The tetraspanin family member CD151 forms complexes with integrins and regulates cell adhesion and migration. While CD151 is highly expressed in megakaryocytes and to a lesser extent in platelets, its physiologic role in platelets is unclear. In this study, we investigate the physical and functional importance of CD151 in murine platelets. Immunoprecipitation/ Western blot studies reveal a constitutive physical association of CD151 with integrin ␣ IIb ␤ 3 complex under strong detergent conditions. Using CD151-deficient mice, we show that the platelets have impaired "outside-in" integrin ␣ IIb ␤ 3 signaling with defective platelet aggregation responses to protease-activated receptor 4 (PAR-4) agonist peptide, collagen, and adenosine diphosphate (ADP); impaired platelet spreading on fibrinogen; and delayed kinetics of clot retraction in vitro. This functional integrin ␣ IIb ␤ 3 defect could not be attributed to altered expression of integrin ␣ IIb ␤ 3 . CD151 ؊/؊ platelets displayed normal platelet alpha granule secretion, dense granule secretion, and static platelet adhesion. In addition, CD151 ؊/؊ platelets displayed normal "in- IntroductionIntegrin ␣ IIb ␤ 3 maintains an inactive conformation on the surface of resting platelets until it is converted to its high-affinity state by agonist induced "inside-out" signaling via G protein-coupled or tyrosine kinase-linked pathways. Once activated, integrin ␣ IIb ␤ 3 can bind its soluble ligands, fibrinogen, and von Willebrand factor (VWF). Subsequently, "outside-in" integrin ␣ IIb ␤ 3 signaling events induce integrin clustering that leads to cytoskeletal reorganization and postoccupancy events including clot retraction, platelet spreading, and platelet aggregation. 1 The importance of integrin ␣ IIb ␤ 3 has been documented in Glanzmann thrombasthenia (GT) patients, who have bleeding complications despite a normal number of platelets. GT patients have dysfunctional platelets, which display quantitative or qualitative defects in integrin ␣ IIb or ␤ 3 . This gives rise to an integrin ␣ IIb ␤ 3 -mediated platelet defect, where the platelets fail to aggregate, bind fibrinogen, or retract fibrin clots. 2,3 This phenotype has been recapitulated in a complete ␤ 3 knock-out (KO) mouse model, which shows the cardinal features of Glanzmann thrombasthenia. 4 The concept that transmembrane receptors including integrins can form multimolecular complexes with tetraspanin-4 superfamily members (TM4SF) was first demonstrated by the ability of integrin ␣ IIb ␤ 3 to associate with a tetraspanin superfamily member, CD9, in stimulated platelets. 5 Subsequently, biochemical associations between tetraspanin superfamily members and other transmembrane receptors have been reported and clarified based upon associations in the presence of detergents with differing stringencies. 6 The tetraspanin superfamily member CD151 (also known as platelet endothelial tetraspan antigen-3/PETA-3) is expressed on the surface of platelets, megakaryocytes, endothelial cells, activated T lymphocytes, Schwann cel...

Carcinoembryonic antigen cell adhesion molecule-1 (CEACAM1) is a surface glycoprotein expressed on various blood cells, epithelial cells, and vascular cells. CEACAM1 possesses adhesive and signaling properties mediated by its intrinsic immunoreceptor tyrosine-based inhibitory motifs that recruit SHP-1 protein-tyrosine phosphatase. In this study, we demonstrate that CEACAM1 is expressed on the surface and in intracellular pools of platelets. In addition, CEACAM1 serves to negatively regulate signaling of platelets by collagen through the glycoprotein VI (GPVI)/Fc receptor (FcR)-␥-chain. ceacam1 ؊/؊ platelets displayed enhanced type I collagen and GPVI-selective ligand, collagen-related peptide (CRP), CRPmediated platelet aggregation, enhanced platelet adhesion on type I collagen, and elevated CRP-mediated alpha and dense granule secretion. Platelets derived from ceacam1 ؊/؊ mice form larger thrombi when perfused over a collagen matrix under arterial flow compared with wild-type mice. Furthermore, using intravital microscopy to ferric chloride-injured mesenteric arterioles, we show that thrombi formed in vivo in ceacam1 ؊/؊ mice were larger and were more stable than those in wild-type mice. GPVI depletion using monoclonal antibody JAQ1 treatment of ceacam1 ؊/؊ mice showed a reversal in the more stable thrombus growth phenotype. ceacam1 ؊/؊ mice were more susceptible to type I collagen-induced pulmonary thromboembolism than wild-type mice. Thus, CEACAM1 acts as a negative regulator of platelet-collagen interactions and of thrombus growth involving the collagen GPVI receptor in vitro and in vivo. (Blood.

We investigated the role of the hematopoietic-specific tetraspanin superfamily member, TSSC6, in platelet function using wild-type mice and TSSC6-deficient mice. TSSC6 is expressed on the surface of murine platelets and is up-regulated by thrombin stimulation, indicating an intracellular pool of TSSC6. Immunoprecipitation/Western blot studies reveal a constitutive physical association of TSSC6 with the integrin ␣ IIb ␤ 3 complex under strong detergent conditions. In vivo evaluation of hemostasis by tail bleeding revealed increased bleeding time, volume of blood lost, and evidence of tail rebleeds in TSSC6 null mice, indicating unstable hemostasis. Using ex vivo techniques, we showed that TSSC6-deficient platelets exhibited impaired kinetics of clot retraction, platelet aggregation at lower doses of PAR-4, and collagen and platelet spreading on fibrinogen in the presence of normal integrin ␣ IIb ␤ 3 expression. TSSC6-deficient platelets showed normal alpha granule secretion, normal "insideout" integrin ␣ IIb ␤ 3 signaling (fluorescein isothiocyanate [FITC]-fibrinogen and JON/A binding), and normal platelet adhesion on fibrinogen. Furthermore, we show that absence of platelet TSSC6 affects the secondary stability of arterial thrombi in vivo upon vascular injury. These data demonstrate that TSSC6 appears to regulate integrin ␣ IIb ␤ 3 "outside-in" signaling events in platelets and is necessary for stability of arterial thrombi in vivo. (Blood.