Peak Thrombin Levels Research Articles

Whole blood storage duration alters fibrinogen levels and thrombin formation.

Whole blood resuscitation for hemorrhagic shock in trauma represents an opportunity to correct coagulopathy in trauma while also supplying red blood cells. The production of microvesicles in stored whole blood and their effect on its hemostatic parameters have not been described in previous literature. We hypothesized that microvesicles in aged stored whole blood are procoagulant and increase thrombin production via phosphatidylserine. Whole blood was obtained from male C57BL/6 male mice and stored in anticoagulant solution for up to 10 days. At intervals, stored whole blood underwent examination with rotational thromboelastography, and platelet-poor plasma was prepared for analysis of thrombin generation. Microvesicles were prepared from 10-day-old whole blood aliquots and added to fresh whole blood or platelet-poor plasma to assess changes in coagulation and thrombin generation. Microvesicles were treated with recombinant mouse lactadherin prior to addition to plasma to inhibit phosphatidylserine's role in thrombin generation. Aged murine whole blood had decreased fibrin clot formation compared with fresh samples with decreased plasma fibrinogen levels. Thrombin generation in plasma from aged blood increased over time of storage. The addition of microvesicles to fresh plasma resulted in increased thrombin generation compared with controls. When phosphatidylserine on microvesicles was blocked with lactadherin, there was no difference in the endogenous thrombin potential, but the generation of thrombin was blunted with lower peak thrombin levels. Cold storage of murine whole blood results in decreased fibrinogen levels and fibrin clot formation. Aged whole blood demonstrates increased thrombin generation, and this is due in part to microvesicle production in stored whole blood. One mechanism by which microvesicles are procoagulant is by phosphatidylserine expression on their membranes.

Blood Biomarkers and Risk of Venous Thromboembolism in Cancer Patients: A Systematic Review and Meta-Analysis

Patients with cancer have an increased risk of venous thromboembolism (VTE). Currently, several VTE risk prediction tools have been developed to identify candidates for primary VTE thromboprophylaxis. However, the availability of reliable and highly discriminatory prediction models for VTE risk assessment in cancer patients is limited, warranting further improvement of VTE risk stratification strategies. The implementation of biomarkers in risk assessment models might lead to refined VTE risk prediction, but it remains to be more comprehensively investigated. In this systematic review and meta-analysis, we aimed to evaluate and summarize all candidate biomarkers and their association with cancer-associated VTE. We conducted an electronic search in Medline, EMBASE and Cochrane central databases for studies that evaluated biomarkers in adult patients with cancer from inception to September 26 th 2022. We included observational and interventional studies reporting on subsequent VTE occurring after a cancer diagnosis. The baseline or index event of the biomarker measurements had to be definitively and uniformly applied to all enrolled patients. Two reviewers independently screened titles, abstracts and full-text articles for inclusion. Median differences (for continuous measures) and Odds Ratios (OR) (for dichotomous cut-off measures) with 95% confidence intervals (95% CI) were estimated and pooled using random-effects models. We assessed each studies risk of bias using the Quality in Prognostic Studies (QUIPS) risk of bias tool. Our search identified 4274 studies that were screened for inclusion (Figure 1). Following the screening, 109 studies (with 520 biomarker measurements) met the inclusion criteria and were included in the systematic review. Of these, 51 studies (with 174 biomarker measurements) were included in the meta-analysis. The majority of the studies were cohort studies (n=47; 32 prospective and 15 retrospective), while the other 4 studies were nested case-control studies. Eighteen studies had a study population of mixed cancer types and the remaining studies had a study population of specific cancer types, including: lung (n=6), gynecologic (n=5), breast (n=3), stomach (n=3), lymphoma (n=2), myeloma (n=2), ovarian (n=2), pancreas (n=2), brain (n=1), colorectal (n=1), gastrointestinal (n=1), genitourinary (n=1), gynecologic or breast (n=1), head and neck (n=1), liver (n=1), and prostate (n=1). We calculated the median difference and 95% CI for 23 different biomarkers. Cancer patients who experienced subsequent VTE events had significantly higher pre-treatment factor VIII activity, peak thrombin, platelet count and prothrombin fragment F1+2 levels compared to patients who did not experience VTE events (Table 1). Pre-treatment d-dimer levels were also higher in cancer patients who developed VTE, however, considerable heterogeneity was observed (Table 1). Contrastingly, pre-treatment hemoglobin, lag time - thrombin generation, prothrombin time and time to peak thrombin levels were significantly lower in those experiencing VTE events compared to patients who did not experience any events (Table 1). D-dimer levels on day-1 after cancer surgery were reported in two studies which, when pooled, revealed that cancer patients with subsequent VTE had higher d-dimer levels post-operatively compared to those with no events (Table 1). Moreover, pre-treatment hemoglobin levels 100 g/L was significantly associated with future VTE risk [OR: 1.42 (95%CI: 1.03-1.97), I 2=0%, n=11] while neutrophil lymphocyte ratio 3 was associated, yet insignificant [OR: 1.60 (95%CI: 0.99-2.60), I 2=43%, n=4]. Pre-treatment platelet count 350 x 10 9/L and white blood count levels 11 x 10 9/L were not significantly associated with future VTE risk [OR: 0.84 (95%CI: 0.66-1.06), I 2=0%, n=9 and 1.32 (0.86-2.03), I 2=56%, n=8, respectively]. From the 51 studies included in the meta-analyses, 27 studies had a low risk of bias, 19 studies had a moderate risk of bias and 5 studies had a high risk of bias. In conclusion, nine blood biomarkers were found to be significantly associated with VTE in cancer patients. Their utility as predictors of VTE in thrombotic risk assessment models may help in optimising VTE prediction and should be further explored in future studies.

Elevated Thrombin Generation Biomarkers and Their Relevance with Decreased Thrombin Generation Potential in Patients with Pulmonary Embolism

