In vitro undetectable PT and Fibrinogen (and in vivo?)

A 81 aged woman came to E.R. of Trieste University Hospital with a traumatic head injury. Blood cells count, liver enzymes and other parameters were normal, but with a photometric clot detection method PT and PT-derived Fibrinogen were undetectable, Fibrinogen-Clauss gave different results (157 to 357 mg/dL) and aPTT-Ratio was normal (0.96). When the instrument detection performance was improved, PT was normal and PT-derived Fibrinogen detectable, but Fibrinogen-Clauss was still very unsteady (352/294/558 mg/dL). When a mixing test was performed with normal pool plasma, PT was corrected, PT-derived Fibrinogen was very low (80 mg/dL) and Fibrinogen-Clauss resulted 360 mg/dL. Fibrinogen-Antigen was 368 mg/dL by a nephelometric immunoassay. In another Lab with a different optical analyzer, PT and aPTT yielded the same results, Fibrinogen-Clauss was 557 mg/dL with 35 IU/ml Thrombin reagent (and a very steep clot formation curve), but 113 mg/dL with 15 IU/ml Thrombin reagent (and a normal curve). With an electromechanical clot detection method, PT-INR and aPTT-Ratio were normal (0.88 and 0.96 respectively), Fibrinogen-Clauss was normal (400 mg/dL) but unsteady. However in a few days our patient healed up perfectly; she declared that her sister had the same performance when she was referred to another Hospital for a check-up, nonetheless they never had any severe bleeding in their life. Samples from our patient\u2019s son and daughter were taken and resulted completely normal for coagulation tests. We hypothetized: 1) a too fast Thrombin formation and/or Fibrinogen consumption, as shown by steep coagulation curves without a stable plateau; 2) an excessive thrombin formation, (in preliminary studies, however, G20210A mutation was absent and F1+2 were normal); 3) a dysfibrinogenemia, (to be studied). Further studies for Endogenous Thrombin Potential about thrombin ipothesis and for genetical pattern about fibrinogen molecule are needed to clarify this case

Cirrhotic thrombocytopenia is a multifactorial condition: evidence of reduced platelet production and incresed platelet destruction

Background: Thrombocytopenia is a common manifestation of liver cirrhosis (LC), but the underlying mechanism is not fully understood. The purpose of our work was to evaluate the platelet kinetics in LC of different etiology by examining platelet production and destruction. Patients: 91 consecutive LC patients (36 HCV, 49 alcoholics, 15 HBV) were enrolled in the study. As controls, 25 cases with idiopathic thrombocytopenic purpura (ITP), 10 with aplastic anemia (AA), and 40 healthy blood donors were studied. Methods: Plasma thrombopoietin (TPO) was measured by ELISA. Absolute reticulated platelet (RP) count was determined by Thiazole Orange method. Plasma glycocalicin (GC) was measured by monoclonal antibodies. Platelet associated and serum circulating antiplatelet antibodies were detected by flow cytometry. The B-cell monoclonality in the PBMC were performed by isotype-specific immunoglobulin fingerprinting. Results: The serum TPO was significantly (p<0.0005) lower in the patients with LC (29.9 \ub1 18.1 pg/ml) than in normals (82.3 \ub1 47.6 pg/ml). The GC index was 1.96 \ub1 1.40 in HCV+ LC (p<0.0005 vs. normals 0.9 \ub1 0.2), 1.79 \ub1 1.51 in alcoholic LC (p<0.006) and 1.71 \ub1 1.69 in HBV + LC (p<0.006). In the patients affected by ITP, the GC index was 12.9 \ub1 4.4 (p<0.000002). The absolute levels of RP were 4.233 \ub1 2.367 109/L in alcoholic LC (p<0.0000000012 vs normals) 4.996 \ub1 3.143 x 109/L in HBV+ LC (p<0.006) and 6.629 \ub1 7.409 x 109/L in HCV+LC (p<0.005). The prevalence of platelet-associated and circulating anti-platelet antibodies was higher in HCV+ LC than in healthy subjects (p<0.0064), than in alcoholic LC (p<0.018) and than in HBV+ LC (p<0.0001). The B-cell monoclonality was found in 8 (27%) of the HCV-positive patients, whereas no monoclonality was found in HBV (p<0.004) or alcoholic patients (p<0.003). Conclusions: Patients with LC present a decreased plasma TPO, an accelerated platelet turnover and a low platelet production. These findings indicate that cirrhotic thrombocytopenia is a multifactorial condition, involving both increased platelet clearance and impaired thrombopoiesis. The HCV-LC is characterized by an increased prevalence of autoimmune phenomena, including anti-platelet antibodies and, as consequence, a platelet turnover more accelerated than in HBV or alcoholic LC

