Haemostatic actions of the folkloric medicinal plant extract Ankaferd Blood Stopper.

Ankaferd Blood Stopper (ABS), a standardized mixture of five plants, has been used historically as a haemostatic agent but its mechanism of action remains unknown. This study investigated the in vitro effects of ABS on haemostatic parameters. When added to plasma or serum, ABS induced the very rapid formation of a protein network and erythrocyte aggregation. The levels of coagulation factors II, V, VII, VIII, IX, X, XI, and XIII were not affected by ABS. Plasma fibrinogen activity and antigen levels were decreased following the addition of ABS, in parallel with the prolonged thrombin time. Total protein, albumin, and globulin levels decreased after the addition of ABS. Our findings suggest that ABS stimulates the formation of an encapsulated protein network that provides focal points for erythrocyte aggregation. ABS has the therapeutic potential to be used for the management of haemorrhage and this agent should be investigated further in clinical trials.

Circulating thrombomodulin levels in familial Mediterranean fever.

Increments in circulating thrombomodulin levels reflect endothelial cell injury. Thrombomodulin can also be synthesized by several inflammatory cells including monocytes, neutrophils, and thrombomodulin itself can modulate the inflammatory response. In this study, we assessed circulating thrombomodulin concentrations in patients with familial Mediterranean fever (FMF). Twenty-five patients with FMF (F/M: 14/11) (mean age: 31.1 +/- 9.7 years) and 25 healthy controls (F/M: 13/12) (mean age: 34.6 +/- 7.0 years) were involved in the study. Thrombomodulin levels were measured by commercially available enzyme-linked immunosorbant assay (ELISA) (Immunoassay of thrombomodulin Diagnostica Stago, Asnieres-Sur-Seine, France). Twenty of the patients were in attack-free period and the remaining five had been during acute FMF attacks. Thrombomodulin levels were higher in the study group (20.9 +/- 12.1 ng/ml) than healthy controls (14.1 +/- 8.4 ng/ml) (p < 0.05). Circulating thrombomodulin levels were also higher in attack-free FMF patients (22.4 +/- 12.9 ng/ml) than controls. This study disclosed for the first time significantly higher increments in the circulating levels of thrombomodulin in FMF. This observation could be a consequence of injured endothelium and/or activated inflammatory cells.

The plasma levels of prostanoids and plasminogen activator inhibitor-1 in primary and secondary thrombocytosis.

An elevated platelet count is a common finding in both hospitalized and ambulatory patients. Thrombosis and bleeding complications are more frequently observed in patients with clonal thrombocytosis than secondary thrombocytosis. The aim of this study was to investigate the behaviors of plasminogen activator inhibitor type 1 (PAI-1), the inhibitor of fibrinolysis; and thromboxane A2 and 6-keto-PGF1 alpha, the products of endoperoxides, in 16 patients affected with clonal thrombocytemia as compared with 16 patients with reactive thrombocytosis and 15 normal controls. In the clonal thrombocytemia group, plasma levels of PAI-1 antigen and activity were significantly higher than both reactive thrombocytosis and control group. Plasma levels of 6-keto-PGF1alpha were significantly higher in the clonal thrombocytemia group than the other two groups and also higher in the reactive thrombocytosis group than the control group, which was also significant. This study confirms that arachidonate metabolism is frequently deranged in patients with thrombocytosis and hypofibrinolysis due to increased PAI-1 plasma levels as shown in the clonal thrombocytosis group. This may explain the thrombotic tendency in myeloproliferative disorders.

Tamoxifen-induced tissue factor pathway inhibitor reduction: a clue for an acquired thrombophilic state?

