Novel insights into mouse models of ectopic proplatelet release

Abstract

Mature bone marrow (BM) megakaryocytes (MKs) produce platelets by extending proplatelets into sinusoidal blood vessels. Defects in this process can lead to thrombocytopenia and increased risk of bleeding. Mice lacking the actin-regulatory proteins Profilin 1 (PFN1), Wiskott-Aldrich Syndrome protein (WASp), Actin Related Protein 2/3 complex (Arp2/3), or adhesion and degranulation-promoting adapter protein (ADAP) display thrombocytopenia and ectopic release of (pro)platelet-like particles into the BM compartment, pointing to an important axis of actin-mediated directional proplatelet formation. The mechanism underlying ectopic release in these mice is still not completely understood. However, we hypothesized that similar functional defects account for this observation. We analyzed WASp-, ADAP-, PFN1-, and ARPC2-knockout mice to determine the role of actin reorganization and integrin activation in directional proplatelet formation. ADAP-, ARPC2-, and PFN1-deficient MKs displayed reduced adhesion to collagen, defective F-actin organization, and diminished β1-integrin activation. WASp-deficient MKs showed the strongest reduction in the adhesion assay of collagen and altered F-actin organization with reduced podosome formation. Our results indicate that ADAP, PFN1, WASp, and ARP2/3 are part of the same pathway that regulates polarization processes in MKs and directional proplatelet formation into BM sinusoids.

Graphical abstract

Figure 1.

Reduced adhesionof BM-derived ADAP-, PFN1-, WASp-, and ARP2/3-deficient MKs on ECM proteins. (A) Cryosections of BM from control (Adap^fl/fl), ADAP-, PFN1-, WASp-, and ARP2/3-deficient mice stained for GPIX (cyan) to visualize MKs, and (pro)PLPs and CD105 (red) to identify sinusoids. The images from the bottom panel are magnified from the regions indicated in the upper panel. Scale bars represent 30 μm (upper panel) and 10 μm (lower panel). (Bi) Gating strategy for the detection of (pro)PLPs and MKs in BM samples based on the size and expression of αIIbβ3. Shown is the dot plot of a Pfn1^fl/fl-PF4-cre BM sample and the corresponding control (Pfn1^fl/fl). (Bii) Quantification of (pro)PLPs in the BM as percentage of the control mean. The percentage of (pro)PLPs in the BM was calculated and normalized to the mean of their respective control MKs. The controls are indicated by dashed lines. (Biii) Quantification of MKs in the BM as percentage of the control mean. The percentage of MKs in the BM was calculated and normalized against the mean of the respective control MKs. The controls are indicated by dashed line. Each data point represents a single mouse. N = 3-9. (C) Normalized adhesion assay of in vitro cultured BM-derived MKs from the respective controls, ADAP-, PFN1-, WASp-, and ARP2/3-deficient mice on Horm collagen (Ci), fibrinogen (Cii), and fibronectin (Ciii). The percentage of adherent mutant MKs after 3.5 hours incubation was calculated and normalized against the mean of their respective control MKs. Controls are indicated with a dashed line. Each dot represents a technical replicate. The experiment was performed at least twice.

Figure 2.

Cultured spread MKs from mutant mice exhibit reduced PLS formation, F-actin content, and β1-integrin activation. (A) Representative confocal images of cultured BM-derived MKs from control (Adap^fl/fl), ADAP-, PFN1-, WASp-, and ARP2/3-deficient mice spread on Horm collagen and stained for F-actin (red), the active form of β1-integrin (cyan), and the nucleus (blue). Scale bars represent 50 μm (left) and 20 μm (right). (B) Mean fluorescence intensity of active β1-integrin, (C) F-actin, and (D) density of PLS in the lowest optical section of spread MKs on Horm collagen, normalized to their respective control cells. Controls are indicated by a dashed line (B-D). (B-D) 35 to 59 MKs per genotype were analyzed. The data points represent individual MKs.