Longitudinal assessment of coagulation potential before, during, and following an in vitro fertilization cycle.

INTRODUCTION: The association between estrogen and hypercoagulability is well-established but little is known about coagulation dynamics during IVF. Our goal was to measure coagulation potential prior to, during, and following an IVF cycle and to investigate differences by conception outcome. MATERIALS AND METHODS: Patients undergoing IVF with fresh embryo transfer at a single academic center using oral contraceptive pills for cycle batching underwent evaluation of thrombin generation using the calibrated automated thrombogram at multiple points during the IVF cycle. Multiple thrombin generation parameters were compared across timepoints and by IVF cycle outcome using ANOVA repeated measures analysis. RESULTS: Of the 17 patients included, 11 conceived. There was a significant increase in peak and total thrombin generation in the entire cohort between the pre-treatment natural follicular phase and following a short course of oral contraceptive pills used for cycle batching. Further increase in these parameters was seen at the time of oocyte retrieval. In the pre-treatment natural follicular phase, patients who conceived had lower peak thrombin generation. There were changes throughout the cycle for factors II, V, VIII, X, XI, XII, antithrombin, and tissue factor pathway inhibitor. Only Factor XI was distinguishable by conception status; values were lower at all visits in patients who conceived. CONCLUSION: Increases in coagulation potential are seen in patients undergoing IVF following a short course of oral contraceptive pills for cycle batching and continue during controlled ovarian hyperstimulation. Those who conceived were seen to have lower peak thrombin generation in the pre-treatment natural follicular phase.

Comparison of Rapid-, Kaolin-, and Native-TEG Parameters in Burn Patient Cohorts With Acute Burn-induced Coagulopathy and Abnormal Fibrinolytic Function.

Although use of thromboelastography (TEG) to diagnose coagulopathy and guide clinical decision-making is increasing, relative performance of different TEG methods has not been well-defined. Rapid-TEG (rTEG), kaolin-TEG (kTEG), and native-TEG (nTEG) were performed on blood samples from burn patients presenting to a regional center from admission to 21 days. Patients were categorized by burn severity, mortality, and fibrinolytic phenotypes (Shutdown [SD], Physiologic [PHYS], and Hyperfibrinolytic [HF]). Manufacturer ranges and published TEG cutoffs were examined. Concordance correlations (Rc) of TEG parameters (R, α-angle, maximum amplitude [MA], LY30) measured agreement and Cohen's Kappa (κ) determined interclass reliability. Patients (n = 121) were mostly male (n = 84; 69.4%), with median age 40 years, median TBSA burn 13%, and mortality 17% (n = 21). Severe burns (≥40% TBSA) were associated with lower admission α-angle for rTEG (P = .03) and lower MA for rTEG (P = .02) and kTEG (P = .01). MA was lower in patients who died (nTEG, P = .04; kTEG, P = .02; rTEG, P = .003). Admission HF was associated with increased mortality (OR, 10.45; 95% CI, 2.54-43.31, P = .001) on rTEG only. Delayed SD was associated with mortality using rTEG and nTEG (OR 9.46; 95% CI, 1.96-45.73; P = .005 and OR, 6.91; 95% CI, 1.35-35.48; P = .02). Admission TEGs showed poor agreement on R-time (Rc, 0.00-0.56) and α-angle (0.40 to 0.55), and moderate agreement on MA (0.67-0.81) and LY30 (0.72-0.93). Interclass reliability was lowest for R-time (κ, -0.07 to 0.01) and α-angle (-0.06 to 0.17) and highest for MA (0.22-0.51) and LY30 (0.29-0.49). Choice of TEG method may impact clinical decision-making. rTEG appeared most sensitive in parameter-specific associations with injury severity, abnormal fibrinolysis, and mortality.

A natural history study of coagulopathy in a porcine 40% total body surface area burn model reveals the time-dependent significance of functional assays.

