Thrombin Generation Peak Height Research Articles

Pharmacodynamic Biomarkers in Infants with Hemophilia A Receiving Emicizumab in HAVEN 7

Background: Emicizumab, a bispecific antibody that mimics the cofactor function of activated factor (F)VIII, demonstrated a favorable safety and efficacy profile in infants with severe hemophilia A (HA) without FVIII inhibitors in HAVEN 7 (NCT04431726; Pipe et al. Blood 2022, 140[S1]: 457-459). It is known that some coagulation factors including FIX and FX, the binding targets of emicizumab, have lower plasma levels during the first 6 months of life. This research investigates whether the developing coagulation system impacts the pharmacodynamics (PD) of emicizumab prophylaxis in infants with HA enrolled to HAVEN 7 from birth to ≤ 12 months of age. Methods: Informed consent from the parents/legally authorized representative and ethics approval were obtained. PD was assessed by activated partial thromboplastin time (aPTT), FVIII activity using a chromogenic assay containing human FIX and FX (considered FVIII-like activity), and by FXIa-triggered thrombin generation (TG) assay. Plasma antigen levels of FIX and FX were determined using immunoassays. Plasma samples from 110 healthy infants (HIs; n=42, 0-3 months old; n=68, >3-24 months old) collected separately were also measured to generate age matched reference ranges. Results: In HAVEN 7, PD biomarkers, and FIX and FX plasma levels were assessed in 55 participants during the first 52 weeks of emicizumab exposure (data cut-off May 22, 2023). At baseline, mean FVIII activity was <1 IU/dL, mean TG peak height was zero, and aPTT was prolonged as expected due to the deficiency of FVIII. By the first post-baseline visit (Week 3), aPTT was normalized to the age matched reference range in most participants and was then generally maintained in the normal range throughout the treatment period. Mean (SD) FVIII-like activity increased to 22.52 (6.05) U/dL at Week 5 after the four loading doses of emicizumab and was sustained at 21-26 U/dL thereafter. Mean (SD) TG peak height increased to 67.37 (27.21) nM at Week 5 and was sustained at 67-88 nM throughout the treatment period. Mean plasma FIX and FX concentrations were not affected by emicizumab treatment. The PD biomarkers, and FIX and FX plasma levels were also analyzed as a function of age group. The loading period (first 4 weeks) was excluded and only “steady state” measurements were considered for the PD markers. All measurements were considered for FIX and FX plasma levels. Mean (SD) FVIII-like activity of 18.14 (5.40) U/dL in the youngest participants (1-2 months old) increased to 23.57 (7.64) U/dL at 3-4 months old and was similar (23-28 U/dL) in the older age groups. Age did not have a significant impact on mean TG peak height. There was no age effect on aPTT. Mean (SD) plasma FIX and FX antigen concentrations were 4.40 (1.09) µg/mL and 6.24 (1.29) µg/mL in the <1 month old participants, increased progressively with age until about 9 months old, and then stayed around 9-10 µg/mL for both FIX and FX. Lower plasma levels of FIX and FX, as well as FVIII activity were also observed in the HIs ≤3 months vs. >3 months of age. There was no significant difference in TG between ≤ 3 months vs. >3 months of age in HIs. FVIII-like activity increased with increasing emicizumab concentrations in a linear fashion up to emicizumab concentrations of 100 µg/mL. Hence, the lower FVIII-like activity in the youngest participants could be partially explained by lower mean “steady state” trough emicizumab concentrations of 48.3 µg/mL at 1 month of age, which then increased to approx. 60 µg/mL at 4-5 months of age and older. TG peak height increased with increasing emicizumab concentrations but showed large variability. Lower FIX and FX plasma levels during the first 9 months of age may also have an impact on the PD results. Even though the levels were lower in the youngest participants, increases in FVIII-like activity and TG were seen at “steady state” emicizumab in all age groups as expected based on previous studies of emicizumab. The sample sizes were small in some of the age groups analyzed. Conclusions: FVIII-like activity and thrombin generation increased, and aPTT was normalized after starting emicizumab prophylaxis in HAVEN 7. Increase with age was seen on FVIII-like activity as well as FIX and FX levels. The PD profiles of emicizumab in infants with HA were consistent with those previously observed in older children and adults with HA. Hence, even with lower levels of FIX and FX observed at the youngest participants, emicizumab showed the expected PD response.

Emicizumab Prophylaxis for the Treatment of Infants with Severe Hemophilia A without Factor VIII Inhibitors: Results from the Interim Analysis of the HAVEN 7 Study

