Platelet Count Plays a Major Role in Regulating Fibrin Formation in Cirrhosis: Clinical Study of Whole Blood Coagulation

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2290-2290
Author(s):  
Charles S Greenberg ◽  
James L. Wells ◽  
Caroline D Vaughn ◽  
Adrian Reuben
Keyword(s):  
Platelet Count ◽  
Blood Coagulation ◽  
Whole Blood ◽  
Thrombin Generation ◽  
Intrinsic Pathway ◽  
Thrombotic Risk ◽  
Normal Volunteers ◽  
Fibrin Formation ◽  
Platelet Number ◽  
Cirrhotic Patients

Abstract Abstract 2290 Recent studies document a paradoxical absence of bleeding in the setting of an abnormal PT and aPTT in cirrhosis. A new equilibrium is established between thrombin generation, fibrin formation and fibrinolysis in cirrhosis that confers protection from spontaneous bleeding and may pose a thrombotic risk. The purpose of this study was to evaluate the coagulation paradox in cirrhosis by using whole blood thromboelastometry. Citrate anticoagulated whole blood is added to a cuvette and coagulation is initiated by adding calcium with either a tissue factor (TF)-based reagent (ExTEM) or an intrinsic pathway activator reagent (InTEM). The transformation of blood into a gel is measured by a computer-based system (ROTEM Instrument) over 30 minutes. We collected samples after obtaining informed consent form patients with cirrhosis. Part of the sample was process for platelet-poor plasma and the PT and aPTT assays performed. The clotting time (CT) which is the time from the start of the assay until initiation of fibrin formation;the clot formation time (CFT), the time from initiation of clotting until a half maximum clot firmness were measured. In addition, maximal clot firmness (MCF) in the presence or absence of cytochalasin an inhibitor of platelet activation was also measured by Fib-TEM assay. The effect of platelet count on these measurements was also evaluated. A total of 54 patients were enrolled with a median MELD score of 16, median PT 19.5sec, median aPTT 46.5sec and median platelet count 77K/CUMM. Only 20% of the population had a platelet count greater than 100K/CUMM. The average CT and CFT measured by the ExTEM were 72sec (normal 38–79sec) and 178sec (normal 34–159sec) respectively. The InTEM assay average CT was 172sec (normal 100–240) and CFT 175sec (normal 20–110). Normal CT was seen in 73% of ExTEM measurements and normal CFT in 53%. CT in InTEM was normal in 93%. In contrast, the InTEM CFT was normal in only 20% of measurements. Changes in CFT, a measure of the rate of thrombin generation and fibrin formation, could be influenced by platelet count. Activated platelets must be able to bind and assemble enzyme complexes that promote thrombin formation. We measured ExTEM and InTEM assays with varying platelet concentrations in normal volunteers. The CFT prolonged in ExTEM assays at 75–90K/CUMM platelets and between 90–120K/CUMM with the InTEM assay for normal volunteers. The platelet count for cirrhotic patient samples that had an abnormal InTEM CFT had platelet count of 64K/CUMM while those with normal InTEM CFT had 136K/CUMM platelets. Cirrhosis patient samples with a normal CFT by ExTEM had an average platelet count of 95K/CUMM and those that had an abnormal CFT by ExTEM had a platelet count of 53K/CUMM. We then looked at whether fibrinogen levels as measured by a Fib TEM assay were normal in cirrhotic patients. We found that 84% had normal fibrinogen levels. When the MCF was abnormal 85% of the samples had platelet counts below 70K, while the others had a low fibrinogen level. Lastly we found 4 patients that had an elevated MCF and these patients had an elevated fibrinogen by FIB-TEM despite having a platelet counts less than 70 K. In conclusion, despite having abnormalities in PT and aPTT assays the ability to initiate and generate a fibrin gel in whole is not abnormal in many cirrhotic patients. If there is a defect in whole blood testing it is more readily detected by the INTEM assay a measure of the intrinsic pathway. The majority of the abnormalities in thrombin formation and generation of maximum fibrin clot are dependent on platelet number. Some patients with elevated fibrinogen levels can compensate for a reduced platelet number. These studies support recent data that suggest platelet count <70K increase the risk of bleeding after invasive procedures in cirrhosis. Future studies will examine whether whole blood coagulation assays can be used to identify cirrhotic patients at risk for bleeding and will require prospective randomized clinical studies. The coagulation paradox in cirrhosis requires evaluation of new assays to define hemorrhagic and thrombotic risk. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Simultaneous Measurement of Thrombin Generation and Fibrin Formation in Whole Blood Applying Continuous Flow Is Predictive for the Amount of Blood Loss during/after Cardiothoracic Surgery

