Differences In Thrombin Generation Research Articles

Extent of peritoneal metastases from colorectal cancer is not associated with changes in thrombin generation or fibrinolysis

Abstract Objectives Cancer cells can activate coagulation and inhibit fibrinolysis. The aim was to investigate the association between the burden of peritoneal metastases from colorectal cancer (PM-CRC) and biomarkers reflecting thrombin generation and fibrinolysis. Methods A cohort of 55 patients with PM-CRC scheduled for cytoreductive surgery. Patients were grouped by the peritoneal cancer index (PCI) assessed intraoperatively into limited PM-CRC (PCI≤15) and extensive PM-CRC (PCI>15). Blood samples were obtained before surgery. Thrombin generation was measured in vivo by thrombin-antithrombin complex (TAT) and prothrombin fragment 1+2 (F1+2), and ex vivo by the endogenous thrombin potential (ETP). Fibrinolysis was analyzed with fibrin clot lysis assay, fibrinogen, and D-dimer. Results Non-significantly decreased thrombin generation by F1+2 (p=0.72), TAT (p=0.32), and ETP (p=0.19) were observed in patients with extensive PM-CRC (n=9) compared with limited PM-CRC (n=46). Non-significantly prolonged 50 % clot lysis time were found in patients with extensive PM-CRC than in patients with limited PM-CRC. Conclusions Minor non-significant differences in thrombin generation and fibrinolysis were found between patients with extensive PM-CRC and limited PM-CRC. Thus, increased peritoneal metastatic burden from colorectal cancer does not seem to affect thrombin generation and fibrinolysis.

Thrombin generation assay in COVID-19 patients shows a hypocoagulable pattern

Patients with COVID-19 often exhibit coagulopathy, which can significantly impact prognosis. Therefore, investigating coagulation in this context is clinically relevant. The thrombin generation assay (TGA) provides comprehensive data on individual clotting patterns. In our study, we utilized a calibrated automated thrombogram to globally assess coagulation in COVID-19 patients. The study included 67 COVID-19 patients (40 hospitalized in the medical ward and 27 in intensive care units) and 45 blood donors for comparison. Our analysis revealed significant differences in TGA parameters (lag time, time-to-peak, thrombin peak, and endogenous thrombin potential) between patients and blood donors, suggesting a hypocoagulable state in the former. Specifically, COVID-19 patients exhibited prolonged lag time and time-to-peak values, as well as lower thrombin peak and endogenous thrombin potential compared to blood donors (Mann-Whitney test: p<0.05); notably, no significant differences in thrombin generation were observed based on the clinical setting. These findings suggest a reduced capacity for thrombin generation, indicating a consumptive coagulopathy in COVID-19 patients and that in this context, thrombosis is primarily attributable to localized effects in the lungs, platelet activation, and/or prothrombotic endothelial dysfunction. The thrombin generation assay is instrumental in defining coagulation patterns in COVID-19 and may also be applicable to other infectious diseases.

Blood clot properties, thrombin generation, and platelet activation in patients with dysglycemia and established atherosclerotic cardiovascular disease: The CASCARA study.

There is a strong link between coronary artery disease (CAD), type 2 diabetes (T2D) on one hand, and altered fibrin clot properties, including increased clot density, and unfavorable fibrin clot structure on the other. T2D-related changes in fibrin clots can increase cardiovascular (CV) disease risk, including future CV events. We aimed to assess fibrin clot properties, thrombin generation, and platelet activation in CAD patients with prediabetes (PD) or T2D, compared to CAD patients without glycemic disorders. We allocated patients to three groups: 1) Those with angiographically established CAD but without glycemic abnormalities (CAD group); 2) individuals with PD and established CAD (CAD+PD group); and 3) patients with T2D and CAD (CAD+T2D group). We conducted comparisons across these groups for thrombin generation, fibrin clot permeability, fibrin clot lysis, and platelet activation. The final analysis included 116 eligible patients: 1) CAD group (n = 31); 2) CAD+PD (n = 42); and 3) CAD+T2D (n = 43). The CAD+T2D patients enrolled had well-controlled T2D (median HbA1c level of 5.90%; IQR: 5.7%-6.3%). We found no significant differences in thrombin generation, fibrin clot properties, or platelet activation markers across the three analyzed groups (all P-values >0.20). However, elevated interleukin-6 (IL-6) levels were noted in both the highest and lowest glucose concentration quartiles. Additionally, a substantial increase in endogenous thrombin potential (ETP) was observed in patients in the highest glycated hemoglobin quintile. Individuals with established CAD and concomitant PD or well-controlled T2D exhibited comparable fibrin clot phenotypes, thrombin generation potential, and platelet activation when compared to CAD patients without dysglycemia.

