Mean Endogenous Thrombin Potential Research Articles

An in Vitro Pilot Study of the Role of Factor IX As an Alternative to Bypassing Agents in the Treatment of Breakthrough Bleeds in Patients with Hemophilia a and Inhibitors Treated with Emicizumab

Introduction: Hemophilia A(HA) is an X linked disorder characterized by a deficiency in Factor VIII. 30% of patients with severe HA develop alloantibodies that inhibit the coagulant activity of FVIII. Emicizumab, a substitution therapy that bridges factor IX and X, is approved in patients with severe HA with and without inhibitors. Indeed, it is now the standard of care prophylactic therapy for patients with inhibitors. Breakthrough bleeds and perioperative management require the use of bypassing agents in addition to emicizumab, with recombinant VIIa deemed the safest of these options. Nevertheless there are still concerns about thrombotic risks and inability to monitor bypassing agents given to patients on emicizumab. Since the rate limiting step for FXa generation in the plasma of patients receiving emicizumab is the concentration of generated FIXa, we conducted an in vitro pilot study to evaluate the hemostatic effect of adding FIX to the plasma of patients on emicizumab prophylaxis. Methods: After informed consent was obtained, blood was collected in citrated tubes prefilled with 1.45mM of corn trypsin inhibitor(CTI) from 25 patients with HA receiving emicizumab prophylaxis. Aligning with previously published data, the added hemostatic effect of rFVIIa was studied in platelet rich plasma (PRP), while all other products were studied in platelet poor plasma (PPP). PRP was spiked with 0, 45, 90 and 270mg/kg of rFVIIa, and PPP was spiked with 0, 10, 20, 30 and 50 IU/kg of activated prothrombin complex concentrate (aPCC); 0, 25, 50 and 100 IU/kg of recombinant FIX(rFIX) and 0, 25, 50 and 100 IU/kg of plasma derived FIX(pdFIX). Thrombin generation was run on these spiked samples using the CAT method. Coagulation was triggered in PRP samples with tissue factor 1pM. PPP samples were activated using 1pM of TF and 1.5pM FIXa. Mean endogenous thrombin potential (ETP, area under the thrombin generation curve) values were compared between the different experiments and with samples obtained from controls. Baseline FIX concentrations were determined in these patients using bovine chromogenic assay. The mean plasma emicizumab concentration (+Standard Deviation) was 44.45 µg/mL (+15.83). Results: As shown in fig 1, ETP was significantly improved with increasing concentrations of aPCC, rFIX and pdFIX in PPP(p<0.005). Fig 2 shows that ETP normalized with the addition of rFVIIa 45mg/kg (p<0.0001) in most of the patients tested. Moreover, we did not see ETP above the upper limit of the normal range when FIX was added, while 50 IU/kg of aPCC and 270 mg/kg of rFVIIa clearly surpassed the upper limit of the normal range, suggesting a risk of hypercoagulability and thrombosis. FIX levels varied between 63.9% and 248.9% with a mean of 134.27%. ETP obtained after adding rFIX 100 IU/kg was significantly greater than that attained with pdFIX (p=0.027), that might be explained by the presence of the activated form of FIX in rFIX concentrates. No statistical significant difference between the doses of 50 IU/Kg of FIX and 100 IU/KG was observed (p>0.05), with a noted tendency to increased ETP with 100IU/KG dose. Conclusion: Our in vitro study demonstrates excess thrombin generation (and potential thrombosis risk) with higher concentrations of rFVIIa and aPCC that are used in the clinical care of patients. While addition of FIX at a range of doses appears to improve thrombin generation without exceeding the upper normal limits. These data suggest that FIX therapy may safely improve hemostasis in patients with HA and inhibitors receiving emicizumab prophylaxis. A clinical study looking at a starting dose of rFIX at 50 IU/KG for mild to moderate bleeds and 100 IU/Kg for more severe bleeds is warranted. In addition to the advantage of monitoring levels with FIX therapy, the longer half-life of FIX offers the potential advantage of controlling hemostasis with single dose administration. Future prospective clinical trials are needed to investigate the efficacy and safety of this therapy in this patient population.

Non‐severe COVID‐19 is associated with endothelial damage and hypercoagulability despite pharmacological thromboprophylaxis

BackgroundHypercoagulability and endothelial dysfunction are hallmarks of coronavirus disease 2019 (COVID‐19) and appear to predict disease severity. A high incidence of thrombosis despite thromboprophylaxis is reported in patients with moderate to severe COVID‐19. Recent randomized clinical trials suggest that therapeutic‐intensity heparin confers a survival benefit in moderate‐severity COVID‐19 compared to standard‐intensity heparin, potentially by harnessing heparin‐mediated endothelial‐stabilizing and anti‐inflammatory effects. ObjectiveWe hypothesized that patients with moderate‐severity COVID‐19 exhibit enhanced hypercoagulability despite standard‐intensity thromboprophylaxis with low molecular weight heparin (LMWH) compared to non‐COVID‐19 hospitalized patients. MethodsPatients with moderate COVID‐19 and a control group (severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2]–negative hospitalized patients) receiving LMWH thromboprophylaxis were recruited. Markers of endothelial damage and plasma thrombin generation parameters were assessed. ResultsTissue plasminogen activator levels were significantly increased in the COVID‐19 group (8.3 ± 4.4 vs. 4.9 ± 2.4 ng/ml; P = .02) compared to non‐COVID‐19–hospitalized patients. Despite thromboprophylaxis, mean endogenous thrombin potential was significantly increased among COVID‐19 patients (1929 ± 448 vs. 1528 ± 460.8 nM*min; P = .04) but lag time to thrombin generation was significantly prolonged (8.1 ± 1.8 vs. 6.2 ± 1.8 mins; P = .02). While tissue factor pathway inhibitor (TFPI) levels were similar in both groups, in the presence of an inhibitory anti‐TFPI antibody, the difference in lag time between the groups was abrogated. ConclusionsCollectively, these data demonstrate that COVID‐19 of moderate severity is associated with increased plasma thrombin generation and endothelial damage, and that hypercoagulability persists despite standard LMWH thromboprophylaxis. These findings may be of clinical interest given recent clinical trial data which suggest escalated heparin dosing in non‐severe COVID‐19 may be associated with improved clinical outcomes.

