Tetraspanin Tspan9 regulates platelet collagen receptor GPVI lateral diffusion and activation

The tetraspanins are a superfamily of four-transmembrane proteins, which regulate the trafficking, lateral diffusion and clustering of the transmembrane proteins with which they interact. We have previously shown that tetraspanin Tspan9 is expressed on platelets. Here we have characterised gene-trap mice lacking Tspan9. The mice were viable with normal platelet numbers and size. Tspan9-deficient platelets were specifically defective in aggregation and secretion induced by the platelet collagen receptor GPVI, despite normal surface GPVI expression levels. A GPVI activation defect was suggested by partially impaired GPVI-induced protein tyrosine phosphorylation. In mechanistic experiments, Tspan9 and GPVI co-immunoprecipitated and co-localised, but super-resolution imaging revealed no defects in collagen-induced GPVI clustering on Tspan9-deficient platelets. However, single particle tracking using total internal reflection fluorescence microscopy showed that GPVI lateral diffusion was reduced by approximately 50% in the absence of Tspan9. Therefore, Tspan9 plays a fine-tuning role in platelet activation by regulating GPVI membrane dynamics

The Adaptor Protein Swiprosin-1/EFhd2 Is Dispensable for Platelet Function in Mice

Background Platelets are anuclear cell fragments derived from bone marrow megakaryocytes that safeguard vascular integrity, but may also cause pathological vessel occlusion. Reorganizations of the platelet cytoskeleton and agonist-induced intracellular Ca2+-mobilization are crucial for platelet hemostatic function. EF-hand domain containing 2 (EFhd2, Swiprosin-1) is a Ca2+-binding cytoskeletal adaptor protein involved in actin remodeling in different cell types, but its function in platelets is unknown. Objective Based on the described functions of EFhd2 in immune cells, we tested the hypothesis that EFhd2 is a crucial adaptor protein for platelet function acting as a regulator of Ca2+-mobilization and cytoskeletal rearrangements. Methods and Results We generated EFhd2-deficient mice and analyzed their platelets in vitro and in vivo. Efhd2-/- mice displayed normal platelet count and size, exhibited an unaltered in vivo life span and showed normal Ca2+-mobilization and activation/aggregation responses to classic agonists. Interestingly, upon stimulation of the immunoreceptor tyrosine-based activation motif-coupled receptor glycoprotein (GP) VI, Efhd2-/- platelets showed a slightly increased coagulant activity. Furthermore, absence of EFhd2 had no significant impact on integrin-mediated clot retraction, actomyosin rearrangements and spreading of activated platelets on fibrinogen. In vivo EFhd2-deficiency resulted in unaltered hemostatic function and unaffected arterial thrombus formation. Conclusion These results show that EFhd2 is not essential for platelet function in mice indicating that other cytoskeletal adaptors may functionally compensate its loss

Mice lacking the SLAM family member CD84 display unaltered platelet function in hemostasis and thrombosis

Background Platelets are anuclear cell fragments derived from bone marrow megakaryocytes that safeguard vascular integrity by forming thrombi at sites of vascular injury. Although the early events of thrombus formation—platelet adhesion and aggregation—have been intensively studied, less is known about the mechanisms and receptors that stabilize platelet-platelet interactions once a thrombus has formed. One receptor that has been implicated in this process is the signaling lymphocyte activation molecule (SLAM) family member CD84, which can undergo homophilic interactions and becomes phosphorylated upon platelet aggregation. Objective The role of CD84 in platelet physiology and thrombus formation was investigated in CD84-deficient mice. Methods and Results We generated CD84-deficient mice and analyzed their platelets in vitro and in vivo. $Cd84^{−/−}$ platelets exhibited normal activation and aggregation responses to classical platelet agonists. Furthermore, CD84 deficiency did not affect integrin-mediated clot retraction and spreading of activated platelets on fibrinogen. Notably, also the formation of stable three-dimensional thrombi on collagen-coated surfaces under flow ex vivo was unaltered in the blood of $Cd84^{−/−}$ mice. In vivo, $Cd84^{−/−}$ mice exhibited unaltered hemostatic function and arterial thrombus formation. Conclusion These results show that CD84 is dispensable for thrombus formation and stabilization, indicating that its deficiency may be functionally compensated by other receptors or that it may be important for platelet functions different from platelet-platelet interactions

The adaptor protein Swiprosin-1/EFhd2 is dispensable for platelet function in mice.