BACKGROUND: Current understanding of hemostatic systems enables us to better explore the enigmatic pathobiology of tamoxifen (TAM)-induced thrombotic diathesis. We have therefore aimed to assess the hemostatic changes in breast cancer patients receiving TAM on an adjuvant basis. PATIENTS AND METHODS: The study population consisted of 43 female patients with hormone receptor-positive breast cancer who received TAM 20 mg/day as part of their adjuvant treatment. Mean age was 52+/-12 years (range 25-74). Twenty-one patients (49%) were premenopausal. Plasma samples were collected prior to and following 6 months of TAM therapy and were assayed for total tissue factor pathway inhibitor (TFPI), free TFPI, lipid-bound TFPI, thrombomodulin, D dimer, activated protein C resistance (APC res), factors VIIa, II, V, VII and X, and global fibrinolytic capacity (GFC). RESULTS: Median total TFPI decreased significantly from 48.5 ng/ml to 36.2 ng/ml (P=0.001), free TFPI from 10 to 7.6 ng/ml (P=0.001) and lipid-bound TFPI from 39.1 to 28.7 ng/ml (P=0.001). There were significant decreases in the levels of factor II (P=0.03), factor V (P=0.001), factor VII (P=0.06), thrombomodulin (P=0.01) and D dimer (P=0.001). However, APC res times were significantly prolonged (P=0.04). The remaining parameters that we have studied were not significantly affected. CONCLUSION: Our findings suggest that TAM tends to activate the coagulation pathway by counteracting major molecules involved in coagulation inhibition, namely TFPI and TM. As reflected by unchanged GFC, the drug appears to impair the expected compensatory activation of the fibrinolytic system, which removes fibrin polymers resulting from coagulation activation.

Plasma basic fibroblast growth factor and bone marrow fibrosis in clonal myeloproliferative disorders.

Basic fibroblast growth factor (bFGF) is an important growth factor involved in clonal hematopoietic expansion, neoangiogenesis, and bone marrow fibrosis, all of which are important pathobiologic features of clonal chronic myeloproliferative disorders (CMPD) and myelodysplastic syndromes (MDS). The aim of this study was to assess circulating bFGF concentrations in patients with CMPD and MDS with respect to the presence of bone marrow fibrosis in histopathologic examination. The study group comprised 18 patients with CMPD (six female, 12 male; median age 50 years), seven patients with MDS (one female, six male; median age 66 years) and 10 healthy adults as controls (four female, six male; median age 29 years). CMPD group included six chronic myelogenous leukemia (CML), seven essential thrombocythemia (ET), three polycythemia vera (PV), two agnogenic myeloid metaplasia (AMM). All seven MDS patients were the FAB subtype of refractory anemia (RA). Bone marrow biopsy sections stained with hematoxylin and eosin (H & E) and for reticulin were examined for the presence of fibrosis. The median plasma bFGF level was 18.2 pg/ml (interquartile range, IQR: 15.2-26.7) in patients with CMPD, 18.0 pg/ml (IQR: 15.8-26.4) in patients with MDS, 13.6 pg/ml (IQR: 9.9-20.0) in the control group. The bFGF levels were significantly higher in patients with CMPD in comparison with the healthy control group (P = 0.031). Circulating bFGF tended to be significantly lower in relation to the development of marrow fibrosis (P = 0.028). The complicated interactions of bFGF and fibrosis in the context of CMPD may be either 'cause' or 'effect'. The bFGF might represent an important link between angiogenesis, fibrosis, and clonal neoplastic hematopoiesis during the development of CMPD.

The predictability of factor V Leiden (FV:Q(506)) gene mutation via clotting-based diagnosis of activated protein C resistance.

After the discovery of activated protein C resistance (APCR) due to factor V Leiden mutation and the causal relationship of the phenomenon with clinical thromboembolism, a wide variety of functional clotting-based assays were developed for testing of APCR in relation to the specific DNA-based analysis of FV:Q(506) Leiden. The aim of this study is to assess a clotting-based APCR assay using procoagulant crotalidae snake venom with respect to the sensitivity, specificity, and predictability for the presence of the factor V Leiden mutation. APCR testing and factor V DNA analyses have been performed concurrently on 319 patient specimens. APCR values of the patients with homozygous factor V Leiden mutation (70.4+/-13.5 s) were significantly lower (p<0.001) in comparison to the subjects with the heterozygous mutation (87.6+/-13.4 s). The assay is highly sensitive (98.7%) and specific (91.9%) for the screening of factor V Leiden mutation. The sensitivity and specificity of the APCR testing reached to 100% below the cut-off value of 120 s among the patients with homozygous factor V Leiden mutation. Therefore, this method could help the desired effective optimal screening strategy for the laboratory search of hereditary thrombophilia focusing on the diagnosis of APCR due to FV:Q(506).

Protein C inhibitor and serum amyloid A in immune thrombocytopaenic purpura.

