Inhibitors of Inorganic Polyphosphate and Nucleic Acids Attenuate in vitro Thrombin Generation in Plasma from Trauma Patients.

BACKGROUND: Inorganic polyphosphate (polyP) is a procoagulant polyanion. We assessed the impact of polyP inhibition on thrombin generation after trauma using the novel polyP antagonists, macromolecular polyanion inhibitor 8 (MPI 8) and universal heparin reversal agent 8 (UHRA-8). METHODS: Plasma thrombin generation (calibrated automated thrombogram, CAT), in 56 trauma patients and 39 controls +/- MPI 8 and UHRA-8 (50 µg/mL), was expressed as lag time (LT, minutes), peak height (PH, nM), and time to peak (ttPeak, minutes), with change in LT (ΔLT) and change in ttPeak (ΔttPeak) quantified. Results expressed in median and quartiles [Q1, Q3], Wilcoxon matched-pairs testing, p < 0.05 significant. RESULTS: Trauma patients had greater baseline PH than controls (182.9 [121.0, 255.2]; 120.5 [62.1, 174.8], p < 0.001). MPI 8 treatment prolonged LT and ttPeak in trauma (7.20 [5.88, 8.75]; 6.46 [5.45, 8.93], p = 0.020; 11.28 [8.96, 13.14]; 11.00 [8.95, 12.94], p = 0.029) and controls (7.67 [6.67, 10.50]; 6.33 [5.33, 8.00], p < 0.001; 13.33 [11.67, 15.33]; 11.67 [10.33, 13.33], p < 0.001). UHRA-8 treatment prolonged LT and ttPeak and decreased PH in trauma (9.09 [7.45, 11.33]; 6.46 [5.45, 8.93]; 14.02 [11.78, 17.08]; 11.00 [8.95, 12.94]; 117.4 [74.5, 178.6]; 182.9 [121.0, 255.2]) and controls (9.83 [8.00, 12.33]; 6.33 [5.33, 8.00]; 16.67 [14.33, 20.00]; 11.67 [10.33, 13.33]; 55.3 [30.2, 95.9]; 120.5 [62.1, 174.8]), all p < 0.001. Inhibitor effects were greater for controls (greater ΔLT and ΔttPeak for both inhibitors, p < 0.001). CONCLUSION: PolyP inhibition attenuates thrombin generation, though to a lesser degree in trauma than in controls, suggesting that polyP contributes to accelerated thrombin generation after trauma.

A murine multiple-injury model for the study of thromboinflammation.

INTRODUCTION: Neutrophil extracellular traps (NETs) contribute to trauma-induced coagulopathy. We aimed to develop a murine multiple-injury model that induces thrombo-inflammatory response, that is, NETosis and accelerated thrombin generation. METHODS: Wild-type male mice (n = 10, aged 8-12 weeks) underwent multiple injuries (gastrocnemius crush, femur fracture, and laparotomy) and were compared with an uninjured control group (n = 10). Mice were euthanized by cardiac puncture performed 3 hours after injury. Whole blood samples were immediately processed to platelet poor plasma for thrombin generation kinetics (calibrated automated thrombogram), myeloperoxidase (MPO), and von Willebrand factor quantification. Immunohistochemistry of lung tissue was performed to assess for citrullinated histone 3 (CitH3) and MPO. A NETosis cluster was defined as 3+ neutrophils staining for CitH3 at 400× magnification (CitH3 cluster). Data were presented either as mean (SD) or median (interquartile range) with p < 0.05 significant. Sham and trauma treated animals were compared by the two-sample Wilcoxon rank-sum test. RESULTS: Animals subjected to multiple injuries had accelerated thrombin generation compared with controls with greater peak height (61.3 [41.2-73.2] vs. 28.4 [19.5-37.5] nM, p = 0.035) and shorter time to peak (3.37 [2.81-3.81] vs. 4.5 [4.08-4.75] minutes, p = 0.046). Markers of neutrophil activation were greater following multiple injuries than in controls (MPO, 961.1 [858.1-1116.8] vs. 481.3 [438.0-648.9] ng/mL; p = 0.004). NETosis, as evidenced by the aforementioned defined number of CitH3 clusters in the lung, was greater in multiple-injury animals than in controls (mean [SD], 3 [2.9] vs. 0.2 [0.7]; p = 0.009). CONCLUSION: This is the first study to demonstrate that NETosis and accelerated thrombin generation can be induced using a murine multiple-injury model, as early as 3 hours following injury.

Estradiol and Dihydrotestosterone Levels in COVID-19 Patients.

