Levels Of Anticoagulant Factors Research Articles

The influence of lower-leg injury and knee arthroscopy on natural anticoagulants and fibrinolysis

BackgroundPatients with lower-leg injuries and those undergoing knee arthroscopy are at increased risk of developing venous thromboembolism. The mechanism is unknown, including the influence of lower-leg injury and knee arthroscopy on natural anticoagulant factors and fibrinolysis. ObjectivesTo study the effect of lower-leg injury and knee arthroscopy on plasma levels of anticoagulant and fibrinolytic factors. MethodsWe applied the following 2 designs to investigate this effect: a cross-sectional study for lower-leg trauma and a before-and-after study for knee arthroscopy. Plasma samples of POT-CAST– and POT-KAST–randomized clinical trial participants (collected shortly after lower-leg trauma or before or after arthroscopy) were analyzed for clot lysis time and levels of antithrombin, tissue factor pathway inhibitor, protein C, free protein S, plasminogen, tissue plasminogen activator, plasminogen activator inhibitor 1, antiplasmin, thrombin activatable fibrinolysis inhibitor, plasmin-antiplasmin, and D-dimer. For the effect of lower-leg injury, samples of 289 patients were compared with preoperative samples of 293 arthroscopy patients, acting as controls using linear regression and adjusting for age, sex, body mass index, comorbidities, and diurnal variation. For the effect of knee arthroscopy, mean changes were calculated for 277 patients using linear mixed models adjusted for diurnal variation. Parameters other than CLT and D-dimer were measured in smaller subsets. ResultsIn lower-leg injury patients, most parameters were stable, whereas D-dimer increased. After arthroscopy, most parameters decreased (especially clot lysis time, D-dimer, plasminogen, and anticoagulant factors), whereas tissue plasminogen activator and thrombin activatable fibrinolysis inhibitor slightly increased. ConclusionIn contrast to lower-leg injury, knee arthroscopy was associated with decreased natural anticoagulant factor levels. Neither lower-leg injury nor knee arthroscopy affected in vivo fibrinolysis.

Intermediate Results of the Cornell Blood Coagulation in Cancer Study (COBLOCA-Study)

Background Venous thromboembolism (VTE) and arterial thrombosis are common complications of cancer and cancer treatment. A history of VTE in cancer patients increases the mortality risk by more than 2-fold compared to patients without prior VTE. The risk of cancer-associated thrombosis depends on the type of cancer, anti-cancer treatment and patient related factors. Some cancers have a high risk of thrombosis including lung, pancreas, gastric and brain. Several cancer-related prediction scores, such as the Khorana score or the PROTECHT score are used to predict the risk of thrombosis. Many mechanisms have been proposed through which cancer-related thrombosis may occur, however it remains difficult to predict thrombotic events in individual patients. Thus, accurate prediction of thrombotic events would be useful in the clinic to identify which patient may benefit from pharmacologic thromboprophylaxis. Therefore, we aim to study whether VTE can be predicted in individual cancer patients using hematological parameters. Methods The Cornell Blood Coagulation in Cancer (COBLOCA) study is an ongoing prospective study of cancer patients seen in a large academic medical center with a follow-up duration of 2 years and a detailed documentation of thrombotic events. We aim to enroll 600 patients with cancer (500 without VTE and 100 with prior VTE). Demographics, type and stage of cancer, treatment, thrombotic events (prior and during study enrollment) and anticoagulation treatment are collected. The levels of fibrinogen, prothrombin, coagulation factor FV, FVII, FVIII, FIX, FX, FXI, FXII, and FXIII, antithrombin, protein C, protein S, von Willebrand Factor antigen and activity, and D-dimer levels were determined in the patient plasma samples. We present an intermediate analysis on the acquired data. Results A total of 76 patients with cancer have been enrolled in the study to date. The association of prior VTE and the levels of hematological variables in patients with cancer were explored in this intermediate analysis. At baseline, 24 patients (32%) had a prior VTE. Twenty-four patients were on anticoagulants at the time of enrollment, of which 54% were on apixaban, 21% on rivaroxaban, 21% on heparin, and 4% on warfarin. Seven patients used aspirin in addition to anticoagulants, and 10 patients only used aspirin. A history of VTE was most common in patients with lung or endometrial cancer. Patients with esophageal, gastric or hematologic neoplasm did not have a history of VTE at enrollment, although 1 patient with gastric cancer developed a VTE during follow-up. From the remaining patients, 67% of patients with pancreatic cancer, 50% of patients with breast, liver and renal cancer, 36% of patients with colon cancer, and 19% with prostate cancer, previously suffered from VTE (Figure 1). At the time of this intermediate analysis, 3 VTE events were reported during the ongoing follow-up period, 1 in a patient with lung cancer, 1 in a patient with gastric cancer and 1 in a patient with duodenal cancer and history of hereditary hemorrhagic telangiectasia. Pro- and anticoagulant factor levels were compared between patients with and without a prior VTE. Von Willebrand Factor (VWF) antigen (+31%) and activity (+36%) levels were significantly increased in cancer patients with a history of VTE. The plasma level of FXI was significantly decreased in cancer patients with prior VTE. Additionally, plasma levels of fibrinogen, prothrombin, FV, FVII, FVIII, FIX, FX, FXII, FXIII, antithrombin, protein C, and protein S showed a trend towards lower levels in patients with a prior VTE, although these differences did not reach statistical significance (Figure 2). Moreover, D-dimer levels were significantly higher in cancer patients with prior VTE (551 μg/mL ± 942 μg/mL) than cancer patients without a history of VTE (332 μg/mL ± 451 μg/mL). Conclusion Both VWF antigen and activity were significantly increased in cancer patients with a history of VTE, indicating that the vessel wall plays an important role in cancer-associated thrombosis. Likewise, elevated D-dimer levels, and reduced levels of available coagulation factors in cancer patients with a history of VTE indicate that these patients might be in a more pro-coagulant state, resulting in (sub-clinical) consumption of coagulation factors and ongoing low grade intra-vascular coagulation. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Role of Arachidonic Acid and Its Metabolites in the Biological and Clinical Manifestations of Idiopathic Nephrotic Syndrome.