Various studies have reported discordant results on the magnitude and direction of burn-induced coagulopathy (BIC), which has recently been associated with multiple organ dysfunction syndrome (MODS) and death. The increased mechanistic understanding of BIC is due, in part, to novel assays that have expanded the armamentarium beyond traditional tests like PT and aPTT. Still, BIC is a dynamic process, and the progression is difficult to define in the thermally-injured. To this end, we aimed to enhance the understanding of burn-induced coagulation abnormalities by employing functional assessments of platelet aggregation, viscoelastic kinetics, and thrombin generation in an extensive burn model in swine. Anesthetized Yorkshire pigs sustained 40% total body surface area (TBSA) full-thickness contact burns and recovered in metabolic cages. Blood was collected at baseline (BL), as well as 6, 24, and 48 h after injury. A significant effect of burn (P < 0.0001) was seen on platelets, with mild thrombocytopenia apparent at 24 h. While slight decreases in aPTT were not significant, rotational thromboelastometry (ROTEM) analysis revealed hypercoagulation 6 and 24 h after burn by a decreased clotting time. Maximum clot firmness increased after burn, but was not statistically significant until 48 h. Hypercoagulation was not supported by platelet aggregation, as the response to ADP was greatly and persistently diminished, and the response to collagen was unchanged. Endogenous thrombin potential was significantly reduced at 6 and 24 h after burn (P < 0.0001), and also correlated with a number of ROTEM parameters and collagen-induced platelet aggregation. In contrast, PT was not correlated with other measured parameters. Taken together, novel coagulation parameters may be more sensitive than PT in characterizing coagulopathy in the setting of burns. The data presented herein makes initial strides to report the natural history of several of these variables over time in a large animal model of extensive burns, indicating early hypercoagulability followed by hypocoagulation. Future work will elucidate the effects of standard of care.

Assessing Factor V Antigen and Degradation Products in Burn and Trauma Patients.

INTRODUCTION: Proposed mechanisms of acute traumatic coagulopathy (ATC) include decreased clotting potential due to factor consumption and proteolytic inactivation of factor V (FV) and activated factor V (FVa) by activated protein C (aPC). The role of FV/FVa depletion or inactivation in burn-induced coagulopathy is not well characterized. This study evaluates FV dynamics following burn and nonburn trauma. METHODS: Burn and trauma patients were prospectively enrolled. Western blotting was performed on admission plasma to quantitate levels of FV antigen and to assess for aPC or other proteolytically derived FV/FVa degradation products. Statistical analysis was performed with Spearman's, Chi-square, Mann-Whitney U test, and logistic regression. RESULTS: Burn (n = 60) and trauma (n = 136) cohorts showed similar degrees of FV consumption with median FV levels of 76% versus 73% (P = 0.65) of normal, respectively. Percent total body surface area (TBSA) was not correlated with FV, nor were significant differences in median FV levels observed between low and high TBSA groups. The injury severity score (ISS) in trauma patients was inversely correlated with FV (ρ = -0.26; P = 0.01) and ISS ≥ 25 was associated with a lower FV antigen level (64% versus. 93%; P = 0.009). The proportion of samples showing proteolysis-derived FV was greater in trauma than burn patients (42% versus. 16%; P = 0.0006). CONCLUSIONS: Increasing traumatic injury severity is associated with decreased FV antigen levels, and a greater proportion of trauma patient samples exhibit proteolytically degraded FV fragments. These associations are not present in burns, suggesting that mechanisms underlying FV depletion in burn and nonburn trauma are not identical.

Placental Maternal Vascular Malperfusion is Associated with Prepregnancy and Early Pregnancy Maternal Cardiovascular and Thrombotic Profiles.