OBJECTIVE: To determine differences in plasma sex hormone levels in male and female coronavirus disease 2019 (COVID-19) patients and healthy volunteers (HVs) because cell entry of severe acute respiratory syndrome coronavirus 2 occurs via the angiotensin-converting enzyme 2 receptor which is downregulated by 17ß-estradiol. PATIENTS AND METHODS: Citrated plasma samples were collected from 101 patients with COVID-19 upon presentation to the emergency department and from 40 HVs between November 1, 2020, and May 30, 2021. Plasma 17ß-estradiol and 5α-dihydrotestosterone (DHT) levels were measured using enzyme-linked immunosorbent assay (pg/mL). Data are presented as median and quartiles (IQR). Wilcoxon rank sum test with a P value less than .05 was considered significant. RESULTS: Patients with COVID-19 (median age, 49 years) included 51 males and 50 females (25 postmenopausal). Hospital admission was required for 58.8% of male patients (n = 30) and 48.0% of female patients (n = 24) (66.7% postmenopausal, n = 16) Healthy volunteers (median age, 41 years) included 20 males and 20 females (9 postmenopausal). Female patients with COVID-19 were found to have decreased 17ß-estradiol levels (18.5 [IQR, 10.5-32.3] pg/mL; 41.4 [IQR, 15.5-111.0] pg/mL, P=.025), and lower 17ß-estradiol to DHT ratios (0.073 [IQR, 0.052-0.159] pg/mL; 0.207 [IQR, 0.104-0.538] pg/mL, P=.015) than female HVs. Male patients with COVID-19 were found to have decreased DHT levels (302.8 [IQR, 249.9-470.8] pg/mL; 457.2 [IQR, 368.7-844.3] pg/mL, P=.005), compared with male HVs. Levels of DHT did not differ between female patients with COVID-19 and female HVs, whereas 17ß-estradiol levels did not differ between male patients with COVID-19 and male HVs. CONCLUSION: Sex hormone levels differ between patients with COVID-19 and HVs, with sex-specific patterns of hypogonadism in males and females. These alterations may be associated with disease development and severity.

Plasma thrombin generation kinetics vary by injury pattern and resuscitation characteristics in pediatric and young adult trauma patients.

BACKGROUND: Thrombin generation kinetics are not well studied in children. This study aimed to assess how thrombin generation kinetics vary in pediatric and young adult (YA) trauma patients by clinical characteristics and injury pattern. METHODS: Prospective cohort study where plasma samples were obtained from pediatric (ages 0-17 years) and YA (ages 18-21 years) trauma patients upon emergency department arrival. Thrombin generation (calibrated automated thrombogram [CAT]) was quantified as lag time (LT, minutes), peak height (PH, nM), time to peak (ttPeak, minutes), and endogenous thrombin potential (ETP, nM × minute). Results are expressed as median and quartiles [Q1, Q3] and compared using Wilcoxon rank sum testing with p < 0.05 considered significant. RESULTS: We enrolled 47 pediatric (median age, 15 [14, 17] years, 78% male, 87% blunt, median Injury Severity Score, 12) and 49 YA (median age 20 [18, 21] years, 67% male, 84% blunt, median Injury Severity Score, 12) patients. Pediatric and YA patients had similar rates of operative intervention (51% vs. 57%), transfusion (25% vs. 20%), and traumatic brain injury (TBI) (53% vs. 49%). Pediatric patients who required an operation had accelerated initiation of thrombin generation, with shorter LT than those who did not (2.58 [2.33, 2.67]; 2.92 [2.54, 3.00], p = 0.034). Shorter LT (2.41 [2.22, 2.67]; 2.67 [2.53, 3.00]) and ttPeak (4.50 [4.23, 4.73]; 5.22 [4.69, 5.75], both p < 0.01) were noted in pediatric patients who required transfusion as compared with those who did not. The YA patients requiring transfusion had shorter LT (2.33 [2.19, 2.74]; 2.83 [2.67, 3.27]) and ttPeak (4.48 [4.33, 5.65]; 5.33 [4.85, 6.28] both p < 0.04) than those who were not transfused. Young adults with TBI had greater ETP than those without (1509 [1356, 1671]; 1284 [1154, 1471], p = 0.032). CONCLUSION: Thrombin generation kinetics in pediatric trauma patients prior to intervention vary with need for operation and transfusion, while thrombin generation kinetics in young adult patients are influenced by TBI and need for operation or transfusion. This is a promising tool for assessing coagulopathy in young trauma patients. LEVEL OF EVIDENCE: Prognostic and Epidemiological; Level III.

TELOMERE LENGTH OF PERIPHERAL BLOOD MONONUCLEAR CELLS IS ASSOCIATED WITH DISCHARGE DISPOSITION IN OLDER TRAUMA PATIENTS.