Background: Currently, many infants with severe hemophilia A (HA) do not receive prophylaxis until at least one year of age, due to the challenges of factor (F)VIII administration. Emicizumab enables initiation of prophylaxis from HA diagnosis via subcutaneous administration, and may mitigate the risk of spontaneous and traumatic bleeding, including intracranial hemorrhages. Aims: This interim analysis of HAVEN 7 (NCT04431726) aims to evaluate the efficacy, safety, pharmacokinetics (PK) and pharmacodynamics (PD) of emicizumab in infants with severe HA without FVIII inhibitors. Methods: HAVEN 7 is a Phase IIIb, multi-center, open-label study of emicizumab in infants ≤12 months with severe HA without FVIII inhibitors. Informed consent from parents/legally authorized representatives and ethics approval have been obtained. Participants receive subcutaneous emicizumab 3mg/kg weekly for 4 weeks, then 3mg/kg every 2 weeks for 52 weeks; at Week 53, participants can continue with this regimen or switch to 1.5mg/kg weekly or 6mg/kg every 4 weeks for the 7-year long-term follow-up period. Efficacy endpoints include the number of treated bleeds; all bleeds; treated spontaneous bleeds; and treated joint bleeds. Annualized bleed rates (ABR) are estimated using a negative binomial regression model. Safety endpoints include adverse events (AEs); thromboembolic events (TEs) and thrombotic microangiopathies (TMAs); and immunogenicity (incidence of anti-drug [emicizumab] antibodies [ADAs]). PK and PD endpoints include plasma trough emicizumab concentration; FIX and FX antigen concentrations; and effect of emicizumab on activated partial thromboplastin time (aPTT), thrombin generation (TG) and FVIII-like activity (measured using a chromogenic assay with human factors). Results: At the interim analysis cut-off date (March 31, 2022), 54 participants (55.6% [n=30] ≥3-≤12 months old; 44.4% [n=24] <3 months) had ≥1 dose of emicizumab; all were male, with a median (range) age of 4.5 (0-11) months. Of these, 30 (55.6%) were minimally treated (≤5 exposure days with hemophilia-related treatments [i.e., FVIII, plasma, cryoprecipitate or whole blood products]) prior to the study, and 24 (44.4%) were previously untreated. Median (range) emicizumab treatment duration was 42.1 (1-60) weeks. Overall, 77 bleeds were reported in 31 participants (57.4%): 88.3% traumatic; 5.2% procedural/surgical; 6.5% spontaneous (Table 1). One participant experienced 12 bleeds (all traumatic; none joint or muscle; none treated). In total, 14 treated bleeds, all traumatic, were reported in 12 participants (22.2%). No intracranial hemorrhage occurred and no participant experienced >2 treated bleeds. Mean model-based ABR (95% confidence interval [CI]) for treated bleeds was 0.4 (0.23-0.65); it was 1.9 (95% CI: 1.35-2.68) and 0.1 (95% CI: 0.01-0.22) for all bleeds and treated joint bleeds, respectively (Table 1). Zero treated bleeds were reported in 77.8% of participants (n=42), while 42.6% of participants (n=23) had no bleeds at all. Fifty participants (92.6%) had ≥1 AE and nine (16.7%) had ≥1 emicizumab-related AE (all injection-site reactions). No AE leading to treatment withdrawal/modification/interruption occurred (Table 2). Eight participants reported 12 serious AEs (SAEs); none was considered emicizumab related. There were no deaths, TEs or TMAs at the time of this interim analysis. PK data were evaluable in 52 participants. Mean trough concentrations of emicizumab increased with loading doses, with concentrations of 63.2µg/mL (95% CI: 59.5-66.8) at Week 5; steady-state concentrations were maintained at 60-65µg/mL thereafter. Emicizumab concentrations were higher than those observed with the same dosing regimen in older people with HA in the HAVEN 1-4 studies (46.7 ± 14.9µg/mL). None of the 48 participants evaluable for immunogenicity analysis tested positive for ADAs. Mean FVIII-like activity increased during loading to 21U/dL and was sustained thereafter. TG peak height increased during loading and continued to increase to reach 100nM from Week 17 onwards. aPTT was normalized by Week 3 (first data point post-baseline) in most participants. Mean FIX and FX concentrations were unaffected by emicizumab. Conclusions: The results of this interim analysis indicate the efficacy and confirm the safety of emicizumab in infants with severe HA without FVIII inhibitors. PK and PD activities were sustained. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Increased BMI and Blood Lipids Are Associated With a Hypercoagulable State in the Moli-sani Cohort.

The coagulation system can be assessed by the thrombin generation (TG) assay, and increased TG peak height, endogenous thrombin potential (ETP), and velocity index are associated with an increased risk of thrombosis. Obesity had been reported to increase TG and is associated with dyslipidemia, which also predisposes to atherosclerotic cardiovascular disease (CVD). However, the effect of the blood lipid profile on TG has not been studied extensively. To gain more insight into the associations of TG, body mass index (BMI) and lipid profile, we studied TG in relation to these parameters in a large Italian population cohort, the Moli-sani study (N = 22,546; age ≥ 35 years; 48% men). TG was measured in plasma samples collected at the enrollment of subjects in the Moli-sani study. TG was triggered with 1 or 5 pM tissue factor, and TG parameters lag time, peak, ETP, time-to-peak (TTP) and velocity index (VI). Additionally, thrombomodulin was added to assess the function of the activated protein C system during TG. In both women and men, overweight (BMI 25–30 kg/m2) and obesity (BMI > 30 kg/m2) were significantly associated with higher ETP, peak and VI (all p < 0.001). High total cholesterol, triglycerides and LDL-cholesterol levels were significantly associated with increased ETP and peak (all p < 0.001). Linear regression analysis revealed that the ETP is positively associated with both plasma LDL and HDL cholesterol levels, whereas the velocity index is positively associated with HDL cholesterol. Additionally, ETP, peak and VI were significantly associated with the plasma triglycerides content. In conclusion, our study shows significant associations of high BMI and blood lipid levels with increased TG parameters, and this hypercoagulability may partly explain the increased risk of CVD in individuals with obesity and/or dyslipidemia.

Coagulation Factor XII Levels and Intrinsic Thrombin Generation in Multiple Sclerosis.