Abstract TP294: Sex Differences in Thrombin Generation in Patients With Acute Ischemic Stroke

Background and Purpose: Sex differences in stroke exist, including variation in stroke risk and outcome. Differences in thrombin generation may contribute to this variation between females and males. To examine this, we assessed sex differences in thrombin generation between females and males with acute ischemic stroke and the relationship to blood cell gene expression. Methods: In 97 patients with acute ischemic stroke thrombin generation was measured by thrombin generation assay. Blood cell gene expression was measured by microarray. Differences in thrombin generation between sexes were identified and the relationship to blood cell gene expression examined. Genes associated with sex differences in thrombin generation were analysed by functional pathway analysis. Results: Females and males had similar overall capacity to generate thrombin. The peak thrombin generated in females was 468.8nM (SD 91.6), comparable to males 479.3 (SD 90.8; p=0.58). Lag time, time to peak thrombin, and endogenous thrombin potential were also similar between females and males. While overall thrombin generation was comparable between females and males with stroke, differences in genes that promote this thrombin generation exist. Females with high peak thrombin had an increase in genes that promote thrombosis, and platelet activation. In contrast, males with high peak thrombin had a decrease in genes involved in thrombus degradation. Conclusion: Females and males with acute ischemic stroke have similar capacity to generate thrombin. However, differences may exist in how this thrombin generation is achieved, with females having increased thrombin signaling, and platelet activation, and males having decreased thrombus degradation. This suggests regulatory differences in thrombosis may exist between females and males that may contribute to sex differences in stroke.

Sex Differences in Thrombin Generation in Patients with Acute Ischemic Stroke.

Sex differences in stroke exist, including variation in stroke risk and outcome. Differences in thrombin generation may contribute to this variation between females and males. To examine this, we assessed sex differences in thrombin generation between females and males with acute ischemic stroke and the relationship to blood cell gene expression. In 97 patients with acute ischemic stroke, thrombin generation was measured by thrombin generation assay. Blood cell gene expression was measured by microarray. Differences in thrombin generation between sexes were identified and the relationship to blood cell gene expression examined. Genes associated with sex differences in thrombin generation were analyzed by functional pathway analysis. Females and males had similar overall capacity to generate thrombin. The peak thrombin generated in females was 468.8nM (SD 91.6), comparable to males (479.3nM;SD 90.8; p = 0.58). Lag time, time to peak thrombin, and endogenous thrombin potential were also similar between females and males. While overall thrombin generation was comparable between females and males with stroke, differences in genes that promote this thrombin generation exist. Females with high peak thrombin had an increase in genes that promote thrombosis, and platelet activation. In contrast, males with high peak thrombin had a decrease in genes involved in thrombus degradation. Females and males with acute ischemic stroke have similar capacity to generate thrombin, however, differences may exist in how this thrombin generation is achieved, with females having increased thrombin signaling, and platelet activation, and males having decreased thrombus degradation. This suggests regulatory differences in thrombosis may exist between females and males that may contribute to sex differences in stroke.

Weight-adjusted dosing of tinzaparin for thromboprophylaxis in obese medical patients

BackgroundThe optimal dose of tinzaparin for prophylaxis in obese medical patients is not well defined. ObjectivesTo evaluate the anti-Xa activity in obese medical patients on tinzaparin prophylaxis adjusted for actual bodyweight. MethodsPatients with a body mass index of ≥30 kg/m2 treated with 50 IU/kg tinzaparin once daily were prospectively included. Anti-Xa and anti-IIa activity; von Willebrand factor antigen and von Willebrand activity; factor VIII activity; D-dimer, prothrombin fragments; and thrombin generation were measured 4 hours after subcutaneous injection between days 1 and 14 after the initiation of tinzaparin prophylaxis. ResultsWe included 121 plasma samples from 66 patients (48.5% women), with a median weight of 125 kg (range, 82-300 kg) and a median body mass index of 41.9 kg/m2 (range, 30.1-88.6 kg/m2). The target anti-Xa activity of 0.2 to 0.4 IU/mL was achieved in 80 plasma samples (66.1%); 39 samples (32.2%) were below and 2 samples (1.7%) above the target range. The median anti-Xa activity was 0.25 IU/mL (IQR, 0.19-0.31 IU/mL), 0.23 IU/mL (IQR, 0.17-0.28 IU/mL), and 0.21 IU/mL (IQR, 0.17-0.25 IU/mL) on days 1 to 3, days 4 to 6, and days 7 to 14, respectively. The anti-Xa activity did not differ among the weight groups (P = .19). Injection into the upper arm compared to the abdomen resulted in a lower endogenous thrombin potential, a lower peak thrombin, and a trend to a higher anti-Xa activity. ConclusionDosing of tinzaparin adjusted for actual bodyweight in obese patients achieved anti-Xa activity in the target range for most patients, without accumulation or overdosing. In addition, there is a significant difference in thrombin generation depending on the injection site.

