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.

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).

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.

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.

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.

Two common genetic thrombotic risk factors: factor V Leiden and prothrombin G20210A in adult Turkish patients with thrombosis.

The prevalence of genetic risk factors for thrombosis varies greatly in different parts of the world, both in patients with thrombosis and in the general population. Factor V Leiden (FVL) and prothrombin G20210A (PT G20210A) mutations are the most common genetic defects leading to thrombosis. We have previously reported that those two thrombotic risk alleles are frequently found in Turkish children with thrombosis. The aim of the present study was to investigate the frequency of FVL and PT G20210A and their clinical manifestations in adult Turkish patients with thrombosis. Between January 1997 and February 2000, 146 patients with documented thrombosis were investigated in our center for the presence of the FVL and PT G20210A mutations. Forty-five of 146 patients with thrombosis (30.8%) were detected to have FVL mutation. Among those cases with the FVL mutation, seven (4.8%) had homozygote and 38 (26%) had heterozygote mutation. The PT G20210A mutation was detected in 10 of the 146 patients with thrombosis (6.8%). Another six cases (4.1%) had both FVL and PT G20210A mutations. The overall frequency of these two common risk alleles in our adult population with thrombosis was 41.6%. Our findings reveal that FVL and PT G20210A mutations are significant genetic risk factors contributing to the pathophysiology of thrombosis in the Turkish population.

Cardiac and great vessel thrombosis in Behçet's disease.

Behçet's disease (BD) is a chronic relapsing systemic vasculitis in which orogenital ulceration is a prominent feature. The disease affects many systems and causes hypercoagulability. We present a 27-year-old male patient who exhibited widespread great vessel thrombosis including right atrial and ventricular thrombi in the setting of right-sided infectious endocarditis and orogenital aphthous ulcerations and erythema nodosum due to BD. We reviewed the enigmatic prothrombotic state of BD, and discuss our prior experiences in this field.

Serum L-selectin and P-selectin levels in lymphomas.

The migration of normal and malignant lymphoid cells is governed by specific adhesion molecules. Selectins comprise a family of adhesion receptors expressed by leukocytes, platelets and endothelial cells. In this study, the serum levels of soluble L-selectin and P-selectin were measured in patients with non-Hodgkin's lymphoma and Hodgkin's disease and found to be significantly elevated in both patient groups compared to healthy controls. This result provides evidence that alterations in the expression and function of adhesion molecules may play an important role in the progression of lymphomas. Further studies are awaited to establish the exact roles of these adhesion molecules in distinct patterns of growth and spread of lymphomas.

Selectin adhesion molecules in Behçet's disease.

OBJECTIVES: The pathogenesis of Behçet's disease (BD) is closely related to endothelial cells, leucocyte functions and autoimmunity. The aim of this study was to investigate circulating selectin adhesion molecules, which are known to play a significant part in the immune response especially by regulating interactions of the leucocytes with endothelium, in BD. METHODS: Plasma E-, L-, and P-selectin concentrations were evaluated in 11 patients with widespread BD (group I), 10 cases with merely mucocutaneous involvement (group II) and 15 age and sex matched healthy control subjects. The patients were newly or previously diagnosed cases not taking any drug for BD. RESULTS: Plasma concentrations of all selectins were significantly higher in group I compared with group II. E-selectin and P-selectin were significantly increased in each subgroup of patients compared with the healthy controls. L-selectin concentrations were higher than the controls only in group I. CONCLUSIONS: Increases in the selectins in BD may be a direct consequence of the leucocyte, endothelium and platelet activations observed during the disease process. However, abnormal/increased selectin expression to various triggers should also be considered. More prominent increases in patients with extensive disease suggest that circulating selectin concentrations are related to disease severity.

The role of natural anticoagulant deficiencies and factor V Leiden in the development of idiopathic portal vein thrombosis.