Characteristics of maternal vascular malperfusion (MVM) are frequently observed in placentas from pregnancies impacted by preeclampsia, intrauterine growth restriction, preterm labor, and intrauterine fetal demise. We sought to evaluate the associations of features of MVM with subclinical measures of cardiovascular health and coagulation potential in healthy young women. Sixty-three healthy young women were recruited and assessed prior to pregnancy on cycle day 9 ± 4, at gestational age 90 ± 6 of early pregnancy, and gestational age 216 ± 5 of late pregnancy. Women were assessed for plasma volume, blood pressure, response to volume loading, cardiac output, and uterine hemodynamics. Platelet-poor plasma was collected to assess thrombin generation on a subset of 33 women at all time points. Following delivery, placentas were collected and analyzed for evidence of MVM. Thrombin generation (TG) was evaluated in the presence of tissue factor (TF) with and without recombinant soluble thrombomodulin (TM). For each, we compared TG lagtime, peak level, and endogenous thrombin potential (ETP). Comparisons were made between dichotomized presence and absence of each individual feature of MVM and cardiovascular and coagulation features. Mean ± standard deviation are presented. Women were 31 ± 4 years of age, body mass index of 24 ± 5 kg/m2, 86% white race, and 80% nulliparous. MVM occurred in 70% of placentas, with infarcts and agglutination (44%), decidual arteriopathy (40%), accelerated villous maturation (32%), placental hypoplasia (29%), and distal villous hypoplasia (17%) documented. Decidual arteriopathy and distal villous hypoplasia were associated with prepregnancy maternal physiology, including decreased plasma volume and subclinical cardiovascular variations. All assessed MVM characteristics had identifiable early pregnancy physiologic characteristics consistent with altered cardiovascular function and decreased uterine response to pregnancy when compared with women who did and did not develop MVM. Accelerated villous maturation was the only MVM feature to differ by thrombin generation parameters in early pregnancy. Thrombin generation potential and blood pressure were elevated in late pregnancy in women who developed decidual arteriopathy. Prepregnancy health status and adaptation to pregnancy play important roles in pregnancy outcomes. Both cardiovascular health and thrombin generation potential may influence early placentation. Longitudinal assessment of subclinical maternal factors may allow for better understanding of the etiologies of MVM lesions, as well as allow for identification of a timeline of the origins of placental pathologies.

Inhalation Injury Is Associated With Endotheliopathy and Abnormal Fibrinolytic Phenotypes in Burn Patients: A Cohort Study.

Burn injury is associated with endothelial dysfunction and coagulopathy and concomitant inhalation injury (IHI) increases morbidity and mortality. The aim of this work is to identify associations between IHI, coagulation homeostasis, vascular endothelium, and clinical outcomes in burn patients. One hundred and twelve patients presenting to a regional burn center were included in this retrospective cohort study. Whole blood was collected at set intervals from admission through 24 hours and underwent viscoelastic assay with rapid thromboelastography (rTEG). Syndecan-1 (SDC-1) on admission was quantified by ELISA. Patients were grouped by the presence (n = 28) or absence (n = 84) of concomitant IHI and rTEG parameters, fibrinolytic phenotypes, SDC-1, and clinical outcomes were compared. Of the 112 thermally injured patients, 28 (25%) had IHI. Most patients were male (68.8%) with a median age of 40 (interquartile range, 29-57) years. Patients with IHI had higher overall mortality (42.68% vs 8.3%; P < .0001). rTEG LY30 was lower in patients with IHI at hours 4 and 12 (P < .05). There was a pattern of increased abnormal fibrinolytic phenotypes among IHI patients. There was a greater proportion of IHI patients with endotheliopathy (SDC-1 > 34 ng/ml) (64.7% vs 26.4%; P = .008). There was a pattern of increased mortality among patients with IHI and endotheliopathy (0% vs 72.7%; P = .004). Significant differences between patients with and without IHI were found in measures assessing fibrinolytic potential and endotheliopathy. Mortality was associated with abnormal fibrinolysis, endotheliopathy, and IHI. However, the extent to which IHI-associated dysfunction is independent of TBSA burn size remains to be elucidated.