Introduction: Little is known regarding peripheral blood mononuclear cell telomere length (PBMC-TL) and response to traumatic injury. The objective of this study was to characterize the role of PBMC-TL in coagulation and clinical outcomes after injury. Methods: Plasma and buffy coats were prospectively collected from trauma patients and healthy volunteers. DNA was purified and PBMC-TL quantified by quantitative polymerase chain reaction. Thrombin generation kinetics were expressed as lag time (in minutes), peak height (in nanometers), time to peak (in minutes), and endogenous thrombin potential (in nM × min). Results are in median and quartiles [Q1, Q3]. P < 0.05 was considered significant (Wilcoxon rank sum testing). Results: Forty-two younger patients (21 [20, 22] years, 69% were male) and 39 older patients (62 [61, 64] years, 79% were male) were included. There was no significant difference in Clinical Frailty Scores between groups. Younger patients had longer total PBMC-TL (0.40 Mb [0.30, 0.49] vs. 0.29 Mb [0.23, 0.33], P < 0.001) and longer average PBMC-TL per chromosome (4.3 kb [3.3, 5.3] vs. 3.2 kb [2.5, 3.7], P < 0.001). When older patients were stratified by 50th percentile of PBMC-TL, there were no differences in thrombin generation; however, those with shorter telomeres were less likely to be discharged home (29% vs. 77%, P = 0.004). Older patients in the bottom quartile of PBMC-TL had shorter lag time (2.78 min [2.33, 3.00] vs. 3.33 min [3.24, 3.89], P = 0.030) and were less likely to be discharged home (22% vs. 90%, P = 0.006) than those in the top quartile of PBMC-TL. Multivariable logistic regression models revealed both increased age and shorter PBMC-TL to be independent predictors of discharge disposition other than home. Conclusion: In older trauma patients, shorter PBMC-TL is associated with accelerated initiation of thrombin generation and lower likelihood of being discharged to home.

Proteomics of Coagulopathy Following Injury Reveals Limitations of Using Laboratory Assessment to Define Trauma-Induced Coagulopathy to Predict Massive Transfusion.

Objective: Trauma-induced coagulopathy (TIC) is provoked by multiple mechanisms and is perceived to be one driver of massive transfusions (MT). Single laboratory values using prothrombin time (INR) or thrombelastography (TEG) are used to clinically define this complex process. We used a proteomics approach to test whether current definitions of TIC (INR, TEG, or clinical judgement) are sufficient to capture the majority of protein changes associated with MT. Methods: Eight level-I trauma centers contributed blood samples from patients available early after injury. TIC was defined as INR >1.5 (INR-TIC), TEG maximum amplitude <50mm (TEG-TIC), or clinical judgement (Clin-TIC) by the trauma surgeon. MT was defined as > 10 units of red blood cells in 24 hours or > 4 units RBC/hour during the first 4 hr. SomaLogic proteomic analysis of 1,305 proteins was performed. Pathways associated with proteins dysregulated in patients with each TIC definition and MT were identified. Results: Patients (n=211) had a mean injury severity score of 24, with a MT and mortality rate of 22% and 12%, respectively. We identified 578 SOMAscan analytes dysregulated among MT patients, of which INR-TIC, TEG-TIC, and Clin-TIC patients showed dysregulation only in 25%, 3%, and 4% of these, respectively. TIC definitions jointly failed to show changes in 73% of the protein levels associated with MT, and failed to identify 26% of patients that received a massive transfusion. INR-TIC and TEG-TIC patients showed dysregulation of proteins significantly associated with complement activity. Proteins dysregulated in Clin-TIC or massive transfusion patients were not significantly associated with any pathway. Conclusion: These data indicate there are unexplored opportunities to identify patients at risk for massive bleeding. Only a small subset of proteins that are dysregulated in patients receiving MT are statistically significantly dysregulated among patients whose TIC is defined based solely on laboratory measurements or clinical assessment.

DNASE-MEDIATED DISSOLUTION OF NEUTROPHIL EXTRACELLULAR TRAPS ACCELERATES IN VITRO THROMBIN GENERATION KINETICS IN TRAUMA PATIENTS.

