Methods and assays for factor VIII activity
Inventors
GILBERT, Gary Eugene • Shi, Jialan • NOVAKOVIC, Valerie A.
Assignees
VETERAN'S AFFAIRS UNITED STATES GOVERNMENT AS REPRESENTED BY DEPARTMENT OF • US Department of Veterans Affairs • Harvard University
Publication Number
US-10114017-B2
Publication Date
2018-10-30
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 relates to methods and assays for measuring factor VIII (fVIII) activity in a sample. The core innovation is based on the discovery that platelets stimulated under physiological conditions, such as by thrombin, comprise binding sites for fVIII that are independent of phosphatidylserine. This differs from the stimulation of platelets by non-physiological agonists like calcium ionophore, which lead to exposure of many fVIII binding sites on the surface of platelets primarily through phosphatidylserine.
The methods and assays disclosed use platelet membrane-comprising compositions, including isolated or thrombin-activated platelets, membrane fractions containing gpIIbIIIa, or phospholipid vesicles, to measure fVIII activity more reliably. These compositions support assembly of the factor Xase complex and binding of fVIII via fibrin(ogen) rather than phosphatidylserine-enriched phospholipid vesicles. The assays provide more physiologically relevant conditions to evaluate fVIII activity, including better clinical diagnosis and monitoring of fVIII deficiency and therapy. Moreover, they improve accuracy of activity assessments for pharmaceutical and engineered fVIII preparations, which current assays evaluate poorly.
Another aspect of the innovation is methods to measure partitioning of fVIII between fibrin(ogen) and von Willebrand factor (vWf). These methods use solid supports with immobilized vWf or fibrin(ogen) to bind fVIII from blood or plasma samples, then separately measure activity bound to each support and activity remaining free in suspension. These methods provide insights into the distribution of fVIII between its plasma complex with vWf and the fibrin(ogen) binding sites, further enhancing understanding and assay accuracy.
Claims Coverage
The patent claims encompass five main inventive features covering methods of measuring fVIII activity using platelet membrane compositions and fibrin, distinguishing fVIII binding states, and kits for these assays.
Method of measuring fVIII activity with fibrin and platelet membrane compositions
A method comprising contacting a sample containing fVIII with fibrin and isolated platelets or a platelet membrane fraction containing gpIIbIIIa, where fibrin binds to the platelet membrane composition and fVIII binds to fibrin, and detecting fVIII activity by plasma-based clotting assays, fibrometer, thromboelastometry device, or through cleavage detection of a chromogenic fXa substrate.
Use of thrombin-activated platelets in fVIII activity measurement
The method wherein the isolated platelets used are thrombin-activated to reflect physiological platelet activation and support fVIII binding via fibrin.
Selective detection of fibrin-bound fVIII using monoclonal antibodies
Distinguishing fVIII activity bound to fibrin from unbound fVIII by addition of monoclonal antibodies that selectively interfere with fVIII binding to fibrin.
Activation of platelet membranes by physiological agonists
The isolated platelets or platelet membrane fractions are activated by contacting them with physiological agonists such as thrombin, peptides stimulating PAR1 and/or PAR4, factor XIa, and/or calcium ions to simulate physiological conditions for fVIII activity assays.
Assay sample specificity and activation step
The assay is performed using plasma as the sample and optionally includes an activation step of platelet membranes with physiological agonists to support accurate fVIII activity measurement.
The claims collectively cover physiologically relevant methods of measuring fVIII activity using fibrin-bound sites on platelet membrane compositions, distinguishing fVIII binding states with selective antibodies, activating platelets via physiological agonists, and kits containing these components for improved clinical and pharmaceutical fVIII activity assessment.
Stated Advantages
Provides more reliable and physiologically relevant measurement of factor VIII activity compared to standard assays using phospholipid vesicles with high phosphatidylserine content.
Improves accuracy in diagnosis of factor VIII deficiency and monitoring of factor VIII therapy, including for engineered factor VIII products.
Enhances reliability of assays measuring factor VIII activity in individuals expressing anti-factor VIII antibodies.
Enables detection and quantification of partitioning of factor VIII between fibrin(ogen) and von Willebrand factor, providing more detailed insight into factor VIII interactions.
Documented Applications
Clinical diagnosis of factor VIII deficiency and monitoring of therapy with factor VIII infusions.
Measuring activity of pharmaceutical preparations of factor VIII, including engineered factor VIII products.
Screening for agents or candidate modifiers of factor VIII activity using assays based on physiologically relevant fVIII binding sites.
Detecting and analyzing the fraction of factor VIII bound to von Willebrand factor versus fibrin(ogen) in blood or plasma samples.
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