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

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

Plasma-based assays distinguish hyperfibrinolysis and shutdown subgroups in trauma-induced coagulopathy.

Trauma patients with abnormal fibrinolysis have increased morbidity and mortality. Knowledge of mechanisms differentiating fibrinolytic phenotypes is important to optimize treatment. We hypothesized that subjects with abnormal fibrinolysis identified by whole blood viscoelastometry can also be distinguished by plasma thrombin generation, clot structure, fibrin formation, and plasmin generation measurements. Platelet-poor plasma (PPP) from an observational cross-sectional trauma cohort with fibrinolysis shutdown (% lysis at 30 minutes [LY30] < 0.9, n = 11) or hyperfibrinolysis (LY30 > 3%, n = 9) defined by whole blood thromboelastography were studied. Noninjured control subjects provided comparative samples. Thrombin generation, fibrin structure and formation, and plasmin generation were measured by fluorescence, confocal microscopy, turbidity, and a fluorescence-calibrated plasmin assay, respectively, in the absence/presence of tissue factor or tissue plasminogen activator (tPA). Whereas spontaneous thrombin generation was not detected in PPP from control subjects, PPP from hyperfibrinolysis or shutdown patients demonstrated spontaneous thrombin generation, and the lag time was shorter in hyperfibrinolysis versus shutdown. Addition of tissue factor masked this difference but revealed increased thrombin generation in hyperfibrinolysis samples. Compared with shutdown, hyperfibrinolysis PPP formed denser fibrin networks. In the absence of tPA, the fibrin formation rate was faster in shutdown than hyperfibrinolysis, but hyperfibrinolysis clots lysed spontaneously; these differences were masked by addition of tPA. Tissue plasminogen activator-stimulated plasmin generation was similar in hyperfibrinolysis and shutdown samples. Differences in LY30, fibrin structure, and lysis correlated with pH. This exploratory study using PPP-based assays identified differences in thrombin generation, fibrin formation and structure, and lysis in hyperfibrinolysis and shutdown subgroups. These groups did not differ in their ability to promote tPA-triggered plasmin generation. The ability to characterize these activities in PPP facilitates studies to identify mechanisms that promote adverse outcomes in trauma. Prognostic/Epidemiological; Level III.

Persistent circulating platelet and endothelial derived microparticle signature may explain on-going pro-thrombogenicity after acute coronary syndrome

AimsMicroparticles (MPs) are submicron vesicles, released from activated, and apoptotic cells. MPs are elevated in the circulation of patients with coronary artery disease (CAD) and have pro-thrombotic potential. However, limited data exists on MP signature over time following an acute coronary event. Methods & resultsCirculating total annexin v + (Anv+) MPs of endothelial (EMP), platelet (PMP), monocyte (MMP), neutrophil (NMP) and smooth muscle cell (SMMP) origin were quantified by flow cytometry. 13 patients with acute coronary syndrome (ACS) were prospectively enrolled and 12 patients with stable angina (SA) were included as a comparator group. A panel of MP was measured at baseline, after percutaneous coronary intervention (PCI) and at days 1, 7, 30 and 6 months. Intra & inter group comparison was made between various time points. MP mediated thrombin generation was measured by recording lag phase, velocity index, peak thrombin and endogenous thrombin potential at these time points and compared with healthy controls. The total AnV+ MP levels were similar in ACS and SA groups at baseline, peaked immediately after PCI and were at their lowest on day 1. PMP & EMP levels remained significantly elevated in ACS patients at 6 months when compared to SA. No such difference was noted with NMP, MMP and SMMP. Patients with coronary artery disease showed abnormal thrombograms when compared to controls. Peak thrombin (nano moles) was significantly higher in CAD when compared to controls (254 IQR [226, 239] in ACS, 255 IQR [219, 328] in SA and 132 IQR [57, 252] in controls; p = 0.006). Differences in thrombin generation between ACS and SA were not significant (p = 1). Furthermore, thrombin parameters remained abnormal in ACS & SA patients at 6 months. ConclusionsTotal MP and individual MP phenotypes were significantly elevated after PCI reflecting endothelial injury. Elevated PMP and EMP levels at 6 months in ACS patients is suggestive of on-going inflammation, endothelial injury and may explain on-going pro-thrombogenicity seen up to 6 months after ACS despite dual antiplatelet therapy.