Background: Pulmonary embolism (PE) is a catastrophic complication in patients with venous thrombosis and is the third leading cardiovascular cause of death after myocardial infraction and stroke. Inflammation, hemostatic dysregulation, and generation of thrombotic mediators contribute to the pathogenesis of PE. Persistent thrombin generation contributes to the overall pathology of PE. The purpose of this study is to investigate endogenous thrombin potential (ETP) in PE patients and demonstrate its relationship with thrombin generation biomarkers, such as prothrombin fragment 1+2 (F1+2), thrombin anti-thrombin complex (TAT) and D-Dimer. Methods: Citrated blood samples from 201 patients with confirmed PE were collected under an IRB approved protocol. Fifty normal human plasma (NHP) samples were used as a reference. Thrombin generation studies were carried out by using fluorogenic substrate method (calibrated automated thrombogram, Diagnostica Stago; Paris, France). Thrombin generation parameters such as peak thrombin, lag time and endogenous thrombin potential (ETP) were compiled. Plasma samples were analyzed for such biomarkers as prothrombin fragment 1+2 (F1+2), thrombin anti-thrombin complex (TAT), and D-Dimer by using commercially available Sandwich ELISA methods. Results were compiled as mean ± SD and applicable statistical analysis was performed by using GraphPad Prism software. Result: PE patients exhibited a wide variation in thrombin generation parameters. ETP in PE patients decreased to 549.2±508.4 nM*min compared to NHP 701.8±214.6 nM*min, thrombin levels in PE patients also decreased to 83.7±80.1 nM compared to NHP 158.5±31.5 nM, while lag time increased 5.4±12.7 mins to 2.6±0.6 mins compared to NHP. Biomarker analysis showed elevated levels of F1+2 (972.3±1508.0 vs 294.2±105.0 pmol/L, 3.3-fold), TAT (34.9±54.3 vs 2.3±1.1ug/L, 15.3-fold), and D-Dimer (6720.0±4810.0 vs 182.6±205.8 ug/ml, 36.8-fold) compared to NHP. Endogenous thrombin potential levels demonstrate moderate and positive correlations with F1+2 (r = 0.22) while weak correlations were noted with TAT (r = 0.14) and D-Dimer (r = 0.05). F1+2 possess strong correlation with TAT (r = 0.55) and moderate for D-Dimer (r = 0.21). TAT also showed strong correlation with D-Dimer (r = 0.45). Thrombin generation parameters and biomarker levels based on PE severity has been shown in figure 1. Marked increase in thrombin generation biomarker levels were noted with increase PE severity. However, for the thrombin generation parameters, there is no differences were noted between the low grade and sub-massive PE, while the ETP and peak thrombin levels decreased, and lag time increased for the massive PE. Conclusion: These studies suggest that persistent thrombin generation in PE patients results in the consumption of prothrombin complex. However, such biomarkers of thrombin generation as F1+2, TAT and D-Dimer are formed, leading to higher circulating levels. Simultaneous measurement of thrombin generation biomarkers and thrombin generation parameters may be useful to determine the pathogenesis of thrombotic complications and optimization of treatment options. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Endogenous Thrombin Generation Potential in Patients with Pulmonary Embolism and Its Relevance to Thrombo-Inflammatory Biomarkers

Background: Pulmonary embolism (PE) is a catastrophic outcome in patients with venous thrombosis and third leading cardiovascular cause of death after myocardial infraction and stroke, estimates around 100,000 deaths annually.Inflammation, hemostatic dysregulation including fibrinolytic imbalance, endothelial compromise, and generation of thrombotic mediators, along with cellular defects and hemodynamic derangement contribute to the pathogenesis of PE.Endogenous thrombin generation contributes to the overall pathology of PE. The purpose of this study is to investigate endogenous thrombin potential (ETP) and the circulating biomarkers of fibrinolytic, inflammatory, and endothelial dysfunction in PE samples. Methods: Citrated blood samples from 400 patients with confirmed PE were collected at Loyola University Medical Center and Gottlieb Memorial Hospital under an IRB approved protocol. Normal human plasma (NHP) comprised of 25 male and 25 female samples were obtained from a commercial source. Thrombin generation studies were carried out using a commercially available a kinetic system. Endogenous GAG's contents were measured by fluorogenic quenching method. Thrombin generation parameters such as peak thrombin, lag time and area under the curve (AUC) were compiled. Plasma samples were retrospectively analyzed for biomarkers, including D-Dimer, PAI-1, tPA, TAFIa, vWF, CRP, IL6, TFPI and VEGF using commercially available sandwich ELISA methods. Results were compiled as mean ± SD and analyzed for significance and correlation by using GraphPad Prism software. Result: On a cumulative basis, PE patients showed a wide variation in thrombin generation parameters (Table 1). Biomarker analysis showed elevated levels of D-Dimer (37.05-fold), CRP (35.34-folds), IL6 (21.62-folds), tPA (4.69-folds), PAI-1a (3.95-folds), VEGF (2.38-folds), TFPI (2.17-folds), GAG's (1.92-folds), vWF (1.89-folds), and TAFIa (1.21-folds). Peak thrombin levels exhibited varying degree of positive and negative correlations with various biomarkers as illustrated in figure 1. Thrombin levels showed significant negative correlations with tPA, endogenous GAG's & TFPI, while positively correlated with VEGF. D-dimer showed positive correlations with CRP & IL6. PAI-1 demonstrated positive correlations with tPA, IL6, and endogenous GAG's. tPA showed negative correlation with TAFI, while positive correlations with endogenous GAG's, and TFPI. vWF showed positive correlations CRP, IL6, and endogenous GAG's. CRP showed positive correlations with IL6. Endogenous GAG's showed positive correlations with TFPI while TFPI showed negative correlation with VEGF. Conclusion: PE patients exhibit a wide variation in endogenous thrombin generation parameters. Overall, peak thrombin and ETP were decreased, whereas the lag time was increased. The increase in thrombo-inflammatory markers is suggestive of upregulation of thrombogenesis, and inflammatory processes in these patients. Thus, the profiling of thrombo-inflammatory biomarkers along with thrombin generation profile may provide additional information on the pathogenesis and severity of PE. However, biomarkers of hemostatic activation, vascular dysfunction and inflammation were increased. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Hemostatic efficacy of marstacimab alone or in combination with bypassing agents in hemophilia plasmas and a mouse bleeding model

BackgroundPatients with hemophilia have deficiencies in intrinsic coagulation factors and can develop inhibitors that limit the effectiveness of replacement coagulation factors. Marstacimab, a human monoclonal antibody, binds and inhibits the human tissue factor pathway inhibitor. Marstacimab is currently under development as a potential prophylactic treatment to prevent bleeding episodes in patients with hemophilia A and B. ObjectiveTo assess the effects of marstacimab alone or in combination with the bypassing agent recombinant factor FVIIa (rFVIIa) or activated prothrombin complex concentrate (aPCC) on thrombin generation and bleeding. MethodsMarstacimab and/or rFVIIa or aPCC were added to hemophilic A or B plasma or nonhemophilic plasma in vitro. Hemostatic activity was measured using the thrombin generation assay. In vivo effects were assessed using a mouse acute bleeding model. Male hemophilia A mice were dosed with marstacimab plus aPCC before tail clip; blood loss was quantified by measuring hemoglobin. ResultsMarstacimab plus rFVIIa or aPCC slightly increased peak thrombin levels compared with either agent alone. This increase was within the reported range for nonhemophilic plasma and did not exceed levels observed in nonhemophilic plasma treated with marstacimab alone. Hemophilia A mice that received 200 U/kg aPCC had significantly reduced bleeding (62%) compared with vehicle‐treated mice (p < 0.05), and marstacimab plus aPCC reduced bleeding by 83.3% compared with vehicle (p= 0.0009). ConclusionsMarstacimab alone or with bypassing agents increased hemostasis in hemophilia plasma without generating excessive thrombin. The hemostatic activity of marstacimab plus aPCC was confirmed in hemophilia A mice.