In Vitro Comparison of Recombinant and Plasma-Derived von Willebrand Factor Concentrate for Treatment of Acquired von Willebrand Syndrome in Adult Extracorporeal Membrane Oxygenation Patients

Coagulopathic bleeding is common during adult extracorporeal membrane oxygenation (ECMO), and acquired von Willebrand syndrome is a contributing factor. We compared ECMO patient blood samples that were treated in vitro with recombinant von Willebrand Factor concentrate and plasma-derived von Willebrand Factor concentrate. Our hypothesis was that recombinant von Willebrand Factor (vWF) would have greater efficacy in increasing vWF function. Secondarily, we hypothesized that recombinant vWF would have less impact on thrombin generation. Thirty ECMO patients and 10 cardiac surgical controls were enrolled in the study. ECMO patient blood samples were treated in vitro with low- and high-dose recombinant vWFs and low- and high-dose plasma-derived vWFs. Whole blood ristocetin-induced platelet aggregation (RIPA), plasma ristocetin cofactor activity (RCo), and thrombin generation were compared between ECMO patient blood samples and control blood samples and between vWF-treated ECMO patient blood samples and nontreated samples. ECMO patient blood samples had severely reduced median RIPA compared to control samples 2 ohms (1-12 [25th-75th percentile]) vs 20 ohms (11-42) (P < .001). Treatment of ECMO patient blood samples with high-dose recombinant vWF significantly increased median RIPA to 10 ohms (2-15) (P < .001), while low-dose recombinant vWF and low- and high-dose plasma-derived vWFs did not significantly increase RIPA; 6 ohms (3-14), 4 ohms (1-13), and 6 ohms (2-10), respectively (P = .25, >.99, and >.99). Treatment with high-dose recombinant vWF and low- and high-dose plasma-derived vWFs significantly increased median plasma RCo to 4.7 international units (IU)/mL (3.7-5.9), 3.3 IU/mL (2.7-4.8), and 3.9 IU/mL (3.4-5.3), respectively, compared to controls 1.8 IU/mL (1.5-2.3) (all P < .001). Treatment with low- and high-dose plasma-derived vWFs significantly increased mean endogenous thrombin potential (6270.2 ± 2038.7 and 6313.1 ± 1913.3) compared to nontreated samples (5856.7 ± 1924.6) (P = .04 and .006), whereas treatment with low- and high-dose recombinant vWFs had no significant effect on mean endogenous thrombin potential (5776.1 ± 2087.3 and 5856.2 ± 1946.4) (P > .99 for both comparisons). In vitro treatment of ECMO patient blood samples with high-dose recombinant vWF was superior to low-dose recombinant vWF and plasma-derived vWF in terms of improving RIPA. In addition, recombinant vWF treatment did not increase endogenous thrombin potential, which may reduce overall thrombotic risk if it used to treat acquired von Willebrand syndrome in ECMO patients.

COVID-19 Is Associated with Endothelial Dysfunction and Enhanced Plasma Thrombin Generation Despite Pharmacological Thromboprophylaxis

BackgroundHospitalised patients with severe COVID-19 (requiring critical care level support) appear to be at increased risk of thrombosis despite standard pharmacological thromboprophylaxis. The magnitude of thrombotic risk in patients with COVID-19 of moderate severity (not requiring critical care) is less clear. The optimal approach to thromboprophylaxis (and the role of intensified thromboprophylaxis) remains to be determined.Evidence of endothelial dysfunction has been widely reported in COVID-19 (particularly in severe COVID) and this may contribute to hypercoagulability.AimTo assess differences in patterns of hypercoagulability and endothelial dysfunction between a group of patients with moderate COVID-19 and a group of age-matched hospitalized patients (SARS-CoV-2 PCR negative) receiving low molecular weight heparin (LMWH) thromboprophylaxis.MethodsBlood was collected from individuals admitted to hospital with COVID-19 of moderate severity (not requiring critical care level support) and a group of age-matched patients admitted with infective/inflammatory illness (SARS-CoV-2 PCR negative). All subjects received standard-dose LMWH thromboprophylaxis, with blood drawn at 12 hours post-dose (and with measurement of anti-FXa activity levels). Circulating levels of endothelial & fibrinolytic markers including ICAM, PAI-1, VCAM, soluble thrombomodulin (sTM), and tissue plasminogen activator (tPA) were determined by ELISA. Thrombin generation (TG) in platelet-poor plasma was assessed by calibrated automated thrombography in the presence of tissue factor (Final concentration, 1pM & 5pM), thrombomodulin (TM) (Final concentration, 6.25nM), and an inhibitory anti-tissue factor pathway inhibitor antibody (anti-TFPI; Final concentration 100μg/mL).Results14 COVID-19 positive subjects and 11 hospitalized controls were recruited. There were no differences in mean age (69.7±4.5 vs 61.6±4.7 years; p= 0.2) or mean Body mass index (25.7±1.1 vs 22.7±1.2 Kg/m2; p=0.1) between groups. No COVID-19 patient or control required critical care support. In the COVID group, radiological evidence of pneumonitis [diffuse (n=3) or peripheral infiltrates (n=7)] was present in the majority of cases. None of the COVID-19 cases were requiring supplemental oxygen at the time of recruitment.All controls were admitted with either respiratory or urinary infection [radiological evidence of pneumonia in 4/11; supplemental oxygen requirement in 2/11, (28-36% FiO2 via nasal cannula)].Plasma levels of sTM, ICAM, PAI-1 & VCAM were similar in both groups. Levels of t-PA were significantly higher in the COVID group (8.31±4.35 vs 4.91±2.37 ng/mL; p= 0.005). Despite similar plasma anti-Xa activity in both groups (0.06 vs 0.04 IU/mL; p=0.2), mean endogenous thrombin potential (ETP) was significantly higher in the COVID group (1929±119.7 vs 1528±138.9 nM*min; p=0.02), although peak thrombin was similar (173.6±26 vs 161.5±31nM). ETP-TM ratio was similar between groups (0.3±0.1 vs 0.2±0.1; p=0.3).Despite increased ETP, the lag time to thrombin generation was significantly prolonged in the COVID group (8.3±0.6 vs 5.8±0.5 mins, p= 0.006). This pattern has previously been observed in vascular diseases associated with altered plasma tissue factor pathway inhibitor (TFPI) activity. In the presence of an anti-TFPI antibody, the difference in lagtime between groups was attenuated (4.7±0.2 vs 3.5±0.1 mins; p= 0.002) and the difference in overall thrombin generation (delta TG) between both groups became significantly increased (Fig.1).ConclusionPlasma thrombin generation is enhanced in patients with non-severe COVID-19 despite pharmacological thromboprophylaxis. Endothelial dysfunction is also observed in this group and appears to modulate parameters of plasma thrombin generation. The clinical implications of these observations are not known although clinical studies of intensified thromboprophylaxis in attenuating thrombotic risk and other complications are ongoing.Fig 1. Inhibition of TFPI activity enhances thrombin generation in COVID-19.In the presence of an inhibitory anti-TFPI antibody, peak plasma thrombin generation was enhanced in COVID-19 in contrast to that observed among SARS-CoV-2 PCR negative hospitalised patients (339.6+25.2 vs 247.4+10.1, p=0.01). [Display omitted] DisclosuresMaguire: Actelion: Research Funding; Bayer Pharma: Research Funding. Ni Ainle: Daiichi-Sankyo: Research Funding; Actelion: Research Funding; Leo Pharma: Research Funding; Bayer Pharma: Research Funding. Kevane: Leo Pharma: Research Funding.