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2813-2813
Author(s):  
Leonie Pelkmans ◽  
Anne Bouwhuis ◽  
Evelien Schurgers ◽  
Theo Lindhout ◽  
Dana Huskens ◽  
...  
Keyword(s):  
Blood Loss ◽  
Whole Blood ◽  
Continuous Flow ◽  
Thrombin Generation ◽  
Cardiothoracic Surgery ◽  
Flow Rates ◽  
Clot Strength ◽  
Fibrin Formation ◽  
Significant Difference ◽  
Fibrinogen Content

Abstract Background. Thrombin is the key enzyme converting fibrinogen into fibrin and assays based on the formation of thrombin and fibrin are currently often used separately both in research and clinical settings. Recently it was shown that thrombin generation and fibrin formation do not always go hand in hand as factor XIIa independently influences the crosslinking of fibrin and thereby the stability of the clot. Objective. The development and clinical validation of an assay that is able to simultaneously measure the generation of thrombin and formation of fibrin in whole blood. Methods and results. We have redesigned an air-bearing rheometer, based on the plate and cone viscositor, rendering it sensitive enough to measure fibrin formation based on the change in viscosity. A confocal fluorescent camera is placed into the rheometer, allowing the measurement of thrombin generation based on the conversion of a thrombin-sensitive substrate as in the original Calibrated Automated Thrombinography (CAT) method. Furthermore, the method allows the application of laminar flow conditions within the range of 100 s-1 until 1200 s-1. For all variables related to fibrin formation and thrombin generation, the intra- and inter-assay variation were below 10% in platelet-poor plasma (PPP). For both parameters endogenous thrombin potential (ETP) and peak we demonstrated an inverse relationship with increasing flow rates (100-1200 s-1) in PPP. This was in line with fibrin formation as maximum clot strength (based on viscosity) was inversely related to an increase in flow rate. Furthermore, as expected, the addition of increasing concentrations of fibrinogen to defibrinated plasma was accompanied with an increase in ETP, peak and maximum clot strength. Our method proved to be sensitive enough to detect an increase of 0.1 mg/ml fibrinogen. Furthermore, in the presence of tissue plasminogen activator, it allows to study fibrinolysis. The system was clinically validated in 70 patients undergoing cardiothoracic surgery. Blood samples were taken pre-bypass before heparinisation. Besides thrombin generation and fibrin formation measurements, clinical parameters together with the blood loss and the amount of transfusion products were recorded until 24 hours after surgery. Thrombin generation parameters obtained with the specially designed rheometer correlated well with the traditional whole blood CAT, whereas fibrin formation parameters correlated well with the fibrinogen content and the rotational thromboelastometry (ROTEM) parameters. Importantly, when patients were divided into two groups based on the median clot strength determined with the rheometer, a significant difference in perioperative and total blood loss was established. Conclusion. We have technically and clinically validated a new method capable of simultaneously measuring thrombin generation and fibrin formation in plasma and whole blood under continuous flow. Our method proved to be sensitive enough to detect differences in fibrinogen content and revealed an effect of different flow rates on both thrombin generation and clot strength. We hereby have not only combined thrombin generation and fibrin formation in whole blood, but by introducing continuous flow, our method is also one step closer to physiology.Importantly,in contrast to the traditionally used methods, our test applied pre-operatively was found to be predictive for the amount of blood loss during and after cardiothoracic surgery. Disclosures No relevant conflicts of interest to declare.