Assessment of Rotational Thromboelastometry and Thrombin Generation Assay to Identify Risk of High Blood Loss and Re-Operation After Cardiac Surgery.

Introduction: We aimed to investigate parameters for prediction of post-operative blood loss and re-operation in patients who underwent cardiopulmonary bypass. Methods: Thrombin generation assay, activated partial thromboplastin time, activated clotting time and rotational thromboelastometry (ROTEM) tests were performed at 4 time points in 65 patients: before skin incision (T1), after heparin injection (T2), after protamine reversal (T3) and before skin closure (T4). Results: Pre-operative endogenous thrombin potential (ETP) and peak thrombin levels were significantly lower in patients with high post-operative blood loss (≥ 800 mL) within 24 h than in those with low blood loss (< 800 mL). Clotting time (CT), maximal clotting firmness, clotting firmness time and alpha angle values of ROTEM measured at T2, T3 or T4 were significant predictors for high post-operative blood loss. An increase in CT-EXTEM over 4 time points was significant in patients who had a re-operation within 48 h compared to their counterparts. Conclusions: This study indicates that pre-operative ETP could predict high post-operative blood loss and that intra-operative ROTEM also helps to stratify risks of high post-operative blood loss and re-operation.

Variations in clot phenotype following injury: The MA-R ratio and fragile clots.

Trauma-induced coagulopathy is a continuum ranging from hypercoagulable to hypercoagulable phenotypes. In single-center studies, the maximum amplitude (MA) to r-time (R) (MA-R) ratio has identified a phenotype of injured patients with high mortality risk. The purpose of this study was to determine the relationship between MA-R and mortality using multicenter data and to investigate fibrinogen consumption in the development of this specific coagulopathy phenotype. Using the Pragmatic Randomized Optimal Platelet and Plasma Ratios data set, patients were divided into blunt and penetrating injury cohorts. MA was divided by R time from admission thromboelastogram to calculate MA-R. MA-R was used to assess odds of early and late mortality using multivariable models. Multivariable models were used to assess thrombogram values in both cohorts. Refinement of the MA-R cut point was performed with Youden index. Repeat multivariable analysis was performed with a binary CRITICAL and NORMAL MA-R. In initial analysis, MA-R quartiles were not associated with mortality in the penetrating cohort. In the blunt cohort, there was an association between low MA-R and early and late mortality. A refined cut point of 11 was identified (CRITICAL: MA-R, ≤11; NORMAL: MA-R, >11). CRITICAL MA-R was associated with mortality in both penetrating and blunt subgroups. In further injury subgroup analysis, CRITICAL patients had significantly decreased fibrinogen levels in the blunt subgroup only. In both blunt and penetrating injury, there was no difference in time to initiation of thrombin burst (lagtime). However, both endogenous thrombin potential and peak thrombin levels were significantly lower in CRITICAL patients. MA-R identifies a trauma-induced coagulopathy phenotype characterized in blunt injury by impaired thrombin generation that is associated with early and late mortality. The endotheliopathy and tissue factor release likely plays a role in the cascade of impaired thrombin burst, possible early fibrinogen consumption and the weaker clot identified by MA-R. Therapeutic/care management, level II.

Thrombin Generation in Patients with Severe Hemophilia A May Be Variable but Predictive of Response to Treatment

Introduction: Patients with severe hemophilia A (HA) may have variable bleeding phenotypes despite having equivalently low factor VIII (FVIII) levels of <1%. The basis for this heterogeneity in the clinical expression of severe hemophilia is poorly understood. Recent evidence indicates that global assays of coagulation may be better predictors of the coagulation capacity and hemostasis compared with traditional coagulation tests. Current prophylaxis goals in patients with hemophilia minimize time spent at baseline. Thus, we hypothesized that evaluating endogenous thrombin potential (ETP) at baseline, 10% and 40% FVIII correction levels would better reflect real-world scenarios and the effects of treatment.Our objective was to study thrombin generation (TG) parameters (ETP, peak thrombin and lag time) at baseline, 10% and 40% factor correction levels in patients with severe hemophilia A. Additionally we aimed to determine if the baseline TG parameters were predictive of response at the spiked levels .Methods: Our study was a single institution cross-sectional study. Patients were recruited from 4/28/2011 - 7/31/2014. A total of 123 patients with HA were recruited out of which 55 patients with severe HA without inhibitors were included in our study. Descriptive characteristics of the study population included in our study are described in Table 1. A hemophilia severity score (HSS) was calculated based on adjusted bleeding scores, factor scores and joint scores. Baseline plasma samples at trough FVIII were obtained from patients and thrombin generation was evaluated using a standard calibrated automated thrombogram (CAT). Full-length recombinant FVIII was added to trough patient plasma for final factor concentrations of 0.1 U/dl and 0.4 U/dl. Spearman's rank correlation coefficients were calculated and p values of <0.05 were considered statistically significant.Results: Samples from 55 patients with severe HA without inhibitors were evaluated. CAT mean lag times were 5.04 minutes (SD 1.92), 8.06 minutes (SD 10.0), 5.87 minutes (SD 4.38); mean ETPs were 485.4 nM/minute (SD 296.6), 822.9 nM/minute (SD 372.2), 1038 nM/minute (SD 254); and mean peak thrombin levels were 37.2 nM (SD 37.9), 77.7 nM (SD 45.9), 138.4 nM (SD 58.5) at baseline, 10% correction and 40% factor correction respectively. There were no significant correlations between HSS score and TG parameters at baseline, 10% or 40%. The correlations ranged from -0.15 to .012. Severe versus non-severe genotype and TG was analyzed in 47 participants in whom genotype status was recorded. In the 29 patients with severe genotypes (62%) there were no comparisons that were statistically significant. However, the peak thrombin generation at 40% correction trended towards significance with levels lower in the patients with severe as compared to those with non-severe genotypes (p=0.07). Additionally we evaluated if TG parameters at baseline were predictive of responses at 10% and 40% correction levels. Baseline ETP significantly predicted ETP with both 10% (p<0.001) and 40% correction (p=0.0018) levels. Baseline peak thrombin was a significant predictor of peak thrombin levels with 10% (p<0.001) and 40% correction (p<0.001). Lagtime at baseline was a significant predictor of lag time at 10% correction (p=0.008) but not 40% correction levels (p=0.737). (Figure 1)Conclusion: Our results confirm previous studies indicating variability in thrombin generation among patients with severe HA (FVIII<1%) at baseline as well as at different correction levels. Since phenotypic heterogeneity is observed in patients with severe HA, this may suggest that TG better correlates with bleeding severity as compared to factor levels. Even though the HSS scores do not correlate with TG parameters this may be reflective of the need for a more robust phenotypic score evaluating bleeding severity in patients with hemophilia. Additionally, baseline TG parameters appear to be responsive to treatment at 10% and 40% factor correction levels, suggesting that baseline measurements maybe sufficient to predict response. The introduction of continuous primary prophylaxis has dramatically changed the natural history of the disease as well as orthopedic status. Therefore, new criteria may be warranted to define the degree of clinical severity of hemophilia to improve patient management and healthcare costs. [Display omitted] DisclosuresDunn:World Federation of Hemophilia USA: Membership on an entity's Board of Directors or advisory committees; Shire: Consultancy, Other: Unrestricted educational grant, Research Funding; Octapharma: Other: Unrestricted educational grant; NovoNordisk: Other: Unrestricted educational grant; Kedrion: Other: Unrestricted educational grant; Biogen: Other: Unrestricted educational grant, Research Funding; CSL Behring: Consultancy, Other: Unrestricted educational grant; Bayer: Consultancy, Other: Unrestricted educational grant; Alnylam: Other: Unrestricted educational grant.

