Tomoaki Doi

HSP27 phosphorylation is correlated with ADP-induced platelet granule secretion

Adenosine diphosphate (ADP) plays a crucial role in hemostasis and thrombosis by activating platelets. ADP has been reported to induce heat-shock protein (HSP) 27 phosphorylation in human platelets. However, the exact role of HSP27 phosphorylation in human platelets has not yet been clarified. In the present study, we investigated the mechanisms and the roles of ADP-induced HSP27 phosphorylation in human platelets. We showed for the first time that both of decreased phosphorylation levels of HSP27 by PD98059, a MEK1/2 inhibitor and SB203580, a p38 MAPK inhibitor were correlated with the suppressed levels of platelet granule secretion but not with platelet aggregation. Furthermore, the inhibition of either the p44/p42 MAPK or p38 MAPK pathways had no effect on ADP-induced platelet aggregation. These results strongly suggest that the ADP-induced phosphorylation of HSP27 via p44/p42 MAPK and/or p38 MAPK is therefore sufficient for platelet granule secretion but not for platelet aggregation in humans.

Thromboxane A2 promotes soluble CD40 ligand release from human platelets

OBJECTIVE The plasma level of soluble CD40 ligand (sCD40L), which induces pro-inflammatory and pro-atherogenic responses, is known to be elevated in atherosclerotic patients. In this study, we investigated the mechanism of sCD40L release from human platelets, focusing on the involvement of thromboxane (TX) A(2). METHODS We measured sCD40L release and TXA(2) production induced by ristocetin, an activator of GPIb/IX/V, from human platelets in vitro. Moreover, plasma sCD40L and TXA(2) levels in the 10 patients with severe carotid artery stenosis who were not taking any anti-platelet medicines were measured and compared with those obtained from non-atherosclerotic controls. RESULTS Ristocetin significantly promoted sCD40L release and TXA(2) generation from platelets in vitro. Aspirin and SC-560, a cyclooxygenase-1 inhibitor, suppressed the ristocetin-induced sCD40L release from platelets in parallel with TXA(2) production. Ozagrel, a TXA(2) synthase inhibitor and PTXA(2), a thromboxane receptor (TP) antagonist also suppressed sCD40L release. U46619, a TP agonist, reversed the suppressive effect of aspirin on sCD40L release. In vivo, plasma levels of sCD40L and TXA(2) in the patients were significantly higher than those in controls. Elevated plasma levels of TXA(2) and sCD40L in the patients were markedly diminished after 7 days of 100mg aspirin administration. CONCLUSION These results strongly suggest that GPIb/IX/V activation induces sCD40L release via TXA(2) from human platelets, and that sCD40L release via TXA(2) generation from platelets in atherosclerotic patients are up-regulated.

αB‐crystallin extracellularly suppresses ADP‐induced granule secretion from human platelets

αB‐crystallin, a low‐molecular‐weight heat shock protein (HSP), has binding sites on platelets. However, the exact role of αB‐crystallin is not clarified. In this study, we investigated the effect of αB‐crystallin on platelet granule secretion. αB‐crystallin attenuated the adenosine diphosphate (ADP)‐induced phosphorylation of p44/p42 mitogen‐activated protein kinase (MAPK) and p38 MAPK. The ADP‐stimulated HSP27 phosphorylation was markedly reduced by αB‐crystallin. αB‐crystallin significantly suppressed the ADP‐induced secretions of both platelet‐derived growth factor (PDGF)‐AB and serotonin. Therefore, our results strongly suggest that αB‐crystallin extracellularly suppresses platelet granule secretion by inhibition of HSP27 phosphorylation via p44/p42 MAPK and p38 MAPK.

Mechanism of collagen-induced release of 5-HT, PDGF-AB and sCD40L from human platelets: Role of HSP27 phosphorylation via p44/p42 MAPK

Collagen plays a crucial role in hemostasis and thrombosis by activating platelets and reportedly induces the phosphorylation of heat shock protein (HSP) 27 in human platelets. However, the exact role of HSP27 phosphorylation in human platelets has not yet been clarified. In the present study, we investigated the mechanism of collagen-induced HSP27 phosphorylation and the role in human platelets. The collagen-effect on the phospholylation of HSP27 was dose-dependent in the range between 0.03 and 1.0 microg/ml. The phosphorylation of p44/p42 mitogen-activated protein kinase (MAPK) was also stimulated by collagen. PD98059, a specific inhibitor of MAPK kinase (MEK1/2), reduced collagen-induced HSP27 phosphorylation as well as p44/p42 MAPK phosphorylation. PD98059 significantly suppressed collagen-induced releases of serotonin (5-HT), platelet-derived growth factor (PDGF)-AB and soluble CD40 ligand (sCD40L) while it had little effect on the platelet aggregation. These results strongly suggest that the collagen-induced phosphorylation of HSP27 via p44/p42 MAPK is sufficient for releases of 5-HT, PDGF-AB and sCD40L from human platelets.