Novel Initiation Mechanism of Blood Coagulation by Intravascular Tissue Factor.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3967-3967
Author(s):  
Bernd Engelmann
Keyword(s):  
Plasma Membrane ◽  
Tissue Factor ◽  
Whole Blood ◽  
Thrombin Generation ◽  
Clinical Chemistry ◽  
Sandwich Elisa ◽  
Blood Component ◽  
Potential Contribution ◽  
Fibrin Formation ◽  
Activated Platelets

Abstract Bernd Engelmann Vascular Biology and Hemostasis, Inst. of Clinical Chemistry, Ludwig-Maximilians-Universität, Munich, Germany Following addition of fibrillar collagen to whole blood in order to mimick the starting process of hemostasis, we unexpectedly observed that tissue factor (TF), the central initiator of coagulation, was exposed within 3–5 min in association with CD15 and CD14 positive blood cells. A series of experiments revealed that the TF presentation was restricted to conjugates of neutrophils (and monocytes) with platelets. To verify the source of the TF, isolated neutrophils and platelets were evaluated for the presence of TF. Using a double sandwich Elisa, the washed platelets were found to contain TF. Conversely, TF was undetectable in the neutrophils. When searching for the intraplatelet location of TF by immunoelectron microscopy (IEM), TF was observed to reside in the alpha-granules and in the surface connected system. No TF was present in the cytoplasma and the dense granules. In response to activation, platelet TF was translocated to the cell surface by fusion of the alpha-granules with the plasma membrane. The externalized TF was found to cluster on platelet filopodia. Inspection of rapidly isolated buffy coat preparations confirmed the absence of TF from the neutrophils. Stimulation of TF-dependent factor Xa formation by the activated platelets was markedly amplified by the isolated neutrophils. This required neutrophil-platelet conjugate formation, as evident from inhibition by antibodies targeting PSGL-1 and CD18. To assess whether the TF triggered coagulation was connected to the platelet recruitment, we evaluated the participation of the ADP system. Disrupting the interaction of ADP with its platelet receptors P2Y12 and P2Y1 suppressed the TF activity in the neutrophil-platelet conjugates. Since the TF exposing filopodia represent preferential sites for the formation of microparticles (MP), we isolated the total pool of circulating MP from whole blood, known to be mainly derived from the platelets. Then, the MP were separated by cell sorting. In MP positive for the platelet specific CD42b, TF could be detected and quantified by western blotting and Elisa. Moderate increases in MP number excessively stimulated blood based TF activity in the presence of platelets and in whole blood. Since activated platelets are known to secrete tissue factor pathway inhibitor (TFPI), an anti-TFPI antibody targeting the Kunitz-2 domain of TFPI was included into the suspensions of the activated platelets. Thereby the TF activity of the isolated platelets was enforced, while the activity in the presence of the neutrophils remained unaffected, suggesting that TFPI partially masks the functional competence of the platelet TF. The potential contribution of platelet-collagen interactions for the activation of coagulation in vivo was analyzed by injecting collagen into the venous blood of mice. Local fibrin formation was documented in pulmonary vessels by EM, and systemic thrombin generation was revealed by increased thrombin-antithrombin complexes. In mice deficient for the P2Y1 ADP receptor, the thrombin generation was markedly reduced, indicating a basic role for the platelet-triggered coagulation during thrombus growth. In conclusion, the intravascular tissue factor enables the entire coagulation system to proceed on the plasma membrane of a single blood component, the surface of the activated platelets. Consequently the coagulation start can be regulated within the platelet aggregate, allowing fibrin formation to be flexibly adjusted to the size of the thrombus and the duration of its development.