The assesment of prothrombotic potential using thrombin generation assay in pediatric patients with nephrotic syndrome: preliminary study.

Nephrotic syndrome (NS) is a common kidney disease associated with an increased risk of thrombotic events. The aim of this study was to assess the prothrombotic potential of patients with NS using the thrombin generation assay (TGA). A total of 35 patients with NS, who were followed in the Division of Pediatric Nephrology in Behcet Uz Children`s Hospital, were included in the study. After the patients with Steroid Resistant NS (n:3) were excluded, 32 patients in total were evaluated for TGA. Patients were primarily classified according to their response to corticosteroid therapy. The control group consisted of 34 healthy volunteers with similar gender and age distribution to the patients. Blood urine nitrogen, creatinine, albumin, triglyceride, cholesterol, 24-hour proteinuria, platelets, erythrocyte sedimentation rate, C-reactive protein and thrombin generation values in activation and remission period of NS were compared. Moreover, TGA values of the patients in their remission period were compared with the values of those in the control group. Endogenous thrombin potential (ETP) and peak thrombin levels were significantly higher in the activation period than remission period of NS. Additionally, after the patients achieved remission, their ETP was still higher than the control group. There was a negative correlation between both ETP and peak thrombin levels of patients with serum albumin, whereas a significant positive correlation was detected with platelet levels. Thromboembolic events were not observed in any of the patients during follow-up. Nephrotic syndrome is strongly associated with hypercoagulopathy as assessed by TGA during active NS. The present study reinforces the usefulness of TGA as a marker of hypercoagulability in pediatric patients with NS. Further studies are needed in this regard.

Peak Thrombin and D-Dimer Levels in Subjects with Severe Hemophilia Receiving Acute Treatment for Bleeding Episodes Experienced during Prophylactic Marstacimab Treatment

Introduction: Marstacimab is a fully humanized monoclonal immunoglobulin G1 that targets the shared K2 domains of tissue factor pathway inhibitor (TFPI)α and (TFPI)β and is currently in phase 3 development. The intended indication is routine prophylaxis treatment to prevent or reduce the frequency of bleeding episodes in patients with hemophilia A or B (with or without inhibitors). Factor replacement or bypass treatment for bleeding events may lead to increased levels of peak thrombin and D-dimer associated with an increased risk of thrombosis . In this post hoc analysis of data from a phase 2 study in patients with hemophilia with and without bleeding episodes, receiving prophylactic marstacimab treatment, peak thrombin and D-dimer levels were investigated to assess the changes in these biomarker levels observed after bleeding episodes. Methods: Individual subject data from the phase 2 study (clinicaltrials.gov identifier: NCT02974855)were used for this analysis. Biomarker data for healthy volunteers who received single doses of marstacimab in a phase 1 dose escalation study (clinicaltrials.gov identifier: NCT02531815) were used as control data, as these subjects represent an intact and uncompromised coagulation system. Study subjects in the phase 2 study received subcutaneous (SC) marstacimab at doses of (1) 150 mg once weekly (QW), with a loading dose of 300 mg, (2) 300 mg QW, and (3) 450 mg QW. All subjects with bleeding episodes were identified, along with on-demand treatment administered for each bleeding episode. Treatments permitted for bleeding episodes included activated coagulation factor VIIa, factor VIII, or factor IX; use of activated prothrombin complex concentrate was prohibited. D-dimer and peak thrombin data collected within 3 days after each bleeding episode were used for this analysis. Time profiles of peak thrombin and D-dimer levels were analyzed to assess the effect of bleed treatment. Biomarker profiles were compared between subjects with and without bleeding episodes, as well as with the data from healthy volunteers (n=41). Results: A total of 15 bleeding episodes were reported in 8 of 26 subjects during the study (excluding screening and follow-up). No subject participating in the study showed any relevant increases in D-dimer levels after receiving on-demand treatment for a bleeding episode while receiving regular prophylaxis with marstacimab, compared with levels seen in subjects who did not experience a bleeding episode. Based on the peak thrombin data (see Figure), 150 nM was observed as the upper limit for 18 of 26 subjects who did not experience any bleeding episodes, which was approximately 50% of the 300 nM observed in healthy volunteer controls treated with 450 mg intravenous marstacimab. Transient increases in peak thrombin of &amp;gt;150 nM were observed at several time points in 3 of 8 subjects who experienced bleeding episodes. The highest peak thrombin level reported was approximately 211 nM in one subject receiving marstacimab 300 mg SC QW and factor VIII concentrate on demand during the study. Conclusions: No transient increases in D-dimer could be attributed to the administration of bleeding episode treatment. The transient increases in peak thrombin levels following on-demand treatment for bleeding episodes did not exceed peak thrombin levels seen in subjects without bleeding events or the levels seen in healthy volunteer controls receiving single doses of marstacimab. Based on peak thrombin and D-dimer levels observed in this post hoc analysis, there does not appear to be any indication of an increased risk of thrombosis post administration of acute on-demand bleeding episode treatment while on prophylactic marstacimab therapy at the doses studied. Disclosures Nayak: Pfizer Inc.: Current Employment, Other. Raje:Pfizer Inc.: Current Employment, Other. Teeter:Pfizer Inc.: Current Employment. Harnisch:Pfizer Inc.: Current Employment, Other. Arkin:Pfizer: Current Employment, Current equity holder in publicly-traded company, Other: own stock/options in the company.