BACKGROUND: Platelets are anuclear cell fragments derived from bone marrow megakaryocytes that safeguard vascular integrity, but may also cause pathological vessel occlusion. Reorganizations of the platelet cytoskeleton and agonist-induced intracellular Ca2+-mobilization are crucial for platelet hemostatic function. EF-hand domain containing 2 (EFhd2, Swiprosin-1) is a Ca2+-binding cytoskeletal adaptor protein involved in actin remodeling in different cell types, but its function in platelets is unknown. OBJECTIVE: Based on the described functions of EFhd2 in immune cells, we tested the hypothesis that EFhd2 is a crucial adaptor protein for platelet function acting as a regulator of Ca2+-mobilization and cytoskeletal rearrangements. METHODS AND RESULTS: We generated EFhd2-deficient mice and analyzed their platelets in vitro and in vivo. Efhd2-/- mice displayed normal platelet count and size, exhibited an unaltered in vivo life span and showed normal Ca2+-mobilization and activation/aggregation responses to classic agonists. Interestingly, upon stimulation of the immunoreceptor tyrosine-based activation motif-coupled receptor glycoprotein (GP) VI, Efhd2-/- platelets showed a slightly increased coagulant activity. Furthermore, absence of EFhd2 had no significant impact on integrin-mediated clot retraction, actomyosin rearrangements and spreading of activated platelets on fibrinogen. In vivo EFhd2-deficiency resulted in unaltered hemostatic function and unaffected arterial thrombus formation. CONCLUSION: These results show that EFhd2 is not essential for platelet function in mice indicating that other cytoskeletal adaptors may functionally compensate its loss

Normal expression levels of specific proteins in EFhd2-deficient platelets.

<p>(A) Analysis of EFhd1, Rac1, mDia1, actin and tubulin expression in <i>Efhd2^+/+</i> and <i>Efhd2^-/-</i> platelets by Western blot. (B) Analysis of phospho-cofilin, phospho-Syk, and phospho-PAK1/2 expression in <i>Efhd2^+/+</i> and <i>Efhd2^-/-</i> platelets by Western blot. Staining of the respective non-phosphorylated proteins served as loading controls.</p

Unaltered aggregation response of <i>Efhd2^-/-</i> platelets.

<p>Washed platelets from <i>Efhd2^+/+</i> (black line) and <i>Efhd2^-/-</i> (gray line) mice were activated with the indicated agonist concentrations and light transmission was recorded on a Fibrintimer 4-channel aggregometer. ADP measurements were performed in prp. Representative aggregation traces of at least 3 individual experiments are depicted.</p

Normal integrin outside-in signaling in <i>Efhd2^-/-</i> platelets.

<p>(A-C) Washed platelets of <i>Efhd2^+/+</i> and <i>Efhd2^-/-</i> mice were allowed to spread on fibrinogen (100 µg/mL) for 30 min after stimulation with 0.01 U/mL thrombin. (A) Statistical evaluation of the percentage of spread platelets at different spreading stages and (B) representative differential interference contrast (DIC) images of 2 individual experiments. 1: roundish, 2: only filopodia, 3: filopodia and lamellipodia, 4: fully spread. (C) Visualization of filamentous actin (red) in spread (30 min) <i>Efhd2^+/+</i> and <i>Efhd2^-/-</i> platelets by cofocal microscopy. (D) Clot retraction of prp upon activation with 4 U/mL thrombin in the presence of 20 mmol/L CaCl₂ at the indicated time points (n = 6).</p

Normal Ca²⁺-mobilization but slightly increased procoagulant activity in EFhd2-deficient platelets upon stimulation.

<p>(A) Maximal increase of cytosolic Ca²⁺ concentration ([Ca²⁺]_i) of <i>Efhd2^+/+</i> (black bars) and <i>Efhd2^-/-</i> platelets (gray bars) after activation with the indicated agonists (thrombin 0.1 U/ml, CRP 4 µg/ml). (B) Flow cytometric analysis of phosphatidylserine (PS) exposure in response to the indicated agonists in <i>Efhd2^+/+</i> and <i>Efhd2^-/-</i> platelets. Washed platelets were stained with Annexin-V-DyLight-488 in the presence of Tyrodes-HEPES buffer containing 3 mmol/L Ca²⁺. The values represent the mean fluorescence intensity (MFI) ± SD for 5 mice per group in 3 independent experiments. Thr: thrombin, CRP: collagen related peptide, CVX: convulxin, RC: rhodocytin, n.s.: not significant, *<i>p</i><0.05.</p