Serotonergic Mechanisms in Haemostasis: A Connection between Major Depression and Thrombotic Risk.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3907-3907
Author(s):  
Irene Lopez-Vilchez ◽  
Montserrat Serra ◽  
Antonio Alonso ◽  
Fulgencio Navalon ◽  
Rosa Hernandez ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cardiovascular Risk ◽  
Major Depression ◽  
Whole Blood ◽  
Selective Serotonin Reuptake Inhibitors ◽  
Thrombotic Risk ◽  
Fibrin Formation ◽  
Elevated Exposure ◽  
Control Samples

Abstract Serotonergic mechanisms implied in both neuronal and platelet functions have been pointed out as a possible link between depressive disorders and cardiovascular risk. Recent studies suggest serotonin (5-HT) as a key element in the generation of a subpopulation of platelets with elevated procoagulant properties (coated-platelets). We have investigated the implication of serotonergic mechanisms in platelet and coagulation activation in samples from healthy donors or from patients suffering major depression. Furthermore, we evaluated the possible prothrombotic role of serotonergic mechanisms and the potential antithrombotic effect of selective serotonin reuptake inhibitors (SSRI) in vitro and compared results with those observed in a group of patients with major depression. Different experimental strategies were used to evaluate platelet function and the activation of coagulation system including standard aggregometry, flow cytometry, perfusion studies with circulating blood to evaluate platelet interaction and fibrin deposition on damaged vessels. Levels of tissue factor (TF) in whole blood and prothrombin fragment F1+2 in plasma samples were also determined. Aggregation studies did not reveal marked differences between control and patients with major depression. The presence of SSRI in plasma decreased up to 30% of the aggregation induced by ADP potentiated with 5-HT. Flow cytometry studies revealed that platelets from patients with major depression showed an enhanced expression of platelet antigens: FV, fibrinogen, CD63, GPIV, GPIb, as well as elevated exposure of anionic phospholipids (p<0.05 vs. controls). Treatment of patients with the SSRI normalized the expression of these antigens vs. control samples. Studies performed under flow conditions only showed a significant increase of the fibrin formation on vascular damaged vessels of patients with major depression vs. controls (71.1±9.5 % vs. 45.8±5.3 %, p<0.05) with corresponding elevations in F1+2 levels. In vitro incubation of blood samples with a SSRI was followed by a 30% of reduction in fibrin formation. Similar tendencies were confirmed in studies performed along clinical treatment of patients with SSRI. A decrease of 20% and 80% of covered surface by fibrin was observed after 8 and 24 weeks of treatment respectively. Levels of whole blood TF of patients suffering depression were found elevated vs. control samples (1.3±0.4 ng/ml vs. 1.0± 0.4 ng/ml respectively) and tended to decrease during the treatment (p<0.05). Our results reveal a more thrombogenic state in patients diagnosed of major depression associated to serotonergic mechanisms. Strategies aimed to control serotonergic mechanisms in platelets may offer a potential therapy for the regulation of the prothrombotic state of patients with major depression, but also in patients with cardiovascular risk.

scholarly journals Continuous Thrombin Generation in Whole Blood: New Applications

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4979-4979
Author(s):  
Shannon M Prior ◽  
Kenneth G Mann ◽  
Kalev Freeman ◽  
Saulius Butenas
Keyword(s):  
Blood Coagulation ◽  
Whole Blood ◽  
Blood Cells ◽  
Thrombin Generation ◽  
Sodium Citrate ◽  
Lag Phase ◽  
Packed Red Blood Cells ◽  
Healthy Donors ◽  
Hemostatic Tests ◽  
Peak Value

