Fibrin Degradation Product D-dimer Research Articles

Abstract 2447: Unraveling the role of the fibrinolytic system in pancreatic cancer progression

Abstract Pancreatic ductal adenocarcinoma (PDAC) exhibits one of the poorest prognoses of all solid cancers and is associated with very low overall and progression-free survival rate. Clinically, PDAC patient tumors show high expression levels of multiple components of the coagulation system (e.g., tissue factor, etc.) and fibrinolytic system, including urokinase plasminogen activator (uPA) and receptor (uPAR). In addition, pancreatic cancer patients show elevated levels of circulating fibrinogen (Fib) and the fibrin degradation product D-dimer in plasma, with increased correlation in patients with distant metastasis. We hypothesized that targeting the plasminogen activation system components would disrupt PDAC tumor progression. Using pancreatic cancer cell lines from the KPC mouse model (K-rasLSL.G12D/+; p53LSL.R172H/+; Elas-CreER), individual components of the fibrinolytic system (uPA and uPAR) were eliminated using CRISPR-Cas9 technology. Knocking out uPA and uPAR in KPC cells resulted in significantly smaller tumors relative to Cas9 control tumors. Compared to WT mice, KPC murine pancreatic cancer cells injected orthotopically in Plg-/- mice had significantly attenuated tumor growth, suggesting the pro-tumor effect of tumor cell-derived uPA/uPAR expression was linked at least in part to plasmin(ogen) activation to enhance PDAC tumor progression. To further investigate the contribution of Plg or Fib in the microenvironment in PDAC progression, we used Plg or Fib specific antisense oligonucleotide (ASO) treatment to specifically deplete plasminogen or fibrinogen levels in mice bearing human PDAC tumors. Treatment with Plg-ASO or Fib-ASO significantly decreased tumor burden in both orthotopic and subcutaneous models bearing human tumors. Bioluminescent imaging revealed that Plg-ASO and Fib-ASO treatment also decreased the spontaneous metastatic burden. Histological analysis of lung and liver tissue from Plg-ASO-treated mice confirmed reduced metastatic burden of the primary tumor, especially to the lungs. Similarly, Fib-ASO-treated mice had significantly lower metastatic burden in both liver and lung tissues. Our data demonstrate that depleting Plg or Fib in circulation reduces tumor growth and metastasis. Collectively, our data suggests the PA system is important to both tumor growth and metastasis. Based on our findings with Plg depletion, the unexpected finding that depletion of the plasmin target fibrin(ogen) in the microenvironment also significantly reduces primary tumor growth and metastasis suggests plasmin(ogen) may function through fibrin(ogen)-independent mechanisms to promote PDAC progression. Citation Format: Nayela N. Chowdhury, Yi Yang, Yingnan Shen, Silpa Gampala, Olivia Babb, Bumsoo Han, Alisa S. Wolberg, Matthew J. Flick, Melissa L. Fishel. Unraveling the role of the fibrinolytic system in pancreatic cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2447.

Performance Evaluation of the Quantitative Point-of-Care LumiraDx D-Dimer Test.

IntroductionFibrin degradation product d-dimer can be a valuable indicator for venous thromboembolism (VTE). The use of d-dimer testing in primary care settings can be limited by restricted access to laboratory services. This performance evaluation compares a quantitative, point-of-care (POC) d-dimer assay (LumiraDx d-Dimer Test) with a reference laboratory-based d-dimer assay.MethodsPlasma samples from patients presenting to secondary care in the UK, USA, and Germany were analyzed centrally using the LumiraDx d-Dimer Test and the reference test (bioMérieux VIDAS d-Dimer Exclusion II immunoassay). Method comparison used Passing–Bablok regression analysis with pre-specified equivalence criteria of r ≥ 0.9 and slope of 0.9–1.1. The NOVEL-3 study (NCT04375982) compared equivalency of fingerstick, venous blood (VB), and plasma samples from the same patient, tested at US primary care clinics next to the patient using the POC LumiraDx d-Dimer device. Measurements obtained from fingerstick and VB samples were compared with results from plasma samples, using Deming regression. The healthy reference range was determined using plasma samples of healthy volunteers, collected by commercial suppliers in Germany and the USA, which were analyzed centrally using the LumiraDx d-Dimer Test and the reference test.ResultsThe LumiraDx d-Dimer Test demonstrated agreement with the bioMérieux VIDAS d-Dimer Exclusion II immunoassay for plasma samples (r = 0.923, slope of 1.016, n = 1767). There was good agreement between fingerstick/VB samples and plasma samples (r = 0.980–0.986, n = 93) measured using the LumiraDx d-Dimer Test. Overall error rates were 1.8%. The healthy reference range 90% percentile for d-dimer was calculated as 533 µg/l fibrinogen equivalent units (FEU).ConclusionsThe quantitative LumiraDx d-Dimer Test is easy to use and can accurately measure d-dimer levels in a range of blood sample types, including fingerstick samples, which could improve assessment of VTE cases in community and hospital near-patient settings.

