Platelet Surface-Associated Activation and Secretion-Mediated Inhibition of Coagulation Factor XII

<div><p>Coagulation factor XII (fXII) is important for arterial thrombosis, but its physiological activation mechanisms are unclear. In this study, we elucidated the role of platelets and platelet-derived material in fXII activation. FXII activation was only observed upon potent platelet stimulation (with thrombin, collagen-related peptide, or calcium ionophore, but not ADP) accompanied by phosphatidylserine exposure and was localised to the platelet surface. Platelets from three patients with grey platelet syndrome did not activate fXII, which suggests that platelet-associated fXII-activating material might be released from α-granules. FXII was preferentially bound by phosphotidylserine-positive platelets and annexin V abrogated platelet-dependent fXII activation; however, artificial phosphotidylserine/phosphatidylcholine microvesicles did not support fXII activation under the conditions herein. Confocal microscopy using DAPI as a poly-phosphate marker did not reveal poly-phosphates associated with an activated platelet surface. Experimental data for fXII activation indicates an auto-inhibition mechanism (<i>k</i>_i/<i>k</i>_a = 180 molecules/platelet). Unlike surface-associated fXII activation, platelet secretion inhibited activated fXII (fXIIa), particularly due to a released C1-inhibitor. Platelet surface-associated fXIIa formation triggered contact pathway-dependent clotting in recalcified plasma. Computer modelling suggests that fXIIa inactivation was greatly decreased in thrombi under high blood flow due to inhibitor washout. Combined, the surface-associated fXII activation and its inhibition in solution herein may be regarded as a flow-sensitive regulator that can shift the balance between surface-associated clotting and plasma-dependent inhibition, which may explain the role of fXII at high shear and why fXII is important for thrombosis but negligible in haemostasis.</p></div

The roles of the platelet surface, MPs, and secretion in fXII activation and contact coagulation.

<p><b>(A)</b> The effect of platelets and/or MP removal on fXIIa formation in 20% plasma (n = 3). After activation, the platelets were diluted to 4×10⁶/mL and centrifuged at 300 g for platelet precipitation and again at 16,000 g for MP precipitation [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116665#pone.0116665.ref025" target="_blank">25</a>]. <b>(B)</b> The effects of 200 μM calpeptin and MDL 28170 on fXIIa formation (in 20% plasma) by A23187-activated platelets after platelet secretion removal. <b>(C)</b> Clotting caused by A23187-activated, secretion-depleted platelets in recalcified plasma (n = 3). A23187-activated, secretion-depleted platelets (100 μL, 6×10⁷/mL) were supplemented with 500 μL of 90% recalcified plasma (see “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116665#sec002" target="_blank">Methods</a>”), and clotting was measured as an increase in optical density at 405 nm; 50 nM fVIIai was used, and CTI was used at 200 μg/mL. <b>(D)</b> Retention of fXIIa by A23187-activated secretion-depleted platelets (n = 3). The platelets were activated with A23187 for 30 min and washed free of platelet secretion (see “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116665#sec002" target="_blank">Methods</a>”). Next, 2×10⁸/mL platelets were incubated with 20% chelated plasma for 1 h before washing again to separate the platelets from the plasma and from soluble fXIIa. The activity of bound fXIIa was estimated using 200 μM S2302 for platelets at 2×10⁷/mL.</p

Comparison of fXII activation by platelets from a normal donor and GPS patients.

<p><b>(A)</b> Comparison of the fXIIa-generating capacities for A23187-activated platelets (at 2×10⁷/mL) from a normal donor (labelled “normal plt”) and three grey platelet syndrome patients (labelled GPS-1, GPS-2, and GPS-3, respectively). <b>(B)</b> Flow-cytometry dot plots demonstrating the significant PS exposure in the analysed platelet preparations upon activation with A23187.</p

Platelet activation of purified and plasma fXII.

<p><b>(A)</b> Flow-cytometry dot-plots demonstrating predominant FITC-fXII binding for PS-positive platelet subpopulations (labelled “PS+”). The result corresponds to 1000 nM FITC-fXII binding. <b>(B)</b> The effect of platelets on activation of purified 200 nM fXII. Platelets were activated by 10 nM thrombin (n = 3). <b>(C)</b> Dose-dependence for platelet-dependent fXIIa formation upon a reaction between thrombin-activated platelets and purified fXII (n = 3). A hyperbolic curve for a representative, typical experiment was fit using <i>K</i>_m^fXII = 64 and [<i>fXIIa</i>]_max = 0.126 nM. The given <i>K</i>_m and [<i>fXIIa</i>]_m values are mean values (±SD) calculated using data from three independent experiments. <b>(D)</b> The effect of different platelet stimulation methods on platelet-dependent fXIIa formation in 20% plasma. The asterisks symbols (*) correspond to p<0.05. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116665#pone.0116665.t001" target="_blank">Table 1</a> represents the p values for comparing fXII activating capacities between activated and non-activated platelets.</p

The role of PS and poly-P in surface-associated fXII activation by platelets.