Studies concerning the role of arachidonic acid (AA) and its metabolites in kidney disease are scarce, and this applies in particular to idiopathic nephrotic syndrome (INS). INS is one of the most frequent glomerular diseases in childhood; it is characterized by T-lymphocyte dysfunction, alterations of pro- and anti-coagulant factor levels, and increased platelet count and aggregation, leading to thrombophilia. AA and its metabolites are involved in several biological processes. Herein, we describe the main fields where they may play a significant role, particularly as it pertains to their effects on the kidney and the mechanisms underlying INS. AA and its metabolites influence cell membrane fluidity and permeability, modulate platelet activity and coagulation, regulate lymphocyte activity and inflammation, preserve the permeability of the glomerular barrier, influence podocyte physiology, and play a role in renal fibrosis. We also provide suggestions regarding dietary measures that are able to prevent an imbalance between arachidonic acid and its parental compound linoleic acid, in order to counteract the inflammatory state which characterizes numerous kidney diseases. On this basis, studies of AA in kidney disease appear as an important field to explore, with possible relevant results at the biological, dietary, and pharmacological level, in the final perspective for AA to modulate INS clinical manifestations.

Oral Contraceptives and Venous Thromboembolism: Focus on Testing that May Enable Prediction and Assessment of the Risk

Combined oral contraceptives (COCs) induce several changes in the levels of coagulation factors. The levels of procoagulant factors are often increased, while levels of anticoagulant factors are decreased. Fibrinolysis is also affected, even if the effect seems to be more counterbalanced by opposite regulation of profibrinolytic and antifibrinolytic factors. These effects on hemostasis are more pronounced with third- or fourth-generation COC compared with second-generation COC. Venous thromboembolism (VTE) risk increases when multiple risk factors, including genetic and environmental, are present simultaneously. COC use causes changes in coagulation that modify the prothrombotic state induced by preexisting hemostatic alterations in a supra-additive manner. Therefore, testing appears to be of importance not only before implementing COC but also to monitor any potential thrombogenicity induced by COC therapy. Inherited genetic factors, such as factor V Leiden, G20210A prothrombin mutation, antithrombin, protein C or protein S deficiencies, non-O blood group, as well as CYP2C9*2 and the rs4379368 mutations, have all been identified as genetic predictive risk factors of VTE in women. Nevertheless, the screening of these genetic biomarkers is not capable of assessing the phenotypic expression of the risk. This review will focus on the different options for screening the thrombogenic status in this population. Specific attention will be given to the endogenous thrombin potential-based activated protein C resistance, a test aiming at assessing the thrombogenicity induced by hormonal therapies and inherited or acquired thrombophilia.

Coagulation Factor Levels and Underlying Thrombin Generation Patterns in Adult Extracorporeal Membrane Oxygenation Patients.

There is a paucity of data on the underlying procoagulant-anticoagulant balance during extracorporeal membrane oxygenation (ECMO). We hypothesized that adult ECMO patients would have an imbalance between procoagulant and anticoagulant factors, leading to an abnormal underlying thrombin generation (TG) pattern. Twenty adult venoarterial (VA) ECMO patients had procoagulant and anticoagulant factor levels measured temporally on ECMO day 1 or 2, day 3, and day 5. In heparin-neutralized plasma, underlying TG patterns, and sensitivity to activated protein C were assessed using calibrated automated thrombogram. TG parameters including lag time, peak TG, and endogenous thrombin potential (ETP) were compared against 5 normal plasma controls (3 males and 2 females) obtained from a commercial supplier. Thrombomodulin (TM) was added to some samples to evaluate for activated protein C resistance. Procoagulant factors (factor [F] II, FV, and FX) were mostly in normal reference ranges and gradually increased during the first 5 ECMO days (P = .022, <.001, <.001). FVIII levels were elevated at all time points and did not change (P = .766). In contrast, FXI was in the low-normal range but did not increase during ECMO (P = .093). Antithrombin (AT) and protein C levels were below normal but increased during the first 5 ECMO days (P = .002 and P = .014). Heparinase-treated samples showed prolonged lag time, increased peak TG, and increased ETP compared to controls; mean difference in lag time on ECMO day 1 or 2 = 6.0 minutes (99% confidence interval [CI], 2.8-9.2), peak TG = 193.4 (99% CI, 122.5-264.3), and ETP = 1170.4 (99% CI, 723.2-1617.6). After in vitro TM treatment, differences in TG parameters were accentuated and ECMO samples appeared insensitive to TM treatment; mean difference in lag time on ECMO day 1 or 2 = 9.3 minutes (99% CI, 6.2-12.4), peak TG = 233.0 (99% CI, 140.9-325.1), and ETP = 1322.5 (99% CI, 764.8-1880.2). Similar differences in TG parameters were observed on ECMO days 3 and 5. Contact activation occurs during ECMO, but procoagulant factor levels are generally preserved. Although heparin-neutralized TG is delayed, peak TG and ETP are supranormal in the setting of high FVIII and low AT and protein C levels. Resistance to TM is also apparent. These changes demonstrate a possible mechanism for hypercoagulability during adult VA ECMO.