Potential Benefit of Soluble Thrombomodulin in Coping with COVID-19/SARS-CoV-2-Induced Coagulopathy

Researchers should investigate the coagulopathy that accompanies Coronavirus Disease 2019 (COVID-19), as the incidence, as well as the mortality associated with coagulopathy, is higher than expected in COVID-19 patients. We read with interest the article published by Tang N et al. suggesting that elevated plasma levels of the fibrin degradation product D-dimer might be a predictive marker for mortality of patients with COVID-19 [1]. The same authors also reported that the use of heparin improved the prognosis of COVID-19 patients with severe coagulopathy and D-dimer levels >6-fold of the normal upper limit [2]. The high plasma levels of D-dimer suggest that the fibrinolytic system is not inhibited; nonetheless, thrombotic events are frequently noted in COVID-19 patients transferred to intensive care units [3]. Curiously, severe cases of COVID-19 are complicated by cytokine release syndrome in conjunction with Hemophagocytic Lymphohistiocytosis (HLH) [4], suggesting that extremely exaggerated hypercoagulability is induced in COVID-19. The pathophysiology of COVID-19-induced coagulopathy has not been fully elucidated. However, recently, postmortem examination of patients with COVID-19 who died of respiratory failure found thrombi in parallel with the deposition of the C5b-9 complex in the microvasculature, indicating terminal complement activation [5]. These pathological findings are reminiscent of the pathogenesis of Transplant-Associated Thrombotic Microangiopathy (TA-TMA) and atypical Hemolytic Uremic Syndrome (aHUS), in which activation of the complement system plays a role. Other possible factors involved in the pathogenesis of COVID-19-induced coagulopathy may be related to the tropism of Severe Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the pathogen that causes COVID-19, to vascular endothelial cells [6]. SARS-CoV-2 infects vascular endothelial cells via the Angiotensin-Converting Enzyme 2 (ACE2) receptor, leading to vascular derangement, so-called endotheliitis, which may result in coagulopathy and TMA. Thrombomodulin (TM) is a cell-surface expressed glycoprotein comprising six distinct domains, including an NH2-terminal lectinlike region, six tandem Epidermal Growth Factor (EGF)-like structures, an O-glycosylation site-rich domain, a transmembrane domain, and a cytoplasmic tail domain [7]. TM binds to thrombin via its EGF-like region and Converts Protein C (PC) to Activated PC (APC), which inhibits coagulant factors FVIIIa and FVa in the presence of protein S, thereby inhibiting further thrombin generation. Interestingly, TM has an anti-inflammatory effect; the lectin-like domain of TM binds and degrades High Mobility Group Box-1 (HMGB1) and attenuates inflammation. Additionally, after interacting with thrombin, TM converts carboxypeptidase Thrombin-Activatable Fibrinolysis Inhibitor (TAFI) to activated TAFI (TAFIa), which inactivates C3 and C5. Furthermore, the fifth domain of EGF-like region of TM (TME5) interacts with the cell-surface chemokine receptor G protein-coupled receptor protein 15 (GPR15) and then exerts cytoprotective effects on vascular endothelial cells in association with upregulation of antiapoptotic MCl-1 proteins, which is mediated by the activation of extracellular signal-regulated kinase [7]. Recombinant human soluble TM (rhTM) comprises extracellular domains of TM. A clinical trial comparing the efficacy and safety of rhTM with Unfractionated Heparin (UFH) in patients with Intravascular Coagulation (DIC) caused by hematological malignancies or infection found that a greater number of patients treated with rhTM recovered from DIC with acceptable toxicity profiles than those who received UFH. Based on these results, the use of rhTM has been approved for the treatment of DIC in Japan since 2008. The use of rhTM effectively treated many patients with DIC with various types of underlying diseases, including transplant-associated complications such as TA-TMA, hepatic sinusoidal obstruction syndrome/veno-occlusive disease and engraftment syndrome [8]. Importantly, endothliitis plays a role in the development of all of these conditions, suggesting the possible cytoprotective roles of rhTM in vascular endothelial cells. Moreover, prompt improvement of DIC caused by HLH developed in association with immune reconstitution syndrome after initiation of highly active antiretroviral therapy for acquired immunodeficiency syndrome was noted in the individual who received rhTM [9]. Thus, the use of anticoagulant rhTM with additional activities for immune systems and vascular endothelial cells could counteract severe COVID-19 with coagulopathy, although a systematic review and meta-analysis of rhTM therapy for sepsis-induced coagulopathy failed to prove the significant improvement of clinical outcomes of patients with sepsis-induced coagulopathy [10].

