Janice Kuhn

Onset of force development as a marker of thrombin generation in whole blood: the thrombin generation time (TGT).

Summary.  Prothrombin activation requires the direct interplay of activated platelets and plasma clotting factors. Once formed, thrombin causes profound, irreversible activation of platelets and reinforces the platelet plug via fibrin formation. Delayed or deficient thrombin production increases bleeding risk. Commonly employed coagulation assays, the prothrombin and partial thromboplastin times, use clot formation as a surrogate marker of thrombin generation. These assays routinely utilize platelet‐poor plasma and completely miss the effects of platelets. Other markers of thrombin generation, prothrombin fragment 1 + 2 (F1 + 2) and thrombin–antithrombin complex, are typically measured after the fact. We report a simple assay, which employs the onset of platelet contractile force (PCF) as a surrogate marker of thrombin generation. PCF generation occurs concomitant with the burst of F1 + 2 release. The time between assay start and PCF onset is termed the thrombin generation time (TGT). TGT is prolonged in clotting factor deficiencies and in the presence of direct and indirect thrombin inhibitors. TGT shortens to normal with clotting factor replacement and shortens with administration of recombinant factor VIIa. TGT is short in thrombophilic states such as coronary artery disease, diabetes and thromboangiitis obliterans and prolongs toward normal with oral and intravenous anticoagulants.

Factor VIIa analog has marked effects on platelet function and clot kinetics in blood from patients with hemophilia A

To evaluate the hemostatic effects of NN1731 and rFVIIa, an ex-vivo study in hemophilia patients used the Hemodyne Hemostasis Analysis System (HAS) to measure platelet contractile force (PCF), clot elastic modulus (CEM), and force onset time (FOT), and the Haemoscope Thrombelastograph (TEG) to measure reaction time (R), kinetics time (K), and maximum amplitude (MA). Blood samples from 10 healthy volunteers and 10 Factor VIII-deficient patients of varying severity (mild, moderate, severe), were spiked with rFVIIa and NN1731 (both 0.64 and 1.28 μg/ml, respectively) and analyzed to characterize platelet function and clot kinetics. There was wide variability in the rFVIIa response. NN1731 had greater and more consistent effects on PCF, CEM, FOT, R, and K relative to rFVIIa, in all hemophilia groups. The lowest NN1731 concentration (0.64 μg/ml) shortened R and FOT, and increased CEM and PCF more than rFVIIa 1.28 μg/ml. NN1731 normalized clotting parameters equivalent to values obtained in healthy volunteers. FOT and R were highly correlated (r2 = 0.96). No correlation was observed between CEM and MA. NN1731 produced less variable, more pronounced and predictable ex-vivo hemostatic effects on PCF, CEM, FOT, R and K than rFVIIa in all hemophilia groups. HAS and TEG assays provided similar estimates of FOT and R, however CEM appeared to be more sensitive than MA to changes in clot firmness.

Monitoring rFVIIa 90 μg kg−1 dosing in haemophiliacs: comparing laboratory response using various whole blood assays over 6 h

Summary.  Recombinant FVIIa is a haemostatic agent administered to patients with severe FVIII or FIX deficiency with inhibitors. Although rFVIIa is effective at stopping bleeding, a reliable assay to monitor its effect is lacking. To characterize the pharmacokinetics and global coagulation effects of rFVIIa for 6 h following a IV dose of 90 μg kg−1. Ten non‐bleeding subjects with severe FVIII or FIX deficiency were infused with a single‐dose of rFVIIa 90 μg kg−1 body weight and blood was collected before and at 0.5, 1, 2, 4 and 6 h postdose. Global haemostasis was characterized throughout the study utilizing whole blood analyses (Hemodyne HAS, TEG, ROTEM). The clearance and half‐life of factor FVII:C was estimated as 39.0 ± 8.8 mL h−1 kg−1 and 2.1 ± 0.2 h respectively. There was good inter‐assay agreement with respect to clot initiation parameters (R, CT and FOT) and these parameters all fell to a mean of approximately 9 min following rFVIIa dosing. The platelet contractile force (PCF) and clot elastic modulus (CEM) were positively correlated to FVII:C (P < 0.0001), and these parameters were dynamic throughout the 6‐h period. The MA and MCF did not correlate to FVII:C nor did they significantly change during the study. Prothrombin F1 + 2 significantly increased following rFVIIa dosing (P < 0.001), but remained steady throughout the study. There was no change in D‐dimer concentrations over time. The FOT, R and CT characterized clot initiation following rFVIIa dosing. The PCF and CEM were correlated to FVII:C and characterized the dynamics of platelet function and clot strength over the rFVIIa dosing interval. The clinical significance of these findings needs additional study.