One of the causes of portal hypertension is portal vein thrombosis (PVT). The aim of this study was to determine whether natural anticoagulant deficiencies, activated protein C resistance (APCR), and factor V Leiden play a role in the development of PVT, leading to cavernous transformation of the portal vein (CTPV). Twenty-three patients with idiopathic CTPV (group 1) seen at Hacettepe University Hospital during the past 12 years were identified and prospectively studied. These 23 patients underwent a detailed hematological evaluation including measurement of protein S, protein C, antithrombin III, activated protein C resistance (APCR), and factor V Leiden gene mutation. Additionally, all patients were tested for anticardiolipin antibodies (ACA), IgG, IgM, and lupus anticoagulant (LA). Natural anticoagulants and APCR were measured using available commercial kits, and factor V Leiden mutation (R506Q) was detected by Mnl I digestion of an amplified factor V DNA fragment. All parameters were measured at least 6 months after the diagnosis of CTPV was established. No patient was on anticoagulant or antiaggregant treatment while tested. The findings in these 23 patients were compared with those in 20 healthy control subjects (group 2), in whom all tests mentioned above were also performed. In 23 patients (group 1), who had no recognizable factor for portal vein thrombosis, considerably natural anticoagulant deficiencies and factor V Leiden mutation positivity were found when we compare them to those healthy controls (group 2). The protein C levels of six patients (26%), the protein S levels of 10 patients (43.5%), and the antithrombin III levels of five patients (26%) were lower than in control subjects. Two patients were found to have combined protein S and antithrombin III deficiency, and one had combined protein S and C deficiency and APCR. APCR was detected in seven of the 23 patients, and six of these seven patients were found to have R506Q factor V Leiden mutations. In group 1, ACA IgG levels were higher in four patients (17%) and ACA IgM level was higher in one (4%) compared with the control group. LA was positive in only one patient in group 1. Natural anticoagulant deficiencies and factor V Leiden mutation are strongly associated with PVT. The natural anticoagulant deficiencies and APCR (almost totally caused by R506Q mutation) produce a favorable medium for thrombus generation. PVT seems to be related to the natural anticoagulant deficiencies and factor V Leiden R506Q mutation. A combination of these defects increases the incidence of PVT and these factors should be evaluated carefully in patients with idiopathic CTPV.

Paget-Schroetter syndrome associated with FV:Q506 and prothrombin 20210A--a case report.

Effort thrombosis of the axillary-subclavian vein (Paget-Schroetter syndrome) develops usually secondary to heavy arm exertion. An underlying chronic venous compressive anomaly at the thoracic outlet or intimal damage of the axillary vein following forceful hyperabduction, external rotation of the shoulder joint has been proposed to explain the pathophysiology of this thrombosis. This condition is usually not attributed to an underlying hypercoagulability such as deficiency of natural coagulation inhibitors. Here, the authors present a case with thrombosis of the axillary-subclavian vein following an effort, with factor V Leiden and prothrombin 20210A mutations. Both factor V Leiden and the genetic variant in the prothrombin gene have been shown to confer an increased risk for venous thrombosis. Although rare, effort thrombosis may develop in a patient with hereditary thrombophilia, so laboratory evaluation should include the common causes of thrombosis.

Circulating thrombomodulin, thrombospondin, and fibronectin in acute myeloblastic leukemias.

In this study, soluble thrombomodulin (TM) was measured as an indicator of endothelial injury in acute myelocytic leukemia (AML) together with fibronectin (FN) and thrombospondin (TS). The study group comprised of 17 (6 men, 11 women; aged 34 +/- 10 years; range 21-61 years) newly diagnosed AML patients. There was infection in 6 patients. Twelve (4 men, 8 women; aged 31 +/- 11 years; range 18-55 years) healthy subjects were studied as the control group. Plasma soluble TM levels were significantly higher in AML patients than in the healthy control group (116.6 +/- 13.7 vs 37.2 +/- 1.75 ng/ml, respectively (mean +/- SEM), p < 0.01). Plasma FN levels were found to be significantly higher in AML patients compared to the control group (15.9 +/- 2.69 vs 8.1 +/- 2.46 ng/ml, respectively (mean +/- SEM). p < 0.01 ). Plasma FN levels in infected patients were significantly lower than in non-infected patients (6.47 +/- 1.3 vs 21.0 +/- 3.1 ng/ml, respectively (mean +/- SD), p < 0.01). Plasma TS levels in the patient group were significantly lower than in the control group (20.6 +/- 1.45 vs 120.8 +/- 18.2 ng/ml, respectively (mean +/- SEM), p < 0.01). In one patient with M7 megakaryoblastic leukemia who also had a high platelet count, plasma TS levels were significantly higher than that in other patients.