Injured tissues favor cancer cell implantation via fibrin deposits on scar zones

AimEvaluation of fibrin role on cancer cells implantation in injured tissues and studying the molecular mechanism of cancer cell interaction with the peritoneal damage. Material and methodsMouse colon cancer (CT26) and human mesothelial cells (HMCs) were used. CT26 cells were implanted on injured peritoneal zones. Icodextrin was used as a lubricant. For in vitro studies, fibrin clots from human plasma were used. The cell-fibrin interaction was observed by optical, electronic, and confocal microscopies. Aprotinin was used as a plasmin inhibitor. Hemostasis impact quantified by (1) the fibrin degradation product D-Dimer and PAR expression in HMCs; (2) the expression of plasminogen activator (PA) and its inhibitor (PAI-1) in cancer cells by qPCR and in supernatants through ELISA after in vitro HMC incubation with 2U of thrombin for 24 h. Results(i) Cancer cell lines were adhered and implanted into the wound area in vivo in both the incision and peeling zones of the peritoneum and on the fibrin network in vitro. (ii) Icodextrin significantly inhibited cancer nodule formation in the scar and the incision or peritoneal damaged zones after surgery. (iii) In in vitro studies, cancer cell interaction with the fibrin clot generated a lysed area, causing an increase in plasmin-dependent fibrinolysis measured by D-dimer levels in the supernatants that was inhibited by aprotinin. (iv) Aprotinin inhibited cell-fibrin interaction and invasion. (v) Thrombin upregulates PAI-1 and downregulates PA expression in HMC. ConclusionInjured tissues favor cancer cell implantation through generated fibrin. Fibrin-cancer cells adhesion can be inhibited by icodextrin.

The Effect of High Storage Temperature on the Stability and Efficacy of Lyophilized Tenecteplase.

Tenecteplase is a thrombolytic protein drug used by paramedics, emergency responders, and critical care medical personnel for the prehospital treatment of blood clotting diseases. Minimizing the time between symptom onset and the initiation of thrombolytic treatment is important for reducing mortality and improving patient outcomes. However, the structure of protein drug molecules makes them susceptible to physical and chemical degradation that could potentially result in considerable adverse effects. In locations that experience extreme temperatures, lyophilized tenecteplase transported in emergency service vehicles (ESVs) may be subjected to conditions that exceed the manufacturer's recommendations, particularly when access to the ambulance station is limited. This study evaluated the impact of heat exposure (based on temperatures experienced in an emergency vehicle during summer in a regional Australian city) on the stability and efficacy of lyophilized tenecteplase. Vials containing 50mg lyophilized tenecteplase were stored at 4.0°C (39.2°F), 35.5°C (95.9°F), or 44.9°C (112.8°F) for a continuous period of eight hours prior to reconstitution. Stability and efficacy were determined through assessment of: optical clarity and pH; analyte concentration using UV spectrometry; percent protein monomer and single chain protein using size-exclusion chromatography; and in vitro bioactivity using whole blood clot weight and fibrin degradation product (D-dimer) development. Heat treatment, particularly at 44.9°C, was found to have the greatest impact on tenecteplase solubility; the amount of protein monomer and single chain protein lost (suggesting structural vulnerability); and the capacity for clot lysis in the form of decreased D-dimer production. Meanwhile, storage at 4.0°C preserved tenecteplase stability and in vitro bioactivity. The findings indicate that, in its lyophilized form, even relatively short exposure to high temperature can negatively affect tenecteplase stability and pharmacological efficacy. It is therefore important that measures are implemented to ensure the storage temperature is kept below 30.0°C (86.0°F), as recommended by manufacturers, and that repeated refrigeration-heat cycling is avoided. This will ensure drug administration provides more replicable thrombolysis upon reaching critical care facilities.