Silica nanoparticles trigger the vascular endothelial dysfunction and prethrombotic state via miR-451 directly regulating the IL6R signaling pathway

BackgroundSafety evaluation is a prerequisite for nanomaterials in a wide range of fields, including chemical industries, medicine or food sciences. Previously, we had demonstrated that SiNPs could trigger the thrombotic effects in vivo, but the underlying mechanisms remain unknown. This study was aimed to explore and verify the role of miR-451a on SiNPs-induced vascular endothelial dysfunction and pre-thrombotic state.ResultsThe color doppler ultrasound results showed that SiNPs had the inhibitory effects on aorta velocity and cardiac output. The histological and ultrastructural analysis manifested that SiNPs could induce the vascular endothelial damage. In addition, the expression level of MDA was elevated while the activity of SOD and GSH-Px were decreased in aortic arch triggered by SiNPs, accompanied with the release of iNOS and decline of eNOS in blood serum. The immunohistochemistry results showed that the positive staining of TF and PECAM-1 were elevated in a dose-dependent manner induced by SiNPs. The activation of coagulation function occurred via shortened TT, PT and APTT while the FIB was elevated markedly induced by SiNPs. Coagulant factors (TF, FXa and vWF) and PLT numbers were increased whereas the levels of anticoagulant factors (ATIII, TFPI and t-PA) were decreased. Microarray analysis showed that the down-regulated miR-451a could target the gene expression of IL6R, which further activated the JAK/STAT signaling pathway triggered by SiNPs. Dual-luciferase reporter gene assay confirmed the directly target relationship between miR-451a and IL6R. Additionally, the chemical mimics of miR-451a led to attenuate the expression of IL6R/STAT/TF signaling pathway in vitro and in vivo induced by SiNPs, while the inhibitor of miR-451a enhanced the activation of IL6R/STAT/TF signaling pathway.ConclusionsIn summary, SiNPs could accelerate the vascular endothelial dysfunction and prethrombotic state via miR-451a negative regulating the IL6R/STAT/TF signaling pathway.

Reversal of hypercoagulability in patients with HCV-related cirrhosis after treatment with direct-acting antivirals.

The long-term impact of sustained virological response (SVR) after direct-acting antivirals (DAAs) on the hypercoagulability associated with HCV cirrhosis is unknown. We longitudinally evaluated the effect of DAAs treatment on cirrhotic coagulopathy. Pro- and anticoagulant factor levels and thrombin generation were assessed in patients with HCV-related cirrhosis at baseline, end of therapy (EOT), at 12, 24 and 48weeks (W) after EOT. Fifty-eight patients were enrolled (86% Child's A). SVR was 100%. Median factor VIII activity significantly decreased at EOT, 12weeks and 24weeks compared with baseline, whereas protein C significantly increased at 24weeks and 48weeks. Cirrhotic patients showed a slight but sustained increase in endogenous thrombin potential (ETP) with a statistically significant difference at EOT, 12weeks, 24weeks and 48weeks compared with baseline. Conversely, thrombomodulin-modified ETP was elevated before treatment and decreased over time to normal levels at 24weeks and 48weeks. The ETP ratio decreased slowly at EOT and 12weeks, and was significantly decreased at 24weeks and 48weeks compared with baseline (P<.001 for both comparisons), being not statistically different from ETP ratio measured in healthy controls. Child's B patients showed a significantly higher ETP ratio compared to Child's A at baseline and did not show any significant improvement in ETP ratio through 12weeks. Two Child's B patients developed PVT with an incidence rate of 1.1% p-yrs (95%CI, 0.18 to 3.58). DAAs therapy in HCV-related cirrhotic patients is associated with significant changes in thrombin generation suggesting a reversal of hypercoagulability particularly in Child's A patients.

Rebalanced Thrombin Generation in Liver Cirrhosis Calls for Tailored Transfusion Protocols