Association of ABO Blood Group with Bleeding Severity in Patients with Bleeding of Unknown Cause

Introduction: Antigens of the ABO blood group system have an impact on the hemostatic balance. The association of blood group non-O and thrombosis risk is well known. In patients with different bleeding manifestations, an overrepresentation of blood group O has been previously reported (Dentali et al, Semin.Thromb. Hemost, 2013). Nevertheless, the independent effect of the ABO blood group on bleeding severity, when considering VWF as a major contributing factor, has not yet been thoroughly investigated. Patients with bleeding of unknown cause (BUC) have a similar bleeding phenotype as patients with a diagnosis of an established bleeding disorder, but the causes underlying their bleeding tendency are currently unclear and might be multifactorial (Gebhart et al, Haemophilia, 2018). Thus, we investigated the prevalence of blood group O and its role as an independent risk factor for increased bleeding severity in a thoroughly characterized cohort of patients with BUC. Methods: For this analysis, we selected all patients with normal results in the assessments of plasmatic coagulation and platelet function consecutively recruited between October 2009 and April 2019 within the Vienna bleeding biobank study (Figure 1; Gebhart et al, Haemophilia, 2018). After informed consent, all patients underwent a structured interview on their previous medical and bleeding history including the assessment of the bleeding severity with the Vicenza bleeding assessment tool (BAT). Biomaterial was processed and stored according to standard operating procedures at the Biobank facility (http://www.biobank.at/). A thorough hemostatic laboratory assessment and measurements of thrombin generation, plasma clot properties, and rotational thromboelastometry (ROTEM), as well as platelet function tests (PFA-100, light transmission aggregometry) were performed. Data on the blood group distribution of 23,145 healthy first-time blood donors were provided by the Austrian Red Cross for comparison. Results: We observed an overrepresentation of blood group O in 199 out of 422 BUC patients (47.2%) compared to 8709 out of 23,145 healthy blood donors (37.6%), odds ratio for blood group O 1.48; 95% CI=1.22-1.79, p&amp;lt;0.001 (Table 1). Blood group O in comparison to non-blood group O was independently associated with an increased bleeding severity (least square mean [95% CI]: 6.2 [5.8-6.6] vs. 5.3 [4.9-5.7], p=0.006) and a higher number of bleeding symptoms (least square (LS) mean [95% CI] 3.5 [3.2-3.7] vs. 3.0 [2.8-3.2], p=0.016), after adjustment for sex, VWF:Ag, VWF:RCo and FVIII activity (Table 2). The prevalence of oral mucosal bleeding was significantly higher in blood group O than in patients with a non-O blood group (26.1% vs. 14.3%), even after adjustment for sex, VWF and FVIII, and multiple testing (p=0.013). When analyzing the influence of blood group O on tests of global hemostatic capacity, results indicated increased clot firmness and reduced lysis in blood group O patients. In ROTEM, the maximum clot firmness was higher (LS mean [95% CI]: 57.4 [56.5-58.3] vs. 55.8 [55.0-56.6] mm, p&amp;lt;0.05) and the maximal lysis lower (LS mean [95% CI]: 13.3 [12.5-14.1] vs. 15.1 [14.4-15.9] %, p&amp;lt;0.05) in patients with blood group O compared to non-O patients, after adjustment for sex, VWF and FVIII, and correction for multiple testing. Also in the analysis of plasma clot properties, the maximum clot absorbance was increased in patients with blood group O after adjustment for sex, VWF and FVIII (LS mean [95% CI]: 0.77 [0.74-0.79] vs 0.71 [0.69-0.74] OD, p&amp;lt;0.05), whereas there was no difference in plasma clot lysis. There was no difference in thrombin generation between BUC patients with blood group O and non-O. We could not identify any differences in platelet function, as assessed by the platelet function analyser-100 and light transmission aggregometry between patients with blood group O in comparison to non-O patients after adjustment for sex, VWF and FVIII, and correction for multiple testing. Conclusion : Blood group O is a risk factor for a more severe bleeding phenotype, especially oral mucosal bleeding, independent of VWF and FVIII levels in patients with BUC. Alterations in global hemostatic capacity in BUC patients with blood group O were identified, whereas platelet function was not different. Our data are important for a better understanding of underlying mechanisms of bleeding in BUC patients, which are most probably multifactorial. Disclosures Jilma: True North Therapeutics: Consultancy, Other: reimbursement for travel costs for scientific presentations; Bioverativ: Consultancy, Other: reimbursement for travel costs for scientific presentations.

