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.

Modeling thrombosis in silico: Frontiers, challenges, unresolved problems and milestones

“…The functions of procoagulant platelets. One of the most mysterious questions in the field is why activated platelets form at least two subtypes: "aggregating" platelets that can actually form the body of a hemostatic plug or thrombus and procoagulant platelets that can accelerate reactions of blood coagulation [32,33,34,6]. As of now, there is a lot of information about signalling pathways leading to these platelet subpopulations and their properties, but no real idea about their physiological or pathological significance.…”

Dynamics of calcium spiking, mitochondrial collapse and phosphatidylserine exposure in platelet subpopulations during activation

“…Platelets are discoid cells 2–3 μm in diameter that circulate in blood at 200–300 × 10 3 cells μL −1 . Their main function is to prevent blood loss via two main mechanisms: (i) the formation of aggregates; and (ii) acceleration of the membrane‐dependent reactions of blood coagulation by externalization of phosphatidylserine (PS) in the outer membrane leaflet, thus providing binding sites for coagulation factors ; membrane‐bound coagulation factors are additionally protected from blood flow . In order to carry out these functions, platelets have to be activated, which can be performed by a variety of agonists, such as thrombin, collagen, and ADP, acting via numerous receptors (protease‐activated receptors PAR1 and PAR4 for thrombin, glycoprotein VI for collagen, P2Y1 and P2Y12 for ADP, and so on ).…”

“…One of the most interesting aspects of platelet activation is that it results in the formation of at least two distinct subpopulations , and that the two main platelet functions are unevenly distributed between them . One subpopulation consists of living, amoeboid, well‐aggregating platelets with no PS on their surface, whereas the other (called ‘coated’, ‘procoagulant’ or ‘necrotic’ platelets ) includes balloon‐shaped dead cells with externalized PS, a cap of α‐granule proteins , and excellent binding of coagulation factors , but no ability to independently form aggregates , and a disrupted cytoskeleton unattached to adhesive receptors, thus additionally downregulating their adhesive properties .…”

Coagulation factors bound to procoagulant platelets concentrate in cap structures to promote clotting

“…Fluorescence intensity was converted to the mean number of molecules per platelet using a calibration curve obtained with green fluorescent protein-conjugated beads prepared and calibrated as described. 20 Linearity of binding quantification was confirmed by control experiments using different ratios of labeled/unlabeled factors (see supplemental Figure 2, available on the Blood Web site).…”