IntroductionMost coagulation factors are produced by the liver, and therefore in liver cirrhosis both pro- and anticoagulant factor levels are reduced. Cirrhosis patients show no clear systemic bleeding or thrombotic phenotype and thrombin generation (TG) is (almost) unaffected, therefore it has previously been proposed that coagulation is rebalanced in cirrhosis because both pro- and anticoagulant processes are affected to a similar extent. Nevertheless, clinical parameters such as the prothrombin time indicate a bleeding risk in cirrhosis patients, and based on these tests patients often receive blood product transfusion during surgery.In this study we investigated thrombin generation and its main underlying pro- and anticoagulant processes (prothrombin conversion and thrombin inactivation) to provide empirical evidence for the hypothesis of rebalanced thrombin generation in liver cirrhosis. In addition, we used in silico experimentation to study the consequences of rebalanced TG for current transfusion practices.Methods25 cirrhosis patients and 25 healthy subjects were enrolled in our study. The group of cirrhosis patients consisted of 22 Child-Pugh A and 3 Child-Pugh B patients. Prothrombin, antithrombin (AT) and α2Macroglobulin (α2M) levels were determined and TG was measured at 5 pM tissue factor by calibrated automated thrombinography. Prothrombin conversion and thrombin inactivation were quantified from each TG curve, and thrombin-antithrombin and thrombin-α2Macroglobulin complex formation was determined.The effect of transfusion of prothrombin complex concentrate (PCC) was simulated by an in silico increase of prothrombin conversion. The increase of prothrombin conversion was simulated by substituting the prothrombin conversion curve of a cirrhosis patient by the average healthy subject prothrombin conversion curve, to normalize prothrombin conversion in patients. Thrombin generation curves were calculated based on this normalized prothrombin conversion curve and the original thrombin inactivation parameters of each cirrhosis patient.ResultsProthrombin and antithrombin are significantly decreased in cirrhosis patients (74 % and 70 %, p<0.001), whereas the α2M level is significantly increased (p<0.001). Thrombin generation in healthy subjects and patients is similar (figure), although prothrombin conversion (65 %, p<0.001) and thrombin inactivation (73 % p<0.001) are markedly reduced in cirrhosis patients. Acquired AT deficiency results in a reduction of thrombin inactivation by AT (64 %, p<0.001), but the substitution of AT by α2M leads to increased thrombin inactivation by α2M (220 %, p<0.001).The effect of prothrombin complex concentrate transfusion on thrombin generation in cirrhosis patients was determined by computational modeling (figure). In silico normalization of prothrombin conversion, as would be achieved by PCC transfusion, causes TG to rise to levels that have been shown to be highly prothrombotic (ETP 2878 ± 1323 nM∙min, p<0.001).ConclusionsDespite large differences in prothrombin conversion and thrombin inactivation, TG in liver cirrhosis patients remains within the normal range (rebalanced), in contrast to the prothrombin time that predicts a bleeding risk in these patients. The transfusion of PCC based on a prolonged prothrombin time would result in a substantial elevation of thrombin generation. This indicates that standard transfusion protocols need to be tailored to the specific needs of cirrhosis patients. [Display omitted] DisclosuresKremers:Synapse bv: Employment. De Laat:Synapse bv: Employment. Wagenvoord:Synapse bv: Employment. Hemker:Synapse bv: Employment.

Procoagulant Phospholipid Activity, Whole Blood Thromboelastography and Thrombin Generation Assay to Detect Hypercoagulability in Thalassemic Children

INTRODUCTIONLife expectancy of thalassemia patients has markedly increased over the last few decades. Nevertheless patients suffer from many complications of this congenital chronic disease.The presence of a highincidence of thrombotic events has led to the identification of a hypercoagulable state in these patients.The thrombotic risk is higher in β-thalassemia intermedia and splenectomized patients. The mechanisms responsible for the increased thrombotic risk are still unclear. Several factors that contribute to the hypercoagulable state in patients with thalassemia have been identified: chronic platelet activation, abnormal red blood cells, microparticles,iron overload, endothelial damage, splenectomy, decreased levels of anticoagulant factors and presence of prothrombotic mutations. We aimed to assess hypercoagulability in children with β-thalassemia. DESIGN AND METHODSSixty eight thalassemia major and 42 thalassemia intermedia patients included our study. The control group consisted of 41 age and sex matched healthy children. Demographic data of patients were recorded from their medical records. None of the thalassemic patients have thrombosis before. To evaluate the relative role of microparticles, blood cells and plasma: coagulation tests (prothrombin time, activated prothrombin time, fibrinogen and d-dimer), serum coagulation factor levels (factor II, V, VII, VIII, IX, X, von willebrand factor, protein C, protein S, antithrombin III), procoagulant phospholipid activity and thrombin generation assay were studied from plasma, thromboelastography from whole blood.The main component of microparticles are negative anionic phospholipids.Procoagulant phospholipid activity is a functional analyse to detect microparticles.Thromboelastography measures indices of the viscoelastic properties of whole blood after activation of coagulation and the thrombin generation assay measures the actual thrombin concentrations before and after the clot is formed. RESULTSThe median age is 144 months ( 11-236 months)in thalassemia major and 142 months (72-202 months) in thalassemia intermedia patients. Plasma factor II, factor V, factor IX, factor X and protein C levels were significantly lower in thalassemia major and intermedia patients than control subjects. Plasma phospholipid activity and whole blood thromboelastography parameters were all consistent with hypercoagulability in thalassemic patients, especially in splenectomized patients. Endogenous thrombin potential (area under the curve in thrombin generation assay) was significantly lower in thalassemic patients than control subjects and in non-splenectomized patients than splenectomized patients contrary to expectations. CONCLUSIONSThe hypercoagulability in thalassemic patients especially in splenectomized patients can be determined with procoagulant microparticle activity and whole blood thromboelastography but not with thrombin generation assay in platelet poor plasma. These findings showthat blood cells and/or platelets may be more important determinants of thrombotic risk rather than plasma abnormalities in thalassemic patients. DisclosuresNo relevant conflicts of interest to declare.