Association of ABO blood group with bleeding severity in patients with bleeding of unknown cause

AbstractBlood group O has been associated with an increased bleeding tendency due to lower von Willebrand factor (VWF) and factor VIII (FVIII) levels. We explored whether blood group O is independently associated with bleeding severity in patients with mild-to-moderate bleeding of unknown cause (BUC) in the Vienna Bleeding Biobank cohort. Bleeding severity was recorded with the Vicenza bleeding score (BS). Blood group O was overrepresented in 422 patients with BUC compared with its presence in 23 145 healthy blood donors (47.2% vs 37.6%; odds ratio, 1.48; 95% confidence interval [CI], 1.22-1.79). The BS and the number of bleeding symptoms were significantly higher in patients with blood group O than in patients with non-O after adjustment for VWF and FVIII levels and sex (least-square [LS] means of BSs: 6.2; 95% CI, 5.8-6.6 vs 5.3; 4.9-5.7; and of number of symptoms: LS, 3.5; 95% CI, 3.2-3.7 vs 3.0; 2.8-3.2, respectively). Oral mucosal bleeding was more frequent in those with blood group O than in those with other blood types (group non-O; 26.1% vs 14.3%), independent of sex and VWF and FVIII levels, whereas other bleeding symptoms did not differ. Patients with blood group O had increased clot density in comparison with those with blood group non-O, as determined by rotational thromboelastometry and turbidimetric measurement of plasma clot formation. There were no differences in thrombin generation, clot lysis, or platelet function. Our data indicate that blood group O is a risk factor for increased bleeding and bleeding severity in patients with BUC, independent of VWF and FVIII levels.

Therapeutic doses of recombinant factor VIIa in hemophilia generates thrombin in platelet‐dependent and ‐independent mechanisms

BackgroundIn hemophilia bypass therapy, a platelet‐dependent mechanism is believed to be primarily responsible for recombinant factor VIIa (rFVIIa)’s hemostatic effect. rFVIIa may also possibly interact with other cells through its binding to endothelial cell protein C receptor (EPCR) or cell surface phospholipids. ObjectivesWe aim to investigate the relative contribution of platelet‐dependent and platelet‐independent mechanisms in rFVIIa‐mediated thrombin generation in hemophilic conditions at the injury site. MethodsPlatelets were depleted in acquired and genetic hemophilia mice using anti‐platelet antibodies. The mice were subjected to the saphenous vein injury, and the hemostatic effect of pharmacological concentrations of rFVIIa was evaluated by measuring thrombin generation at the injury site. ResultsAdministration of anti‐mouse CD42 antibodies to mice depleted platelets by more than 95%. As expected, hemophilia mice, compared with wild‐type mice, generated only a small fraction of thrombin at the injury site. The depletion of platelets in hemophilia mice further reduced thrombin generation. However, when pharmacological doses of rFVIIa were administered to hemophilia mice, substantial amounts of thrombin were generated even in the platelet‐depleted hemophilia mice. No differences in thrombin generation were detected among FVIII−/−, EPCR‐deficient FVIII−/−, and EPCR‐overexpressing FVIII−/− mice depleted of platelets or not. Evaluation of platelets by flow cytometry as well as immunoblot analysis showed no detectable expression of EPCR. ConclusionsOur data suggest that pharmacological concentrations of rFVIIa generate thrombin in hemophilia in both platelet‐dependent and platelet‐independent mechanisms.

Laboratory evidence for hypercoagulability in cirrhotic patients with history of variceal bleeding

