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

Abstract INTRODUCTION: Life 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 METHODS: Sixty 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. RESULTS: The 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. CONCLUSIONS: The 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. Disclosures No relevant conflicts of interest to declare.

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Simultaneous Replacement of Endothelial Thrombomodulin and Plasma Protein C: A Novel Therapeutic Strategy for Sepsis-Induced Disseminated Intravascular Coagulation

Blood ◽

10.1182/blood.v128.22.2613.2613 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2613-2613

Author(s):

Colin F Greineder ◽

Ian Johnston ◽

Carlos Hipolito Villa ◽

Douglas B. Cines ◽

Mortimer Poncz ◽

...

Keyword(s):

Fluorescence Microscopy ◽

Fusion Protein ◽

Whole Blood ◽

Protein C ◽

Thrombin Generation ◽

Fibrin Deposition ◽

Platelet Poor Plasma ◽

Thrombin Generation Assay ◽

Tnf Α ◽

Microscopy Images

Abstract Sepsis induces a procoagulant state, which in its most extreme form can result in disseminated intravascular coagulation (DIC), a syndrome of widespread microvascular thrombosis, ischemia, and organ dysfunction. Suppression of the protein C (PC) pathway has long been thought to be an important part of the pathophysiology, leading to development of a number of candidate therapeutics, including soluble human thrombomodulin (shTM, or ART123), currently being evaluated in a phase III clinical trial. While conferring less risk of bleeding toxicity than previous pharmacologic interventions into the protein C pathway, shTM fails to reproduce the spatial localization of its endogenous counterpart, preventing optimal interaction with PC bound to its endothelial receptor. To test the importance of localization to the endothelial membrane, we compared the antithrombotic activity of shTM to that of hTM/R6.5, a fusion protein therapeutic that anchors recombinant human TM to endothelial ICAM-1. In modified thrombin generation assays, in which platelet-poor plasma was exposed to cytokine-activated human endothelial cells, cell bound hTM/R6.5 was more potent than shTM mixed into the plasma. A similar result was found in a human whole blood microfluidic assay of sepsis-induced microvascular thrombosis, in which endothelial bound hTM/R6.5 more effectively inhibited fibrin deposition than its soluble counterpart. To investigate its mechanism of action, the fusion protein was tested in the setting of moderate PC deficiency, a common occurrence in patients with DIC. While PC depletion alone had no effect on fibrin deposition in this model, it significantly reduced the efficacy of hTM/R6.5, which was restored by supplementation using plasma-derived PC concentrate. These results indicate that endothelial bound TM acts not only as a direct inhibitor of thrombin, but derives antithrombotic activity from the activation of PC, such that it may be insufficient to reverse the coagulopathy of patients with significant deficiency in plasma PC. Likewise, the data match the clinical experience with PC supplementation, which appears to be of nominal benefit in sepsis or DIC, despite repeated demonstration of efficacy in patients with severe congenital PC deficiency, who have normal levels of endothelial TM and EPCR. The synergistic effect observed with simultaneous replacement of plasma PC and endothelial TM, however, suggest that this combination may represent a novel, translational therapeutic strategy. Figure (a) Thrombin generation assay using fluorescent thrombin substrate. Thrombin was generated following addition of platelet poor plasma to static monolayers of tissue factor expressing, TNF-α activated HUVEC. Treatment of cells with hTM/R6.5, followed by washing to remove non-specifically bound fusion protein, was more effective than addition of shTM to the plasma. Inset shows AUC (area under curve) analysis: * = p < 0.05 vs. TNF only, # = p < 0.05 for hTM/R6.5 vs. shTM at each concentration. (b) Fibrin generation (measured using AF647-conjugated fibrin antibody) in whole blood microfluidic assay. 3D-confluent endothelial monolayers were grown within flow chambers and activated with TNF-α prior to the infusion of whole blood at 5 dynes/cm2. hTM/R6.5 shows more prolonged inhibition of coagulation than shTM. Inset shows fluorescence microscopy images at 30 min after infusion of whole blood and AUC analysis: * = p < 0.05 vs. TNF only. (c) Fibrin deposition in control vs. PC deficient blood. PC supplementation with plasma-derived concentrate restores the anti-thrombotic activity of hTM/R6.5. Inset shows fluorescence microscopy images for each condition at 7.5 min after infusion of whole blood. AUC analysis is also shown: ** = p < 0.05 vs. all other conditions. Figure. (a) Thrombin generation assay using fluorescent thrombin substrate. Thrombin was generated following addition of platelet poor plasma to static monolayers of tissue factor expressing, TNF-α activated HUVEC. Treatment of cells with hTM/R6.5, followed by washing to remove non-specifically bound fusion protein, was more effective than addition of shTM to the plasma. Inset shows AUC (area under curve) analysis: * = p < 0.05 vs. TNF only, # = p < 0.05 for hTM/R6.5 vs. shTM at each concentration. (b) Fibrin generation (measured using AF647-conjugated fibrin antibody) in whole blood microfluidic assay. 3D-confluent endothelial monolayers were grown within flow chambers and activated with TNF-α prior to the infusion of whole blood at 5 dynes/cm2. hTM/R6.5 shows more prolonged inhibition of coagulation than shTM. Inset shows fluorescence microscopy images at 30 min after infusion of whole blood and AUC analysis: * = p < 0.05 vs. TNF only. (c) Fibrin deposition in control vs. PC deficient blood. PC supplementation with plasma-derived concentrate restores the anti-thrombotic activity of hTM/R6.5. Inset shows fluorescence microscopy images for each condition at 7.5 min after infusion of whole blood. AUC analysis is also shown: ** = p < 0.05 vs. all other conditions. Disclosures No relevant conflicts of interest to declare.

