Thrombin generation: phenotypic quantitation

Summary.  An individual's ability to generate thrombin following tissue factor stimulus was evaluated in 13 healthy male donors in a 6-month study. Thrombin generation in whole blood collected by phlebotomy, contact pathway suppressed by the presence of 100 µg mL⁻¹ corn trypsin inhibitor, was initiated by the addition of 5 p m tissue factor/10 n m phospholipid. Reactions were quenched at 20 min by the addition of an ethylenediaminetetraacetic acid (EDTA), benzamidine, FPRck cocktail. Thrombin generation was determined by an ELISA for thrombin–antithrombin III (TAT) complex formation. Results showed that the levels of TAT observed varied from 245 to 775 n m . Thrombin production was consistent within each individual, CVi = 11.6%, but varied significantly within the group, CVg = 25.2%, and correlated inversely with an individual's clotting time ( r  = − 0.54, P  = 0.07). No correlations were individually observed between TAT and C-reactive protein, antithrombin III, factors II, V, VII, VIII, IX and X, fibrinogen and prothrombin time. However, computer simulations, which integrated each individual's coagulation factor levels using the Speed Rx method (Hockin et al. , J Biol Chem 2002; 277: 18322), predicted maximum active thrombin levels (ranging from calculated values of 220–500 n m ) consistent with the empirically determined values. Overall, these data suggest that thrombin generated in whole blood exclusively by tissue factor stimulation can be used as an integrative phenotypic marker to determine an individual's response to a tissue factor challenge.     

Thrombin generation in vascular tissue

BACKGROUND: Classically, it is thought that the vast majority of thrombin is generated on the surface of platelets, however, thrombotic events occur in patients despite treatment with potent antiplatelet agents. METHODS AND RESULTS: In freshly harvested left internal mammary artery (IMA) sections, addition of CaCl2 and platelet-poor plasma (PPP) were sufficient to stimulate a profound burst of thrombin and this effect was inhibited by antitissue factor antibodies. Ultracentrifugation of PPP to remove platelet microparticles had no effect on thrombin generation. Both the extrinsic and factor VIII-dependent pathways were necessary for IMA-supported thrombin generation as PPP derived from individuals deficient in factors V, VII, VIII or X did not support thrombin production. Small amounts of thrombin were generated utilizing factor IX (FIX)-deficient plasma, however, thrombin was not generated by aorta from FIX-deficient mice when FIX-deficient plasma was used. The addition of non-lipidated tissue factor (0.6 pM) and CaCl2 to actively proliferating cultured human aortic smooth muscle cells (SMC) resulted in a pronounced burst of thrombin generation occurring between 3 and 15 min after treatment. In the absence of tissue factor, thrombin was generated but at a slower rate and with a peak value 26% of that observed in the presence of tissue factor. CONCLUSION: Significant thrombin generation can occur on vascular tissue in the absence of platelets or platelet microparticles and on the surface of non-apoptotic SMC.     

Thrombin generation in patients with a first acute myocardial infarction

Summary. Background: Despite improved treatment options, myocardial infarction is still an important cause of morbidity and mortality. One of the contributing mechanisms in the acute myocardial infarction (AMI) is plasma hypercoagulability. Methods: We investigated hypercoagulability in 135 (first) patients with AMI using thrombin generation (TG) testing. TG testing was performed in plasmas, drawn upon admission and before medication administration, and subsequently after 4 days, 3 and 6 months. Further, we evaluated determinants of thrombin generation using multiple regression analysis of major coagulation proteins and inhibitors. Admission TG results were also related to 1‐year outcome: cardiovascular death, recurrent myocardial infarction, a second coronary intervention [percutaneous coronary intervention (PCI) or coronary artery bypass grafting (CABG)] and ischemic stroke. Results: At day 0, the TG parameters peak height, endogenous thrombin potential (ETP) and lag time were increased compared with a reference population. Peak height and lag time stayed persistently increased in patients. The lowest half of the ETP values was statistically not significantly associated with an occurrence of endpoints. The lowest half of the ETP values combined with the upper half of the D‐dimer values were associated with endpoints; odds ratio 5.8 (1.1–30.7). Tissue factor pathway inhibitor (TFPI) seems to be an important determinant of TG in AMI and healthy persons. Conclusions: TG reflects acute hypercoagulability during AMI and partly also in the 6‐month period after the acute event. TG shows a trend of an inverse association with risk of recurrent ischemic cardiovascular complications. Unraveling mechanisms in TG might improve our understanding of the pathophysiology of AMI and direct future improvements in medical care.     