Release of Phosphorylated HSP27 (HSPB1) from Platelets Is Accompanied with the Acceleration of Aggregation in Diabetic Patients

We investigated the relationship between HSP27 phosphorylation and collagen-stimulated activation of platelets in patients with diabetes mellitus (DM). Platelet-rich plasma was prepared from blood of type 2 DM patients. The platelet aggregation was analyzed in size of aggregates by an aggregometer using a laser scattering method. The protein phosphorylation was analyzed by Western blotting. Phosphorylated-HSP27 and PDGF-AB released from platelets were measured by ELISA. The phosphorylated-HSP27 levels at Ser-78 and Ser-82 induced by collagen were directly proportional to the platelet aggregation. Total HSP27 levels in platelets were decreased concomitantly with the phosphorylation. The released HSP27 levels were significantly correlated with the phosphorylated levels of HSP27 in the platelets stimulated by 0.3 μg/ml collagen. The low dose collagen-stimulated release of HSP27 was detected but relatively small in healthy donors. The released levels of PDGF-AB were in parallel with the levels of released HSP27. Area under the curve (AUC) of small aggregation (9-25 μm) induced by 0.3 μg/ml collagen was inversely proportional to the levels of released HSP27. AUC of large aggregation (50-70 μm) was directly proportional to the levels of released HSP27. Exogenous recombinant phosphorylated- HSP27 hardly affected the aggregation or the released levels of PDGF-AB induced by collagen. These results strongly suggest that HSP27 is released from human platelets accompanied with its phosphorylation induced by collagen, which is correlated with the acceleration of platelet aggregation in type 2 DM patients.

Antithrombin III suppresses ADP-induced platelet granule secretion: Inhibition of HSP27 phosphorylation

Antithrombin III (AT-III), an anti-coagulant, has recently been reported to directly affect human platelet functions. However, the exact mechanism of AT-III in platelets remains to be clarified. We have previously shown that adenosine diphosphate (ADP)-induced phosphorylation of heat shock protein 27 (HSP27) via p44/p42 mitogen-activated protein kinase (MAPK) and p38 MAPK is correlated with platelet granule secretion. In the present study, we investigated the relationship between AT-III and the ADP-induced platelet granule secretion. The ADP-induced secretion of platelet-derived growth factor (PDGF)-AB and serotonin (5-HT) were significantly suppressed by AT-III. The ADP-induced soluble CD40 ligand (sCD40L) release was inhibited by either PD98059, a MEK inhibitor, or SB203580, a p38 MAPK inhibitor. AT-III also inhibited the sCD40L release. AT-III markedly attenuated the ADP-induced phosphorylation levels of p44/p42 MAPK and p38 MAPK. Furthermore, the ADP-induced HSP27 phosphorylation was suppressed by AT-III. These results strongly suggest that AT-III directly acts on platelets and suppresses ADP-induced platelet granule secretion due to inhibiting HSP27 phosphorylation via p44/p42 MAPK and p38 MAPK.

(-)-Epigallocatechin gallate selectively inhibits adenosine diphosphate‑stimulated human platelet activation: Suppression of heat shock protein 27 phosphorylation via p38 mitogen‑activated protein kinase

(‑)‑Epigallocatechin gallate (EGCG) is a major component of green tea. It has been demonstrated that EGCG has an antithrombotic effect by inhibiting platelet aggregation. However, the detailed mechanisms underlying the effects of EGCG remain to be elucidated. The present study examined the effects of EGCG on human platelet activation by various stimulators and the exact underlying mechanisms. EGCG suppressed adenosine diphosphate (ADP)‑stimulated platelet aggregation dose dependently between 30 and 70 µM. By contrast, EGCG failed to affect platelet aggregation stimulated by collagen, U46619 (a TP agonist) or ristocetin (an activator of GPIb/IX/V). EGCG attenuated the ADP‑induced phosphorylation of p38 mitogen‑activated protein (MAP) kinase and heat shock protein 27 (HSP27). The ADP‑stimulated release of platelet‑derived growth factor (PDGF)‑AB and the soluble CD40 (sCD40) ligand was inhibited by EGCG. These findings suggest that EGCG selectively inhibits ADP‑stimulated human platelet activation and that EGCG reduces the release of PDGF‑AB and the sCD40 ligand due to suppressing HSP27 phosphorylation via p38 MAP kinase.