AimWe aimed to assess the relationship between procoagulant imbalance and the occurrence of variceal bleeding in patients with liver cirrhosis. MethodsWe compared the results of chromogenic assay for the functional evaluation of the Protein C anticoagulant pathway (ThromboPath®), thromboelastometry and the levels of factor VII, VIII, and antithrombin in two groups of cirrhotic patients: Group 1 (n = 25) — patients with moderate or large esophageal or gastric varices, who had never experienced acute gastrointestinal bleeding and Group 2 (n = 24) — patients with a history of variceal bleeding. ResultsDespite the differences in MELD score and the results of basic laboratory tests indicating more severe cirrhosis and suggesting a greater risk of bleeding in Group 2, the results of thromboelastometry did not differ significantly between groups. The ThromboPath® test results [ThP B: 67.8 ± 13.4 versus 59.09 ± 12.4%, p = 0.023] and factor VII level [69.04 ± 24.16 vs 53.54 ± 25.06, p = 0.032] confirmed greater plasma procoagulant activity in Group 1 compared to Group 2. However, there were no statistically significant differences in thrombin generation after activation of the protein C. Plasma of patients in Group 2 was more resistant to anticoagulation with protein C compared to Group 1 (PICI%: 65.58 ± 7.24 versus 55.64 ± 13.07%, p = 0.001). ConclusionThe results of our study confirm the lack of influence of coagulation disorders on the occurrence of variceal bleeding. Moreover, the results of ThromboPath® assay indicate hypercoagulability in patients with a history of variceal bleeding and more severe liver cirrhosis, compared to patients who have never bled.

Four-factor prothrombin complex concentrate improves thrombin generation and prothrombin time in patients with bleeding complications related to rivaroxaban: a single-center pilot trial

BackgroundDirect oral anticoagulants (DOACs) pose a great challenge for physicians in life-threatening bleeding events. The aim of this study was to test the efficacy of reversing the DOAC rivaroxaban using four-factor PCC (prothrombin complex concentrate), a non-specific reversing agent.MethodsPatients with life-threatening bleeding events during rivaroxaban treatment were included and administered 25 U kg−1 of PCC. Blood samples were collected immediately prior to as well as after PCC treatment at predefined time intervals. The primary endpoint was defined as the difference in thrombin generation (TG) parameters ETP (endogenous thrombin potential) and Cmax (peak thrombin generation) prior to and ten minutes subsequent to PCC treatment.ResultsThirteen patients, of whom the majority suffered from intra-cranial haemorrhage (ICH) or subdural haemorrhage (SDH), were included and administered PCC. The results show that the ETP (TG) significantly (p = 0.001) improved by 68% and Cmax (TG) by 54% (p = 0.001) during PCC treatment. In addition, the Quick value (prothrombin time: QuickPT) significantly improved by 28% and the activated partial thromboplastin time (aPTT) was decreased by 7% ten minutes after PCC administration. Cmax was reduced at baseline, but not ETP, aPTT or QuickPT. Lag time until initiation (TG, tlag), thromboelastometry clotting time (CTEXTEM) and time to peak (TG, tmax) correlated best with measured rivaroxaban levels and were out of normal ranges at baseline, but did not improve after PCC administration. In 77% of the patients bleeding (ICH/SDH-progression) ceased following PCC administration. During the study three participants passed away due to other complications not related to PCC treatment. The possibility of thrombosis formation was also evaluated seven days after administering PCC and no thromboses were found.ConclusionsThis study shows that use of PCC improved ETP, Cmax, QuickPT and aPTT. However, of these parameters, only Cmax was reduced at the defined baseline. It can be concluded that CTEXTEM, tlag and tmax correlated best with the measured rivaroxaban levels. The study drug was found to be safe. Nonetheless, additional studies specifically targeting assessment of clinical endpoints should be performed to further confirm these findings.Clinical trial registrationEudraCT trial No. 2013–004484-31.

Thrombin Generation Testing in Patients with Myelofibrosis.

Primary myelofibrosis (PMF) is a chronic clonal myeloid disorder. Together with essential thrombocythemia (ET) and polycythemia vera (PV), it belongs to a group of Philadelphia chromosome-negative myeloproliferative neoplasms. Thrombotic events are serious complications negatively influencing the quality and length of these patients' lives. The confirmed risk factors for venous thromboembolism are age over 60 years, a positive history of thromboembolism, presence of common cardiovascular risks, JAK2 V617F mutation and, according to some authors, leukocytosis. Various opinions on the role of thrombocythemia have been published. The present study was undertaken to evaluate the benefit of thrombin generation test and its potential use in predicting the risk of thrombosis in MF patients. The analysis included plasma samples obtained from 36 patients diagnosed with MF in our center from 2004 to 2016 (JAK2 V617-positive 53%; CALR-positive 31%; MPL-positive 14%; triple negative 2%) and a control group comprising 20 healthy volunteer blood donors. Thrombin generation was measured in platelet-rich plasma using the TECHNOTHROMBIN® TGA kit (Technoclone, Austria) and the fully automated system Ceveron® Alpha (Technoclone). The results were correlated with clinical and laboratory parameters of the patients. There were differences in thrombin generation as expressed by endogenous thrombin potential (ETP) between patients and healthy controls, with ETP being lower in the patient group (p = 0.0003). Analysis confirmed a significant correlation between thrombin generation and platelet counts, with higher thrombin generation in patients with thrombocythemia > 400 x 109/L (p = 0.04). ETP values were consistently higher in earlier disease stages and lower in CALR-mutated myelofibrosis. In MF patients, thrombin generation is mainly influenced by platelet counts and, to a lesser extent, by mutation status, activity, and progression of the disease. Thrombin generation test results have confirmed that thrombocythemia is a potential risk factor for thrombotic complications.