Platelet production and destruction in liver cirrhosis

BACKGROUND &#38; AIMS: Thrombocytopenia is common in liver cirrhosis (LC) but the mechanisms are not fully understood. The purpose of our work was to evaluate platelet kinetics in LC with different etiologies by examining platelet production and destruction. METHODS: Ninety-one consecutive LC patients (36 HCV, 49 alcoholics, 15 HBV) were enrolled. As controls, 25 subjects with idiopathic thrombocytopenic purpura, 10 subjects with aplastic anemia, and 40 healthy blood donors were studied. Plasma thrombopoietin (TPO) was measured by ELISA. Reticulated platelets (RP) were determined using the Thiazole Orange method. Plasma glycocalicin (GC) was measured using monoclonal antibodies. Platelet associated and serum antiplatelet antibodies were detected by flow cytometry. B-cell monoclonality in PBMC was assessed by immunoglobulin fingerprinting. RESULTS: Serum TPO was significantly lower in LC (29.9\ub118.1 pg/ml) compared to controls (82.3\ub147.6 pg/ml). The GC levels were higher in LC (any etiology) than in healthy cases. Conversely, the absolute levels of RP were lower in LC (any etiology) than in healthy controls. The platelet-associated and serum anti-platelet antibodies were higher in HCV+ LC compared to healthy subjects (p<0.0064), alcoholic LC (p<0.018), and HBV+ LC (p<0.0001). B-cell monoclonality was found in 27% of the HCV+LC, while it was not found in HBV+ or alcoholic LC. CONCLUSIONS: Patients with LC present decreased plasma TPO, accelerated platelet turnover, and reduced platelet production. This indicates that LC thrombocytopenia is a multifactorial condition involving both increased platelet clearance and impaired thrombopoiesis

Serum Response Factor depletion affects the proliferation of the hepatocellular carcinoma cells HepG2 and JHH6

For hepatocellular carcinoma (HCC), a leading cause of cancer death world-wide, there is no effective therapy especially for the advanced stage of the disease. Thus, we started the investigations about a novel anti HCC approach based on the depletion of the transcription factor serum response factor (SRF) in HCC cell lines; SRF choice was based on its recently proposed contribution to HCC tissue development and on its important role in cell proliferation. SRF depletion, obtained by a siRNA (siSRF797), was studied in two HCC cell lines, i.e. HepG2 and JHH6 assigned to high and lowhepatocytic differentiation grade on the base of the capacity to synthesize albumin. In the HCC cell lines examined, siSRF797 reduced both the mRNA and protein levels of SRF without inducing unspecific interferon response or cytotoxicity. Moreover, SRF depletion induced the reduction of S-phase cells and a decrease in cell number and vitality. Particularly in HepG2, cell growth impairment was paralleled by the decrease of the levels of the transcription factor E2F1 together with some of its regulated genes. In HepG2 but not in JHH6, SRF depletion was associated with apoptosis. Finally, in both HepG2 and JHH6, the combined administration of siSRF797 and bortezomib, a proteasome inhibitor whose therapeutic potential for HCC is considered attractive, further reduced cell viability compared to either siSRF797 or bortezomib treatment alone. In conclusion, SRF depletion affects the expansion of the high and low differentiation grade HCC cells HepG2 and JHH6. These results can pave the way to understand the role of SRF in HCC development and possibly to identify novel anti HCC therapeutic strategies