Coated platelets and severe haemophilia A bleeding phenotype: Is there a connection?

Coated platelets are a subpopulation of platelets that possess highly prothrombotic properties. Previous observational data suggest that bleeding phenotype in severe haemophilia A is associated with coated platelet levels. Haemophilia A patients with higher coated platelet levels may have a mild bleeding phenotype; those with lower levels may have a more severe bleeding phenotype.

Modulation of the activated protein C pathway in severe haemophilia A patients: The effects of thrombomodulin and a factor V-stabilizing fab

The thrombomodulin (TM)/activated protein C (APC) system is a key regulator of haemostasis, limiting amplification and propagation of the formed blood clot to the injury site. Dampening APCs inhibition of factor V (FV) and factor VIII (FVIII) may be a future strategy in developing next‐generation therapeutic targets for haemophilia treatment.

Overcoming delayed in-vitro response to rFVIIa: effects of rFVIIa and rFVIIa analogue (vatreptacog alfa) concentration escalation in whole blood assays.

In a previous pharmacokinetic/pharmacodynamic study in nonbleeding hemophilia patients, variability in laboratory response to recombinant factor VIIa (rFVIIa) 90 &mgr;g/kg was noted, and the patients were described as delayed or rapid laboratory responders based on time to clot formation. The current study determined whether in-vitro experiments could reproduce previous in-vivo findings; whether the delayed laboratory response to rFVIIa 90 &mgr;g/kg is improved by spiking with high-dose rFVIIa or rFVIIa analogue (vatreptacog alfa); whether a dose–response is observed with our method. In-vitro experiments were conducted in our previous patient cohort using rFVIIa 1.28 and 3.84 &mgr;g/ml and vatreptacog alfa 0.28 and 0.56 &mgr;g/ml. Whole blood studies were conducted using the Hemodyne Hemostasis Analysis System (platelet contractile force, clot elastic modulus, force onset time) and rotational thromboelastometry (clotting time, maximum clot firmness). Spiking with rFVIIa 1.28 &mgr;g/ml showed the same distribution of delayed and rapid laboratory response as observed previously. Increasing in-vitro rFVIIa concentrations improved the coagulation parameters; however, there remained delayed and rapid responders. Vatreptacog alfa improved the coagulation parameters at all concentrations tested, and the 0.56 &mgr;g/ml concentration normalized the force onset time, platelet contractile force, clot elastic modulus and clotting time parameters. A dose–response was observed with both assays. There was good agreement between the laboratory responses obtained after intravenous administration of rFVIIa 90 &mgr;g/kg and in-vitro spiking studies. Escalating rFVIIa and vatreptacog alfa concentrations improved coagulation parameters in all patients compared to rFVIIa 1.28 &mgr;g/ml. Vatreptacog alfa produced more pronounced coagulation effects at lower concentrations than rFVIIa; and the 0.56 &mgr;g/ml concentration completely normalized responses in all patients.

Evaluating the thrombin generation profiles of four different rFVIII products in FVIII-deficient plasma using FIXa and FXIa activation

The thrombin generation assay (TGA) can be used to monitor factor replacement therapy in patients with haemophilia. The TGA assay is typically performed using tissue factor as the reaction activator; however, activating with FIXa or FXIa can enhance assay sensitivity when FVIII < 1%.

Successful multi-modal immune tolerance induction for factor IX deficiency with inhibitors and allergic reactions

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