In Vivo and Ex Vivo Thrombin Generation in Noncomorbid Patients with Suspected Deep Venous Thrombosis

2019 ◽  
Vol 7 (2) ◽  
pp. 291-292
Author(s):  
Evi Kalodiki ◽  
Fredrik Wexels ◽  
Ola Dahl ◽  
Jeanine Walenga ◽  
Walter Jeske ◽  
...  
Keyword(s):  
Venous Thrombosis ◽  
Deep Venous Thrombosis ◽  
Thrombin Generation ◽  
Ex Vivo

Animal Models of Thrombosis Help Predict the Human Therapeutic Concentration of PRT54021, a Potent Oral Factor Xa Inhibitor.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 901-901 ◽  
Author(s):  
Keith Abe ◽  
Gail Siu ◽  
Susan Edwards ◽  
Pei Hua Lin ◽  
Bing Yan Zhu ◽  
...  
Keyword(s):  
Animal Models ◽  
Venous Thrombosis ◽  
Thrombin Generation ◽  
Ex Vivo ◽  
Vena Cava ◽  
Factor Xa ◽  
Fold Change ◽  
In Vivo Models ◽  
Antithrombotic Activity ◽  
Fxa Inhibitor

Abstract Factor Xa (fXa) inhibition has resulted in the emergence of a new class of antithrombotics. Pharmacodynamic monitoring of these agents has proven problematic. The present study was designed to determine the target concentration of an oral fXa inhibitor required for clinical trials using both thrombin generation assays and three in vivo models and determine whether clotting assays such as activated partial thromboplastin time (aPTT) and prothrombin time (PT) would be suitable for monitoring human dosing. PRT54021 (PRT021) is a potent inhibitor of human fXa (Ki=117pM). PRT021 and fondaparinux, an indirect fXa inhibitor, both significantly inhibited TAT and F1.2 generation in human whole blood. Compared to a therapeutic level of fondaparinux (200nM), PRT021 (200nM) was more potent in suppressing both markers. Multiple doses of PRT021 were evaluated in three animal models. The first model, which measured clot accretion on cotton threads placed in rabbit abdominal vena cava, compared inhibition of thrombus mass by PRT021 to that of supratherapeutic doses of enoxaparin (a LMW heparin). The second model compared the ability of PRT021 to maintain vessel patency under arterial flow conditions in FeCl3 induced thrombosis in rat carotid artery to that achieved by enoxaparin or clopidogrel (an antiplatelet agent). The third model investigated inhibition of 111In labeled platelet deposition on dacron grafts and expansion chambers placed in femoral arteriovenous shunts in baboons. PRT021 and enoxaparin were administered as IV infusions and clopidogrel was dosed orally for three days. Ex vivo PT and aPTT were measured in all models. The models encompass stringent criteria of arterial and venous thrombosis and PRT021 produced dose-responsive antithrombotic activity in each of the three models. The efficacy of PRT021 compared favorably to supratherapeutic levels of enoxaparin and clopidogrel. Unlike in the rodent models, efficacy in primates was attained at a much lower dose with minimal prolongation of PT. Species specificity was also demonstrated by in vitro extensions of PT and aPTT in rat, rabbit, baboon and human plasma. A 2X change of PT was attained at concentrations of 8.9, 1.6, 1 and 0.4μM respectively. The data indicate that doses of PRT021 that inhibit thrombin generation in human blood and that provide anticoagulation similar to baboon dosed at 0.49mg/kg may be sufficient to prevent venous thrombosis in humans. Comparative modeling of extents of change in PT to levels of antithrombotic efficacy also leads us to predict that human therapeutic activity for PRT021 may be attained without concurrent changes in ex vivo clotting parameters. The targeted concentration is currently being tested in Phase II trials for its ability to prevent venous thromboembolism in orthopedic surgery patients. Model of Thrombosis Agent, Dose Antithrombotic Activity aPTT fold change PT fold change Rabbit vena cava PRT021,3mg/kg 76% inhibition 2.22 2.34 Rabbit vena cava Enoxaparin, 1.6mg/kg 96% inhibition 2.06 2.01 Rat carotid PRT021,19.1mg/kg 90% patency 1.69 2.20 Rat carotid Enoxaparin, 7.6mg/kg 70% patency 3.49 1.19 Rat carotid Clopidogrel, 3mg/kg/day 80% patency 1.03 1.01 Baboon arteriovenous PRT021,0.49mg/kg 90% inhibition (venous), 32% inhibition (arterial) 1.29 1.17

scholarly journals Thrombin Generation Assay and Its Application in the Clinical Laboratory