High levels of procoagulant factors mediate the association between free thyroxine and the risk of venous thrombosis: the MEGA study

Thyroid hormone affects the coagulation system, but its effect on clinical disease is not clear. We determined the associations of levels of free thyroxine (FT4), thyroid-stimulating hormone (TSH) and anti-thyroid peroxidase antibodies (antiTPO) with levels of coagulation factors and the risk of venous thrombosis. In a large population based case-control study (Multiple Environmental and Genetic Assessment of risk factors for venous thrombosis study) on the etiology of venous thrombosis, we determined the levels of FT4, TSH, antiTPO, factor FII, FVII, FVIII, FIX, FX, von Willebrand factor (VWF), antithrombin, protein C, protein S and fibrinogen in 2177 cases and 2826 controls. High levels of FT4 were associated with increased concentrations of procoagulant factors, and not with levels of anticoagulant factors. High levels of FT4 were also associated with the risk of venous thrombosis, up to an odds ratio (OR) of 2.2 (95% confidence interval [CI] 1.0-4.6) for levels above 24.4 pm relative to FT4 levels between 15.5 and 18.9 pm. In 11 cases and one control, clinical hyperthyroidism had been diagnosed within a year of the thrombotic event, leading to an OR of 17.0 (95% CI 2.2-133.0) for thrombosis. The ORs approached unity after adjustment for FVIII and VWF, which suggests that the effect was mediated by these factors. Low TSH levels were also, but less evidently, associated with thrombosis, whereas there was no association between antiTPO and venous thrombosis risk. High levels of FT4 increase the concentrations of the procoagulant proteins FVIII, FIX, fibrinogen, and VWF, and by this mechanism increase the risk of venous thrombosis.

Modeling thrombin generation: plasma composition based approach

Thrombin has multiple functions in blood coagulation and its regulation is central to maintaining the balance between hemorrhage and thrombosis. Empirical and computational methods that capture thrombin generation can provide advancements to current clinical screening of the hemostatic balance at the level of the individual. In any individual, procoagulant and anticoagulant factor levels together act to generate a unique coagulation phenotype (net balance) that is reflective of the sum of its developmental, environmental, genetic, nutritional and pharmacological influences. Defining such thrombin phenotypes may provide a means to track disease progression pre-crisis. In this review we briefly describe thrombin function, methods for assessing thrombin dynamics as a phenotypic marker, computationally derived thrombin phenotypes versus determined clinical phenotypes, the boundaries of normal range thrombin generation using plasma composition based approaches and the feasibility of these approaches for predicting risk.

Acute arterial thrombosis following chemotherapy in a patient with a gastric carcinoma : case report - online article

The pathogenesis of in situ thrombosis in cancer patients is not well known. Possible factors include endothelial damage, decreasing levels of anticoagulant factors and increasing levels of pro-coagulants. In the literature, the incidence of arterial thrombosis in cancer patients is reported to be 3.8%; 5-fluorouracil is mentioned as a rare causative agent, whereas cisplatin is thought to be the most common agent responsible for in situ thrombosis. In this report we present a 43-year-old male patient with bilateral popliteal artery embolism after 5-fluorouracil/cisplatin/taxotare combination chemotheraphy for gastric carcinoma. He had no additional risk factors such as smoking or any persistent organic arterial disease. He had sinus cardiac rhythm on electrocardiography and there were no abnormalities on echocardiography that could have been source of emboli. Surgical thrombectomy was performed with effective anticoagulation. After the operation, our medical oncologist discontinued 5-fluorouracil. At follow up, there was no evidence of thrombosis, with normal vascular flow rate.

Towards An Improvement of the Coagulation Imbalance in Sickle Cell Disease After Hematopoietic Stem Cell Transplantation: A Preliminary Study