Abstract Background. Hemostatic tests have been utilized to clarify the blood coagulation potential of both healthy and diseased individuals. They include tests for whole blood and plasma clotting times, coagulation and fibrinolytic factors and, recently, thrombin generation (TG). TG assays provide explicit information and remain the most physiologically-relevant hemostatic tests ex vivo. We have currently been using a TG assay in whole blood, described by Ninivaggi et al. (2012), for the evaluation of several hemostatic agents and disorders of blood coagulation. Methods. Whole blood from either healthy donors or trauma patients was drawn into 3.2% sodium citrate [± 0.1 mg/mL corn trypsin inhibitor (CTI) prior to the assay; prevents contact pathway activation] or into 3.2% sodium citrate/0.1 mg/mL CTI. Selected agents were added to blood prior to recalcification and TG was monitored in a fluorogenic assay. Packed red blood cells (pRBC) from healthy donors were reconstituted with platelet-poor plasma (PPP) and treated with 0.1 mg/mL CTI. TG was monitored in the absence of any exogenous initiation, in the presence of 5 pM TF initiation and also in the presence of synthetic phospholipids. Results. Whole Blood A. Blood from 10 healthy donors was collected into both citrate (with CTI added prior to the assay) and CTI/citrate and TG was monitored ± 5 pM relipidated tissue factor (TF) initiation. In the absence of TF, blood collected into citrate led to a lag phase ~3-fold longer than that in the presence of TF and peak thrombin ~1.4-fold lower. Blood collected into CTI/citrate, in the absence of TF, led to a lag phase ~6.4-fold longer than that in the presence of TF and peak thrombin ~4.3-fold lower. B. Titrations of relipidated TF and FXIa into citrated blood containing CTI led to concentration-dependent changes in the duration of the lag phase. C. Several TG antagonists were evaluated in citrated blood containing CTI triggered with 5 pM TF. a. 0.5 U/mL unfractionated heparin prolonged the lag phase ~2-fold and suppressed the peak thrombin ~3-fold. b. Hemophilia A and B were "induced" via the addition of inhibitory antibodies to FVIII and FIX, respectively, and although the lag phase was not altered both additions led to significant TG suppression with peak thrombin dropping ~3-fold. c. Several thrombin and FXa inhibitors were evaluated at their pharmacologic concentrations. Fondaparinux slightly prolonged the lag phase, whereas dabigatran increased it by 2.6-fold. Rivaroxaban doubled the lag phase while bivalirudin was slightly less efficient. None of these inhibitors had a pronounced effect on the rate and peak value of TG. d. Activated protein C (APC) at 10 nM slightly prolonged the lag phase and suppressed the peak thrombin. D. Citrate blood from a trauma patient was tested for endogenous activity. In the presence of CTI and absence of an initiator, α-TF antibody did not alter TG, indicating the absence of functional TF. α-FXIa antibody prolonged the lag phase and suppressed TG and α-FIXa antibody completely abolished TG, indicating the presence of active FXIa and FIXa. Packed Red Blood Cells (pRBC) In the absence of an initiator, no TG was observed in pRBC/CTI reconstituted with PPP. The addition of 5 pM TF led to TG after ~8 min with a peak value slightly lower than in blood (described in A).The addition of phospholipids did not change the TG profile. Conclusions. This continuous whole blood TG assay requires 15 µL of blood for a triplicate analysis of a single condition and has the potential for the evaluation of TG in disorders relevant to blood coagulation and for the monitoring of treatments administered in response to these disorders. Disclosures Mann: Haematologic Technologies, Inc.: Employment, Equity Ownership; Baxter: Consultancy; Bayer: Consultancy; Biogen IDEC: Consultancy; CSL Behring: Consultancy; Merck: Consultancy; Pfizer: Consultancy; The Medicines Company: Consultancy; XO1: Consultancy; Vascular Solutions: Consultancy; Stago: Consultancy.

Platelets and Phospholipids in Tissue Factor-initiated Thrombin Generation

2001 ◽  
Vol 86 (08) ◽  
pp. 660-667 ◽  
Author(s):  
Saulius Butenas ◽  
Richard Branda ◽  
Cornelis van ’t Veer ◽  
Kevin Cawthern ◽  
Kenneth Mann
Keyword(s):  
Platelet Count ◽  
Tissue Factor ◽  
Whole Blood ◽  
Thrombin Generation ◽  
Phase 1 ◽  
Platelet Counts ◽  
Initiation Phase ◽  
Platelet Concentration ◽  
American Society

