Protein expression in FlnA-null platelets and MKs. FlnA-null and control blood platelets (A) or MKs from day 3 cultures (B) were probed with fluorescein isothiocyanate-labeled antibodies for surface expression of GPIbα, GPIbβ, GPIX, GPV, and CD61 and analyzed by flow cytometry. Results are expressed as mean fluorescence intensity (MFI) and are mean plus or minus SD (n = 4). *P < .05. Expression of VWFR subunits is decreased on FlnA-null blood platelets but normal on FlnA-null MKs. Lysates of control and FlnA-null blood platelets (C) and MKs from day 3 cultures (D) were subjected to SDS-PAGE and probed with antibodies directed against FlnA, GPIbα, CD61, MMP9, and ADAM17; GAPDH was used as loading control. Blots shown are representative of 4 independent experiments. The increased degradation of GPIbα in the FlnA-null platelets correlated with high expression levels of MMP9 and ADAM17. (E) Lysates of mouse FLCs (day 0), immature (day 2), and mature (day 3) MKs and blood platelets (mPlt) were probed for FlnA, GPIbα, GPIbβ, and actin; GAPDH was used as loading control. The anti-GPIbβ antibody detects GPIbβ bound to GPIbα and the membrane-anchored truncated remainder of GPIbα (t-GPIbα) in nonreducing conditions. Maturation of MKs leads to increased expression of FlnA, GPIbα, GPIbβ, and actin. (F) Linkage of GPIb to the MK and platelet cytoskeleton. Triton X-100 soluble (S) and insoluble (P) fractions were collected by centrifugation of control MK and platelet lysates at 100 000g for 30 minutes at 4°C and probed for FlnA, GPIbα, and GPIbβ (in nonreducing conditions) and actin. At day 3, FlnA, GPIbα, and GPIbβ are tethered to F-actin in control MKs and platelets. GPIb is not tethered to the actin cytoskeleton of FlnA-null MKs (G) or platelets (H). Blots shown are representative of 3 independent experiments.