Factor XII (FXII) activation initiates the intrinsic (contact) coagulation pathway. It has been recently suggested that FXII could act as an autoimmunity mediator in multiple sclerosis (MS). FXII depositions nearby dentritic cells were detected in the central nervous system of MS patients and increased FXII activity has been reported in plasma of relapsing remitting and secondary progressive MS patients. FXII inhibition has been proposed to treat MS. To investigate in MS patients multiple FXII-related variables, including the circulating amount of protein, its pro-coagulant function, and their variation over time. To explore kinetic activation features of FXII in thrombin generation (TG). In plasma from 74 MS patients and 49 healthy subjects (HS), FXII procoagulant activity (FXII:c) and FXII protein (FXII:Ag) levels were assessed. Their ratio (FXII:ratio) values were derived. Intrinsic TG was evaluated by different triggers. Higher FXII:Ag levels (p = 0.003) and lower FXII:ratio (p < 0.001) were detected in MS patients compared with HS. FXII variables were highly correlated over four time points, which supports investigation of FXII contribution to disease phenotype and progression. A significant difference over time was detected for FXII:c (p = 0.031). In patients selected for the lowest FXII:ratio, TG triggered by ellagic acid showed a trend in lower endogenous thrombin potential (ETP) in MS patients compared with HS (p = 0.042). Intrinsic triggering of TG by nucleic acid addition produced longer time parameters in patients than in HS and substantially increased ETP in MS patients (p = 0.004) and TG peak height in HS (p = 0.008). Coherently, lower FXII:ratio and longer lag time (p = 0.02) and time to peak (p = 0.007) point out a reduced response of FXII to activation in part of MS patients. In MS patients, factor-specific and modified global assays suggest the presence of increased FXII protein level and reduced function within the intrinsic coagulation pathway. These novel findings support further investigation by multiple approaches of FXII contribution to disease phenotype and progression.

Plasma treated with amotosalen and ultraviolet A light retains activity for hemostasis after 5 days post‐thaw storage at 1 to 6oC

Plasma thawed and stored at 1 to 6° C for up to 5 days (thawed plasma [TP]) provides rapid availability in emergencies and reduces plasma waste, but it carries risks of coagulation factor loss or activation, bacterial outgrowth, and viral contamination. We characterized changes in amotosalen/ultraviolet A (UVA) light pathogen-reduced, fresh-frozen plasma (FFP) and plasma frozen within 24 hours (PF24) with post-thaw storage. Amotosalen/UVA light-treated FFP and PF24 were thawed after approximately 3 to more than 12 months of frozen storage and held at 1 to 6° C for 5 days. Global assessments of coagulation and hemostatic, antithrombotic, and activation markers indicative of function were assessed. Day 5, thawed amotosalen/UVA light-treated FFP and PF24 contained levels of Factors II, V, VIII, IX, X, von Willebrand factor ristocetin cofactor (vWF:RCo), fibrinogen, antithrombin III (ATIII), protein C, and protein S similar to the levels measured in Day 5 TP, as described in the Circular of Information. Thrombin generation was robust on Day 5 (amotosalen/UVA: FFP = 1866 ± 402 nM/minute; PF24 = 1800 ± 277 nM/minute). Most factor activities on Day 5, including von Willebrand factor-cleaving protease (ADAMTS-13), were more than 90% of Day 0 values, except for known labile Factors V and VIII and protein S. All units contained greater than 0.4 IU/mL protein S and α2 plasmin inhibitor on Day 5. Global functional indices, including thrombin-antithrombin complexes, nonactivated thromboplastin time, and thrombin-generation peak height, did not indicate activation of the coagulation cascade, although isolated units showed raised levels of Factor VIIa and Complement 3a. Amotosalen/UVA light-treated FFP and PF24 demonstrated retention of procoagulant and antithrombotic activity after 5 days post-thaw storage at 1 to 6° C.

A new era of treatment for patients with haemophilia A?

Treatment and prevention of bleeding episodes in patients with severe haemophilia A require frequent intravenous injection of factor VIII. Inhibitory antibodies against factor VIII occur in approximately 30 % of these patients during the first exposure days and immune tolerance induction to eradicate the inhibitor is challenging. Prevention of bleeds in patients with haemophilia A and inhibitors is less effective and there is ongoing research for alternative treatment options. A promising approach in 2016 is the development of emicizumab (ACE910), a bi-specific IgG antibody to factor IXa and factor X, that mimics the cofactor function of factor VIII. Due to the different structure of this antibody it cannot be neutralized by factor VIII inhibitors and has the possibility to achieve haemostasis in patients with severe haemophilia A with and without inhibitors. First studies in healthy volunteers and in patients showed a shortened activated partial thromboplastin time and increased peak height of thrombin generation in a dose-dependent manner. The half-life of the drug was 4 to 5 weeks. There were no clinical signs of thrombosis and no laboratory abnormalities indicating hypercoagulability. In a first study with 18 patients with severe haemophilia A with and without inhibitors a remarkable reduction in the annualised bleeding rate occurred. Safety of the drug has to be proven in ongoing research. Mimicking the cofactor activity of factor VIII by a bispecific antibody for the treatment of severe haemophilia A is so far safe and seems to be effective and is one highlight in haemostasis 2016.