Clinical value of blood D-dimers in lower extremity deep venous thrombosis in patients with severe combined abdominal trauma

Aim. To establish sensitivity and specificity of deep vein thrombosis detection in patients with severe combined abdominal trauma by studying cross-linked fibrin degradation products (D-dimer) using erythrocyte agglutination SimpliRED® technology. Materials and methods . We analyzed the treatment results of 105 patients with severe combined abdominal trauma who underwent surgery inLvivThoraco-AbdominalTraumaCenter (Surgical Department No. 1), Traumatology and Neurosurgery Departments of the Lviv City Emergency Hospital in 2012–2017. Results. The sensitivity and specificity of lower extremity deep venous thrombosis detection in patients with severe combined abdominal trauma was established by studying D-dimers as well as their dynamics. Mandatory inclusion criteria for the study were: negative result of the D-dimer test upon admission, absence of instrumental and clinical signs of lower extremity deep venous thrombosis. The examination was performed prior to the operation or mini-invasive methods of diagnostics (laparoscopy) and on day 3 and 7 after. The high value of the method is explained by its maximum adaptation to the conditions of urgent surgery; it does not require specific inhibitors, does not depend on blood sampling technology or platelet count and can be performed in a few minutes. A positive result, which indicated the active fibrinolysis, was evaluated within 2 min by erythrocyte agglutination in a test cell and the latter absence in a control one. The D-dimer test was positive in 78 (74.3 %) patients in the study group. The peak of positive test results was observed on the 5th day after surgery. In all the injured with lower extremity deep venous thrombosis, a strong positive test result was revealed, which was manifested by erythrocyte agglutination at the 5–20 sec and the presence of D-dimers for a long time (an average of 19.3 ± 7.1 days). In the low-risk group of deep vein thrombosis and embolism by J. Caprini, the positive test was detected in 6 (5.7 %) patients, in the moderate and high-risk groups – in 28 (26.7 %) and 44 (41.9 %), respectively. Conclusions. The method of D-dimers detection in blood by SimpliRED ® technology showed a rather high sensitivity in patients with severe combined abdominal trauma of 82.3 %, but low specificity of 32.3 %. In the absence of prophylaxis, the incidence of deep vein thrombosis in patients with severe combined abdominal trauma was 25.7 %. The method of ultrasonic compression angioscanning of the lower extremity veins verified the proximal vein thrombosis predominance – 74.1 %. The risk of thrombus fragmentation and potential pulmonary thromboembolism was detected in 13.7 % of patients.

Biphasic activation of complement and fibrinolysis during the human nasal allergic response

Biphasic activation of complement and fibrinolysis during the human nasal allergic response

Incidental finding of pathological coagulation parameters

Pathological coagulation parameters may reflect life-threatening hemorrhagic or thromboembolic diseases but may also be a laboratory result without any clinical significance, result from in vitro phenomena or preanalytical errors. This article gives an overview of potential pitfalls in coagulation diagnostics, lists the differential diagnoses of pathological coagulation parameters and describes further steps in the diagnostic approach to clarify pathological results. The focus lies on coagulation parameters that are frequently determined in routine clinical investigations, e.g. platelet count, prothrombin time, activated partial thromboplastin time (aPTT) and fibrinogen. Besides heparin, fondaparinux, danaparoid, and vitamin K antagonists, direct factor Xa inhibitors and direct thrombin inhibitors are nowadays available for therapeutic anticoagulation. This article gives an overview of the influence of anticoagulants on coagulation parameters which depends on the dose, the time of the last administration, as well as the method used for the determination of coagulation parameters. Moreover, common reasons for elevation of the fibrin degradation product D-dimer are presented. The clinical utility of D-dimer assays is limited by their poor specificity. Elevated D-dimer concentrations can be found in various diseases and also under normal physiological circumstances (e.g. in the elderly). Thus, the most useful clinical application of D-dimer is evidence of normal values to essentially rule out venous thromboembolism.