2016 ◽  
Vol 62 (5) ◽  
pp. 699-707 ◽  
Author(s):  
Armando Tripodi
Keyword(s):  
Liver Disease ◽  
Thrombin Generation ◽  
Clinical Laboratory ◽  
Ex Vivo ◽  
Myeloproliferative Neoplasms ◽  
Oral Anticoagulants ◽  
Relative Strength ◽  
Nonalcoholic Fatty Liver ◽  
Thrombin Generation Assay

Abstract BACKGROUND A gap exists between in vivo and ex vivo coagulation when investigated by use of the coagulation tests prothrombin time (PT) and activated partial thromboplastin time (APTT). The thrombin generation assay (TGA) has been developed to fill this gap. CONTENT TGA evaluates thrombin generation (resulting from the action of the procoagulant driver) and decay (resulting from the action of the anticoagulant driver), thus assessing the balance between the two. Coagulation of the test plasma (platelet poor or platelet rich) is activated by small amounts of tissue factor and phospholipids, and the reaction of thrombin generation is continuously monitored by means of a thrombin-specific fluorogenic substrate. Among the parameters derived from the thrombin-generation curve, the most important is the endogenous thrombin potential, defined as the net amount of thrombin that test plasmas can generate on the basis of the relative strength of the pro- and anticoagulant drivers. TGA is therefore the candidate assay to investigate hypo- or hypercoagulability. SUMMARY From my analysis of the literature, I draw the following conclusions. There is strong evidence that TGA is helpful to elucidate coagulation mechanisms in various clinical conditions that until recently were poorly understood (chronic liver disease; diabetes; inflammatory bowel disease, myeloproliferative neoplasms, nonalcoholic fatty liver disease). TGA is a promising laboratory tool for investigating hemorrhagic coagulopathies and monitoring replacement therapy in hemophiliacs, predicting the risk of recurrent venous thromboembolism after a first event, and monitoring patients on parenteral or oral anticoagulants. These applications require clinical trials in which TGA results are combined with specific clinical end points.

scholarly journals Dynamic Hemostasis and Fibrinolysis Assays in Intensive Care COVID-19 Patients and Association with Thrombosis and Bleeding—A Systematic Review and a Cohort Study

2021 ◽  
Author(s):  
Christine Lodberg Hvas ◽  
Julie Brogaard Larsen ◽  
Kasper Adelborg ◽  
Steffen Christensen ◽  
Anne-Mette Hvas
Keyword(s):  
Intensive Care ◽  
Thrombin Generation ◽  
Ex Vivo ◽  
Clot Lysis ◽  
Healthy Controls ◽  
Rotational Thromboelastometry ◽  
Thrombin Generation Assay ◽  
Primary Hemostasis ◽  
Normal Platelet

AbstractPatients admitted to the intensive care unit (ICU) with coronavirus disease 2019 (COVID-19), the infectious pathology caused by severe acute respiratory syndrome coronavirus 2, have a high risk of thrombosis, though the precise mechanisms behind this remain unclarified. A systematic literature search in PubMed and EMBASE identified 18 prospective studies applying dynamic coagulation assays in ICU COVID-19 patients. Overall, these studies revealed normal or slightly reduced primary hemostasis, prolonged clot initiation, but increased clot firmness. Thrombin generation assay parameters generally were equivalent to the control groups or within reference range. Fibrinolysis assays showed increased clot resistance. Only six studies related their findings to clinical outcome. We also prospectively included 51 COVID-19 patients admitted to the ICU. Blood samples were examined on day 1, 3–4, and 7–8 with platelet function tests, rotational thromboelastometry (ROTEM), in vivo and ex vivo thrombin generation, and clot lysis assay. Data on thrombosis, bleeding, and mortality were recorded during 30 days. Primary hemostasis was comparable to healthy controls, but COVID-19 patients had longer ROTEM-clotting times and higher maximum clot firmness than healthy controls. Ex vivo thrombin generation was similar to that of healthy controls while in vivo thrombin generation markers, thrombin–antithrombin (TAT) complex, and prothrombin fragment 1 + 2 (F1 + 2) were higher in ICU COVID-19 patients than in healthy controls. Impaired fibrinolysis was present at all time points. TAT complex and F1 + 2 levels were significantly higher in patients developing thrombosis (n = 16) than in those without. In conclusion, only few previous studies employed dynamic hemostasis assays in COVID-19 ICU-patients and failed to reveal a clear association with development of thrombosis. In ICU COVID-19 patients, we confirmed normal platelet aggregation, while in vivo thrombin generation was increased and fibrinolysis decreased. Thrombosis may be driven by increased thrombin formation in vivo.