In immune thrombocytopaenic purpura (ITP), phagocytic cells prematurely destroy platelets opsonized by anti-platelet auto-antibodies, while residual platelets rescued from these autoimmune attacks are hyperfunctioning. The exact pathobiological basis of this phenomenon is unknown. Protein C inhibitor (PCI), a platelet alpha-granule pro-coagulant molecule, is released on activation of platelets. Serum amyloid A (SAA; an acute phase protein), however, inhibits platelet aggregation and modulates platelet adhesion. We aimed to assess circulating soluble plasma PCI and SAA concentrations in 17 patients with newly diagnosed ITP and ten healthy volunteers. Plasma PCI concentrations tended to be higher in ITP patients, despite absolute thrombocytopaenia, than in normal controls. SAA levels were significantly higher in ITP patients compared with the control group. We conclude that secretion of the alpha-granule PCI content of platelets could result from platelet activation, and that PCI may be the link between platelet microparticles and haemostatically active ITP platelets. Increased concentrations of SAA and PCI may interfere with the disordered and compensatory pro-coagulant mechanisms of ITP.

Elevated serum soluble Fas levels in the various stages of hepatitis C virus-induced liver disease.

We aimed to determine serum soluble Fas antigen (sFas) levels at various stages of hepatitis C virus (HCV)-induced liver disease, and investigate correlations between serum sFas levels and clinical, biochemical and pathologic features. Sixty-five patients were categorized into five groups: 1, chronic active hepatitis C, elevated alanine aminotransferase (ALT), HCV-polymerase chain reaction (PCR) positive; 2, responders to interferon + ribavirin therapy; 3, cirrhosis; 4, chronic hepatitis C, normal ALT, HVC-PCR positive; and 5, sustained responders. Group 6 comprised 15 control individuals. Serum sFas levels were measured by enzyme-linked immunosorbent assay. Significant differences in serum sFas levels were found between the following groups: 1 and 2; 1 and 3; 1 and 4; 1 and 6; and 3 and 6. Serum sFas levels did not correlate with ALT, histological activity or HCV-PCR positivity within group 1. Serum sFas levels appear to increase in advanced stages of HCV-induced liver disease, as a result of host-related immunological factors.

Bloodstream thrombopoietin in rheumatoid arthritis with thrombocytosis.

Thrombopoietin (TPO) is the major regulator of growth and differentiation of megakaryocytes. Recent studies have shown that TPO may also act as an acute-phase reactant, and it has been suggested as a component of inflammatory reactions. In this study our objective was to investigate serum TPO levels in patients with rheumatoid arthritis, a complex chronic inflammatory disorder not uncommonly associated with thrombocytosis. Bloodstream TPO concentrations were assessed in 13 RA patients with platelet counts between 450 and 650 x 10(9)/l, 10 RA patients with platelet counts >650 x 10(9)/l, 15 RA patients with normal platelet counts and 12 healthy controls. RA patients with normal platelet counts had TPO levels comparable with healthy controls. TPO concentrations in patients with mild thrombocytosis were significantly elevated, whereas patients with markedly increased thrombocyte counts had prominently decreased TPO levels. These results indicate that TPO seems to be associated with reactive thrombocytosis in RA patients with active disease. In patients with extremely increased thrombocytosis serum TPO levels might be regulated by increased platelet mass via receptor-mediated uptake and metabolism.

Rational use of the PFA-100 device for screening of platelet function disorders and von Willebrand disease.

Two hundred and five patients referred for evaluation of platelet functions and 126 healthy controls were tested with the PFA-100 instrument. A cut-off value of 150 s for collagen/epinephrine (CEPI) closure time (CT) produced most acceptable sensitivity (90%), specificity (85.2%), and positive (82.6%) and negative (91.6%) predictivity values for screening of platelet function disorders and von Willebrand disease (vWD). All patients with vWD and Glanzmann thrombasthenia could be detected by PFA-100. Both CEPI and collagen/adenosine diphosphate (CADP) CTs were elevated in all of these cases. Sensitivity of the device was 81.6% for patients with platelet secretion defects. CADP CT was normal in 63.9% of the patients in this subgroup. Specificity (47%) and positive predictivity (57%) of the instrument were diminished in patients with low hemoglobin concentrations. Depending on the results, an algorithm was developed for screening of platelet function disorders and vWD with PFA-100.

