Abraham Kornberg

Modulation of the maturation of human leukemic promyelocytes (HL-60) to granulocytes or macrophages.

The relationship between the effects of two types of inducers on the maturation of a line of human promyelocytic cells (HL-60) was studied. The dual potentiality of these promyelocytes was demonstrated by the ability of isolated colonies to differentiate into granulocytes, following induction by dimethylsulphoxide (DMSO) or express properties specific to macrophages following exposure to 12-O-tetradecanoylphorbol-13-acetate (TPA). The differentiation process involved an irreversible step which occurred 48 h after exposure to DMSO, and a few h after exposure to TPA. This implies that the presence of the inducer in the culture medium is no more required for completion of the differentiation programme. Removal of the inducer prior to this step resulted in reversal of all the inducer-mediated effects. During the period of non-commitment the specific pathway of maturation was still undetermined; cells in which differentiation was initiated by exposure to DMSO were able to develop into macrophages after substitution of DMSO by TPA. Moreover, pre-exposure to DMSO and other inducers of granulocyte differentiation resulted in higher sensitivity to TPA, as indicated by the cell response to low TPA concentrations and more rapid expression of macrophage specific properties. These results suggest that the early stages in HL-60 differentiation are probably common to the granulocyte and the macrophage pathways.

Comparison of native prothrombin antigen with the prothrombin time for monitoring oral anticoagulant prophylaxis.

BackgroundOral anticoagulation is most frequently monitored using the prothrombin time, but an alternative approach is measurement of native, fully carboxylated, prothrombin antigen (NPA). We have correlated results of the prothrombin time and NPA with development of venous thrombosis or bleeding complications in a clinical trial of warfarin prophylaxis following total hip arthroplasty to determine the potential value of NPA measurement for monitoring oral anticoagulation. Methods and ResultsPatients in one arm of a prospective, randomized trial received warfarin prophylactically beginning 10 to 14 days before total hip arthroplasty in a dose adjusted to prolong the international normalized ratio (INR) to 1.5 on the day of surgery and 2.5 after surgery. NPA was measured by ELISA, and the prothrombin time was measured using rabbit brain thromboplastin. Samples were tested from 97 patients, and data from 81 patients who had adequate venography were analyzed to correlate test results with occurrence of thrombosis. The prothrombin time and INR were less sensitive than NPA to the lowest intensities of anticoagulation, with the prothrombin time index increasing from 1.0 to 1.3 and the INR increasing from 1.0 to 2.0, whereas the NPA concentration decreased fourfold, from 200 to 50 pg/mL. There was little correlation between either the prothrombin time index or the INR and the development of thrombosis, whereas NPA concentrations were significantly higher on the day of surgery and on postoperative days 1, 3, 5, and 7 in patients who developed venous thrombosis. Higher concentrations of NPA were associated with an increased risk of venous thrombosis, but there was no relation between thrombosis and the prothrombin time index or INR There was no significant correlation between surgical blood loss and prothrombin time index, INR, or NPA concentration. However, patients who received the largest number of transfusions on the day of surgery had significantly lower NPA concentrations than patients who required no transfusion. ConclusionThese results indicate that the NPA concentration more accurately reflects the antithrombotic effect of warfarin than does prothrombin time and may be superior in monitoring prophylactic oral anticoagulation.

Protein Z levels and central retinal vein or artery occlusion

Abstract:  Objectives: Central retinal vein occlusion (CRVO) and central retinal artery occlusion (CRAO) are common disorders associated with risk factors for atherosclerosis. Protein Z is a cofactor for the inactivation of activated factor X (Xa) by the protein Z dependent protease inhibitor. Protein Z deficiency was recently linked to increased risk of arterial thrombosis. We investigated whether CRVO and CRAO are associated with low protein Z levels. Patients and methods: Patients with CRVO, CRAO or recurrent branch retinal vein occlusion were recruited to the study. Protein Z level, lupus anticoagulant (LAC), anticardiolipin antibodies (ACA) and activated protein C resistance (APCR) were determined in plasma from patients (n = 36) and healthy controls (n = 42). Results: Thirty patients in the study group had traditional risk factors for retinal vessel occlusion and six patients had none. There was no significant difference in protein Z levels between the whole study group patients and controls (1995 ± 810 vs. 2010 ± 603 ng/mL, P = 0.922). However, patients with no risk factors for retinal vessel occlusion had significantly lower protein Z levels than controls (1379 ± 682 vs. 2010 ± 603 ng/mL, P = 0.022). Positive LAC was found in six patients and one control subject (P = 0.04). There were three patients and one control subject with abnormal APCR (P = 0.3) and none with positive ACA. Low protein Z level (lower than fifth percentile of control) was not associated with the presence of LAC or APCR. Conclusion: Low protein Z level may be another risk factor for retinal vessel occlusion in patients without traditional risk factors for these disorders.

