Studies on the actin-binding protein HS1 in platelets

Abstract

Background: The platelet cytoskeleton mediates the dramatic change in platelet morphology that takes place upon activation and stabilizes thrombus formation. The Arp2/3 complex plays a vital role in these processes, providing the protrusive force for lamellipodia formation. The Arp2/3 complex is highly regulated by a number of actin-binding proteins including the haematopoietic-specific protein HS1 and its homologue cortactin. The present study investigates the role of HS1 in platelets using HS1-/- mice.

Results: The present results demonstrate that HS1 is not required for platelet activation, shape change or aggregation. Platelets from HS1-/- mice spread normally on a variety of adhesion proteins and have normal F-actin and Arp2/3 complex distributions. Clot retraction, an actin-dependent process, is also normal in these mice. Platelet aggregation and secretion is indistinguishable between knock out and littermates and there is no increase in bleeding using the tail bleeding assay.

Conclusion: This study concludes that HS1 does not play a major role in platelet function. It is possible that a role for HS1 is masked by the presence of cortactin.

Figure 1

Domain organisation of HS1^-/- and genotyping of knockout mice. (A) Schematic representation of mouse cortactin and HS1 proteins. N – terminal acidic domain, R1, R2, etc – Cortactin repeats, CC – coiled coil helical domain, PRD – proline rich domain, SH3 – C-terminal Src homology domain. Numbers indicate amino acid number. (B) Genotyping of HS1 knockout mice by PCR. WT – wild type, HS1^+/- – heterozygote, HS1^-/- – homozygote. (C) Western blot of platelet extracts from WT and HS1^-/- mice probed with α-HS1 (top panel) and α-tubulin (bottom panel).

Figure 2

Analysis of platelet spreading and F-actin organization. Washed platelets (2 × 10⁷ platelets/ml) from WT and HS1^-/- mice were added to cover slips coated with collagen (100 μg/ml), CRP (10 μg/ml) or fibrinogen (100 μg/ml) ± thrombin (1 U/ml) and allowed to settle for 45 or 90 min at 37°C. Spread platelets were fixed in formalin and imaged using DIC. Representative images of platelets at 45 min are shown in (A). Mean platelet area was measured. No significant difference was observed between WT and HS1^-/- platelets on any surface at 45 min (B). Platelets spread on fibrinogen ± thrombin for 45 min (C) were labeled with α-p34 for Arp2/3 complex (top panel), and rhodamine phalloidin for F-actin (middle panel). The merged images are shown in the bottom panel. (D) Spread platelets were also labeled with α-cortactin (top panel) and rhodamine phalloidin (middle panel). Merged images are shown in the bottom panel. Protein extracts from WT and HS1^-/- mice were blotted (E) for cortactin, WASp, Scar/WAVE1, Scar/WAVE 2 and N-WASp. Blots were also probed for tubulin to check loading. Scale bars = 5 μM.

Figure 3

Clot retraction assays in WT and HS1^-/- PRP. (A) Time course of clot contraction and (B) the volume of serum excluded at each time point. No significant difference was observed between WT and HS1^-/- platelet rich plasma at any time point (n = 3).

Figure 4

Aggregation and secretion response of WT and HS1^-/- platelets. (A) – (C) Representative aggregation traces for WT and HS1^-/- platelets in response to maximal and threshold concentrations of thrombin, collagen and CRP. (D – I) Percentage aggregation and ATP secretion of WT and HS1^-/- platelets was determined after 2 min stimulation with varying concentrations of thrombin (D & G), collagen (E & H) and CRP (F & I). For % aggregation the mean ± SEM of 3 different mice is presented. For ATP secretion, mean ± SEM from 1 representative experiment is shown (WT – Black bars, HS1^-/- – White bars). No significant difference was observed between WT and HS1^-/- platelets for any of the agonists tested.

Figure 5

In vitro flow assays. Whole blood from WT (A) or HS1^-/- (B) mice was flowed over collagen at a shear rate of 1000 s^-1 for 4 min. Aggregates were fixed in formalin and imaged using DIC. Flow experiments were also carried out with DiOC₆ labeled platelets (representative image shown in C) and the mean surface area of aggregates was calculated from fluorescence images (D). Means ± SEM are plotted (n = 3). No significant difference was observed between surface area coverage of WT and HS1^-/- platelets (P = 0.669). Scale bars = 20 μM

Figure 6

Bleeding time measurements in WT and HS1^-/- mice. HS1^-/- mice show no significant difference in bleeding as determined by volume of blood lost in 10 min following removal of the terminal 3 mm of the tail (P = 0.852). Closed circles represent individual data points, horizontal bars the mean and vertical bars the SEM. (n = 12 for WT and 10 for HS1^-/-).