SummaryThe influence of platelets on tissue factor (TF)-initiated thrombin generation in a reconstituted model of blood coagulation and in whole blood was evaluated. No thrombin generation was observed over 15 min in the reconstituted model when either TF or platelets and phospholipids were omitted. At 25 pM TF, the rates of thrombin generation were platelet and PCPS concentration-dependent and achieved maximum (1.0 nM/s) in the physiological range of platelet concentration. Similar rates were achieved in the absence of platelets when 1-2 μM phospholipid was used. However, the maximum rates of thrombin generation (5.2-6.0 nM/s) and the shortest initiation phase (1 min) were attained between 25 and 100 μM phospholipid. In the reconstituted model, an increase in platelet concentration from 0.125 × 108/ml to 0.5 × 108/ml decreased the duration of the initiation phase (in the absence of phospholipids) from 4.3 min to 2 min. Further increases in platelet concentration did not affect this phase. Sequential whole blood studies were conducted in blood of a chemotherapy patient who developed reduced platelet counts. The TF (12.5 pM) initiated clotting of patient’s blood was accelerated from ~10 min to 5 min when the platelet concentration increased from 0.05 × 108/ml to 0.11 × 108/ml. Clotting times were essentially unchanged for platelet concentrations exceeding 0.5 × 108/ml (range 0.5-3.1 × 108/ml). Similarly, clotting of whole blood obtained from healthy volunteers was not affected by the platelet count, which varied from 1.5 × 108/ml to 3.1 × 108/ml (4.0 ± 0.5 min). The data obtained in both models are consistent with in vivo observations that clinical bleeding is most likely to occur at platelet counts <0.1 × 108/ml.Portions of this work were presented at the 40th Annual Meeting of the American Society of Hematology, December 4-8, 1998, Miami Beach, Florida (abstracts #140 and #738).

Mechanism Of Peak Protraction In Thrombin Generation After Administration Of Direct Factor Xa Inhibitors

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1141-1141
Author(s):  
Saartje Bloemen ◽  
Bas De Laat ◽  
H. Coenraad Hemker ◽  
Raed Al Dieri
Keyword(s):  
Tissue Factor ◽  
Whole Blood ◽  
Thrombin Generation ◽  
Platelet Rich Plasma ◽  
Factor Xa ◽  
Strong Inhibition ◽  
Intrinsic Pathway ◽  
Extrinsic Pathway ◽  
Double Peak ◽  
Platelet Poor Plasma

Abstract Introduction Thrombin generation testing is becoming an increasingly important tool to measure the variables of the clotting system. Under normal conditions the curve is bell-shaped, yet after addition of direct, reversible factor Xa (FXa) inhibitors, the shape of the curve changes. Direct FXa inhibitors cause a strong inhibition (in vitro and ex vivo) on the peak, whereas the ETP is affected to a smaller extent, due to a protraction of the thrombin generation process. This leads to a long plateau or a ‘double peak’. Aim To investigate the mechanism behind the protraction of the thrombin generation curve. Materials & Methods Thrombin generation (TG) was determined by using Calibrated Automated Thrombinography (CAT) in platelet poor plasma (PPP), platelet rich plasma and whole blood. The effect of direct FXa inhibitors was investigated with both a parenteral (otamixaban) and an oral agent (rivaroxaban). The two inhibitors were tested at different concentrations, including the plasma concentrations that would be found after therapeutic dosing (otamixaban: 100, 250 and 400 nM and rivaroxaban: 200, 400 and 800 nM). Thrombin generation was performed in control plasma, factor VII (FVII), FVIII, FIX, FXI and TFPI (tissue factor pathway inhibitor) deficient plasmas, activated with tissue factor (TF), FIXa or kaolin. Results Thrombin generation curves in PPP in the presence of direct FXa inhibitors display a protracted shape with a strong inhibition of the peak. When the intrinsic pathway is bypassed (e.g. in FVIII or FIX deficient plasma), the first part of the peak is no longer present. When the extrinsic pathway is evaded (e.g. by activating with FIXa), the second part of the peak disappears. This leads us to believe that when a direct FXa inhibitor is present, the first part of thrombin generation is due to the intrinsic tenase complex and the second part can be attributed to the extrinsic tenase (prothrombinase). When TFPI deficient plasma is activated with TF, the curve returns to its normal bell-shape. So, TFPI is an important contributor to the second peak. When TG was activated with kaolin, we could not distinguish an effect. In platelet rich plasma and whole blood, a stronger effect on peak height than on ETP is found, however the distinct shape that can be discerned in platelet poor plasma is not seen. Conclusions Addition of direct FXa inhibitors to PPP results in a protracted TG curve, which could also be described as a double peak. The first peak can be attributed to the actions of the intrinsic pathway (FVIII and FIX), the second part of the peak is due to mechanisms in the extrinsic pathway. The protraction of the peak can also be restored by activating TFPI deficient plasma with TF, so this also contributes to the effect of the extrinsic pathway. The inhibition of the negative feedback on the TF-FVIIa complex by the TFPI-FXa complex will prevent the shutting off of the extrinsic pathway. Disclosures: No relevant conflicts of interest to declare.