Circulating Syndecan-1 and Tissue Factor Pathway Inhibitor, Biomarkers of Endothelial Dysfunction, Predict Mortality in Burn Patients.

OBJECTIVE: The aim of this study is to evaluate the association between burn injury and admission plasma levels of Syndecan-1 (SDC-1) and Tissue Factor Pathway Inhibitor (TFPI), and their ability to predict 30-day mortality. BACKGROUND: SDC-1 and TFPI are expressed by vascular endothelium and shed into the plasma as biomarkers of endothelial damage. Admission plasma biomarker levels have been associated with morbidity and mortality in trauma patients, but this has not been well characterized in burn patients.Methods: This cohort study enrolled burn patients admitted to a regional burn center between 2013 and 2017. Blood samples were collected within 4 h of admission and plasma SDC-1 and TFPI were quantified by ELISA. Demographics and injury characteristics were collected prospectively. The primary outcome was 30-day in-hospital mortality. RESULTS: Of 158 patients, 74 met inclusion criteria. Most patients were male with median age of 41.5 years and burn TBSA of 20.5%. The overall mortality rate was 20.3%. Admission SDC-1 and TFPI were significantly higher among deceased patients. Plasma SDC-1 >34 ng/mL was associated with a 32-times higher likelihood of mortality [OR: 32.65 (95% CI, 2.67-399.78); P = 0.006] and a strong predictor of mortality (area under the ROC [AUROC] 0.92). TFPI was associated with a nine-times higher likelihood of mortality [OR: 9.59 (95% CI, 1.02-89.75); P = 0.002] and a fair predictor of mortality (AUROC 0.68). CONCLUSIONS: SDC-1 and TFPI are associated with a higher risk of 30-day mortality. We propose the measurement of SDC-1 on admission to identify burn patients at high risk of mortality. However, further investigation with a larger sample size is warranted.

Computational model of tranexamic acid on urokinase mediated fibrinolysis.

Understanding the coagulation process is critical to developing treatments for trauma and coagulopathies. Clinical studies on tranexamic acid (TXA) have resulted in mixed reports on its efficacy in improving outcomes in trauma patients. The largest study, CRASH-2, reported that TXA improved outcomes in patients who received treatment prior to 3 hours after the injury, but worsened outcomes in patients who received treatment after 3 hours. No consensus has been reached about the mechanism behind the duality of these results. In this paper we use a computational model for coagulation and fibrinolysis to propose that deficiencies or depletions of key anti-fibrinolytic proteins, specifically antiplasmin, a1-antitrypsin and a2-macroglobulin, can lead to worsened outcomes through urokinase-mediated hyperfibrinolysis.

Burn-Induced Coagulopathies: a Comprehensive Review.

Burn-induced coagulopathy is not well understood, and consensus on diagnosis, prevention, and treatments are lacking. In this review, literature on burn-induced (and associated) coagulopathy is presented along with the current understanding of the effects of burn injury on the interactions among coagulation, fibrinolysis, and inflammation in the acute resuscitative phase and reconstructive phase of care. The role of conventional tests of coagulopathy and functional assays like thromboelastography or thromboelastometry will also be discussed. Finally, reported methods for the prevention and treatment of complications related to burn-induced coagulopathy will be reviewed.

Utilizing Plasma Composition Data to Help Determine Procoagulant Dynamics in Patients with Thermal Injury: A Computational Assessment.

