Thrombospondin receptor (CD36) has been recently identified in platelets and various cell lines as the receptor for thrombospondin, an adhesive protein required for irreversible aggregation of platelets as well as other adhesive processes. Thrombospondin receptor, one of major glycosylated platelet membrane proteins, is thought to play an important role as a cell adhesion molecule in blood coagulation system as well as intercellular signaling. In this work, thrombospondin receptor was purified to homogeneity from human platelet by wheat germ agglutinin (WGA)-affinity chromatography and size exclusion chromatography on Ultrogel-AcA44. The molecular weight of the purified thrombospondin receptor was about 88 kDa on SDS-PAGE and its identity was confirmed by immunoblot analysis and immunodiffusion assay.

Site-1 protease (S1P) ablation in the osterix-lineage in mice drastically reduces bone development and downregulates bone marrow-derived skeletal stem cells. Here we show that these mice also suffer from spina bifida occulta with a characteristic lack of bone fusion in the posterior neural arches. Molecular analysis of bone marrow-derived non-red blood cell cells, via single-cell RNA-Seq and protein mass spectrometry, demonstrate that these mice have a much-altered bone marrow with a significant increase in neutrophils and Ly6C-expressing leukocytes. The molecular composition of bone marrow neutrophils is also different as they express more and additional members of the stefin A (Stfa) family of proteins. In vitro, recombinant Stfa1 and Stfa2 proteins have the ability to drastically inhibit osteogenic differentiation of bone marrow stromal cells, with no effect on adipogenic differentiation. FACS analysis of hematopoietic stem cells show that despite a decrease in hematopoietic stem cells, S1P ablation results in an increased production of granulocyte-macrophage progenitors, the precursors to neutrophils. These observations indicate that S1P has a role in the lineage specification of hematopoietic stem cells and/or their progenitors for development of a normal hematopoietic niche. Our study designates a fundamental requirement of S1P for maintaining a balanced regenerative capacity of the bone marrow niche.