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Procoagulant imbalance in Klinefelter syndrome assessed by thrombin generation assay and whole blood thromboelastometry

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/clinem/dgaa936 ◽

2020 ◽

Author(s):

Indirli Rita ◽

Emanuele Ferrante ◽

Erica Scalambrino ◽

Eriselda Profka ◽

Marigrazia Clerici ◽

...

Keyword(s):

Whole Blood ◽

Protein C ◽

Thrombin Generation ◽

Klinefelter Syndrome ◽

Platelet Rich Plasma ◽

Coagulation Factors ◽

Cross Sectional Study ◽

Cross Sectional ◽

Thrombin Generation Assay ◽

Increased Risk

Abstract Context Klinefelter syndrome (KS) is a condition at increased risk of thrombosis compared to 46,XY men. Objective To investigate the coagulation balance of KS patients by thrombin generation assay (TGA) and thromboelastometry. Design Observational, cross-sectional study. Setting Three tertiary endocrinological centers in Milan, Italy. Patients or other participants 58 KS patients and 58 age-matched healthy controls were included. Anticoagulant or antiplatelet therapy and known coagulation disorders were exclusion criteria. Interventions TGA was performed in platelet-poor plasma (PPP) and platelet-rich plasma (PRP). Whole-blood thromboelastometry and activities of coagulation factors were assessed. Main Outcome Measures Endogenous thrombin potential (ETP), i.e. the area under the thrombin generation curve, assessed with and without thrombomodulin (ETP-TM + and ETP-TM -), and their ratio (ETP-ratio) were considered as indexes of procoagulant imbalance. Results Patients with KS displayed higher PPP-ETP-TM + (mean 1528vs.1315nMxmin; p<0.001), PPP-ETP-ratio (0.78vs.0.70, p<0.001), factor (F)VIII (135%vs.107%; p=0.001), fibrinogen (283vs.241 mg/dL; p<0.001) and FVIII/protein C ratio (1.21vs.1.06; p<0.05) compared to controls. Protein C was comparable in the two groups. Similar results were observed in PRP. ETP-ratio was positively associated with FVIII (rho=0.538, p<0.001) in KS. Thromboelastometry parameters confirmed evidence of hypercoagulability in KS. Conclusions Patients with KS display a procoagulant imbalance expressed by increased thrombin generation both in PPP and PRP, which is at least in part explained by increased FVIII levels. The procoagulant imbalance, which was confirmed by thromboelastometry, may be responsible for the thrombotic events observed in these patients. Further investigation on the benefit/risk ratio of antithrombotic prophylaxis is warranted.