ABSTRACT: Introduction: Neutrophil extracellular traps (NETs) trigger thrombin generation. We aimed to characterize the effects of deoxyribonuclease (DNAse) on NET components (cell-free DNA [cfDNA] and histones) and thrombin generation after trauma. Methods: Citrated plasma samples were collected from trauma patients and healthy volunteers. Thrombin generation (calibrated automated thrombogram) was measured as lag time (LT, in minutes), peak height (in nM), and time to peak thrombin generation (in minutes). Citrullinated histone 3 (CitH3) and 4 (CitH4) were measured by enzyme-linked immunosorbent assay; cfDNA by PicoGreen (all in nanograms per milliliter). Samples analyzed +/- DNAse (1,000 U/mL). Results expressed as median and quartiles [Q1, Q3], Wilcoxon testing, P < 0.05 significant. Results: We enrolled 46 patients (age, 48 [31, 67] years; 67% male) and 21 volunteers (age, 45 [28, 53] years; 43% male). Deoxyribonuclease treatment of trauma plasma led to shorter LT (3.11 [2.67, 3.52] min; 2.93 [2.67, 3.19] min), shorter time to peak thrombin generation (6.00 [5.30, 6.67] min; 5.48 [5.00, 6.00] min), greater peak height (273.7 [230.7, 300.5] nM; 288.7 [257.6, 319.2] nM), decreased cfDNA (576.9 [503.3, 803.1] ng/mL; 456.0 [393.5, 626.7] ng/mL), decreased CitH3 (4.54 [2.23, 10.01] ng/mL; 3.59 [1.93, 7.98] ng/mL), and increased H4 (1.30 [0.64, 6.36] ng/mL; 1.75 [0.83, 9.67] ng/mL), all P < 0.001. The effect of DNAse was greater on trauma patients as compared with volunteers for LT (ΔLT, -0.21 vs. -0.02 min, P = 0.007), cfDNA (ΔcfDNA -133.4 vs. -84.9 ng/mL, P < 0.001), and CitH3 (ΔCitH3, -0.65 vs. -0.11 ng/mL, P = 0.004). Conclusion: Deoxyribonuclease treatment accelerates thrombin generation kinetics in trauma patient samples as compared with healthy volunteers. These findings suggest that NETs may contribute to the hypercoagulable state observed in trauma patients.

Resolution of acute pulmonary embolism using anticoagulation therapy alone in coronavirus disease 2019.

OBJECTIVE: To investigate the radiographic resolution of acute pulmonary embolism (PE) using contrast-enhanced computed tomography (CECT) examinations in patients diagnosed with acute PE while hospitalized with coronavirus disease 2019 (COVID-19) and to understand the mid-term and long-term implications of anticoagulation therapy. METHODS: We identified patients with acute PE per CECT and at least one follow-up CECT from March 11, 2020, to May 27, 2021, using a prospective registry of all hospitalized patients with COVID-19 infection receiving care within a multicenter Health System. Initial and follow-up CECT examinations were reviewed independently by two radiologists to evaluate for PE resolution. The Modified Miller Score was used to assess for thrombus burden at diagnosis and on follow-up. RESULTS: Of the 6070 hospitalized patients with COVID-19 infection, 5.7% (348/6070) were diagnosed with acute PE and 13.5% (47/348) had a follow-up CECT examination. The mean ± standard deviation time to follow-up imaging was 44 ± 48 days (range, 3-161 days). Of 47 patients, 47 (72.3%) had radiographic resolution of PE, with a mean time to follow-up of 48 ± 43 days (range, 6-239 days). All patients received anticoagulation monotherapy for a mean of 149 ± 95 days and this included apixaban (63.8%), warfarin (12.8%), and rivaroxaban (8.5%), among others. The mean Modified Miller Score at PE diagnosis and follow-up was 4.8 ± 4.2 (range, 1-14) and 1.4 ± 3.3 (range, 0-16; P < .0001), respectively. Nine patients (19%) died at a mean of 13 ± 8 days after follow-up CECT (range, 1-27 days) and at a mean of 28 ± 16 days after admission (range, 11-68 days). Seen of the nine deaths (78%) deaths were associated with progression of COVID-19 pneumonia. CONCLUSIONS: Hospitalized patients with COVID-19 have a clinically apparent 5.7% rate of developing PE. In patients with follow-up imaging, 72.3% had radiographic thrombus resolution at a mean of 44 days while on anticoagulation. Prospective studies of the natural history of PEs with COVID-19 that include systematic follow-up imaging are warranted to help guide anticoagulation recommendations.

Euglobulin clot lysis time reveals a high frequency of fibrinolytic activation in trauma.

