Effects of all-trans retinoic acid or chemotherapy on the molecular regulation of systemic blood coagulation and fibrinolysis in patients with acute promyelocytic leukemia.

We studied the pathogenesis of the bleeding disorder in acute promyelocytic leukemia by measuring procoagulant, profibrinolytic, and proinflammatory mediators in peripheral blood and bone marrow cells from 25 previously untreated patients. Patients were induced with either all-trans retinoic acid (ATRA) or chemotherapy. Plasma levels of fibrinopeptide A (FPA), fibrin d-dimer, thrombin antithrombin (TAT) complex, prothrombin fragment 1.2 (F1.2), urokinase-type plasminogen activator (uPA), tissue-type plasminogen activator (t-PA) and plasminogen activator-inhibitor 1 (PAI-1) were measured before and after therapy, as was the cellular expression of the genes for tissue factor (TF) and interleukin-1 beta (IL-1 beta). The mean plasma levels of fibrin d-dimer, F1.2, TAT and FPA were markedly elevated prior to therapy and declined during the first 30 days of treatment with either ATRA or chemotherapy, but more rapidly and to a greater extent in patients treated with ATRA. ATRA treatment was associated with a significant decrease in TF gene expression in bone marrow cells during the first 30 days of treatment, whereas IL-1 beta gene expression, which decreased in the cells of six patients treated with either chemotherapy or ATRA, actually increased in the remaining six patients treated with either chemotherapy or ATRA. In patients with APL, treatment with either chemotherapy or ATRA rapidly ameliorates the coagulopathy, as indicated by an abrupt decline in markers of clotting activation. An increase in cytokine gene expression (e.g. IL-1 beta) may provide an explanation for the persistent hypercoagulability observed in some patients with APL, regardless of therapeutic approach. Our data confirms and extends earlier observations by others that ATRA is more effective than chemotherapy alone in rapidly reducing the procoagulant burden of APL tumor cells. However, our data also suggests that cytokine expression in some patients may be accelerated by either chemotherapy or ATRA. The implications of this observation for understanding the retinoic acid syndrome will require further studies.

Activation of coagulation and angiogenesis in cancer: immunohistochemical localization in situ of clotting proteins and vascular endothelial growth factor in human cancer.

Thrombin-catalyzed, cross-linked fibrin (XLF) formation is a characteristic histopathological finding in many human and experimental tumors and is thought to be of importance in the local host defense response. Although the pathogenesis of tumor-associated fibrin deposition is not entirely clear, several tumor procoagulants have been described as likely primary stimuli for the generation of thrombin (and XLF) in the tumor microenvironment (TME). In a previous study of a variety of human tumors we have shown that tissue factor (TF) is the major procoagulant. However, the relative contribution to fibrin deposition in the TME of tumor cell TF and host cell TF (eg, macrophage-derived) was not established. In addition, recent evidence has implicated TF in the regulation of the synthesis of the pro-angiogenic factor vascular endothelial growth factor (VEGF) by tumor cells. In the current study we used in situ techniques to determine the cellular localization of XLF, TF, VEGF, and an alternative tumor procoagulant, so-called cancer procoagulant (CP), a cysteine protease that activates clotting factor X. In lung cancer we have found XLF localized predominantly to the surface of tumor-associated macrophages, as well as to some endothelial cells and perivascular fibroblasts in the stromal area of the tumors co-distributed with TF at the interface of the tumor and host cells. Cancer pro-coagulant was localized to tumor cells in several cases but not in conjunction with the deposition of XLF. TF and VEGF were co-localized in both lung cancer and breast cancer cells by in situ hybridization and immunohistochemical staining. Furthermore, a strong relationship was found between the synthesis of TF and VEGF levels in human breast cancer cell lines (r2 = 0.84; P < 0.0001). Taken together, these data are consistent with a highly complex interaction between tumor cells, macrophages, and endothelial cells in the TME leading to fibrin formation and tumor angiogenesis.

Hb Catonsville (glutamic acid inserted between Pro-37(C2)alpha and Thr-38(C3)alpha). Nonallelic gene conversion in the globin system?

Hb Catonsville is an unstable variant in which glutamic acid is inserted into the alpha-globin chain between Pro-37(C2) and Thr-38(C3). The peptide sequence data are consistent with the DNA sequence of the polymerase chain reaction-amplified fragment of the variant globin gene, which shows the insertion of the triplet codon--GAA--into the mutant alpha-globin gene. In the normal alpha-globin gene cluster the codon for glutamic acid is GAG rather than GAA. Thus, there are two features unique to Hb Catonsville, one the insertion of a single residue into the interior of the alpha-globin chain, and two the presence of the alternate codon for glutamic acid. The experimental evidence suggests that Hb Catonsville may be an example of nonhomologous nonallelic gene conversion, an observation not previously reported in this gene family. The mutation occurs in the critical alpha 1 beta 2 interface of the hemoglobin tetramer and leads to a variant with high oxygen affinity, a reduced cooperativity, and Bohr effect.

Hemoglobin Evanston: alpha 14(A12) Trp leads to Arg. A variant hemoglobin associated with alpha-thalassemia-2.