INTRODUCTION: The development of methods that generate individualized assessments of the procoagulant potential of burn patients could improve their treatment. Beyond its role as an essential intermediate in the formation of thrombin, factor (F)Xa has systemic effects as an agonist to inflammatory processes. In this study, we use a computational model to study the FXa dynamics underlying tissue factor-initiated thrombin generation in a small cohort of burn patients. MATERIALS AND METHODS: Plasma samples were collected upon admission (Hour 0) from nine subjects (five non-survivors) with major burn injuries and then at 48 hours. Coagulation factor concentrations (II, V, VII, VIII, IX, X, TFPI, antithrombin (AT), protein C (PC)) were measured and used in a computational model to generate time course profiles for thrombin (IIa), FXa, extrinsic tenase, intrinsic tenase and prothrombinase complexes upon a 5 pM tissue factor stimulus in the presence of 1 nM thrombomodulin. Parameters were extracted from the thrombin and FXa profiles (including max rate (MaxRIIa and MaxRFXa) and peak level (MaxLIIa and MaxLFXa)). Procoagulant potential was also evaluated by determining the concentration of the complexes at select times. Parameter values were compared between survivors and non-survivors in the burn cohort and between the burn cohort and a simulation based on the mean physiological (100%) concentration for all factor levels. RESULTS: Burn patients differed at Hour 0 (p < 0.05) from 100% mean physiological levels for all coagulation factor levels except FV and FVII. The concentration of FX, FII, TFPI, AT and PC was lower; FIX and FVIII were increased. The composition differences resulted in all nine burn patients at Hour 0 displaying a procoagulant phenotype relative to 100% mean physiological simulation (MaxLIIa (306 ± 90 nM vs. 52 nM), MaxRIIa (2.9 ± 1.1 nM/s vs. 0.3 nM/s), respectively p < 0.001); MaxRFXa and MaxLFXa were also an order of magnitude greater than 100% mean physiological simulation (p < 0.001). When grouped by survival status and compared at the time of admission, non-survivors had lower PC levels (56 ± 18% vs. 82 ± 9%, p < 0.05), and faster MaxRFXa (29 ± 6 pM/s vs. 18 ± 6 pM/s, p < 0.05) than those that survived; similar trends were observed for all other procoagulant parameters. At 48 hours when comparing non-survivors to survivors, TFPI levels were higher (108 ± 18% vs. 59 ± 18%, p < 0.05), and MaxRIIa (1.5 ± 1.4 nM/s vs. 3.6 ± 0.7 nM/s, p < 0.05) and MaxRFXa (13 ± 12 pM/s vs. 35 ± 4 pM/s, p < 0.05) were lower; similar trends were observed with all other procoagulant parameters. Overall, between admission and 48 hours, procoagulant potential, as represented by MaxR and MaxL parameters for thrombin and FXa, in non-survivors decreased while in survivors they increased (p < 0.05). In patients that survived, there was a positive correlation between FX levels and MaxLFXa (r = 0.96) and reversed in mortality (r= -0.91). CONCLUSIONS: Thrombin and FXa generation are increased in burn patients at admission compared to mean physiological simulations. Over the first 48 hours, burn survivors became more procoagulant while non-survivors became less procoagulant. Differences between survivors and non-survivors appear to be present in the underlying dynamics that contribute to FXa dynamics. Understanding how the individual specific balance of procoagulant and anticoagulant proteins contributes to thrombin and FXa generation could ultimately guide therapy and potentially reduce burn injury-related morbidity and mortality.

Assessment of Coagulation Homeostasis in Blunt, Penetrating, and Thermal Trauma: Guidance for a Multicenter Systems Biology Approach.

INTRODUCTION: Provisioning care for traumatically injured patients makes conducting research very proximal to injury difficult. These studies also inherently have regulatory barriers to overcome. Here we outline a protocol for acute-phase enrollment of traumatically injured patients into a prospective observational clinical trial with precise and comprehensive sample acquisition in support of a systems biology approach to a research study. METHODS: Experts in trauma, burn, blood coagulation, computational biology, and integrative systems biology developed a prospective study that would capture the natural history of coagulation pathology after traumatic injury. Blood was sampled at admission and serial time points throughout hospitalization. Concurrently, demographic and outcomes data were recorded and on-site point-of-care testing was implemented. Protocols were harmonized across sites and sampling protocols validated through demonstration of feasibility and sample quality assurance testing. A novel data integration platform was developed to store, visualize, and enable large-scale analysis of empirical and clinical data. Regulatory considerations were also addressed in protocol development. RESULTS: A comprehensive Manual of Operations (MOO) was developed and implemented at 3 clinical sites. After regulatory approval, the MOO was followed to collect 5,348 longitudinal samples from 1,547 patients. All samples were collected, processed, and stored per the MOO. Assay results and clinical data were entered into the novel data management platform for analyses. CONCLUSION: We used an iterative, interdisciplinary process to develop a systematic and robust protocol for comprehensive assessment of coagulation in traumatically injured patients. This MOO can be a template for future studies in the acute setting.

