Engagement of platelet membrane glycoprotein (GP) Ib-IX-V by von Willebrand factor triggers Ca11-dependent activation of aIIbb3, resulting in (patho)physiological thrombus formation. It is demonstrated here that the cytoplasmic domain of GPIb-IX-V associates with cytosolic calmodulin. First, an anti-GPIba antibody coimmunoprecipitated GPIb-IX and calmodulin from platelet lysates. Following plat...

Glycoprotein Ib (GPIb), the receptor for von Willebrand factor, is a two-chain member constituent of the platelet/megakaryocytic lineage. Studies on its expression have been hampered by the difficulties in obtaining purified megakaryocytes in a sufficient number. We report a suspension liquid culture procedure that allowed isolation of more than 1 x 10(6) megakaryocytes with a purity ranging fr...

We have recently shown that the platelet integrin alpha(IIb)beta(3) is activated by von Willebrand factor (vWF) binding to its platelet receptor, glycoprotein Ib-IX (GPIb-IX), via the protein kinase G (PKG) signaling pathway. Here we show that GPIb-IX-mediated activation of integrin alpha(IIb)beta(3) is inhibited by dominant negative mutants of Raf-1 and MEK1 in a reconstituted integrin activat...

We have recently shown that several components from the platelet plasma membrane were also present at different rates in the alpha-granule membrane. This is the case for the glycoprotein (GP) IIb-IIIa (CD41), CD36, CD9, PECAM1, and Rap1b, while the GPIB-IX-V complex was considered to escape the rule. In this investigation, we studied the subcellular localization of GPIb, GPIX, and GPV in the re...

At sites of vascular injury, von Willebrand factor (VWF) mediates platelet adhesion through binding to platelet glycoprotein Ib (GPIb). Previous studies identified clusters of charged residues within VWF domain A1 that were involved in binding GPIb or botrocetin. The contribution of 28 specific residues within these clusters was analyzed by mutating single amino acids to alanine. Binding to a p...

BACKGROUND We previously reported that an enzyme-linked immunospot (ELISPOT) assay for detecting anti-GPIIb/IIIa antibody-secreting B cells is a sensitive method for identifying patients with immune thrombocytopenia (ITP). Here we assessed the clinical significance of measuring circulating B cells producing antibodies to GPIb, another major platelet autoantigen. METHODS Anti-GPIb and anti-GPI...

Domains 3 and 5 of high-molecular-weight kininogen (HK) have been shown to bind to platelets in a zinc-dependent reaction. However, the platelet-binding proteins responsible for this interaction have not been identified. We have focused on the platelet-binding site for the heavy chain (domain 3), which we approached using a domain 3-derived peptide ligand and isolated binding proteins by affini...

Platelet-specific antigen Sib(a) has been highly implicated in the pathogenesis of refractoriness to human leukocyte antigen (HLA)-matched platelet transfusions in Japan. We provide evidence that the Sib(a) antigen is located on the glycoprotein (GP) Ib alpha and has a close association with the molecular weight (mol wt) polymorphism of GPIb. In modified antigen-capture ELISA (MACE), anti-Sib(a...

Glycoproteins GPVI and GPIb-IX-V stimulate robust tyrosine phosphorylation of Syk and PLCg2 (phospholipase Cg2) in washed platelets, but only the former stimulates pronounced activation of phospholipase. Using phospho-specific antibodies, we demonstrate that GPVI, but not GPIb-IX-V, stimulates significant tyrosine phosphorylation of Syk at the autophosphorylation site pY525/526, a marker of Syk...