Summary
Thromboelastography (TEG) was first described by Hartert in 1948, and was designed to monitor viscoelastic clot strength in whole blood in real time. The current TEG method and Rotational ...Thromboelastometry (ROTEM) were subsequently developed from the original principles. Both of the modern methods provide data by measuring changes in the viscoelastic strength of a small sample of clotting blood in response to a constant rotational force. The important advantage of these techniques is to visually observe and quantify blood coagulation including the propagation, stabilization and dissolution phases of clot formation under low shear conditions. Analysis of the results provides detailed kinetic data on fibrin generation, clot strength and fibrinolysis. These TEG/ROTEM analyses therefore enable evaluation of global clotting function and the monitoring of haemostatic treatment in various clinical situations, not only in patients with genetic bleeding disorders, such as haemophilia, but also in patients undergoing cardiac surgery, liver transplantation or suffering from traumatic injury. Some evidence suggests that haemostatic management using TEG/ROTEM leads to a reduction in total transfusions of whole blood or clotting factors. Wider clinical application of this technology seems likely.
Emicizumab is a humanized bispecific antibody that mimics the cofactor function of factor VIII. In a dose-escalation study in Japanese persons with hemophilia A, including those with factor VIII ...inhibitors, emicizumab markedly reduced the number of bleeding episodes.
Hemophilia A is a serious bleeding disorder caused by a deficiency of clotting factor VIII. Approximately 50% of patients have severe hemophilia A,
1
defined as less than 1% residual factor VIII activity (<1 IU per deciliter).
2
Such patients have severe bleeding from early childhood, and without appropriate treatment, recurrent bleeding into joints can lead to irreversible hemoarthropathy.
3
,
4
Standard treatment for hemophilia A includes regular prophylaxis and episodic treatment with recombinant or plasma-derived factor VIII. The goals of prophylaxis with factor VIII are to increase factor VIII activity to at least a moderate level (1 to 5 IU per deciliter) . . .
Background
Factor VIII (FVIII) is activated by thrombin‐catalyzed cleavage at three sites. Previous reports indicated that the A2 domain contained thrombin‐interactive sites responsible for cleavage ...at Arg372. We have also found that the A1 domain of FVIII bound to the anion‐binding exosite I of thrombin. The present study focused, therefore, on thrombin interaction with A1 residues 337‐372 containing clustered acidic and hirugen‐like sequences.
Aim
To identify specific thrombin‐interactive site(s) within the A1 acidic region of FVIII.
Methods and Results
The synthetic peptide of residues 337‐353 with sulfated Tyr346 (337‐353S) significantly blocked thrombin‐catalyzed FVIII activation and cleavage at Arg372, while a corresponding peptide of residues 354‐372 had no significant effect. Treatment with 1‐ethyl‐3‐(3‐dimethylaminopropyl)‐carbodiimide to cross‐link thrombin and 340‐350S suggested that the 344‐349 clustered acidic region was involved in thrombin interaction. Alanine‐substituted FVIII mutants, Y346A and D347A/D348A/D349A, depressed thrombin‐catalyzed activation and cleavage at Arg372, with peak activation at ~ 50% and cleavage rates of ~ 10% to 20% compared to wild type (WT). The peak level of thrombin‐catalyzed activation and the cleavage rate at Arg372 using FVIII mutants with 337‐346 residues substituted with hirugen‐sequences (MKNNEEAEDY337‐346GDFEEIPEEY) were ~ 1.5‐ and ~ 2.5‐fold of WT, respectively. Surface plasmon resonance‐based analysis demonstrated that the Kd for active‐site modified thrombin interactions using Y346A and D347A/D348A/D349A mutants was ~ 3‐ to 6‐fold higher than that of WT, and that the hirugen‐hybrid mutant facilitated association kinetics ~ 1.8‐fold of WT.
Conclusion
Residues 346‐349 with sulfated Tyr provided a thrombin‐interactive site responsible for activation and cleavage at Arg372. A hirugen‐hybrid A1 mutant showed more efficient thrombin‐catalyzed cleavage at Arg372.