Activation of the fibrinolytic system plays a central role in the host response to trauma. There is significant heterogeneity in the degree of fibrinolysis activation at baseline that is usually assessed by whole blood thromboelastography (TEG). Few studies have focused on plasma markers of fibrinolysis that could add novel insights into the frequency and mechanisms of fibrinolytic activation in trauma. Global fibrinolysis in plasma was assessed using a modified euglobulin clot lysis time (ECLT) assay in 171 major trauma patients and compared to commonly assessed analytes of fibrinolysis. The median ECLT in trauma patients was significantly shorter at 8.5 h (IQR, 1.3-19.5) compared to 19.9 h (9.8-22.6) in healthy controls (p < 0.0001). ECLT values ≤2.5th percentile of the reference range were present in 83 (48.5%) of trauma patients, suggesting increased fibrinolytic activation. Shortened ECLT values were associated with elevated plasmin-antiplasmin (PAP) complexes and free tissue plasminogen activator (tPA) levels in plasma. Sixteen (9.2%) individuals met the primary outcome for massive transfusion, here defined as the critical administration threshold (CAT) of 3 units of packed red cells in any 60-minute period within the first 24 h. In a univariate screen, plasma biomarkers associated with CAT included D-dimer (p < 0.001), PAP (p < 0.05), free tPA (p < 0.05) and ECLT (p < 0.05). We conclude that fibrinolytic activation, measured by ECLT, is present in a high proportion of trauma patients at presentation. The shortened ECLT is partially driven by high tPA levels and is associated with high levels of circulating PAP complexes. Further studies are needed to determine whether ECLT is an independent predictor of trauma outcomes.

Exploring the utility of a novel point-of-care whole blood thrombin generation assay following trauma: A pilot study.

INTRODUCTION: Plasma thrombin generation kinetics as measured by the calibrated automated thrombogram (CAT) assay is a predictor of symptomatic venous thromboembolism after trauma. We hypothesized that data from a new prototype assay for measurement of thrombin generation kinetics in fresh whole blood (near patient testing of thrombin generation), will correlate with the standard CAT assay in the same patients, making it a potential tool in the future care of trauma patients. METHODS: Patients were enrolled from June 2018 to February 2020. Within 12 hours of injury, blood samples were collected simultaneously for both assays. Variables compared and correlated between assays were lag time, peak height, time to peak, and endogenous thrombin potential. Data are presented as median with interquartile range (IQR). Spearman and Pearson correlations were estimated and tested between both assays; a P value of <0.05 was considered to be significant. RESULTS: A total of 64 trauma patients had samples analyzed: injury severity score = 17 (IQR), 10-26], hospital length of stay = 7.5 (IQR), 2-18) days, age = 52 (IQR, 35-63) years, 71.9% male, and 42.2% of patients received a transfusion within 24 hours of injury. Thrombin generation parameters between plasma and whole blood were compared and found that all parameters of the two assays correlate in trauma patients. CONCLUSION: In this pilot study, we have found that a novel point-of-care whole blood thrombin generation assay yields results with modest but statistically significant correlations to those of a standard plasma thrombin generation assay. This finding supports studying this device in a larger, adequately powered study.

Quantification of von Willebrand factor and ADAMTS-13 after traumatic injury: a pilot study.

BACKGROUND: Von Willebrand factor (VWF) is an acute phase reactant synthesized in the megakaryocytes and endothelial cells. VWF forms ultra-large multimers (ULVWF) which are cleaved by the metalloprotease ADAMTS-13, preventing spontaneous VWF-platelet interaction. After trauma, ULVWF is released into circulation as part of the acute phase reaction. We hypothesized that trauma patients would have increased levels of VWF and decreased levels of ADAMTS-13 and that these patients would have accelerated thrombin generation. METHODS: We assessed plasma concentrations of VWF antigen and ADAMTS-13 antigen, the Rapid Enzyme Assays for Autoimmune Diseases (REAADS) activity of VWF, which measure exposure of the platelet-binding A1 domain, and thrombin generation kinetics in 50 samples from 30 trauma patients and an additional 21 samples from volunteers. Samples were analyzed at 0 to 2 hours and at 6 hours from the time of injury. Data are presented as median (IQR) and Kruskal-Wallis test was performed between trauma patients and volunteers at both time points. RESULTS: REAADS activity was greater in trauma patients than volunteers both at 0 to 2 hours (190.0 (132.0-264.0) vs. 92.0 (71.0-114.0), p<0.002) and at 6 hours (167.5 (108.0-312.5.0) vs. 92.0 (71.0-114.0), p<0.001). ADAMTS-13 antigen levels were also decreased in trauma patients both at 0 to 2 hours (0.84 (0.51-0.94) vs. 1.00 (0.89-1.09), p=0.010) and at 6 hours (0.653 (0.531-0.821) vs. 1.00 (0.89-1.09), p<0.001). Trauma patients had accelerated thrombin generation kinetics, with greater peak height and shorter time to peak than healthy volunteers at both time points. DISCUSSION: Trauma patients have increased exposure of the VWF A1 domain and decreased levels of ADAMTS-13 compared with healthy volunteers. This suggests that the VWF burst after trauma may exceed the proteolytic capacity of ADAMTS-13, allowing circulating ULVWF multimers to bind platelets, potentially contributing to trauma-induced coagulopathy. LEVEL OF EVIDENCE: Prospective case cohort study.

