Prediction of platelet production during chemotherapy of acute leukemia

Mean megakaryocyte ploidy, mean platelet volume, and platelet count were measured during 17 courses of chemotherapy for acute nonlymphocytic leukemia. During the myelosuppression from chemotherapy, all three variables fell; during recovery, megakaryocyte ploidy rose 1–2 days before platelet volume, which in turn rose 1–2 days before platelet count. Serial platelet volumes and counts of these patients were compared to the nomogram of the inverse nonlinear relation between platelet count and platelet size in reference subjects. Platelet volume became inappropriately small before platelet count fell substantially and remained small through most of the thrombocytopenic nadir. The end of the nadir was predictable 1–2 days after platelet volume increased to lie congruent with the reference nomogram. Changes in thrombopoiesis appear to occur sequentially in megakaryocyte ploidy, platelet volume, and platelet count. Changes in platelet count, and therefore the appearance of duration of the thrombocytopenic nadir, can be predicted by 1–2 days with platelet volume and 3–4 days with megakaryocyte ploidy. As platelet count rose, despite the continuing predominance of “young” platelets, MPV fell, suggesting that megakaryocyte stimulation as well as platelet age affects platelet size.     

Bone marrow effects of anagrelide therapy in patients with myelofibrosis with myeloid metaplasia

In a prospective study investigating the therapeutic role of anagrelide in myelofibrosis with myeloid metaplasia, 20 patients received anagrelide in daily oral doses of 0.5-3 mg. 17 patients were evaluable and received anagrelide for a median of 2 years (range 0.5-4 years). No patient had a clinically appreciable benefit. Bone marrow (BM) examinations at baseline and after 6 and 12 months of treatment were available for 17, 17 and 12 patients, respectively. In all evaluable cases, BM megakaryocyte number increased after 6 months of anagrelide treatment. Also, Ulex europaeus agglutinin-1 staining of megakaryocytes revealed a left-shifted maturation pattern in most patients with a platelet response to anagrelide. However, megakaryocyte staining intensity for transforming (TGF-beta) and platelet-derived (PDGF) growth factors was not affected consistently by treatment. No patient had a >/=2 grade change in either BM fibrosis or osteosclerosis. These in-vivo data support our previous in-vitro observations that anagrelide interferes with megakaryocyte maturation rather than proliferation. Lack of a positive treatment effect is consistent with the finding that anagrelide did not significantly alter megakaryocyte expression of TGF-beta and PDGF.     

Expression of adhesion antigens of human bone marrow megakaryocytes, circulating megakaryocytes and blood platelets.

There is evidence that mature megakaryocytes migrate into sinusoids, enter the blood and fragment in the vascular bed. We wondered whether differences in expression of adhesion antigens could be associated with the egress of megakaryocytes from bone marrow into the peripheral blood or the fragmentation into platelets. Megakaryocytes from human marrow were purified by counterflow centrifugal elutriation followed by a glycoprotein Ib-dependent agglutination procedure. Megakaryocytes from central venous blood and pulmonary arteries were purified by counterflow centrifugal elutriation alone. Adhesion antigens were labelled in an immunohistochemical assay. Both bone marrow megakaryocytes and platelets from healthy volunteers stained > 75% positive for CD36, CD41, CD42, Cdw49b (alpha subunit VLA2), Cdw49e (alpha subunit VLA5), Cdw49f (alpha subunit VLA6) and CD62. Circulating megakaryocytes, although > 75% positive for CD41, had, unlike platelets and bone marrow megakaryocytes, a reduced and remarkable heterogeneous (5-100% positive) labelling with antibodies against Cdw49b, Cdw49e, Cdw49f. These results could be confirmed by comparing the bone marrow megakaryocytes, circulating megakaryocytes and platelets from 7 patients that were recovered and processed at the same time. Morphologically mature, circulating megakaryocytes have, unlike bone marrow megakaryocytes, a heterogeneous expression of adhesion antigens, especially of Cdw49b, Cdw49e, and Cdw49f.     

Selective enrichment in human megakaryocyte progenitors by the B203.13 surface differentiation antigen

The B203.13 monoclonal antibody specifically recognizes a subset (8–18.5%) of human CD34⁺ haemopoietic progenitors, which are significantly ( P  < 0.05) enriched in megakaryocyte progenitors (CFU-meg). Moreover, B203.13 expression was progressively lost as maturation proceeded along the megakaryocytic lineage. In fact: (i) CFU-meg derived from CD34⁺/B203.13⁺ showed a greater number of cells/colony with respect to CD34⁺/B203.13⁻; (ii) after 10 d of liquid cultures with 100 ng/ml of thrombopoietin, only a minority of CD34-derived cells positive for CD41 coexpressed B203.13; (iii) in situ immunocytochemical staining revealed that B203.13 was expressed exclusively on immature megakaryoblasts; (iv) circulating platelets did not express B203.13.     

