A CD9, alphaIIbbeta3, integrin-associated protein, and GPIb/V/IX complex on the surface of human platelets is influenced by alphaIIbbeta3 conformational states.
A noncovalently associated complex comprising of CD9, the fibrinogen (Fg) receptor alphaIIbbeta3, integrin-associated protein (IAP), and glycoprotein (GP) Ib/V/IX complex was isolated from Chaps-solubilized human platelets. The CD9 complex was immunoprecipitated by mAbs specific for CD9 (mAb7), IAP (BRIC126), GPIb (SZ1), GPIX (GR-P), beta3 (AP3) and alphaIIb (C3). Additionally, the association between CD9 and alphaIIbbeta3 was demonstrated by ELISA. In this system, CD9 did not bind to vitronectin receptor (alphavbeta3) suggesting that CD9/alphaIIbbeta3 association was alphaIIb-subunit or alphaIIbbeta3-complex dependent. D3, an alphaIIbbeta3-activating mAb that is also an anti-LIBS (ligand-induced binding site), immunoprecipitated primarily alphaIIbbeta3 with GPIb and IAP. CD9 was not detected in D3 immunoprecipitates. D3 binding induced platelet aggregation via direct alphaIIbbeta3 activation and was upregulated by the alphaIIbbeta3 antagonist eptifibatide. In contrast, AP3 and C3 exhibited neither effect. In addition, D3 also inhibited whole blood clot retraction, in contrast to AP3 and C3, suggesting that conformational constraints on alphaIIbbeta3 by D3 binding not only influenced the CD9 complex but also affected alphaIIbbeta3 post receptor occupancy events. The CD9 complex was immunoprecipitated in the presence of eptifibatide, demonstrating that alphaIIbbeta3 receptor occupancy was not sufficient to cause complex dissociation. CD9 complex isolation was also independent of platelet activation, although a twofold increase in the quantity of CD9 complex was seen after platelet activation by alpha-thrombin in the presence of CaCl2 compared with that present in EDTA. Stirred platelets showed fibrinogen-mediated aggregation by alpha-thrombin in the presence of CaCl2 but not with EDTA, suggesting that fibrinogen crosslinking of CD9 complexes via alphaIIbbeta3 could be partially responsible for this increase. These findings imply that the platelet CD9 complex is independent of platelet activation although it is dependent upon the conformation state of alphaIIbbeta3.
Longhurst
C M
CM
Department of Medicine, University of Tennessee, Memphis, TN, USA.
White
M M
MM
Wilkinson
D A
DA
Jennings
L K
LK
D000911:Antibodies, Monoclonal; D015703:Antigens, CD / Q000737:chemistry / Q000378:metabolism*; D001792:Blood Platelets / Q000276:immunology* / Q000378:metabolism*; D002462:Cell Membrane / Q000276:immunology / Q000378:metabolism; D000077542:Eptifibatide; D006801:Humans; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D008562:Membrane Glycoproteins; D010455:Peptides / Q000494:pharmacology; D015539:Platelet Activation; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D060245:Tetraspanin 29
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; C554869:CD9 protein, human; D046911:Macromolecular Substances; D008562:Membrane Glycoproteins; D010455:Peptides; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D019038:Platelet Glycoprotein GPIb-IX Complex; D060245:Tetraspanin 29; D000077542:Eptifibatide