Neutrophil Extracellular Trap Formation and Syndecan-1 Shedding Are Increased After Trauma.

BACKGROUND: Damage-associated molecular patterns (DAMPs) stimulate endothelial syndecan-1 shedding and neutrophil extracellular traps (NET) formation. The role of NETs in trauma and trauma-induced hypercoagulability is unknown. We hypothesized that trauma patients with accelerated thrombin generation would have increased NETosis and syndecan-1 levels. METHODS: In this pilot study, we analyzed 50 citrated plasma samples from 30 trauma patients at 0 h (n = 22) and 6 h (n = 28) from time of injury (TOI) and 21 samples from healthy volunteers, for a total of 71 samples included in analysis. Thrombin generation was quantified using calibrated automated thrombogram (CAT) and reported as lag time (LT), peak height (PH), and time to peak (ttPeak). Nucleosome calibrated (H3NUC) and free histone standardized (H3Free) ELISAs were used to quantify NETs. Syndecan-1 levels were quantified by ELISA. Results are presented as median [interquartile range] and Spearman rank correlations. RESULTS: Plasma levels of H3NUC were increased in trauma patients as compared with healthy volunteers both at 0 h (89.8 ng/mL [35.4, 180.3]; 18.1 ng/mL [7.8, 37.4], P = 0.002) and at 6 h (86.5 ng/mL [19.2, 612.6]; 18.1 ng/mL [7.8, 37.4], P = 0.003) from TOI. H3Free levels were increased in trauma patients at 0 h (5.74 ng/mL [3.19, 8.76]; 1.61 ng/mL [0.66, 3.50], P = 0.002) and 6 h (5.52 ng/mL [1.46, 11.37]; 1.61 ng/mL [0.66, 3.50], P = 0.006). Syndecan-1 levels were greater in trauma patients (4.53 ng/mL [3.28, 6.28]; 2.40 ng/mL [1.66, 3.20], P < 0.001) only at 6 h from TOI. H3Free and syndecan-1 levels positively correlated both at 0 h (0.376, P = 0.013) and 6 h (0.583, P < 0.001) from TOI. H3NUC levels and syndecan-1 levels were positively correlated at 6 h from TOI (0.293, P = 0.041). TtPeak correlated inversely to H3 NUC (-0.358, P = 0.012) and syndecan-1 levels (-0.298, P = 0.038) at 6 h from TOI. CONCLUSIONS: Our pilot study demonstrates that trauma patients have increased NETosis, measured by H3NUC and H3Free levels, increased syndecan-1 shedding, and accelerated thrombin generation kinetics early after injury.

Thrombin Generation Kinetics are Predictive of Rapid Transfusion in Trauma Patients Meeting Critical Administration Threshold.

INTRODUCTION: We hypothesize that a patient (pt) with accelerated thrombin generation, time to peak height (ttPeak), will have a greater odds of meeting critical administration threshold (CAT) criteria (> 3 packed red blood cell [pRBC] transfusions [Tx] per 60 min interval), within the first 24 h after injury, independent of international normalized ratio (INR). METHODS: In a prospective cohort study, trauma patients were enrolled over a 4.5-year period and serial blood samples collected at various time points. We retrospectively stratified pts into three categories: CAT+, CAT- but receiving some pRBC Tx, receiving no Tx within the first 24 h. Blood collected prior to Tx was analyzed for thrombin generation parameters and prothrombin time (PT)/INR. RESULTS: A total of 484 trauma pts were analyzed: injury severity score = 13 [7,22], age = 48 [28, 64] years, and 73% male. Fifty pts met criteria for CAT+, 64 pts CAT-, and 370 received no Tx. Risk factors for meeting CAT+: decreased arrival systolic blood pressure (OR 2.82 [2.17, 3.67]), increased INR (OR 2.09, [1.66, 2.62]) and decreased time to peak OR 2.27 [1.74, 2.95]). These variables remained independently associated with increased risk of requiring Tx in a multivariable logistic model, after adjusting for sex and trauma type. CONCLUSIONS: Pts in hemorrhagic shock, who meet CAT+ criteria, are characterized by accelerated thrombin generation. In our multivariable analysis, both ttPeak and PT/INR have a complementary role in predicting those injured patients who will require a high rate of Tx.