Plasma thrombospondin in immune thrombocytopenic purpura.

Patients with immune thrombocytopenic purpura (ITP) rarely suffer life-threatening haemorrhages despite significant thrombocytopenia, probably because large numbers of hyperfunctioning platelets are present. Thrombospondin is a platelet alpha-granule protein and its plasma level may reflect platelet activation. We assessed circulating thrombospondin levels in 12 newly diagnosed ITP patients (one man; 11 women, aged 36 +/- 16 years) before they were treated for ITP. Twelve healthy people (four men; eight women, aged 31 +/- 11 years) acted as controls. Plasma thrombospondin concentrations were measured using enzyme-linked immunoassays. Thrombospondin concentrations tended to be higher, despite thrombocytopenia, in ITP patients (158.8 +/- 28.2 ng/ml) compared with controls (120.7 +/- 18.2 ng/ml). The difference was not statistically significant, but the relatively high circulating thrombospondin concentrations we observed suggest that residual platelets could be activated in ITP, thus indicating a more benign clinical course compared with aplastic thrombocytopenia.

Changes in the concentration and distribution of tissue factor pathway inhibitor in Behçet's disease and systemic lupus erythematosus: effect on the prethrombotic state.

BACKGROUND: Tissue factor pathway inhibitor (TFPI) is an anticoagulant which modulates the tissue factor (TF) dependent pathway, acting on the factor VIIa/TF complex, factor Xa, and thrombin. Although most TFPI is found in association with plasma lipoproteins and platelets, the functional pool is bound to vascular endothelium and is released into the circulation on stimulation with heparin or low molecular weight heparin (LMWH). OBJECTIVE: To assess the vascular endothelial TFPI pool in patients with Behçet's disease (BD) or systemic lupus erythematosus (SLE). METHODS: Plasma TFPI concentrations were determined before, and 20 and 60 minutes after subcutaneous LMWH injection in 15 newly diagnosed patients with BD and 12 with SLE, and in 12 healthy controls. RESULTS: Baseline median TFPI was 149.5 ng/ml in healthy subjects, and the percentage change in TFPI at 20 minutes (((value at 20th min - baseline value)/baseline value) x 100) was 575.2. TFPI concentrations in patients with BD were initially normal at baseline (136.0 ng/ml), but the percentage change (44.7) was significantly lower than in the patients with SLE and the controls. Baseline TFPI concentrations in patients with SLE (83.0 ng/ml) were lower than in the control group, but the TFPI response to stimulation with LMWH reached a level (626.4%) comparable to that of the controls. CONCLUSION: Depletion of the functional endothelial pool in BD and low circulating concentrations of TFPI despite an intact pool in SLE may be important in the pathogenesis of thrombosis in these vasculitic syndromes.

Soy protein diet significantly improves endothelial function and lipid parameters.

BACKGROUND: Replacement of animal protein with soy protein in the diet is associated with decreased cholesterol levels. However, the effects of soy protein diet on endothelial function are not well known. HYPOTHESIS: The aim of the study was to investigate the effects of soy protein diet on plasma lipids and endothelial function parameters assessed by two different methods. METHODS: Twenty hypercholesterolemic, nonsmoker male patients (age 50.1+/-11.8 years), with a normal body mass index, were included. After calculating their daily requirements, a diet with 25-30% of energy from fats. 10-12% from proteins, and the rest from carbohydrates was instituted. Sixty percent of the animal source proteins of the diet were substituted by soy. The anthropometric measures, lipid parameters, and endothelial functions of the subjects were assessed at baseline and 6 weeks after soy protein diet. Flow-mediated endothelium-dependent dilatation (EDD) and plasma thrombomodulin (TM) levels were evaluated as endothelial function parameters. RESULTS: After diet, plasma total cholesterol, low-density lipoprotein cholesterol, apolipoprotein B, and triglyceride levels decreased significantly (p <0.001, p < 0.001, p = 0.039, and p = 0.001, respectively). The mean plasma TM levels were also significantly reduced with diet (p = 0.004). Studies of the brachial artery indicated a borderline dilatation in baseline brachial artery diameter (p = 0.05), however the diameter at reactive hyperemia was significantly larger after diet (p<0.001), resulting in a significant improvement of EDD (p = 0.002). CONCLUSION: Soy protein diet significantly improves plasma lipid profile in patients with hypercholesterolemia. Furthermore, the endothelial function, as judged by two different methods (EDD and plasma TM levels), also improves with soy protein diet.