Stability Of Thrombin Generation In Cirrhotic Patients

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2361-2361
Author(s):  
Thomas Sinegre ◽  
Armand Abergel ◽  
Géraldine Lamblin ◽  
Marc G Berger ◽  
Aurélien Lebreton
Keyword(s):  
Protein C ◽  
Thrombin Generation ◽  
Activated Protein C ◽  
Final Concentration ◽  
Control Group ◽  
Healthy Controls ◽  
Thrombotic Risk ◽  
Compensated Cirrhosis ◽  
Cirrhotic Patients ◽  
The Stability

Abstract Introduction Cirrhotic patients have a significant impairment of hemostasis. Most procoagulant and anticoagulant factors decrease but there are still exceptions such as factor VIII. Since many years, new approaches allowed to consider the cirrhotic patients as patients exposed to a thrombotic risk in many circumstances. In this concern, global tests as Thrombin Generation Assay (TGA) have an interest and many studies using thrombomodulin (TM) demonstrated a resistance to activated protein C (aPC) pathway. These assays evaluate the variations of both procoagulant factors and anticoagulant factors. However, the stability of thrombin generation in these patients is unknown. The aim of this study was to evaluate the stability of thrombin generation parameters (with and without TM and aPC) in cirrhotic patients compared to healthy volunteers. Patients and Methods 8 cirrhotic patients and 15 healthy controls were included in this study. For each patient, the follow-up period covers three months including three blood samples (D0, W6 and W12). Patients included in the study are cirrhotic (Prothrombin Time < 70% and / or liver dysmorphia and / or Fibroscan> 20 kPa and / or histology and / or association of portal hypertension and liver failure) of alcoholic origin. They are exclusively male, free of hepatocellular carcinoma and not anticoagulated. Thrombin Generation was performed in platelet-poor plasma in the presence / absence of TM (3.6 nmol/L) and in the presence/absence of aPC (1 nmol/L). The thrombin generation test was carried out according to the principle described by Hemker using Fluoroskan Ascent®, and the analysis software ThrombinoscopeTM. Reagent contains phospholipids (4 microM / L) and tissue factor (5 pM/L). TM is soluble thrombomodulin purified from rabbit lungs at final concentration 3.6 nmol/L. Human aPC was used at final concentration of 1 nmol / L (IC 50). The results are expressed as ratios: with and without TM and with and without PCa. Lag Time (LT), Endogenous Thrombin Potential (ETP), Peak Height (PH) and Time to Peak (TP) were analyzed. Statistical analysis compared the coefficient of variation (CV) between the control group and cirrhotic patients for each ratio (with and without TM with and without PCa) and that for each parameter by the Mann-Whitney test. Results Patients included in the study have chronic liver disease. Seven of them have compensated cirrhosis (CHILD A) and one cirrhotic patient has complicated cirrhosis (infection). The average age of cirrhotic is 50 (range 37-57). For both TM or aPC, all parameters of TGA are stable in cirrhotic patients compared with healthy controls. Regarding ETP, the most used parameter, CV of the ratio with and without TM is 12% (2-24) for the controls and 13% (2-22) for the cirrhotic patients while CV of the ratio with and without aPC is 11% (3-24) for the controls and 11% (6-21) for the cirrhotic patients. Similar matches were found for PH: 12% (3-25) for the controls versus 12% (1-22) for the cirrhotic patients with TM and 12% (1-20) for the controls versus 11% (5-19) for the cirrhotic patients. Similar results were found for LT and TP. Conclusions The Thrombin generation - performed with Thrombomodulin and activated Protein C and expressed with a ratio - in patient with cirrhosis is similarly stable than the thrombin generation in the control population. These observations can be used in the management and the design of further studies involving cirrhotic patients. Now, it is possible to follow the cirrhotic patients with TGA in order to detect the occurrence of a hypercoagulability state. However patients included in the study were mostly compensated cirrhosis. Further studies are needed to evaluate the stability of thrombin generation in the other subgroups of cirrhotic patients. Disclosures: No relevant conflicts of interest to declare.