Reduction of coagulability state one year after bariatric surgery

BackgroundObese patients are in a hypercoagulable state relative to normal-weight patients. Low-grade inflammation may be a key factor for this condition. ObjectivesOur study aimed to compare the coagulability state of morbidly obese patients before and 1 year after bariatric surgery (BS) using the Thrombin Generation (TG) test, a validated method to assess coagulation in vitro. SettingUniversity hospital. MethodsAll patients undergoing BS between September 1, 2014 and April 30, 2015 were eligible for this prospective study (n = 42). Two distinct reagents were used for TG initiation based on the tissue factor concentration (Reagents LOW and HIGH). The main outcomes were endogenous thrombin potential (ETP) and peak height of TG. The rate of follow-up after one year was 97%. ResultsOne year after surgery, %weight loss was 32.5±8.4%; CRP decreased from 9.0 (3.7–12.9) to 1.1 (0.3–2.8) mg/mL (P<.001) and fibrinogen from 4.2±.8 to 3.5±.8 g/L (P<.001). The ETP (%) decreased from (108.0 (95.0–117.0) to 78.0 (71.0–98.0) (P<.001) (LOW reagent) and from 113.0 (103.0–134.0) to 96.0 (86.0–107.0) (P<.001) (HIGH reagent). Peak height (%) decreased from (117.0 (92.0–139.0) to 82.0 (70.0–111.0) (P = .003) (LOW reagent) and from 106.0 (96.0–118.0) to 97.0 (87.8–105.2) (P = .003) (HIGH reagent). ConclusionOur study shows a significant reduction in TG potential one year after BS in morbidly obese patients. Reduction of low grade inflammation may be one of the underlying mechanisms.

A first-in-human phase 1 study of ACE910, a novel factor VIII–mimetic bispecific antibody, in healthy subjects

AbstractACE910 is a recombinant humanized bispecific antibody that binds to activated factor IX and factor X and mimics the cofactor function of factor VIII (FVIII). This first-in-human study examined the safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of ACE910 in healthy male adults. A total of 40 Japanese and 24 white subjects were randomized to receive a single subcutaneous injection of ACE910 (Japanese: 0.001, 0.01, 0.1, 0.3, or 1 mg/kg; white: 0.1, 0.3, or 1 mg/kg; n = 6 per dose group) or placebo (n = 2 per dose group). ACE910 exhibited a linear PK profile and had a half-life of ∼4 to 5 weeks. In FVIII-neutralized plasma, ACE910 shortened activated partial thromboplastin time and increased peak height of thrombin generation in a dose-dependent manner. All adverse events were nonserious and did not lead to any subject's withdrawal. Neither clinical findings nor laboratory abnormalities indicating hypercoagulability were observed. Two of 48 subjects receiving ACE910 (1 Japanese and 1 white) were positive for anti-ACE910 antibodies (anti-drug antibodies [ADAs]). One subject tested positive for ADAs both before and after ACE910 administration, whereas the other became ADA positive after receiving ACE910. The PK and PD profiles of ACE910 were similar in healthy Japanese and white subjects and suggest that ACE910 will be an effective and convenient prophylactic treatment of hemophilia A. This trial was registered at www.clinicaltrials.jp as #JapicCTI-121934.

Restoration of Procoagulant Factor Levels after Cardiac Surgery Causes Thrombin Generation to Rise to Thrombotic Levels

IntroductionCardiac surgery with cardiopulmonary bypass (CPB) causes hemodilution and coagulation factor consumption. The administration of unfractionated heparin accelerates thrombin inactivation and leads to elevated consumption of antithrombin (AT). The heparin-mediated release of tissue factor pathway inhibitor (TFPI) from the vessel wall reduces prothrombin activation. CPB surgery thus affects both pro- and anticoagulant processes, which are reflected in changes in a patient's thrombin generation potential.In this study we investigated how cardiac surgery changes thrombin generation (TG), prothrombin conversion and thrombin inactivation. In addition, we studied the consequences of these changes in pro- and anticoagulant processes for transfusion management using in silico experimentation.MethodsThirty patients undergoing cardiac surgery with CPB were included and samples were collected before and after surgery and protamine administration. AT and prothrombin levels were determined and TG was measured at 5 pM tissue factor by calibrated automated thrombinography. Prothrombin conversion and thrombin inactivation were quantified from each TG curve.The effect of transfusion of prothrombin complex concentrate (PCC) was simulated by an in silico increase of prothrombin conversion. The effect of PCC administration in combination with AT was modeled as an increase of prothrombin conversion and antithrombin levels. The effect of in silico transfusion of PCC on coagulation was quantified by TG parameters.ResultsCPB surgery causes a reduction of plasma coagulation factor levels: AT (47%, p<0.001), prothrombin (63%, p<0.001), and decreases TG peak height by 71% (p<0.001). After surgery prothrombin conversion is significantly impaired (402 ± 218 vs 701 ± 181 nM; p<0.001), which is partly compensated for by lower thrombin inactivation (63%, p<0.001), resulting in a reduction of TG.We modeled the effect of the transfusion of prothrombin complex concentrate with or without added antithrombin on TG. If the prothrombin conversion capacity was increased to pre-surgery levels in the absence of antithrombin, TG peak height increases to 454 ± 72 nM (figure A). A slight increase of prothrombin conversion (up to 83% of the pre-surgery level) brings TG peak height into the normal range (386±61 nM), but the ETP remains elevated (3278 ± 1141 nM). The optimal way to restore thrombin generation is the simultaneous infusion of PCC and antithrombin. Our in silico experiments indicate that this restores both the peak height (362 ± 69 nM) and the ETP (1601 ± 369 nM∙min) to the pre-surgery level (figure B).ConclusionsCardiac surgery with CPB causes the hemostatic balance to shift toward bleeding due to a reduction of prothrombin conversion. This is only partially compensated by a reduction of thrombin inactivation, resulting in reduced TG after surgery.Post-surgery bleeding can be (and often is) remedied by the transfusion of PCC. However, full restoration of the prothrombin conversion capacity results in a TG profile that predicts a prothrombotic state, as an ETP value in the highest quartile is associated with a 5 times elevated thrombosis risk according to Winckers et al. Equilibrated restoration of the hemostatic capacity asks for the simultaneous supplementation of both pro- and anticoagulant factors, i.e. PCC containing AT or fresh frozen plasma. [Display omitted] DisclosuresKremers:Synapse bv: Employment. Bloemen:Synapse bv: Employment. De Laat:Synapse bv: Employment. Wagenvoord:Synapse bv: Employment. Al Dieri:Synapse bv: Employment. Hemker:Synapse bv: Employment.