scholarly journals Neutrophils in Deep Venous Thrombosis

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. SCI-29-SCI-29
Author(s):  
Thomas W. Wakefield
Keyword(s):  
Venous Thrombosis ◽  
Deep Venous Thrombosis ◽  
Extracellular Dna ◽  
Strong Positive Correlation ◽  
Plasma Dna ◽  
Vein Wall ◽  
Control Serum ◽  
Significant Difference ◽  
Wells Score

Abstract Neutrophils are involved in venous thrombogenesis, through P-selectin and neutrophil endothelial traps (NETS). In order to determine the importance of neutrophils to thrombogenesis, neutrophil depletion was performed in our rat model of stasis-induced deep venous thrombosis (DVT). Animals were treated with control serum or rabbit anti-rat PMN serum administered perioperatively and sacrificed at two and seven days. At two days, neutropenic rats had 1.6-fold larger thrombi (P = 0.04) and 1.4-fold higher femoral venous pressures by manometry (P = 0.008) without a difference in thrombus neovascularization. By seven days, DVT sizes were similar, but vein wall injury persisted in neutropenic rats with a two-fold increase in vein wall stiffness by microtensiometry (P < 0.05), as well as a 1.2-fold increased thickness (P = 0.04). Vein wall and intrathrombus uPA by Western immunoblotting, as well as intrathrombus MMP-9 gelatinase activity was significantly less in neutropenic rats than controls (P < 0.001). Conversely, MMP-2 was significantly elevated in neutropenic inferior vena cava (IVC) at two days after DVT.1 P-selectin inhibition has been found to limit venous thrombosis in mice. Animals with high circulating levels of sP-selectin (^CT) were compared to selectin gene-deleted animals (PKO, EPKO) and wild-type (WT) mice. ^CT mice showed a significant 50% increase in thrombosis in our IVC ligation model while EPKO mice had the smallest thrombi. A significant difference was noted between ^CT and EPKO for neutrophils, monocytes, and total inflammatory cells at day two. Microparticle (MP) analysis revealed that in the ^CT, WT and PKO mice, a mixed leukocyte (MAC-1) and platelet (CD41) MP population was present. EPKO mice (with the smallest thrombi) revealed primarily a platelet-derived MP population, suggesting the importance of leukocyte-derived MPs in venous thrombogenesis. Of interest, the ^CT mice with the highest TM showed an elevated level of mean channel fluorescence for MAC-1 antibody, indicative of leukocyte derived MPs.2 Through processes that also involve the initial activation of leukocytes and platelets, neutrophils initiate and amplify thrombosis through the formation of NETS, which are extracellular fragments of DNA containing histones and antimicrobial proteins.3,4 In vitro and in vivo, NETs provide a scaffold and stimulus for thrombus formation.5 In order to investigate if plasma DNA (surrogate for NETS) is elevated in patients with DVT and to determine correlations with other biomarkers of DVT, we studied patients presenting to our diagnostic vascular laboratory. From December 2008 to August 2010, patients were divided into three distinct groups: (1) DVT positive, patients symptomatic for DVT confirmed by ultrasound (n = 47); (2) DVT negative, patients with leg pain but negative by ultrasound (n = 28); and (3) control healthy non-pregnant volunteers without signs or symptoms of active or previous DVT (n = 19). Blood was collected for biomarkers and the Wells score risk of DVT was assessed. Results showed that circulating DNA was significantly elevated in DVT patients, compared with both DVT-negative patients (57.7 vs. 17.9 ng/mL; P < 0.01) and controls (57.7 vs. 23.9 ng/mL; P < 0.01). There was a strong positive correlation with C-reactive protein (P < 0.01), D-dimer (P < 0.01), VWF (P < 0.01), Wells score (P < 0.01) and MPO (P < 0.01), along with a strong negative correlation with ADAMTS13 (P < 0.01) and the ADAMTS13/VWF ratio. The logistic regression model showed a strong association between plasma DNA and the presence of DVT (ROC curve 0.814) suggesting a role for DNA in venous thrombogenesis.6 References: 1. Varma MR, Varga AJ, Knipp BS, Sukheepod P, Upchurch GR, Kunkel SL, Wakefield TW, Henke PK. Neutropenia impairs venous thrombosis resolution in the rat. J Vasc Surg. 2003;38:1090-1098 2. Myers DD, Hawley AE, Farris DM, Wrobleski SK, Thanaporn P, Schaub RG, Wagner DD, Kumar A, Wakefield TW. P-selectin and leukocyte microparticles are associated with venous thrombogenesis. J Vasc Surg.2003;38:1075-1089 3. Fuchs TA, Brill A, Duerschmied D, Schatzberg D, Monestier M, Myers DD, Wrobleski SK, Wakefield TW, Hartwig JH, Wagner DD. Extracellular DNA traps promote thrombosis. Proceedings of the National Academy of Sciences. 2010;107:15880-15885 4. von Brühl M-L, Stark K, Steinhart A, Chandraratne S, Konrad I, Lorenz M, Khandoga A, Tirniceriu A, Coletti R, Köllnberger M. Monocytes, neutrophils, and platelets cooperate to initiate and propagate venous thrombosis in mice in vivo. The Journal of Experimental Medicine. 2012;209:819-835 5. Massberg S, Grahl L, von Bruehl M-L, Manukyan D, Pfeiler S, Goosmann C, Brinkmann V, Lorenz M, Bidzhekov K, Khandagale AB. Reciprocal coupling of coagulation and innate immunity via neutrophil serine proteases. Nature Medicine. 2010;16:887-896 6. Diaz JA, Fuchs TA, Jackson TO, Kremer Hovinga JA, Lämmle B, Henke PK, Myers Jr DD, Wagner DD, Wakefield TW. Plasma DNA is elevated in patients with deep vein thrombosis. J Vasc Surg. 2013;1:341-348 Disclosures No relevant conflicts of interest to declare.

