Methods and assays for factor VIII activity
Inventors
GILBERT, Gary Eugene • Shi, Jialan • NOVAKOVIC, Valerie A.
Assignees
US Department of Veterans Affairs • Harvard University
Publication Number
US-11067572-B2
Publication Date
2021-07-20
Expiration Date
2034-11-11
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Abstract
The methods and compositions described herein relate to the measurement of factor VIII (fVIII) levels and/or activity.
Core Innovation
The invention provides methods, assays, and kits for measuring factor VIII (fVIII) activity in a sample under more physiologically relevant conditions. It is based, in part, on the discovery that platelets stimulated under physiological conditions, such as by thrombin, expose binding sites for fVIII that are independent of phosphatidylserine, contrasting with stimulation by non-physiological agonists like calcium ionophore that expose many phosphatidylserine-dependent fVIII binding sites. This leads to assays that replace phospholipid vesicles with platelet membrane-comprising or mimetic compositions, providing more reliable measurement of fVIII activity for clinical diagnosis and monitoring of therapy.
The methods involve contacting a sample containing fVIII with fibrin or fibrinogen (fibrin(ogen)) and a platelet membrane-comprising composition under conditions permitting fibrin(ogen) binding to the platelet membrane and fVIII binding to fibrin(ogen), then detecting fVIII activity. Platelet membranes can include isolated platelets, thrombin-activated platelets, platelet membrane fractions containing gpIIbIIIa, or phospholipid vesicles. Detection can use chromogenic factor Xa substrates, plasma-based clotting assays, fibrometers, or thromboelastometry devices. The assays also enable measurement of the partitioning of fVIII between fibrin(ogen) and von Willebrand factor (vWf) by contacting blood or plasma with solid supports bearing immobilized fibrin(ogen) or vWf and measuring bound and unbound fVIII activity.
The background problem being addressed is that traditional fVIII activity assays rely on phospholipid vesicles with high phosphatidylserine content, stimulated by non-physiological agonists, which do not reflect physiologic platelet activation and yield varying and sometimes unreliable activity measurements—particularly for engineered fVIII products and individuals with anti-fVIII antibodies. This limits the accuracy of clinical diagnosis and therapeutic monitoring for hemophilia A, which results from defective or absent fVIII causing bleeding disorders treated by intravenous factor VIII infusion.
Claims Coverage
The claims include one independent claim directed to a kit for measuring fVIII activity, encompassing compositions and components for performing physiological fibrin(ogen)- and platelet membrane-based assays, and several dependent claims specifying additional features and methods related to measuring fVIII activity using this kit.
Kit comprising fibrin(ogen), platelet membrane-comprising composition, coagulation factors, and solid support with anti-fVIII antibodies
A kit that includes fibrin or fibrinogen, a platelet membrane-comprising composition, Factor IXa, Factor X, a solid support having monoclonal antibodies that specifically bind fVIII immobilized thereupon, and packaging materials therefor.
Inclusion of chromogenic fXa substrate to enable activity detection
The kit further comprises a chromogenic fXa substrate to facilitate detection of fVIII activity via cleavage by activated factor Xa.
Platelet membrane composition association with fibrin(ogen)
The fibrin or fibrinogen is provided in association with the platelet membrane-comprising composition to allow physiological binding and activity measurement.
Activated gpIIbIIIa in the platelet membrane composition
The platelet membrane-comprising composition comprises activated gpIIbIIIa incorporated into a membrane composition, reflecting platelet receptor functionality for fibrinogen binding relevant to fVIII activity.
Method instructions for measuring fVIII activity under physiologically relevant conditions
The kit includes instructions for a method that contacts a sample with fibrin(ogen) and a platelet membrane composition under conditions permitting binding of fibrin(ogen) to the membrane and fVIII binding to fibrin(ogen), in the presence of Factor IXa and Factor X, and detecting fVIII activity.
Detection of fVIII activity using chromogenic factor Xa substrate cleavage
Instructions describe detecting fVIII activity by monitoring cleavage of a chromogenic factor Xa substrate by factor Xa generated in the assay.
Activity detection via plasma-based clotting, fibrometer, or thromboelastometry
Instructions further describe detection of fVIII activity using plasma-based clotting assays, fibrometer measurements, or thromboelastometry devices to assess coagulation supported by the assay components.
The claims collectively cover a kit combining fibrin(ogen), platelet membrane components including activated gpIIbIIIa, coagulation factors IXa and X, and detection systems such as chromogenic substrates and solid supports with anti-fVIII antibodies, alongside methods for measuring fVIII activity under physiologically relevant conditions that improve assay reliability and clinical relevance.
Stated Advantages
Replacement of phospholipid vesicles with platelet membrane-comprising compositions provides more reliable measurement of fVIII activity for clinical diagnosis and monitoring.
More accurate assays of pharmaceutical and engineered fVIII products, overcoming poor predictive value of current assays.
Enhanced reliability of measuring fVIII activity in individuals expressing anti-fVIII antibodies due to physiologically relevant assay conditions.
The activated platelet membrane-based assay provides a broader dynamic range and greater sensitivity to inhibitory antibodies compared to standard assays.
Documented Applications
Measurement of factor VIII activity in clinical samples for diagnosis of factor VIII deficiency and monitoring of therapeutic factor VIII infusions.
Measurement of the partitioning of factor VIII binding between fibrin(ogen) and von Willebrand factor in blood or plasma samples.
Screening assays to identify agents that modulate factor VIII activity or factor VIII interaction with fibrin(ogen), including recombinant fVIII products.
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