Expression and Function of the Collagen Receptor GPVI during Megakaryocyte Maturation

Abstract

In this report, the expression and function of the platelet collagen receptor glycoprotein VI (GPVI) were studied in human megakaryocytes during differentiation and maturation of mobilized blood and cord blood derived CD34 ؉ cells. By flow cytometry, using an anti-GPVI monoclonal antibody or convulxin, a GPVI-specific ligand, GPVI was detected only on CD41؉ cells including some CD41 ؉ /CD34؉ cells, suggesting expression at a stage of differentiation similar to CD41. These results were confirmed at the mRNA level using reverse transcriptionpolymerase chain reaction. GPVI expression was low during megakaryocytic differentiation but increased in the more mature megakaryocytes (CD41 high ). As in platelets, megakaryocyte GPVI associates with the Fc receptor ␥ chain (FcR␥). The FcR ␥ chain was detected at the RNA and protein level at all stages of megakaryocyte maturation preceding the expression of GPVI. The other collagen receptor, ␣ 2 ␤ 1 integrin (CD49b/CD29), had a pattern of expression similar to GPVI. Megakaryocytic GPVI was recognized as a 55-kDa protein by immunoblotting and ligand blotting, and thus it presented a slightly lower apparent molecular mass than platelet GPVI (58 kDa). Megakaryocytes began to adhere to immobilized convulxin via GPVI after only 8 -10 days of culture, at a time when megakaryocytes were maturing. At this stage of maturation, they also adhered to immobilized collagen by ␣ 2 ␤ 1 integrin-dependent and -independent mechanisms. Convulxin induced a very similar pattern of protein tyrosine phosphorylation in megakaryocytes and platelets including Syk, FcR␥, and PLC ␥2 . Our results showed that GPVI is expressed early during megakaryocytic differentiation but functionally allows megakaryocyte adherence to collagen only at late stages of differentiation when its expression increases.At sites of vascular injury, platelets adhere and are activated by contact with collagen fibers exposed with the subendothelium matrix leading to thrombus formation. Several collagen receptors are present on platelets: integrin ␣ 2 ␤ 1 (CD49b/ CD29), GPIV 1 (CD36), GPVI, p65, and a recently cloned type III collagen receptor (1, 2). The direct interaction between collagen and integrin ␣ 2 ␤ 1 adheres platelets at the collagen surface, but platelet activation and thrombus formation and attachment are subsequently mainly supported by GPVI. In addition to collagen, two GPVI-specific ligands have been described: collagen-related peptides (3) and convulxin, a snake venom protein (4). GPVI has recently been characterized as a new member of the immunoglobulin superfamily of cell-associated receptors, in which expression is restricted to the hematopoietic lineage (5-7). It is coexpressed as a noncovalent complex with the common immunoglobulin receptor ␥ (FcR␥) chain, which acts as the signaling subunit of the complex (8, 9) As a consequence, signaling pathways coupled to GPVI dimerization are identical to those coupled to other immune receptors; tyrosine phosphorylation of the FcR ␥ chain on its immunoreceptor tyrosine-...

Myosin-II inhibition and soft 2D matrix maximize multinucleation and cellular projections typical of platelet-producing megakaryocytes

“…The bone surface is high in collagen-I (collagen hi ) and stiff, with an estimated elasticity E ECM for osteoid of ∼34 kPa (2), whereas the marrow space is collagen lo and very soft, approximated here with E ECM = 0.3 kPa (21). MKs express two collagen receptors: GPVI and integrin-α2β1 (22,23). Previous results indicate that collagen suppresses maturation of MKs in vitro (24), and so we hypothesized that a low collagen, compliant ECM favors MK polyploidization.…”

Discoidin Domain Receptor 1 Protein Is a Novel Modulator of Megakaryocyte-Collagen Interactions

“…In this regard, previous works pointed out the role of collagen receptors ␣2␤1 and GPVI in regulating MK function and platelet production on type I collagen; however, the expression and function of other matrix receptors on MKs are still unknown (11)(12)(13)(14). Discoidin domain receptors (DDR1 and DDR2) are tyrosine-kinase collagen receptors that are stimulated by fibril- lar and basement membrane collagens and mediate cell adhesion and migration in different tissues.…”

Proplatelet formation is selectively inhibited by collagen type I through Syk-independent GPVI signaling

“…Although these receptors are expressed by megakaryocytes from relatively early stages in their maturation (Lagrue-Lak-Hal et al, 2001), their function on PPF is ill-defined. Moreover, other collagen receptors, such as leukocyte-associated immunoglobulin-like receptor-1 (LAIR1) (Steevels et al, 2010) and discoidin domain receptor 1 (DDR1) (Abbonante et al, 2013) have been proposed as inhibitory collagen receptors on early megakaryocytes, whose expression decreases during maturation and thus are absent from circulating platelets.…”