P2765Efficacy of venous recanalization by noninvasive pulsed cavitational ultrasound therapy on swine model of acute deep venous thrombosis

2019 ◽  
Vol 40 (Supplement_1) ◽  
Author(s):  
L Khider ◽  
G Goudot ◽  
C Del Giudice ◽  
T Mirault ◽  
P Bruneval ◽  
...  
Keyword(s):  
Venous Thrombosis ◽  
Deep Venous Thrombosis ◽  
Surrounding Tissue ◽  
Ultrasound Therapy ◽  
Swine Model ◽  
High Morbidity ◽  
Linear Probe ◽  
Venous Recanalization

Abstract Background Post thrombotic syndrome is a frequent complication of deep venous thrombosis and is associated with high morbidity and hospitalization. Treatments currently available are invasive, involve use of endovenous procedures with stents and balloons, and frequently require general anesthesia. Pulsed cavitational ultrasound therapy (PCUT) emerged recently as a new technique to destroy remotely soft tissue. We recently demonstrated that PCUT was able to recanalize non-invasively in in vitro model of acute venous thrombosis (human blood clot). Purpose We aim to test the safety and efficacy of venous recanalization by noninvasive PCUT in vivo swine model of acute venous thrombosis. Methods All the experiments were performed on White large X Landrace swine. We induced an acute femoral deep venous thrombosis by using occlusive balloons introduced from jugular and popliteal vein combined with local injection of 50 IU of human thrombin. A 3 cm length occlusive thrombosis was obtained after 2 hours. Swines were divided in three groups: one with PCUT without follow-up (n=11), one with PCUT and follow-up at 14 days (n=8), and a control group also followed for 14 days (n=5). Acutely and during the follow up all swines received curative anticoagulation. To achieve PCUT, a 2.25 MHz transducer centered by a linear probe was used and cavitation was obtained in the center of the vein (Figure). After manual determination of thrombus location, a robotic arm was used to automatically move the transducer along the thrombus. Effectiveness of recanalization was evaluated by echo-Doppler and phlebography. Safety was assessed by Doppler ultrasound of the insonified area (artery, veins and surrounding tissue) and by histological analysis (local femoral vein and artery and lungs for pulmonary embolism). Results Among the 24 swines, we obtained 22 occlusive venous thromboses and 2 partial. The median length of the thrombus was 26±4.4 mm with vein diameter of 8.5±1.6 mm. Acutely, thrombosis recanalization was systematically obtained among the 19 swine with PCUT with median treatment duration of 33 minutes with residual diameter of 2.9±0.9 mm. No extravasation of contrast material or hematoma was observed after the therapy. After a 14-day follow-up, 75% of recanalisation remain permeable vs. 0% of vein permeable in the no therapy group (p=0.008). Residual diameter was 2.6±1.2 mm, which correspond to 50% of the venous diameter. No vein or artery damage and no embolism or pulmonary infarction was observed in all pigs. Figure 1 Conclusion We have demonstrated in vivo the safety and the efficacy of PCUT for non-invasive venous recanalization, persistent after 2 weeks. Acknowledgement/Funding French society of cardiology