Thrombocytopenia following liver transplantation is associated with platelet consumption and thrombin generation

Thrombocytopenia frequently occurs immediately after orthotopic liver transplantation. We have investigated the cause of this phenomenon in a cohort of 45 consecutive liver transplant recipients. The median preoperative platelet count (range) of 129 × 10⁹/l (14–719) fell to 56 × 10⁹/l (23–334) by the fourth postoperative day. The median preoperative reticulated platelet percentage (range) of 6.7% (2.2–23.9) increased to 16.4% (4.6–40.8) on day 7. There was a significant rise in prothrombin fragment F1.2 by the first postoperative day which was followed by rises in fibrinogen and fibrin degradation products. There was no increase in platelet-associated immunoglobulin or markers of endothelial activation. We conclude that there is an increased rate of platelet consumption associated with thrombin generation that reflects the magnitude of liver transplant surgery.     

Platelets release novel thiol isomerase enzymes which are recruited to the cell surface following activation

The thiol isomerase enzymes protein disulphide isomerase (PDI) and endoplasmic reticulum protein 5 (ERp5) are released by resting and activated platelets. These re‐associate with the cell surface where they modulate a range of platelet responses including adhesion, secretion and aggregation. Recent studies suggest the existence of yet uncharacterised platelet thiol isomerase proteins. This study aimed to identify which other thiol isomerase enzymes are present in human platelets. Through the use of immunoblotting, flow cytometry, cell‐surface biotinylation and gene array analysis, we report the presence of five additional thiol isomerases in human and mouse platelets and megakaryocytes, namely; ERp57, ERp72, ERp44, ERp29 and TMX3. ERp72, ERp57, ERp44 and ERp29 are released by platelets and relocate to the cell surface following platelet activation. The transmembrane thiol isomerase TMX3 was also detected on the platelet surface but does not increase following activation. Extracellular PDI is also implicated in the regulation of coagulation by the modulation of tissue factor activity. ERp57 was identified within platelet‐derived microparticle fractions, suggesting that ERp57 may also be involved in the regulation of coagulation as well as platelet function. These data collectively implicate the expanding family of platelet‐surface thiol isomerases in the regulation of haemostasis.     

Susceptibility of rat non-alcoholic fatty liver to the acute toxic effect of acetaminophen

Background and Aim: Acetaminophen overdose is the most frequent cause of acute liver failure. Non‐alcoholic fatty liver disease is the most common chronic condition of the liver. The aim was to assess whether non‐alcoholic steatosis sensitizes rat liver to acute toxic effect of acetaminophen. Methods: Male Sprague–Dawley rats were fed a standard diet (ST‐1, 10% kcal fat) and high‐fat gelled diet (HFGD, 71% kcal fat) for 6 weeks and then acetaminophen was applied in a single dose (1 g/kg body weight). Animals were killed 24, 48 and 72 h after acetaminophen administration. Serum biochemistry, activities of mitochondrial complexes, hepatic malondialdehyde, reduced and oxidized glutathione, triacylglycerol and cholesterol contents, and concentrations of serum and liver cytokines (TNF‐α, TGF‐β1) were measured and histopathological samples were prepared. Results: The degree of liver inflammation and hepatocellular necrosis were significantly higher in HFGD fed animals after acetaminophen administration. Serum markers of liver injury were elevated only in acetaminophen treated HFGD fed animals. Concentration of hepatic reduced glutathione and ratio of reduced/oxidized glutathione were decreased in both ST‐1 and HFGD groups at 24 h after acetaminophen application. Mild oxidative stress induced by acetaminophen was confirmed by measurement of malondialdehyde. Liver content of TNF‐α was not significantly altered, but hepatic TGF‐β1 was elevated in acetaminophen treated HFGD rats. We did not observe acetaminophen‐induced changes in activities of respiratory complexes I, II, and IV and activity of caspase‐3. Conclusion: Liver from rats fed HFGD is more susceptible to acute toxic effect of acetaminophen, compared to non‐steatotic liver.     