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An overview of genetic risk factors in thrombophilia

Srpski arhiv za celokupno lekarstvo ◽

10.2298/sarh10s1079d ◽

2010 ◽

Vol 138 (suppl. 1) ◽

pp. 79-81 ◽

Cited By ~ 2

Author(s):

Valentina Djordjevic ◽

Ljiljana Rakicevic ◽

Dragica Radojkovic

Keyword(s):

Risk Factors ◽

Genetic Risk ◽

Protein C ◽

Significant Risk ◽

Genetic Risk Factors ◽

Genetic Defects ◽

Exact Nature ◽

Factor V Leiden Mutation ◽

Thrombotic Risk ◽

Factor Ii

Thrombophilia is a multifactorial disorder, involving both genetic and acquired risk factors that affect the balance between procoagulant and anticoagulant factors and lead to increased tendency to thrombosis. The concept that thrombophilia could be associated with genetic defects was first proposed in 1965 after the discovery of familiar antihrombin III deficiency. Further family studies showed that deficiency of protein C or protein S also increased thrombotic risk. In the coming years the advent in DNA technology, especially the invention of PCR reaction, played an important role in the identification of the exact nature of these deficiencies and opened new possibilities in the genetic research of thrombophilia. The breakthrough came with the discovery of activated protein C resistance and Factor V Leiden mutation. Shortly afterwards a mutation in the 3? untranslated region of Factor II gene (FII G20210A) associated with increased concentration of factor II in plasma, was described. Large epidemiologic studies have conformed that these two common mutations represent significant risk factors for thrombophilia. In the last decade several prothrombotic genetic risk factors have been described, including genes variants associated with increased levels of coagulation factors, defects of natural coagulation inhibitors, defects of the fibrinolytic system and hyperhomocysteinemia. These genetic defects or their combination have been extensively studied in an attempt to elucidate the possible association with increased thrombotic tendency. The large-scale DNA analysis systems are now becoming available, opening a new era in the genetic studies of thrombophilia. New technology will enable many genes to be studied in a single patient bringing us closer to the ?personalized? medicine.

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Protein C deficiency screening using a thrombin-generation assay

Thrombosis and Haemostasis ◽

10.1160/th06-09-0530 ◽

2007 ◽

Vol 97 (01) ◽

pp. 165-166 ◽

Cited By ~ 5

Author(s):

Nathalie Hézard ◽

Lobna Bouaziz-Borgi ◽

Marie-Geneviève Remy ◽

Bernadette Florent ◽

Philippe Nguyen

Keyword(s):

Protein C ◽

Thrombin Generation ◽

Protein C Deficiency ◽

Thrombin Generation Assay ◽

Deficiency Screening

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Protein C deficiency screening using a thrombin generation assay – An upgrade

Thrombosis and Haemostasis ◽

10.1160/th07-04-0314 ◽

2007 ◽

Vol 98 (09) ◽

pp. 691-692 ◽

Cited By ~ 4

Author(s):

Joost van Veen ◽

Peter Cooper ◽

Steve Kitchen ◽

Michael Makris ◽

Alex Gatt

Keyword(s):

Protein C ◽

Thrombin Generation ◽

Protein C Deficiency ◽

Thrombin Generation Assay ◽

Deficiency Screening

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Native Whole Blood Thrombin Generation Assay Evaluates Therapeutic Efficacy Of Plasma and Platelet-Derived FVIII

Blood ◽

10.1182/blood.v122.21.1093.1093 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1093-1093

Author(s):

Christina K Baumgartner ◽

Qizhen Shi ◽

Robert R. Montgomery

Keyword(s):

