Jan Stasko

Circulating vascular endothelial growth factor in the normo- and/or microalbuminuric patients with type 2 diabetes mellitus.

Relationship between serum vascular endothelial growth factor (VEGF) level and parameters of endothelial injury and/or dysfunction in patients with diabetes mellitus type 2 with or without microalbuminuria was investigated. Eighty-four diabetic patients were divided in two subgroups (42 each): normoalbuminuric (NAU) and microalbuminuric (MAU). Forty-two blood donors were in control group. Serum VEGF and plasma von Willebrand factor, soluble thrombomodulin, plasminogen activator inhibitor 1, thrombin-activatable fibrinolysis inhibitor (TAFI) and tissue plasminogen activator (t-PA) were measured using enzyme-linked immunosorbent assay in all subjects. VEGF was significantly higher in NAU compared to controls. The difference between MAU and controls was not statistically significant, but there was a trend toward significance. Only TAFI correlated with VEGF in MAU. An observed significant increase of serum VEGF level already in NAU suggests that serum VEGF could be a sensitive predictor of endothelial dysfunction in type 2 diabetes.

Recombinant Activated Factor VII in Patients at High Risk of Bleeding

Abstract Currently, recombinant activated factor VII (rFVIIa) (NovoSeven ® ) is indicated for the treatment of spontaneous and surgical bleeding in congenital haemophilia A and B patients with inhibitors to factors VIII (FVIII) and IX (FIX) >5 Bethesda units (BU) worldwide, and in patients with acquired haemophilia, congenital FVII deficiency and Glanzmanns thrombasthenia in Europe. Until April 2003, almost three-quarters of a milion doses of rFVIIa have been administered proving its efficacy and excellent safety record. According to results from initial clinical trials and a large number of case reports, the rFVIIa may be effective not only in treating haemophilia patients but also in treatment of bleeding in patients on oral anticoagulation or heparin, patients with liver diseases, von Willebrand disease (vWD), thrombocytopenia, various platelet defects, congenital or acquired deficiency of FVII, and in subjects without any pre-existing coagulopathy with diffuse life-threatening bleeding triggered by surgery or trauma. This review will briefly summarize rFVIIa mode of action in haemostasis, the current clinical experience with rFVIIa and focus on the alternative use of rFVIIa in patients at the high risk of bleeding in both spontaneous cases and clinical trials reports. † stasko@jfmed.uniba.sk

The relationship among TAFI, t-PA, PAI-1 and F1 + 2 in type 2 diabetic patients with normoalbuminuria and microalbuminuria.

Disturbances of coagulation and fibrinolysis in type 2 diabetes mellitus (DM2) contribute to increased rates of macrovascular complications such as myocardial infarction and ischemic stroke. The aim of the study was to investigate the relationship among plasminogen activator inhibitor 1 (PAI-1), thrombin-activable fibrinolysis inhibitor (TAFI), tissue plasminogen activator (t-PA), prothrombin fragments 1 + 2 (F1 + 2), glycemic control, hypertension, sex and body mass index (BMI) in DM2 patients with normoalbuminuria and microalbuminuria. Forty-two normoalbuminuric (NAU), 42 microalbuminuric (MAU) patients with DM2 and 42 blood donors as control group were enrolled. TAFI, PAI-1, t-PA and F1 + 2 were assessed by enzyme-linked immunosorbent assay (ELISA) in all patients. TAFI was significantly increased in the MAU group, PAI-1 and F1 + 2 were increased in both groups and t-PA was not elevated in either group compared to controls. We found positive correlations in the NAU: TAFI and fibrinogen (r = 0.65, P = 0.02), PAI-1 and triglycerides (r = 0.67, P = 0.01), in the MAU: TAFI and F1 + 2 (r = 0.48, P = 0.02), TAFI and systolic blood pressure (r = 0.53, P = 0.01), PAI-1 and BMI (r = 0.43, P < 0.05). We found decreased fibrinolysis in DM2 patients presented with increased PAI-1 in both NAU and MAU. Hypofibrinolysis in MAU is further accented by the elevation of TAFI. TAFI-mediated inhibition of fibrinolysis in DM2 is regulated independently from PAI-1. Patient[Combining Acute Accent]s sex does not affect diabetes-related changes in hemostasis and fibrinolysis.