Is resistance futile? The role of activated thrombin‐activatable fibrinolysis inhibitor resistance in bleeding in factor XI deficiency

Is resistance futile? The role of activated thrombin‐activatable fibrinolysis inhibitor resistance in bleeding in factor XI deficiency

Reduced fibrinolytic resistance in patients with factor XI deficiency. Evidence of a thrombin‐independent impairment of the thrombin‐activatable fibrinolysis inhibitor pathway

Essentials Plasma of factor XI-deficient patients (FXI-dp) displays enhanced fibrinolysis. We investigated the role of thrombin activatable fibrinolysis inhibitor (TAFI) in 18 FXI-dp. FXI-dp generated less activated TAFI (TAFIa) on clotting challenge and were resistant to TAFIa. TAFI activation and TAFIa resistance correlated with bleeding score and bleeding phenotype. Background Factor XI (FXI) deficiency, a rare disorder with unpredictable bleeding, has been associated with reduced fibrinolytic resistance as a result of abnormal fibrin density. Objective We investigated the involvement of thrombin-activatable fibrinolysis inhibitor (TAFI) in the increased lysability of FXI-deficient (FXI-def) clots and the role of thrombin. Patients/Methods Eighteen patients with FXI deficiency (1-58%) and 17 matched controls were investigated for fibrinolytic resistance to t-PA, thrombin generation, TAFI activation and response to TAFIa. Results When clotting was induced by 0.5 pm tissue factor (TF), FXI-def plasmas displayed less thrombin and TAFIa generation and shorter lysis time than controls. A 100-fold higher TF concentration (to bypass FXI) abolished the difference in thrombin generation but not in lysis time between patients and controls. Normalization of FXI levels by a FXI concentrate increased thrombin generation but had no effect on the lysis time of FXI-def plasma. Moreover, when clots were induced by purified thrombin and high concentrations of FXa inhibitor, FXI-def plasma still generated less TAFIa and displayed a shorter lysis time than controls. Finally, upon TAFIa addition, the lysis time of FXI-def plasma was prolonged significantly less than that of control plasma, suggesting a TAFIa resistance. TAFIa generation and TAFIa resistance were correlated with the bleeding score, displaying a considerable capacity to discriminate between patients with and without bleeding. Conclusions TAFI pathway impairment, largely caused by a hitherto unknown TAFIa resistance, appears to be one main cause of decreased fibrinolytic resistance in FXI deficiency and might be clinically useful for assessing the bleeding risk of FXI-def patients.

Comparison by sex between thrombin generation and fibrin network characteristics in a healthy population

BackgroundThe aim of the present work was to compare sex differences in thrombin generation and fibrin network characteristics in a young healthy population, and correlate thrombin generation parameters with fibrin network characteristics. MethodsSixty individuals aged 21y (18–26), 50% men and 50% women were selected. Thrombin generation was performed with the Technothrombin® TGA kit. Plasma fibrin formation kinetic was followed by turbidity at 350nm, and the fibrin elastic modulus was measured with the Hemodyne®. In addition, the prothrombin polymorphism G20210A was assessed. ResultsThrombin generation in men was: lag time (LT): 12.5±3.0min, peak thrombin: 257±135nmol/l, and endogenous thrombin potential (ETP): 3459±449nmol/l·min, while in women the LT was shortened (9.7±2.8min, p<0.05) and thrombin formation increased (peak thrombin: 345±178nmol/l and ETP: 4017±429nmol/l·min, p<0.05). Prothrombin and fibrinogen plasma levels were similar between sexes, and the GG genotype of prothrombin G20210A polymorphism (rs1799963) had a frequency of 1. In women, fibrin formation was slightly faster and the elastic modulus was higher than men. ConclusionsThe differences in thrombin generation between women and men were not related to prothrombin concentration, prothrombin polymorphism G20210A or fibrinogen concentration.