Bone marrow necrosis in a girl with Hodgkin’s disease

Bone marrow necrosis (BMN) is a rare finding in children with malignancy occurring most commonly in children with acute lymphoblastic leukemia. This article describes the first case of a girl who developed BMN during treatment for Hodgkin's disease. During the second cycle of chemotherapy, she experienced sudden profound bone pain in the lumbosacral region associated with elevated levels of lactate dehydrogenase (LDH), fibrin degradation products (D-Dimer), and alkaline phosphatase as well as pancytopenia and leukoerythroblastosis. MRI studies showed multiple confluent areas with low signal intensity and rim contrast enhancement in all vertebral bodies. Bone marrow biopsy revealed focal necrosis within hypocellular bone marrow. The patient responded quickly to symptomatic treatment with analgetics and heparin; however, elevations of LDH and D-Dimer persisted for 1.5 and 8 months, respectively. Clinicians should be aware of this rare condition to establish the diagnosis and to continue oncologic treatment as early as possible.

Assessing thrombolytic efficacy in vitro: Clot mass loss versus fibrinogen protein fragment concentration

Ultrasound (US) acts synergistically with recombinant tissue plasminogen activator (rt-PA) to accelerate thrombolysis. A correlation between clot mass loss and production of the fibrin degradation product D-dimer was investigated using an in vitro clot thrombolysis model. Fully retracted clots formed from 1.5-ml human whole blood were suspended in plasma at 37°C and treated with rt-PA, or rt-PA, Definity®, and pulsed 120-kHz US for 30 min. Clots in plasma alone served as controls. Thrombolytic efficacy was assessed as percent clot mass loss. Samples from plasma surrounding the clot and from macerated clots were analyzed for D-dimer using an enzyme linked immunosorbent serologic assay. A statistically significant enhancement in clot mass loss was observed for clots exposed to rt-PA, Definity®, and US compared to rt-PA alone. Clots treated with rt-PA exhibited a higher concentration of D-dimer both in the clots and plasma. However, clots treated with rt-PA, Definity®, and US did not exhibit an enhanced leve...

Day/Night rhythm of hemostatic factors in obstructive sleep apnea.

To investigate the hypothesis that day/night patterns of prothrombotic activity differ between patients with obstructive sleep apnea (OSA) and individuals with no OSA. Prothrombotic markers' day/night rhythms recorded over one 24-h period. General clinical research center. 38 untreated OSA patients as verified by polysomnography (apnea-hypopnea index > or = 10/h sleep) and 22 non-OSA controls. Blood samples were collected every 2 h to measure plasma levels of fibrinolysis-inhibiting plasminogen activator inhibitor (PAI)-1 and the primary fibrin degradation product D-dimer. Day/night variation in hemostasis factors was examined using a cosinor analysis. Mesor (mean) PAI-1 over the 24-h period was higher (P = 0.015), and mesor of D-dimer was lower (P = 0.001) in patients with OSA than in the non-OSA controls. These group differences stayed significant when controlling for age and gender. After further adjustment for body mass index, mean arterial pressure, and smoking, the relationship between OSA and PAI-1 became non-significant, but the relationship between OSA and D-dimer continued to be significant (P = 0.006). In the fully adjusted analysis, the amplitude (peak) for D-dimer was lower in OSA patients than in non-OSA controls (P = 0.048). The acrophase (time of the peak) for PAI-1 and D-dimer did not significantly differ between groups. The relatively higher average level of PAI-1 and lower average level of D-dimer across the 24-h in OSA patients might reflect decreased fibrinolytic capacity and fibrin degradation, respectively. The findings provide some evidence for a prothrombotic state in OSA, but were only partially independent of metabolic variables.

Concentrated Ambient Ultrafine Particle Exposure Induces Cardiac Changes in Young Healthy Volunteers

Exposure to ambient ultrafine particles has been associated with cardiopulmonary toxicity and mortality. Adverse effects specifically linked to ultrafine particles include loss of sympathovagal balance and altered hemostasis. To characterize the effects of acute exposure to ambient ultrafine particles in young healthy humans. Nineteen healthy nonsmoking male and female subjects between the ages of 18 and 35 were exposed to filtered air or to an atmosphere in which captured ultrafine (<0.16 microm) particles were concentrated by a factor of up to 20-fold over ambient levels with the use of particle concentrators fitted with size-selective outlets (ultrafine concentrated ambient particles [UFCAPs]). Subjects underwent bronchoalveolar lavage 18 hours after each exposure. Cardiovascular endpoints measured included pulmonary function, clinical chemistry, and hematological parameters, as well as heart rate variability and repolarization indices. Exposure to UFCAPs was statistically associated with an increase in frequency domain markers of heart rate variability, specifically indicative of elevated vagal input to the heart. Consistent with this finding were increases in the variance associated with the duration of the QT interval. In addition, UFCAP exposure resulted in a significant increase in blood levels of the fibrin degradation product D-dimer as well as a modest elevation in the inflammatory chemokine IL-8 recovered in the lavage fluid. These findings show mild inflammatory and prothrombic responses and are suggestive of alterations in cardiac repolarization induced by UFCAP inhalation.