Interconnectedness of global hemostasis assay parameters in simultaneously evaluated thrombin generation, fibrin generation and clot lysis in normal plasma

BackgroundFluorogenic thrombin generation (TG) assays and turbidity-based fibrin generation (FG)- and fibrinolysis (FL)-resistance assays have been sought to assess bleeding and clotting disorders. Theoretically, TG, FG and FL tests should provide overlapping information because thrombin is responsible for FG and induces protection from FL. The relationships between TG, FG and FL parameters remain poorly investigated, partly because existing experimental systems do not permit simultaneous detection of both TG and FG in the same sample of plasma, and are instead tested in separate experiments. Objectives and methodsWe evaluated the potential benefits of a combined TG/FG/FL assay by testing responses of normal plasma to a wide range of tissue factor (TF) and tissue plasminogen activator (tPA) concentrations. Correlations between multiple parameters extracted from the TG and FG/FL curves were also compared. ResultsRate of FG correlated well with TG peak height at all TF concentrations, but correlations between TG and FL parameters depended on the TF concentration. Without thrombomodulin, all FG/FL parameters at high TF could be predicted from TG parameters and no FL protection was observed. With thrombomodulin and high TF, TF-dependent FL protection did not correlate with TF-dependent TG. The fluorogenic thrombin substrate did not interfere with optical density readings, and meaningful tPA concentrations did not interfere with TG readings. ConclusionsIn normal plasma, TG, FG and FL parameters may provide interchangeable information. Evaluation of FL-resistance may provide additional data under special assay conditions, but the value of this information should be studied under disease conditions.

Tissue factor‐independent inhibition of thrombin generation by tissue factor pathway inhibitor‐α

Tissue factor pathway inhibitor-α (TFPIα) inhibits factor Xa by forming a binary TFPI-FXa complex in a reaction that is stimulated by protein S. TF-FVIIa forms a quaternary complex with TFPIα and FXa, which shuts off the initiation of coagulation via the extrinsic pathway. To investigate whether direct inhibition of FXa by TFPIα independently of TF plays a role in downregulating coagulation. Inhibition of FXa by TFPIα in plasma was determined by measuring thrombin generation triggered with FXa, the FX activator from Russell's viper venom (RVV-X), FXIa, or FIXa. TF-independent anticoagulant activities of TFPIα and its cofactor, protein S, were quantified: (i) after neutralization of TFPIα and protein S with anti-TFPI or anti-protein S antibodies; and (ii) in TFPI-depleted or protein S-depleted plasmas supplemented with varying amounts of TFPIα or protein S. Both anti-TFPI and anti-protein S antibodies enhanced thrombin generation in plasma triggered with RVV-X, FXa, FIXa, or FXIa. Anti-TFPI and anti-protein S antibodies decreased the lag time and increased the peak height of thrombin generation to the same extent, indicating that inhibition of FXa by TFPIα requires the presence of protein S. TFPIα and protein S titrations in TFPI-depleted or protein S-depleted plasma in which thrombin formation was initiated with triggers other than TF also revealed TF-independent anticoagulant activity of TFPIα, which was completely dependent on the presence of protein S. Direct inhibition of FXa by TFPIα contributes to the downregulation of coagulation.