P-622 Prothrombotic biomarkers during controlled ovarian stimulation for assisted reproductive techniques

Abstract Study question Does the evolution of prothrombotic biomarkers over time differ between antagonist and long agonist stimulation protocols for assisted reproductive techniques (ART) ? Summary answer The hypercoagulable state was higher and persistent in the agonist and antagonist with hCG triggering groups compared to the antagonist with GnRH agonist triggering group. What is known already Controlled ovarian stimulation (COS) for ART is associated with supra-physiological serum estradiol levels, a hypercoagulable state and an increased risk of venous thrombosis. Most thromboembolic events associated with COS occur in the context of ovarian hyperstimulation syndrome (OHSS). The use of hCG for final follicular maturation increases the risk of OHSS. In antagonist protocols, GnRH agonist triggering is known to prevent or reduce OHSS and is therefore widely used in women at risk. The impact of the different IVF protocols on pro-thrombotic biomarkers is unknown. Study design, size, duration In this prospective observational cohort study, infertile women undergoing COS for ART in 2017-2019 at the University Hospitals of Geneva and Lausanne (Switzerland) were included. We evaluated changes in key coagulation parameters (D-dimers, factor VIII, fibrinogen activity, protein S and protein C) and thrombin generation, our primary outcome, (using 5 pM of tissue factor) by calibrated automated thrombinography before stimulation (T1), on the day of ovulation triggering (T2) and seven days after triggering (T3). Participants/materials, setting, methods COS was started without hormonal pre-treatment. Protocols were prescribed according to the standards used in each centre taking into account the risk of OHSS (agonist protocol with hCG trigger in women without OHSS risk (Group 1); antagonist protocol in women at risk of OHSS with hCG trigger (Group 2;) or GnRH agonist trigger (Group 3;); variation of endogenous thrombin potential (ETP) was measured and compared among groups using mixed effects linear regression model. Main results and the role of chance A total of 64 women were included: 24 were in group 1, 16 in group 2, and 24 in group 3. The mean age (SD) was 37.8 (2.8), 35.9(5.2) and 34(4.6) years in groups 1, 2 and 3 respectively. As expected, women in group 1 had a statistically lower level of anti-müllerian hormone (p = &amp;lt; 0.001), a lower antral follicular count (p = &amp;lt; 0.001) and lower number of MII oocytes and embryos obtained (p = &amp;lt; 0.001). Mean serum estradiol levels were 1836 (1160), 1628 (815) and 3754 (2165) ng/L at T2, and 945 (471), 1061 (495) and 413 (729) ng/L at T3, in group 1 to 3, respectively. In multivariable regression analysis, the levels in group 3 were statistically higher at T2 and lower at T3 (overall time*group interaction: p &amp;lt; 0.001). The mean ETP was similar between all groups at T1, and increased in all groups at T2 (1442, 1426 and 1486 nM/min in groups 1, 2 and 3, respectively) (p = 0.013). Overall, ETP evolution over time was statistically different between groups, with the lowest increase of ETP between T1 and T3 in group 3. Protein C and protein S levels were stable, while D-dimers, fibrinogen and factor VIII increased at T2 and T3 in all groups. Limitations, reasons for caution Stimulation protocols were prescribed according to the clinical profile and OHSS risks; groups therefore differ substantially in regards to age and ovarian reserve. Thromboembolic events are rare events after COS, we therefore evaluated biological markers of hypercoagulability and not clinical events. Wider implications of the findings Women with GnRH agonist triggering protocol did not increase mean ETP in the week after ovulation, while women with hCG triggering did. This different prothrombotic profile was independent of the variation of the other coagulation parameters investigated. This effect of ovulation triggering should be confirmed by further studies. Trial registration number NCT04188444

P–622 Prothrombotic biomarkers during controlled ovarian stimulation for assisted reproductive techniques

Abstract Study question Does the evolution of prothrombotic biomarkers over time differ between antagonist and long agonist stimulation protocols for assisted reproductive techniques (ART) ? Summary answer The hypercoagulable state was higher and persistent in the agonist and antagonist with hCG triggering groups compared to the antagonist with GnRH agonist triggering group. What is known already Controlled ovarian stimulation (COS) for ART is associated with supra-physiological serum estradiol levels, a hypercoagulable state and an increased risk of venous thrombosis. Most thromboembolic events associated with COS occur in the context of ovarian hyperstimulation syndrome (OHSS). The use of hCG for final follicular maturation increases the risk of OHSS. In antagonist protocols, GnRH agonist triggering is known to prevent or reduce OHSS and is therefore widely used in women at risk. The impact of the different IVF protocols on pro-thrombotic biomarkers is unknown. Study design, size, duration In this prospective observational cohort study, infertile women undergoing COS for ART in 2017–2019 at the University Hospitals of Geneva and Lausanne (Switzerland) were included. We evaluated changes in key coagulation parameters (D-dimers, factor VIII, fibrinogen activity, protein S and protein C) and thrombin generation, our primary outcome, (using 5 pM of tissue factor) by calibrated automated thrombinography before stimulation (T1), on the day of ovulation triggering (T2) and seven days after triggering (T3). Participants/materials, setting, methods COS was started without hormonal pre-treatment. Protocols were prescribed according to the standards used in each centre taking into account the risk of OHSS (agonist protocol with hCG trigger in women without OHSS risk (Group 1); antagonist protocol in women at risk of OHSS with hCG trigger (Group 2;) or GnRH agonist trigger (Group 3;); variation of endogenous thrombin potential (ETP) was measured and compared among groups using mixed effects linear regression model. Main results and the role of chance A total of 64 women were included: 24 were in group 1, 16 in group 2, and 24 in group 3. The mean age (SD) was 37.8 (2.8), 35.9(5.2) and 34(4.6) years in groups 1, 2 and 3 respectively. As expected, women in group 1 had a statistically lower level of anti-müllerian hormone (p = &amp;lt;0.001), a lower antral follicular count (p = &amp;lt;0.001) and lower number of MII oocytes and embryos obtained (p = &amp;lt;0.001). Mean serum estradiol levels were 1836 (1160), 1628 (815) and 3754 (2165) ng/L at T2, and 945 (471), 1061 (495) and 413 (729) ng/L at T3, in group 1 to 3, respectively. In multivariable regression analysis, the levels in group 3 were statistically higher at T2 and lower at T3 (overall time*group interaction: p &amp;lt; 0.001). The mean ETP was similar between all groups at T1, and increased in all groups at T2 (1442, 1426 and 1486 nM/min in groups 1, 2 and 3, respectively) (p = 0.013). Overall, ETP evolution over time was statistically different between groups, with the lowest increase of ETP between T1 and T3 in group 3. Protein C and protein S levels were stable, while D-dimers, fibrinogen and factor VIII increased at T2 and T3 in all groups. Limitations, reasons for caution Stimulation protocols were prescribed according to the clinical profile and OHSS risks; groups therefore differ substantially in regards to age and ovarian reserve. Thromboembolic events are rare events after COS, we therefore evaluated biological markers of hypercoagulability and not clinical events. Wider implications of the findings: Women with GnRH agonist triggering protocol did not increase mean ETP in the week after ovulation, while women with hCG triggering did. This different prothrombotic profile was independent of the variation of the other coagulation parameters investigated. This effect of ovulation triggering should be confirmed by further studies. Trial registration number NCT04188444