Emicizumab reduces bleeding events in patients with severe hemophilia A (HA). The coagulation potential of emicizumab at a clinical dose appears to correspond to about 15 IU/dL of factor VIII ...activity (FVIII:C), the equivalent of converting from a severe to mild phenotype. However, the clinical and laboratory characteristics of HA patients receiving emicizumab (Emi-PwHA) compared with patients with mild HA (PwMHA) remain to be determined. We reviewed clinical data from Emi-PwHA (n = 63) and PwMHA (n = 15) and evaluated comprehensive coagulation function using Ca
2+
-triggered rotational thromboelastometry (ROTEM) and ellagic acid/tissue factor-triggered clot waveform analysis (modified CWA). The median FVIII:C in PwMHA was 13.0 (IQR 8.5–17.0) IU/dL. Bleeding patterns in both groups were similar and classified into three categories: (1) spontaneous bleeding, post-traumatic, (2) bleeding within 1–2 days, and (3) delayed bleeding after 1–2 weeks. The coagulation potential in Emi-PwHA with and without breakthrough bleeds was comparable. Furthermore, coagulation function in Emi-PwHA was equivalent to PwMHA, although time between treatment and hospitalization for breakthrough bleeds in PwMHA appeared to be longer than those in Emi-PwHA. The coagulation potential and bleeding patterns appeared to be similar in Emi-PwHA and PwMHA, indicating that emicizumab-driven coagulation potential reflected mild HA.
Background
Sepsis is a common underlying disease associated with disseminated intravascular coagulation (DIC). We have recently determined hemostatic pathological states at diagnosis through ...simultaneous assessment of coagulation and fibrinolysis potentials in sepsis‐associated DIC using clot‐fibrinolysis waveform analysis. Here we aimed to investigate hemostatic pathological states, focusing on the balance between coagulation and fibrinolysis dynamics during the clinical course in pediatric sepsis‐associated DIC.
Methods
Coagulation and fibrinolysis potential functions in three pediatric patients with sepsis‐associated DIC during their clinical course were quantified using clot‐fibrinolysis waveform analysis. A maximum coagulation velocity (|min1|) and maximum fibrinolysis velocity (|FL‐min1|) was calculated as a ratio relative to normal plasma.
Results
In case 1, coagulation‐enhanced and fibrinolysis‐depressed state (|min1|‐ratio 2.22 and |FL‐min1|‐ratio 0.42) was observed on day 1. This discrepancy significantly reduced after anticoagulant therapy and plasma exchange on day 2. A well‐balanced hemostatic state (0.70 and 0.62, respectively) was restored on day 7. In case 2, fibrinolysis‐impaired state (|min1|‐ratio 1.09 and |FL‐min1|‐ratio 0.21) was seen on day 1. The |min1| ratio was slightly prolonged and the |FL‐min1| ratio was severely decreased. Both were restored on day 7 and returned to normal levels on day 12. In case 3, twofold coagulation‐ and fibrinolysis‐enhanced states (|min1|‐ratio 1.99 and |FL‐min1|‐ratio 1.11) were seen on day 1. However, both potentials rapidly decreased on day 2 (0.49 and 0.0, respectively). She died on day 5.
Conclusions
The hemostatic pathological states in sepsis‐associated DIC depend on disease progression. Comprehensive assessment of coagulation‐fibrinolysis potentials over time may therefore be helpful in considering optimal treatment plans for sepsis‐associated DIC.
A combined product of plasma-derived factor (F)VIIa and FX (pd-FVIIa/FX; Byclot
®
) is currently available for the hemostatic treatment of hemophilia A and B patients with inhibitors in Japan. ...Limited information is available, however, on its coagulant effect in acquired hemophilia A (AHA). In the present study, we assessed the coagulant effect of pd-FVIIa/FX on impairment of coagulation potentials in AHA. The bypassing agents, pd-FVIIa/FX, recombinant FVIIa (rFVIIa), and activated prothrombin complex concentrates (aPCC) were spiked with normal plasma preincubated with anti-FVIII monoclonal antibody (AHA-model plasma), and added to plasmas from AHA patients. Clot waveform analysis (CWA) triggered by the mixture of tissue factor and ellagic acid was subsequently performed. In the AHA-model, pd-FVIIa/FX improved all of the CWA parameters in a dose-dependent manner, irrespective of epitope specificity, with significant improvements relative to rFVIIa and aPCC. The coagulant effect of pd-FVIIa/FX at 1.6 µg/mL (corresponding to 120 µg/kg infusion) at the maximum therapeutic dose was outside the normal range. Moreover, the addition of pd-FVIIa/FX led to a greater improvement in the coagulant potentials in AHA plasmas than those of rFVIIa and/or aPCC. These data suggest that pd-FVIIa/FX significantly improves the impaired coagulant potentials in AHA and is potentially therapeutic.