Thrombin generation and heparin-induced thrombocytopenia

Summary.  Background : Heparin-induced thrombocytopenia (HIT) is a severe complication of heparin therapy. IgG antibodies targeting the platelet factor 4-heparin complex activate platelets and generate microparticles with procoagulant activity. Objectives : To determine whether the thrombin generation assay is capable of detecting procoagulant activity induced by patient platelet-poor plasma (PPP) in donor platelet-rich plasma (PRP). Patients and methods : We explored two groups of patients; group 1 ( n  = 23): patients with a positive clinical and biological diagnosis of HIT; group 2 ( n  = 25): patients with a negative clinical and biological diagnosis of HIT. Mixtures of donor PRP and patient PPP (1:1) were incubated either with unfractionated heparin 0.2 U mL⁻¹ or with physiological saline. Thrombin generation was assessed by calibrated thrombinography. The effect of heparin on the mixtures was evaluated according to the ratio of the values with and without heparin (wH/woH) of the five thrombogram parameters. Results : With low heparin concentrations, plasma of group 1 activates donor platelets and generates procoagulant activity. A set of three ratios outside the cut-off values corresponds to the 'HIT thrombogram profile', characterized by a highly specific aspect of the thrombogram wH in relation to the thrombogram woH. None of the group 2 patients presented a HIT thrombogram profile. The results of thrombinography correlate well with the results of the platelet aggregation test. Conclusion : Our studies illustrate the central paradox of HIT, namely enhancement of thrombin generation in the presence of heparin. The HIT thrombogram profile as it is defined in this study can detect the procoagulant activity of HIT IgG antibodies.     

Thrombin generation and venous thromboembolism

Venous thromboembolism is a chronic and potential fatal disease. Determination of recurrence risk is time-consuming and costly, and sometimes not feasible: many patients carry more than one risk factor, the relevance of some factors with regard to risk of recurrence is unknown, and existence of thus far unknown risk factors must be considered. A laboratory assay that measures multifactorial thrombophilia would be useful to identify patients at risk of thrombosis. The process of thrombin generation is the central event of the hemostatic process. Thrombin generation is increased in patients at risk of thrombosis including those with antithrombin deficiency or those who are taking hormonal contraceptives. Risk of first and recurrent venous thrombosis is higher in patients with increased thrombin generation. Thus, by use of a simple global marker of coagulation stratification of patients according to their risk of thrombosis is possible. Future studies are needed to improve the management of patients with VTE and increased thrombin generation.     

Thrombin generation profiles in deep venous thrombosis

BACKGROUND: Reliable markers and methods to predict risk for thrombosis are essential to clinical management. OBJECTIVE: Using an integrated approach that defines an individual's comprehensive coagulation phenotype might prove valuable in identifying individuals at risk for experiencing a thrombotic event. METHODS: Using a numerical simulation model, we generated tissue factor (TF) initiated thrombin curves using coagulation factor levels from the Leiden Thrombophilia Study population and evaluated thrombotic risk, by sex, age, smoking, alcohol consumption, body mass index (BMI) and oral contraceptive (OC) use. We quantitated the initiation, propagation and termination phases of each individuals' comprehensive TF-initiated thrombin generation curve by the parameters: time to 10 nm of thrombin, maximum time, level and rate (MaxR) of thrombin generated and total thrombin. RESULTS: The greatest risk association was obtained using MaxR; with a 2.6-fold increased risk at MaxR exceeding the 90th percentile. The odds ratio (OR) for MaxR was 3.9 in men, 2.1 in women, and 2.9 in women on OCs. The association of risk with thrombin generation did not differ by age (OR:2.8 OR:2.5), BMI (OR:2.9 OR:2.3) or alcohol use. In both numerical simulations and empirical synthetic plasma, OC use created extreme shifts in thrombin generation in both control women and women with a prior thrombosis, with a larger shift in thrombin generation in control women. This suggests an interaction of OC use with underlying prothrombotic abnormalities. CONCLUSIONS: Thrombin generation based upon the individual's blood composition is associated with the risk for thrombosis and may be useful as a predictive marker for evaluating thrombosis on an individual basis.     