Hb Evanston (alpha 14 Trp leads to Arg) was detected on cellulose acetate at pH 8.4 as a band with an electrophoretic mobility similar to that of Hb S. In addition, a band migrating cathodic to Hb A2 suggested the presence of a variant Hb A2 with a substitution in the alpha-chain, a fact that was later confirmed by structural analysis. An unusual feature of Hb Evanston is its low percentage; less than 10% occurs in the hemolysate. Studies indicate that the variant is not unstable, but there appears to be a defect in globin-chain synthesis. Gene mapping also shows that it is associated with the alpha-thalassemia-2 gene. The variant has high oxygen affinity with normal cooperativity and a normal Bohr effect. The combination of Hb Evanston with alpha-thalassemia-2 produced anemia in this black family.

Hemolytic disease of the newborn caused by a new deletion of the entire beta-globin cluster.

We describe a new type of gamma delta beta-thalassemia in four generations of a family of Scotch-Irish descent. The proposita presented with hemolytic disease of the newborn, which was characterized by a microcytic anemia. Initial restriction endonuclease analysis of the DNA showed no grossly abnormal patterns, but studies of polymorphic restriction sites and gene dosage revealed an extensive deletion that removed all the beta- and beta-like globin genes from the affected chromosome. In situ hybridization of chromosome preparations with radioactive beta-globin gene probes showed that only one 11p homolog contained the beta-globin gene cluster in the affected family members.

Alpha-thalassemia in Ashkenazi Jews.

Members of a Jewish family of Polish origin were found to have hypochromic, microcytic erythrocytes. By restriction endonuclease analysis of DNA, the propositus, a brother, and an aunt were found to have a single alpha-globin gene on each chromosome 16. Five family members have one chromosome bearing two alpha-genes (5' and 3') with a single alpha-gene on the homologous chromosome. Gene mapping indicated that the chromosome bearing a single alpha-gene arose via an unequal crossover between misaligned 5' and 3' alpha-genes and was introduced into the family from three separate sources. In addition, a Jewish man of Hungarian origin was found to have alpha-thalassemia trait with single alpha-genes on both chromosomes 16 and a survey of 25 Jewish subjects yielded one man of German origin with an alpha-gene deletion. Alpha-thalassemia should be considered in the differential diagnosis of disease in Jewish persons with microcytic, hypochromic erythrocytes.

Evaluation of a commercial kit for microchromatographic quantitation of hemoglobin A2 in the presence of hemoglobin S.

Commercial microcolumns introduced in 1976 by Helena Laboratories ("Hb A2 Quik Column") and by Isolab, Inc. ("Quik-Sep") provide a rapid, simple, accurate method for quantitation of hemoglobin A2 (Hb A2). However, these kits cannot be used for the quantitation of Hb A2 in the presence of slow-moving variants such as Hb S. Recently, Isolab, Inc., produced a new kit ("Quik-Sep Improved Hb A2 Test") for quantitation of both Hb A2 and Hb S. We compared results obtained with the new Isolab kit to results obtained with the original Tris/HCl method for quantitation of Hb A2 and Hb S. Blood was drawn from persons with sickle cell trait (A/S), sickle cell anemia (S/S), sickle cell/beta+ thalassemia (S/beta+ thal) and sickle cell/beta 0 thalassemia (S/beta 0 thal) and percentages of Hb A2 and Hb S were determined by each method. We found no significant differences in Hb A2 percentages by the two methods, and the coefficients of variation were similar. Both methods showed only slight overlap of Hb A2 values from subjects with some form of beta thalassemia and those with A/S or S/S. However, the Tris/HCl method consistently gave values for Hb S that were higher and closer to those expected, suggesting that the Isolab kit does not accurately quantitate Hb S.

Microchromatographic methods for hemoglobin A2 quantitation compared.

On 20 consecutive work days during four weeks, one technologist performed 24 microchromatographic determinations of hemoglobin A2 (Hb A2) by each of four methods: the Efremov procedure requiring Tris/HCl buffer, the original Huisman technique with use of glycine developer, and two commercial test kits in which a modified glycine developer is used. The bloood samples tested were obtained from 12 adults with no hematological abnormality and from 12 beta-thalassemia carriers previously diagnosed by familial and hematologic studies. Results by the first method and the two commercial kits (one from Helena Laboratories and one from Isolab, Inc.) did not differ significantly in precision for either the normal or beta-thalassemia trait samples. For both sample types, the second method yielded larger coefficients of variation than those obtained with the other methods. Moreover, the second method was the only one with which values overlapped for normal samples and samples with above-normal Hb A2 concentrations.

Trimodality in the proportion of hemoglobin G Philadelphia in heterozygotes: evidence for heterogeneity in the number of human alpha chain loci.

The extent of variability in the number of human hemoglobin (Hb) alpha chain loci has not yet been conclusively determined. There is evidence that in some populations individuals may possess two alpha-chain loci, while in other populations only one locus is present. Electrophoresis of peripheral blood from 53 heterozygotes for Hb G Philadelphia (alpha 68 Asn leads to Lys) revealed that the proportion of Hb G is trimodally distributed, with modes at approximately 20, 30, and 40% Hb G. Familial, hematologic, and statistical studies suggest that hte proportion of Hb G is not random but is genetically controlled and inversely correlated with mean cell volume. Two alternative genetic models are proposed to explain these findings: one assums alpha-thalassemia, while the other postulates variability in the number of alpha-chain loci in the American Black population. Biosynthetic studies of blood from 15 subjects revealed balanced synthesis of alpha and beta globin chains in heterozygotes from all three classes, strongly supporting variable gene dosage rather than alpha-thalassemia as the mechanism underlying the observed trimodality in the proportion of Hb G. Incompatibilities between out results and current concepts of alpha-thalassemia are discussed in the context of differences between Black compared with Oriental and Italian forms of Hb H disease.