Laboratory Testing for Heparin-Induced Thrombocytopenia

Journal of Thrombosis and Thrombolysis -     

Guideline review: Guideline review: EASL clinical practice guidelines: drug-induced liver injury (DILI)

The European Association for the Study of the Liver has produced extensive guidelines for the investigation and management of drug-induced liver injury. Here, we provide a commentary and overview of some of the principle disease investigations and management that arise from these guideline recommendations.     

The relation of megakaryocyte ploidy to platelet volume

To determine how alterations of megakaryocyte proliferation will affect platelet production, we measured mean platelet volume (MPV), platelet volume heterogeneity, platelet count, and mean megakaryocyte ploidy in 42 patients. In normal subjects, mean platelet volume and megakaryocyte ploidy were related inversely but nonlin-early to platelet count, whereas mean platelet volume and platelet volume heterogeneity were related directly. In patients with immune thrombocytopenic purpura (low platelet count, MPV above normal, and increased megakaryocyte ploidy), and in those with reactive thrombocytosis (high platelet count, low MPV and megakaryocyte ploidy), the relation of MPV to megakaryocyte ploidy, platelet volume heterogeneity, and platelet count resembled or extended the relations found in normal subjects. By contrast, in patients with aplastic anemia or megaloblastic anemia, or in patients who were undergoing chemotherapy for leukemia, heterogeneity was increased abnormally at any MPV, and both MPV and megakaryocyte ploidy were substantially lower, at any platelet volume, than in normals or the above other groups. The most common ploidy class was 8N in all patients, and the mean megakaryocyte ploidy correlated directly and linearly with mean platelet volume. The data show that bone marrow with megakaryocytes of higher ploidy produces platelets that are both larger and more heterogeneous.     

Inherited thrombocytopenias—recent advances in clinical and molecular aspects

Since the beginning of the century, our knowledge of inherited thrombocytopenias greatly advanced, and we presently know 30 forms with well-defined genetic defects. This great advancement changed our view of these disorders, as we realized that most patients have only mild thrombocytopenia with inconspicuous bleeding or no bleeding tendency at all. However, better knowledge of inherited thrombocytopenias also revealed that some of the most prevalent forms expose to the risk of acquiring during infancy or adulthood additional disorders that endanger the life of patients much more than hemorrhages. Thus, inherited thrombocytopenias are complex disorders with quite different clinical features and prognosis. Identification of novel genes whose mutations result in low platelet count greatly advanced also our knowledge of the megakaryocyte biology and proved beyond any doubt that the defective proteins play an essential role in platelet biogenesis or survival in humans. Based on the study of inherited thrombocytopenias, we better understood the sequence of molecular events regulating megakaryocyte differentiation, maturation, and platelet release. Since nearly 50% of patients have as yet unidentified genetic or molecular mechanisms underlying their inherited thrombocytopenia, further studies are expected to reveal new clinical entities and new molecular mechanisms of platelet production.     

Elevated levels of basic fibroblast growth factor in megakaryocytes and platelets from patients with idiopathic myelofibrosis

Idiopathic myelofibrosis, or agnogenic myeloid metaplasia, is a chronic myeloproliferative disorder characterized by clonal expansion and marrow fibrosis. Although marrow fibrosis appears to be a reactive process, it substantially contributes to impaired haemopoiesis. During the last few years the implication of megakaryocyte-derived growth factors in its pathogenesis has been documented.
We previously reported increased expression of TGF-β in patients with idiopathic myelofibrosis. In the present study we show that circulating megakaryocytic cells from such patients expressed high levels of basic fibroblast growth factor (bFGF). An increased expression of bFGF was also detected in patients' platelets. Under culture conditions, bFGF present in megakaryocytic cells was not exported into the medium, consistent with the fact that bFGF is devoid of a secretion peptide signal. Interestingly, this lack of bFGF secretion was observed in all patients but one, who was in an accelerated phase of the disease and presented an important percentage of circulating megakaryoblasts.     

Thrombocytopenia associated with environmental exposure to polyurethane

Few chemicals in the environment have been implicated as causes of isolated thrombocytopenia, and the evidence is usually less than convincing because the patients were not rechallenged with the chemical in vivo. In the present paper, a child is reported with the onset of thrombocytopenia in temporal association with environmental exposure to polyurethane. Five years after the initial thrombocytopenia had resolved, an inadvertent in vivo rechallenge with environmental polyurethane resulted in recurrence of the thrombocytopenia. This recurrence, together with the fact that only 1-4% of cases of idiopathic thrombocytopenic purpura in children recur, provided strong evidence for a causal role for the polyurethane exposure in this patient's thrombocytopenia. In summary, environmental exposure to polyurethane should be considered in the differential diagnosis of acquired thrombocytopenia in childhood.     