Increased Thrombin Generation and Fibrin Formation In Premature Aging BMAL1 Deficient Mice

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3575-3575 ◽  
Author(s):  
Marisa Ninivaggi ◽  
Hilde Kelchtermans ◽  
Marijke Kuipers ◽  
Bianca Hemmeryckx ◽  
Johan Heemskerk ◽  
...  
Keyword(s):  
Whole Blood ◽  
Thrombin Generation ◽  
Target Genes ◽  
Conflicts Of Interest ◽  
Premature Aging ◽  
Specific Substrate ◽  
Wild Type ◽  
Thrombotic Risk ◽  
Increased Risk ◽  
Deficient Mice

Abstract Introduction The occurrence of venous thromboembolism is strongly age-dependent and has a substantial morbidity and mortality. While the incidence is<1 per 1000 per year up to an age of 50, it thereafter increases rapidly end exponentially. The cause of this increased risk is still unclear, however immobility, decreased muscular tone, aging of the veins and the presence of other or more risk factors and diseases than young individuals all contribute to this increased risk. Whether age-induced circulatory changes and/or increased levels of blood coagulation levels are responsible for the increased thrombogenicity remains unclear. We recently developed a method in which these problems were overcome enabling the measurement of thrombin generation (TG) in a small aliquot of blood. The advantage of the use of this whole blood assay is that the small volume enables us to apply this assay to mice and the use of whole blood enables us to include the effect of blood cells on TG. Objectives By modifying the classic plasma Calibrated Automated Thrombogram (CAT) assay, we were able of measuring TG in whole blood in mice. The objective was to validate this assay in mice blood and to examine the rate and extent TG in a mouse model of premature aging. Methods TG was assayed in 20- to 28-week-old brain and muscle ARNT-like protein-1 (Bmal1)-deficient (knockout, KO) mice and wild-type (WT) littermates. The Bmal1-KO mice have an impaired circadian behavior and demonstrate loss of rhythmicity in the expression of their target genes. They are known to have a reduced lifespan and display symptoms of premature aging. Mice blood samples were taken from the orbital sinus using a capillary anticoagulated with citrate (3.8%). Coagulation and therefore TG was initiated with a solution containing calcium, tissue factor (TF) and a thrombin specific substrate. Varying the TF concentration revealed an optimal of 0.5 pM. At the end of the TG assay, the samples were fixated, prepared for and analysed by scanning electron microscopy (SEM). Results The intra-assay variations (CV%) in mice blood of the endogenous thrombin potential (ETP), peak height, lagtime, time-to-peak and velocity index were 10% or less (n=24). The whole blood TG test showed that Bmal1-KO mice have a significantly (p<0.001) higher ETP (437±7 nM.min; mean±SD, n=5) when compared with wild type mice (ETP=220±45 nM.min; mean±SD, n=5). The peak heights also differed significantly (p=0.027). However, differences in lagtime, TTP and velocity index were not significantly different (p-values were respectively 0.45, 0.15 and 0.74). Applying SEM we found that Bmal1 deficient mice display a more dense fibrin network with smaller pores compared to WT-mice. Conclusions The whole blood TG assay in mice blood revealed to be reproducible and we demonstrated that aging Bmal1-KO mice have a hypercoagulable state when compared with WT mice indicating that a higher thrombotic risk in elderly is partially due to a more procoagulation state. Disclosures: No relevant conflicts of interest to declare.

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