Abstract 4840 BackgroundSeveral components of the clotting system are modified towards hypercoagulability in sickle cell disease (SCD). To date, bone marrow transplantation is the only validated curative treatment of SCD. To our knowledge, whether hypercoagulable state reverse to normal following Hematopoietic Stem Cell Transplantation (HSCT) has never been explored. ObjectiveTo study changes in pro- and anticoagulant factors, the global haemostatic potential and microparticle (MP) levels following HSCT in SCD children. Patients and MethodsFour children with severe SCD were explored before and 3 months after HSCT. They were 2 females (6 and 11 years old) and 2 males (6 and 12 years old). Platelet-poor plasma was prepared from 0.109 M citrated blood after a double centrifugation. Before HSCT, patients were suffering from cerebrovascular disease (2), recurrent acute chest syndrome (2) and frequent vaso-occlusive crises (3).Determination of pro- and anticoagulant factor levels were performed using routine assays on the STAR ® automate. The global haemostatic potential was explored using the Calibrated Automated Thrombogram® method of thrombin generation. Thrombin generation was triggered using 5pM tissue factor and 4μM synthetic phospholipids (PL) with and without 6nM thrombomodulin (TM). Additional experiments were performed using 5pM tissue factor alone. Procoagulant activity of MPs was determined using a capture-based method. Total annexin V positive (A5 MP), red blood cells (RBC MP) and platelets (PLT MP) microparticle levels were measured using a FascCanto II flow cytometer. Calibration was performed using Megamix® beads. RBC MPs and PLT MPs were detected using labeled antibodies directed against glycophorin A and CD42 respectively. ResultsResults of pro-and anticoagulant factor levels are presented in the figure. Those of D-Dimer levels, thrombin generation and microparticle levels are presented in the table.Three months after HSCT, levels of procoagulant factors VII, V, X, and II, protein C activity, free protein S and antithrombin were higher whereas D-dimers, A5 MP, RBC MP and PLT MP levels were lower in all patients as compared to before HSCT. For 3 of them, endogenous thrombin potential (ETP) and peak height were lower in the presence of thrombomodulin as well as factor VIII levels, and the procoagulant activity of MPs. Reduction of ETP in the presence of thrombomodulin was higher after HSCT.When thrombin generation was triggered without addition of exogenous phospholipids, the peak was lower in all patients whereas ETP was reduced in 2 patients.Table:D-Dimer levels, thrombin generation parameters and microvesicle levels in 4 SCD children before and 3 months after successful allogeneic hematopoietic stem cell transplantationParametersBefore HSCT3 months after HSCTD-Dimers (μg/ml)0.72 (0.52-0.91)0.29 (0.15-0.46)ETP with TM (nM.min)183 (95-271)97 (59-204)Peak with TM (nM)50.8 (25.2-76.4)30.8 (14.5-63.4)Reduction of ETP with TM (%)75.6 (62.6-88.5)85.4 (74.7-91.7)ETP without PL780.5 (714.5-846.5)681 (566.3-862.5)Peak without PL81.4 (71.5-91.3)51.6 (43.5-69.9)Procoagulant MP (nM)12.1 (8.4-46.6)6.5 (5-9.7)Total A5 MP (events)1912 (476-4678)903 (340-1273)RBC MP (events)408 (216-2316)165 (136-202)PLT MP (events)1515 (847-3823)837 (449-1062)Data are expressed as median (range). [Display omitted] ConclusionAlthough the number of patients included is too small to allow statistical analyses, the results of this preliminary study showed that there is a trend toward the resolution of the hypercoagulable state of SCD 3 months after successful allogeneic HSCT. Indeed, after this procedure, we observed a trend toward the reduction of the consumptive coagulopathy as shown by higher levels of pro- and anticoagulant factors and lower D-dimers levels. We also observed a trend toward an improvement of the coagulation imbalance as shown by lower thrombin generation, higher response to thrombomodulin and reduced levels of procoagulant MP deriving both from red blood cells and platelets. These findings need to be confirmed with more cases and longer follow-up especially when patients are no more under Ciclosporin A. Disclosures:No relevant conflicts of interest to declare.

L-Asparaginase and the effect of age on coagulation and fibrinolysis in childhood acute lymphoblastic leukemia

Alterations in haemostasis are frequently observed in children with acute lymphoblastic leukemia (ALL). It was the objective of this study to analyse age-related disturbances in coagulation and fibrinolysis parameters during the induction phase of the antileukemic treatment. Sixty-four children were classified by age into three groups (1-5, 6-10, 11-16 years), and studied during induction treatment of ALL including four weeks of dexamethasone, followed by two weeks tapering of dexamethasone during which 6,000 IU/m(2) native L-Asparaginase (total 4 doses) was administered intravenously twice weekly. Blood samples were collected immediately before each L-Asparaginase infusion to analyze procoagulant (fibrinogen, factor [F] II, FV, FVII, F IX, F X) and anticoagulant factors (antithrombin [AT], protein C, protein S), parameters of thrombin generation (F1+2, TAT) and fibrinolysis (alpha2-antiplasmin, plasminogen, PAP, D-dimer). Children were in a hypercoagulable state after four weeks of dexamethasone due to upregulation of coagulation parameters. Upregulation was highest in the two youngest age groups. During L-Asparaginase treatment the 11- to 16-year-olds showed lower values in procoagulant and, even more, in anticoagulant factor levels compared to the younger children. Activation markers of thrombin generation and fibrinolysis did not change over time during the study period. Decreased synthesis of alpha2-antiplasmin and plasminogen during L-Asparaginase treatment resulted in less potential of clot lysis by plasmin in children older than 11 years of age. In conclusion, a more severe decline of anticoagulant and fibrinolytic parameters in children between 11 and 16 years of age underline that these children are at higher risk of thrombosis during ALL induction treatment.

Age-Related Changes in Hemostasis in Children with Acute Lymphoblastic Leukemia; Influences of Peg-Asparaginase Upfront and Native E Coli Asparaginase during Remission Induction Therapy.

