CLEC-2–deficient platelets do not interact with PDPN. (A) Gene targeting strategy for Clec-2. The deleted sequence in exon 1 encodes the entire intracellular domain of the CLEC-2 receptor, including the YxxL motif required to activate SYK, as well as the transcriptional and translational start sites. (B) Clec-2^−/− platelets lack CLEC-2 protein. Immunoblotting was used to measure CLEC-2 and actin proteins in cell lysate derived from brain (left lane) and platelet-rich plasma (right lanes). CLEC-2 levels in Clec-2^+/− platelets were reduced by approximately 50% compared with those in Clec-2^+/+ platelets (second and third lanes from left, lysate from 0.75 mL of blood loaded). BecauseClec-2^−/− mice die in the perinatal period, CLEC-2 levels were measured in platelets derived from lethally irradiated wild-type mice that had been reconstituted with Clec-2^+/+ or Clec-2^−/− fetal liver cells (right 4 lanes, lysate from 0.1 mL of blood loaded in each lane). (C) In situ hybridization of E14.5 mouse embryos for Clec-2. Clec-2 expression is indicated in red; DAPI nuclear staining is shown in blue. (D) PDPN-Fc proteins bind Clec-2^+/+ but not Clec-2^−/− platelets. (E) PDPN-Fc proteins activate Clec-2^+/+ but not Clec-2^−/− platelets. Platelet activation was measured by the binding of allophycocyanin-conjugated fibrinogen. Platelet activation by the glycoprotein VI receptor agonist convulxin is shown as a positive control.