A Review of Pathophysiology, Clinical Features, and Management Options of COVID-19 Associated Coagulopathy.

ABSTRACT: There is increasing evidence that novel coronavirus disease 2019 (COVID-19) leads to a significant coagulopathy, a phenomenon termed "COVID-19 associated coagulopathy." COVID-19 has been associated with increased rates of both venous and arterial thromboembolic events, a source of significant morbidity and mortality in this disease. Further evidence suggests a link between the inflammatory response and coagulopathy associated with COVID-19. This presents a unique set of challenges for diagnosis, prevention, and treatment of thrombotic complications. In this review, we summarize and discuss the current literature on laboratory coagulation disruptions associated with COVID-19 and the clinical effects of thromboembolic events including pulmonary embolism, deep vein thrombosis, peripheral arterial thrombosis, and acute ischemic stroke in COVID-19. Endothelial injury and augmented innate immune response are implicated in the development of diffuse macro- and microvascular thrombosis in COVID-19. The pathophysiology of COVID-19 associated coagulopathy is an important determinant of appropriate treatment and monitoring of these complications. We highlight the importance of diagnosis and management of dysregulated coagulation in COVID-19 to improve outcomes in COVID-19 patients with thromboembolic complications.

Biomarkers of thromboinflammation correlate to COVID-19 infection and admission status in emergency department patients.

Background: COVID-19-associated coagulopathy is incompletely understood. Objectives: To characterize thrombin generation, Von Willebrand Factor (VWF), neutrophil extracellular traps (NETs), and their role in COVID-19 risk stratification in the emergency department (ED). Patients/methods: Plasma samples from 67 ED COVID-19 patients were compared to 38 healthy volunteers (HVs). Thrombin generation (calibrated automated thrombogram, CAT) was expressed as lag time (LT, min), peak height (PH, min), and time to peak (ttPeak, min). Citrullinated nucleosomes and histones were quantified with ELISA, VWF antigen and activity (IU/dL) through latex immunoassay, Factor VIII (IU/dL) through one-stage optical clot detection, and VWF multimers with Western blot densitometry. Wilcoxon testing and multivariable logistic regression were performed. Results presented as median [Q1, Q3]; p < 0.05 significant. Results: COVID-19 patients had longer LT (4.00 [3.26, 4.67]; 2.95 [2.67, 3.10], p < 0.001) and ttPeak (7.33 [6.33, 8.04]; 6.45 [6.00, 7.50], p = 0.004), greater VWF antigen (212 [158, 275]; 110 [91, 128], p < 0.001) and Factor VIII levels (148 [106, 190]; 106 [86, 129], p < 0.001), with decreased high molecular weight multimers (Normalized multimer ratio 0.807 [0.759, 0.869]; 0.891 [0.858, 0.966], p < 0.001), than HVs. COVID-19 patients requiring admission from the ED had longer LT and ttPeak with greater VWF antigen and Factor VIII levels than those not admitted. Two and three variable models of CAT parameters and VWF correlated with COVID-19 and admission status (C-statistics 0.677 to 0.922). Conclusions: Thrombin generation kinetics and VWF levels, independent of NETs, may have a role in predicting admission need for COVID-19 patients.

Development and application of global assays of hyper- and hypofibrinolysis.

Numerous methods for evaluation of global fibrinolytic activity in whole blood or plasma have been proposed, with the majority based on tissue-type plasminogen activator (t-PA) addition to initiate fibrinolysis. We propose that such an approach is useful to reveal hypofibrinolysis, but t-PA concentrations should be kept to a minimum. In this paper, we describe a low-concentration t-PA plasma turbidity assay to evaluate several congenital factor deficiencies, including plasminogen activator inhibitor-1 (PAI-1) and plasminogen deficiency, as well as hemophilia A and B. In addition, we demonstrate a threshold dependency on endogenous PAI-1 levels. To assess endogenous hyperfibrinolysis, we suggest that assays that avoid t-PA addition are preferable, with assays based on euglobulin fractionation remaining a viable choice. We describe a euglobulin fraction clot lysis time (ECLT) assay with spectrophotometric readout and other modifications, and evaluate it as a tool to measure hyperfibrinolysis in inherited clotting factor deficiency states. We demonstrate that the ECLT is predominantly driven by residual amounts of PAI-1, t-PA, and α2-antiplasmin. These assays should be further evaluated for the detection of hypo- or hyperfibrinolysis in acquired thrombotic or hemorrhagic disorders.

Endogenous Procoagulant Activity in Trauma Patients and Its Relationship to Trauma Severity.