Hemodilution and Endothelial Cell Regulation of Whole Blood Coagulation.

Background: Beyond localized damage to the circulatory system and surrounding tissue, trauma stresses endothelial cells throughout the vasculature, potentially leading to hemorrhagic or thrombotic complications away from the injury site. Objective: Use a whole blood endothelial cell model to define the effects of crystalloid fluid therapy on protein C pathway regulation of tissue factor-initiated coagulation. Methods: Tissue factor-initiated coagulation was studied in the presence of EA.hy926 cells. Blood was diluted to 70% or 40% using normal saline or lactated ringers. Analyses of coagulation dynamics included clot times, thrombin formation (thrombin-antithrombin complex), FV activation/inactivation, fibrinogen consumption, FXIII activation, and platelet activation. Results: In all donors, the onset of thrombin generation was not altered in 70% blood using either diluent; with the blood component reduced to 40%, clot time was prolonged two-fold when normal saline was utilized but was unchanged with lactated ringers. The timing of the activations of FV, fibrinogen, and platelets paralleled the effects of dilution on clot times. Extensive inactivation of FVa was observed in undiluted blood and where lactated ringers was the diluent but not in trials with 40% blood/60% normal saline. Conclusion: Feedback inhibition of tissue factor-initiated coagulation by the protein C pathway is not compromised by hemodilution with crystalloids.

Computational Model for Hyperfibrinolytic Onset of Acute Traumatic Coagulopathy.

The onset of acute traumatic coagulopathy in trauma patients exacerbates hemorrhaging and dramatically increases mortality. The disease is characterized by increased localized bleeding, and the mechanism for its onset is not yet known. We propose that the fibrinolytic response, specifically the release of tissue-plasminogen activator (t-PA), within vessels of different sizes leads to a variable susceptibility to local coagulopathy through hyperfibrinolysis which can explain many of the clinical observations in the early stages from severely injured coagulopathic patients. We use a partial differential equation model to examine the consequences of vessel geometry and extent of injury on fibrinolysis profiles. In addition, we simulate the efficacy of tranexamic acid treatment on coagulopathy initiated through endothelial t-PA release, and are able to reproduce the time-sensitive nature of the efficacy of this treatment as observed in clinical studies.

Microparticles formed during storage of red blood cell units support thrombin generation.

BACKGROUND: Intact red blood cells (RBCs) appear to support thrombin generation in in vitro models of blood coagulation. During storage of RBC units, biochemical, structural, and physiological changes occur including alterations to RBC membranes and release of microparticles, which are collectively known as storage lesion. The clinical consequences of microparticle formation in RBC units are unclear. This study was performed to assess thrombin generation via the prothrombinase complex by washed RBCs and RBC-derived microparticles as a function of RBC unit age. METHODS: Well-characterized kinetic and flow cytometric assays were used to quantify and characterize microparticles isolated from leukocyte-reduced RBC units during storage for 42 days under standard blood banking conditions. RESULTS: Stored RBCs exhibited known features of storage lesion including decreasing pH, cell lysis, and release of microparticles demonstrated by scanning electron microscopy. The rate of thrombin formation by RBC units linearly increased during storage, with the microparticle fraction accounting for approximately 70% of the prothrombinase activity after 35 days. High-resolution flow cytometric analyses of microparticle isolates identified phosphatidylserine-positive RBC-derived microparticles; however, their numbers over time did not correlate with thrombin formation in that fraction. CONCLUSION: Red blood cell-derived microparticles capable of supporting prothrombinase function accumulate during storage, suggesting an increased potential of transfused units as they age to interact in unplanned ways with ongoing hemostatic processes in injured individuals, especially given the standard blood bank practice of using the oldest units available.