Thrombin generation in morbid obesity: significant reduction after weight loss

Summary. Background: Patients with morbid obesity (MO; body mass index > 40 kg m^?2) suffer from an increased risk of cardiovascular disease, stroke, venous thromboembolism and all‐cause mortality. Objectives: Because weight loss by bariatric surgery reduces cardiovascular and all‐cause mortality, we hypothesized that the plasmatic clotting system might be involved in cardiovascular risk. Patients/Methods: Thirty‐six MO patients [mean age 42 (±13) years; 29 female) were investigated before and 2 years after bariatric surgery. Thrombin generation was measured with a commercially available assay (Technothrombin‐TGA,Technoclone). Metabolic parameters and parameters of the hemostatic system, such as tissue factor (TF), TF pathway inhibitor (TFPI), plasminogen activator inhibitor‐1 (PAI‐1) and prothrombinfragment 1.2 (F1.2), were determined. To investigate associations of changing parameters, deltas were calculated. Results: Metabolic parameters improved with a mean weight loss of 41 (±19) kg. Postoperatively, the lag phase was significantly extended compared with preoperative values [median (25th–75th percentile), 7 (4–12) vs. 12 (7–19) min, P = 0.005]. Peak thrombin decreased after weight loss from 345 (232–455) to 282 (111–388) nm (P = 0.015) and the area under the curve from 3962 (3432–5023) to 3227 (2202–4030) nm thrombin (P < 0.001). TF, PAI‐1 and F1.2 significantly decreased after weight loss. Analyses of the deltas showed a significant correlation between peak thrombin and total cholesterol (r = 0.50), triglycerides (r = 0.46) and HbA1c (r = 0.55). Moreover, an inverse correlation was found between insulin resistance and the lag phase (r = ?0.46). Conclusion: Thrombin generation, a marker of the overall coagulation potential, decreased significantly with weight reduction. This might, at least in part, explain the decreased risk of cardiovascular disease after bariatric surgery.     

TRANSFUSION PRACTICE: Toward extended phenotype matching: a new operational paradigm for the transfusion service

BACKGROUND: Conventional pretransfusion testing uses hemagglutination to ensure donor‐recipient compatibility for ABO/D status and recipient alloantibodies. While screening large numbers of donor units for multiple antigens by hemagglutination is impractical, novel methods of DNA analysis permit the rapid determination of an extended human erythrocyte antigen (xHEA) phenotype. A prospective observational study was conducted at four hospital transfusion services to test an alternative paradigm of identifying xHEA‐typed units for patients in three cohorts by utilizing DNA analysis and a novel inventory management model. STUDY DESIGN AND METHODS: xHEA typing of recipient samples and donor units of known ABO/D status was performed by HEA analysis (BeadChip, BioArray Solutions). xHEA‐typed units were assigned to pending transfusion requests using an inventory management system designed to simulate blood order processing. The fraction of requests fulfilled, or “fill fraction” (FF) was determined at four levels of matching stringency. RESULTS: For alloimmunized patients, all but one participating site observed an FF of more than 95% when matching for ABO, D, and known alloantibodies and an FF of more than 90% when additionally matching for C, c, E, e, and K; the site handling the most challenging requests still observed FFs of 62 and 51%, respectively. FF was found to correlate positively with the ratio of available donor units to units requested and negatively with the degree of recipient alloimmunization. CONCLUSION: This study demonstrates that substantial fill fractions can be achieved by selecting existing donor units for xHEA analysis and operating an inventory management system for efficient allocation of units to recipients.     

凝血酶生成试验在遗传性凝血因子缺陷症患者出血评估中的价值

目的 探讨遗传性凝血因子V(FV)、X(FX)和XI(FXI)缺陷及FV和凝血因子VⅢ(FVⅢ)联合缺陷(F5F8D)患者的血浆凝血因子活性、凝血酶生成曲线各参数和临床出血症状之间的关系.方法 采集遗传性FV(n=24)、FX(n=14)和FXI(n=18)缺陷及F5F8D(n=8)患者及携带者的外周血进行常规出凝血检...     

