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Fibrinogen αC-subregions critically contribute blood clot fibre growth, mechanical stability, and resistance to fibrinolysis

• Authors : McPherson, Helen R; Duval, Cedric; Baker, Stephen R ...

• Source: eLife ; volume 10 ; ISSN 2050-084X

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Author response: Fibrinogen αC-subregions critically contribute blood clot fibre growth, mechanical stability, and resistance to fibrinolysis

• Authors : McPherson, Helen R; Duval, Cedric; Baker, Stephen R

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Decision letter: Fibrinogen αC-subregions critically contribute blood clot fibre growth, mechanical stability, and resistance to fibrinolysis

• Authors : Iqbal, Jameel; Zaidi, Mone

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Figure 8. Heparan sulfate removal of perlecan facilitates platelet adhesion. ; The indicated substrates were coated alone or in combination onto wells in 96-well plates (2.5 µg/ml collagen and 10 µg/ml for all other substrates) overnight. Where indicated, wells were treated with 5 mU/ml heparinase III. Platelets from (A) humans or (B) mice were allowed to adhere for 1 hr and adhesion was quantified colorimetrically with 4-nitrophenyl phosphate(pNPP). (A) Human, platelets; n = 4–5 individual donors from 3 to 4 independent experiments; P-values were calculated using one-way ANOVA with Sidak’s post-hoc test. (B) Mouse platelets; n = 4 samples/condition/genotype from two independent experiments. Owing to severe thrombocytopenia, platelets from up to five mice were pooled for one KO sample. P-values for differences between WT and Mpig6b–/–mice were calculated using two-way ANOVA with Sidak’s post-hoc test. (C, D) Adhesion of (C) human or (D) WT and G6b–/–platelets on fibrinogen and perlecan. (i) Mean surface area of individual platelets quantified by KNIME software analysis. In panel (C) (i) n = 5 donors from two independent experiments. P-values were calculated using one-way ANOVA with Sidak’s post-hoc test. Total number of cells analyzed: fibrinogen, 1957; perlecan, 239; perlecan + heparinase III, 686. In panel (D) (i) n = 5–7 mice/condition/genotype from 2 to 3 independent experiments. P-values were calculated using two-way ANOVA with Sidak’s post-hoc test. Total number of cells analyzed: 134–176 for perlecan conditions, and 913–1277 for fibrinogen conditions. *, p

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Figure 5. Cleavage of the βI domain disulfide impairs fibrinogen binding. ; (A) Expression of wild-type (64.8% of cells) and β3 C177,184S disulfide mutant αIIbβ3 integrin (82.9% of cells) on BHK cells from staining with fluorescene-conjugated anti-CD61 antibody and flow cytometry. Transfection with empty vector served as negative control. (B) Binding of soluble fibrinogen-AF647 (0.1 mg/mL) to BHK cells expressing wild-type or C177,184S mutant αIIbβ3 integrin. Binding is expressed as the % of CD61+ cells that bound fibrinogen. Data points and errors (SD) are from four separate experiments. (C) Adhesion of BHK cells expressing wild-type or C177,184S mutant αIIbβ3 integrin to immobilized fibrinogen. Experiments were performed in triplicate, three fields were analyzed per well and errors are SD. *p

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Author response image 1. ERp5 triggers αIIbβ3 integrin de-adhesion from fibrin. ; Platelet adhesion to immobilized fibrin in the absence of presence of 2 µM ERp5 was measured. Fibrin was immobilized in two ways: immobilized fibrinogen was incubated with thrombin and residual thrombin inactivated with hirudin (fbg/IIa), or pre-formed fibrin was directly immobilized (fibrin). Adhesion was measured in the first 4 minutes at a fluid shear rate of 1000 s-1. The bars and errors (1 SD) are from three measurements each from three different healthy donor platelets. *, p < 0.05, **, p < 0.01; assessed by unpaired, two-tailed Student’s t-test. Additional experimental details are as described in the manuscript for platelet adhesion to fibrinogen.

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Figure 1. ERp5 triggers αIIbβ3 integrin de-adhesion from fibrinogen. ; (A) ERp5 does not induce αIIbβ3 integrin activation on platelets, or influence integrin activation by ADP. The activated αIIbβ3 is measured by PAC-1 antibody binding. The data points and errors (1 SD) are from measurements of four different healthy donor platelets. (B–D) Platelet adhesion to fibrinogen under flow is impaired by ERp5. Part B are representative images of platelet adhesion to immobilized fibrinogen in the absence of presence of 2 µM ERp5. Parts C and D are kinetics of platelet adhesion to fibrinogen in the absence or presence of 2 µM ERp5 at fluid shear rates of 1000 s−1 or 3000 s−1, respectively. The data points and errors (1 SD) are from three measurements each from three different healthy donor platelets. *p

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Figure 2. Mechanical force accelerates ERp5-induced fibrinogen de-adhesion. ; (A) Biomembrane-Force-Probe (BFP) detection of αIIbβ3–fibrinogen (FBG) binding in a purified system. The probe bead was coated with fibrinogen and streptavidin (SA) for attachment of the bead to biotinylated red blood cell (RBC). Fibrinogen is the focus for interaction with αIIbβ3 on the target bead. The effect of soluble ERp5 on this interaction was measured. (B) Force versus time traces from two representative test cycles. The target bead was driven to approach and contact the probe bead, then retracted. In a 'no bond' event (black), the cycle ended after the probe–target separation. In a 'bond' event (red), the target was clamped (marked by *) at a preset force until dissociation. Lifetime is measured from the point when the clamped force (i.e. 30 pN) was reached to the point when the bond dissociated, signified by a force drop to zero. (C) Soluble ERp5 has no significant effect on αIIbβ3–fibrinogen adhesion frequency. The adhesion frequencies represent mean ± SEM of n = 3 independent experiments in the absence or presence of 2 µM ERp5. For each experiment, five random probe–target pairs with 50 touches were analyzed and averaged. (D) Lifetime of fibrinogen–αIIbβ3 integrin bonds vs. clamp force in the absence or presence of 2 µM ERp5. Results represent mean ±SEM of 10–30 measurements at each force bin. *p

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Figure 7. ERp5 regulation of fibrinogen release from activated αIIbβ3 integrin. ; Activation of αIIbβ3 integrin results in transition from a bent (low affinity) to an extended (high affinity) structure. While much is known about the structure and function of resting and activated integrins, little is known about how integrins disengage from their ligands. Our findings indicate that ERp5 regulates fibrinogen release from extended αIIbβ3 integrin by reducing the βI domain Cys177-Cys184 disulfide bond (light blue oval). Cleavage of the disulfide is controlled by both ligand binding and mechanical force. It is feasible that the disulfide bond could reform and the integrin reengage fibrinogen (dotted arrow).

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Figure 10. G6b knockout megakaryocytes show enhanced spreading on perlecan. ; Adhesion of WT and Mpig6b–/–MKs on perlecan. (i) Mean surface area of MKs was quantified with ImageJ. n = 4–6 mice/condition/genotype from three independent experiments; total cell numbers analyzed per condition/genotype were 77–188 for conditions with perlecan only and 1671–2866 for conditions with fibrinogen. P values were calculated using two-way ANOVA with Sidak’s post-hoc test, ***, p

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