Circulating erythroid progenitors in patients with 'spent' polycythaemia vera and myelofibrosis with myeloid metaplasia.

Summary. The ability of circulating progenitor cells from patients with polycythaemia vera (PV) and myelofibrosis with myeloid metaplasia (MMM) to develop erythroid colonies was studied in cultures with and without erythropoietin. In all normal controls, patients with secondary polycythaemia and MMM, erythroid colonies developed only after the addition of erythropoietin. Only in patients with PV, both in the active and spent phases of the disease, erythroid colonies developed in the absence of erythropoietin. The results indicate the perpetuation of erythropoietindependent, as well as erythropoietin‐independent progenitors in both phases of this disease. Although spent PV often clinically resembles MMM, there is a basic difference in the behaviour of the circulating erythroid progenitors in these diseases which may serve as a useful tool in discriminating MMM from spent PV, when there is no history of active PV.

Clinical manifestations and laboratory findings in patients with lupus anticoagulants

Clinical and laboratory features were evaluated in 48 patients with lupus anticoagulants and the efficiency of three different assays in the detection of lupus anticoagulants was compared. The diagnosis of lupus anticoagulants was based on a prolonged activated partial thromboplastin test not corrected in a mixture of 1:1 with normal plasma and lack of specific inhibitors against coagulation factors. Platelet neutralization procedure was positive for lupus anticoagulants in 98% of the patients, tissue thromboplastin inhibition ratio in 79%, and kaolin clotting time index in 77%. At least one of the assays was positive in 100% of the cases. The largest minority of the patients (31%) suffered from systemic lupus erythematosus. The others had a variety of non‐immunological disorders. In the 13 patients who had been operated on, only 1 with renal failure developed hemorrhagic complications after renal biopsy due to thrombocytopathy. The incidence of recurrent spontaneous miscarriage, immune thrombocytopenia and positive direct antiglobulin test, anti‐nuclear and anti‐DNA antibodies and VDRL was significantly higher in patients with lupus anticoagulants and systemic lupus erythematosus compared to patients with lupus anticoagulants but without systemic lupus erythematosus.

Effect of venom sac extract of the Oriental hornet (Vespa orientalis) on coagulation factors.

Venom sac extract of the Oriental hornet significantly prolongs the prothrombin time and the activated partial thromboplastin time both in vitro in human plasma and in vivo in cats. Activity of factors VIII and IX in plasma is reduced to less than 1% within 5 min even with 1 microgram of venom sac extract per ml. The activity of purified factor VIII, as well as semipurified factors IX and X, in factor IX complex was also significantly reduced after incubation with the venom. The decrease of factors II, V, VII, X, XI and XII activity to 9%, 11%, 11%, 29%, 1.7% and 0.7% of normal, respectively, is dose- and time-dependent. Thrombin time, plasma fibrinogen and fibrin degradation products are not affected. The anticoagulant activity is not reversed by dialysis and is abolished completely by heating; it resides mainly in fractions with mol.wts above 5000. The venom has a proteolytic activity on 14C-globin which is partially inhibited by trasylol and ethylenediaminetetraacetic acid. Thus, the venom sac extract exhibits both serine and metaloprotease activities which may affect the activity of the plasma coagulation factors.