Thrombin Generation As a Predictive Biomarker for Venous Thromboembolism in Patients with Pancreatic and Lung Cancer Undergoing Systemic Therapy

Introduction: Venous thromboembolism (VTE) is a leading cause of death in cancer patients, but routine thromboprophylaxis is untenable due to an unacceptably high rate of bleeding complications and judicious patient selection is critical. The Khorana score (KS) predicts the risk for chemotherapy-associated VTE, but the score has limitations because it is assessed only at one time point prior to initiation of treatment. Thrombin generation (TG) is a novel biomarker that assesses global coagulation activation and predicted increased VTE risk in cancer patients in the Vienna Cancer and Thrombosis Study (CATS). In CATS, patients with high peak TG had a 3- fold increased risk for VTE compared to those with low peak TG. A limitation of the study was that it enrolled a heterozygous cancer patient population, and peak TG was not reported by tumor type. Additionally, TG was only measured at enrollment into the study, not over time. The primary aim of our study was to determine if assessment of TG (both peak TG and endogenous thrombin potential (ETP)) improves VTE risk prediction compared to the KS alone during systemic therapy for lung and pancreas cancer. A secondary objective was to evaluate the impact of systemic therapy on peak TG and ETP over time.Methods: This is an ongoing prospective study enrolling adults with newly diagnosed locally advanced or metastatic adenocarcinoma of the lung or pancreas who will receive their cancer therapy at Dartmouth Hitchcock Medical Center. Those with a history of active VTE or use of full dose anticoagulant medication within 30 days prior to enrollment are excluded. After informed consent, KS is calculated and blood is collected in sodium citrate tubes at 3 different time points (at initiation of therapy, and at the beginning of the 2nd and 3rd cycles of systemic therapy). Platelet-poor plasma is prepared by centrifugation and stored at -80°C until analysis by calibrated automated thrombogram (CAT; Thrombinoscope BV, Maastricht, Netherlands) using 1 pM tissue factor and 4 uM phospholipids to trigger coagulation reactions. Measurements are performed in triplicate for each specimen, and raw data are converted to peak TG and ETP. Information about symptomatic VTE events within 6 months of study entry is collected from the medical record.Results: We present the results for 15 patients who have completed study-related procedures to date, including 6 lung and 9 pancreatic cancer patients with a median age 61 and 69 years, respectively. The majority of patients had metastatic disease and all patients received systemic therapy. The KS breakdown for the cohort is: KS1, 7%; KS2, 60%; KS3 27%; KS4 7%. Mean peak TG were 341, 324, 641 and 890 nmole, and mean ETP were 1459, 1685, 2155, 2051 nmole/min for KS 1,2,3 and 4, respectively, and both peak TG and ETP decreased over time after cancer therapy. VTE occurred in 4 patients (27%), and the median KS was 2 for patients with and without VTE. Mean peak TG prior to initiation of chemotherapy was similar in patients with and without VTE (359 and 389 nmole, respectively) but mean ETP was higher in patients with VTE (1959 vs 1760 nmole/min, respectively).Conclusions: Though our sample size to date is small, preliminary results suggest that there is a direct correlation between KS and peak TG and ETP, and that ETP at diagnosis may be a more reliable predictor VTE than KS in lung and pancreatic cancer patients. Peak TG and ETP decreased progressively after initiation of systemic therapy, suggesting that the degree of coagulation activation is proportional to tumor burden. Though preliminary, these data suggest that TG is a promising biomarker of VTE risk. Enrollment to the study is continuing.Funding: This study was funded by Northern New England Clinical Oncology Society. DisclosuresNo relevant conflicts of interest to declare.

Coagulation Kinetics in Adolescent Females Initiating Combined Oral Contraceptives