TFPI Expresses Anticoagulant Activity Independent Of TF-FVIIa

Tissue factor pathway inhibitor (TFPI) is a Kunitz-type serine protease inhibitor that down-regulates the extrinsic coagulation pathway by inhibiting the tissue factor-factor VIIa complex (TF-FVIIa). Inhibition of TF-FVIIa by TFPI is greatly enhanced by FXa. In order to inhibit TF-FVIIa, TFPI first binds to and inhibits Factor Xa (FXa) thus forming a binary TFPI-FXa complex in a reaction that is stimulated by protein S. The TFPI-FXa complex subsequently forms a quaternary complex with TF-FVIIa and blocks the initiation of coagulation. Although TFPI can directly inhibit FXa, this inhibition is generally considered to be of limited physiological significance because the plasma TFPI concentration (0.25-0.5 nM) is several orders of magnitude lower than the plasma concentration of other FXa inhibitors e.g. antithrombin (AT = 2.5-5 μM).We investigated whether TFPI expresses anticoagulant activity independent of TF-FVIIa i.e. to test whether direct inhibition of FXa by TFPI contributes to the down-regulation of coagulation.In model systems, the inhibition of FXa by TFPI and AT was determined in the presence of calcium chloride, phospholipids and protein S using a chromogenic substrate to monitor FXa activity. FXa inhibition by TFPI in plasma was determined by measuring thrombin generation curves triggered with FXa, with the FXa activator from Russell's viper venom (RVV-X) and with FXIa or FIXa in normal pooled plasma. These experiments were performed in the presence of a cocktail of anti TF/anti FVIIa antibodies to inhibit any contribution of traces of TF that might be present in plasma to initiate coagulation. The TF-independent contributions of TFPI and/or protein S to the down-regulation of thrombin generation were quantified 1) after neutralization of TFPI and protein S present in plasma with an anti-TFPI antibody cocktail or anti-protein S antibodies and 2) via TFPI and protein S titrations in TFPI- and in protein S-depleted plasma.In model systems containing purified proteins, physiological concentrations TFPI (0.25 nM) appeared to be more effective in FXa inhibition (t1/2 = 2.5 min) than physiological concentrations AT (2.5 μM) which inhibited FXa with t1/2 = 3.2 min. Both anti-TFPI and anti-protein S antibodies enhanced thrombin generation that was triggered in plasma with RVV-X, FXa, FIXa or FXIa. Depending on the trigger and trigger concentration used anti-TFPI and anti-protein S antibodies decreased the lag time and/or increased the peak height of thrombin generation. At high trigger concentrations neither anti-TFPI nor anti-protein S antibodies enhanced thrombin generation. TFPI and protein S titrations in TFPI- and protein S-depleted plasma in which thrombin formation was initiated with triggers other than TF confirmed that both TFPI and protein S express TF-independent anticoagulant activity. TFPI titrations in protein S-depleted plasma triggered with RVV-X or FXIa showed that the TF-independent anticoagulant activity of TFPI was greatly reduced in the absence of protein S. Supplementation of the protein S-depleted plasma with protein S restored the anticoagulant effect of TFPI.We demonstrated that TFPI, despite its low plasma concentration, expresses anticoagulant activity in the absence of TF. Kinetic experiments in model systems at plasma TFPI concentrations show that direct inhibition of FXa by TFPI can account for the TF-independent anticoagulant effect of TFPI on thrombin generation. Blocking the anticoagulant activity of protein S with anti-protein S antibodies also enhances thrombin generation with triggers other than TF. The fact that neutralization of TFPI and of protein S with specific antibodies resulted in a similar stimulation of thrombin generation suggests that TFPI is strongly dependent on the presence of protein S for expression of TF-independent anticoagulant activity in plasma. The ability of TFPI to inhibit thrombin generation initiated via the intrinsic coagulation pathway is an important observation particularly in the light of recent reports which indicate that the intrinsic coagulation pathway plays a relevant role in thrombus formation and in cardiovascular disease. Disclosures:Rosing:Baxter Innovations GmbH, Vienna, Austria: Consultancy, Research Funding. Hartmann:Baxter Innovations GmbH, Vienna, Austria: Employment. Scheiflinger:Baxter Innovations GmbH, Vienna, Austria: Employment. Dockal:Baxter Innovations GmbH, Vienna, Austria: Employment.

Thrombin Generation Test Can Predict Bleeding Tendency In Patients With Severe Factor XI Deficiency

IntroductionFactor XI (FXI) is a rare bleeding disorder defined as severe deficiency when FXI activity level is less than 20IU/dL. Unlike hemophilia A or B, patients with severe FXI deficiency do not bleed spontaneously and their bleeding tendency is unpredictable and poorly correlated with FXI level. Therefore, almost all patients with severe FXI deficiency are being treated similarly unrelated to their inert bleeding tendency. Lately there is a growing interest in introducing global coagulation tests to assess the risk of bleeding in trauma patients as well as in patients with congenital bleeding disorders. Thrombin generation (TG) test is a global assay that can provide information regarding hemostasis in healthy individuals or in patients with congenital and acquired bleeding disorders. Our group had previously shown that recalcification induced TG is a useful tool to determine the optimal dose of recombinant factor VIIa for patients with severe FXI deficiency and inhibitors going through major surgery (Livnat et al. Thromb Haemost 2009). AimIn the present study we aimed to characterize the capability of TG to serve as an ideal tool to define upfront bleeders and non-bleeders among FXI deficient patients and find the optimal conditions of TG that could distinguish between bleeders and non-bleeders thus eventually leading to efficient personalized treatment. MethodsCase control study composed of 16 unrelated patients with FXI levels range >1-8dL-1and 14 healthy controls. For TG assay blood was taken from all participants simultaneously in both buffered citrate and corn trypsin inhibitor (CTI) tubes after obtaining informed consent. TG was performed in platelet poor plasma (PPP) in the presence of 4 µM phospholipids and initiated by recalcification in the presence and absence of 1pM tissue factor (TF). Three TG parameters were analyzed: lag time, thrombin peak and endogenous thrombin potential (ETP). ResultsTable 1 summarizes FXI activity, FXI genotype, thrombin peak height and bleeding status (i.e, bleeding following challenges when prophylactic treatment was not given) of patients in the study group. As expected, FXI levels poorly correlated with bleeding tendency. Good correlation between FXI levels, bleeding tendency and TG peak height was found when blood was taken in citrated tubes and not in CTI containing tubes. While the normal range of peak height in recalcification-induced TG (without TF) was 421±161 nM, no TG was initiated with recalcification in PPP of FXI patients with less than 1%. FXI levels 2-4% were sufficient to induce TG with recalcification but thrombin peak height was remarkable lower in comparison to controls. In FXI levels above 5%, the thrombin peak height induced by recalcification varied between low to normal range. Interestingly, when TG was initiated by 1pM TF the TG peak of non-bleeders reached normal values (normal peak height in the presence of 1pM TF=411±121), while in the bleeders the peak was reduced unrelated to FXI levels (range 74-205). ConclusionsIn summary TG induced by recalcification in the presence of low TF but not when performed in CTI tubes may efficiently distinguish between bleeders and non-bleeders in FXI deficient patients going through major trauma unrelated to patients' FXI level. This observation permits to consider less aggressive prophylactic treatment to patients with reduced risk for bleeding thus lowering the risk of thrombosis due to over treatment. Disclosures:No relevant conflicts of interest to declare.