Erythropoietin and granulocyte colony-stimulating factor increase plasminogen activator inhibitor-1 release in HUVEC culture

UNLABELLED Recombinant human erythropoietin (rHuEPO) is considered to be a mitogenic and chemotactic agent in cultured endothelial cells (ECs). The effect of recombinant granulocyte-macrophage (rGM-CSF) and granulocyte (rG-CSF) colony-stimulating factors on the proliferation of human umbilical vein endothelial cells (HUVECs) has been controversial. METHODS HUVEC proliferation and the release of endothelial markers in HUVEC culture stimulated by rHuEPO, rGM-CSF and G-CSF were measured. RESULTS We found the dose-dependent stimulating effect of rHuEPO and rGM-CSF on HUVEC proliferation, but we did not achieve this with rG-CSF. rHuEPO like rG-CSF was an effective agent in stimulating the plasminogen activator inhibitor (PAI)-1 release from HUVECs to a cultured medium, while rGM-CSF failed. CONCLUSION We suggest that rHuEPO showed prothrombotic changes in HUVEC culture. Our results support the idea of suspected rHuEPO direct prothrombotic role in haemodialysed patients treated with rHuEPO.

Fibronectin, plasminogen activator inhibitor type 1 (PAI-1) and uterine artery Doppler velocimetry as markers of preeclampsia

Objective: The purpose of this study was to examine plasma levels of fibronectin and plasminogen inhibitor type 1 (PAI-1), and alterations in uterine artery (UtA) waveforms throughout normotensive and preeclamptic pregnancies and to analyze its predictive value for the detection of preeclampsia within the second trimester of pregnancy. Material and methods: Blood samples were collected from 102 healthy, nulliparous women between the 24th and 26th gestational week. Preeclampsia developed in 13 patients; 89 normotensive control subjects were matched from the same cohort. Plasma samples were assayed for fibronectin and PAI-1 by enzyme-linked immunosorbent assay. Color pulsed Doppler examinations of UtA were performed after blood sampling. Trends were compared between two groups. Results: Maternal plasma fibronectin and PAI-1 levels and average PI, RI and S/D ratios of patients with preeclampsia were significantly higher (p < 0.05). The best cut-off values for predicting preeclampsia of fibronectin, PAI-1, PI, RI, S/D ratio based on ROC curve analysis were 290 mg/ml, 77.3 ng/ml, 1,0615, 0.605 and 2,59 respectively. The areas under the curve equal to 0.705, 0.753, 0.689, 0.695 and 0.699 for fibronectin, PAI-1 and uterine artery Doppler PI, RI, S/D ratio were determined for the prediction of preeclampsia. Conclusions: Fibronectin, PAI-1 and UtA Doppler are potentially useful predictors of preeclampsia. Maternal plasma PAI-1 combinated with fibronectin had the highest predictive values in our study.

Soluble P-Selectin During a Single Hemodialysis Session in Patients With Chronic Renal Failure and Erythropoietin Treatment:

In several studies, hemodialysis (HD) patients treated with recombinant human erythropoietin (rHuEPO) because of renal anemia showed increased levels of soluble adhesion molecules. The purpose of the study was to investigate the changes of soluble P-selectin (sSELP) and its relationship to platelet activation during a single HD session in patients with long-term rHuEPO treatment. Fifty-two HD patients with chronic renal failure were involved—26 with rHuEPO treatment (EPO group) and 26 without (non-EPO group). Thirty healthy subjects served as the control group. The sSELP, β-thromboglobulin, and platelet factor 4 plasma levels were measured before and after a single 4-hour HD session on a cuprophane dialyzer. The basal β-thromboglobulin and platelet factor 4 plasma levels were significantly increased in both HD groups compared with healthy controls but did not change after a single HD session, except for a significant decrease of platelet factor 4 in the non-EPO group. The predialysis sSELP plasma levels did not differ significantly compared with those of the healthy controls, but there was a significant increase of sSELP levels after a single HD session in both groups (EPO, P < .005; non-EPO, P < .05, respectively). These results suppose that the increased sSELP level was released from platelets during the course of a single HD session. The more significant increase of the sSELP plasma levels in EPO group during HD indicates that platelets are more activated in patients with long-term rHuEPO treatment, and this fact could partially explain the suspected tendency for thrombosis in these patients.

Is Gas6 Protein Associated With Sticky Platelet Syndrome

The aim of this study was to detect the prevalence of the polymorphisms of growth arrest—specific gene 6 (Gas6; Gas6 c. 834 + 7G > A) in patients with sticky platelet syndrome (SPS). Sticky platelet syndrome is a hereditary, autosomal dominant thrombophilia characterized by platelet hyperaggregation after low concentrations of platelet inducers—adenosine diphosphate (ADP) and epinephrine (EPI). The cause of SPS still remains unknown, but in recent years it was suggested that Gas6 protein may have a potential role in the pathogenesis of SPS. To assess the Gas6 polymorphisms (Gas6 c. 834 + 7G > A), 128 patients with SPS were included in the study and examined by polymerase chain reaction (PCR) method. GG genotype was detected in 63 (49.2%) patients, GA genotype in 53 (41.4%) patients, and AA genotype in 12 (9.4%) patients. The results in controls did not differ significantly compared to patients with SPS. Our findings did not prove allele A to be less associated with thrombosis and that ‘‘prothrombotic’’ allele G may be associated with higher risk of thrombosis. We cannot support the idea that Gas6 protein and Gas6 polymorphisms may be associated with thrombosis in SPS.