Objectives: The objective of this pilot study was to evaluate the effect of combined oral contraceptives (COC) on rotational thromboelastometry (ROTEM®) and Technothrombin®TGA (thrombin generation assay) coagulation kinetics, as a surrogate marker of thrombotic risk, in adolescent females. The epidemiology of risk factors associated with contraception-related venous thromboembolism (VTE) in adolescents is not well defined. Estrogen contained in COC produces activated protein C resistance thus increasing the risk of thrombosis. The risk of COC-related VTE is significantly impacted by duration of COC use. The highest risk of COC-related VTE is during the first 6 to 12 months in first-time users. Furthermore, the risk of contraception-related VTE is higher for obese women as compared with non-obese women. Recent evidence found that endogenous thrombin potential (ETP) increased during COC initiation, but have not followed users longitudinally.Methods: This longitudinal cohort included adolescents, ages 12-21 years, initiating COC containing 35 mcg ethinyl estradiol/0.25 mg norgestimate. Balanced recruitment was utilized to strive for equal numbers of obese and non-obese subjects and varied gynecologic age (i.e. years from menarche). Exclusion criteria included personal/family history of thrombophilia or venous thromboembolism, personal history of bleeding diatheses, contraindication to COC, tobacco use, hemoglobin < 10 g/dL, and anti-platelet or anticoagulant medication use. Citrated whole blood samples were collected at baseline (pre-COC), 1, 3, 6, and 12 months for coagulation studies (TGAÒ and ROTEMÒ) and FSH suppression to document COC adherence. TGA parameters included: ETP and peak thrombin production. ROTEMÒkinetic parameters evaluated included: clotting time (CT); viscosity amplitude at 10 & 20 minutes (A10 & A20); and maximum clot firmness (MCF). Changes in parameters were explored over 6 months of COC exposure for coagulation kinetics and 12 months for ETP.Results: Interim analyses included 15 subjects out of a planned 30 total (86.7% White; mean age 16.8 ± 2.8 years; mean BMI 25.1 kg/m2, BMI range 16.2-43.3 kg/m2, 33.3% obese). Post-COC, mean ETP was elevated above baseline (3176.8 nM*min) as early as 1 month and was highest at 6 months (3869.6 & 3890.8 nM*min; p<0.05) (Figure 1A).Mean peak thrombin production was increased from baseline to 6 months (179.5 vs 224.7 nM; p<0.05). From baseline to 3 months, there was a significant (p<0.05) increase in mean A10 (48.0 vs. 52.1 mm), A20 (53.8 vs. 58.6 mm), and MCF (54.0 vs. 58.6 mm) (Figures 1B,1C).There was a significant decrease in mean CT from baseline to 6 months (252.6 vs. 210.6 seconds; p<0.05).Conclusions: After COC initiation, adolescents demonstrated hypercoagulable alterations in TGA and all ROTEM® kinetic parameters. These alterations were evident as early as 1 month but continued to increase over the next 5 months. Continued study will assess if hypercoagulable alterations persist at 12 months. Further study will explore the impact of obesity status at COC initiation on thrombotic risk, as assessed by these global coagulation assays. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Maternal thrombin generation and D-dimer levels in obesity and pregnancy: results from the maternal thrombin generation in obesity and pregnancy (MaTOPs) study.

Venous thromboembolic disease (VTE) risk increases five-fold antenatally and 14-fold during the puerperium. Obesity significantly increases this risk. The D-dimer assay and more novel Calibrated Automated Thrombogram (CAT) assay laboratory tests display potential for use in VTE risk stratification in pregnancy, although to date, research in the performance characteristics of these tests in obese and nonobese pregnant populations is limited. The aim of this study was to compare D-dimer and thrombin generation levels in obese and nonobese pregnant women. Pregnant women were recruited and categorised, as obese (BMI ≥30) or nonobese (BMI 18.5-25). Blood was collected at 26-28 weeks' gestation, 36-40 weeks' gestation and 6-12 weeks postpartum and D-dimer concentrations and endogenous thrombin potential (ETP) were determined. Student's t-test was used to analyse differences in mean D-dimer and ETP. At 36-40 weeks, obese pregnant women had higher D-dimer concentrations (P = 0.001) but lower ETP levels compared with nonobese women (P = 0.044). D-dimer was higher in nonobese than in obese women at 6-12 weeks postpartum (P = 0.026). There was no difference in mean D-dimer (P = 0.825) and mean ETP (P = 0.424) between obese and nonobese women at 26-28 weeks. No difference was observed in mean ETP at 6-12 weeks postpartum (P = 0.472). ETP was lower in both obese and nonobese women postpartum than during pregnancy. D-dimer was lower in obese women but not in nonobese women postpartum. D-dimer concentrations and ETP were not significantly different during multiple time points in pregnancy and postpartum between obese and nonobese pregnant women suggesting limited utility in VTE risk assessment in obese pregnant women.

Thrombin generation in children with febrile neutropenia.

Febrile neutropenia (FN) is a common and serious complication in patients with leukemia. Hemostasis and inflammation are two interrelated systems in response to infection. We aimed to investigate the course of thrombin formation in febrile neutropenia attack of children with acute lymphoblastic leukemia (ALL). Thrombin generation was monitored in children treated for ALL at diagnosis of febrile neutropenia (FN) (t < sub > 0 < /sub > ), at 48 < sup > th < /sup > hour of FN (t1) and after recovery from neutropenia (t < sub > 2 < /sub > ). Twenty-nine patients and 50 healthy children as control were enrolled into the study. Mean endogenous thrombin potential (ETP) and mean peak value of thrombin results at t < sub > 1 < /sub > were significantly higher than at t < sub > 0 < /sub > , t < sub > 2 < /sub > and control groups, respectively. A positive but statistically nonsignificant correlation between ETP values at t < sub > 1 < /sub > and duration of neutropenia was observed. Although thrombin generation is enhanced both due to chemotherapy or malignancy itself, our results revealed that thrombin formation also increased in neutropenic infection of children with leukemia.

Effect of low or high doses of low‐molecular‐weight heparin on thrombin generation and other haemostasis parameters in critically ill patients with COVID‐19

The clinical picture of the coronavirus 2 (SARS-CoV-2)/COVID-19-related acute respiratory syndrome is often associated with a coagulopathy. An elevated D-dimer has been linked with an unfavorable prognosis in COVID-19 patients.

Rivaroxaban and Dabigatran for Suppression of Mechanical Heart Valve–Induced Thrombin Generation

Patients with mechanical heart valves (MHVs) require warfarin to prevent thromboembolism. Dabigatran was less effective than warfarin in patients with MHVs, which prompted a black box warning against the use of direct oral anticoagulants for this indication. However, rivaroxaban and apixaban, which inhibit factor Xa, have not been evaluated in patients with MHVs. To determine whether rivaroxaban and apixaban would be effective, we used MHV-induced thrombin generation assays to compare them withwarfarin either alone or in combination with dabigatran. Thrombin generation in the absence or presence of MHV leaflets or sewing ring segments (SRSs) was quantified. Studies were done in control plasma; plasma from patients on warfarin; plasma containing varying concentrations of rivaroxaban, apixaban, or dabigatran alone; or plasma containing rivaroxaban plus dabigatran. Mean endogenous thrombin potential (ETP) increased 1.2-fold, 1.5-fold, and 1.8-fold in the presence of leaflets, Teflon (Terumo Aortic (Sunrise, FL)) SRSs, or Dacron (Terumo Aortic (Sunrise, FL)) SRSs, respectively. Rivaroxaban and apixaban reduced ETP at concentrations above 50 ng/mL but were less effective than warfarin. When rivaroxaban and dabigatran were combined, they suppressed ETP in a more than additive manner. Whereas warfarin suppresses MHV-induced thrombin generation, MHVs induce the generation of factor Xa in concentrations that overwhelm clinically relevant concentrations of rivaroxaban or apixaban. When used in combination, rivaroxaban and dabigatran are more effective than either agent is alone, suggesting that concomitant inhibition of factor Xa and thrombin is better than inhibition of either clotting enzyme alone.