Increased systemic coagulation activity in patients with rheumatic mitral stenosis: assessment of the clinical and echocardiographic determinants.

In this study, we aimed to determine systemic coagulation activity in patients with rheumatic mitral stenosis and to define determinants of a possible prethrombotic state. Peripheral venous plasma level of thrombin-antithrombin III complex was measured in 84 consecutive patients with rheumatic mitral stenosis who had no left atrial thrombus by transesophageal echocardiography. The patients had significantly higher thrombin-antithrombin III complex values (mean +/- SD = 9.6+/-15.9 ng/ ml) compared with the healthy subjects (2.1+/-1.8 ng/ml) (P<0.001). Among many clinical and echocardiographic variables, severe mitral regurgitation (odds ratio = 6.7, P<0.001) and left atrial spontaneous echo contrast (odds ratio = 22.8, P<0.001) appeared as significant predictors of the increased systemic coagulation activity in multivariate logistic regression analysis. In conclusion, systemic coagulation activity is increased in the patients with rheumatic mitral stenosis, and coexistence of severe mitral regurgitation and presence of left atrial spontaneous echo contrast are determinants of this increment.

Evaluation of the haemostatic system during ketoacidotic deterioration of diabetes mellitus.

Clinical observations have indicated the frequent development of thrombotic complications during diabetic ketoacidosis (DKA). This study aimed to examine whether haemostatic changes that could lead to a thrombotic tendency occur during ketoacidosis. Plasma levels of in vivo haemostatic markers reflecting activation degrees of the coagulation system, fibrinolytic system, platelets and endothelium were assayed in 34 patients with DKA, both at diagnosis and 1 week after recovery. We found coagulation system and platelet activation and endothelial injury/activation in the patients at diagnosis of DKA. Although significant improvements were observed after recovery, only platelet activity was completely normalized. Fibrinolytic activity was also increased, both at diagnosis and after recovery, compared to the control group. However, although coagulation activity was prominently increased at diagnosis compared to the recovery period, there was no change in fibrinolytic activity in the same periods; on the contrary, the fibrinolytic capacity of the endothelium was diminished at diagnosis of DKA compared to the recovery period, suggesting the presence of relative hypofibrinolysis during DKA. Indications for a role of hyperglycaemia in the emergence of haemostatic disturbances during DKA were observed.

Thrombopoietin, interleukin-6, and P-selectin at diagnosis and during post-steroid recovery period of patients with autoimmune thrombocytopenic purpura.

Plasma concentrations of the most potent megakaryocytopoietic cytokines, thrombopoietin (TPO) and interleukin-6 (IL-6), and the platelet activation marker P-selectin were evaluated in 24 patients with autoimmune thrombocytopenic purpura (ATP) who responded to conventional steroid treatment, at diagnosis and after steroid-induced recovery. Baseline TPO concentration (median [interquartile range]=0 [17.52] pg/ml) was significantly decreased and IL-6 (38 [19.75] pg/ml) and P-selectin (485 [393.75] ng/ml) were significantly elevated compared with healthy subjects (100 [68] pg/ml, 8 [7] pg/ml and 166 [69] ng/ml, respectively). Following steroid treatment, all values approached normal, i.e., TPO (20 [18.75] pg/ml) was increased and IL-6 (19.5 [13] pg/ml) and P-selectin (248 [172.5] ng/ml) were decreased, significantly. The decrease of TPO in ATP is suggested to occur due to increased megakaryocyte mass and, consequently, TPO clearance. The non-lineage-specific cytokine IL-6 may be elevated to compensate for megakaryocytopoiesis/thrombopoiesis. The elevation of P-selectin may reflect compensatory platelet hyperactivation; however, this molecule also might be a marker of platelet destruction.