Inhibition of thrombin‐mediated factor V activation contributes to the anticoagulant activity of fibrinogen γ′

Besides its role in blood clotting, fibrinogen exerts a poorly understood anticoagulant function by binding thrombin and modulating its activity. In particular, the γA/γ' fibrinogen isoform binds with high affinity to thrombin exosite II through the anionic carboxyl-terminal end of the γ' chain. This interaction down-regulates thrombin-mediated factor VIII (FVIII) activation, but its effect on FV activation is unknown. To investigate the overall anticoagulant activity of fibrinogen and particularly of fibrinogen γ' in plasma, and to verify whether the fibrinogen γ' carboxyl-terminal peptide affects thrombin-mediated FV activation. Thrombin generation was measured by calibrated automated thrombography in whole and defibrinated plasma and in plasma supplemented with the (sulfated) fibrinogen γ' carboxyl-terminal peptide (0-500μmol L(-1) ). The effect of the peptide on thrombin-mediated FV activation was studied in model systems and in plasma. Total fibrinogen prolonged the lag time of thrombin generation at low tissue factor (TF) concentrations. The fibrinogen γ' peptide dose-dependently prolonged the lag time and decreased the peak height of thrombin generation at low TF, whereas a scrambled control peptide was ineffective. These effects persisted in the presence of an anti-FVIII antibody, suggesting that the peptide may also inhibit thrombin-mediated activation of FV. This was confirmed in model systems and in plasma. Total fibrinogen and the fibrinogen γ' peptide have an overall anticoagulant effect on thrombin generation determined at low TF. Inhibition of thrombin-mediated FV activation by the fibrinogen γ' peptide is a novel mechanism of the anticoagulant activity of fibrinogen γ'.

The in-vitro effect of fibrinogen, factor XIII and thrombin-activatable fibrinolysis inhibitor on clot formation and susceptibility to tissue plasminogen activator-induced fibrinolysis in hemodilution model

Patients suffering major traumatic or surgical bleeding are often exposed to hemodilution resulting in dilutional coagulopathy. The aim of this study was to evaluate in vitro the effects of fibrinogen, factor XIII and thrombin-activatable fibrinolysis inhibitor (TAFI) on clot formation and resistance to fibrinolysis in hemodilution conditions. Citrated whole blood from 36 healthy volunteers was diluted to 30 and 60% with lactated Ringer's solution. Blood samples were subsequently supplemented with fibrinogen, FXIII, TAFI or their combinations. Rotation thromboelastometry (ROTEM) in whole blood and thrombin generation in plasma were performed in the presence of CaCl₂ and tissue factor/EXTEM reagent, and fibrinolysis was induced by tissue plasminogen activator (tPA). Hemodilution was expressed by decrease of peak height in thrombin generation and α-angle and maximum clot firmness (MCF) in ROTEM. Fibrinogen, FXIII or TAFI did not correct the decrease in thrombin generation peak height. In ROTEM, spiking of diluted blood with fibrinogen stimulated clot propagation. In tPA-treated blood fibrinogen, FXIII and TAFI increased clot firmness and inhibited fibrinolysis. Stronger protection against fibrinolysis was achieved combining FXIII with TAFI. Hemodilution was associated with inhibition of thrombin generation; however, this effect was not sensitive to blood spiking with fibrinogen, FXIII and TAFI. In ROTEM, these hemostasis agents improved clot strength and decreased clot susceptibility to tPA in nondiluted and to more extent in diluted blood. The maximal protection against fibrinolysis was caused by TAFI. Combining FXIII with TAFI exerted synergistic inhibitory effect on fibrinolysis.

Effect of rivaroxaban, in contrast to heparin, is similar in neonatal and adult plasma

Neonates have lower levels of clotting factors as well as inhibitors. Effects of heparin in neonatal plasma differ from those in adult plasma, and dosage recommendations cannot be extrapolated from adult trials. Riveroxaban is an oral direct factor Xa inhibitor that can achieve an anticoagulant effect without dependence on anti-thrombin. We performed comparative thrombin generation measurements in neonatal cord and adult plasma with different concentrations of unfractionated heparin and rivaroxaban to evaluate the potential of rivaroxaban in neonatal anticoagulation. The impact of heparin or rivaroxaban on the neonatal and adult hemostatic system was determined measuring calibrated automated thrombin generation and activated partial thromboplastin time in platelet-poor plasma pools of 15 adult samples or 15 neonatal cord samples and addition of seven increasing concentrations of heparin or rivaroxaban, respectively, to the pooled samples. Lag time, time to peak and peak height of thrombin generation in neonatal cord samples were significantly less affected by different heparin concentrations than in adult samples, whereas the impact on reduction of endogenous thrombin potential was higher in neonatal cord samples. The impact of rivaroxaban on thrombin generation parameters showed better comparability between neonatal cord and adult samples. Both anticoagulants showed the same differences in activated partial thromboplastin time between adult and neonatal plasma at each concentration. Rivaroxaban shows a very similar pattern in neonatal cord and adult plasma in suppressing thrombin generation and prolonging activated partial thromboplastin time values, suggesting that dose finding may be easier with rivaroxaban in neonates.

