Homozygous loss of mouse tetraspanin CD82 enhances integrin αIIbß3 expression and clot retraction in platelets.

Integrin αIIbß3 is critical for platelet-mediated blood clotting. Tetraspanins are well-established regulators of integrins and genetic loss of tetraspanin CD151 or TSSC6 in mice leads to increased bleeding due to inadequate integrin αIIbß3 outside-in signaling. Conversely, mild but enhanced integrin αIIbß3 activation and hyperaggregation is observed in CD9 and CD63 null mice respectively. CD82 is reportedly expressed in platelets; however its function is unknown. Using genetically engineered CD82 null mice, we investigated the role of the tetraspanin CD82 in platelet activation. Loss of CD82 resulted in reduced bleed times in vivo. CD82 was present on the surface of both human and mouse platelets, and its levels did not change upon platelet activation or degranulation. No differences in platelet activation, degranulation, or aggregation in response to ADP or collagen were detected in CD82 null mice. However, the kinetics of clot retraction was enhanced, which was intrinsic to the CD82-null platelets. Integrin αIIbß3 surface expression was elevated on the platelets from CD82 null mice and they displayed enhanced adhesion and tyrosine kinase signaling on fibrinogen. This is the first report on CD82 function in platelets; which we found intrinsically modulates clot retraction, integrin αIIbß3 expression, cell adhesion, and tyrosine signaling.

Loss of ATE1-mediated arginylation leads to impaired platelet myosin phosphorylation, clot retraction, and in vivo thrombosis formation.

Protein arginylation by arginyl-transfer RNA protein transferase (ATE1) is emerging as a regulator protein function that is reminiscent of phosphorylation. For example, arginylation of ß-actin has been found to regulate lamellipodial formation at the leading edge in fibroblasts. This finding suggests that similar functions of ß-actin in other cell types may also require arginylation. Here, we have tested the hypothesis that ATE1 regulates the cytoskeletal dynamics essential for in vivo platelet adhesion and thrombus formation. To test this hypothesis, we generated conditional knockout mice specifically lacking ATE1 in their platelets and in their megakaryocytes and analyzed the role of arginylation during platelet activation. Surprisingly, rather than finding an impairment of the actin cytoskeleton structure and its rearrangement during platelet activation, we observed that the platelet-specific ATE1 knockout led to enhanced clot retraction and in vivo thrombus formation. This effect might be regulated by myosin II contractility since it was accompanied by enhanced phosphorylation of the myosin regulatory light chain on Ser19, which is an event that activates myosin in vivo. Furthermore, ATE1 and myosin co-immunoprecipitate from platelet lysates. This finding suggests that these proteins directly interact within platelets. These results provide the first evidence that arginylation is involved in phosphorylation-dependent protein regulation, and that arginylation affects myosin function in platelets during clot retraction.

Clot retraction is mediated by factor XIII-dependent fibrin-αIIbß3-myosin axis in platelet sphingomyelin-rich membrane rafts.

Membrane rafts are spatially and functionally heterogenous in the cell membrane. We observed that lysenin-positive sphingomyelin (SM)-rich rafts are identified histochemically in the central region of adhered platelets where fibrin and myosin are colocalized on activation by thrombin. The clot retraction of SM-depleted platelets from SM synthase knockout mouse was delayed significantly, suggesting that platelet SM-rich rafts are involved in clot retraction. We found that fibrin converted by thrombin translocated immediately in platelet detergent-resistant membrane (DRM) rafts but that from Glanzmann's thrombasthenic platelets failed. The fibrinogen γ-chain C-terminal (residues 144-411) fusion protein translocated to platelet DRM rafts on thrombin activation, but its mutant that was replaced by A398A399 at factor XIII crosslinking sites (Q398Q399) was inhibited. Furthermore, fibrin translocation to DRM rafts was impaired in factor XIII A subunit-deficient mouse platelets, which show impaired clot retraction. In the cytoplasm, myosin translocated concomitantly with fibrin translocation into the DRM raft of thrombin-stimulated platelets. Furthermore, the disruption of SM-rich rafts by methyl-ß-cyclodextrin impaired myosin activation and clot retraction. Thus, we propose that clot retraction takes place in SM-rich rafts where a fibrin-αIIbß3-myosin complex is formed as a primary axis to promote platelet contraction.

