The tetraspanin membrane protein CD151 is a broadly expressed molecule noted for its strong molecular associations with integrins, especially ␣3␤1, ␣6␤1, ␣7␤1, and ␣6␤4. In vitro functional studies have pointed to a role for CD151 in cell-cell adhesion, cell migration, platelet aggregation, and angiogenesis. It has also been implicated in epithelial tumor progression and metastasis. Here we describe the generation and initial characterization of CD151-null mice. The mice are viable, healthy, and fertile and show normal Mendelian inheritance. They have essentially normal blood and bone marrow cell counts and grossly normal tissue morphology, including hemidesmosomes in skin, and expression of ␣3 and ␣6 integrins. However, the CD151-null mice do show phenotypes in several different tissue types. An absence of CD151 leads to a minor abnormality in hemostasis, with CD151-null mice showing longer average bleeding times, greater average blood loss, and an increased incidence of rebleeding occurrences. CD151-null keratinocytes migrate poorly in skin explant cultures. Finally, CD151-null T lymphocytes are hyperproliferative in response to in vitro mitogenic stimulation.

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...

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.