Longitudinal Assessment of Thrombin Generation in Patients with Hemophilia Receiving Fitusiran Prophylaxis: Phase II Study Results

Longitudinal Assessment of Thrombin Generation in Patients with Hemophilia Receiving Fitusiran Prophylaxis: Phase II Study Results

MG1113, a specific anti–tissue factor pathway inhibitor antibody, rebalances the coagulation system and promotes hemostasis in hemophilia

BackgroundReplacement therapy is the most common treatment for reduction of bleeding and control of episodic bleeding in individuals with hemophilia. Despite the proven effectiveness of factor replacement therapy, repeated intravenous administration is a heavy burden to individuals with hemophilia. ObjectivesTo reduce the burden, therapeutic agents that can be subcutaneously administered need to be developed, and an anti–tissue factor pathway inhibitor (TFPI) antibody may be a suitable candidate for this purpose. MethodsMG1113 is an IgG4 monoclonal antibody that binds to Kunitz‐2 domain (KD2) of TFPI. To confirm the coagulation potential of MG1113, several tests were conducted using factor VIII (FVIII)‐ or IX (FIX)‐deficient plasma. For the ex vivo spiking test, platelet‐poor plasma samples from 14 individuals with hemophilia were spiked with MG1113. The in vivo efficacy was determined using blood loss tests, modified prothrombin time (mPT), and free TFPI quantification after intravenous or subcutaneous administration of MG1113 into hemophilia A (HA)‐induced rabbits. ResultsRadiographic crystallography demonstrated the specific binding site between MG1113 and KD2. In FVIII‐deficient plasma and the plasma of individuals with hemophilia, peak thrombin and endogenous thrombin levels were increased by MG1113 in a concentration‐dependent manner. Rotational thromboelastometry assay revealed that clotting time, clot formation time, and maximum clot firmness were normalized in MG1113‐treated blood of patients. Intravenous or subcutaneous injection of MG1113 into HA‐induced rabbits resulted in rebalancing of blood loss, mPT, and free TFPI levels. ConclusionsThese results indicate that subcutaneous administration of MG1113 neutralizes the function of TFPI and regulates bleeding in individuals with hemophilia.

ENDOGENOUS THROMBIN GENERATION POTENTIAL IN PATIENTS WITH PULMONARY EMBOLISM AND ITS RELEVANCE TO THROMBOTIC AND INFLAMMATORY BIOMARKERS

SESSION TITLE: Pulmonary Vascular Disease Posters SESSION TYPE: Original Investigation Posters PRESENTED ON: October 18-21, 2020 PURPOSE: Pulmonary embolism (PE) is a catastrophic outcome in patients with venous thrombosis. In the United States around 900,000 people (1 to 2 per 1,000) patients are affected each year with the mortality rate ranging from 10 – 30 %. Inflammation, hemostatic dysregulation and vascular complications contribute to the pathogenesis of PE. Endogenous thrombin generation contributes to the overall pathology of PE. The purpose of this study is to investigate endogenous thrombin potential (ETP) and the circulating biomarkers of inflammation, hemostatic activation and endothelial dysfunction in plasma samples obtained from the patients at the onset of PE. METHODS: Citrated blood samples from 78 patients with confirmed PE were collected at Loyola University Medical Center and Gottlieb Memorial Hospital under an IRB approved protocol. Normal human plasma (NHP) comprised of 25 male and 25 female samples were obtained from a commercial source (George King Biomedical, Overland park, KS.) Thrombin generation studies were carried out using a commercially available a kinetic system (Diagnostica Stago, Paris, France). Thrombin generation parameters such as peak thrombin, lag time and area under the curve (AUC) were compiled. Frozen samples were retrospectively analyzed for biomarkers, including D-Dimer, PAI-1, TNFa, IL6, IL1b, VEGF and TFPI using commercially available sandwich ELISA methods. Results were compiled as mean ± SD and analyzed for significance and correlation. Applicable statistical analysis was carried out by using GraphPad Prism software. RESULTS: On a cumulative basis, PE patients showed a wide variation in thrombin generation parameters. Peak thrombin levels (91.1 nM vs 160.0 nM) and AUC (554.7 nM*min vs 700.9 nM*min) were decreased in PE patients while lag time was increased (6.41 min vs 2.7 min). Biomarkers analysis showed elevated levels of D-Dimer (7521.3 ng/ml) in comparison to NHP (192.8 ng/ml), PAI (67.3 ng/ml) in comparison to NHP (10.5 ng/ml), TNF-a (3.2 pg/ml) in comparison to NHP (1.9 pg/ml), IL6 (99.9 pg/ml) in comparison to NHP (1.24 pg/ml), IL1b (1.6 pg/ml) in comparison to NHP (0.79 pg/ml) and TFPI (139.1ng/ml) in comparison to NHP (62.3 ng/ml) while and VEGF (31.24 pg/ml) levels were decreased in comparison to NHP (45.5 pg/ml. The biomarker levels did not show any correlation with peak thrombin levels with the exception of VEGF and TFPI. Samples with higher thrombin generation (>150 nM) showed marked elevation of all of the biomarkers studied. CONCLUSIONS: PE patients exhibit a wide variation in endogenous thrombin generation parameters. However, biomarkers of hemostatic activation, vascular dysfunction and inflammation were increased. CLINICAL IMPLICATIONS: Measurement of thrombin generation parameters along with the biomarkers of inflammation and endothelial dysfunctions may be useful in the overall management of PE patients. DISCLOSURES: No relevant relationships by Emily Bontekoe, source=Web Response No relevant relationships by Yevgeniy Brailovsky, source=Web Response No relevant relationships by Amir Darki, source=Web Response No relevant relationships by Jawed Fareed, source=Web Response No relevant relationships by ambar Farooqui, source=Web Response No relevant relationships by Debra Hoppensteadt, source=Web Response No relevant relationships by Omer Iqbal, source=Web Response No relevant relationships by Fakiha Siddiqui, source=Web Response

Decreased Thrombin Generation Potential in Lymphoma Patients is associated with Increased D‐dimer, CRP, vWF and TNF‐α. Interrelationship between Thrombogenesis and Inflammation