Protein S As a Potential Treatment for FIX-Derived Deep Vein Thrombosis

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2277-2277
Author(s):  
Vijaya Satish Sekhar Pilli ◽  
Willium Plautz ◽  
Rinku Majumder ◽  
Paolo Simioni
Keyword(s):  
Deep Vein Thrombosis ◽  
Thrombin Generation ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Protein S ◽  
Factor Ix ◽  
Wild Type ◽  
Vein Thrombosis ◽  
Deep Vein

Abstract Background: Every year, 0.1-0.2% of the USA population experiences deep vein thrombosis (DVT). Two causes of DVT are increased Factor IX (FIX) levels and hyperactivating mutations in FIX (FIX Padua variant- R338L and Malmo variant T148A). In principle, inhibition of activated FIX (FIXa) should alleviate DVT. Previous in vitro studies demonstrated that the anticoagulant Protein S (PS) inhibits the intrinsic pathway mediated by wild type FIXa, making PS an attractive candidate to treat DVT. Aims: To establish Protein S as a remedy for FIX-mediated DVT/Padua/Malmo Methods: Anisotropy, clotting assays, thrombin generation assays, co-localization, co-immunoprecipitation, and bleeding assays. Results: We further explored the physiological relevance of the PS-FIXa interaction and PS-mediated inhibition of FIXa by ex vivo (co-immunoprecipitation) and in vivo (co-localization) studies. Because PS can inhibit FIXa in vivo, we used competitive, direct anisotropy assays and co-immunoprecipitation assays to measure the efficiency PS and hyperactive FIXa (R338L) interaction. Interestingly, the results demonstrated that FIXa R338L has lost its affinity towards PS compared with wild type FIXa. The same finding was obtained by ex vivo thrombin generation assays and FXa generation assays supplemented with various concentrations of PS. Thus, to be inhibited, hyperactive FIX requires a greater amount of PS compared with wild type FIXa. We are further confirming this finding with mouse models. Conclusion: Addition of PS to plasma inhibits both wild type and R338L FIXa and extends clotting time. Previous studies showed that the addition of PS has no significant negative effects. Thus, we conclude that PS supplementation potentially constitutes a novel and effective treatment for FIX-mediated DVT. Disclosures No relevant conflicts of interest to declare.

Effect of New Synthetic Heparin Mimetics on Whole Blood Thrombin Generation In Vivo and In Vitro in Rats

2002 ◽  
Vol 87 (02) ◽  
pp. 238-244 ◽  
Author(s):  
J.P. Hérault ◽  
A. Bernat ◽  
C. Gaich ◽  
J.M. Herbert
Keyword(s):  
Venous Thrombosis ◽  
Charge Density ◽  
Whole Blood ◽  
Thrombin Generation ◽  
Platelet Rich Plasma ◽  
Lag Time ◽  
Drug Candidate ◽  
Platelet Factor

SummaryThe effect of new heparin mimetics (synthetic oligosaccharides) was studied in vitro with regard to thrombin generation (TG) in rat platelet rich plasma (PRP) and whole blood (WB) and in vivo on stasis-induced venous thrombosis in the rat.TG in PRP and in WB was highly dependent on platelet count and strongly influenced by the haematocrit. The peak of TG appeared to be significantly higher in WB than in PRP whereas the endogenous thrombin potential (ETP) was not significantly different under either condition.The effect of hirudin, the synthetic pentasaccharide SR90107/ Org31540 (SP) and heparin were measured on TG in PRP and WB. We then compared the effect of two new synthetic heparin mimetics (SR121903A and SanOrg123781) with potent and comparable antithrombin (AT) mediated activity against factor Xa and thrombin. These two compounds were made of a pentasaccharide with a high affinity to AT, prolonged at the non-reducing end by an oligosaccharide chain recognised by thrombin. In SR121903A, the charge density and charge distribution was analogous to that of heparin whereas in SanOrg123781 the charges were only located on the last 5 saccharides of the non-reducing end of the molecule. In PRP and in WB, SR121903A acted on the lag time and on the AUC whereas SanOrg123781 inhibited thrombin formation with no effect on the lag time. SanOrg123781 was more potent in inhibiting TG than SR121903A. This difference was due to the structures of the compounds that differed in their ability to be neutralised by platelet factor 4. The antithrombotic effect of the two compounds was examined in a venous thrombosis model in rats. We observed that SanOrg123781 was more active than SR121903A and heparin.Taken together, these results indicate that the activity of oligosaccharides is greatly influenced by the global charge density of the molecule and show that SanOrg123781 is a potent and promising antithrombotic drug candidate.

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