Alterations in hemostasis are frequently observed in children with ALL. Although increased thrombin generation is already present at diagnosis, thromboembolism is reported only after initiation of antileukemic therapy and mainly in children older than 10 years, indicating a relationship between disease, age and antileukemic treatment. During their development children will undergo physiological changes in hemostasis. These age-related changes might be related to the risk for symptomatic thrombo-embolic disease. The current report presents the results of a prospective study on coagulation and fibrinolysis parameters in 121 children with newly diagnosed ALL. The DCOG (Dutch Childhood Oncology Group) ALL-9 remission induction protocol consists of weekly vincristine injections and oral dexamethasone 6 mg/m2 during 4 weeks. From day 29, the dexamethasone dose is tapered and E. coli Asparaginase Paronal® is introduced, twice weekly at 6000 IU/m2 iv for 2 weeks. 64 children were studied according to this protocol, 57 children additionally received 1000 IU/m2 PEG-Asparaginase iv before the start of remission induction chemotherapy. Blood samples were collected at diagnosis (day -5) and 5 days after the PEG-Asparaginase administration at the start of regular induction chemotherapy (day 0), and immediately before each Paronal® infusion to analyze procoagulant (fibrinogen, F II, F V, F VII, F IX, F X) and anticoagulant factors (AT, prot C, prot S) and parameters of thrombin generation (F1+2, TAT) and fibrinolysis (a2-antiplasmin, plasminogen, PAP, D-dimer). At diagnosis a situation of enhanced thrombin generation was found (elevated levels of F1+2 and TAT) and signs of consumption coagulopathy (increased D-dimer together with hypofibrinogenemia). The effect of one dose of 1000 IU/m2 PEG-Asparaginase resulted five days later in a decrease of procoagulant and anticoagulant proteins; thrombin generation decreased, fibrinolysis did not increase further. 1000 IU/m2 PEG-Asparaginase induced an asparagine depletion lasting 4 weeks. The asparagine depletion did not result in a persistent inhibition of coagulation protein synthesis as all coagulation parameters at day 29 and at day 43 did not differ between patients who did or did not receive PEG-Asparaginase window-treatment. If we divided the children in three age groups (1–5, 6–10 and 11–16 years) the 11–16 year old children showed a greater decline in procoagulant and in anticoagulant factor levels compared to the younger children during Paronal® treatment. Also the fibrinolytic potential (a2-antiplasmin and plasminogen) was significantly more decreased in children older than 11 years during Paronal® treatment. Activation markers concerning thrombin generation and fibrinolysis did not change along time after day 29. Conclusion: Age-related changes in hemostasis underline the increased risk of thrombosis during ALL induction treatment for children between 11 and 16 years of age compared to younger children.

The plasma hemostatic proteome: thrombin generation in healthy individuals.

The range of plasma concentrations of hemostatic analytes in the population is wide. In this study these components of blood coagulation phenotype are integrated in an attempt to predict clinical risk. We modeled tissue factor (TF)-induced thrombin generation in the control population (N = 473) from the Leiden Thrombophilia Study utilizing a numerical simulation model. Hypothetical thrombin generation curves were established by modeling pro- and anticoagulant factor levels for each individual. These curves were evaluated using parameters which describe the initiation, propagation and termination phases of thrombin generation, i.e. time to 10 nm thrombin (approximate clot time), total thrombin and the maximum rates and levels of thrombin generated. The time to 10 nm thrombin varied over a 3-fold range (2.9-9.5 min), maximum levels varied over a approximately 4-fold range (200-800 nm), maximum rates varied approximately 4.8-fold (90-435 nm min(-1)) and total thrombin varied approximately 4.5-fold (39-177 microm s(-1)) within this control population. Thrombin generation curves, defined by the clotting factor concentrations, were distinguished by sex, age, alcohol consumption, body mass index (BMI) and oral contraceptive (OC) use (OC > sex > BMI > age). Our results show that the capacity for thrombin generation in response to a TF challenge may represent a method to identify an individual's propensity for developing thrombosis.

Alterations in the Coagulation Profile in Renal Pig-to-Monkey Xenotransplantation

Five monkey recipients of a porcine renal xenograft were studied to determine the relationship between fibrin formation in acute humoral xenograft rejection (AHXR) and procoagulant and anticoagulant factor levels to establish whether changes in coagulation parameters could be used to predict AHXR and determine whether AHXR is associated with overt disseminated intravascular coagulopathy (DIC) in this model.Variable degrees of compensated consumptive coagulopathy were observed in each primate. Elevated thrombin-antithrombin (TAT), F1+2 and D-dimer levels consistent with thrombin generation and fibrin formation were recorded. There was no consumption of the main clotting inhibitors (including antithrombin) or a progressive, severe drop in fibrinogen levels and platelet counts, although grafts were left in situ. After transplantation, D-dimer levels remained persistently high, so they were of limited value in defining this coagulopathy. At post mortem, no cases of multiorgan involvement typical of overt DIC were observed. The lack of a rapid postoperative recovery of clotting inhibitor levels after transplantation was invariably associated with early poor outcome.This study shows that AHXR is associated with various degrees of compensated consumptive coagulopathy in our pig-to-primate model. No clear relationship was found between coagulation parameter levels and graft outcome.

Procoagulant and anticoagulant factor abnormalities following the fontan procedure: increased factor VIII may predispose to thrombosis

Objective Using age-matched controls, this study prospectively evaluated coagulation factor abnormalities and hemodynamic variables in children who had undergone the Fontan operation. Methods Coagulation factors were assayed in 20 children (mean age 6.4 ± 2.9 years), at a mean 3.7 ± 2.3 years after the Fontan procedure; 24 healthy children (mean age 6.8 ± 2.8 years) were assayed as controls. Concentration of factors II, V, VII, VIII, IX, X; ATIII; plasminogen; proteins C and S; fibrinogen; serum albumin; and liver enzymes were measured. Normal reference intervals based on the control patients were determined using 95% confidence limits. Patient demographic, hemodynamic variables, and elapsed time after the Fontan procedure were evaluated as possible predictors of coagulation abnormalities. Results Concentrations of protein C; factors II, V, VII, X; plasminogen; and ATIII were significantly lower in Fontan patients compared with age-matched controls ( P < .01); factor VIII was significantly elevated in 6 patients (35%), 2 of whom had a thromboembolic event. A higher superior vena cava pressure was predictive of an elevated factor VIII level ( P = .003). No other specific hemodynamic variables were predictive of a procoagulant or anticoagulant abnormality. Conclusion Procoagulant and anticoagulant factor levels were significantly lower in patients after the Fontan operation independent of hemodynamic variables peculiar to the Fontan circulation. Increased factor VIII level requires further evaluation as a cause of thrombosis in patients with Fontan physiology and may also indicate a subset of these patients in whom anticoagulation is indicated.