Antithrombin effect on coagulation and fibrinolytic profiles after living donor liver transplantation: a pilot study

Early after liver transplantation, patients are in a hypercoagulable state because of an imbalance between coagulation and fibrinolysis because of the slow recovery of depleted anticoagulant proteins. Antithrombin (AT) is used in anticoagulant protocols to prevent thrombosis. The subjects of the present study were 17 men and eight women that underwent living donor liver transplantation. The initial 15 cases were administered AT concentrate (1500 U/day) on postoperative days (POD) 1 through 3 (AT group) and the following 10 consecutive cases were not administered AT (control). AT, thrombin-AT complex, plasmin-alpha2 plasmin inhibitor complex, thrombomodulin, fibrin degradation product D-dimer (FDP-DD) level, prothrombin time international normalized ratio, activated partial thromboplastin time, and platelet counts were measured. In the AT group, AT activity was maintained at levels >80% for 5 days after transplantation. In the control group, AT activity did not return to normal during the first 2 weeks after the operation. FDP-DD levels were significantly higher in the control group than in the AT group (P < 0.05). Six patients in the control group and three patients in the AT group required transfusions with platelet concentrate (P < 0.05). AT supplementation might reduce FDP-DD levels and prevent decreased platelet counts in the early stages after liver transplantation.

Hemostasis Activation in Thrombophilic Subjects With or Without a History of Venous Thrombosis

Thrombophilia is considered to increase the risk of venous thrombosis (VT) due to hemostasis activation. To determine the level of hemostasis activation in thrombophilic subjects with or without a history of VT, hemostasis activation markers prothrombin fragment 1 and 2 (F1+2), thrombin-antithrombin complex (TAT), and cross-linked fibrin degradation products (D-dimer) were measured in 94 subjects with (patients) and 101 subjects without a history of VT (controls). A total of 34.8% of patients and 14.8% of controls (P= .002) had at least 1 thrombophilic defect (protein C deficiency, activated protein C [APC] resistance, presence of lupus anticoagulants, or prothrombin G20210A polymorphism). The subjects were divided into 4 subgroups: patients with (TF(+) patients) and without (TF(-) patients) thrombophilia, and controls with (TF(+) controls) and without (TF(-) controls) thrombophilia. Hemostasis activation was comparable between all patients and controls (TAT: 2.1 vs 2.6 microg/L; F1+2: 1.0 vs 0.9 nmol/L; D-dimer: 36 vs 37 microg/L, respectively) and between TF(+) and TF(- ) patients. However, TF(+) controls had a significantly higher prevalence of increased hemostasis activation markers compared with TF(-) controls (TAT>4.4 microg/L, 38.4 vs 7.3%; F1+2>1.1 nmol/L, 53.8 vs 22.0%; D-dimer >78 microg/L, 30.7 vs 8.8% of subjects, respectively; all P< .05). After stratification for thrombophilic defects, hemostasis activation was associated with APC resistance in controls and with protein C deficiency in patients. To conclude, thrombophilia was associated with hemostasis activation in controls. We assumed that, in patients, the differences in hemostasis activation between subjects with or without thrombophilia were blurred due to undetermined and unidentified thrombophilic defects.