Timed feeding of mice modulates light-entrained circadian rhythms of reticulated platelet abundance and plasma thrombopoietin and affects gene expression in megakaryocytes

Circadian ( c . 24 h) rhythms of physiology are entrained to either the environmental light-dark cycle or the timing of food intake. In the current work the hypothesis that rhythms of platelet turnover in mammals are circadian and entrained by food intake was explored in mice. Mice were entrained to 12 h light-dark cycles and given either ad libitum (AL) or restricted access (RF) to food during the light phase. Blood and megakaryocytes were then collected from mice every 4 h for 24 h. It was found that total and reticulated platelet numbers, plasma thrombopoietin (TPO) concentration and the mean size of mature megakaryocytes were circadian but not entrained by food intake. In contrast, a circadian rhythm in the expression of Arnt1 in megakaryocytes was entrained by food. Although not circadian, the expression in megakaryocytes of Nfe2 , Gata1 , Itga2b and Tubb1 expression was downregulated by RF, whereas Ccnd1 was not significantly affected by the feeding protocol. It is concluded that circadian rhythms of total platelet number, reticulated platelet number and plasma TPO concentration are entrained by the light-dark cycle rather than the timing of food intake. These findings imply that circadian clock gene expression regulates platelet turnover in mammals.     

Herbal extracts as hepatoprotectants against acetaminophen hepatotoxicity

Many plant-derived natural products have the potential to be hepatoprotective and therefore can be used to treat acute and chronic liver diseases. The challenge is to identify the most promising compounds and evaluate their protective mechanism. In a recently published article, Wang et al evaluated extracts of the plant Gentiana manshurica Kitagawa (GM) in a model of acetaminophen hepatotoxicity. The authors concluded that GM is hepatoprotective against acetaminopheninduced liver injury due to its antioxida...     

大蒜油对对乙酰氨基酚所致小鼠急性肝损伤的保护作用

目的 观察大蒜油对对乙酰氨基酚(AP)所致小鼠急性肝损伤是否有保护作用.方法 将75只小鼠随机分为阴性对照组、模型组以及大蒜油低、中、高三个剂量组(分别为25、50、100 mg/kg),给药后2 h除阴性对照组其他各组小鼠均灌胃AP 240 mg/kg;24 h后小鼠摘眼球取血,离心,测定血清中丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)的活性,同时比较各组小鼠的肝脏系数和肝脏病理组织学变化.结果 与模型组相比,大蒜油低、中、高剂量组的肝脏系数明显减小,血清ALT、AST活性显著降低(P＜0.01),同时肝脏组织病理学检查发现模型组肝细胞发生气球样变,甚至坏死,炎细胞浸润,而大蒜油组无明显病理学改变.结论 大蒜油对AP所致小鼠急性肝损伤有明显的保护作用.     

Effects of vascular endothelial growth factors and their receptors on megakaryocytes and platelets and related diseases

It is well known that vascular endothelial growth factors (VEGFs) and their receptors (vascular endothelial growth factor receptors, VEGFRs) are expressed in different tissues, and VEGF‐VEGFR loops regulate a wide range of responses, including metabolic homeostasis, cell proliferation, migration and tubuleogenesis. As ligands, VEGFs act on three structurally related VEGFRs (VEGFR1, VEGFR2 and VEGFR3 [also termed FLT1, KDR and FLT4, respectively]) that deliver downstream signals. Haematopoietic stem cells (HSCs), megakaryocytic cell lines, cultured megakaryocytes (MKs), primary MKs and abnormal MKs express and secrete VEGFs. During the development from HSCs to MKs, VEGFR1, VEGFR2 and VEGFR3 are expressed at different developmental stages, respectively, and re‐expressed, e.g., VEGFR2, and play different roles in commitment, differentiation, proliferation, survival and polyplodization of HSCs/MKs via autocrine, paracrine and/or even intracrine loops. Moreover, VEGFs and their receptors are abnormally expressed in MK‐related diseases, including myeloproliferative neoplasms, myelodysplastic syndromes and acute megakaryocytic leukaemia (a rare subtype of acute myeloid leukaemia), and they lead to the disordered proliferation/differentiation of bone marrow cells and angiogenesis, indicating that they are closely related to these diseases. Thus, targeting VEGF‐VEGFR loops may be of potential therapeutic value.