Thrombin generation before and after multicomponent blood collection

BACKGROUND: Apheresis technology has made tremendous progress up to the development of automated blood component collection, which offers increased efficiency in donor blood use, but the concern about the contact of donor blood with artificial surfaces remains. Activation of the hemostatic system is a major issue in this context and is controversial. The aim of this study was to estimate the effect of apheresis on continuous thrombin generation (TG), representing a new tool to examine the overall function of the plasmatic clotting system. STUDY DESIGN AND METHODS: Twenty-six voluntary blood donors, fulfilling the law requirement for apheresis donation, participated in the study. Two units of platelets (6 x 10(11)) and 1 unit of red cells (250 mL; hematocrit level, 80%) were collected using two types of cell separators (Amicus, Fenwal, Inc.; and Trima Accel, Gambro BCT). Each donor underwent collection on both apheresis systems with at least 8 weeks in between. Samples of blood were collected before, immediately after, and 48 hours after apheresis. TG was measured using a slow fluorogenic substrate by means of calibrated automated thrombography (CAT). RESULTS: CAT data changed only slightly, and no significant changes were seen before, immediately after, and 48 hours after apheresis (p > 0.05). The variables did not differ significantly between the two different apheresis systems (p > 0.05). CONCLUSION: Using a CAT-based technique, no change in variables of continuous TG were observed, suggesting that multicomponent blood collection did not lead to severe alterations in the hemostatic system of the donors.     

Thrombin generation before and after multicomponent blood collection

The development of apheresis technology has increased efficiency in donor blood use by collecting specific blood components in several combinations. The question of donor safety raised by the contact of donor blood with foreign, only in part biocompatible surfaces remains. The aim of this study was to estimate the effect of multicomponent blood collection on thrombin generation performing an overall function test of coagulation. DONORS, METHODS: 26 blood donors were included. Per apheresis two units of platelets and one unit of RBCs were collected by two cell separators (Amicus and Trima Accel). Each donor underwent the procedure on both apheresis systems. Samples were collected before, immediately after, and 48 hours after apheresis. Thrombin generation was measured by means of calibrated automated thrombography (CAT). RESULTS: CAT-data changed only slightly and no significant changes were seen before, immediately after, and 48 hours after apheresis. The parameters did not differ significantly between the two different apheresis devices. CONCLUSION: No change in parameters of continuous thrombin generation occurred, suggesting that apheresis did not lead to severe alterations in the haemostatic system.     

TRANSFUSION PRACTICE: A clinical scenario‐based survey of transfusion decisions for intensive care patients with delayed weaning from mechanical ventilation

BACKGROUND: Despite evidence supporting the use of restrictive hemoglobin (Hb) transfusion triggers in critically ill patients, translation of this evidence into practice remains inconsistent. It was hypothesized that clinicians believe that longer‐term ventilated patients require a higher Hb, particularly when ischemic heart disease coexists. STUDY DESIGN AND METHODS: A scenario was developed describing an anemic patient recovering from multiple organ failure, but failing weaning trials after 6 days of mechanical ventilation. Clinicians were asked to state their Hb transfusion trigger and target Hb range assuming no history of ischemic heart disease (Scenario 1), known stable chronic ischemic heart disease (Scenario 2), or evidence of myocardial ischemia during weaning trials (Scenario 3). A prospective cross‐sectional postal survey of clinicians practicing intensive care in the United Kingdom was undertaken. RESULTS: A total of 184 responses were obtained (52% response rate), which varied widely. Median (first, third quartile) transfusion trigger Hb levels were 8 (7, 8), 9 (8, 9.5), and 9.5 (9, 9.5) g/dL for Scenarios 1 to 3, respectively (p < 0.001 across and between each group). The target Hb was more than 9 g/dL for 47, 80, and 94% of respondents for Scenarios 1 to 3, respectively, and more than 10 g/dL for 14, 44, and 65% of respondents for Scenarios 1 to 3, respectively (p < 0.001 across the groups). CONCLUSIONS: In response to scenarios, clinicians in the United Kingdom believe that a more liberal transfusion practice is required for patients failing weaning trials after 6 days of mechanical ventilation than the current evidence base supports.     

TRANSFUSION PRACTICE: Coagulopathy screening and early plasma treatment for the prevention of intraventricular hemorrhage in preterm infants

BACKGROUND: Despite the improvement in the assistance and treatment of preterm infants, intraventricular hemorrhage (IVH) remains a frequent complication in these patients. Our aim was to demonstrate the hypothesis that a coagulopathy screening and the early treatment with fresh‐frozen plasma (FFP) of proven coagulopathy may contribute to decrease the occurrence of IVH in infants with gestational age of less than 29 weeks. STUDY DESIGN AND METHODS: This study compared two cohorts of infants who received FFP (10 mL/kg) after the evidence of pathologic coagulation tests performed within 2 hours after birth (screening group, n = 127) or after the development of bleedings and evidence of pathologic coagulation tests (no‐screening group, n = 91). RESULTS: The screening strategy decreased the relative risk (RR) of developing IVH (RR = 0.65; 95% confidence interval, 0.44‐0.98) compared to no‐screening strategy, but the effect was limited to infants born at 23 to 26 weeks of gestation in whom screening strategy lowered IVH occurrence (34.5% vs. 61.1%, p = 0.008). CONCLUSIONS: A coagulopathy screening strategy decreases the risk of developing IVH in preterm infants but this effect is limited to infants born at 23 to 26 weeks of gestation.     