Elevation of E-Selectin Concentrations may Correlate with Potential Endothelial Dysfunction in Individuals with Hypopituitarism During Therapy with Growth Hormone

Increased mortality due to cardiovascular disease has been described in adult patients with untreated growth hormone (GH) deficiency. GH replacement therapy has been demonstrate to improve vascular reactivity and reverses early atherosclerotic changes in GH deficient adults. The objective of this study was the assessment of fibrinolytic markers, soluble adhesion molecules, inflammatory cytokines and endothelial function in hypopituitary adults with GH deficiency and with GH replacement therapy. We studied 20 GH deficient patients, 10 men and 10 women (aged, 43.4 +/- 8.4 years) under GH replacement therapy compared with a control group matched for age and body mass index, 9 men and 16 women. All subjects, patients and controls, were life-long non-smokers, normotensive and non-diabetic. The following variables were recorded: anthropometrical and body composition variables, serum concentrations of glucose, insulin and C-peptide; thrombin anti-thrombin fragments and fibrin degradation product D-dimer that were determined by an enzyme-linked-immunosorbent assay (ELISA); IGF-I by radioimmunoassay; C-reactive protein by highly sensitive immunonephelometry; E-selectine, P-selectine, soluble intercellular cell adhesion molecule-1, soluble vascular cell adhesion molecule-1, interleukin-6 and monocyte chemoattractant protein-1 by ELISA. The assessment of endothelial function in vivo was measured by Doppler. Patients with GH deficiency had higher hip/waist ratio and C-peptide and triglycerides concentrations than controls. Our results demonstrated no difference in fibrinolytic markers among patients and controls. E-selectin concentrations were higher in patients than in controls, 22.5+/-11.4 vs. 10.7+/-6.2 microg/L, p = 0.0001. P-selectin, soluble intercellular cell adhesion molecule-1, soluble vascular cell adhesion molecule-1, interleukin-6, monocyte chemoattractant protein-1 and C-reactive protein were similar in the 2 groups. Vascular reactivity and carotid intima-media thickness were also similar in patients and controls. In this study we have demonstrated in adults with GH deficiency under GH substitution elevation of E-selectin concentrations that may correlate with potential endothelial dysfunction suggesting that the protective effect of GH in these patients may be enhancing other mechanisms.

O-36: Similarities in the rate of induction of changes in hemostasis variables by a transdermal contraceptive patch (ORTHO EVRA®) vs oral contraceptives in healthy female subjects

O-36: Similarities in the rate of induction of changes in hemostasis variables by a transdermal contraceptive patch (ORTHO EVRA®) vs oral contraceptives in healthy female subjects

Peripheral fibrinolytic markers, soluble adhesion molecules, inflammatory cytokines and endothelial function in hypopituitary adults with growth hormone deficiency

Increased mortality due to cardiovascular disease has been described in adult patients with untreated GH deficiency (GHD). GH replacement has been demonstrate to improve vascular reactivity and reverse early atherosclerotic changes in adults with GHD. Assessment of fibrinolytic markers, soluble adhesion molecules, inflammatory cytokines and endothelial function in hypopituitary adults with GHD at baseline and 1 year after GH replacement therapy. We studied 10 patients with GHD (five men and five women; aged 45.6 +/- 10.4 years) at baseline and 1 year after GH replacement therapy compared with a control group (nine men and 16 women) matched for age and body mass index (BMI). All subjects, patients and controls, were life-long nonsmokers, normotensive and nondiabetic. The following variables were recorded: anthropometric and body composition variables, serum concentrations of glucose, insulin and C-peptide; thrombin anti-thrombin (TAT) fragments and fibrin degradation product D-dimer, which were determined by an enzyme-linked immunosorbent assay (ELISA); IGF-I by radioimmunoassay (RIA); C-reactive protein (CRP) by highly sensitive immunonephelometry; E-selectin, P-selectin, soluble intercellular cell adhesion molecule-1 (sICAM-1), soluble vascular cell adhesion molecule-1 (sVCAM-1), interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) by ELISA. The assessment of endothelial function in vivo was measured with a Doppler device. Patients with GHD without GH substitution had higher hip/waist ratio and body fat than controls. Insulin, C-peptide and triglyceride concentrations were also higher. Our results demonstrated no difference in fibrinogen and in TAT fragment concentrations among patients and controls. E-selectin concentrations were higher in patients than in controls (26.1 +/- 11 vs. 10.7 +/- 6.2 microg/l, P = 0.0001). P-selectin, sICAM-1, sVCAM-1, IL-6, MCP-1 and CRP were similar in the two groups. Vascular reactivity and carotid intima-media thickness (IMT) were also similar in patients and controls. After 1 year of GH treatment we found no changes in biochemical parameters, fibrinolytic markers, soluble adhesion molecules, inflammatory cytokines and endothelial function. Adults with GHD show some subtle changes in soluble adhesion molecules but our data suggest no beneficial effects of GH over these markers in relationship to endothelial function. Factors other than GH treatment, such as differences in age, degree of obesity, the presence of diabetes mellitus and arterial hypertension or tobacco consumption, could explain the observed increase in markers of vascular risk in GH-deficient patients.