IntroductionInflammation and other vascular factors contribute to the pathogenesis of thrombotic complications in lymphoma patients. Biomarkers of hemostatic activation, vascular dysfunction and inflammation are upregulated in lymphoma. It has been reported that thrombin generation and inflammatory biomarkers are increased in cancer while the thrombin generation potential is decreased. This study compares the thrombin generation potential and its relevance to the generation of various biomarkers of hemostatic activation process and inflammatory responses.Materials and MethodCitrated blood samples from 90 patients with confirmed diagnosis of non‐Hodgkin lymphoma (NHL), Hodgkin lymphoma (HL) and Chronic lymphocytic leukemia/Small lymphocytic lymphoma (CLL/SLL) were collected from University of Belgrade Clinic, Belgrade, Serbia. 50 samples of normal human plasma (NHP) was obtained from George King Biomedical (Overland park, KS). Thrombin generation studies were carried out using a commercially available a kinetic system. Thrombin generation parameters such as peak thrombin (PT), lag time (LT) and area under the curve (AUC) were compiled. Plasma levels of D‐Dimer, CRP, vWF and TNF‐α were measured by using commercially available Sandwich ELISA assays. Results were compiled in terms of mean ± SD. Correlation analysis were carried out by using Prism, Graphpad.ResultsOn a cumulative basis, lymphoma patients showed an increase in lag time (2.9 ± 1.17) in comparison to the NHP (2.7± 0.61). The peak thrombin levels was decreased (119.0 ± 46.2) in the lymphoma patients in comparison to NHP (159.7 ± 31.1). The AUC was decreased (670.2 ± 290.01) in the lymphoma patients in comparison to NHP (700.9 ± 198.0). When the lymphoma patients were sub grouped, peak thrombin levels were similar in HL (129.9), NHL (114.7) and CLL (127.4) and were decreased compared to NHP (159.7). However the AUC was increased in the HL (769), decreased in NHL (642.4) and was comparable for CLL (715.4) compared to NHP (700.9). Lymphoma patients also exhibited elevated levels of D‐Dimer (878 ± 1205 ng/ml) in comparison to NHP (280 ng/ml), CRP (15.2 ± 26.6 ug/ml) in comparison to NHP (0.9 ug/ml), vWF (271.8 ± 189.7 %) in comparison to NHP (122%) and TNF‐α (16.4 ± 20.64 pg/ml) in comparison to NHP (4.2 pg/ml). In the HL group D‐Dimer and CRP correlated well with AUC and PT, in this group vWF also showed a good correlation with PT. In the NHL group, CRP correlated well with LT.ConclusionLymphoma patients represent a heterogenous group in which both the hypercoagulable state and inflammatory responses simultaneously occur as evident by increase in D‐Dimer and inflammatory biomarkers. The decrease in thrombin generation potential in these patients may be due to the consumption of prothrombin and other coagulation factors. Wide variations in circulating levels of various biomarkers and thrombin generation parameters between various lymphoma groups are noted. Profiling of thrombin generation and inflammatory biomarkers may be helpful in thrombotic risk stratification of lymphoma patients and their anti‐thrombotic management.

Differential Augmentation of Thrombin Generation by Andexanet Alfa in Lymphoma Patients

IntroductionThe prevalence of thrombosis in lymphoma patients is reportedly high and ranges from 3–10%. Vascular malfunction and inflammatory processes further contribute to the thrombotic activation processes in these patients. Andexanet alfa is an antidote for factor Xa inhibitors and its usage has been reported with thrombotic complications. This study was designed to compare effect of andexanet alfa (AA) on the thrombin generation potential and its relevance to the generation of thrombin.Materials and MethodCitrated blood samples from 78 patients with confirmed diagnosis of non‐Hodgkin lymphoma (NHL), Hodgkin lymphoma (HL) and Chronic lymphocytic leukemia/Small lymphocytic lymphoma (CLL/SLL) were collected from the Clinic of Hematology Unit, University of Belgrade, Belgrade, Serbia. 50 samples of normal human plasma (NHP) was obtained from George King Biomedical (Overland park, KS). NHP was prepared for referencing purposes. Individual samples were supplemented with andexanet alfa at 100 ug/ml. Thrombin generation studies were carried out using a commercially available a kinetic fluorogenic substrate method (calibrated automated thrombogram; CAT). Thrombin generation parameters such as peak thrombin (PT), lag time (LT) and area under the curve (AUC) were compiled. Results were compiled in terms of mean ±SD.ResultsOn a cumulative basis, lymphoma patients showed an increase in LT (2.9 1.15) in comparison to NHP (2.06 .02) which decreases with AA (1.94 0.1). The PT levels were decreased (120.34 44.3) in comparison to NHP (159.20 4.8) and increases with AA (153.70 37.2). The AUC was also decreased (668.85 284.3) in comparison to NHP (756.46 34.5) which increases with AA (758.32 226.4). When the lymphoma patients were sub‐grouped, peak thrombin levels were similar in HL (128.16), NHL (113.99) and CLL (129.77) were decreased compared to normal human plasma (159.2) which increases with AA, in HL (180.75), NHL (154.16) and CLL (142.46) compared to AA control (153.7). The AUC was increased in the HL (769), decreased in NHL (642.4) and was comparable for CLL (715.4) compared to normal human plasma (756.46). However, with AA the AUC values increases for HL (938.31) and decreases for NHL (744.52) and CLL (725.26) in comparison with AA control (758.32). Variations in lag time were noted in the three groups.ConclusionLymphoma patients represent a heterogenous group of patients in which both the hypercoagulable state and inflammatory responses simultaneously occur. Thrombin is constantly generated in some of these patients leading to the formation of fibrin. The observed decrease in thrombin generation potential in these patients may be due to the consumption of prothrombin and other coagulation factors. Differential increase in thrombin generation in post andexxa supplemented samples suggest that the use of this agent may potentially be associated with thrombotic complications.

Decreased Thrombin Generation Potential in Lymphoma Patients Is Associated with Increased D-Dimer, CRP, Vwf and TNF-α. Interrelationship between Thrombogenesis and Inflammation