Probing the Dynamics of Clot-Bound Thrombin at Venous Shear Rates.

In closed system models of fibrin formation, exosite-mediated thrombin binding to fibrin contributes to clot stability and is resistant to inhibition by antithrombin/heparin while still susceptible to small, active-site inhibitors. Each molecule of fibrin can bind ∼1.6 thrombin molecules at low-affinity binding sites (Kd = 2.8 µM) and ∼0.3 molecules of thrombin at high-affinity binding sites (Kd = 0.15 µM). The goal of this study is to assess the stability of fibrin-bound thrombin under venous flow conditions and to determine both its accessibility and susceptibility to inhibition. A parallel-plate flow chamber (7 × 50 × 0.25 mm) for studying the stability of thrombin (0-1400 nM) adhered to a fibrin matrix (0.1-0.4 mg/mL fibrinogen, 10 nM thrombin) under a variety of venous flow conditions was developed using the thrombin-specific, fluorogenic substrate SN-59 (100 µM). The flow within this system is laminar (Re < 1) and reaction rates are driven by enzyme kinetics (Pe = 100, Da = 7000). A subpopulation of active thrombin remains stably adhered to a fibrin matrix over a range of venous shear rates (46-184 s-1) for upwards of 30 min, and this population is saturable at loads >500 nM and sensitive to the initial fibrinogen concentration. These observations were also supported by a mathematical model of thrombin adhesion to fibrin, which demonstrates that thrombin initially binds to the low-affinity thrombin binding sites before preferentially equilibrating to higher affinity sites. Antithrombin (2.6 µM) plus heparin (4 U/mL) inhibits 72% of the active clot-bound thrombin after ∼10 min at 92 s-1, while no inhibition is observed in the absence of heparin. Dabigatran (20 and 200 nM) inhibits (50 and 93%) clot-bound thrombin reversibly (87 and 66% recovery). This model illustrates that clot-bound thrombin stability is the result of a constant rearrangement of thrombin molecules within a dense matrix of binding sites.

Reference ranges for rotational thromboelastometry in male Sprague Dawley rats.

BACKGROUND: A functional coagulation assay was used to investigate the extrinsic pathway of coagulation on citrated whole blood samples from healthy adult male Sprague Dawley rats using the mini cup and pin system. METHODS: Reference values for coagulation parameters from forty-three animals were calculated using data obtained from the ROTEM® delta hemostasis analyzer with the EXTEM test. RESULTS: The following ranges, presented as the 2.5-97.5 percentiles, were established: CT [18-77], CFT [20-80], α [73-86], MCF [53-70], and ML [1-22], along with others. CONCLUSIONS: These reference ranges can be used in future studies in rats to identify clinically significant coagulopathies.

In-depth analysis of clotting dynamics in burn patients.