Endothelial and platelet markers in diabetes mellitus type 2.

Diabetes mellitus (DM) is an extremely common disorder which carries a risk of vascular impairment. DM type 2 (DM2) can be characterized by the dysfunction of haemostasis manifesting by stimulated coagulation process, disorder of platelet function and decreased fibrinolytic activity. These all are the reasons why DM2 is the most common acquired thrombophilia. Endothelial dysfunction along with platelet hyperactivity are unquestionably involved in the hyperactivation of platelets and clotting factors in DM. As a natural consequence of continuous investigation, many markers of endothelial dysfunction and diabetic thrombocytopathy have been identified and considered for implementation in clinical practice. Endothelial function can be assessed by the evaluation of endothelial markers, circulating molecules synthesised in various amounts by the endothelium. These markers precede the signs of evident microangiopathy. Platelets have an ethiopathogenic relation to the microangiopathy in DM. Their increased activity was confirmed in both types of DM. Predictors of endothelial and platelet disorder could improve the screening of individuals at increased risk, thus leading to the early diagnosis, appropriate treatment, as well as to the effective prevention of the complications of DM2. In the article we deal with the mechanisms involved in the pathogenesis of endothelial and platelet functional abnormalities, endothelial and platelet markers of DM2 considered for implementation in clinical practice and possibilities of their detection.

Platelet aggregation abnormalities in patients with fetal losses: the GP6 gene polymorphism.

OBJECTIVE To evaluate the GP6 gene polymorphism in patients with sticky platelet syndrome (SPS) and fetal loss. DESIGN Genetic association study. SETTING Perinatal center. PATIENT(S) Twenty-seven patients with SPS, manifested as fetal loss, and 42 control subjects without SPS and no history of fetal loss and thrombosis. INTERVENTION(S) SPS was diagnosed by platelet aggregometry (PACKS-4 aggregometer; Helena Laboratories). Seven single-nucleotide polymorphisms (SNPs) of the GP6 gene were evaluated. MAIN OUTCOME MEASURE(S) Occurrence of SNPs of the GP6 gene in SPS patients versus control subjects. RESULT(S) We found a higher occurrence of three SNPs of the GP6 gene in SPS patients versus control subjects (rs1671153: 0.204 vs. 0.048, odds ratio [OR] 5.116, 95% confidence interval [CI] 1.536-17.03; rs1654419: 0.204 vs. 0.071, OR 3.326, 95% CI 1.149-9.619; rs1613662: 0.204 vs. 0.071, OR 3.326, 95% CI 1.149-9.619). The haplotype analysis showed a significantly higher occurrence of two haplotypes (CTGAG in haplotype 5: 0.185 vs. 0.059, OR 3.568, 95% CI 1.142-11.14; and CGATAG in haplotype 6: 0.204 vs. 0.048, OR 4.961, 95% CI 1.488-16.53). CONCLUSION(S) Our results, especially the higher occurrence of haplotypes CTGAG and CGATAG in SPS patients, support the idea that GP6 gene polymorphism may be associated with platelet hyperaggregability, a possible cause of fetal loss.

Different models of inheritance in selected genes in patients with sticky platelet syndrome and fetal loss.

INTRODUCTION The aim of this study was to evaluate the genetic variability of selected single nucleotide polymorphisms (SNPs) within GAS6 and PEAR1 genes and explore the association between selected SNPs and risk for fetal loss in women with sticky platelet syndrome (SPS). MATERIALS AND METHODS We examined 23 female patients with SPS and history of spontaneous abortion, and 42 healthy women who served as controls. The diagnosis of SPS was established by light transmission aggregometry according to methods and criteria developed by Mammen et al. We also assessed four SNPs within the GAS6 gene (rs7400002, rs1803628, rs8191974, rs9550270) and two SNPs within PEAR1 gene (rs12041331, rs12566888). RESULTS We identified two SNPs within PEAR1 gene (rs12041331, rs12566888) and one SNP within GAS6 gene (rs9550270) that have higher occurrence in SPS patients with history of abortion. An increased risk for abortion was observed in carriers of the rs7400002 within GAS6 gene. Conversely, we found that the T allele of PEAR1 c. -9-4663G > T polymorphism appears to be protective for fetal loss. CONCLUSION Our results support the idea that genetic variability of GAS6 and PEAR1 genes may be associated with platelet hyperaggregability. The study also suggests a possible polygenic type of SPS heredity.