Introduction: The prevalence of thrombosis in lymphoma patients is reportedly high and ranges from 3 - 10%, and further increased at advanced stages of the disease especially in hgNHL. Inflammation and other vascular factors contribute to the pathogenesis of these thrombotic complications. Biomarkers of hemostatic activation, vascular dysfunction and inflammation are elevated in lymphoma. We have previously reported that thrombin generation biomarkers such as D-Dimer, thrombin anti-thrombin complex (TAT) and prothrombin fragment (F1.2) along with inflammatory biomarkers are increased in cancer patients. Interestingly, despite an increase in thrombin generation markers, thrombin generation potential in these patients is decreased. This study was designed to compare the thrombin generation potential and its relevance to the generation of various biomarkers of hemostatic activation process and inflammatory responses. Methods: Citrated blood samples from 90 patients with confirmed diagnosis of non-Hodgkin lymphoma (NHL), Hodgkin lymphoma (HL) and Chronic lymphocytic leukemia/Small lymphocytic lymphoma (CLL/SLL) were collected from the Clinic of Hematology Unit, University of Belgrade, Belgrade, Serbia. 50 samples of normal human plasma (NHP) was obtained from George King Biomedical (Overland park, KS). NHP was prepared for referencing purposes. Thrombin generation studies were carried out using a commercially available a kinetic fluorogenic substrate method (calibrated automated thrombogram; CAT). Thrombin generation parameters such as peak thrombin (PT), lag time (LT) and area under the curve (AUC) were compiled. Plasma levels of D-Dimer, CRP, vWF and TNF- α were measured by using commercially available Sandwich ELISA assays. Results were compiled in terms of mean ± SD. Correlation analysis carried out by using Prism, Graphpad. Results: On a cumulative basis, lymphoma patients showed an increase in lag time (2.9 1.17) in comparison to the NHP (2.7). The peak thrombin levels was decreased (119.0 46.2) in the lymphoma patients in comparison to NHP (159.7 31.1). The AUC was decreased (670.2 290.01) in the lymphoma patients in comparison to NHP (700.9 ). When the lymphoma patients were sub grouped, peak thrombin levels were similar in HL (129.9), NHL (114.7) and CLL (127.4) and were decreased compared to NHP (159.7). However the AUC was increased in the HL (769), decreased in NHL (642.4) and was comparable for CLL (715.4) compared to NHP (700.9). Variations in lag time were noted in the three groups. The lymphoma patients also exhibited elevated levels of D-Dimer (878 1205 ng/ml) in comparison to NHP (280 ng/ml), CRP (15.2 26.6 ug/ml) in comparison to NHP (0.9 ug/ml), vWF (271.8 189.7 %) in comparison to NHP (122%) and TNF-α (16.4 20.64 pg/ml) in comparison to NHP (4.2 pg/ml). Interestingly, when the lymphoma group were subdivided into various groups, the D-Dimer level was highest in HL following the order HL&gt;NHL&gt;CLL. CRP and vWF levels were highest in HL and comparable in both NHL and CLL. Wide scatter in TNF-α data was noted with CLL exhibiting highest levels with the ranked order CLL&gt;NHL&gt;HL. The composite data on thrombin generation parameters and biomarkers is shown on the table 1A. In the HL group D-Dimer and CRP correlated well with AUC and PT, in this group vWF also showed a good correlation with PT. In the NHL group, CRP correlated well with LT. In the CLL group none of the biomarkers correlated well with the thrombin generation parameters. On a cumulative basis, D-Dimer, CRP, vWF and TNF-α had no significant relevance to thrombin generation parameters. The composite regression coefficience data is shown on table 1B. Conclusion: Lymphoma patients represent a heterogenous group in which both the hypercoagulable state and inflammatory responses simultaneously occur as evident by increase in D-Dimer and inflammatory biomarkers. While the overall thrombin generation potential in the plasma of these patients is decreased, thrombin is constantly generated in some of these patient leading to the formation of fibrin. The observed decrease in thrombin generation potential in these patients may be due to the consumption of prothrombin and other coagulation factors. Wide variations among these lymphoma groups are noted. Profiling of thrombin generation and inflammatory biomarkers may be helpful in thrombotic risk stratification of lymphoma patients and their anti-thrombotic management. Disclosures No relevant conflicts of interest to declare.

Analysis of the role of thrombomodulin in all-trans retinoic acid treatment of coagulation disorders in cancer patients

BackgroundClinical studies have shown that all-trans retinoic acid (RA), which is often used in treatment of cancer patients, improves hemostatic parameters and bleeding complications such as disseminated intravascular coagulation (DIC). However, the mechanisms underlying this improvement have yet to be elucidated. In vitro studies have reported that RA upregulates thrombomodulin (TM) expression on the endothelial cell surface. The objective of this study was to investigate how and to what extent the TM concentration changes after RA treatment in cancer patients, and how this variation influences the blood coagulation cascade.ResultsIn this study, we introduced an ordinary differential equation (ODE) model of gene expression for the RA-induced upregulation of TM concentration. Coupling the gene expression model with a two-compartment pharmacokinetic model of RA, we obtained the time-dependent changes in TM and thrombomodulin-mRNA (TMR) concentrations following oral administration of RA. Our results indicated that the TM concentration reached its peak level almost 14 h after taking a single oral dose (110 frac{mg}{m^2} ) of RA. Continuous treatment with RA resulted in oscillatory expression of TM on the endothelial cell surface. We then coupled the gene expression model with a mechanistic model of the coagulation cascade, and showed that the elevated levels of TM over the course of RA therapy with a single daily oral dose (110 frac{mg}{m^2} ) of RA, reduced the peak thrombin levels and endogenous thrombin potential (ETP) up to 50 and 49%, respectively. We showed that progressive reductions in plasma levels of RA, observed in continuous RA therapy with a once-daily oral dose (110 frac{mg}{m^2} ) of RA, did not affect TM-mediated reduction of thrombin generation significantly. This finding prompts the hypothesis that continuous RA treatment has more consistent therapeutic effects on coagulation disorders than on cancer.ConclusionsOur results indicate that the oscillatory upregulation of TM expression on the endothelial cells over the course of RA therapy could potentially contribute to the treatment of coagulation abnormalities in cancer patients. Further studies on the impacts of RA therapy on the procoagulant activity of cancer cells are needed to better elucidate the mechanisms by which RA therapy improves hemostatic abnormalities in cancer.

Reversal of Factor Xa Inhibitors by Andexanet Alfa May Increase Thrombogenesis Compared to Pretreatment Values

Recombinant coagulation factor Xa (FXa), inactivated Zh-zo, also known as andexanet alfa (AA), is a modified version of human FXa that has been developed to neutralize FXa inhibitors. We studied the reversal effect of AA for these inhibitors in various anticoagulant and thrombin generation (TG) assays. Individual aliquots of normal human plasma containing 1 µg/mL of apixaban, betrixaban, edoxaban, and rivaroxaban, were supplemented with saline or AA at a concentration of 100 µg/mL. Clotting profiles include prothrombinase-induced clotting time, activated partial thromboplastin time, and prothrombin time. Factor Xa activity was measured using an amidolytic method. Thrombin generation was measured using a calibrated automated thrombogram. Differential neutralization of all 4 anticoagulants was noted in the activated clotting time and other clotting tests. The FXa activity reversal profile varied with an observed decrease in apixaban (22%), betrixaban (56%), edoxaban (28%), and rivaroxaban (49%). Andexanet alfa also led to an increased TG in comparison to saline. The peak thrombin was higher (40%), area under the curve (AUC) increased (15%), whereas the lag time (LT) decreased (17%). Andexanet alfa added at 100 µg/mL to various FXa supplemented systems resulted in reversal of the inhibitory effects, restoring the TG profile; AUC, LT, and peak thrombin levels were comparable to those of unsupplemented samples. Andexanet alfa is capable of reversing anti-Xa activity of different oral FXa inhibitors but overshoots thrombogenesis in both the saline and FXa inhibitor supplemented systems. The degree of neutralization of Xa inhibitor is specific to each agent.