Assessment of Bleeding Phenotype in Hemophilia Α By a Novel Point-of-Care Global Assay

Introduction: Patients with Hemophilia A have considerable phenotypic heterogeneity with respect to clinical severity based on their baseline factor levels. As clinical bleeding risk is helpful to individualize factor replacement therapy in hemophilia patients, previous studies have utilized direct and indirect methods of thrombin generation to classify individual bleeding phenotypes, however, with variable results. An easy to use, point-of-care, global assay to assess bleed phenotype, can be a useful tool in the clinical setting to determine intensity of prophylaxis therapy for patients with hemophilia. We have previously introduced a novel, point-of-care (POC), dielectric microsensor, ClotChip, and demonstrated its sensitivity to factor replacement in patients with severe hemophilia A. We aim to further test the ability of ClotChip in assessment of a bleeding phenotype, as described by a bleeding score, in patients with hemophilia A. Methods: After IRB approval, 28 patients with hemophilia A of varying severity and well-characterized bleeding history, were enrolled in this study at the time of trough factor levels. The bleeding history was extracted from patient charts and included number of bleeds (joint and soft-tissue), annual factor usage in terms of units/kg, and number of target joints. These parameters were used to generate a bleeding score (range: 0 - 24), and patients were divided in to 2 categories with scores between 0 - 12 (n=14) and &gt; 12 (n=14). Healthy volunteers (n=17) were accrued as controls. Whole blood samples were obtained by venipuncture into collection tubes containing 3.2% sodium citrate. Samples were then tested with the ClotChip within 2 hours of collection. ClotChip is based on the electrical technique of dielectric spectroscopy (DS) and features a low-cost (material cost &lt; $1), small- sized (26mm × 9mm × 3mm), and disposable microfluidic biochip with miniscule sample volume (&lt; 10 µL). The ClotChip readout was taken as the temporal variation in the real part of blood dielectric permittivity at 1 MHz. Our previous studies have shown that the ClotChip readout is sensitive to the global coagulation process and the time to reach a peak in permittivity (Tpeak) is a sensitive parameter to assess coagulation factor defects. Thrombin generation assay (TGA) using low tissue factor concentration was also performed on blood samples according to the manufacturer's direction. TGA was not available for 4 hemophilia and 2 control samples. Endogenous thrombin potential (ETP) parameter of TGA was used in this study to assess thrombin generation. Data are reported as mean ± standard deviation (SD). Analysis of variance (ANOVA) was used to test for statistical significance between groups with P &lt; 0.05. Spearman's correlation test was used to derive correlation statistics. Results: ClotChip exhibited a mean Tpeak of 2186s ± 1560s for hemophilia patients in the group with higher bleeding scores (i.e. score &gt;12), a mean Tpeak of 931s ± 496s for the group with lower bleeding scores (i.e. score &lt;12) and a mean Tpeak of 441s ± 74s for the healthy group (Figure 1A). A significant difference in Tpeak was found between the group with higher bleeding scores compared to the group with lower bleeding scores (P = 0.002) as well as between higher bleeding scores and the healthy group (P &lt; 0.0001). However, no significant difference in the TGA ETP parameter was detected between the groups with higher bleeding scores (mean ETP: 470 ± 814) and lower bleeding scores (mean ETP: 471 ± 897) (Figure 1B). ETP exhibited a statistical difference between the healthy group (mean ETP: 3462 ± 575) and both hemophilia groups (P &lt; 0.0001). We also carried out studies to investigate the correlative power of the ClotChip Tpeak parameter to the TGA ETP parameter when including additional blood samples that were collected at various times during a hemophilia patient's prophylaxis regimen. The ClotChip Tpeak parameter exhibited strong negative correlation to the TGA ETP parameter (Spearman's rs= -0.73, P &lt; 0.0001). Conclusions: Our studies suggest that a novel dielectric microsensor (ClotChip) could be useful in assessing bleeding phenotype in hemophilia A patients, allowing rapid assessment of hemostasis using a miniscule amount of whole blood (&lt;10 µL) at the POC. Further studies are needed to determine if ClotChip data can be used to individualize prophylactic factor replacement regimens in hemophilia A patients. Disclosures Maji: XaTek, Inc: Patents &amp; Royalties: 9,995,701. Suster:XaTek, Inc: Consultancy, Patents &amp; Royalties: 9,995,701. Mohseni:XaTek, Inc: Consultancy, Patents &amp; Royalties. Ahuja:XaTexk Inc.: Consultancy, Patents &amp; Royalties, Research Funding; Rainbow Children's Foundation: Research Funding; Bayer: Consultancy; Biovertiv Sanofi: Consultancy; Genentech: Consultancy.

Effects of Andexanet Alfa on Thrombin Generation in Bleeding Associated with Factor Xa Inhibitors

Effects of Andexanet Alfa on Thrombin Generation in Bleeding Associated with Factor Xa Inhibitors

Thrombin Generation in Chinese Pregnant Women and the Effect of Insulin Use on Thrombin Generation in Patients with GDM.

Pregnancy is a hypercoagulable state associated with an increased risk of venous thrombosis. Calibrated automated thrombogram (CAT) is a test to monitor the thrombin generation (TG), a laboratory marker of thrombosis risk, and increases during normal pregnancy, but it is still unclear whether TG is related to the use of insulin in pregnant women with gestational diabetes mellitus (GDM). We performed thrombin generation by CAT on 135 normal pregnant women, including 43 in first trimester, 32 in second trimester, 60 in third trimester, respectively; 68 pregnant women with GDM were also enrolled, 19 patients with GDM using insulin to control blood glucose and 49 patients control their blood glucose through diet and exercise with noninsulin treatment. The overall CAT parameters were calculated using descriptive statistics method with mean ± standard deviation. Mean endogenous thrombin potential, peak thrombin generation, and StartTail time increased significantly with the pregnancy. There was no significant difference in TG test parameters except StartTail time(P = .003) in insulin-treated GDM group when compared to those without insulin in the GDM group. The normal ranges for CAT parameters in pregnant women were determined. Thrombin generation increased significantly in first trimester and remains stable in second and third trimester. The use of insulin in patient with GDM did not affect thrombin generation test. Our study helps to establish the reference range of thrombin generation in Chinese normal pregnant population and provide more basis to predict the risk of thrombus complicating during pregnancy.