Rac regulates collagen-induced HSP27 phosphorylation via p44/p42 MAP kinase in human platelets.

We previously reported that the collagen-induced phosphorylation of heat shock protein (HSP) 27 via p44/p42 mitogen-activated protein (MAP) kinase is sufficient to induce the secretion of platelet-derived growth factor (PDGF)-AB and the release of soluble CD40 ligand (sCD40L) from human platelets. It has been shown that Rac, which belongs to the Rho family of small GTPases, is involved in the collagen-induced platelet aggregation. In this study, we investigated the role of Rac in the collagen-stimulated release of PDGF-AB and sCD40L in human platelets. Human blood was donated from healthy volunteers and platelet-rich plasma was obtained from the blood samples. The samples were then treated with 1.0 µg/ml collagen for 0, 1, 3, or 5 min and Rac1 activity was determined using the Rac1 Activation Assay kit. We found that collagen stimulated the activation of Rac in human platelets in a time-dependent manner. However, pre-treatment with NSC23766, a selective inhibitor of Rac-guanine nucleotide exchange factor interaction, reduced the collagen-induced platelet aggregation. NSC23766 markedly attenuated not only the collagen-induced p44/p42 MAP kinase phosphorylation, but also the phosphorylation of HSP27 at three serine residues (Ser-15, Ser-78 and Ser-82). In addition, the collagen‑induced release of PDGF-AB and sCD40L was significantly suppressed by NSC23766 in a dose-dependent manner. These results strongly suggest that Rac regulates the collagen-induced HSP27 phosphorylation via p44/p42 MAP kinase in human platelets, resulting in the stimulation of PDGF-AB secretion and the release of sCD40L.

Sphingosine 1-phosphate (S1P) suppresses the collagen-induced activation of human platelets via S1P4 receptor

Sphingosine 1-phosphate (S1P) is as an extracellular factor that acts as a potent lipid mediator by binding to specific receptors, S1P receptors (S1PRs). However, the precise role of S1P in human platelets that express S1PRs has not yet been fully clarified. We previously reported that heat shock protein 27 (HSP27) is released from human platelets accompanied by its phosphorylation stimulated by collagen. In the present study, we investigated the effect of S1P on the collagen-induced platelet activation. S1P pretreatment markedly attenuated the collagen-induced aggregation. Co-stimulation with S1P and collagen suppressed collagen-induced platelet activation, but the effect was weaker than that of S1P-pretreatment. The collagen-stimulated secretion of platelet-derived growth factor (PDGF)-AB and the soluble CD40 ligand (sCD40L) release were significantly reduced by S1P. In addition, S1P suppressed the collagen-induced release of HSP27 as well as the phosphorylation of HSP27. S1P significantly suppressed the collagen-induced phosphorylation of p38 mitogen-activated protein kinase. S1P increased the levels of GTP-bound Gαi and GTP-bound Gα13 coupled to S1PPR1 and/or S1PR4. CYM50260, a selective S1PR4 agonist, but not SEW2871, a selective S1PR1 agonist, suppressed the collagen-stimulated platelet aggregation, PDGF-AB secretion and sCD40L release. In addition, CYM50260 reduced the release of phosphorylated-HSP27 by collagen as well as the phosphorylation of HSP27. The selective S1PR4 antagonist CYM50358, which failed to affect collagen-induced HSP27 phosphorylation, reversed the S1P-induced attenuation of HSP27 phosphorylation by collagen. These results strongly suggest that S1P inhibits the collagen-induced human platelet activation through S1PR4 but not S1PR1.

Efficacy of Granulocyte and Monocyte Adsorption Apheresis for Treatment of Palmoplantar Pustulosis

Palmoplantar pustulosis (PPP) is characterized by neutrophilic pustules with erythema, which are limited to the hands and feet. Although granulocyte and monocyte adsorption apheresis (GMA) has shown remarkable effects on generalized pustular psoriasis, there are few reports of PPP treated with GMA. We treated three refractory PPP patients using GMA weekly for 5 weeks. The skin eruptions were assessed by a 5‐grade score for scales, pustules, and erythema. GMA decreased the total grade from 9 to 2 in patients 1 and 2, and from 7 to 3 in patient 3. The GMA effects were estimated to be excellent in all three patients. Pustule formation and pain disappeared in all cases. The treatment effect lasted for at least 5 months after GMA. GMA was also effective for relieving the arthralgia in one patient, but it recurred at 6 weeks. Based on these findings, GMA could be an effective therapy for refractory PPP.