Decreased Thrombin Generation Potential Is Associated with Increased Thrombin Generation Markers in Sepsis Associated Coagulopathy

Introduction: Sepsis associated coagulopathy (SAC) is commonly seen in patients which eventually leads to dysfunctional hemostasis and disseminated intravascular coagulation (DIC). Thrombin generation plays an important role in the overall pathophysiology of this process. Previous studies have reported an increase in thrombin generation markers such as thrombin antithrombin complex (TAT) and prothrombin fragment (F1.2). (Hoppensteadt et al. Clin Appl Thromb Hemost. 2014 Mar;20(2):129-35). Sepsis eventually results in consumption coagulopathy in which some of the clotting factors are consumed. The purpose of this study is to determine the thrombin generation potential of baseline blood samples obtained from sepsis associated coagulopathy patients and demonstrate their relevance to thrombin generation markers.Materials and Methods: Baseline citrated blood samples were prospectively collected from 49 patients with sepsis and suspected DIC. DIC scores were determined according to the ISTH scoring system (PTINR, fibrinogen, D-dimer and platelet count). Healthy control (n=50) represented citrated plasma obtained from a commercial supplier (George King Biomediacl, Overland Park, KS). Thrombin generation studies were carried out using a commercially available florigenic substrate methods with thrombin calibrator and PPP reagent (calibrated automated thrombogram; CAT). Such parameters as peak thrombin, lag time and area under the curve were compiled. TAT and F1.2 were measured using commercially available ELISA methods (Seimens, Indianpolis, IN). Functional antithrombin levels were measured using a chromogenic substrate method. All results were calculated in terms of mean ± SD. Applicable statistical methods were used to correlate the thrombin generation parameters with thrombin generation markers and antithrombin.Results: The peak thrombin levels were lower (82±40nM) in the SAC patients in comparison to higher levels observed in the normal plasma (133±10nM). The AUC was lower (561 ±280) in the SAC group in comparison to the normal (624±18). The SAC group showed much longer lag time (4.1±2.1) in comparison to the normal (2.1 ± 2.2). Wide variations in the results were observed in these parameters in the SAC group. The compsoite data is compiled in the table. the F1.2 levels in the DIC group were much higher (570±48 pmol) in comparison to the normal (210±25 pmol). The TAT levels also increased in the SAC group (27.9 ±5.1 ng/ml) in comparison to the normal (2.8 ±0.8 ng/ml). The functional antithrombin levels were decreased in the SAC group (64 ±11%). No correlation was observed between thrombin generation parameters and thrombin generation markers.Conclusion: These results validate the earlier observations that thrombin generation such as F1.2 and TAT are decreased in patients with SAC. However thrombin generation parameters are significantly elevated in this group in comparison to normals. This may be due to the consumption of prothrombin due to the activation of the coagulation system. The SAC group also showed wide variations in both the thrombin generation parameters and the F1.2 and TAT. These variations may be due to the differences in the endogenous pathophysiology state in the SAC patients. The decreased functional AT levels observed in the SAC group are due to the formation of the complex between generated thrombin and antithrombin. Simultaneous profiling of thrombin generation and thrombin generation markers may be helpful in the risk stratification of these patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Extended Storage of Thawed Cryoprecipitate: Responsible and Rational Use of a Scarce Resource

BackgroundCryoprecipitate (cryo) is a frozen plasma derivative consisting of fibrinogen, factor (F) XIII, FVIII and von Willebrand factor (vWF), and was originally intended to replace FVIII in hemophilia. Today, cryo is primarily used as fibrinogen replacement in acquired coagulopathy related to trauma, obstetric hemorrhage, and DIC. Due to the lability of FVIII, guidelines mandate that cryo be given within 6 hours of thawing. Fibrinogen is much more stable; however, and these time constraints may unnecessarily contribute to delayed product utilization and increased waste. Viscoelastic assays are increasingly used to assess coagulopathy and may be better indicators of global coagulation function than standard coagulation assays. Here we evaluated the performance of thawed, refrigerated cryoprecipitate over extended storage times.MethodsCryo (six donor pools, n=8) was purchased from South Texas Blood and Tissue, thawed on first day of testing, and stored in aliquots refrigerated at 4°C for up to 35 days. Assays were performed in 37°C rewarmed product at baseline post-thaw and at 4, 24, and 72h, and then weekly to 35 days. Assays for factor levels were conducted in cryo diluted 1:10 in PBS and using a hematology analyzer (Stago) for fibrinogen and FVIII or by ristocetin cofactor assay (RCo) for vWF (Siemens). Coagulation and thrombin generation of stored cryo was tested in ROTEM and Calibrated Automated Thrombogram (CAT) by mixing cryo with frozen aliquots of cryo-poor plasma. A massive transfusion protocol (MTP) was simulated by combining cryo with red cells, fresh frozen plasma, and fresh apheresis platelets (1:1:1:1 ratio); this simulation was tested for coagulation with ROTEM and for vWF-mediated fluorescent-labeled platelet adhesion to collagen in a microfluidics flow system (Bioflux).ResultsIn simulated MTP, global coagulation function as measured by EXTEM and FIBTEM G-values from ROTEM were above normal reference ranges for whole blood for the entire 35d period (EXTEM smallest median G=13592 dyn/cm2, ref. range=4800-12250; FIBTEM smallest median G=2041, ref. range=495-1667). There was a slight decline in G value over storage duration, but it was not statistically significant (Fig 1). Lysis index at 30 min (LI30) in the EXTEM of simulated MTP did not statistically deviate over time (Fig 2).The MTP FIBTEM A10 value, shown to be correlated with fibrinogen concentration, remains high over 35 days, with only some values on D35 dropping into the normal range (Fig 3; means at D0=35.75mm and D35=26.63; ref. range=9-24). A10 and G were highly correlated (r=.812, p=.008).FVIII activity in cryo declined from 9-fold above average plasma levels over 35 days to approximately 25% of its starting activity. Surprisingly, the drop in FVIII was not correlated with clotting time (CT) in the EXTEM (r=-.335, p=.739), and CT was either faster than or within normal references ranges for whole blood at all time points (Fig 4; slowest median CT=49.5s, ref. range=42-72). The change in CT was correlated with measured peak thrombin levels (Fig 5; r=-.883, p=.002), although FVIII was not correlated with peak thrombin generation (r=.403, p=.282).vWF activity of cryo initially increased as has been previously reported but declined to baseline level by D7 and further through D35. vWF activity was correlated with the rate of platelet adhesion to a collagen surface under arterial flow (Fig 6; r=.677, p=.045).DiscussionCryo is mainly used in resuscitation of severe hemorrhage in MTP. It contributes to hemostasis by providing vWF to support platelet adhesion and primary hemostasis, FVIII to augment thrombin generation, and fibrinogen as clot substrate. Standard coagulation assays such as PT and aPTT or isolated factor activity assays are inadequate for evaluating the contribution of cryo to global MTP performance. The data presented here, derived from multiple functional tests including ROTEM, CAT, ristocetin cofactor assay, and Bioflux, show that thawed cryoprecipitate stored at 4°C is functionally viable for up to 35 days; as part of a massive transfusion protocol. It supports clot strength, thrombin generation, and platelet adhesion. Adoption of an extended shelf life for thawed cryo would reduce waste and increase availability for treatment of hemorrhage. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.