Background It has been observed that trauma patients have elevated plasma procoagulant activity that could be assigned to an elevated concentration of tissue factor (TF). However, in many instances there is a discrepancy between the levels of TF and the procoagulant activity observed. We hypothesized that factor XIa (FXIa) could be responsible for this additional activity and that the presence and levels of both proteins could correlate with trauma severity. Methods Citrate plasma from 98 trauma patients (47 blunt, 17 penetrating, and 34 thermal) were evaluated in clotting assays for the presence of FXIa and TF activity using respective inhibitory antibodies. Results When the three trauma patient groups were divided into two cohorts (Injury Severity Score [ISS] > 25 and ISS ≤ 25), higher frequencies and concentrations of both TF and FXIa were observed for all the more severe injury subgroups. Conclusions The majority of trauma patients have active FXIa in their plasma, with a significant fraction having active TF as well. Additionally, both TF and FXIa frequency and concentration directly relate to trauma severity. These data suggest the use of these two proteins as potential markers for the stratification of trauma patients.

Thrombin generation profiles as predictors of symptomatic venous thromboembolism after trauma: A prospective cohort study.

BACKGROUND: Reliable biomarkers predictive of venous thromboembolism (VTE) after acute trauma are uncertain. The objective of the study was to identify risk factors for symptomatic VTE after trauma, including individual plasma coagulome characteristics as reflected by thrombin generation. METHODS: In a prospective, case-cohort study, trauma patients were enrolled over the 4.5-year period, 2011 to 2015. Blood was collected by venipuncture into 3.2% trisodium citrate at 0, 6, 12, 24, and 72 hours after injury and at hospital discharge. Platelet poor plasma was stored at -80 °C until analysis. Thrombin generation, as determined by the calibrated automated thrombogram (CAT) using 5 pM tissue factor (TF)/4 µM phospholipid (PS), was reported as peak height (nM thrombin) and time to peak height (ttPeak [minutes]). Data are presented as median [IQR] or hazard ratio with 95% CI. RESULTS: Among 453 trauma patients (injury severity score = 13.0 [6.0, 22.0], hospital length of stay = 4.0 [2.0, 10.0] days, age = 49 [28, 64] years, 71% male, 96% with blunt mechanism, mortality 3.2%), 83 developed symptomatic VTE within 92 days after injury (35 [42%] after hospital discharge). In a weighted, multivariate Cox model that included clinical and CAT characteristics available within 24 hours of admission, increased patient age (1.35 [1.19,1.52] per 10 years, p < 0.0001), body mass index ≥30 kg/m (4.45 [2.13,9.31], p < 0.0001), any surgery requiring general anesthesia (2.53 [1.53,4.19], p = 0.0003) and first available ttPeak (1.67 [1.29, 2.15], p < 0.00001) were independent predictors of incident symptomatic VTE within 92 days after trauma (C-statistic = 0.799). CONCLUSION: The individual's plasma coagulome (as reflected by thrombin generation) is an independent predictor of VTE after trauma. Clinical characteristics and ttPeak can be used to stratify acute trauma patients into high and low risk for VTE. LEVEL OF EVIDENCE: Prognostic, level III.

Risk factors for venous thromboembolism after acute trauma: A population-based case-cohort study.

BACKGROUND: Predictors of venous thromboembolism (VTE) after trauma are uncertain. OBJECTIVE: To identify independent predictors of VTE after acute trauma. METHODS: Using Rochester Epidemiology Project (REP) resources, we identified all Olmsted County, MN residents with objectively-diagnosed incident VTE within 92days after hospitalization for acute trauma over the 18-year period, 1988-2005. We also identified all Olmsted County residents hospitalized for acute trauma over this time period and chose one to two residents frequency-matched to VTE cases on sex, event year group and ICD-9-CM trauma code predictive of surgery. In a case-cohort study, demographic, baseline and time-dependent characteristics were tested as predictors of VTE after trauma using Cox proportional hazards modeling. RESULTS: Among 200 incident VTE cases, the median (interquartile range) time from trauma to VTE was 18 (6, 41) days. Of these, 62% cases developed VTE after hospital discharge. In a multiple variable model including 370 cohort members, patient age at injury, male sex, increasing injury severity as reflected by the Trauma Mortality Prediction Model (TMPM) Mortality Score, immobility prior to trauma, soft tissue leg injury, and prior superficial vein thrombosis were independent predictors of VTE (C-statistic=0.78). CONCLUSIONS: We have identified clinical characteristics which can identify patients at increased risk for VTE after acute trauma, independent of surgery. Almost two thirds of all incident VTE events occurred after initial hospital discharge (18day median time from trauma to VTE) which questions current practice of not extending VTE prophylaxis beyond hospital discharge.