Abstract
Factor VIII (FVIII) is activated by thrombin-catalyzed cleavage at Arg
372
, Arg
740
, and Arg
1689
. Our previous studies suggested that thrombin interacted with the FVIII C2 domain ...specific for cleavage at Arg
1689
. An alternative report demonstrated, however, that a recombinant (r)FVIII mutant lacking the C2 domain retained >50% cofactor activity, indicating the presence of other thrombin-interactive site(s) associated with cleavage at Arg
1689
. We have focused, therefore, on the A3 acidic region of FVIII, similar to the hirugen sequence specific for thrombin interaction (54–65 residues). Two synthetic peptides, spanning residues 1659–1669 with sulfated Tyr
1664
and residues 1675–1685 with sulfated Try
1680
, inhibited thrombin-catalyzed FVIII activation and cleavage at Arg
1689
. Treatment with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide to cross-link thrombin with either peptide showed possible contributions of both 1664–1666 and 1683–1684 residues for thrombin interaction. Thrombin-catalyzed activation and cleavage at Arg
1689
in the alanine-substituted rFVIII mutants within 1663–1666 residues were similar to those of wild type (WT). Similar studies of 1680–1684 residues, however, demonstrated that activation and cleavage by thrombin of the FVIII mutant with Y1680A or D1683A/E1684A, in particular, were severely or moderately reduced to 20 to 30% or 60 to 70% of WT, respectively. Surface plasmon resonance-based analysis revealed that thrombin interacted with both Y1680A and D1683A/E1684A mutants with approximately sixfold weaker affinities of WT. Cleavage at Arg
1689
in the isolated light-chain fragments from both mutants was similarly depressed, independently of the heavy-chain subunit. In conclusion, the 1680–1684 residues containing sulfated Tyr
1680
in the A3 acidic region also contribute to a thrombin-interactive site responsible for FVIII activation through cleavage at Arg
1689
.
Emicizumab is a bispecific antibody to factor (F) IX/IXa and FX/FXa, which mimics FVIIIa cofactor function. Emicizumab prophylaxis significantly decreases bleeding events for patients with hemophilia ...A (PwHA). However, global hemostatic monitoring in emicizumab-treated PwHA remains poorly investigated. Using rotational thromboelastometry (ROTEM), we evaluated coagulation potentials of whole blood samples from seven emicizumab-treated PwHA who participated in ACE001JP/ACE002JP studies. Dose-dependent coagulation-enhancing effects of emicizumab to whole blood from PwHA mixed with an anti-FVIII C2 antibody in vitro were evident by non-activated ROTEM analysis (NATEM). The relationship between FVIII levels and NATEM parameters in PwHA not participating in the clinical trials demonstrated that CT + CFT inversely correlated with FVIII levels. These parameters were defined as NATEM-based grading of coagulation potential; ‘T1’ (FVIII < 1 IU/dL), ‘T2’ (1 ≤ , < 12 IU/dL) and ‘T3’ (12 ≤ , < 60 IU/dL). Coagulation function in emicizumab-treated PwHA was determined to be ‘T2’ or ‘T3,’ and was dependent on plasma emicizumab concentration. Improvement of NATEM-based grades corresponded with significant reduction of bleeding episodes, except for target joints, and differences were due to the time to reach the coagulation-steady state in individual patients. The NATEM analysis may be useful for intra- and inter-individual evaluation of coagulation function in emicizumab-treated PwHA.
Summary
Emicizumab, a humanised bispecific antibody recognising factors (F) IX/IXa and X/Xa, can accelerate FIXa-catalysed FX activation by bridging FIXa and FX in a manner similar to FVIIIa. ...However, details of the emicizumab–antigen interactions have not been reported so far. In this study, we first showed by surface plasmon resonance analysis that emicizumab bound FIX, FIXa, FX, and FXa with moderate affinities (
K
D
= 1.58, 1.52, 1.85, and 0.978 μM, respectively). We next showed by immunoblotting analysis that emicizumab recognised the antigens’ epidermal growth factor (EGF)-like domains. We then performed
K
D
-based simulation of equilibrium states in plasma for quantitatively predicting the ways that emicizumab would interact with the antigens. The simulation predicted that only a small part of plasma FIX, FX, and emicizumab would form antigen-bridging FIX–emicizumab–FX ternary complex, of which concentration would form a bell-shaped relationship with emicizumab concentration. The bell-shaped concentration dependency was reproduced by plasma thrombin generation assays, suggesting that the plasma concentration of the ternary complex would correlate with emicizumab’s cofactor activity. The simulation also predicted that at 10.0–100 μg/ml of emicizumab–levels shown in a previous study to be clinically effective–the majority of plasma FIX, FX, and emicizumab would exist as monomers. In conclusion, emicizumab binds FIX/FIXa and FX/FXa with micromolar affinities at their EGF-like domains. The
K
D
-based simulation predicted that the antigen-bridging ternary complex formed in circulating plasma would correlate with emicizumab’s cofactor activity, and the majority of FIX and FX would be free and available for other coagulation reactions.
Institution where the work was carried out: Research Division, Chugai Pharmaceutical Co., Ltd.
Supplementary Material to this article is available online at www.thrombosis-online.com.
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