TRANSFUSION PRACTICE: Liver donor's age and recipient's serum creatinine predict blood component use during liver transplantation

BACKGROUND: Excessive use of blood components during liver transplantation should be avoided because it has been associated with poor outcomes and it may stress blood bank resources. STUDY DESIGN AND METHODS: To determine preoperative predictors of excessive transfusion requirements in patients undergoing liver transplantation, the clinical records of 126 consecutive adult patients undergoing primary liver transplantation were retrospectively reviewed. Outcome variables included number of red blood cells (RBCs), plasma, and plateletpheresis components intraoperatively transfused. Univariate analyses of the following predictor variables were performed: recipient age, sex, ethnicity, height/weight, Model for End Stage Liver Disease score, year of transplant, previous abdominal surgery, hepatoma, wait‐list time, standard recipient laboratory values obtained immediately before transplantation, cold ischemia time, donor age, sex, and height/weight. Multivariate analysis using logistic regression was used to build a model that best predicted how many blood components should be available before transplant. RESULTS: Donor age of more than 50 years old (odds ratio [OR], 2.8 95% confidence interval [CI], 1.3‐6.0), and recipient serum creatinine (SCr) level of more than 1.3 mg/dL (OR, 3.8 95% CI, 1.6‐8.9) were the only variables found to be predictive of RBC use in multivariate analysis. This model accurately predicted the use of more than 10 units of RBCs 79% of cases. Having both adverse factors present resulted in using more than one box in 80% of cases as compared to 44% of cases where only one or no adverse factor was present (p = 0.002). Further analyses showed a direct correlation between the number of RBCs transfused and plasma (r = 0.93) and plateletpheresis components (r = 0.74) transfused. [Corrections added after online publication 22‐Jul‐2009: OR updated from 3.8 to 2.8; CI from 1.6‐8.9 to 1.3‐6.0; OR from 2.8‐3.8.] CONCLUSION: Liver donor's age and recipient's SCr are important in preoperatively predicting blood use during liver transplantation.     

Thrombin generation in haemophilia A patients with factor VIII inhibitors after infusion of recombinant factor VIIa

Background Development of factor VIII inhibitors is a serious complication in haemophilia A patients. Recombinant factor VIIa (rVIIa) is clinically effective, but its effects on haemostatic system need still to be fully elucidated. Material and methods In an open controlled study, we measured thrombin generation (peak thrombin) in venous blood and prothrombin fragment F1 + 2 (F1 + 2) and d ‐dimer in venous and in shed blood in five haemophilia A patients with inhibitors before and after rVIIa infusion. A total of five healthy individuals who did not receive rVIIa served as controls. Results At baseline, patients had lower mean (min‐max) peak thrombin levels than controls [0·12 (0·0–0·6) vs. 186·9 (116·0–254·4) nM, P = 0·001]. After infusion, peak thrombin levels increased in average to 40·7 (28·3–51·6) nM, which translates into 80·2% (95% CI 65·4–88·6%) lower levels compared to that of controls. Mean (min‐max) F1 + 2 levels in venous blood did not differ significantly between patients and controls [160·7 (89·8–331·3) vs. 160·8 (104·4–242·3) pmol L^?1], but increased in average (min‐max) by 39·4% (14·1–58·5%) after infusion. In blood emerging from incisions made to determine the bleeding (shed blood), F1 + 2 levels were lower in patients than controls [1383·3 (906·4–2044·6) vs. 2981·7 (1610·0–4539·6) pmol L^?1; P = 0·04], but were not affected by rVIIa; d ‐dimer levels were significantly higher in haemophiliacs than in controls and remained unchanged after infusion. Conclusions Haemophilia A patients with factor VIII inhibitors have low thrombin generation. After rVIIa, the extent of coagulation activation as measured by levels of F1 + 2 is increased, but thrombin generation is restored to only 20%. Peak thrombin levels could reflect the effects of rVIIa on coagulation mechanisms, and their relevance with regard to the clinical coagulation defect of haemophilia A patients with factor VIII inhibitors might be evaluated.