADP, 2.5-5 micrometer epinephrine and 40 microlite 1/ml soluble collagen. Addition of human fibrinogen to the Tyrode-suspending buffer was required for ADP and epinephrine, but was not necessary for trypsin or collagen. Release of (14C)5HT was obtained with trypsin and collagen using the same concentrations as used in aggregation. GFP stored at room temperature for 48 h were still responsive to trypsin and collagen, whereas aggregability and (14C)5HT release induced by ADP and epinephrine were already impaired 5 h after collection of blood. CP-CPK, an ADP-removing reagent, blocked aggregation and release induced by low trypsin concentrations, suggesting that ADP plays an intermediate role in the mechanism by which trypsin activates platelets. Trypsin appears to be a valuable reagent for studying platelet physiology, particulary following storage.

Some observations on the in vivo effect of propranolol on platelet aggregation and release.

Platelet function was investigated in four normal volunteers, one patient with a mild form of von Willebrand disease, and one with a thrombocytopathy, all taking propranolol. No effect on platelet function attributable to this drug could be demonstrated in any of these subjects. It is suggested that propranolol administered in conventional doses does not impair platelet hemostatic function.

Coagulation abnormalities associated with the use of anabolic steroids.

According to a number of recent reports, persons using anabolic steroids may be subject to an increased risk of thromboembolism. We evaluated the effect of anabolic steroid use on the coagulation and fibrinolytic systems of 16 male bodybuilders to determine whether alterations occurred that would predispose them to a hypercoagulable state. No attempt was made to regulate or guide steroid use. Paired blood samples, both with and without steroid use, were obtained from six individuals, and the remaining subjects provided single samples obtained either during steroid use or nonuse. No differences were noted in most parameters, but we did find a significant increase in protein C antigen (p = 0.008) and free protein S antigen (p = 0.015), with a decreased euglobulin lysis time (p = 0.021) during steroid use. We also found a reduction in total cholesterol levels (p = 0.035) during steroid use. At least some of these findings suggest an activated fibrinolytic state, a known effect of anabolic steroids. The results do not support the presence of a hypercoagulable state. If anabolic steroids do produce a thrombotic tendency, they may do so through alterations in other hemostatic mechanisms or changes in lipid fractions, or more sensitive coagulation assays may be required for detection.

Factor VIII epitopes recognized with inhibitory monoclonal antibodies.

To test whether anti-idiotypic immunoregulation of factor VIII(FVIII)-inhibiting antibodies could be feasible in hemophiliacs, we assayed the minimal number and range of immunogenic, functional FVIII epitopes to which a series of murine anti-FVIII monoclonal antibodies (MAb) were directed. Rabbit anti-idiotypic sera to 7 MAb were prepared and used in competitive immunoradiometric assays to test Id similarities among 36 murine MAb. At least 12 immunogenic and functional epitopes were found on the FVIII molecule. The large range of target loci for FVIII inhibitors will make their immunoregulation by anti-idiotypic methods difficult.

In vitro cell density-dependent clonal growth of EGF-responsive murine neural progenitor cells under serum-free conditions.

Neural progenitor cell populations responsive to epidermal growth factor (EGF) have been shown to have proliferative potential and give rise to neurons, astrocytes, and oligodendrocytes. We have characterized EGF-responsive neural progenitor cells that give rise to bilineage neuronal/glial colonies (colony-forming unit neuron-glia; CFU-NeGl) and unilineage neuronal colonies (CFU-Ne). Clonality was confirmed utilizing mixtures of brain cells from Balb/c and ROSA26 (transgenic for beta-galactosidase) mice. With a few exceptions, colonies showed either all blue cells or all clear cells after staining with X-Gal. Clonal growth was analyzed after 10-11 days in relation to cell density by determining colony size and plating efficiency. Growth was density dependent (no growth below 10,000 cell/ml) and thus single cell cloning was not accomplished. An average plating efficiency of 4% was found for EGF-responsive neural cells derived from day 15-18 murine embryos when cultured at 12,500 to 200,000 cells/ml. Similar results were obtained with 1-day-old postnatal neural cells. When colonies were categorized by size, the relative number of colonies over 50 cells appeared to be maximum at 50,000 plated cells/ml. After 11 days in culture, 94, 96, and 78% of the colonies contained cells that expressed nestin, neurofilament, and GFAP, respectively. Double-label experiments revealed that > 62% of the colonies contained both GFAP and neurofilament expressing cells. These studies establish the existence of at least two populations of clonal neural progenitors: CFU-Ne and CFU-NeGl in fetal and postnatal murine brain.

Hypothesis for the control of clotting factor VIII inhibitory antibodies by decreasing potency of helper T-cell-recognized epitopes in factor VIII.

The study of the immunobiology of FVIII inhibitors may lead to new therapies for this potentially severe complication of haemophilia A and to new principles for the use of therapeutic proteins. In order to characterize the idiotype-anti-idiotype networks regulating FVIII inhibitors, we developed rabbit anti-idiotypic sera to 7 murine inhibitors and found at least 12 independent FVIII loci to which inhibitors could be raised. Rabbit antisera to the FVIII peptide, Ser1687-Thr1695, characterized one functional site to which about 46% of patients' inhibitor sera reacted. The multiplicity of inhibitor-recognized epitopes in FVIII makes it impractical, at the present time, to develop clinically useful specific anti-idiotypic therapies for FVIII inhibitors. Alternatively, one might induce genomic mutations in recombinant FVIII molecules to decrease immunogenicity of epitopes recognized by T helper cells. Methods to design such altered therapeutic proteins are presented, based on changing the longitudinal hydrophobic strip-of-helix which is in or near many T-cell-presented epitopes.

Potential and distribution of transplanted hematopoietic stem cells in a nonablated mouse model.

Increasingly, allogeneic and even more often autologous bone marrow transplants are being done to correct a wide variety of diseases. In addition, autologous marrow transplants potentially provide an opportune means of delivering genes in transfected, engrafting stem cells. However, despite its widespread clinical use and promising gene therapy applications, relatively little is known about the mechanisms of engraftment in marrow transplant recipients. This is especially so in the nonablated recipient setting. Our data show that purified lineage negative rhodamine 123/Hoechst 33342 dull transplanted hematopoietic stem cells engraft into the marrow of nonablated syngeneic recipients. These cells have multilineage potential, and maintain a distinct subpopulation with "stem cell" characteristics. The data also suggests a spatial localization of stem cell "niches" to the endosteal surface, with all donor cells having a high spatial affinity to this area. However, the level of stem cell engraftment observed following a transplant of "stem cells" was significantly lower than that expected following a transplant of the same number of unseparated marrow cells from which the purified cells were derived, suggesting the existence of a "nonstem cell facilitator population," which is required in a nonablated syngeneic transplant setting.