BACKGROUND: Studies associating coagulopathic changes with burn injury have relied on limited tests such as partial thromboplastin time (PTT) and international normalized ratio (INR). Understanding the clotting dynamics and associated risk factors after burn injury could influence management. This work aimed to identify real-time changes in coagulation after burn injury not indicated by PTT or INR alone. MATERIALS AND METHODS: Nine burn-injured patients at a regional burn center were enrolled for blood collection at admission and set intervals over 96 h. Patient demographics, management, and laboratory data (PTT and INR) were collected. Plasma assays determined factors II, V, VII, VIII, IX, X, XI, antithrombin, and protein C functional activity as well as PAP, D-Dimer, fibrin monomer, TFPI, IL-1b, IL-6, IL-10, IL-12p.70, and TNF-α concentrations. RESULTS: Overall, five patients died. These patients had higher mortality scores and were more acidotic. All patients had normal coagulation studies (INR < 1.5, PTT < 45 s) within 24 h of admission, and only two were abnormal after. Increased factor VIII and IX activity were identified in seven patients at admission. Decreased antithrombin and protein C activity were seen in all patients. Patients had increased PAP, D-Dimer, and fibrin monomer concentrations throughout their hospital course. At admission, increased fold changes were seen in IL-6 (2.5-117) and IL-10 (2.4-32), whereas IL-1b and TNF-α levels were depressed in all patients. CONCLUSIONS: Extensive changes not identified by PTT or INR were seen after burn injury that may explain perturbed coagulation in these patients. This approach further characterizes the impact thermal injury has on coagulation.

Modeling thrombin generation: plasma composition based approach.

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

Interchangeability of rotational elastographic instruments and reagents.

BACKGROUND: Viscoelastic measurements are frequently being used in clinical and research settings for a rapid assessment of the hemostatic processes of blood clot formation and degradation. These measurements are being performed on either of two instruments (TEG and ROTEM) using their proprietary reagents. Standardization between the instruments and the reagents has been lacking but is necessary to compare results across instruments. In this study, we perform a crossover analysis between the TEG and ROTEM instruments using proprietary reagents from each manufacturer. METHODS: We tested three sets of reagents as follows: (1) in-tem and ex-tem (Tem International GmbH); (2) kaolin and RapidTEG (Haemonetics); (3) a well-characterized control recombinant tissue factor-phospholipid reagent. Blood was drawn from six healthy donors, and each reagent was run concurrently in the TEG and ROTEM instruments. The volume of commercial reagent and calcium used was adjusted for crossover measurements to maintain the same concentration of each reagent in the blood. The outputs of clot time, rate of clot formation, and maximum firmness of the clot of the ROTEM and the TEG tracings were evaluated. RESULTS: The in-tem and RapidTEG reagents showed no disparity between instruments for any parameter. Significant differences between the instruments were found in the α angle and maximum firmness of the clot for ex-tem and kaolin reagents as well as in the clot time and maximum firmness of the clot for the recombinant tissue factor-phospholipid reagent. CONCLUSION: Although significant differences were observed for some parameters, the magnitudes were small compared with the differences between tests or the normal range variation in parameter values observed for these tests. These findings indicate that the instruments are more interchangeable than previously reported.

From principle to practice: bridging the gap in patient profiling.

The standard clinical coagulation assays, activated partial thromboplastin time (aPTT) and prothrombin time (PT) cannot predict thrombotic or bleeding risk. Since thrombin generation is central to haemorrhage control and when unregulated, is the likely cause of thrombosis, thrombin generation assays (TGA) have gained acceptance as "global assays" of haemostasis. These assays generate an enormous amount of data including four key thrombin parameters (lag time, maximum rate, peak and total thrombin) that may change to varying degrees over time in longitudinal studies. Currently, each thrombin parameter is averaged and presented individually in a table, bar graph or box plot; no method exists to visualize comprehensive thrombin generation data over time. To address this need, we have created a method that visualizes all four thrombin parameters simultaneously and can be animated to evaluate how thrombin generation changes over time. This method uses all thrombin parameters to intrinsically rank individuals based on their haemostatic status. The thrombin generation parameters can be derived empirically using TGA or simulated using computational models (CM). To establish the utility and diverse applicability of our method we demonstrate how warfarin therapy (CM), factor VIII prophylaxis for haemophilia A (CM), and pregnancy (TGA) affects thrombin generation over time. The method is especially suited to evaluate an individual's thrombotic and bleeding risk during "normal" processes (e.g pregnancy or aging) or during therapeutic challenges to the haemostatic system. Ultimately, our method is designed to visualize individualized patient profiles which are becoming evermore important as personalized medicine strategies become routine clinical practice.