Plasma fibronectin- and thrombospondin-adhesive molecules during acute attacks and attack-free periods of familial Mediterranean fever.

We assessed plasma concentrations of fibronectin (FN) and thrombospondin (TSP) during acute attacks and attack-free periods of patients with familial Mediterranean fever (FMF). Seven female and three male FMF patients (mean age 34+/-7 years) were enrolled in the study. Plasma samples were obtained during acute FMF attacks and after 3 months of freedom from attacks. Erythrocyte sedimentation rate, C-reactive protein, and white blood cell count were evaluated concurrently. Plasma levels of FN and TSP were assayed by enzyme-linked immunosorbent assay (ELISA). Both FN and TSP concentrations were found to increase during acute attacks. Levels of adhesive molecules decreased during attack-free periods (P < 0.05). Significant correlations were found between FN and TSP levels and the concentrations of acute-phase response indicators (P< 0.05). This study disclosed for the first time significantly higher increments in the plasma levels of FN and TSP during acute FMF attacks than in attack-free periods. Therefore, the two matrix glycoproteins may play precipitating and/or regulatory roles in the inflammatory processes of these attacks.

Extent of coronary atherosclerosis and homocysteine affect endothelial markers.

The aim of the study was to evaluate the effects of the presence, extent, and clinical stability of coronary artery disease on endothelial function parameters, C-reactive protein and homocysteine levels. Fifty-eight patients with angiographically documented coronary artery disease and 25 patients with normal coronary arteries were evaluated for risk factors, plasma homocysteine, C-reactive protein, and soluble adhesion molecule levels. Vascular cell adhesion molecule-1 and sE-selectin were significantly higher in the group with coronary artery disease than in healthy subjects (p = 0.005 and p = 0.031, respectively). Patients with unstable angina had significantly higher C-reactive protein (p < 0.001), troponin I (p < 0.01), and leukocyte counts (p < 0.05) than those with stable angina. sE-selectin levels were correlated with the extent of coronary atherosclerosis (r = 0.444, p < 0.05), and plasma homocysteine levels were associated with vascular cell adhesion molecule-1 (r = 0.479, p < 0.05) in unstable cases. These results suggest that vascular cell adhesion molecule-1 and sE-selectin are useful for determining the presence of coronary atherosclerosis, whereas C-reactive protein, troponin 1, and leukocyte count are predictors of clinical stability.

Selectins in normal pregnancy, pre-eclampsia and missed abortus.

Selectins, are known to be increased in the serum of patients with pre-eclampsia, indicating that these molecules are possible markers of endothelial cell injury. In this study, we investigated P, E and L selectin levels in normal pregnancy, pre-eclampsia, and missed abortus. Plasma P and L selectins levels were significantly higher in normal pregnancy and pre-eclampsia than healthy controls; but plasma concentrations of E selectins were not different between these groups. Plasma P selectin was significantly higher in pre-eclampsia than normal pregnancy. Plasma concentrations of all selectins were significantly higher in missed abortus than healthy control. L selectin levels were higher in pre-eclampsia and missed abortus than normal pregnancy. We found the levels of selectins were increased in pre-eclampsia and missed abortus. Although selectins were suspected to play a role in the pathogenesis of pre-eclampsia, in conjunction with previous studies, we thought that elevated selectin levels are a non-specific consequence of endothelial injury rather than being a cause.

Thrombopoietin and mean platelet volume in coronary artery disease.