Antithrombin-independent thrombin inhibitors, but not direct factor Xa inhibitors, enhance thrombin generation in plasma through inhibition of thrombin-thrombomodulin-protein C system

There is increasing concern that some anticoagulants can paradoxically increase thrombogenesis under certain circumstances. Previously, we demonstrated that at certain doses a direct thrombin inhibitor, melagatran, worsens the coagulation status induced by tissue factor (TF) injection in a rat model. We utilised an in vitro thrombin generation (TG) assay to determine if direct thrombin inhibitors could enhance TG in human plasma, and whether inhibition of the negative-feedback system [thrombin-thrombomodulin (TM)-protein C] contributed to the TG enhancement. TG in human plasma was assayed by means of the calibrated automated thrombography. In this assay, direct factor Xa (FXa) inhibitors such as edoxaban and antithrombin (AT)-dependent anticoagulants such as heparin did not increase, but simply suppressed TG. AT-independent thrombin inhibitors (melagatran, lepirudin, and active site blocked thrombin (IIai)) increased peak levels of TG (2.0, 1.6, and 2.2-fold, respectively) in the presence of 12 nM recombinant human soluble TM (rhsTM). Melagatran and lepirudin at higher concentrations began to suppress TG. In the absence of rhsTM, the enhancement of peak TG by melagatran decreased to 1.2-fold. Furthermore, in protein C-deficient plasma, AT-independent thrombin inhibitors failed to enhance TG. In addition, a human protein C neutralising antibody increased the peak height of TG in the presence of rhsTM. These results suggest that AT-independent thrombin inhibitors may activate thrombogenesis by suppression of the thrombin-induced negative-feedback system through inhibition of protein C activation. In contrast, direct FXa inhibitors are more useful than AT-independent thrombin inhibitors in terms of lower possibility of activation of the coagulation pathway.

Coagulation factors and the protein C system as determinants of thrombin generation in a normal population.

Thrombin generation is a powerful tool to probe overall plasma coagulability. To determine which plasma factors influence the various parameters of the thrombin generation curve, for example lag time, peak height and endogenous thrombin potential (ETP), under different experimental conditions. Plasma levels of coagulation factors and inhibitors, as well as thrombin generation at 1 pm tissue factor (TF) +/- thrombomodulin (TM) and at 13.6 pm TF +/- activated protein C (APC), were determined in plasma from 140 healthy individuals. Data were analysed by multiple regression models. Thrombin generation increased with age and was higher in females than in males. Under all conditions, the lag time was mainly dependent on the levels of free tissue factor pathway inhibitor (TFPI), free protein S (PS), factor VII (FVII), FIX and fibrinogen. The major determinants of thrombin generation (ETP and peak height) at 1 pm TF were fibrinogen, FXII (despite inhibition of contact activation), free TFPI and antithrombin (AT), both in the absence and in the presence of TM. Thrombin generation in the presence of TM was also dependent on protein C levels. At 13.6 pm TF, thrombin generation was determined by prothrombin, AT, fibrinogen, free TFPI and FV levels in the absence of APC, and by free TFPI, free PS and FX levels in the presence of APC. The lag time, ETP and peak height of thrombin generation depend on the levels of multiple coagulation factors and inhibitors. The specific assay determinants vary with the experimental conditions.

Evidence of Different Subspecies of Microparticles (MP) Derived from Red Blood Cells (RMP) and Comparison of Their Phenotypes and Procoagulant Activity.

BACKGROUND: The potential roles of cell derived microparticles (MP) such as those derived from platelets (PMP), endothelium (EMP), leukocytes (LMP), and red cells (RMP) have been receiving increasing attention in disorders of hemostasis/thrombosis and inflammation and they are emerging as valuable biomarkers. However among these MP, little is known about RMP. Our recent clinical studies indicate that RMP play a role in hemostasis and thrombosis in patients with thrombocytopenia and in thrombocytosis. However, the phenotypes and procoagulant activity of their subspecies remain unknown. We report evidence for heterogeneity of RMP following differential centrifugation.METHODS: RMP were prepared by exposure of washed RBC to the calcium ionophore, A23187, and the RBC were removed by low-speed centrifugation. The RMP were washed twice at 20,000xg for 15 min. Procoagulant activity of RMP was measured by the calibrated automated thrombogram (CAT) system (Hemker et al Pathophysiol Haemost Thromb. 2002; 32:249) using thrombin substrate Z-Gly-Gly-Arg-AMC on a fluorescence plate reader. The lag time and peak height (nM) of thrombin generation were recorded. Markers used for labeling RMP were PE-labeled anti-glycophorin (GlyP), FITC-anti-tissue factor (TF), FITC-annexin V (AnV), and/or FITC-lectin Ulex europeaus I (Ulex).RESULTS: In thrombin generation assay, RMP induced a long lag time (24±3 min) but high thrombin peak (330±37 nM). These data were consistent with the flow cytometric finding that RMP carried very little TF (<0.1%) but very high AnV binding (88±6%). By high speed centrifugation (15,000xg for 10 min), two populations of RMP were studied: the larger RMP in the pellet expressed GlyP, AnV and Ulex while the smaller or lighter RMP remaining in the supernatant, did not express GlyP and AnV but do express Ulex. The smaller RMP accounted for 30–40% of total Ulex+ RMP. These two subspecies (large and small) of RMP showed distinct thrombin generation profiles. The lag time and peak height of thrombin generation for large RMP (GlyP+/AnV+/Ulex+) was 23–28 min and 300–335 nM, respectively, which is close to values of whole RMP. On the other hand, the smaller RMP (Ulex+/GlyP−/AnV−) produced much longer lag time (31–38 min) and lower peak (60–75 nM), indicating that the majority of the procoagulant activity of RMP is associated with larger RMP.SUMMARY: The present study demonstrates that RMP are rich in anionic phospholipids and effective in generating thrombin in vitro. We have identified 2 distinct subpopulations of RMP by differential centrifugation: One larger RMP express binding of anti-GlyP, AnV and Ulex, and carry the majority of procoagulant activity. The smaller RMP expressing only Ulex binding exhibit much weaker procoagulant activity. The roles of these two species of RMP remain to be elucidated. We speculate that smaller RMP may represent the nanovesicles described by Allen et al [Biochem J 188:881, 1980] and that Ulex may be a novel and convenient means for the study of these small vesicles.