Gene Therapy ◽

Replacement Therapy ◽

Whole Blood ◽

Therapeutic Efficacy ◽

Thrombin Generation ◽

Generation Rate ◽

Gene Therapy Approach ◽

Thrombin Generation Assay ◽

Recombinant Fviii ◽

Dose Dependent

Abstract Factor VIII (FVIII) gene therapy is a promising approach to potentially permanently and cost-effectively correct the bleeding phenotype of hemophilia A patients and improve patients quality of life. Our group has developed a successful gene therapy approach in which FVIII expression is targeted to platelets. Platelet expressed FVIII protects hemophilic mice from lethal blood loss after vessel injury. Most importantly this therapy does not induce FVIII inhibitory antibodies and is even successful in the treatment of mice with pre-existing high titer inhibitors. Therefore this approach is among the first to hold promise for patients who develop inhibitory antibodies against FVIII that render FVIII replacement therapy ineffective. Levels of platelet expressed FVIII achieved by gene therapy may vary between individuals due to differences in ex vivotransduction and gene expression efficiency. We determined hemostatic efficacy over a wide therapeutic dose range with a novel native whole blood thrombin generation assay. Tracking the correction of abnormal bleeding phenotypes during the treatment of patients with hemostatic disorders is crucial to evaluate success of therapy. Global coagulation assays in contrast to single clotting factor assays are desirable to better understand the overall hemostatic condition of patients. Here we evaluated thrombin generation using a modified protocol of a recently described whole blood assay. In our native assay we initiated coagulation without the addition of tissue factor. Sole recalcification of whole blood resulted in thrombin generation with high reproducibility. Lag time (LT) determined in blood from C57BL/6 WT mice was 6 ± 0.2 min (Mean ± SEM) , thrombin generation rate was 58 ± 6 nM/min and thrombin peak was 188 ± 7 nM. In contrast, FVIII deficient blood had negligible thrombin generation with 39 ± 7 min LT, 1.4 ± 0.3 nM/min thrombin generation rate and 12 ± 3 nM thrombin peak. Spiking hemophilic blood with increasing concentrations of recombinant FVIII ex vivo resulted in a dose dependent increase in thrombin generation. Reconstitution of hemophilic blood with FVIII to a 1%, 10% and 100% level shortened LT to 19 ± 1, 12 ± 0.3 and 9 ± 0.5 min, respectively. To evaluate efficacy of platelet-derived FVIII we utilized a newly developed transgenic mouse model that expresses high levels of FVIII in platelets. Homozygous mice express platelet FVIII levels corresponding to 20% endogenous FVIII in whole blood. We combined different ratios of FVIII deficient blood with blood from platelet FVIII expressing transgenic mice. At low ratios of transgenic blood, similar to ex vivospiking with recombinant FVIII, thrombin generation parameters were dose-dependent. Remarkably, a corresponding dose of as low as 0.2% platelet-derived FVIII significantly elevated thrombin generation above FVIII deficient blood and had comparable therapeutic efficacy as a 5-fold higher dose of recombinant FVIII (LT, 18 ± 2 vs 19 ± 1). Similarly, efficacy of 1.5% of platelet-derived FVIII compared with the 6.7-fold higher, 10% dose of recombinant FVIII (LT, 13 ± 1 vs 12 ± 0.3). Further increase of thrombin generation was noticed with platelet FVIII expressing transgenic blood ratios corresponding to 2% and 5% FVIII levels (LT, 11 ± 0.3 and 8.7 ± 0.3 min, respectively). Interestingly, our native assay showed that the platelet FVIII expressing transgenic blood ratio corresponding to a FVIII level of only 5% was sufficient to induce maximal thrombin generation, similar to that obtained with undiluted transgenic blood (LT, 8.7 ± 0.6 min). A similar FVIII dose-dependency was identified for additional thrombin generation parameters including endogenous thrombin potential, thrombin peak, peak time and thrombin generation rate. We conclude that this native whole blood thrombin generation assay could be used to track therapeutic efficacy of hemophilia A treatment. Using this assay, our data indicate that similar to FVIII replacement therapy our previously established platelet targeted FVIII gene therapy approach enhances hemostasis over a wide therapeutic dose level. This is of great importance because levels of platelet expressed FVIII achieved upon gene therapy in mice vary. In agreement with our previous reports our data from native whole blood thrombin generation assay confirm that at lower FVIII dose levels platelet targeted FVIII gene therapy might be more efficient than factor replacement therapy. Disclosures: No relevant conflicts of interest to declare.