Decrease in anticoagulant factors in patients with prostate cancer treated with diethylstilbestrol diphosphate

Estrogen has been highly evaluated as one of the most potent endocrine agents for the treatment of prostate cancer. Unfortunately, a high risk of cardiovascular complications is a clinically important adverse effect of estrogen therapy, and occasionally the complications are fatal. In recent years a high incidence (55%) of thrombotic events has been reported in patients with congenital protein S (PS) deficiency. The aim of this study is to determine the relationship between cardiovascular complications of estrogen therapy and anticoagulant factor levels in the serum of patients with prostate cancer. Study 1 employed 99 patients with prostate cancer: 39 were untreated, 25 were treated with LH-RH agonist therapy alone, and 35 were treated with oral diethylstilbestrol diphosphate (DESdP) 300 mg per day. We measured the serum levels of anticoagulant factors, parameters antithrombin III (ATIII), protein C (PC), PS, coagulant and fibrinolytic factors in all patients. In study 2, the adverse effects of DESdP therapy on the serum levels of anticoagulant factors were examined in 8 patients with advanced prostate cancer. In study 1, the ATIII and PS levels of the patients treated with estrogen therapy were significantly lower than those in either the untreated patients or the patients treated with an LHRH agonist alone. Especially, both PS antigen (51.5 +/- 16.0%) and PS activity (42.9 +/- 16.0%) were markedly lower in estrogen-treated patients than in the untreated patients (102 +/- 20.8%, 100.6 +/- 20.7%, respectively) or the patients treated with an LH-RH agonist alone (97.9 +/- 16.8%, 91.5 +/- 17.7%, respectively, both p < 0.0001). PS was decreased to below the normal lower limit of normal in 82% (24/35) of the patients on estrogen therapy. In study 2, all 8 cases showed a significant decrease in PS after DESdP therapy. Our results showed that the PS levels in the oral DESdP group were almost the same as in patients with congenital PS deficiency. We conclude that decreased PS may play a role in the development of cardiovascular complications in prostate cancer patients on estrogen therapy.

Inherited Thrombophilic Risk Factors and Venous Thromboembolism: Distinct Role in Peripheral Deep Venous Thrombosis and Pulmonary Embolism

To investigate whether the FII A(20210) mutation is associated with isolated pulmonary embolism (PE).Case-control study.Five thrombosis centers in southern Italy.Six hundred forty-seven consecutive referred patients with objectively documented venous thrombosis and 1,329 control subjects.Medical histories were collected. The G-to-A transition at nucleotide 1691 within the factor V gene (FV Leiden) and the G-to-A transition at nucleotide position 20210 within the prothrombin gene locus (FII A(20210)), levels of anticoagulant factors, and levels of antiphospholipid antibodies were determined by standard techniques. Patients with deep venous thrombosis (DVT) of the lower extremities (n = 346) or with additional PEs (n = 175) showed similar prevalences of FV Leiden mutation (24.3% and 16.6%, respectively) and FII A(20210) mutation (14.2% and 12.6%, respectively), and similar deficiencies of natural anticoagulants (4.9% and 2.3%, respectively). In both groups, the frequencies of FV Leiden and/or FII A(20210) mutation were higher than those observed among 1,329 apparently healthy control subjects (4.8% and 4.4%, respectively; p < 0.0001). Among patients with isolated PE (n = 126), prevalences of FV Leiden (7.1%) and FII A(20210) mutation (8.7%) were similar to those of control subjects. Inherited thrombophilic abnormalities were less frequent among patients with PE only (15.6%) than among those with DVT only (37.0%; p < 0.001) or whose conditions were complicated by PE (28. 0%; p = 0.020). Adjusting for age and sex, FV Leiden mutation, FII A(20210) mutation, or both mutations were associated with DVT with PE (FV Leiden mutation: odds ratio [OR], 3.0; 95% confidence interval [CI], 1.6 to 5.5; FII A(20210) mutation: OR, 2.6; 95% CI, 1. 3 to 5.2; and both mutations: OR, 82.1; 95% CI, 7.5 to 901.2) or without PE (FV Leiden mutation: OR, 6.1; 95% CI, 4.0 to 9.3; FII A(20210) mutation: OR, 2.8; 95% CI, 1.7 to 4.8; and both mutations: OR, 167.5; 95% CI, 21.6 to 1,297.7), but not with isolated PE (FV Leiden mutation: OR, 1.2; 95% CI, 0.5 to 2.8; FII A(20210) mutation: OR, 1.2; 95% CI, 0.5 to 3.1; and both mutations: OR, 22.1; 95% CI, 1. 3 to 370.2).FII A(20210) mutation is associated with DVT in the lower extremities alone or when complicated by PE, but it is not associated with isolated PE.