<p><b>(A)</b> The effect of 20 μg/mL annexin V or 1.5 μM prothrombin on 280 nM fXII activation (in the presence of 2.5 mM CaCl₂) by A23187-activated (secretion-depleted) platelets (n = 3). <b>(B)</b> Comparison of the fXIIa-generating capacities for A23187-activated platelets (at 2×10⁶/mL) and 4 μM PS- or PC-liposomes (depicted as PS and PC, respectively) in 20% chelated plasma. The mean values (±SD) were calculated from three independent experiments (n = 3). <b>(C)</b> Confocal microscopy analysis of poly-P localisation in activated platelets with 2.5 mM calcium. DAPI was used as poly-P marker [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116665#pone.0116665.ref027" target="_blank">27</a>], and AlexaFluor-647-annexin V was used as a marker for procoagulant PS-positive platelets (labelled “PS+”). PS-negative activated platelets are labelled “PS-” (n = 3). <b>(D)</b> Confocal microscopy analysis of poly-P localisation in activated platelets without 2.5 mM calcium. FITC-anti-fibrin(ogen) antibody was used as an activated platelet marker (n = 3). The images in (C) and (D) were collected at an ∼2 μm z-depth over the fibrinogen surface to reduce non-specific fibrinogen immunofluorescence.</p

FXII activation model.

<p><b>(A)</b> Computational model schematic for fXII activation on the platelet surface. Soluble fXII first binds the surface and is then activated; fXIIa can inhibit further activation through an auto-inhibition mechanism [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116665#pone.0116665.ref026" target="_blank">26</a>]. The reaction rate constants (<i>k</i>_i) for the model were estimated based on the data in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116665#pone.0116665.g001" target="_blank">Fig. 1C</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116665#pone.0116665.s004" target="_blank">S4C Fig.</a>, and the surface area of an activated platelet was considered 50 μm². <b>(B)</b> Platelet-dependent fXIIa formation in buffer as a function of the fXII concentration added. The black circles correspond to the experimental data (±SD for three independent experiments, n = 3); the curve corresponds to the computational model. The consistency between the data and model curve confirms the fXII auto-inhibition hypothesis. <b>(C)</b> The dose-dependence of fXIIa formation in 20% plasma for A23187-activated platelets after platelet secretion removal. The platelets were activated at 2×10⁸/mL and diluted to the indicated concentrations for further reaction with plasma. The black squares represent the experimental data (n = 3), and the curve represents the computational model. The consistency between the data and model curve confirms the reliability of the model.</p

Inhibition of fXIIa activity through platelet secretion.

<p><b>(A)</b> The effect of platelet secretion removal on platelet-related fXII activation (means±SD for three independent experiments, n = 3). A portion of the A23187-activated platelet suspension was diluted to 4×10⁶/mL and mixed 1:1 with 40% chelated plasma to measure the initial fXIIa-generating capacity in the presence of platelet secretion (which was estimated at 100%). The remaining platelets were washed free from platelet secretion, diluted, and analysed. <b>(B)</b> The effect of platelet secretion on 2 nM fXIIa activity (n = 3). The reaction in buffer A without platelets was compared with the reaction in the presence of 2×10⁶/mL activated platelets or platelet secretion (at an equivalent dilution). <b>(C)</b> Western blot analysis of purified C1-INH (labelled “C1-INH”) and platelet secretion (labelled “Secretion”). Supernatant containing platelet secretion was incubated with 25 nM fXIIa, and the total protein was concentrated 20-fold. After resolution using 4–10% SDS-PAGE gels and subsequent Western blotting, the bands were developed using the ECL method. The figures on the left depict the molecular masses of markers measured in kDa (n = 4).</p

Computer simulation of C1-inhibitor washout.

<p>(<b>A</b>) We used a scheme that depicts the platelet aggregate to test the C1-INH washout hypothesis. The platelet concentration inside the thrombi was considered 1 per 15 fl. The initial C1-inhibitor distribution in the aggregate for the simulations is shown in grey, and at values ranging from 0.1 to 10 μm/s, the flow penetrated the aggregate. At t = 0, C1-INH at 100 μM (estimated from ref. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116665#pone.0116665.ref033" target="_blank">33</a>]) and fXII at 450 nM appeared simultaneously. Factor XII could be activated on the platelet surface, and C1-INH could diffuse through the aggregate (D = 10 μm²/s, based on the molecular weight) and move due to the flow. The fXIIa diffusion was assumed negligible because fXII activation is surface-associated ([<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116665#pone.0116665.ref010" target="_blank">10</a>] and this study), and typically, fXIIa is tightly bound to the activation surface [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116665#pone.0116665.ref039" target="_blank">39</a>]. The C1-INH action was described using a mass action equation with the reaction constant [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116665#pone.0116665.ref034" target="_blank">34</a>] 0.00366 μM^-1s^-1. (<b>B</b>) Time-course of the distance-averaged [C1-INH] for various flow velocities. (<b>C</b>) Time-course of the surface-averaged [fXIIa] for various flow velocities. The C1-INH and fXIIa spatial distributions were governed by a set of differential equations, which were solved using the finite volume solver available within the Virtual Cell environment.</p