Increased thrombin generation in splanchnic vein thrombosis is related to the presence of liver cirrhosis and not to the thrombotic event

IntroductionIn recent years there have been increasing evidence associating liver disease with hypercoagulability, rather than bleeding. The aim of the study was to evaluate the haemostatic potential in patients with liver disease. Patients and methodsWe measured thrombin generation in the presence and absence of thrombomodulin in patients with portal vein thrombosis (PVT, n=47), Budd-Chiari syndrome (BCS, n=15) and cirrhosis (n=24) and compared the results to those obtained from healthy controls (n=21). Fifteen patients with PVT and 10 patients with BCS were treated with warfarin and were compared to an equal number of patients with atrial fibrillation matched for prothrombin time-international normalized ratio. We assessed resistance to thrombomodulin by using ratios [marker measured in the presence/absence of thrombomodulin]. ResultsThere were no differences in thrombin generation between patients on warfarin treatment and their controls. Cirrhotic patients generated more thrombin in the presence of thrombomodulin and exhibited thrombomodulin resistance compared to controls [p=0.006 for endogenous thrombin potential (ETP) and p<0.001 for peak thrombin and both ratios ETP and peak] and patients with non-cirrhotic PVT (p=0.001, p=0.006, p<0.001, p<0.001 for ETP, peak, ratio ETP, ratio peak, respectively). The patients with cirrhotic PVT exhibited higher ETP (p=0.044) and peak (p=0.02) in the presence of thrombomodulin than controls, as well as thrombomodulin resistance (ETP and peak ratios: p=0.001). ConclusionsHypercoagulability and thrombomodulin resistance in patients with cirrhosis were independent of the presence of splanchnic vein thrombosis. The hypercoagulability in patients with cirrhotic PVT could have implications for considering longer or more intensive treatment with anticoagulants in this group.

Alpha-2-Macroglobulin Is a Major Determinant Of a Lower Thrombin Generation In Infants and Children Compared To Adults

▪ IntroductionDevelopmental Haemostasis, the concept describing maturation of the haemostatic system is based on established age specific differences in the quantity and function of haemostatic proteins. Interestingly, infants and children have a lower incidence of thromboembolic events than adults. Understanding the precise mechanism of thromboprotection in infants and children may therefore provide novel prevention and treatment strategies for adults. We hypothesized that a significant component of the thromboprotective mechanism is contributed to by the age-specific differences in thrombin generation. AimThis study was designed to investigate differences in thrombin generation in different age groups from infants to adults, to obtain more insight into age related differences in thromboembolic rates. Materials and MethodsPlasma samples from 86 infants, children and adults were tested individually and the results were grouped according to age: 1 month to 1 year (N=13), 1-5 years (N=20), 6-10 years (N=19), 11-16 years (N=16) and adults (N=18). Thrombin generation (TG) was measured using Calibrated Automated Thrombinography (CAT) triggered with tissue factor (TF) at 5pM and 1pM final concentration. Parameters measured from the TG curve included: lag time, maximal thrombin concentration (peak), time to peak, as well as the area under the curve (endogenous thrombin potential, ETP). The raw data from TG was also used to calculate the overall, as well as alpha-2-macroglobulin (A2M) dependent decay constants. A2M function was measured with a thrombin inhibition assay developed in house. ResultsBoth ETP and peak thrombin in infants and children was consistently significantly lower than in adults at both 1pM and 5pM tissue factor concentrations. The difference in ETP ranged from 27% to 35% (p<0.0001), while the difference in peak thrombin generated ranged from 28% to 37% (p<0.005). The thrombin decay constant (total inhibition of thrombin) was 15% (p<0.01) higher in infants and children compared to adults. The A2M-specific decay constant was on average 93% (p<0.001) higher in the younger populations and represented 14% of the total thrombin inhibition, compared to 9% in adults. The functional A2M-levels were 54% (p<0.05) higher in infants and children compared to adults; and correlated significantly with age (r=0.564, p<0.001). Discussion and ConclusionsThis study confirms that TG in children (including adolescents) is lower than in adults and shows for the first time that this is predominantly caused by higher thrombin inhibition, largely due to an increase of A2M activity.Considering that the levels of A2M are increased by 50%, Antithrombin is decreased by approximately 37%, whilst the levels of Prothrombin, Fibrinogen and Heparin Cofactor II remain the same in infants and children compared to adults; therefore A2M is an important inhibitor of thrombin in early life and during childhood.Age-specific differences in thrombin generation as demonstrated by our study possibly play an important role in the thromboprotective mechanism functioning in infants and children. Disclosures:No relevant conflicts of interest to declare.