Fibrinogen‐ and Fibrin‐Degradation Products during Fibrinolytic Therapya

The administration of a fibrinolytic agent accelerates dissolution of an occluding vascular thrombus by stimulating local fibrinolysis and plasmic degradation of its fibrin matrix. However, systemic activation of fibrinolysis also occurs, and this results in a variable degree of proteolytic degradation of plasma proteins including fibrinogen. Therefore, plasmic degradation products of both fibrinogen and fibrin circulate during fibrinolytic therapy. The ability to identify degradation products derived fi-om the thrombus separately fi-om those originating fkom plasma fibrinogen could be of significant value in monitoring the course of fibrinolytic therapy. Approaches to identification of fibrin-specific degradation products have been based on structural differences in degradation products of fibrinogen compared to those of fibrin. Plasmin action on fibrinogen results in initial cleavages of the carboxyl terminus of the Aar chains, and also release of the peptide Bola2 from the amino terminus of the BB chain, resulting in formation of fragment X. With further plasmin action, the coiledtoil regions linking the central and lateral domains are cleaved forming the intermediate fragment Y and terminal fragments D and E.1 The extent of fibrinogen degradation is variable during lytic therapy and is greater with streptokinase than with “fibrin-specific” agents such as tPA, but the extent of proteolysis is limited, and fragment X is the predominant derivative fbrmed.2 Plasmic degradation products of fibrin differ from those of fibrinogen owing to structural changes that accompany the conversion of fibrinogen to fibrin. One such change is the factor XIIIacatalyzed formation of isopeptide bonds between pairs of adjacent y-chains.3 Since the y-chain cross-link in stabilized fibrin renders this portion of the molecule resistant to plasmin, these bonds are retained in degradation products. A large number of macromolecular plasmic derivatives of cross-linked fibrin have been identified,’ the smallest of which is fragment DD, consisting of the D regions of two adjacent fibrin monomen cross-linked through y-chains.4-7

Danazol and Myelodysplasic Syndromes

Excerpt To the editor: The successful treatment with danazol of three patients with myelodysplastic syndromes and severe thrombocytopenia was described recently by Cines and coworkers (1). After ad...

Bone Marrow Expression of CCL3 Is Not Correlated with the Extent of Lytic Bone Lesions

marked contrast to the investigations mentioned above, Haaber et al. [8] analyzed CCL3 and CCL4 expression in highly purified plasma cells from 170 MM patients using quantitative RT-PCR and did not find evidence for significant human myeloma plasma cell expression of CCL3, as expression was only observed in 6 of 170 MM patients. The authors concluded then that their data do not support that MM plasma cells play an important role in the production of CCL3 or CCL4, but the tumor cells may stimulate other cell components in the bone marrow (BM) to produce these cytokines. In the present study, we addressed the latter issue and the whole controversy by comparing the gene expression profile (GEP) in BM samples from patients with various BM malignancies either with existent or absent lytic bone lesions. The use of unsorted BM samples exhibited the expression by the global cell population existing in vivo in each case, including tumor and non-tumor cells (the latter might be stimulated to secrete CCL3 by tumor cells, as offered by Haaber et al. [8] ). The immediate fixation of the samples following aspiration eliminated the bias related to delayed fixation [9] . The chemokine CCL3/MIP-1α is expressed in multiple myeloma (MM) cells and directly stimulates osteoclast formation [1] . MIP-1α also enhances adhesive interactions between MM cells and marrow stromal cells with increasing expression of RANKL and IL-6, which further increase bone destruction and tumor burden [2–4] . The chemokine receptor CCR1 is responsive to CCL3/MIP1α in human and mouse osteoclast precursors, and CCR1 activation leads to the formation of osteolytic lesions and facilitates tumor growth. In cell culture, CCL3 and CCR1 stimulate tumor growth both directly and indirectly via upregulation of cell adhesion and cytokine secretion. Targeting either ligand or receptor reverses these effects, leading to in vivo tumor burden control and prevention of osteolysis in murine and humanized mouse models [5] . Several inhibitors of CCL3/MIP-1α and CCR1 are currently under preclinical investigation and CCR1 blockade was shown recently to produce a profound decrease in tumor burden and osteolytic damage in the murine 5TGM1 model of MM bone disease [6] . In newly diagnosed MM patients, serum levels of CCL3 are elevated and correlate with the extent of bone disease, bone resorption and disease prognosis [7] . Nevertheless, in Received: July 1, 2013 Accepted: October 22, 2013 Published online: February 18, 2014

Blastic Transformation of Chronic Granulocytic Leukemia and other Myeloproliferative Disorders

Myeloproliferative disorders such as polycythemia vera (PV) and agnogenic myeloid metaplasia (AMM) with myelofibrosis (MF) progress to acute leukemia in about 30% of cases [1–3]. However, careful cytochemical, surface marker, and ultra-structural studies have not been performed in all these cases [4].