Coagulation and fibrinolytic profiles and appropriate use of heparin after living‐donor liver transplantation

Heparin is widely used to reduce the incidence of vascular thrombosis after liver transplantation. Appropriate use of heparin based on changes in coagulation and fibrinolytic profiles, however, has not yet been discussed in detail. We performed living-donor liver transplantation for 128 adult patients. In this series, dalteparin (25 IU/kg/d) was administered until post-operative day (POD) 2. On POD 3, the anticoagulant drug was changed to heparin (unfractionated heparin sodium, 5000 U/d), the dose of which was changed according to the level of activated clotting time (ACT) targeted between 130 and 160 s. The plasma level of plasmin-alpha2 plasmin inhibitor complex, thrombin-antithrombin III complex (TAT), and fibrin degradation product D-dimer (FDP-DD) were monitored in the 21 patients. Predictors for heparin doses were analyzed among clinical parameters (n = 128). Four patients (3%) were complicated with thrombosis despite the above-mentioned anticoagulation protocol. Transfusion and/or relaparotomy for hemostasis were necessary for bleeding in 19 patients (15%). The TAT level markedly elevated until POD 3 and FDP-DD peaked later. The required heparin dose to maintain adequate ACT levels increased linearly until POD 8, and kept constant thereafter, which correlated with the weight of the liver graft (p = 0.01). Thus, frequent monitoring of the heparin dosage is necessary to keep the ACT level in the target range in the first post-operative week. High hemorrhage complications in our series indicate that the lower target ACT range may be preferable in the second post-operative week.

Ultrasound affects distribution of plasminogen and tissuetype plasminogen activator in whole blood clots in vitro

Ultrasound of 2 MHz frequency and 1.2 W/cm(2) acoustic intensity was applied to examine the effect of sonication on recombinant tissue-type plasminogen activator (rt-PA)-induced thrombolysis as well as on the distribution of plasminogen and t-PA within whole blood clots in vitro. Thrombolysis was evaluated quantitatively by measuring clot weight reduction and the level of fibrin degradation product D-dimer (FDP-DD) in the supernatant. Weight reduction in the group of clots treated both with ultrasound and rt-PA was 35.2% +/-6.9% which is significantly higher (p<0.0001) than in the group of clots treated with rt-PA only (19.9% +/-4.3%). FDP-DD level in the supernatants of the group treated with ultrasound and rt-PA increased sevenfold compared to the group treated with rt-PA alone, (14895 +/-2513 ng/ml vs. 2364 +/-725 ng/ml). Localization of fibrinolytic components within the clots was accomplished by using gel-entrapping technique and immunohistochemistry. Spatial distributions of t-PA and plasminogen showed clearly that ultrasound promoted the penetration of rt-PA into thrombi significantly (p<0.0001), and broadened the zone of lysis from 8.9 +/-2.6 microm to 21.2 +/-7.2 microm. We speculate that ultrasound enhances thrombolysis by affecting the distribution of rt-PA within the clot.

Hemostatic Side Effects of High-Dose Methotrexate in Childhood Acute Lymphoblastic Leukemia

The purpose of this study is to investigate the hemostatic side effects of HDMTX. Between 2001 and 2002, 20 children with acute lymphoblastic leukemia at the Dr. Sami Ulus Children's Hospital, Department of Pediatric Hematology and Oncology, treated according to the St. Jude ALL XIII protocol were eligible to this study. Methotrexate at a dose of 2 g/m2was infused over 24 hours. Coagulation screening studies included prothrombin time (PT), APTT, fibrinogen, fibrin degradation product (D-Dimer), factor II, factor V, factor VII, factor VIII, factor IX, factor X, PC, PS, AT-III determinations before HDMTX therapy (PreT), 1 day after (PostT1D), and 1 week after (PostT1W) the end of the HDMTX infusion. We found that PT and APTT were prolonged, PC, PS, and AT-III levels were decreased with a slight increase in D-Dimer 1 day after the administration of HDMTX and all of them returned to the normal levels by 7 days. In addition we found that FVII, FIX, FX were significantly decreased 1 day after therapy and normalised by 7 days.