PDK1 regulates platelet activation and arterial thrombosis.

The effects of phosphoinositide-dependent protein kinase 1 (PDK1), a master kinase in the phosphoinositide 3-kinase/Akt pathway, on platelet activation are unknown. Accordingly, platelet-specific PDK1-deficient mice were characterized to elucidate the platelet-related function(s) of PDK1. We found that PDK1 deficiency caused mild thrombocytopenia. The aggregation of PDK1(-/-) platelets was diminished in response to low levels of thrombin, U46619, and adenosine 5'-diphosphate. Further results demonstrated that PDK1 regulates thrombin-induced platelet activation by affecting αIIbß3-mediated outside-in signaling. This result provided an explanation for the diminished spreading of PDK1(-/-) platelets on immobilized fibrinogen (Fg) and the decreased rate of clot retraction in platelet-rich plasma (PRP) containing PDK1(-/-) platelets. PDK1 deficiency diminished agonist-induced Akt Ser473 phosphorylation and thoroughly abolished Akt Thr308 and Gsk3ß Ser9 phosphorylation in response to agonist treatment and platelet spreading, respectively. A Gsk3ß inhibitor fully restored the aggregation of PDK1(-/-) platelets in response to low levels of thrombin, normal spreading of PDK1(-/-) platelets on Fg, and normal clot retraction in PRP containing PDK1(-/-) platelets. Those results indicated that Gsk3ß is one of the major downstream effectors of PDK1 in thrombin-induced platelet activation and αIIbß3-mediated outside-in signaling. In addition, in vivo data demonstrated that PDK1 is an important regulator in arterial thrombosis formation.

JAM-A protects from thrombosis by suppressing integrin αIIbß3-dependent outside-in signaling in platelets.

Mounting evidence suggests that agonist-initiated signaling in platelets is closely regulated to avoid excessive responses to injury. A variety of physiologic agonists induce a cascade of signaling events termed as inside-out signaling that culminate in exposure of high-affinity binding sites on integrin α(IIb)ß(3). Once platelet activation has occurred, integrin α(IIb)ß(3) stabilizes thrombus formation by providing agonist-independent "outside-in" signals mediated in part by contractile signaling. Junctional adhesion molecule A (JAM-A), a member of the cortical thymocyte marker of the Xenopus (CTX) family, was initially identified as a receptor for a platelet stimulatory mAb. Here we show that JAM-A in resting platelets functions as an endogenous inhibitor of platelet function. Genetic ablation of Jam-A in mice enhances thrombotic function of platelets in vivo. The absence of Jam-A results in increase in platelet aggregation ex vivo. This gain of function is not because of enhanced inside-out signaling because granular secretion, Thromboxane A2 (TxA2) generation, as well as fibrinogen receptor activation, are normal in the absence of Jam-A. Interestingly, integrin outside-in signaling such as platelet spreading and clot retraction is augmented in Jam-A-deficient platelets. We conclude that JAM-A normally limits platelet accumulation by inhibiting integrin outside-in signaling thus preventing premature platelet activation.

Signal-dependent protein synthesis by activated platelets: new pathways to altered phenotype and function.

New biologic activities of platelets continue to be discovered, indicating that concepts of platelet function in hemostasis, thrombosis, and inflammation require reconsideration as new paradigms evolve. Studies done over 3 decades ago demonstrated that mature circulating platelets have protein synthetic capacity, but it was thought to be low level and inconsequential. In contrast, recent discoveries demonstrate that platelets synthesize protein products with important biologic activities in a rapid and sustained fashion in response to cellular activation. This process, termed signal-dependent translation, uses a constitutive transcriptome and specialized pathways, and can alter platelet phenotype and functions in a fashion that can have clinical relevance. Signal-dependent translation and consequent protein synthesis are examples of a diverse group of posttranscriptural mechanisms in activated platelets that are now being revealed.