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Comparison of platelet-derived and plasma factor VIII efficacy using a novel native whole blood thrombin generation assay

Journal of Thrombosis and Haemostasis ◽

10.1111/jth.13169 ◽

2015 ◽

Vol 13 (12) ◽

pp. 2210-2219 ◽

Cited By ~ 5

Author(s):

C. K. Baumgartner ◽

G. Zhang ◽

E. L. Kuether ◽

H. Weiler ◽

Q. Shi ◽

...

Keyword(s):

Factor Viii ◽

Whole Blood ◽

Thrombin Generation ◽

Thrombin Generation Assay ◽

Plasma Factor

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ACUTE INTERRUPTION OF ORAL ANTICOAGULANT THERAPY: A THROMBOTIC RISK?

10.1055/s-0038-1643265 ◽

1987 ◽

Author(s):

J Rouvier ◽

H Vidal ◽

J Gallino ◽

M Boccia ◽

A Scazziota ◽

...

Keyword(s):

Anticoagulant Therapy ◽

Vitamin K ◽

Oral Anticoagulant ◽

Protein C ◽

Oral Anticoagulant Therapy ◽

Factor Vii ◽

Factor X ◽

Functional Protein ◽

Thrombotic Risk ◽

Factor Ii

It is still on discussion how oral anticoagulant therapy must be interrupted. A progressive diminution of drug intake have been proposed in order to avoid a MreboundM of vitamin K-dependent procoagulant factors. At the present, it is well known that coumarin drugs affect not only the biologic activity of factors II, VII, IX and X but also Protein C (PC), an inhibitor of coagulation kinetics, and their cofactor Protein S. With the aim to determine the recovery level of PC in relation with the others vitamin K-dependent factors, the effect of suppression of anticoagulant therapy in patients under chronic treatment with acenocoumarin was studied.Quick time, functional factors II, VII, X (one stage methods), functional PC (Francis method) and immunological Factor II and Protein C (Laurell) were determined before and 36 hours after suspension of acenocoumarin administration.Results showed that: 1) Recovery levels of functional Protein C (increased from 28.55% ±2.57 to 72.64% ±5.9) were significantly higer than functional Factor II (22.09% ±2.34 to 30.73% ±8.64), Factor VII (22.55% ±2.01 to 40.73% ±4.85) and Factor X (23.27% ±2.66 to 39.18% ±3.19). Statistical analysis (Newmann-Keuls test) showed at least a p<0.01 between PC increase and factors II, VII or X increment.2) No significant differences were seen between immunological levels of Factor II before and after suspension of acenocoumarin.3) Levels of immunological PC in patients under anticoagulant therapy were higer than functional PC. After acenocoumarin suppression, not correlation was seen between immunological and functional Protein C recovery.It is concluded that acute suppression of acenocoumarin does not induce a thrombotic tendency because the recuperation of functional Protein C is more important than factors II, VII and X recovery.

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Comparison of a New Automated Amidolytic Assay of Thrombin Generation with “ Prothrombin Time” and “Thrombotest”.

10.1055/s-0039-1687509 ◽

1979 ◽

Author(s):

J.J.C. Jonker ◽

L.H.M. van Riel ◽

M.M.P. Paulssen

Keyword(s):

Prothrombin Time ◽

Whole Blood ◽

Thrombin Generation ◽

Chromogenic Substrate ◽

Automated Method

An automated method was developed for the determination of thrombin generation in citrated whole blood during activation by thromboplastin. The thrombin generated was allowed to split the chromogenic substrate Tos-Gly-Pro-Arg-pNA yielding p-Nitroaniline. The comparison with this method and “Prothrombin time” in 50 samples was significant (r = 0,88 P < 0,001) and also in 503 samples with “Thrombotest” (r = 0,73 P < 0,001). This assay may serve as a specific, precise and fast and cheaper alternative for “prothrombin time” and “Thrombotest” in large Thrombosis Services.

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