Individual thrombin generation and spontaneous bleeding rate during personalized prophylaxis with Nuwiq®(human‐cl rhFVIII) in previously treated patients with severe haemophilia A

For individuals with haemophilia A, prophylaxis with factor VIII (FVIII) is typically directed towards trough activity >1IU/dL; however, some patients still experience spontaneous bleeding events (sBEs). Aims were to evaluate relationships of endogenous thrombin potential (ETP) and FVIII:C with occurrence of clinical bleeding. GENA-21 was a prospective, open-label, phase IIIb study investigating the safety and efficacy of Nuwiq® (human-cl rhFVIII) in previously treated adults with severe haemophilia A. The study included a 72-hour pharmacokinetic (PK) evaluation phase and a 6-month personalized prophylaxis phase in which treatment was guided by PK parameters. This subanalysis assessed FVIII:C by one-stage assay and ETP by thrombin generation assay in blood samples. Baseline mean ETP was lower in the 7 patients who experienced sBEs during personalized prophylaxis versus 25 who did not (n=32 with data from PK phase and prophylaxis phase; P=.0002). During personalized prophylaxis (n=49), only patients with lower median trough ETP experienced sBEs (8/49 patients; ROC AUC=0.9421; P<.0001); there was no significant relationship for FVIII:C in predicting sBEs (ROC AUC=0.5838; P=.4750). Directly following infusion of human-cl rhFVIII, ETP was lower in patients who experienced sBEs versus those who did not (P=.0002), whereas FVIII:C did not differ significantly between these groups. In adults with severe haemophilia A and reduced thrombin generation, increased frequency of spontaneous bleeding was observed irrespective of trough FVIII levels. Thus, personalized prophylaxis should take into account variables other than FVIII:C. Large prospective trials are needed to verify ETP as a marker for spontaneous bleeding.

Effect of double-filtration plasmapheresis for antibody-mediated rejection on hemostasis parameters and thrombin generation

IntroductionDonor-specific alloantibodies (DSAs) cause kidney-allograft loss in chronic antibody-mediated rejection (CAMR). Treatment relies on blocking antibody-producing cells and removing DSAs by apheresis: e.g., double-filtration plasmapheresis (DFPP). Materials and methodsTo determine the impact of DFPP (6 or 8 sessions/patient) on clotting factors and natural anticoagulants, and on thrombin generation, we performed a prospective and observational study in five CAMR kidney-transplant patients who received DFPP plus rituximab therapy. Thrombin generation was performed in poor platelet plasma (PPP) with 5 pM tissue factor without and with 2 nM recombinant human thrombomodulin. ResultsAfter the first DFPP session, median levels of high molecular-weight proteins (fibrinogen, FV, FVIII, FXI, FXIII, von Willebrand factors and α2-MG) decreased significantly to <50% of baseline values, whereas levels of low molecular-weight factors (<100 kDa) were not significantly modified, except for protein S and TFPI. Of note, binding-protein (BP) S, i.e., C4BP, was significantly decreased. Over the course of successive DFPP sessions, both high and lower molecular-weight proteins (<100 kDa) with longer half-lives (>2 days, prothrombin and factor XII) were significantly decreased. DFPP also highly affected thrombin generation in the absence of thrombomodulin but not significantly in the presence of thrombomodulin. After the first DFPP session, mean endogenous thrombin potential (ETP) and peak thrombin (PH) significantly decreased when the thrombin generation assay was performed without thrombomodulin (respectively, 1084 nM·min for ETP and 210 nM for PH after the first DFPP session compared to 1616 nM·min and 264 nM at baseline). In the presence of thrombomodulin, there was only a slight decrease in ETP and PH (respectively 748 nM·min, and 172 nM after the first DFPP session compared to 822 nM·min and 179 nM at baseline). After the last session, median ETP and PH decreased respectively to 646 nM·min and 143 nM without thrombomodulin, and, to 490 nM·min and 117 nM with thrombomodulin. ConclusionsDFPP significantly removed high molecular-weight proteins from the haemostatic system and profoundly decreased levels of protein S and TFPI. Overall thrombin-generation balance was only moderately affected in the presence of thrombomodulin. Nevertheless, high depletion of fibrinogen, FXIII and Von Willebrand Factor may expose patients to an increased risk of bleeding.

Reversal of apixaban anticoagulation by four‐factor prothrombin complex concentrates in healthy subjects: a randomized three‐period crossover study

Background Currently, there is no approved reversal agent for direct activated factor Xa (FXa) inhibitors; however, several agents are under investigation, including prothrombin complex concentrates (PCCs). Objective This open-label, randomized, placebo-controlled, three-period crossover study assessed the effect of two four-factor PCCs on apixaban pharmacodynamics and pharmacokinetics in 15 healthy subjects. Methods Subjects received apixaban 10 mg twice daily for 3 days. On day 4, 3 h after apixaban, subjects received a 30-min infusion of 50 IU kg-1 Cofact, Beriplex P/N (Beriplex), or saline. Change in endogenous thrombin potential (ETP), measured with a thrombin generation assay (TGA), was the primary endpoint. Secondary endpoints included changes in other TGA parameters, prothrombin time (PT), International Normalized Ratio (INR), activated partial thromboplastin time, anti-FXa activity, apixaban pharmacokinetics, and safety. Results Apixaban-related changes in ETP and several other pharmacodynamic measures occurred following apixaban administration. Both PCCs reversed apixaban's effect on ETP; the differences in adjusted mean change from pre-PCC baseline to end of infusion were 425 nm min (95% confidence interval [CI] 219.8-630.7 nm min; P < 0.001) for Cofact, and 91 nm min (95% CI - 31.3 to 212.4 nm min; P > 0.05) for Beriplex. Both PCCs returned ETP to pre-apixaban baseline levels 4 h after PCC infusion, versus 45 h for placebo. For both PCCs, mean ETP peaked 21 h after PCC initiation, and then slowly decreased over the following 48 h. Both PCCs reversed apixaban's effect on TGA peak height, PT, and INR. Apixaban pharmacokinetic and anti-FXa profiles were consistent across treatments. Conclusions Cofact and Beriplex reversed apixaban's steady-state effects on several coagulation assessments.

Generation of Platelet Microparticles after Cryopreservation of Apheresis Platelet Concentrates Contributes to Hemostatic Activity.

