Exposure of platelet fibrinogen receptors by a monoclonal antibody to CD9 antigen

Abstract

We found that a monoclonal antibody to CD9 antigen, PMA2, induces fibrinogen binding to platelets and examined the mechanism for this. That PMA2 recognized the CD9 antigen was confirmed by its immunoblot- reactivity with a 24,000-dalton protein, reactivity with platelets and common acute lymphoblastic leukemia (ALL) cells, and competitive binding with the ALB6 antibody known as the CD9 antibody. At saturation, PMA2 bound to approximately 46,000 sites per platelet. The binding of 125I-fibrinogen to platelets occurred in a PMA2 concentration-dependent manner and was blocked by EDTA or an anti- glycoprotein (GP)IIb-IIIa monoclonal antibody. PMA2-stimulated platelets caused ATP secretion and thromboxane B2 synthesis under non- stirred conditions. The role of secreted ADP and thromboxane in fibrinogen-binding and subsequent platelet aggregation was studied using creatine phosphate/creatine phosphokinase (CP/CPK) and aspirin. CP/CPK or aspirin alone reduced fibrinogen binding to 20% to 30%; however, this binding was sufficient to support full platelet aggregation. Combined treatment with CP/CPK and aspirin abolished fibrinogen binding and aggregation. These results demonstrate that the binding of IgG molecules to the CD9 antigen exposes fibrinogen receptors through both secreted ADP and thromboxane and that either one of both can expose the receptors to an extent sufficient to aggregate platelets.

Tetraspanins as regulators of the tumour microenvironment: implications for metastasis and therapeutic strategies

“…Most tissues including haematopoietic, osteoclast precursor cells and epithelial cells, except red blood cells and pancreas Sincock et al, 1997;Nakamura et al, 2001) • Regulation of cell adhesion and motility in normal and cancerous cells (Ikeyama et al, 1993;Kotha et al, 2008;Powner et al, 2011). • Platelet activation and aggregation (Boucheix et al, 1983;Hato et al, 1988;Slupsky et al, 1989) • Regulates paranodal junction formation (Ishibashi et al, 2004). • Sperm-egg fusion (Higginbottom et al, 2003;Rubinstein et al, 2006) • Regulatory role on the cell-surface expression and function of MT1-MMP (Lafleur et al, 2009) and CD9P-1 (Chambrion and Le Naour, 2010;Schroder et al, 2013) • Expresses on CD4+ T-cells and involved in T-cell activation (Tai et al, 1996;Kobayashi et al, 2004) CD63 MLA1 TSPAN30 Ocular melanomaassociated antigen…”

CD9 of mouse brain is implicated in neurite outgrowth and cell migration in vitro and is associated with the α6/β1 integrin and the neural adhesion molecule L1

“…In search of novel cell surface constituents that may promote neurite outgrowth and migration of neuronal cell bodies, we have generated monoclonal antibodies and tested them for their ability to inhibit neurite outgrowth and migration of neuronal cell bodies on astrocytes which are the functional partners of neurons in vivo. We have characterized a cell surface constituent involved in outgrowth of neurites and astrocytic processes and migration of neuronal cell bodies and identified it as the mouse homologue of CD9, a cell surface marker of hemopoietic cells that has been implicated in aggregation of platelets (Hato et al, 1988;Jennings et al, 1990;Griffith et al, 1991;Kroll et al, 1992). CD9 is a signal transducer in that it activates Ca2+ entry into neural cells and appears to cooperate with the 016/@l integrin to promote laminindependent neurite outgrowth, possibly in a cooperative manner.…”

analysis of CD9, CD32 and p67 signalling: use of degranulated platelets indicates direct involvement of CD9 and p67 in integrin activation

“…Since FcgRII expression is increased following platelet activation (from a range of 1000-2000 receptors/platelet on resting platelets to values >2000 receptors/platelet on activated cells (McCrae et al, 1990)), any change in antigen density as a result of platelet degranulation (Berndt et al, 1993) is very unlikely to account for the observed weak response of degranulated platelets to FcgRII engagement as compared with their much stronger and apparently equal responses to both SYB-1 and LeoA1. Resting platelets express 1200 and 35 000-65 000 molecules/cell of PTA-1 (Scott et al, 1989) and CD9 (Hato et al, 1988) respectively. It is possible that the degranulation process altered expression of these antigens, but the observed strong response to PTA and SYB-1 could not have been the result of Fc receptor signalling given the limited number of these receptors on degranulated cells, and given the weak signal generated by FcgRII crosslinking.…”

“…Since the response of washed (non-degranulated) platelets to stimulation with CD9 and crosslinked IV.3 mAbs involves the induction of an intracellular Ca 2+ rise (Anderson & Anderson, 1990;Hato et al, 1988;Ozaki et al, 1991), we compared the pattern of Ca 2+ fluxes specifically induced by each of these mAbs in degranulated platelets. Fig 2 shows that SYB-1, LeoA1, and crosslinked IV.3 were indeed able to induce cytosolic Ca 2+ fluxes within degranulated platelets.…”