title: Hysteresis-like binding of coagulation factors X/Xa to procoagulant activated platelets and phospholipids results from multistep association and membrane-dependent multimerization.
abstract: Binding of coagulation factors X (fX) and Xa (fXa) to activated platelets is required for the formation of membrane-dependent enzymatic complexes of intrinsic tenase and prothrombinase. We carried out an in-depth characterization of fX/fXa binding to phospholipids and gel-filtered, thrombin-activated platelets. Flow cytometry, surface plasmon resonance, and computational modeling were used to investigate interactions of fX/fXa with the membranes. Confocal microscopy was employed to study fXa binding to platelet thrombi formed in flowing whole blood under arterial conditions. Binding of fX/fXa to either vesicles or procoagulant platelets did not follow a traditional one-step reversible binding model. Their dissociation was a two-step process resulting in a plateau that was up to 10-fold greater than the saturation value observed in the association experiments. Computational modeling and experimental evidence suggested that this was caused by a combination of two-step association (mainly for fX) and multimerization on the membrane (mainly for fXa). Importantly, fX formed multimers with fXa, thereby improving its retention. The same binding/dissociation hysteresis was observed for annexin V known to form trimers on the membranes. Experiments with platelets from gray syndrome patients showed that alpha-granular factor Va provided an additional high-affinity binding site for fXa that did not affect the hysteresis. Confocal microscopy observation of fXa binding to platelet thrombi in a flow chamber and its wash-out confirmed that this phenomenon persisted under physiologically relevant conditions. This suggests its possible role of "locking" coagulation factors on the membrane and preventing their inhibition in plasma and removal from thrombi by flow.
authors.affiliation: Federal Research and Clinical Centre of Pediatric Hematology, Oncology and Immunology, Moscow 117198, Russia.
authors.forename: Nadezhda A
authors.initials: NA
authors.lastname: Podoplelova
authors.affiliation: Laboratory of Molecular Mechanisms of Hemostasis, Center for Theoretical Problems of Physicochemical Pharmacology, Moscow 119991, Russia; Faculty of Physics, Moscow State University, Moscow 119992, Russia.
authors.forename: Anastasia N
authors.initials: AN
authors.lastname: Sveshnikova
authors.affiliation: U.S. Food and Drug Administration, Center for Biologics Evaluation and Research, Silver Spring, MD 20993, USA.
authors.forename: James H
authors.initials: JH
authors.lastname: Kurasawa
authors.affiliation: U.S. Food and Drug Administration, Center for Biologics Evaluation and Research, Silver Spring, MD 20993, USA.
authors.forename: Andrey G
authors.initials: AG
authors.lastname: Sarafanov
authors.affiliation: Department of Pediatric Onco-Hematology, APHM, La Timone Hospital, Aix-Marseille University, Marseille, France.
authors.forename: Herve
authors.initials: H
authors.lastname: Chambost
authors.affiliation: National Research Center for Hematology, Moscow 125167, Russia.
authors.forename: Sergey A
authors.initials: SA
authors.lastname: Vasil'ev
authors.affiliation: Federal Research and Clinical Centre of Pediatric Hematology, Oncology and Immunology, Moscow 117198, Russia.
authors.forename: Irina A
authors.initials: IA
authors.lastname: Demina
authors.affiliation: Federal Research and Clinical Centre of Pediatric Hematology, Oncology and Immunology, Moscow 117198, Russia; Laboratory of Molecular Mechanisms of Hemostasis, Center for Theoretical Problems of Physicochemical Pharmacology, Moscow 119991, Russia; Faculty of Physics, Moscow State University, Moscow 119992, Russia.
authors.forename: Fazly I
authors.initials: FI
authors.lastname: Ataullakhanov
authors.affiliation: Aix-Marseille Université, 13005 Marseille, France.
authors.forename: Marie-Christine
authors.initials: MC
authors.lastname: Alessi
authors.affiliation: Federal Research and Clinical Centre of Pediatric Hematology, Oncology and Immunology, Moscow 117198, Russia; Laboratory of Molecular Mechanisms of Hemostasis, Center for Theoretical Problems of Physicochemical Pharmacology, Moscow 119991, Russia; Faculty of Physics, Moscow State University, Moscow 119992, Russia; Faculty of Biological and Medical Physics, Moscow Institute of Physics and Technology, Dolgoprudny 141700, Russia. Electronic address: mapanteleev@yandex.ru.
authors.forename: Mikhail A
authors.initials: MA
authors.lastname: Panteleev
mesh_terms: D001704:Biopolymers / Q000378:metabolism*; D001792:Blood Platelets / Q000378:metabolism*; D003198:Computer Simulation; D005170:Factor X / Q000378:metabolism*; D015951:Factor Xa / Q000378:metabolism*; D006801:Humans; D010743:Phospholipids / Q000378:metabolism*; D015539:Platelet Activation; D020349:Surface Plasmon Resonance
chemical_list: D001704:Biopolymers; D010743:Phospholipids; D005170:Factor X; D015951:Factor Xa
keywords: Annexin V; Blood coagulation; Blood platelets; Coagulation factor X; Membrane-dependent reaction; Phosphatidylserine