Objective:In the last decade, substantial evidence has accumulated about the use of cryopreserved platelet concentrates, especially in trauma. However, little reference has been made in these studies to the morphological and functional changes of platelets. Recently platelets have been shown to be activated by cryopreservation processes and to undergo procoagulant membrane changes resulting in the generation of platelet-derived microparticles (PMPs), platelet degranulation, and release of platelet-derived growth factors (PDGFs). We assessed the viabilities and the PMP and PDGF levels of cryopreserved platelets, and their relation with thrombin generation.Materials and Methods:Apheresis platelet concentrates (APCs) from 20 donors were stored for 1 day and cryopreserved with 6% dimethyl sulfoxide. Cryopreserved APCs were kept at -80 °C for 1 day. Thawed APCs (100 mL) were diluted with 20 mL of autologous plasma and specimens were analyzed for viabilities and PMPs by flow cytometry, for thrombin generation by calibrated automated thrombogram, and for PDGFs by enzyme-linked immunosorbent assay testing.Results:The mean PMP and PDGF levels in freeze-thawed APCs were significantly higher (2763±399.4/µL vs. 319.9±80.5/µL, p<0.001 and 550.9±73.6 pg/mL vs. 96.5±49 pg/mL, p<0.001, respectively), but the viability rates were significantly lower (68.2±13.7% vs. 94±7.5%, p<0.001) than those of fresh APCs. The mean endogenous thrombin potential (ETP) of freeze-thawed APCs was significantly higher than that of the fresh APCs (3406.1±430.4 nM.min vs. 2757.6±485.7 nM.min, p<0.001). Moreover, there was a significant positive poor correlation between ETP levels and PMP levels (r=0.192, p=0.014).Conclusion:Our results showed that, after cryopreservation, while levels of PMPs were increasing, significantly higher and earlier thrombin formation was occurring in the samples analyzed despite the significant decrease in viability. Considering the damage caused by the freezing process and the scarcity of evidence for their in vivo superiority, frozen platelets should be considered for use in austere environments, reserving fresh platelets for prophylactic use in blood banks.

Cryopreserved Platelets: From in Vitro Thrombin Generation Potential to in Vivo Safety

Background: Cryopreservation of platelets (PLTs) at -80°C with dimethyl sulfoxide (DMSO) can extend their shelf life up to 2 years. Cryopreserved PLTs (CRY-PLTs) are reported to have a greater in vivo hemostatic effect than liquid-stored PLTs. Aims of this study were: i. to evaluate the thrombin generation potential of buffy coat derived cryopreserved PLTs (CRY- BC PLT) in comparison with fresh buffy coat derived platelets concentrates; ii. to determine the efficacy and safety of CRY-PLTs transfusion in hematological patients with severe thrombocytopenia.Materials and methods: BC PLTs were obtained from 5 buffy coats and pooled. The final PLTs concentrates were leukoreduced by filtration and transferred to a 650 mL patented cryopreservation kit (Promedical ®) which allowed mixing with DMSO 25% in a closed system and following removal of supernatant without further manipulations. BC-PLTs were washed prior freezing, suspended in homologous plasma from 1 of the 5 donors to a final concentration of 200 mL and frozen at - 80°. CRY- BC PLTs were preserved at -80°C with 6% DMSO. A system of 3 accessory bags directly connected to the mother bag was adopted for the in vitro study, to avoid repeated freezing/thawing of samples. In vitro assays were performed before freezing and at 3,6 and 9 months after thawing. Before assay, CRY-BC PLT were thawed at 37°C and diluted in plasma to adjust to 300× 109/L PLTs. Fresh BC PLTs underwent the same dilution to adjust to 300 ×109/L PLTs. Thrombin generation (TGA) was tested in CRY BC-PLTs and compared to TG potential of fresh BC PLTs. TGA was triggered by the addition of 0.5 pmol/L of recombinant human tissue factor. Endogenous thrombin potential (ETP) and peak height (PH) were determined. Flow Cytometry assays for PLTs activation markers and thromboelastography were also determined on each sample.CRY-BC PLTs, separately prepared according to the above described method for in vivo study, were infused in five hematological patients with acute leukemia (AL) and severe thrombocytopenia (PLTs <10 ×109/L) participating to the trial NCT02032134.CRY-BC PLTs were transfused to control epistaxis (n=2) and for prophylaxis (n=3). Patients were observed up to 7 days after infusion and the occurrence of any side effect was registered. An increase in PLTs count was observed only in one case, under prophylaxis, but bleeding was successfully controlled or prevented in all cases. Plasma from patients transfused with CRY-BC PLTs was tested for TGA pre-treatment and 24 hours after treatmentResults Fourty nine BC-PLTs from 245 healthy volunteer donors (145 males and 100 females, mean age: 48.16.±18.91) were prepared, cryopreserved and analyzed up to 9 months after storage. Cryopreserved PLTs show a good thrombin generation potential that is stably maintained up to 9 months after cryopreservation [ETP (nM min): 529.25±98.64 at T0, 558.82±114.67 at T3, at 548.57±93.38 T6 and 533.04±103.15 at T9 months, respectively; PH(nM): 132.77±44.9 at T0, 103.4±44.9 at T3, 108.0±36.7 at T6 and 132.0±44.6 at T9 months, respectively]. At TGA, fresh BC-PLTs (n=35) had a mean ETP of 760.13±130.11, PH was 138.9±40.2. Thrombin generation of CRY-BC PLTs is comparable to fresh BC-PLTs, even if slightly decreased. Infusion of CRY-BC PLT (1U) was effective in controlling mucosal bleeding (epistaxis) in two patients with AL and severe thrombocytopenia. CRY-PLT were also effective when administered for prophylaxis in 3 patients with very low platelets count secondary to chemotherapy. In vivo, thrombin generation is stably maintained up to 24 hours after infusion of 1 Unit of CRY-BC PLTs, without any adverse effect (mean ETP pre-treatment was: 414.13±160.60, 24 hours after transfusion: 326.95±152.54). CRY-BC PLTs were safe and they did not determine any thrombotic event.At flow-cytometry, CRY-BC PLTs expressed higher activation markers (CD62P,CD63) than fresh BC PLTs. CRY-BC PLTs are able to significantly decrease the time to clot formation and clot strength, as measured also by thromboelastography. CRY-BCPLTs activation/deterioration is accompanied by an effective hemostatic in vivo function.Conclusions: Cryopreserved PLTs have an enhanced hemostatic activity and a good thrombin generation potential. They are effective and safe in preventing and controlling bleeding, being available in emergency/urgency settings also for patients with acute leukemia and severe thrombocytopenia. DisclosuresReina:Promedical: Consultancy.