BACKGROUND: Large platelets are shown to be hemostatically more active. It has been suggested that mean platelet volume (MPV) is increased during acute myocardial infarction (AMI) and unstable angina pectoris (USAP). However, the underlying mechanism of the phenomenon remains unclear. HYPOTHESIS: In this study, platelets, MPV, and thrombopoietin (TP) levels were investigated in patients with coronary artery disease (CAD) and healthy controls. METHODS: Twenty patients with AMI and 20 patients with USAP were included in this study. Seventeen healthy adult subjects served as controls. Venous blood samples of the subjects were drawn within 12 h after admission. Thrombopoietin levels were measured by ELISA and platelet counts and MPV were assayed by autoanalyzer. RESULTS: Patients with AMI and USAP had higher platelet counts than those in the control group. Although the platelet counts were slightly higher in AMI than in USAP, this did not reach statistical significance. Mean platelet volume and levels of TP were found to be elevated in patients with AMI and USAP compared with control subjects (p < 0.001). Thrombopoietin levels were higher in AMI than USAP, but this was not statistically significant. There was a positive correlation between TP levels and MPV values (p < 0.05). CONCLUSION: Increased TP levels may increase both platelet counts and platelet size, resulting in hemostatically more active platelets, which may contribute to the development and progression of CAD.

Platelet and leukocyte deactivation after intracoronary stent placement in patients receiving combined antiplatelet therapy.

Activated platelets and leukocytes have been demonstrated to play a role in the development of stent thrombosis, and coronary angioplasty has been shown to result in activation of platelets, leukocytes, and endothelial cells. We aimed to evaluate the effects of intracoronary stent placement and aspirin plus ticlopidine treatment on platelets, leukocytes, and endothelial cells via observing the serial changes in the circulating soluble forms of adhesion molecules in 54 patients with coronary artery disease, who had elective coronary angioplasty and stent implantation for a single lesion of the left anterior descending artery. After stent placement, intravenous heparin infusion was administered only for 24 hours, and aspirin plus ticlopidine treatment was applied for 1 month. Venous blood samples were drawn before stent placement, and repeated 24 and 48 hours after the procedure. Patients were excluded if they had had recent cardiovascular events or any illness that might influence platelet, leukocyte, and endothelial cell function. The plasma level of sL-selectin was significantly decreased 48 hours after coronary stenting (636+/-110 ng/mL vs 567+/-93 ng/mL; P = 0.001, respectively). Likewise, the plasma level of sP-selectin was also decreased significantly 48 hours after the procedure (260+/-61 ng/mL vs 233+/-83 ng/mL, P = 0.01). The sE-selectin level was found to be significantly increased 24 hours (31+/-9 ng/mL vs 39+/-12 ng/mL, P = 0.0001) and 48hours(31+/-9 ng/mL vs 42+/-15 ng/mL, P = 0.001) after coronary stenting. The results of our study suggest that significant platelet and leukocyte deactivation take place in patients treated with combined antiplatelet therapy after stenting; endothelial cell activation also occurs during this treatment.

Cytokines, endothelium, and adhesive molecules in pathologic thrombopoiesis.

Clonal thrombocytosis (CT) associated with myeloproliferative disorders (MPD) is believed to be secondary to autonomous unregulated platelet production. Secondary or reactive thrombocytosis (RT) can be observed in a number of clinical circumstances and may be related to persistent production of some thrombopoietic factors acting on megakaryocytes (MK). The goal of this study is to assess the serum concentrations of these cytokines in control subjects and patients with MPD associated with thrombocythemia, RT, and autoimmune thrombocytopenic purpura (ATP). Eleven patients with MPD, five with chronic myeloid leukemia (CML), three with polycythemia vera (PCV), two with essential thrombocythemia (ET), one with myelofibrosis, 15 with RT, eight with ATP, and 12 healthy volunteers were enrolled in the study. Serum interleukin (IL)-1beta, IL-6, tumor necrosis factor-alpha (TNF), fibronectin, intracellular adhesion molecule-1 (ICAM-1), and thrombomodulin (TM) were measured in these groups. Interleukin- 1beta, IL-6, and TNF levels were high in patients with RT and ATP, suggesting that these cytokines act on early uncommitted progenitors, promoting commitment along the MK lineage and leading to thrombocytosis or compensation for thrombocytopenia. TM was significantly increased in patients with MPD compared to all other groups, probably indicating the presence of subclinical endothelial damage. Fibronectin levels were high in MPD and RT patients. This finding can be secondary to high platelet turnover in these patients. We found that ICAM-1 levels were high in patients with clonal thrombocytosis. ICAM-1 can be one of the factors initiating the events ultimately leading to clonal thrombocytosis. Thrombocythemia associated with MPD is an autonomous phenomenon not regulated by cytokines.