Mitogen-activated protein kinases (MAPKs) are expressed in platelets and are activated downstream of physiological agonists. Pharmacological and genetic evidence indicate that MAPKs play a significant role in hemostasis and thrombosis, but it is not well understood how MAPKs are activated upon platelet stimulation. Here, we show that apoptosis signal-regulating kinase 1 (ASK1), a member of the MAP3K family, is expressed in both human and murine platelets. ASK1 is rapidly and robustly activated upon platelet stimulation by physiological agonists. Disruption of ( ) resulted in a marked functional defect in platelets. platelets showed an impaired agonist-induced integrin αβ activation and platelet aggregation. Although there was no difference in Ca rise, platelet granule secretion and thromboxane A (TxA) generation were significantly attenuated in platelets. The defective granule secretion observed in platelets was a consequence of impaired TxA generation. Biochemical studies showed that platelet agonists failed to activate p38 MAPK in platelets. On the contrary, activation of c-Jun-terminal kinases and extracellular signal-regulated kinase 1/2 MAPKs was augmented in platelets. The defect in p38 MAPK results in failed phosphorylation of cPLA in platelets and impaired platelet aggregate formation under flow. The absence of Ask1 renders mice defective in hemostasis as assessed by prolonged tail-bleeding times. Deletion of also reduces thrombosis as assessed by delayed vessel occlusion of carotid artery after FeCl-induced injury and protects against collagen/epinephrine-induced pulmonary thromboembolism. These results suggest that the platelet Ask1 plays an important role in regulation of hemostasis and thrombosis.

Pregnancy-specific glycoproteins (PSGs) are immunoglobulin superfamily members encoded by multigene families in rodents and primates. In human pregnancy, PSGs are secreted by the syncytiotrophoblast, a fetal tissue, and reach a concentration of up to 400 ug/ml in the maternal bloodstream at term. Human and mouse PSGs induce release of anti-inflammatory cytokines such as IL-10 and TGFβ1 from monocytes, macrophages, and other cell types, suggesting an immunoregulatory function. RGD tri-peptide motifs in the majority of human PSGs suggest that they may function like snake venom disintegrins, which bind integrins and inhibit interactions with ligands. We noted that human PSG1 has a KGD, rather than an RGD motif. The presence of a KGD in barbourin, a platelet integrin αIIbβ3 antagonist found in snake venom, suggested that PSG1 may be a selective αIIbβ3 ligand. Here we show that human PSG1 binds αIIbβ3 and inhibits the platelet – fibrinogen interaction. Unexpectedly, however, the KGD is not critical as multiple PSG1 domains independently bind and inhibit αIIbβ3 function. Human PSG9 and mouse Psg23 are also inhibitory suggesting conservation of this function across primate and rodent PSG families. Our results suggest that in species with haemochorial placentation, in which maternal blood is in direct contact with fetal trophoblast, the high expression level of PSGs reflects a requirement to antagonise abundant (3 mg/ml) fibrinogen in the maternal circulation, which may be necessary to prevent platelet aggregation and thrombosis in the prothrombotic maternal environment of pregnancy.

Background: Immune complexes (ICs) bind to and activate platelets via FcγRIIA, causing patients to experience thrombocytopenia, as well as an increased risk of forming occlusive thrombi. Although platelets have been shown to mediate IC-induced pathologies, the mechanisms involved have yet to be fully elucidated. We identified that apoptosis signal-regulating kinase 1 (ASK1) is present in both human and mouse platelets and potentiates many platelet functions. Objectives: Here we set out to study ASK1's role in regulating IC-mediated platelet functions in vitro and IC-induced pathologies using an in vivo mouse model. Methods: Using human platelets treated with an ASK1-specific inhibitor and platelets from FCGR2A/Ask1-/transgenic mice, we examined various platelet functions induced by model ICs in vitro and in vivo. Results: We found that ASK1 was activated in human platelets following cross-linking of FcγRIIA using either anti-hCD9 or IV.3 + goat-anti-mouse. Although genetic deletion or inhibition of ASK1 significantly attenuated anti-CD9-induced platelet aggregation, activation of the canonical FcγRIIA signaling targets Syk and PLCγ2 was unaffected. We further found that anti-mCD9-induced cPla 2 phosphorylation and TxA 2 generation is delayed in Ask1 null transgenic mouse platelets leading to diminished δ-granule secretion. In vivo, absence of Ask1 protected FCGR2A transgenic mice from thrombocytopenia, thrombosis, and systemic shock following injection of anti-mCD9. In whole blood microfluidics, platelet adhesion and thrombus formation on fibrinogen was enhanced by Ask1. Conclusions: These findings suggest that ASK1 inhibition may be a potential target for the treatment of IC-induced shock and other immune-mediated thrombotic disorders.

Apoptosis signal-regulating kinase 1 (ASK1) is a mitogen-activated protein kinase kinase kinase (MAPKKK) that regulates activation of the c-Jun N-terminal kinase (JNK)- and p38-stress response pathways leading to apoptosis in nucleated cells. We have previously shown that ASK1 is expressed in platelets and regulates agonist-induced platelet activation and thrombosis. However, the mechanism by which platelet agonists cause activation of ASK1 is unknown. Here, we show that in platelets agonist-induced activation of p38 is exclusively dependent on ASK1. Both thrombin and collagen were able to activate ASK1/p38. Activation of ASK1/p38 was strongly dependent on thromboxane A2 (TxA2) and ADP. Agonist-induced ASK1 activation is blocked by inhibition of phospholipase C (PLC) β/γ activity or by chelating intracellular Ca2+. Furthermore, treatment of platelets with thapsigargin or Ca2+ ionophore robustly induced ASK1/p38 activation. In addition, calcium and integrin-binding protein 1 (CIB1), a Ca2+-dependent negative regulator of ASK1, associates with ASK1 in resting platelets and is dissociated upon platelet activation by thrombin. Dissociation of CIB1 corresponds with ASK1 binding to tumor necrosis factor (TNF) receptor associated factor 6 (TRAF6) and the autophosphorylation of ASK1 Thr838 within the catalytic domain results in full activation of ASK1. Furthermore, genetic ablation of Cib1 in mice augments agonist-induced Ask1/p38 activation. Together our results suggest that in resting platelets ASK1 is bound to CIB1 at low Ca2+ concentrations. Agonist-induced platelet activation causes an increase in intracellular Ca2+ concentration that leads to the dissociation of CIB1 from ASK1, allowing for proper dimerization through ASK1 N-terminal coiled-coil (NCC) domains.

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